metastasis of breast cancer

Metastasis of Breast Cancer

Cancer Metastasis – Biology and Treatment

Series Editors

Richard J. Ablin, Ph.D., University of Arizona, College of Medicine and The Arizona CancerCenter, AZ, U.S.A.Wen G. Jiang, M.D., Wales College of Medicine, Cardiff University, Cardiff, U.K.

Advisory Editorial Board

Harold F. Dvorak, M.D.Phil Gold, M.D., Ph.D.Danny Welch Ph.D.Hiroshi Kobayashi, M.D., Ph.D.Robert E. Mansel, M.S., FRCS.Klaus Pantel Ph.D.

Recent Volumes in this Series

Volume 4: Proteases and Their Inhibitors in Cancer MetastasisEditors: Jean-Michel Foidart and Ruth J. MuschelISBN 1-4020-0923-2

Volume 5: MicrometastasisEditor: Klaus PantelISBN 1-4020-1155-5

Volume 6: Bone Metastasis and Molecular MechanismsEditors: Gurmit Singh and William OrrISBN 1-4020-1984-X

Volume 7: DNA Methylation, Epigenetics and MetastasisEditor: Manel EstellerISBN 1-4020-3641-8

Volume 8: Cell Motility in Cancer Invasion and MetastasisEditor: Alan WellsISBN 1-4020-4008-3

Volume 9: Cell Adhesion and Cytoskeletal Molecules in MetastasisEditors: Anne E. Cress and Raymond B. Nagle

Volume 10: Metastasis of Prostate CancerEditors: Richard J. Ablin and Malcolm D. Mason

ISBN 1-4020-5846-2

VOLUME 11

Volume 11: Metastasis of Breast Cancer

ISBN 1-4020-5866-7

ISBN 1-4020-5128-X

Editors: Robert E. Mansel, Oystein Fodstad and Wen G. Jiang

Cancer

Edited by

and

Metastasis of Breast

Cardiff University School of Medicine

University of South Alabama

Cardiff University School of Medicine

Robert E. Mansel

Oystein Fodstad

W en G. Jiang

A C.I.P. Catalogue record for this book is available from the Library of Congress.

Published by Springer,P.O. Box 17, 3300 AA Dordrecht, The Netherlands.

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ISBN 978-1-4020-5866-0 (HB)ISBN 978-1-4020-5867-7 (e-book)

TABLE OF CONTENTS

Chapter 1 …………………………………………… 1 Metastasis of breast cancer: an introduction Robert E. Mansel, Oystein Fodstad, and Wen G. Jiang

Chapter 2 …………………………………………… 7 The genetic control of breast cancer metastasis Rajeev S. Samant, Oystein Fodstad, and Lalita A. Shevde

Chapter 3 …………………………………………… 31 BRCA1 in initiation, invasion, and metastasis of breast cancer: a perspective from the tumor microenvironment

Shaun D. McCullough, Yanfen Hu, and Rong Li

Chapter 4 …………………………………………… 47 Cell motility and breast cancer metastasis

Marc E. Bracke, Daan De Maeseneer, Veerle Van Marck, Lara Derycke, Barbara Vanhoecke, Olivier De Wever, and Herman T. Depypere

Chapter 5 …………………………………………… 77 Tight junctions and metastasis of breast cancer

Tracey A. Martin

Chapter 6 …………………………………………… 111

Lalita A. Shevde and Judy A. King

List of Contributors …………………………………..……… ix

and metastasis Cell adhesion molecules in breast cancer invasion

Chapter 7 …………………………………………… 137 Endocrine resistance and breast cancer invasion Stephen Hiscox, Julia Gee, and Robert I. Nicholson

Chapter 8 …………………………………………… 151 The role of aromatase and other oestrogen producing enzymes in mammary carcinogenesis

Mohamed Salhab and Kefah Mokbel

Chapter 9 …………………………………………… 171 The role of the HGF regulatory factors in breast cancer

Christian Parr and Wen G. Jiang

Chapter 10 …………………………………………… 203 The insulin-like growth factor-1 ligand in breast cancer management

Yoon M. Chong, Ash Subramanian, and Kefah Mokbel

Chapter 11 …………………………………………… 219 Lymphangiogenesis and metastatic spread of breast cancer

Mahir A. Al-Rawi and Wen G Jiang

Chapter 12 …………………………………………… 241 Breast cancer secreted factors alter the bone microenvironment

Valerie A. Siclari, Theresa A. Guise, and John M. Chirgwin

Chapter 13 …………………………………………… 259 Cyclooxygenease-2 and breast cancer

Gurpreet Singh-Ranger and Kefah Mokbel

279 Prognostic and predictive factors in human breast cancer

Soe Maunglay, Douglas C. Marchion, and Pamela N. Münster

vi

…………………………………………… Chapter 14

Table of contents

Chapter 15 …………………………………………… 307 Molecular imaging in metastatic breast cancer

C.P. Schröder, G.A.P. Hospers, P.H.B. Willemse, P.J. Perik, E.F.J. de Vries, P.L. Jager, W.T.A. van der Graaf, M.N. Lub-de Hooge, and E.G.E. de Vries

Chapter 16 …………………………………………… 321 Detection of disseminated tumor cells in the bone marrow and blood of breast cancer patients

Volkmar Müller and Klaus Pantel

Chapter 17 …………………………………………… 333 Sentinel lymph node biopsy in early-stage breast cancer

Amit Goyal and Robert E. Mansel

Chapter 18 …………………………………………… 355 Surgical management of patients with metastatic breast cancer

Adam I. Riker, SuHu Liu, Mona Hagmaier, Matthew J. D. D’lessio, and Charles E. Cox

Chapter 19 …………………………………………… 373 Therapeutic aspect of metastatic breast cancer: chemotherapy

Robert C.F. Leonard and Thinn P. Pwint

Chapter 20 …………………………………………… 389

Allan Lipton

Chapter 21 …………………………………………… 405 Hormonal therapies of metastatic breast cancer: the past and the present

Jürgen Geisler and Per Eystein Lønning

Index …………………………………………… 425

viiTable of contents

in breast cancer The diagnosis and treatment of bone metastases

LIST OF CONTRIBUTORS

Al-Rawi, Mahir A., MB, BCh, PhD, Metastasis and Angiogenesis Research Group, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, UK

Bracke, Marc, PhD, Professor, Laboratory of Experimental Cancer Research, Department of Experimental Cancer Research, Radiotherapy and Nuclear Medicine, University Hospital, De Pintelaan 185, B-9000 Gent, Belgium

Chirgwin, John M., PhD, Professor of Internal Medicine, Division of Endocrinology, University of Virginia PO Box 801401, Charlottesville VA 22908-1401, USA

Cox, Charles E., M.D., University of South Florida, Moffitt Cancer Center, 13902 Magnolia Drive, Tampa, FL 33612, USA; Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama, USA

D’Alessio, Matthew J., MD, University of South Alabama-Mitchell

De Maeseneer, Daan, Laboratory of Experimental Cancer Research,

Medicine, University Hospital, De Pintelaan 185, B-9000 Gent, BelgiumDepartment o f Experimental Cancer Research, Radiotherapy and Nuclear

George’s Hospital, London, SW17 0QT, UK

Cancer Institute, 301 North University Blvd., MSB 2015, Mobile, AL36688, USA

Chong, Yoon M., Department of Breast & Endocrine Surgery, St.

ix

de Vries, E.F.J., Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, Groningen, The Netherlands

de Vries, Elisabeth G.E., MD, PhD, Department of Medical Oncology, University of Groningen and University Medical Center, P.O. Box 30.001, 9700 RB, Groningen, The Netherlands.

De Wever, Olivier, Laboratory of Experimental Cancer Research, Depart-

Medicine, University Hospital, De Pintelaan 185, B-9000 Gent, Belgium

Depypere, Herman T., Department of Gynaecological Oncology, University Hospital, De Pintelaan 185, B-9000 Gent, Belgium

Derycke, Lara, Laboratory of Experimental Cancer Research, Depart-


Fodstad, Oystein, PhD, Director of Research, Professor of Cancer Biology and Pharmacology, Michell Cancer Institute, University of South Alabama, 307 N. University Blvd., MSB 2015, Mobile, AL 36688, USA

Gee, Julia, Tenovus Centre for Cancer Research, Welsh School of Phar-

Geisler, Jürgen, Consultant, Section of Oncology, Department of Medicine, Haukeland University Hospital, N-5021 Bergen, Norway

Goyal, Amit, MB, MD, Lecturer, University Department of Surgery, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, UK

Guise, Theresa A., Division of Endocrinology, University of Virginia PO Box 801401, Charlottesville VA 22908-1401, USA

List of contributorsx

ment of Experimental Cancer Research, Radiotherapy, and Nuclear


macy, Cardiff University, Heath Park, Cardiff, CF10 3XF, UK

Hagmaier, Mona, PA, University of South Alabama-Mitchell Cancer Institute, 301 North University Blvd., MSB 2015, Mobile, AL 36688, USA

Hiscox, Stephen, Tenovus Centre for Cancer Research, Welsh School of

Hospers, G.A.P., Departments of Medical Oncology. University Medical Centre Groningen, Groningen, The Netherlands

Hu, Yanfen, Department of Biochemistry and Molecular Genetics, 1300 Jefferson Avenue, School of Medicine University of Virginia, Charlottesville, VA 22908, USA

Jager, P.L., Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, Groningen, The Netherlands

Jiang, Wen G., MB, BCh, MD, Professor of Surgery and Tumour Biology, Metastasis and Angiogenesis Research Group, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, UK

King, Judy, MD, PhD, Associated Professor of Pathology, Department of Pathology, University of South Alabama Medical School, Mobile, Alabama, USA

Wales Cancer Institute, Singleton Hospital, Swansea,Wales, UK

Li, Rong, PhD, Associate Professor, Department of Biochemistry and Molecular Genetics, 1300 Jefferson Avenue, School of Medicine University of Virginia, Charlottesville, VA 22908, USA

Lipton, Allan, MD, Pennsylvania State University, College of Medicine,

Hershey, PA 17033, USA

Liu, SuHu, MD, PhD, University of South Alabama-Mitchell Cancer Institute, 301 North University Blvd., MSB 2015, Mobile, AL 36688, USA

List of contributors xi

Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA

Leonard, Robert C.F., Professor of Clinical Oncology, South West

Pharmacy, Cardiff University, Heath Park, Cardiff, CF10 3XF, UK

Medicine, Haukeland University Hospital, N-5021 Bergen, Norway

Lub-de Hooge, M.N., Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, Groningen, The Netherlands

Mansel, Robert E., MS, FRCS, CBE, Professor of Surgery, University Department of Surgery, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, UK

Marchion, Douglas C., PhD, Experimental Therapeutics and Breast Medical Oncology, Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center, 12902 Magnolia Dr, Tampa, FL 33612, USA

Martin, Tracey A., PhD, Lecturer, Metastasis and Angiogenesis Research Group, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, UK

Maunglay, Soe, MD, Experimental Therapeutics and Breast Medical Oncology, Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center, 12902 Magnolia Dr, Tampa, FL 33612, USA

McCullough, Shaun D. Department of Biochemistry and Molecular Genetics, 1300 Jefferson Avenue, School of Medicine University of Virginia, Charlottesville, VA 22908, USA

Mokbel, Kefah, MB, BS, MS, FRCS, Consultant Breast Surgeon at St. George’s and The Princess Grace Hospitals, Professor at Brunel Institute

Nottingham Place, London, W1U 5NY, UK

Oncology and Experimental Therapeutics, H. Lee Moffitt Cancer Center, 12902 Magnolia Drive, MRC 4E, Tampa, FL 33612, USA

List of contributorsxii

Center Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany

Lønning, Per Eystein, Professor, Section of Oncology, Department of

(St. George’s Medical School), The Princess Grace Hospital, 42–52 of Cancer Genetics & Pharmacogenomics, Reader in Breast Surgery

Munster, Pamela N., MD, Associate Professor, Division of Breast

Müller, Volkmar, MD, Department of Gynecology, University Medical

Nicholson, Richard I., PhD, Professor of Cancer Pharmacology School of Pharmacy, Cardiff University, Redwood Building, Cardiff, UK

Pantel, Klaus, MD, PhD, Professor Dr, Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Martinistraße 52, D-20246, Hamburg, Germany

Parr, Christian, PhD, Metastasis and Angiogenesis Research Group, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, UK

Perik, P.J., Departments of Medical Oncology. University Medical Centre Groningen, Groningen, The Netherlands

Pwint, Thinn P., South West Wales Cancer Institute, Singleton Hospital, Swansea, Wales, UK

Riker, Adam, MD, Chief, Surgical Oncology, Associate Professor, Mitchell Cancer Institute, University of South Alabama, 307

Salhab, Mohemmad, St. George’s Hospital, London, SW17 0QT, UK

Samant, Rajeev S., PhD, Cancer Institute, University of South Alabama, 307 N. University Blvd., MSB 2015, Mobile, AL 36688, USA

Schröder, C.P., Departments of Medical Oncology. University Medical Centre Groningen, Groningen, The Netherlands

Shevde, Lalita A., PhD, Cancer Institute, University of South Alabama, 307 N. University Blvd., MSB 2015, Mobile, AL 36688, USA

Siclari, Valerie A., Division of Endocrinology, University of Virginia PO Box 801401, Charlottesville VA 22908-1401, USA

List of contributors xiii

N. University Blvd., MSB 2015, Mobile, AL 36688, USA

Singh-Ranger, Gurpreet, St. George’s Hospital, London, SW17 0QT, UK

van der Graaf, W.T.A., Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, Groningen, The Netherlands

van Marck, Veerle, Laboratory of Experimental Cancer Research, Depart-


Vanhoecke, Barbara, Laboratory of Experimental Cancer Research,


Willemse, P.H.B., Departments of Medical Oncology. University Medical Centre Groningen, Groningen, The Netherlands

List of contributorsxiv

St. George’s Hospital, London, SW17 0QT, UK Subramanian, Ash., Department of Breast & Endocrine Surgery,


Department of Experimental Cancer Research, Radiotherapy, and Nuclear

1 R.E. Mansel et al. (eds.), Metastasis of Breast Cancer, 1–5. © 2007 Springer.

Chapter 1

METASTASIS OF BREAST CANCER: AN INTRODUCTION

Breast cancer is the leading female cancer in Europe and in the USA

and amongst the cancer types with high incidence in the rest of the world.

the female population in industrialised countries. The incidence of breast

group. Metastasis, the spread of breast cancer to other locations in the body, is the main reason that leads to the mortality in the patients. The past decades have seen a significant progress in understanding the molecular and cellular mechanisms of cancer metastasis and development of new diagnostic, prognostic and predictive tools. Some of the new discoveries have been translated into clinical practice. This book aims at providing the current knowledge in the area of molecular and cellular basis of breast cancer metastasis, biological factors that influence the metastatic process, developments in the diagnosis of metastatic breast cancer, and current thinking in the management of metastatic breast cancer.

Robert E. Mansel1, Oystein Fodstad2, and Wen G. Jiang1

Cancer derives from a collection of multiple genetic aberrations, and the same can be said as to the development of metastasis. The metastatic charac-teristics, which may be predisposed or acquired during the development

1Metastasis and Angiogenesis Research Group, Cardiff University School of MedicinHeath Park, Cardiff CF14 4XN, UK; 2Michell Cancer Institute, University of South Alabama, University of South Alabama, 307 N. University Blvd., MSB 2015, Mobile, AL 36688, USA

In the UK and USA, approximately one in every ten women will contract the disease in their lifetime and it is amongst the leading cause of death in

cancer increases with age and is generally peaked in the 50–60 age

2

or metastasis- related genes. The past decades have witnessed the identifi-cation of an increasing number of such genes. New technologies, such as differential display, microarrays, and other high-throughput technologies have aided the discovery. Some of the key genes and genetic control mechanisms are discussed by Samant et al. in chapter 2. Beyond the tradi-tionally recognised metastasis-related genes, some of the genes previously known only linked to the development and initiation of breast cancer have also shown to be involved in invasion and metastasis. One example, the BRCA1 gene, is discussed by Li et al. in chapter 3. The process of metastasis is collectively known as the metastatic cascade, during which a number of steps have to be completed by breast cancer cells in order to successfully establish a metastatic focus at a dis-tant location. The process, although intimately linked to genetic mecha-nisms, is also orchestrated by the interaction between cancer cells and its surrounding environment. The interaction between cancer cells and the surrounding matrix is extensively discussed by Bracke et al. in chapter 4. In addition, the chapter has indepth discussion of the role of cell motility and invasion in the metastasic spread of breast cancer. A number of cellular structures are known to participate in the control

ones include the cytoskeletal system, cell adhesion (both cell–cell and

Martin in chapter 5 and Shevde and King in chapter 6. The role of matrix and interaction between cancer cells and extracellular matrix has also been documented in chapter 4. Both intrinsic and exogenous factors have important influence over the metastatic potential of breast cancer cells. Three of these factors/complexes

(HGF) IGF-1, and cyclooxygenase-2 (COX-2), respectively HGF is one of the typical examples that cancer cells can be regulated by exogenous factors generated by other cell types than cancer cells. HGF, generated mainly by stromal fibroblasts in breast tumours, can be activated by a complex enzymatic cascade and ultimately acts on both cancer cells and endothelial cells. The responses from the respective cell types lead to increased metastatic potential and angiogenesis. Chapter 9, however, has emphasised the impact and importance of the regulators of HGF, include-ing matriptases, HGF activator (HGFA), and the HGFA inhibitors which work in orchestration in the activation and inactivation of pro-HGF. The importance of the IGF-I axis in the biology of breast cancer and predict-ing the clinical outcome have been discussed in chapter 10. The role of

Mansel, Fodstad, and Jiang

mechanisms by which cancer cells metastasise. Those well-established

cell–matrix), and matrix-related mechanisms. Two of the specific struc-

have been discussed in chapters 9, 10, and 13 – hepatocyte growth factor

COX-2 in breast cancer has been hotly debated in recent years. In chapter 13, Mokbel et al. have discussed the role of COX-2 in the metabolism of

of the disease, is also governed by a number of genetic mechanisms,

tures, cell–cell adhesion and tight junctions, have been discussed by

3

arachidonic acid and prostaglandins, the biological role of COX-2 in breast cancer both in vitro and in vivo, the clinical value of using COX-2 as a prognostic predictor, and ways to interfere COX-2 in breast cancer therapies. The metastatic spread of breast cancer cells follows two main routes: the vascular and the lymphatic. The vascular route, which is also intimately linked to the angiogenesis process, has been extensively documented in the past decade in reviews and books. The current volume has a focus on the lymphatic route of cancer spread. Recent development in the genera-tion of new lymphatic vessels, lymangiogenesis, has been documented by Al-Rawi and Jiang in chapter 11. Novel lymphatic markers and

Goyal and Mansel have discussed nodal metastatis, focusing on sentinel node metastasis and the implications in cancer treatment. The role of oestrogen and oestrogen receptors in breast cancer development has been very well established in the past decade. However, the impact of the female hormone in the invasion and metastasis of breast cancer is beginning to be dissected. In chapter 7, Hiscox et al. have discus-sed the impact of the hormone, hormone receptor, and hormone resistance in the invasion and metastasis of breast cancer. Links between hormone resistance and aberration in cell adhesion complex and growth factor signalling are being established and may have important implications in the understanding of endocrine regulation and the metastatic cascade.

Mokbel et al. in chapter 8, in which the role of aromatase in the meta-bolism of female hormones as well as the clinical and prognostic value of the enzymes are discussed. One of the main destinations for metastatic breast cancer cells in the body is the bone. Bony metastasis is the leading metastatic event in clinical breast cancer. Although much is yet to be learned in the biology of bone metastasis, Siclari et al. (chapter 12) have extensively discussed the factors secreted by breast cancer cells in the development of bone metastasis. In

number of new molecules have been documented to actively participate in the bone metastasis, as discussed in chapter 12. For example, the CCN family, interleukins (IL-8, IL-11, and IL-18) have been shown to play a key part in the development of bone metastasis. These new discoveries may also help in tailoring treatment of these debilitating metastases. Further to these development in understanding the biology of bone metastasis, the

1. Metastasis of breast cancer

lymphangiogenic factors have also been documented. In chapter 17,

The other hormone-related enzyme, aromatase, has been documented by

addition to the well-known molecules such as PTHrP and RANKL, a

clinical front on the current methods of diagnosis and strategies of treat-ment of bone metastasis from breast cancer have been given by Lipton (chapter 20).

Prognosis and predictive factors for metastatic breast cancer have also been a topic of discussion. While the clinical and pathological factors

4

have their clinical value and implications, recent years have witnessed the rapid progress in searching for new markers including molecular markers. In chapter 14, Münster et al. have provided a thorough review of the traditional and latest factors that have been used in predicting and profiling metastatic breast cancer. While the value of nodal involvement, tumour size, tumour grade, age, histology, and proliferation markers have held their value, the authors have documented the latest development in bone marrow micrometastasis, Her-2, and uPA. Furthermore, the chapter has also provided an updated view on the development of molecular profiling including using DNA microarrays, gene pattern arrays, which may hold fresh information in this important area of metastasis research. In chapter 15, Schröder et al. have explored the exciting new horizon in the diagnosis of metastatic breast cancer, molecular imaging. New methods (e.g., PET and FDG-PET) and labelling techniques have been explored. Recent work on labelling HER-2 and ER as imaging tool has also been explored. The role of micrometastasis in breast cancer metastasis has gained increasing recognition in the past decade. Müller and Pantel (chapter 16) have provided a current view of the methods and molecular markers used in the detection of disseminating tumour cells in the blood of patients with breast cancer. The recent development in analysing the disseminating

that tumour cells acquire the genetic changes relevant to their metastatic capacity early in tumorigenesis, challenging the traditional view that tumour cells acquire their metastatic genotype and phenotype late during tumour development. Investigations into the disseminating cancer cells in bone marrow and the blood have provided prognostic information and may prove valuable in decision making in the clinic. In chapter 17, recent development in sentinel node metastasis has been explored. Current methods of detecting sentinel node and details of current studies have been analysed. Critically, a model of training for performing sentinel node biopsy has been suggested, pointing to an important aspect of supervised training in the success in conducting sentinel node biopsies. The impor-tance of sentinel node in decision making, prognosis, and predicting clinical outcome has also been discussed in chapters 14 and 18.

Management of metastatic breast cancer has been covered by Riker

(chapter 21), from surgical, chemotherapeutic, and hormonal aspects,

Mansel, Fodstad, and Jiang

Diagnosis of metastatic breast cancer has been a long-debated issue.

tumour cells in primary and in early-stage breast cancer have indicated

(chapter 18), Pwint and Leonard (chapter 19), and Geisler and Lønning

respectively, together with Lipton (chapter 20), who deals with the diagnosis and management of the bony metastasis. In chapter 18, the pat-tern of breast cancer metastasis has been overviewed. Most importantly, possible surgical options to the difficult secondary lesions, such as those seen in the liver, bones, brain, and lungs, have been documented. In

5

chemotherapy in dealing with metastatic breast cancer. Endocrine therapy has been an important pillar in the management of breast cancer. In chapter 20, the current strategy in the management of bone metastasis has been documented, with emphasis on the use of biphosphonates and radiotherapy. In chapter 21, Geisler and Lønning have comprehensively reviewed the available endocrine therapies, ranging from ovarian sup-pression option to the latest anti-hormone methods, in the management

emphasised by the authors. Although chemotherapy of breast cancer is improved and targeted therapies have been introduced for the treatment of breast cancer, the role of endocrine therapy within the adjuvant and metastatic setting has not been weakened, as the authors stated. This is

aramatase inhibitors, tamoxifen, and other new anti-hormone modalities. Metastasis of breast cancer has been a challenge from biological

knowledge to this important area of breast cancer.

chapter 19, Leonard and Pwint have discussed the current strategies of

metastatic breast cancer in pre- and postmenopausal women have been of metastatic breast cancer. Different strategies for the treatment of

clearly supported by the recent results from the large-scale studies of

research to clinical management. The current book brings a wealth of

1. Metastasis of breast cancer

7

© 2007 Springer.

Chapter 2

THE GENETIC CONTROL OF BREAST CANCER METASTASIS

Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36688, USA

Abstract: Metastasis of breast cancer is a complex event involving coordinated cross-talk of several proteins. Genes that control the resultant metastasis can be broadly classified as metastasis promoter genes (MPGs) and metastasis suppressor genes (MSGs). There is an explosion of information in the studies that focus on these genes; however, thus far, a very few of them are actually tested clinically and/or in vivo functionally. In this chapter we will focus on the metastasis controlling genes that have been tested for clinical relevance or functional properties in breast cancer metastasis models.

Keywords: metastasis suppressing genes (MSGs), functional validation, gene discovery

1. INTRODUCTION

Breast cancer is capable of having unusually long latency. It is also capable of spreading at a variety of secondary sites that include vital organs such as brain, lungs, and bones (1). Metastasis is the spread and concomitant growth of the cancer at a discontinuous site. The chances of survival from metastatic breast cancer are less than 5%. Thus effective prevention and treatment of metastasis is a major focus of research in breast cancer. Several cells are shed by the primary tumor in the cir-culation, however only a subset of cells seems to form metastasis. There are several models explaining the origin of metastasis. A unifying fact that has emerged thus far is it is controlled by the genetic makeup of

control breast cancer metastasis, one can notice two distinct groups: metastasis promoting genes (MPGs) and metastasis suppressor genes (MSGs). MSGs suppress the spread and growth of the cancer at a

Rajeev S. Samant, Oystein Fodstad, and Lalita A. Shevde

the cancer cells (2, 3). In the published literature regarding genes that

R.E. Mansel et al. (eds.), Metastasis of Breast Cancer, 7–30.

the human genome project and gene profiling studies has lead to a substantial addition in the number of genes in these categories. However in this chapter we discuss only the human genes that are tested in vivo functionally (using animal models) or those that have been validated in multiple controlled clinical studies.

Breast cancer is a term broadly applied to infiltrating ductal, infiltrating lobular, medullary, tubular, and mucinous carcinomas. Despite improved understanding of the molecular mechanisms of metastasis, the genetic information available is not categorized to a definite subtype. Thus, there still remains a gap between integrating the relevance of the findings and a definite subtype/category. The experimental findings for one subtype (or cell line) may not hold true for another subtype of breast cancer. Hence, although a metastasis gene expression signature is identified, the patient subpopulation that relates to these genes is not clear.

There are several key questions in the genetic control of breast cancer.

1. What genetic changes are necessary and sufficient for cells to become metastatic?

2. Are there global metastasis controlling genes? 3. Are metastases clonal? 4. Does the metastasis signature exist in primary tumor? 5. What controls the gene expression change with the onset of

metastasis? 6. 7. Are there specific genes that direct the metastasis to a specific

secondary site? 8. What are the host factors and secondary site microenvironment

that contribute to metastasis? The answers to these questions will have a profound impact on the

diagnosis, prognosis, and treatment (therapeutic intervention) of breast cancer.

2. RELATED GENES

1970s but was advocated aggressively after a decade. Metastasis promoting genes were identified as genes that promote breast tumor aggressiveness, this included invasion and migration. However successful colonization at the secondary site (that will lead to macroscopic metastasis) was only

8 Samant, Fodstad, and Shevde

Do the metastasis-associated genes have a normal physiologic role?

IDENTIFICATION OF METASTASIS-

The concept of metastasis-associated genes was launched in the early

secondary site without altering tumor formation whereas MPGs do the opposite (4–6). The phenomenal outburst of information concomitant to

There are several innovative tools and tricks used for the gene identification.

The more traditional approach compared cell lines that differ in metastatic potential using karyotyping to look for additions/ deletions/translocations. These techniques point to a locus or a region on a chromosome that bears a metastasis-related gene. However there is a very involved discovery process for the identification of the exact gene. A more recent approach is to monitor differential gene expression using differential display or subtractive hybridization. Contemporary methods involve the use of recently developed microarray technologies. The success is limited due to the vast amount of data obtained and false positives.

Also needless to say that differential gene expression is not the only way to regulate gene function, posttranslational modifications such as

butions and these are apparent with the advent of modern proteomic techniques.

3. BREAST CANCER METASTASIS CONTROLLING GENES

These are the proteins that are implicated to influence critical steps in the metastasis of breast cancer resulting in promotion of metastasis. These critical steps and the genes involved are summarized in Table 1.

3.1.1. Immune evasion

Cancer cells can grow by escaping from the attack of immune cells, thus disrupting the host immune system, which is progressively sup-pressed as a result of tumor progression and metastasis. The molecular mechanisms by which cancer cells evade the host immune system have been investigated in mouse models and clinical samples.

Tumor cells employ several mechanisms to evade immune response including loss of tumor antigen, alteration of HLA class I antigen, defec-tive death receptor signaling, lack of costimulation, immunosuppressive cytokines, and immunosuppressive T cells (9). Gutierrez et al. showed that FasL expression by breast tumor plays a central role in the induction

92. Genetic control of breast cancer metastasis

1.

2.

3.

phosphorylation, glycosylation, acetylation, etc. have significant contri-

3.1. Metastasis-Promoting Genes (MPGs)

discernable using xenograft studies or mouse mammary tumor model studies. On the other hand search for metastasis suppressing genes had started in mid- to late 1980s and the field really flourished at the turn of the millennium (7, 8).

lymphocyte apoptosis and impairs expression of NKG2D and T-cell activation. A study by Ueno et al. reports that compared with healthy female controls, breast cancer patients, especially those with liver metastases, have higher circulating sFas levels (13).

Table 1. Critical steps and genes involved in breast cancer metastasis

Steps in breast cancer metastasis

Genes involved

1 Immune evasion Fas and FasL 2 Adhesion Selectins, integrins, lectins, and cadherins 3 Invasion (proteolysis) Metalloproteinases, uPA, serine

proteinases, and cathepsins. 4 Motility Autotaxin, and hepatocyte growth factor

(HGF) 5 Chemo attractants (tumor

environment) Osteonectin (SPARC), CXCR4, and CCR7

6 Cytoskeletal rearrangement S100A4 7 Cell survival Osteopontin 8 Gene regulation (chromatin

remodeling) MTA1

9 Molecules with mechanisms COM1, RKIP

3.1.2. Adhesion

Metastatic cells need to detach from the primary site and attach at the secondary site. Thus it needs an intricate expression control of various adhesion molecules on the cell surface in space and time (14). Specific families of adhesion molecules whose expression correlates with meta-stasis include selectins, integrins, lectins, and cadherins. Details about these molecules have been discussed by Shevde and King in chapter 6.

3.1.3. Invasion (Proteolysis)

The degradation of the extracellular matrix is mediated by a number of families of extracellular proteinases. These families include the serine proteinases, such as the plasminogen-urokinase plasminogen activator

like cathepsin D and L (24–27), and the zinc-dependent matrix metallo-proteinases (MMPs). There are many observations from various research groups highlighting the central role of MMP-driven extracellular matrix


yet to be confirmed

(uPA) (15,16) and leukocyte elastases (17–23), the cysteine proteinases,

of apoptosis of infiltrating Fas-immune cells providing a mechanism for tumor immune privilege (10). It was also observed that FasL in breast tissue is functionally active and that tamoxifen inhibits FasL expression, allowing the killing of cancer cells by activated lymphocytes (11). Fas exists in two forms, transmembrane and soluble (sFas). A study by Bewick et al. suggests that plasma levels of sFas may be a valuable clinical pro-gnostic factor in predicting outcome for patients with metastatic breast cancer undergoing high-dose chemotherapy (12). sFas induces host

3.1.4. Motility There are several secreted signals that decide motility in cancers. One

of the key factors that affect motility is the autocrine motility factor, autotaxin.

Autotaxin

Autotaxin (ATX) is a novel metastasis-enhancing motogen and angio-genesis factor. Yang et al. found that the expression of ATX mRNA was closely linked to invasiveness of breast cancer. This was supported by immunohistochemical analysis of the breast tissues. MDA-MB-435S breast cancer cells, that express higher amount of ATX mRNA, show greater relative invasiveness to fibroblast-conditioned medium than MCF7, MDA-MB-231, and HBL-100 breast cancer cells. Furthermore, ATX-transfected MCF7 cells showed increased motility and invasive-ness compared to vector-transfected MCF7 cells (34).

Hepatocyte growth factor (HGF) or scatter factor (SF)

Hepatocyte growth factor (HGF) has been reported as the cause of many biological events, including cell proliferation, movement, invasive-ness, morphogenesis, and angiogenesis. Sheen-Chen et al. reported that breast cancer patients with more advanced TNM staging were shown to have higher serum soluble HGF. Thus, preoperative serum soluble HGF levels might reflect the severity of invasive breast cancer (35). This is sup-ported by a paper by Taniguchi et al. that reports a significant increase in the circulating level of HGF in primary breast cancer patients as com-pared to healthy controls. Additionally, 82.9% patients with recurrent breast cancer had an increase in the serum HGF level (36). Yamashita et al. measured immunoreactive (ir)-HGF concentration in tumor extracts of 258 primary human breast cancers and found that breast cancer patients with high ir-HGF concentration had a significantly shorter relapse-free and overall survival rate when compared to those with low ir-HGF concentration. Thus hepatocyte growth factor is a strong and independent predictor of recurrence and survival in human breast cancer (37). There are several cell line and animal model studies that support


cancer dissemination. High levels of two MMPs (i.e., MMP-2 and stro-melysin-3) have been found to correlate with poor outcome in patients with breast cancer, (28–30). Batimastat reduced both lung colonization and spontaneous metastasis of a highly malignant rat mammary cancer

by antisense oligodeoxynucleotides prevented invasion of an artificial (31). In mouse mammary cancer cell lines, inhibition of stromelysin-1

remodeling in mammary gland development, breast cancer, and breast

basement membrane (32). The ratio of active to latent form of MMP-2 increased with tumor progression in invasive breast cancers (33).

This resulted in increased adhesion of tumor cell lines to bone marrow-derived endothelial cells and transendothelial migration of cancer cells (41). Martin et al. showed that HGF decreased transepithelial resistance and increased paracellular permeability of two human breast cancer cell lines, MDA-MB-231 and MCF7. HGF modulates the levels of several tight junction molecules including occludin, claudin-1 and -5, JAM-1 and -2 in these cells. Thus, HGF disrupts tight junction function in human breast cancer cells by effecting changes in the expression of tight junction molecules (42). Using multiple approaches including ribozymes

serine protease inhibitors of HGF activity (43), the Jiang laboratory has demonstrated that HGF plays a crucial role in cancer metastasis (48).

3.1.5. Chemo attractants (Tumor environment)

Osteonectin


(43, 44), NK4 (a variant form of HGF) (45-47), and novel Kunitz-type

SPARC (secreted protein acidic and rich in cysteine), also known as osteonectin is a secreted glycoprotein which is detected in a number of normal and neoplastic human tissues in vivo. It is an extracellular matrix (ECM)-associated protein which is postulated to regulate cell migration, adhesion, proliferation, and matrix mineralization. Early studies by Graham et al. report that loss of ER expression may lead to overexpression of osteonectin and contribute to a poorer differentiated, more invasive phenotype (49). SPARC is also reported to decrease levels of TIMP-2, causing an increase in the activation of MMP-2 in breast cancer cells (50). Additionally, osteonectin is indirectly controlled by c-Jun and can increase invasion and motility of MCF7 breast cancer cells (51). Campo McKnight et al. showed that osteonectin isolated from conditioned media of several breast cancer cell lines enhances the migration of breast cancer cells to vitronectin (52). Jacob et al. showed that the purified active factor from bone and from marrow stromal-cell-conditioned medium is a low glycosylated osteonectin that specifically promotes the invasive

this patient data. HGF stimulates tumor growth and tumor angiogenesis of human breast cancers in the mammary fat pads of athymic nude mice (38) and also promotes spontaneous metastasis of human metastatic breast carcinoma MDA-MB-435 cells (39). Mechanistic insight about HGF was developed when Matteucci et al. reported that HGF enhanced CXCR4 mRNA and protein expression in MCF7 (low invasive) carcinoma cells; while in response to hypoxia, CXCR4 induction was observed in both MCF7 and MDA-MB-231 (highly invasive) carcinoma cells. Thus HGF and hypoxia may contribute to breast carcinoma cell invasiveness by inducing the chemokine receptor CXCR4 (40). Studies by Mine et al. demonstrated that HGF stimulated breast cancer cells by upregulating CD44 expression via the tyrosine kinase signaling pathway.

in breast cancer and as such has a significant bearing on patient prognosis and long-term survival.

Chemokine receptors

Chemokine receptors are defined by their ability to induce directional migration of cells toward a gradient of a chemotactic cytokine (chemotaxis). In particular, the chemokine CXCL12 and its receptor CXCR4 have prominent roles in primary and metastatic breast cancer (56, 57). Binding of CXCL12 to CXCR4 induced activation of the Akt pathway, MAPK pathway, and the Jak-Stat pathway, culminating in increased motility, invasion, and survival (58). Abrogating expression of CXCR4 and CXCR3 functionally inhibits growth and metastasis of breast cancer in murine models (59). The clinical significance of CXCR4 in breast cancer is widely reported. CXCR4 associated with increased risk of metastasis to the liver (60–62), CXCR1 was associated with metastasis to the brain (60–62). Patients with chemokine receptor CCR6 positivity were more likely to develop a first metastasis in the pleura. In addition, chemokine receptor CCR7 expression was associated with the occurrence of skin metastases (61). Thus expression of chemokine receptors in the primary tumor predicts the site of metastatic relapse in

that expression of CXCR4 is associated with axillary lymph node status in patients with early breast cancer (63). Similar findings were also

role in the breast cancer metastasis.

S100A4

The calcium-binding S100A4 protein has been associated with increased metastatic capacity of cancer cells, and recent studies have suggested an


patients with axillary node positive breast cancer. Su et al. demonstrated

3.1.6. Cytoskeleton rearrangement

reported by Kang et al. (64). Thus chemokine receptors play a deciding

associated with higher grade of the tumor and poor prognosis.

In clinical specimens, high expression of osteonectin in breast tumor tissues was seen in ductal as well as lobular tumors. Increased expression of osteonectin was seen in Grade 3 and TNM2 and TNM4 tumors. Node-positive tumors also exhibited higher levels of SPARC than node-negative tumors. It was also noted that SPARC was present in high

ability of bone-metastasizing breast cancer cells but not that of nonbone-metastasizing tumor cells (53). These reports are contrasted by a study by Dhanesuan et al. who conclude that SPARC, in fact, is inhibitory to human breast cancer cell proliferation, and does not stimulate migration (54).

levels in NPI2 and NPI3 tumors. Over a 6-year follow-up period, high levels of SPARC was seen to be significantly associated with the overall

correlation with disease-free survival (55). Thus, overall, SPARC appears to play a crucial role in tumor development and aggressiveness

survival of the patients (P = 0.0198). However, there was no significant

3.1.7. Cell survival

Osteopontin Osteopontin (OPN) is a secreted, integrin-binding phosphoprotein that

is produced by a limited number of normal tissues, including bone and other mineralized tissues. OPN expression specifically within the tumor

detected in the plasma of late-stage breast cancer patients (74, 75). Since OPN is expressed by both tumor infiltrating lymphocytes as well as the tumor cells themselves, OPN expression specifically within the tumor cells correlates with patient survival (73).

OPN signaling acts to enhance malignancy by giving the cells a survival/growth advantage. OPN also augments attributes that confer increased aggressiveness by activating expression of genes and functions that contribute to metastasis. In concert with growth factor receptor pathways, such as EGFR and c-met, OPN can accentuate effects of EGF and HGF/scatter factor respectively (76, 77). A recent study reports that OPN induces multiple changes in gene expression that reflect the six

sufficiency in growth signals, insensitivity to antigrowth signals, evading apoptosis, tissue invasion and metastasis, sustained limitless replicative

enhanced incidence of bone metastases by breast cancer cells with combined overexpression of OPN and interleukin-11, which could be

further increased by the overexpression of CTGF (79). Moreover, a specific splice variant of OPN is associated with conferring an aggressivephenotype upon breast cancer cells (80). Thus, in a nutshell, OPN poten-tiates the attributes of tumor cell survival and aggressiveness.

‘‘hallmarks of cancer’’ in a model of breast cancer progression: self-

cells reciprocally correlated with patient survival (72, 73). OPN is

growth genetic instability, and angiogenesis (78). Kang et al. showed

inverse correlation between the expression level of S100A4 and survival of breast cancer patients (65, 66). Functionally, the introduction of S100A4 into MCF7 cells enables the MCF7 cells to grow tumors in mice in the absence of estrogen, i.e., S100A4 confers estrogen-independence upon the breast cancer cells (67). The C-terminal region of S100A4 is important for its metastasis-inducing properties, deletion of the last 15

nonmalignant tumors in neu transgenic mice and in malignant tumors from neu/S100A4 double transgenic mice (69). Clinically, S100A4 expression is an indicator of a poor prognosis for T1N0M0 breast cancer (70). High levels of S100A4 expression in combination with either Met or OPN correlate with adverse prognosis and low survival (70, 71). While there is no single mechanism attributed to S100A4 to increase aggressiveness of cancer cells, the increased levels are undisputedly

amino acids of S100A4 reduced motility/invasion (68). S100A4 regulates cell motility and invasion in epithelial cells lines isolated from


associated with higher grade of the tumor and poor prognosis.

loss of E-cadherin and decreased cytoplasmic beta-catenin. MTA2 expression is correlated with ERalpha protein expression in invasive breast tumors (87). MTA2 binds to ERalpha and represses its activity in human breast cancer cells. Furthermore, MTA2 inhibits ERalpha-mediated colony formation and renders breast cancer cells resistant to estradiol and the growth-inhibitory effects of the antiestrogen tamoxifen (88). Recent studies have also shown that growth factor stimulation of breast cancer cells induces the expression of MTA1 and its interaction with and repression of the estrogen receptor (ER) transactivation func-

hormone independence. Furthermore, the status of the ER pathway modu-lates the expression of MTA3 as well as epithelial-to-mesenchymal transition in human breast tumors (81, 89)

3.1.9. Molecules with mechanism yet to be confirmed

COM1

human breast carcinoma cells upon formation of experimental metastatic tumors. Using primary carcinomas and uninvolved adjacent breast tissue

mRNA were significantly upregulated in the tumors compared to the normal breast tissues (90, 91). Jiang et al. compared a cohort of breast cancer tumors (n-120) with matched normal nonneoplastic mammary

tion, leading to enhanced anchorage-independent growth in vitro and

COM1 was identified as a novel factor which was upregulated in

from breast cancer patients Ree et al. found that the levels of com1

3.1.8. Gene regulation (Chromatin remodeling)

Metastasis-associated genes (MTAs) Metastasis-associated genes (MTAs) represent a rapidly growing

novel gene family. At present, there are three different known genes (MTA1, MTA2, and MTA3) and six reported isoforms (MTA1, MTA1s, MTA1-ZG29p, MTA2, MTA3, MTA3L). MTA1, MTA2, and MTA3 are components of the nucleosome remodeling and deacetylation complex, which is associated with adenosine triphosphate-dependent chromatin remodeling and transcriptional regulation. MTA proteins, as a part of the NuRD complex (nuclear remodeling and deacetylation complex), are thought to modulate transcription by influencing the status of chromatin remodeling (81–84). MTA1mRNA expression directly correlates with metastatic potential (85, 86); however, the function of the MTA1 gene product in tumor progression and metastasis remains unknown. Altered expression of MTA1 has been observed in both premalignant lesion and malignant breast carcinoma, but an elevated nuclear expression was observed in ER-negative carcinomas. MTA3 exclusively expressed in a subset of cells of ER-positive premalignant lesions but not in carcinomas (87). MTA2 expression seems to be unrelated to ER status. Loss of MTA3 expression and more nuclear localization of MTA1 occurred with


PCR. They have reported that COM1 is a nuclear protein, whose expression is reduced in human breast cancer tissues and cancer cell

tumors correlate with the prognosis of the patients and with the long-term overall survival in association with ER status (92, 93). Thus there is an apparent controversy regarding COM1. The mechanism by which COM1 acts is still debatable. However, Bratland et al. compared the growth-regulatory mechanisms of nontumorigenic and estrogen-dependent MCF7 cells with those of the tumorigenic and tamoxifen-resistant subline MCF7/LCC2 in the presence of Vitamin D3. Proliferation of MCF7/LCC2 cells, which revealed constitutive COM1 expression, was

tissues (n = 32) for COM1 using conventional and real-time quantitative

lines. The loss of COM1 protein is primarily from the nuclear compartment in cancer cells. The expression levels of COM1 in breast

inhibited by Vitamin D3. Furthermore, when the com1-negative MCF7 cells were stably transfected with COM1, the resulting MCF7/COM1 cells showed a significant decrease in colony formation (94). These

3.2. The MSG field was launched by the discovery of nm23 (95, 96). This

field realized its momentum at the turn of the last millennium. To date

results indicate that rather than promoting growth, COM1 may participate in the regulatory pathway involved in cellular growth inhibitionwhen recruited by inhibitory signals (94).

Metastasis Suppressor Genes (MSGs)

there are at least 13 metastasis suppressor genes functionally characterized: Nm23, KAI-1, KISS-1, TXNIP (VDUP1), CRSP3, MKK4, Src-suppressed C kinase substrate (SSeCKS) the likely rodent ortholog of human Gravin/AKAP12, RhoGDI2, E-cadherin (encoded by CAD1), Drg-1 (a.k.a. RTP, cap43, and rit42), Tissue inhibitors of metalloproteases (TIMPs), RKIP, and BRMS1; however, not all of them have been characterized for involvement in suppression of breast cancer metastasis (97).

We must mention that most of these studies are based on using human breast cancer xenografts in athymic mice. There are two ways of verifying the functional impact of the metastasis suppressor genes in

cardiac injection (98, 99). 3.2.1. Breast Cancer Metastasis 1 (BRMS1) BRMS1 has been shown to suppress metastasis of a variety of meta-static human breast cancer lines (100, 101). The murine ortholog of BRMS1

(cells injected via tail vein and pulmonary metastasis scored). There are some new models of breast cancer cells metastasizing to bone via intra-

orthotopic mammary fat pad site) or experimental metastasis model animal models; the spontaneous metastasis model (xenograft at the


a transcription co-repressor complex. There is a group of homologous was shown to have similar properties (102). BRMS1 is a member of

involved in chromatin modulation (103). BRMS1 is implicated in regulat-

expression by targeting nuclear factor-kappaB activity (104). DeWald and others have implicated BRMS1 reduction of phosphoinositide signaling in MDA-MB-435 breast carcinoma cells (105). However till date there is no convincing patient study that substantiates the exact role of this protein or loss of expression of BRMS1.

3.2.2. KiSS1

carcinoma MDA-MB-435 cells after transfection with the MSG KiSS-1, implicating its importance in breast cancer (106). Expression of KiSS-1 in breast cancer cells is regulated by direct interaction of transcription

hormone-related protein regulates KiSS-1 in breast cancer cells (108). Studies by Stark and colleagues revealed significantly reduced mRNA expression of MSG KISS-1, KAI1, BRMS1, and MKK4 in breast cancer

ing gap junctions and Cicek et al. have shown that BRMS1 inhibits gene

Lee et al. demonstrated suppression of metastasis in human breast

However, there are conflicting reports about the role of KiSS-1 in breast cancer. When Martin et al. determined the expression and distri-bution of KiSS-1 and its receptor in human breast cancer tissues to identify a possible link between expression levels and patient prognosis, contrary to the intuitive extrapolation from the observations of the ini-tial mouse model studies stated above, levels of expression of KiSS-1 were higher in tumor compared to background tissues and significantly

factors AP-2alpha and SP1 (107). Dittmer et al., showed that parathyroid

brain metastasis (109).

increased in node positive tumors compared to node negative. KiSS-1 expression was also increased with increasing grade and TNM status. There were no such trends with the KiSS-1 receptor. Expression of KiSS-1 was higher in patients who had died from breast cancer than those who had remained healthy whereas expression of the receptor was

more aggressive phenotype (110). This work suggests that KiSS-1 plays a role beyond the initial metastasis repressor in this cancer type.

NM23 is known to be a family of eight proteins occurring in all cellular compartments (110). In vitro correlates of suppression include reduced invasion, motility, and soft agar colonization, and induction of differentiation. Differentiation remains one of the key correlates of altered NM23 expression in multiple model systems. Both in vitro and in vivo studies support a role for this gene in breast differentiation. NM23-H1 transfectants of the human MDA-MB-435 breast carcinoma cell line formed acinar structures, secreted the basement membrane proteins

reduced. Thus, overexpression of KiSS-1 in breast cancer cells results in

3.2.3. NM23


proteins that are BRMS1-like proteins and are collectively or independently

laminin and type IV collagen to the basal side of the acinus, and produced sialomucin in three-dimensional cultures in the laboratory of Bissell (110). A knockout mouse for NM23-M1 exhibited growth retarda-tion and pronounced mammary defects. In virgin mice, ductal elongation and branching was poor and the mammary gland failed to fill the fat pad. These morphological differences were overcome in pregnancy, but a functional defect persisted in feeding pups (111). The breast cancer data support the conclusion that altered NM23 expression levels may be of functional significance in humans.

CD82, also known as KAI1, was identified as a prostate cancer MSG on human chromosome 11p1.2. The product of CD82 is KAI1, a 40- to 75-kDa tetraspanin cell-surface protein also known as the leukocyte cell-surface marker, CD82. Phillips et al., demonstrated a correlation between reduction of metastasis in the MDA-MB-435 model system and increased expression of the Kai-1 protein (111). Downregulation of KAI1 has been found to be clinically associated with metastatic progression in a variety of cancers. Stark et al. revealed significantly reduced mRNA expression of KAI1, in breast cancer brain metastasis (109). Yang et al., showed that

3.2.4. KAI1

KAI1 protein levels were inversely correlated with the metastatic potential of breast cancer cells. Furthermore, examination of KAI1 protein expres-sion in specimens from 81 patients with breast cancer showed high levels of KAI1 protein in normal breast tissues and noninvasive breast cancer

demonstrated significantly lower KAI1 expression (112).

(ductal carcinoma in situ). In contrast, KAI1 expression was reducedin most of the infiltrating breast tumors. More malignant tumors

3.2.5. MKK4 MKK4, located in close proximity to p53 gene, is thought to be a tumor and a MSG. A low-rate MKK4 gene alteration has been found in a few tumor types, including breast and pancreatic cancers (113). Also, the expression of MKK4 is significantly reduced in breast cancer brain metastases (109). A suppressor activity for prostate and ovarian tumor metastasis has also been suggested (114) (115). However, ectopic expression of MKK4 by adenoviral delivery in MKK4-negative cancer lines stimulated the cell proliferation and invasion, whereas knockdown of MKK4 expression by small interference RNA in an MKK4-positive breast cancer cell line, MDA-MB-231, resulted in decreased anchorage-independent growth, suppressed tumor growth in mouse xenograft model, and increased cell susceptibility to apoptosis brought by stress signals such as serum deprivation (109). These results argue that MKK4 functions as a pro-oncogenic molecule instead of a suppressor in breast tumors.


3.2.6. TXNIP

expressed in the breast cancer cell line MCF7, is localized predominantly in the nucleus and exhibits growth suppressive activity. TBP-2 protein localizes to the nucleus in cells treated with an anticancer drug, suberoylanilide hydroxamic acid (116). Estrogen represses TXNIP in MCF7 human breast cancer cells. This repression can be blocked by treatment with the histone deacetylase inhibitor, trichostatin A (117). A

high-fat n-6 diet caused a decrease in the expression of VDUP1 and was associated with a higher number of adenocarcinomas and aggressive tumor phenotype in experimental breast cancer (in rats) (118).

3.2.7. E-cadherin E-cadherin is the prototype member of the cadherin family of calcium-dependent cell–cell adhesion molecules. It is expressed in normal adults in luminal epithelial cells, and is lost concomitantly with tumor pro-

tein 1 (VDUP1) is an endogenous molecule interacting with thioredoxin(TRX), negatively regulating TRX function and being implicated inthe suppression of tumor development and metastasis. TBP-2 ectopically

connection with diet was identified by Escrich et al. who reported that a

gression in breast cancers. In fact, E-cadherin expression is irreversibly lost in >85% of invasive lobular breast cancers. Loss of E-cadherin appears to be an early event in these tumors, since even noninvasive lobular carcinoma in situ frequently lacks E-cadherin (119, 120). This may result from loss of heterozygosity (LOH) at 16q22.1, involving the E-cadherin gene CDH1 (approximately 50%) (121), frequently in combination with mutation (50%) (119, 120, 122) or epigenetic silencing of the remaining CDH1 allele (123–128). The status of the estrogen receptor (ER) can also have regulatory effects on E-cadherin. Absence of the ER results in decreased levels of a metastasis-associated protein,

Thioredoxin-binding protein-2 (TBP-2)/vitamin D3 upregulated pro-

MTA3, which plays a role in chromatin remodeling as part of a larger repressive complex, Mi-2/NuRD. This complex normally represses the transcription factor Snail, which in turn represses E-cadherin. Loss of estrogen signaling reverses the repression of Snail, resulting in its increase and subsequent repression of E-cadherin (129–133). Loss of E-cadherin correlates with ER negativity, supporting this as one possible mechanism for E-cadherin loss in some breast cancers. In general, while E-cadherin expression correlates inversely with histological grade and thus differentiation, its expression is not well correlated with survival. In some studies reduced E-cadherin correlates with shorter metastasis-free

other reports indicate that heterogenous staining of the tumor for E-cadherin is a poor indicator. In contrast, other studies suggested that E-cadherin presence was actually a marker of poor survival. In fact, cells

periods and poor prognosis in node negative patients (124, 134), while


3.2.8. Drg-1 The expression of the Drg-1 (differentiation-related gene-1) protein is significantly reduced in breast tumor cells, particularly in patients with lymph node or bone metastasis as compared to those with localized breast cancer. In studies by Bandopadhyay et al. Drg-1 expression also exhibited significant inverse correlation with the disease-free survival rate of patients and emerged as an independent prognostic factor. The downregulation of the Drg-1 gene appeared to be largely at the RNA level, and the DNA methylation inhibitor, 5-Azacytidine, significantly elevated the Drg-1 gene expression in various breast tumor cell lines. Furthermore, they found that overexpression of the Drg-1 gene suppresses the invasiveness of breast cancer cells in vitro, and this suppression was also achieved by treatment of cells with 5-Azacytidine

(139). Moreover, combination of the two markers, PTEN and Drg-1,

of the most aggressive forms of breast cancer, inflammatory breast cancer (IBC) and invasive micropapillary carcinoma (IMPC), often overexpressE-cadherin (135–137). Thus, evaluating E-cadherin expression alone inbreast cancers is more useful for distinguishing lobular from ductalcarcinomas than predicting clinical outcome.

upregulates the tumor metastasis suppressor gene Drg-1 in breast cancer (138). Further studies by the same group demonstrated that PTEN

emerged as a significantly better predictor of prostate and breast cancer patient survival than either marker alone. Thus these results strongly suggest functional involvement of the Drg-1 gene in suppressing the metastatic advancement of human breast cancer.

3.2.9. TIMP Breast cancer cells need to cross the basement membrane (BM) tissue boundaries. MMPs are enzymes with proteolytic activity towards extracellular matrix components (ECM) of the BM, which are blocked by physiological tissue inhibitors of metalloproteinases (TIMPs). Cancer metastasis occurs as a result of an imbalance between MMPs, and their inhibitors. In cultured breast cancer cell lines, transfection of TIMP-4 into the invasive human breast cancer cell line MDA-MB-435 reduced invasion in an in vitro model system (29, 140) and overexpression of TIMP-2 in MDA-231 cells reduced osteolytic lesions after injection of these cells into nude mice (141). Giannelli et al. found that pro-MMP-9 and TIMP-1 serum concentrations are inversely correlated in breast cancer patients. Their results show that after surgery, when the breast cancer tissue was removed, pro-MMP-9 concentrations dramatically

in primary breast carcinomas are associated with development of distant

decreased and TIMP-1 concentrations strongly increased (142). Ree et al. showed that high levels of messenger RNAs for TIMP-1 and TIMP-2


infiltrative breast carcinomas, showed a correlation of TIMP-2 with proliferative activity and patient survival in breast cancer (142). It is

carcinoma.

3.2.10. RKIP RKIP was described as a physiologic endogenous inhibitor protein of

the Raf-1/mitogen-activated protein kinase (MAPK) kinase/extracellular signal-regulated kinase (ERK) pathway. RKIP interferes with the Raf-1-mediated phosphorylation and activation of MAPK kinase via its ability to disrupt the interaction between the two kinases. Treatment with chemotherapeutic agents induces RKIP expression, sensitizing the breast and prostate cancer cells to apoptosis. This is corroborated by a similar effect of ectopic expression of RKIP in breast cancer cells that are

endogenous RKIP by expression of antisense and small interfering RNA (siRNA) confers resistance on sensitive cancer cells to anticancer drug-induced apoptosis. In a large clinical cohort comprising 103 patients with metastatic and nonmetastatic breast cancer, RKIP expression was high in breast duct epithelia and retained to varying degrees in primary breast tumors. However, in lymph node metastases, RKIP expression was highly

by tumor tissues may be a determinant of the progression in breast possible, that the imbalance between MMPs and TIMPs produced

resistant to the effects of DNA-damaging agent. This sensitization canbe reversed by upregulation of survival pathways. Downregulation of

significantly reduced or lost (143, 144). RKIP expression is independent of other markers for breast cancer progression and prognosis.

4. CONCLUSIONS

Metastasis, the spread of cancer cells from the primary tumor to distant organs and their treatment-resistant proliferation in multiple locations, remains a major clinical and biological challenge.

The genetics of breast cancer metastasis is a very broad and complex field of study. It is relatively new and expanding. There are several

metastases (90). Another study conducted by Nakopoulou et al. using

studies for relevance, and their mechanisms of action need to be elucidated. Interestingly there is not a unique signaling pathway that has emerged as a key. This further emphasizes the need for more exhaustive studies. A better understanding of the molecular mechanisms that regulate the process of metastasis and of the complex interactions between the metastatic cells and host factors can provide a biological foundation for the design of more effective therapy.

potential candidates identified; however, functional validation, patient


ACKNOWLEDGMENT We wish to acknowledge all our colleagues and collaborators whose

R.S.S. is a recipient of Susan G. Komen Breast Cancer Foundation research grant # BTCR0503488.

REFERENCES

1. metastatic breast cancer: twenty-year data from two SEER registries. BMC Cancer 2004, 4:60.

2. Welch DR, Steeg PS, Rinker-Schaeffer CW. Molecular biology of breast cancer metastasis. Genetic regulation of human breast carcinoma metastasis. Breast Cancer Res 2000, 2(6):408–416.

3. Chambers AF, Groom AC, MacDonald IC. Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer 2002, 2(8):563–572.

4. Steeg PS, Ouatas T, Halverson D, Palmieri D, Salerno M. Metastasis suppressor genes: basic biology and potential clinical use. Clin Breast Cancer 2003, 4(1):51–62.

5. Bogenrieder T, Herlyn M. Axis of evil: molecular mechanisms of cancer metastasis. Oncogene 2003, 22(42):6524–6536.

6. Hoon DS, Kitago M, Kim J, Mori T, Piris A, Szyfelbein K, Mihm MC, Jr., Nathanson SD, Padera TP, Chambers AF et al. Molecular mechanisms of metastasis. Cancer Metastasis Rev 2006, 25(2):203–220.

7. Berger JC, Vander Griend D, Stadler WM, Rinker-Schaeffer C. Metastasis suppressor genes: signal transduction, cross-talk and the potential for modu-lating the behavior of metastatic cells. Anticancer Drugs 2004, 15(6):559–568.

work has allowed us to compile this chapter. We have tried to put several research groups work in perspective and we wish to apologize if there is any work or reference that we may have missed. L.A.S. is a recipient of Susan G. Komen Breast Cancer Foundation research grant # BTCR0402317.

8. Berger JC, Vander Griend DJ, Robinson VL, Hickson JA, Rinker-Schaeffer CW. Metastasis suppressor genes: from gene identification to protein function and regulation. Cancer Biol Ther 2005, 4(8):805–812.

9. Kim R, Emi M, Tanabe K, Arihiro K. Tumor-driven evolution of immuno-suppressive networks during malignant progression. Cancer Res 2006, 66(11): 5527–5536.

10. Gutierrez LS, Eliza M, Niven-Fairchild T, Naftolin F, Mor G. The Fas/Fas-ligand system: a mechanism for immune evasion in human breast carcinomas. Breast Cancer Res Treat 1999, 54(3):245–253.

11. Mor G, Kohen F, Garcia-Velasco J, Nilsen J, Brown W, Song J, Naftolin F. Regulation of fas ligand expression in breast cancer cells by estrogen:

Tai P, Yu E, Vinh-Hung V, Cserni G, Vlastos G. Survival of patients with

functional differences between estradiol and tamoxifen. J Steroid Biochem Mol Biol 2000, 73(5):185–194.

12. Bewick M, Conlon M, Parissenti AM, Lee H, Zhang L, Gluck S, Lafrenie RM. Soluble Fas (CD95) is a prognostic factor in patients with metastatic breast cancer undergoing high-dose chemotherapy and autologous stem cell transplantation. J Hematother Stem Cell Res 2001, 10(6):759–768.

13. Ueno T, Toi M, Tominaga T. Circulating soluble Fas concentration in breast cancer patients. Clin Cancer Res 1999, 5(11):3529–3533.


14. Behrens J. The role of cell adhesion molecules in cancer invasion and metastasis. Breast Cancer Res Treat 1993, 24(3):175–184.

15. Gandhari M, Arens N, Majety M, Dorn-Beineke A, Hildenbrand R. Urokinase-type plasminogen activator induces proliferation in breast cancer cells. Int J Oncol 2006, 28(6):1463–1470.

16. Arens N, Gandhari M, Bleyl U, Hildenbrand R. In vitro suppression of

antisense strategies. Int J Oncol 2005, 26(1):113–119. 17. Yamashita J, Akizuki M, Jotsuka T, Harao M, Nakano S. Neutrophil elastase

predicts trastuzumab responsiveness in metastatic breast cancer. Breast J 2006, 12(3):288.

18. Yui S, Tomita K, Kudo T, Ando S, Yamazaki M. Induction of multicellular 3-D spheroids of MCF-7 breast carcinoma cells by neutrophil-derived cathepsin G and elastase. Cancer Sci 2005, 96(9):560–570.

19. Foekens JA, Ries C, Look MP, Gippner-Steppert C, Klijn JG, Jochum M. Elevated expression of polymorphonuclear leukocyte elastase in breast cancer tissue is associated with tamoxifen failure in patients with advanced disease. Br J Cancer 2003, 88(7):1084–1090.

20. Foekens JA, Ries C, Look MP, Gippner-Steppert C, Klijn JG, Jochum M. The prognostic value of polymorphonuclear leukocyte elastase in patients with primary breast cancer. Cancer Res 2003, 63(2):337–341.

21. Yamashita J, Ogawa M, Shirakusa T. Free-form neutrophil elastase is an independent marker predicting recurrence in primary breast cancer. J Leukoc Biol 1995, 57(3):375–378.

22. Pei D, Majmudar G, Weiss SJ. Hydrolytic inactivation of a breast carcinoma

23. Finlay TH, Tamir S, Kadner SS, Cruz MR, Yavelow J, Levitz M. alpha 1-Antitrypsin- and anchorage-independent growth of MCF-7 breast cancer cells. Endocrinology 1993, 133(3):996–1002.

24. Thomssen C, Schmitt M, Goretzki L, Oppelt P, Pache L, Dettmar P, Janicke F, Graeff H. Prognostic value of the cysteine proteases cathepsins B and cathepsin L in human breast cancer. Clin Cancer Res 1995, 1(7):741–746.

25. Schmitt M, Wilhelm O, Janicke F, Magdolen V, Reuning U, Ohi H, Moniwa N, Kobayashi H, Weidle U, Graeff H. Urokinase-type plasminogen activator

urokinase plasminogen activator in breast cancer cells–a comparison of two

cell-derived serpin by human stromelysin-3. J Biol Chem 1994, 269(41): 25849–25855.

(uPA) and its receptor (CD87): a new target in tumor invasion and metastasis. J Obstet Gynaecol 1995, 21(2):151–165.

26. Rozhin J, Sameni M, Ziegler G, Sloane BF. Pericellular pH affects distribution

27. Schmitt M, Janicke F, Moniwa N, Chucholowski N, Pache L, Graeff H. Tumor-associated urokinase-type plasminogen activator: biological and clinical significance. Biol Chem Hoppe Seyler 1992, 373(7):611–622.

28. Duffy MJ, McCarthy K. Matrix metalloproteinases in cancer: prognostic markers and targets for therapy (review). Int J Oncol 1998, 12(6):1343–1348.

29.

30. Talvensaari-Mattila A, Paakko P, Hoyhtya M, Blanco-Sequeiros G, Turpeenniemi-Hujanen T. Matrix metalloproteinase-2 immunoreactive protein:

and secretion of cathepsin B in malignant cells. Cancer Res 1994, 54(24): 6517–6525.

Duffy MJ, Maguire TM, Hill A, McDermott E, O’Higgins N. Metallo- proteinases: role in breast carcinogenesis, invasion and metastasis. BreastCancer Res 2000, 2(4):252–257.

a marker of aggressiveness in breast carcinoma. Cancer 1998, 83(6):1153–1162.

31. Eccles SA, Box GM, Court WJ, Bone EA, Thomas W, Brown PD. Control of lymphatic and hematogenous metastasis of a rat mammary carcinoma by the


matrix metalloproteinase inhibitor batimastat (BB-94). Cancer Res 1996, 56(12):2815–2822.

32. Lochter A, Srebrow A, Sympson CJ, Terracio N, Werb Z, Bissell MJ. Misregulation of stromelysin-1 expression in mouse mammary tumor cells accompanies acquisition of stromelysin-1-dependent invasive properties. J Biol Chem 1997, 272(8):5007–5015.

33. Davies B, Miles DW, Happerfield LC, Naylor MS, Bobrow LG, Rubens RD, Balkwill FR. Activity of type IV collagenases in benign and malignant breast disease. Br J Cancer 1993, 67(5):1126–1131.

34. Yang SY, Lee J, Park CG, Kim S, Hong S, Chung HC, Min SK, Han JW, Lee HW, Lee HY. Expression of autotaxin (NPP-2) is closely linked to invasiveness of breast cancer cells. Clin Exp Metastasis 2002, 19(7):603–608.

35. Sheen-Chen SM, Liu YW, Eng HL, Chou FF. Serum levels of hepatocyte growth factor in patients with breast cancer. Cancer Epidemiol Biomarkers Prev 2005, 14(3):715–717.

36. Taniguchi T, Toi M, Inada K, Imazawa T, Yamamoto Y, Tominaga T. Serum concentrations of hepatocyte growth factor in breast cancer patients. Clin Cancer Res 1995, 1(9):1031–1034.

37. Yamashita J, Ogawa M, Yamashita S, Nomura K, Kuramoto M, Saishoji T, Shin S. Immunoreactive hepatocyte growth factor is a strong and independent predictor of recurrence and survival in human breast cancer. Cancer Res 1994, 54(7):1630–1633.

38. Lamszus K, Jin L, Fuchs A, Shi E, Chowdhury S, Yao Y, Polverini PJ, Laterra J, Goldberg ID, Rosen EM. Scatter factor stimulates tumor growth and tumor angiogenesis in human breast cancers in the mammary fat pads of nude mice. Lab Invest 1997, 76(3):339–353.

39. Meiners S, Brinkmann V, Naundorf H, Birchmeier W. Role of morphogenetic factors in metastasis of mammary carcinoma cells. Oncogene 1998, 16(1): 9–20.

40. Matteucci E, Locati M, Desiderio MA. Hepatocyte growth factor enhances CXCR4 expression favoring breast cancer cell invasiveness. Exp Cell Res 2005, 310(1):176–185.

41. Mine S, Fujisaki T, Kawahara C, Tabata T, Iida T, Yasuda M, Yoneda T, Tanaka Y. Hepatocyte growth factor enhances adhesion of breast cancer cells to endothelial cells in vitro through up-regulation of CD44. Exp Cell Res 2003, 288(1):189–197.

42. Martin TA, Watkins G, Mansel RE, Jiang WG. Hepatocyte growth factor disrupts tight junctions in human breast cancer cells. Cell Biol Int 2004, 28(5):361–371.

43. Parr C, Jiang WG. Hepatocyte growth factor activation inhibitors (HAI-1 and HAI-2) regulate HGF-induced invasion of human breast cancer cells. Int J Cancer 2006, 119(5):1176–1183.

44. Jiang WG, Grimshaw D, Lane J, Martin TA, Abounader R, Laterra J, Mansel RE. A hammerhead ribozyme suppresses expression of hepatocyte growth factor/scatter factor receptor c-MET and reduces migration and invasiveness of breast cancer cells. Clin Cancer Res 2001, 7(8):2555–2562.

45. Martin TA, Parr C, Davies G, Watkins G, Lane J, Matsumoto K, Nakamura T, Mansel RE, Jiang WG. Growth and angiogenesis of human breast cancer in a nude mouse tumour model is reduced by NK4, a HGF/SF antagonist. Carcinogenesis 2003, 24(8):1317–1323.

46. Martin TA, Mansel RE, Jiang WG. Antagonistic effect of NK4 on HGF/SF induced changes in the transendothelial resistance (TER) and paracellular permeability of human vascular endothelial cells. J Cell Physiol 2002, 192(3):268–275.


47. Hiscox S, Parr C, Nakamura T, Matsumoto K, Mansel RE, Jiang WG. Inhibition of HGF/SF-induced breast cancer cell motility and invasion by the HGF/SF variant, NK4. Breast Cancer Res Treat 2000, 59(3):245–254.

48. Parr C, Watkins G, Mansel RE, Jiang WG. The hepatocyte growth factor regulatory factors in human breast cancer. Clin Cancer Res 2004, 10(1 Pt 1): 202–211.

49. Graham JD, Balleine RL, Milliken JS, Bilous AM, Clarke CL. Expression of osteonectin mRNA in human breast tumours is inversely correlated with oestrogen receptor content. Eur J Cancer 1997, 33(10):1654–1660.

50. Gilles C, Bassuk JA, Pulyaeva H, Sage EH, Foidart JM, Thompson EW. SPARC/osteonectin induces matrix metalloproteinase 2 activation in human breast cancer cell lines. Cancer Res 1998, 58(23):5529–5536.

51. Briggs J, Chamboredon S, Castellazzi M, Kerry JA, Bos TJ. Transcriptional upregulation of SPARC, in response to c-Jun overexpression, contributes to increased motility and invasion of MCF7 breast cancer cells. Oncogene 2002, 21(46):7077–7091.

52. Campo McKnight DA, Sosnoski DM, Koblinski JE, Gay CV. Roles of osteonectin in the migration of breast cancer cells into bone. J Cell Biochem 2006, 97(2):288–302.

53. Jacob K, Webber M, Benayahu D, Kleinman HK. Osteonectin promotes prostate cancer cell migration and invasion: a possible mechanism for metastasis to bone. Cancer Res 1999, 59(17):4453–4457.

54. Dhanesuan N, Sharp JA, Blick T, Price JT, Thompson EW. Doxycycline-inducible expression of SPARC/Osteonectin/BM40 in MDA-MB-231 human breast cancer cells results in growth inhibition. Breast Cancer Res Treat 2002, 75(1):73–85.

55. Watkins G, Douglas-Jones A, Bryce R, Mansel RE, Jiang WG. Increased levels of SPARC (osteonectin) in human breast cancer tissues and its association with clinical outcomes. Prostaglandins Leukot Essent Fatty Acids 2005, 72(4): 267–272.

56. Luker KE, Luker GD. Functions of CXCL12 and CXCR4 in breast cancer. Cancer Lett 2006, 238(1):30–41.

57. Zlotnik A. Chemokines and cancer. Int J Cancer 2006, 119(9):2026–2029. 58. Lee BC, Lee TH, Avraham S, Avraham HK. Involvement of the chemokine

receptor CXCR4 and its ligand stromal cell-derived factor 1alpha in breast cancer cell migration through human brain microvascular endothelial cells. Mol Cancer Res 2004, 2(6):327–338.

59. Liang Z, Yoon Y, Votaw J, Goodman MM, Williams L, Shim H. Silencing of CXCR4 blocks breast cancer metastasis. Cancer Res 2005, 65(3):967–971.

60. Zlotnik A. Involvement of chemokine receptors in organ-specific metastasis. Contrib Microbiol 2006, 13:191–199.

61. Andre F, Cabioglu N, Assi H, Sabourin JC, Delaloge S, Sahin A, Broglio K, Spano JP, Combadiere C, Bucana C et al. Expression of chemokine receptors predicts the site of metastatic relapse in patients with axillary node positive primary breast cancer. Ann Oncol 2006, 17(6):945–951.

62. Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, McClanahan T, Murphy E, Yuan W, Wagner SN et al. Involvement of chemokine receptors in breast cancer metastasis. Nature 2001, 410(6824):50–56.

63. Su YC, Wu MT, Huang CJ, Hou MF, Yang SF, Chai CY. Expression of CXCR4 is associated with axillary lymph node status in patients with early breast cancer. Breast 2006, 15(4):533–539.

64. Kang H, Watkins G, Douglas-Jones A, Mansel RE, Jiang WG. The elevated level of CXCR4 is correlated with nodal metastasis of human breast cancer. Breast 2005, 14(5):360–367.


66. Platt-Higgins AM, Renshaw CA, West CR, Winstanley JH, De Silva Rudland S, Barraclough R, Rudland PS. Comparison of the metastasis-inducing protein S100A4 (p9ka) with other prognostic markers in human breast cancer. Int J Cancer 2000, 89(2):198–208.

67. Grigorian MS, Ambartsumian NS, Georgiev GP, Lukanidin EM. (Expression of mts1 gene in human breast cancer MCF-7 cells increases their malignancy). Mol Biol (Mosk) 1999, 33(4):651–656.

68. Zhang S, Wang G, Liu D, Bao Z, Fernig DG, Rudland PS, Barraclough R. The C-terminal region of S100A4 is important for its metastasis-inducing properties. Oncogene 2005, 24(27):4401–4411.

69. Jenkinson SR, Barraclough R, West CR, Rudland PS. S100A4 regulates cell motility and invasion in an in vitro model for breast cancer metastasis. Br J Cancer 2004, 90(1):253–262.

70. Lee WY, Su WC, Lin PW, Guo HR, Chang TW, Chen HH. Expression of S100A4 and Met: potential predictors for metastasis and survival in early-stage breast cancer. Oncology 2004, 66(6):429–438.

71. de Silva Rudland S, Martin L, Roshanlall C, Winstanley J, Leinster S, Platt-Higgins A, Carroll J, West C, Barraclough R, Rudland P. Association of S100A4 and osteopontin with specific prognostic factors and survival of patients with minimally invasive breast cancer. Clin Cancer Res 2006, 12(4):1192–1200.

72. Tuck AB, O’Malley FP, Singhal H, Harris JF, Tonkin KS, Kerkvliet N, Saad Z, Doig GS, Chambers AF. Osteopontin expression in a group of lymph node negative breast cancer patients. Int J Cancer 1998, 79(5):502–508.

73. Tuck AB, Chambers AF. The role of osteopontin in breast cancer: clinical and experimental studies. J Mammary Gland Biol Neoplasia 2001, 6(4):419–429.

74. Bramwell VH, Doig GS, Tuck AB, Wilson SM, Tonkin KS, Tomiak A, Perera F, Vandenberg TA, Chambers AF. Serial plasma osteopontin levels have prog-nostic value in metastatic breast cancer. Clin Cancer Res 2006, 12(11 Pt 1): 3337–3343.

75. Harris JF. Elevated plasma osteopontin in metastatic breast cancer associated with increased tumor burden and decreased survival. Clin Cancer Res 1997, 3(4):605–611.

76. Tuck AB, Hota C, Wilson SM, Chambers AF. Osteopontin-induced migration of human mammary epithelial cells involves activation of EGF receptor and multiple signal transduction pathways. Oncogene 2003, 22(8):1198–1205.

77. Tuck AB, Elliott BE, Hota C, Tremblay E, Chambers AF. Osteopontin-induced, integrin-dependent migration of human mammary epithelial cells involves activation of the hepatocyte growth factor receptor (Met). J Cell Biochem 2000, 78(3):465–475.

Singhal H, Bautista DS, Tonkin KS, O’Malley FP, Tuck AB, Chambers AF,

78. Cook AC, Tuck AB, McCarthy S, Turner JG, Irby RB, Bloom GC, Yeatman TJ, Chambers AF. Osteopontin induces multiple changes in gene expression that reflect the six “hallmarks of cancer” in a model of breast cancer progression. Mol Carcinog 2005, 43(4):225–236.

79. Kang Y, Siegel PM, Shu W, Drobnjak M, Kakonen SM, Cordon-Cardo C, Guise TA, Massague J. A multigenic program mediating breast cancer metastasis to bone. Cancer Cell 2003, 3(6):537–549.

80. He B, Mirza M, Weber GF. An osteopontin splice variant induces anchorage independence in human breast cancer cells. Oncogene 2006, 25(15):2192–2202.

81. Kumar R, Wang RA, Bagheri-Yarmand R. Emerging roles of MTA family members in human cancers. Semin Oncol 2003, 30(5 Suppl 16):30–37.

65. Rudland PS, Platt-Higgins A, Renshaw C, West CR, Winstanley JH, Robertson L, Barraclough R. Prognostic significance of the metastasis-inducing protein S100A4 (p9Ka) in human breast cancer. Cancer Res 2000, 60(6):1595–1603.


in epithelial cancer cell invasion, proliferation and nuclear regulation. Clin Exp Metastasis 2003, 20(1):19–24.

85. Toh Y, Pencil SD, Nicolson GL. Analysis of the complete sequence of the novel metastasis-associated candidate gene, mta1, differentially expressed in mammary adenocarcinoma and breast cancer cell lines. Gene 1995, 159(1): 97–104.

86. Toh Y, Pencil SD, Nicolson GL. A novel candidate metastasis-associated gene, mta1, differentially expressed in highly metastatic mammary adenocarcinoma cell lines. cDNA cloning, expression, and protein analyses. J Biol Chem 1994, 269(37):22958–22963.

87. Zhang H, Stephens LC, Kumar R. Metastasis tumor antigen family proteins during breast cancer progression and metastasis in a reliable mouse model for human breast cancer. Clin Cancer Res 2006, 12(5):1479–1486.

88. Cui Y, Niu A, Pestell R, Kumar R, Curran EM, Liu Y, Fuqua SA. Metastasis-associated protein 2 is a repressor of estrogen receptor alpha whose overexpression leads to estrogen-independent growth of human breast cancer cells. Mol Endocrinol 2006, 20(9):2020–2035.

89. Kumar R. Another tie that binds the MTA family to breast cancer. Cell 2003, 113(2):142–143.

90. Ree AH, Pacheco MM, Tvermyr M, Fodstad O, Brentani MM. Expression of a novel factor, com1, in early tumor progression of breast cancer. Clin Cancer Res 2000, 6(5):1778–1783.

91. Ree AH, Tvermyr M, Engebraaten O, Rooman M, Rosok O, Hovig E, Meza-Zepeda LA, Bruland OS, Fodstad O. Expression of a novel factor in human breast cancer cells with metastatic potential. Cancer Res 1999, 59(18): 4675–4680.

92. Jiang WG, Watkins G, Douglas-Jones A, Mokbel K, Mansel RE, Fodstad O. Expression of Com-1/P8 in human breast cancer and its relevance to clinical outcome and ER status. Int J Cancer 2005, 117(5):730–737.

93. Jiang WG, Davies G, Fodstad O. Com-1/P8 in oestrogen regulated growth of breast cancer cells, the ER-beta connection. Biochem Biophys Res Commun 2005, 330(1):253–262.

94. Bratland A, Risberg K, Maelandsmo GM, Gutzkow KB, Olsen OE, Moghaddam A, Wang MY, Hansen CM, Blomhoff HK, Berg JP et al. Expression of a novel factor, com1, is regulated by 1,25-dihydroxyvitamin D3 in breast cancer cells. Cancer Res 2000, 60(19):5578–5583.

95. Steeg PS, Bevilacqua G, Kopper L, Thorgeirsson UP, Talmadge JE, Liotta LA, Sobel ME. Evidence for a novel gene associated with low tumor metastatic potential. J Natl Cancer Inst 1988, 80(3):200–204.

96. Steeg PS, Bevilacqua G, Pozzatti R, Liotta LA, Sobel ME. Altered expression of NM23, a gene associated with low tumor metastatic potential, during adenovirus 2 Ela inhibition of experimental metastasis. Cancer Res 1988, 48(22):6550–6554.

97. paradigm. Cancer Lett 2003, 198(1):1–20.

98. Mundy G. Preclinical models of bone metastases. Semin Oncol 2001, 28 (4 Suppl 11):2–8.

99. Price JE, Zhang RD. Studies of human breast cancer metastasis using nude mice. Cancer Metastasis Rev 1990, 8(4):285–297.

Shevde LA, Welch DR. Metastasis suppressor pathways–an evolving

82. Nicolson GL, Moustafa AS. Metastasis-Associated genes and metastatic tumor progression. In Vivo 1998, 12(6):579–588.

83. Nicolson GL. Breast cancer metastasis-associated genes: role in tumour progression to the metastatic state. Biochem Soc Symp 1998, 63:231–243.

84. Nicolson GL, Nawa A, Toh Y, Taniguchi S, Nishimori K, Moustafa A. Tumor metastasis-associated human MTA1 gene and its MTA1 protein product: role


102. Samant RS, Debies MT, Shevde LA, Verderame MF, Welch DR. Identification and characterization of the murine ortholog (brms1) of breast-cancer metastasis suppressor 1 (BRMS1). Int J Cancer 2002, 97(1):15–20.

103. Meehan WJ, Samant RS, Hopper JE, Carrozza MJ, Shevde LA, Workman JL, Eckert KA, Verderame MF, Welch DR. Breast cancer metastasis suppressor 1 (BRMS1) forms complexes with retinoblastoma-binding protein 1 (RBP1) and the mSin3 histone deacetylase complex and represses transcription. J Biol Chem 2004, 279(2):1562–1569.

104. Cicek M, Fukuyama R, Welch DR, Sizemore N, Casey G. Breast cancer metastasis suppressor 1 inhibits gene expression by targeting nuclear factor-kappaB activity. Cancer Res 2005, 65(9):3586–3595.

105. DeWald DB, Torabinejad J, Samant RS, Johnston D, Erin N, Shope JC, Xie Y, Welch DR. Metastasis suppression by breast cancer metastasis suppressor 1 involves reduction of phosphoinositide signaling in MDA-MB-435 breast carcinoma cells. Cancer Res 2005, 65(3):713–717.

106. Mitchell DC, Abdelrahim M, Weng J, Stafford LJ, Safe S, Bar-Eli M, Liu M. Regulation of KiSS-1 metastasis suppressor gene expression in breast cancer cells by direct interaction of transcription factors activator protein-2alpha and specificity protein-1. J Biol Chem 2006, 281(1):51–58.

107. Lee JH, Welch DR. Suppression of metastasis in human breast carcinoma MDA-MB-435 cells after transfection with the metastasis suppressor gene, KiSS-1. Cancer Res 1997, 57(12):2384–2387.

108. Dittmer A, Vetter M, Schunke D, Span PN, Sweep F, Thomssen C, Dittmer J. Parathyroid hormone-related protein regulates tumor-relevant genes in breast cancer cells. J Biol Chem 2006, 281(21):14563–14572.

109. Stark AM, Tongers K, Maass N, Mehdorn HM, Held-Feindt J. Reduced metastasis-suppressor gene mRNA-expression in breast cancer brain metastases. J Cancer Res Clin Oncol 2005, 131(3):191–198.

110. Martin TA, Watkins G, Jiang WG. KiSS-1 expression in human breast cancer. Clin Exp Metastasis 2005, 22(6):503–511.

111. Arnaud-Dabernat S, Bourbon PM, Dierich A, Le Meur M, Daniel JY. Knockout mice as model systems for studying nm23/NDP kinase gene functions. Application to the nm23-M1 gene. J Bioenerg Biomembr 2003, 35(1):19–30.

112. Yang X, Wei L, Tang C, Slack R, Montgomery E, Lippman M. KAI1 protein is down-regulated during the progression of human breast cancer. Clin Cancer Res 2000, 6(9):3424–3429.

113. Su GH, Hilgers W, Shekher MC, Tang DJ, Yeo CJ, Hruban RH, Kern SE. Alterations in pancreatic, biliary, and breast carcinomas support MKK4 as a genetically targeted tumor suppressor gene. Cancer Res 1998, 58(11): 2339–2342.

114. Ichikawa T, Hosoki S, Suzuki H, Akakura K, Igarashi T, Furuya Y, Oshimura M, Rinker-Schaeffer CW, Nihei N, Barrett JC et al. Mapping of metastasis suppressor genes for prostate cancer by microcell-mediated chromosome transfer. Asian J Androl 2000, 2(3):167–171.

115. Yamada SD, Hickson JA, Hrobowski Y, Vander Griend DJ, Benson D, Montag A, Karrison T, Huo D, Rutgers J, Adams S et al. Mitogen-activated protein

100. Seraj MJ, Samant RS, Verderame MF, Welch DR. Functional evidence for a novel human breast carcinoma metastasis suppressor, BRMS1, encoded at chromosome 11q13. Cancer Res 2000, 60(11):2764–2769.

101. Samant RS, Seraj MJ, Saunders MM, Sakamaki TS, Shevde LA, Harms JF, Leonard TO, Goldberg SF, Budgeon L, Meehan WJ et al. Analysis of mecha-nisms underlying BRMS1 suppression of metastasis. Clin Exp Metastasis 2000, 18(8):683–693.


118. Escrich E, Moral R, Garcia G, Costa I, Sanchez JA, Solanas M. Identification of novel differentially expressed genes by the effect of a high-fat n-6 diet in experimental breast cancer. Mol Carcinog 2004, 40(2):73–78.

119. Berx G, Cleton-Jansen AM, Strumane K, de Leeuw WJ, Nollet F, van Roy F, Cornelisse C. E-cadherin is inactivated in a majority of invasive human lobular breast cancers by truncation mutations throughout its extracellular domain. Oncogene 1996, 13(9):1919–1925.

120. Berx G, Cleton-Jansen AM, Nollet F, de Leeuw WJ, van de Vijver M, Cornelisse C, van Roy F. E-cadherin is a tumour/invasion suppressor gene mutated in human lobular breast cancers. Embo J 1995, 14(24):6107–6115.

121. Cleton-Jansen AM. E-cadherin and loss of heterozygosity at chromosome 16 in breast carcinogenesis: different genetic pathways in ductal and lobular breast cancer? Breast Cancer Res 2002, 4(1):5–8.

122. Becker KF, Reich U, Schott C, Becker I, Berx G, van Roy F, Hofler H. Identification of eleven novel tumor-associated E-cadherin mutations. Mutations in brief no. 215. Online. Hum Mutat 1999, 13(2):171.

123. Berx G, Van Roy F. The E-cadherin/catenin complex: an important gatekeeper in breast cancer tumorigenesis and malignant progression. Breast Cancer Res 2001, 3(5):289–293.

124. Cowin P, Rowlands TM, Hatsell SJ. Cadherins and catenins in breast cancer. Curr Opin Cell Biol 2005, 17(5):499–508.

125. Droufakou S, Deshmane V, Roylance R, Hanby A, Tomlinson I, Hart IR. Multiple ways of silencing E-cadherin gene expression in lobular carcinoma of the breast. Int J Cancer 2001, 92(3):404–408.

126. Mielnicki LM, Asch HL, Asch BB. Genes, chromatin, and breast cancer: an epigenetic tale. J Mammary Gland Biol Neoplasia 2001, 6(2):169–182.

127. Strathdee G. Epigenetic versus genetic alterations in the inactivation of E-cadherin. Semin Cancer Biol 2002, 12(5):373–379.

128. Thoreson MA, Reynolds AB. Altered expression of the catenin p120 in human cancer: implications for tumor progression. Differentiation 2002, 70(9-10): 583–589.

129. Fujita N, Jaye DL, Kajita M, Geigerman C, Moreno CS, Wade PA. MTA3, a Mi-2/NuRD complex subunit, regulates an invasive growth pathway in breast cancer. Cell 2003, 113(2):207–219.

130. Peinado H, Ballestar E, Esteller M, Cano A. Snail mediates E-cadherin repression by the recruitment of the Sin3A/histone deacetylase 1 (HDAC1)/ HDAC2 complex. Mol Cell Biol 2004, 24(1):306–319.

131. Peinado H, Marin F, Cubillo E, Stark HJ, Fusenig N, Nieto MA, Cano A. Snail and E47 repressors of E-cadherin induce distinct invasive and angiogenic properties in vivo. J Cell Sci 2004, 117(Pt 13):2827–2839.

132. Bolos V, Peinado H, Perez-Moreno MA, Fraga MF, Esteller M, Cano A. The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors. J Cell Sci 2003, 116(Pt 3):499–511.

kinase kinase 4 (MKK4) acts as a metastasis suppressor gene in human ovarian carcinoma. Cancer Res 2002, 62(22):6717–6723.

116. Nishinaka Y, Nishiyama A, Masutani H, Oka S, Ahsan KM, Nakayama Y, Ishii Y, Nakamura H, Maeda M, Yodoi J. Loss of thioredoxin-binding protein-2/vitamin D3 up-regulated protein 1 in human T-cell leukemia virus type I-dependent T-cell transformation: implications for adult T-cell leukemia leukemogenesis. Cancer Res 2004, 64(4):1287–1292.

117. Deroo BJ, Hewitt SC, Peddada SD, Korach KS. Estradiol regulates the thioredoxin antioxidant system in the mouse uterus. Endocrinology 2004, 145(12):5485–5492.


137. De La Cruz C, Moriya T, Endoh M, Watanabe M, Takeyama J, Yang M, Oguma M, Sakamoto K, Suzuki T, Hirakawa H et al. Invasive micropapillary carcinoma of the breast: Clinicopathological and immunohistochemical study. Pathol Int 2004, 54(2):90–96.

138. Bandyopadhyay S, Pai SK, Hirota S, Hosobe S, Takano Y, Saito K, Piquemal D, Commes T, Watabe M, Gross SC et al. Role of the putative tumor metastasis suppressor gene Drg-1 in breast cancer progression. Oncogene 2004, 23(33):5675–5681.

139. Bandyopadhyay S, Pai SK, Hirota S, Hosobe S, Tsukada T, Miura K, Takano Y, Saito K, Commes T, Piquemal D et al. PTEN up-regulates the tumor metastasis suppressor gene Drg-1 in prostate and breast cancer. Cancer Res 2004, 64(21):7655–7660.

140. Wang M, Liu YE, Greene J, Sheng S, Fuchs A, Rosen EM, Shi YE. Inhibition of tumor growth and metastasis of human breast cancer cells transfected with tissue inhibitor of metalloproteinase 4. Oncogene 1997, 14(23):2767–2774.

141. Yoneda T, Sasaki A, Dunstan C, Williams PJ, Bauss F, De Clerck YA, Mundy GR. Inhibition of osteolytic bone metastasis of breast cancer by combined treatment with the bisphosphonate ibandronate and tissue inhibitor of the matrix metalloproteinase-2. J Clin Invest 1997, 99(10):2509–2517.

142. Giannelli G, Erriquez R, Fransvea E, Daniele A, Trerotoli P, Schittulli F, Grano M, Quaranta M, Antonaci S. Proteolytic imbalance is reversed after therapeutic surgery in breast cancer patients. Int J Cancer 2004, 109(5):782–785.

143. Hagan S, Al-Mulla F, Mallon E, Oien K, Ferrier R, Gusterson B, Garcia JJ, Kolch W. Reduction of Raf-1 kinase inhibitor protein expression correlates with breast cancer metastasis. Clin Cancer Res 2005, 11(20):7392–7397.

144. Chatterjee D, Bai Y, Wang Z, Beach S, Mott S, Roy R, Braastad C, Sun Y, Mukhopadhyay A, Aggarwal BB et al. RKIP sensitizes prostate and breast cancer cells to drug-induced apoptosis. J Biol Chem 2004, 279(17):17515–17523.

133. Batlle E, Sancho E, Franci C, Dominguez D, Monfar M, Baulida J, Garcia De Herreros A. The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol 2000, 2(2):84–89.

134. Knudsen KA, Lin CY, Johnson KR, Wheelock MJ, Keshgegian AA, Soler AP: Lack of correlation between serum levels of E- and P-cadherin fragments and the presence of breast cancer. Hum Pathol 2000, 31(8):961–965.

135. Kuroda N, Sugimoto T, Takahashi T, Moriki T, Toi M, Miyazaki E, Hiroi M, Enzan H. Invasive micropapillary carcinoma of the breast: an immuno-histochemical study of neoplastic and stromal cells. Int J Surg Pathol 2005, 13(1):51–55.

136. Nagi C, Guttman M, Jaffer S, Qiao R, Keren R, Triana A, Li M, Godbold J, Bleiweiss IJ, Hazan RB. N-cadherin expression in breast cancer: correlation

Breast Cancer Res Treat 2005, 94(3):225–235. with an aggressive histologic variant–invasive micropapillary carcinoma.


31 R.E. Mansel et al. (eds.), Metastasis of Breast Cancer, 31–46. © 2007 Springer.

Chapter 3

A PERSPECTIVE FROM THE TUMOR MICROENVIRONMENT

Shaun D. McCullough, Yanfen Hu, and Rong Li Department of Biochemistry and Molecular Genetics, Health Science Center, Universityof Virginia, Charlottesville, VA 22908, USA

Abstract: Women who inherit cancer-predisposing mutations in the BRCA1 gene have about 80% lifetime chance of developing breast cancer. BRCA1 mutation-associated tumors are often diagnosed as high-grade, typically display a basal epithelial phenotype, and proliferate rapidly. While somatic mutations of BRCA1 are rarely found in sporadic breast cancer cases, 30–40% of the sporadic cases show reduced BRCA1 expression, supporting the notion that impaired BRCA1 function may contribute to the develop-ment of both familial and sporadic forms of breast cancer. Furthermore, low levels of BRCA1 expression have been linked with the occurrence of distant metastases in sporadic disease. Since cloning of the gene more than a decade ago, BRCA1 has been implicated in a large array of cellular functions, most notably DNA damage repair. However, the relationship between the known molecular functions of BRCA1 and the clinico-pathological features of BRCA1-associated tumors remains elusive. Why do BRCA1 mutations predominantly affect female breast and ovaries? Why do BRCA1-associated cancers tend to have a poor prognosis? How can the knowledge of BRCA1 function be translated into more targeted and efficacious therapies? In this review, we will discuss these important issues in light of some recent findings from laboratory and preclinical studies, which point to a need to look “outside the box” of epithelial cells by elucidating BRCA1 functions in the context of the unique tumormicroenvironment.

Keywords: receptor, tumor microenvironment.

AND METASTASIS OF BREAST CANCER: BRCA1 IN INITIATION, INVASION,

BRCA1, DNA repair, transcription, estrogen, tissue-specificity, estrogen

1. BRCA1: A TISSUE-SPECIFIC TUMOR

SUPPRESSOR GENE

Breast cancer susceptibility gene BRCA1 was identified in 1994 through genetic linkage analysis and positional cloning (1, 2). Germ-line muta-tions of BRCA1 occur at a frequency of approximately 1 in 250 women, and these mutations account for 45% of the familial breast cancer and 80–90% of the hereditary cases where both breast and ovarian cancers occur (breast-ovarian cancer syndrome) (3–5). Genetic analysis of BRCA1- associated tumor specimens strongly indicate that BRCA1 functions as a tumor suppressor, as the tumors invariably lose the wild-type copy of BRCA1 and retain the inherited mutant copy (loss of heterozygosity; LOH). However, in contrast to mutations of other well-defined tumor

less, reduced expression of BRCA1 mRNA, and protein has been observed in a significant percentage (30–40%) of sporadic breast/ovarian cancer cases; and this is particularly true in tumors with high nuclear grade (6–8). Furthermore, promoter hypermethylation-mediated gene silencing of the BRCA1 locus occurs in 10–15% of sporadic breast and ovarian cancer cases (9–11), supporting the notion that BRCA1 may also play a role in suppression of sporadic breast cancer. In a recent comprehensive analysis of cancer risks among BRCA1 mutation-carriers, it was shown that this group of women has 80% chance of developing breast cancer in their lifetime (12). Interestingly, the same study also found that physical exercise and lack of obesity in adolescence significantly delay the onset of BRCA1-associated breast cancer, which underscores the importance of nongenetic factors in cancer prevention.

Figure 1. Diagram of the BRCA1 protein. The structural motifs including the RING and BRCT domains are highlighted. Also listed is a subset of BRCA1-interacting proteins.

region are rarely found in sporadic breast or ovarian cancers. Neverthe-suppressor genes such as p53, somatic mutations in the BRCA1 coding

McCullough, Hu, and Li32

2. STRUCTURAL AND FUNCTIONAL FEATURES OF THE BRCA1 PROTEIN

The human BRCA1 gene encodes a 1863-amino acid protein, which contains a highly conserved RING finger domain at the amino terminus and two BRCT repeats at the carboxyl terminus (Fig. 1). The vast majority of cancer-predisposing mutations of BRCA1 give rise to truncated and presumably nonfunctional proteins (3). Approximately 10% of mutations result in change of a single amino acid, many of which are located in the RING and BRCT domains. The molecular functions of the BRCA1 protein have been a subject of intense research for more than a decade. The ubiquitously expressed protein is implicated in a large array of cellular events, including DNA repair, transcription, chromatin remodeling, ubiquitination, DNA damage checkpoint, mitotic spindle checkpoint, and control of centrosome duplication (7, 13–21).

Among all the reported functions of BRCA1, its role in the DNA damage response has been most extensively investigated (13, 14, 16, 18). A wealth of evidence indicates that BRCA1 is physically associated with multiple proteins involved in DNA repair and checkpoint control, and their nuclear co-localization is one of the hallmarks in the activation of DNA damage response (22–26). BRCA1 is phosphorylated by several key protein kinases involved in the DNA damage checkpoint control, including ATM, ATR, and CHK2 (27–29), and is thought to act as a signal-transducing molecule that links upstream sensors of DNA damage with the downstream effectors. BRCA1-deficient human and murine cells are hypersensitive to various types of genotoxic insults, including DNA double-strand breaks (30–34). Chromosomal instability due to com-promised functions of BRCA1 in DNA repair and DNA damage checkpoint most likely contribute in a significant manner to BRCA1 mutation-associated cancer susceptibility.

In addition to DNA repair, the role of BRCA1 in gene regulation has also been well explored (7, 13, 15, 21). Although BRCA1 is not a sequence-specific DNA binding protein, it can be associated with a number of site-specific transcription factors (35–41), chromatin-modifying protein complexes (42–45), and the RNA polymerase II (RNAPII) holo-enzyme itself (42, 46–48). Ectopic expression and siRNA knockdown experiments have led to the identification of a number of BRCA1 target genes including p21CIP, GADD45, pS2/TFF1, MAD2, OPN, and ANG1 (35, 39, 40, 49–56). Many of the BRCA1-regulated genes are important

players in cell cycle regulation, mitotic checkpoint, cell migration, and

3. BRCA1 in initiation, invasion, and metastasis 33

angiogenesis, and their aberrant expression due to the loss of BRCA1 activity in transcription may lead to the BRCA1 mutation-associated tumorigenesis.

So far the only known enzymatic activity of BRCA1 is its ubiquitin (Ub) E3 ligase activity. The N-terminal RING domain of BRCA1 inter-acts with another structurally similar RING finger protein BARD1, and

the RING domain of BRCA1 abolish the Ub E3 ligase activity of the BRCA1/BARD1 complex, providing a compelling link between ubiquity-

the BRCA1/BARD1 complex remain to be elucidated. However, recent studies have indicated that ubiquitination of the largest subunit of RNA polymerase II by BRCA1/BARD1 is responsible for DNA damage-induced inhibition of RNA processing (58, 59). In addition, BRCA1/BARD1 has been shown to ubiquitinate γ-tubulin, which is involved in the control of proper centrosome duplication and chromosomal segregation (60).

The construction of whole-body and tissue-specific BRCA1 knockout mice has allowed for a better understanding of the role that Brca1 plays in both embryonic development and tumorigenesis in vivo. Whole-body BRCA1 knockout mice fail to develop properly and die in utero before day 7.5 of gestation (61). Characterization of the embryonic lethal pheno-type in the BRCA1 null embryos suggested that they exhibited defects in cellular proliferation (61). Further studies with this knockout mouse model indicated that loss of functional p53 delayed embryonic lethality

participate in a common genetic pathway (62). Relatively recent work

affected both cell growth and metastatic potential in MEFs isolated from the knockout mice (63). In this system, loss of BRCA1 results in p53-dependent senescence, therefore allowing clonal selection for cells that can bypass senescence through loss of functional p53. Interestingly, the

+/+

immortalized clone was shown to be p53-negative. Once immortalized, the BRCA1 null MEFs proliferated at a significantly greater rate and exhibited greater metastatic potential than immortalized control MEFs. The results from these studies begin to reconcile the seeming paradox between the accepted function of BRCA1 as a tumor suppressor and the

with the findings from the laboratory research, studies of human clinical

nation and breast cancer. The exact in vivo ubiquitination substrates of

in vitro (19, 57). Importantly, missense cancer-predisposing mutations in the BRCA1/BARD1 heterodimer confers strong Ub E3 ligase activity

by Cao et al. demonstrated how the interplay between BRCA1 and p53

controls and nearly every a much lower frequency than BRCA1

slow growth phenotype of BRCA1 mutant/null cells in culture. Consistent

immortalization of BRCA1-null MEFs was observed to occur with

samples indicate that BRCA1 mutation-associated breast cancers exhibit

34 McCullough, Hu, and Li

in BRCA1 null mice to day 9.5 of gestation, suggesting that BRCA1 and p53

inactivating mutations in the p53 gene with a greater frequency than their sporadic counterparts (64).

3. THE MOLECULAR BASIS OF THE TISSUE SPECIFICITY OF BRCA1-ASSOCIATED TUMORS

The exact molecular basis for the tissue-specificity of BRCA1-related tumors remains elusive. Furthermore, it is unclear why somatic mutations of BRCA1 are rare in sporadic cancer cases. The highly tissue-specific character of BRCA1-associated tumors stands in stark contrast with the ubiquitous nature of BRCA1 expression, as well as the generality and multiplicity of its reported functions. As reviewed above, compelling evidence strongly implicates BRCA1 in maintenance of genome stability. However, it remains unclear as to why deficiency of BRCA1 function in DNA damage response, a cellular event thought to be universally impor-tant in all cell types and both genders, would specifically increase the risk of breast and ovarian cancers in women. Several models have been proposed to explain the tissue-specific nature of BRCA1-associated tumors. For example, it has been suggested that BRCA1-deficient breast and ovarian epithelial cells may be more refractory to apoptosis than those in other tissues, thus allowing the former to accumulate additional genetic instability (65). Alternatively, the tissue-specific nature of BRCA1- associated tumors may arise from a higher frequency of LOH in the breast and ovarian epithelial cells (66). While maintenance of genetic stability is obviously an important part of the tumor suppressor function of BRCA1, it remains to be seen whether loss of this activity alone could fully account for the tissue- and gender-specific nature of BRCA1-associated tumors.

The action of estrogen is critical to both normal mammary gland development and breast cancer (67–69). Aberrant changes of the expres-sion and/or activity of ERα and its coregulators have been associated with breast carcinogenesis (70, 71). In light of the fact that cancer-predisposing mutations of BRCA1 predominantly affect the breast and


3.1 Possible tissue-specific genetic instability

3.2 Modulation of ERα activity by BRCA1instability

specificity” could be explained by a potential link between BRCA1 and estrogen action. In support of this notion, the wild-type BRCA1 protein has been implicated in the regulation of ERα-mediated gene expres-sion. Initial studies by Rosen et al. demonstrated that the exogenous expression of BRCA1 resulted in downregulation of estrogen-stimulated expression of an estrogen-responsive reporter construct in human breast, prostate, and cervical carcinoma cell lines (72). Additional studies by this and other groups have shown that BRCA1 is physically associated with ERα-regulated promoters such as pS2 and regulates expression of the corresponding endogenous gene expression in breast cancer cell lines (40, 55, 73). Additional in vitro characterization has indicated that BRCA1 and ERα physically interact with each other through the amino-

may promote estrogen-dependent cell growth and neoplasia in the breast tissue. However, the tissue culture-based findings would have to be reconciled with the clinical observation that most BRCA1-associated breast tumors are basal-like and ERα-negative (see below).

In addition to dysregulated transcriptional activity of ERα, prolonged estrogen exposure is also a well-documented risk factor for breast cancer (68, 74–78). Ovaries, specifically ovarian granulosa cells, are the primary source of estrogen in premenopausal women. This explains why early menarche and late menopause are associated with increased risks of breast cancer (79). Aromatase (Cyp19) is expressed in a restricted number of steroidogenic tissues including ovaries. The enzyme catalyzes the con-version from androgen to estrogen, the rate-limiting step in estrogen bio-synthesis (80). Recently published work from our laboratories suggests that expression of BRCA1 in ovarian granulosa cells is inversely correlated with that of aromatase during steroidogenesis (81). Importantly, small interfering RNA (siRNA)-mediated knockdown of BRCA1 or its partner BARD1 resulted in elevated aromatase expression and its enzymatic activity in ovarian granulosa cells (81). In an independent study, Dubeau et al. made an intriguing observation that ovarian granulosa cell-specific Brca1 knockout mice develop ovarian and uterine tumors that still contain

ovary, two major estrogen-responsive tissues, the conundrum of “tissue-

terminal region of BRCA1 and the ligand-binding domain (LBD) of ER-α in an estrogen-independent manner (40). Therefore, loss of the tran-scriptional corepressor function of BRCA1 in BRCA1-deficient cells

the wild-type Brca1 gene (82). These in vitro and in vivo findings point

3.3 BRCA1 and regulation of estrogen biosynthesis


to a cell nonautonomous role of BRCA1 in modulating the endocrine and/or paracrine actions of estrogen.

Figure 2. Proposed impact of BRCA1 on different cell types within the mammary tumor

At menopause, ovarian estrogen production ceases and extragonadal sites such as adipose tissue become the prominent sources of estrogen (80, 83). In addition to the alteration in the source of estrogen, the capacity of estrogen as a signaling molecule changes from an endocrine to a localized paracrine/autocrine role (84). Indeed, elevated intratumoral aromatase expression and estrogen production are linked to the develop-ment of postmenopausal breast cancer (85, 86). This involves an intricate paracrine loop between tumor and the surrounding adipose stromal cells (ASCs): tumor cell-derived factors such as interleukin 6 (IL-6) and pro-staglandin E2 (PGE2) stimulate aromatase expression and hence estrogen production in ASCs, which in turn promote estrogen-dependent growth of tumor cells (87-89). Such a “vicious cycle” is thought to facilitate breast cancer progression in the unique mammary tissue microenviron-ment. This also serves as the rationale for using aromatase inhibitors, such as letrozole, as efficacious agents for the treatment of postmeno-pausal breast cancer (90). In addition to the modulation of aromatase expression in ovarian granulosa cells (81), BRCA1 also appears to repress aromatase gene expression in ASCs (91, 92). Therefore, by

microenvironment. E2 and T stand for 17beta estradiol and testosterone, respectively.

blunting estrogen production in ovaries and mammary microenviron-ment, BRCA1 may reduce estrogen-mediated gene expression and


suppress the initiation of estrogen-dependent tumorigenesis (Fig. 2). This function of BRCA1 in stromal cells may occur in parallel with the BRCA1-mediated repression of ERα transcriptional activity in mam-mary epithelial cells. Given the known carcinogenic effect of estrogen and its metabolites (93), elevated local estrogen levels due to BRCA1 deficiency in stromal cells may also contribute to genetic instability, thus compounding the consequence of impaired DNA repair capability in BRCA1-defective epithelial cells within the same microenvironment.

4.

The relevance of estrogen/ERα to the etiology of BRCA1-associated tumors has been a long-standing clinical conundrum. BRCA1-associated tumors are largely ERα-negative (6) and their gene expression profile resembles that from basal epithelial cells in the mammary gland (94, 95). On the other hand, prophylactic oophorectomy, which removes the major source of circulating estrogen in premenopausal women, significantly reduces risk of breast cancer in BRCA1-mutation carriers (96, 97). Consistent with the findings in human (96, 97), oophorectomy decreases the incidence of mammary tumor formation in the MMTV-BRCA1-/- mouse model (98). In addition, tamoxifen has been shown to be effective in reducing the risk of contralateral tumors in BRCA1-mutation carriers (99). Epidemiological evidence also suggests that hormonal exposure and obesity in adolescence, which are well-known risk factors for sporadic breast cancer, can significantly affect breast cancer onset for BRCA1-mutation carriers (12).

How could one reconcile the ERα-negative feature of BRCA1-associated tumors with the apparent impact of estrogen exposure on the disease risk? One possible explanation for the aforementioned paradox is that ERα-positive BRCA1-deficient cells may evolve to become ERα-negative tumors during the disease progression. Consistent with this notion,

OF BRCA1-RELATED BREAST CANCER

4.1

early-stage mammary tumors from MMTV-BRCA1-/- knockout mice are largely ERα-positive, whereas late-stage tumors usually lack ERα expres-sion (100) (Chuxia Deng, NIH, personal communication). Therefore, it is

MOLECULAR BASIS FORCLINICOPATHOLOGICAL FEATURES

Is BRCA1-associated tumorigenesis estrogen-dependent?


possible that modulation of estrogen production and/or the transcript-tional activity of ERα by wild-type BRCA1 in stromal and epithelial

positive cells in the same microenvironment could influence the behavior of BRCA1-deficient, ERα-negative preneoplastic cells through a para-crine mechanism. Obviously, an in-depth investigation of the BRCA1–

BRCA1-associated tumors are usually diagnosed as high-grade infiltrating ductal carcinoma (99). Patients with BRCA1-associated breast tumors tend to have a poorer prognosis than those with sporadic tumors, suggesting that loss of BRCA1 function may lead to a more aggressive progression of breast cancer. Interestingly, a recent report suggests a high incidence of brain metastasis in BRCA1-associated cancer cases (101). Contrary to what has been observed in sporadic breast cancer, BRCA1 mutation-associated poor prognosis often occurs in node-negative cases, where tumors do not spread to axillary lymph nodes (6). It was postulated that BRCA1-associated tumors might choose metastatic routes other than the lymphatic system, perhaps through newly formed blood vessels surrounding the tumors (6). Just as proposed for the initiation of BRCA1-associated breast tumors, the exact pattern and route

immortalized mammary epithelial cell line MCF10A disrupts normal acinar morphogenesis in vitro (104). Reduction of BRCA1 in the MCF10A cell line led to aberrant cell proliferation and failure to respond to extra-cellular matrix (ECM)-dependent differentiation signals. Of particular interest is the observation in this study that treatment of BRCA1-depleted MCF10A cells with conditioned medium from control counterparts

of BRCA1-associated tumors. In an alternative scenario, normal ERα-cells, respectively, may play a critical role in suppressing the initiation

estrogen connection will be of great importance to more targeted prevention and treatment of BRCA1-associated cancers. The same research may alsoshed light on the functional consequences of reduced BRCA1 expressionassociated with many sporadic breast cancers (6).

for the progression and spreading of these tumors may also be determined by an intricate interaction between BRCA1-deficient tumor cellsand the surrounding stroma. Is there any evidence in support of sucha hypothesis?

partially restored the ability of these BRCA1-depleted cells to complete three-dimensional acinar morphogenesis in vitro. These results are consistent with the possibility that mammary epithelial cells secrete an

4.2 Why do BRCA1-associated cancers have a poor prognosis?

Using a recently popularized three-dimensional cell culture system that mimics the in vivo mammary microenvironment (102, 103), Furuta et al. showed that BRCA1 depletion by shRNA interference in the


autocrine/paracrine factor in a BRCA1-dependent fashion to promote normal differentiation. In support of this notion, a follow-up study from the same group found that BRCA1 directly represses transcription of angiopoietin (ANG1), the product of which acts in a paracrine manner to promote endothelial cell survival and vascularization (56). In an independent study, BRCA1 was shown to repress ERα-dependent tran-scription and secretion of vascular endothelial growth factor (VGEF) in breast cancer cells (105). Of clinical importance, both studies demon-strated that cancer-predisposing mutants of BRCA1 fail to reduce the

these studies raise a distinct possibility that loss of BRCA1 in mammary epithelial cells may have a significant impact on the behavior of the stromal cells in the tumor microenvironment, which in turn may influence the metastatic outcome of the BRCA1-associated cancer (Fig. 2).

Cytogenetic analyses of clinical samples also shed some intriguing light on the genetic instability of BRCA1-associated tumor and the surrounding stroma in the same microenvironment. In a recent report,

carriers was similar between the breast tumor cells and the associated stroma (106). Further, LOH at the BRCA1 locus of several patients was only observed in the breast tumor stroma (106). These observations suggest a role for stromal BRCA1 in suppressing tumor progression that

compartment may be similar to that of stromal p53 mutations recently demonstrated in breast and prostate tumors (107, 108). Lastly, it has been recently reported that malignant human breast cancer epithelial cells can fuse with and transform mouse stroma (109). Therefore, it will be of interest to see whether the increased genetic instability due to loss of BRCA1 in the microenvironment may result in fusion of the epithelial and stroma components.

5. CONCLUSION

Since the identification of the BRCA1 tumor suppressor gene more than a decade ago, intense research in the field has implicated BRCA1 in a disparate array of cellular processes. Despite the explosive knowledge of BRCA1 in the literature, there exists a disconnect between the uni-versal nature of BRCA1 functions and the highly tissue-specific impact of the BRCA1 mutations on tumorigenesis. Although BRCA1 mutation

carriers have a high risk of developing breast cancer, the genetic and nongenetic modifiers that influence the penetrance of BRCA1 mutations remain largely unexplored. Furthermore, the atypical clinicopathological

may be independent of LOH at the BRCA1 locus in the epithelium.The potential tumor-promoting effect of BRCA1 loss in the stromal

Weber et al. found that, on a total-genome scale, LOH in BRCA1 mutation

expression of these angiogenesis-related genes (56, 105). Therefore,


features of BRCA1-associated cancer suggests an involvement of BRCA1 in suppressing specific metastatic routes for cancer progression, although a direct role of BRCA1 mutations in metastasis remains to be discerned. A comprehensive understanding of these outstanding issues on BRCA1-related cancer biology will go a long way to help develop more targeted and effective prevention and treatment of the disease. A careful exami-nation of the current literature has led us to the proposal of an integrative study of BRCA1 in the context of the unique mammary gland/tumor microenvironment. Historically, studies of BRCA1 have been conducted in breast epithelial/carcinoma cell lines. By looking “outside the box” of epithelial cells and interrogating the impact of BRCA1 in both mammary epithelial and stromal cells, we may be able to understand the etiology of BRCA1 mutation-associated tumors in a systemic way. Given the loss of BRCA1 expression in many sporadic breast cancer cases, continued work in this direction also promises to have a broad application to breast cancer therapies.

ACKNOWLEDGMENTS

NIH grants (CA118578 and CA93506). Due to limited space, we apolo-gize to those authors whose excellent work was not cited in this review.

REFERENCES

1. Friedman LS et al. Confirmation of BRCA1 by analysis of germline mutations linked to breast and ovarian cancer in ten families. Nat Genet, 1994; 8: 399–404.

2. Miki Y et al. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science, 1994; 266(5182): 66–71.

3. Rahman N and MR. Stratton. The genetics of breast cancer susceptibility. Annu Rev Genet, 1998; 32: 95–121.

4. Welcsh PL and M-C King. BRCA1 and BRCA2 and the genetics of breast and ovarian cancer. Hum Mol Genet, 2001; 10: 705–713.

5. Nathanson KL, R Wooster, and BL Weber. Breast cancer genetics: what we know and what we need. Nat Med, 2001; 7: 552–556.

6. Narod SA and WD Foulkes. BRCA1 and BRCA2: 1994 and beyond. Nature Reviews Cancer, 2004; 4: 665–676.

7. Rosen EM et al. BRCA1 gene in breast cancer. J Cell Physiol, 2003; 196: 19–41. 8. Thompson ME et al. Decreased expression of BRCA1 accelerates growth and

is often present during sporadic breast cancer progression. Nat Genet, 1995; 9: 444–450.

associated with reduced gene expression in human somatic cells. FASEB J, 2000; 14: 1585–1594.

9. Magdinier F et al. Regional methylation of the 5’ end CpG island of BRCA1 is


Work in Yanfen Hu and Rong Li’s laboratories was supported by

12. King MC, Marks JH, and Mandell JB. Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2. Science, 2003; 302(5645): 643–646.

13. Scully R and DM Livingston. In search of the tumor-suppressor functions of BRCA1 and BRCA2. Nature, 2000; 408: 429–432.

14. Zheng L et al. Lessons learned from BRCA1 and BRCA2. Oncogene, 2000; 19(53): 159–175.

15. Monteiro ANA. BRCA1: exploring the links to transcription. TIBS, 2000; 25: 469–474.

16. Venkitaraman AR. Cancer susceptibility and the functions of BRCA1 and BRCA2. Cell, 2002; 108: 171–182.

17. Deng CX. Role of BRCA1 in centrosome duplication. Oncogene, 2002; 21: 6222–6227.

18. Jasin M. Homologous repair of DNA damage and tumorigenesis: The BRCA connection. Oncogene, 2002; 21: 8981–8993.

19. Baer R. and T Ludwig. The BRCA1/BARD1 heterodimer, a tumor suppressor complex with ubiquitin E3 ligase activity. Curr Opin Genet Dev, 2002; 12: 86–91.

20. Starita LM, and JD Parvin. The multiple nuclear functions of BRCA1: transcription, ubiquitination and DNA repair. Curr Opin Cell Biol, 2003; 15: 345–350.

21. Lane TF. BRCA1 and transcription. Cancer Biol. and Ther, 2004; 3: 75–80. 22. Paull TT et al. A critical role for histone H2AX in recruitment of repair factors

to nuclear foci after DNA damage. Curr Biol, 2000; 10: 886–895. 23. Scully R et al. Dynamic changes of BRCA1 subnuclear location and phos-

phorylation state are initiated by DNA damage. Cell, 1997; 90: 425–435. 24. Zhong Q et al. Association of BRCA1 with the hRad50-hMre11-p95 complex

and the DNA damage response. Science, 1999. 285: 747–750. 25. Dong Y et al. Regulation of BRCC, a holoenzyme complex containing BRCA1

and BRCA2, by a signalosome-like subunit and its role in DNA repair. Mol. Cell, 2003; 12: 1087–1099.

26. Wang Y et al. BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. Genes & Dev, 2000; 14: 927–939.

27. Cortez D et al. Requirement of ATM-dependent phosphorylation of brca1 in the DNA damage response to double-strand breaks. Science, 1999; 286(5442): 1162–1166.

28. Tibbetts RS et al. Functional interactions between BRCA1 and the checkpoint kinase ATR during genotoxic stress. Genes & Dev, 2000; 14: 2989–3002.

29. Lee J-S et al. hCds1-mediated phosphorylation of BRCA1 regulates the DNA damage response. Nature, 2000; 404: 201–204.

30. Moynahan ME et al. Brca1 controls homology-directed DNA repair. Mol Cell, 1999; 4(4): 511–518.

31. Abbott DW et al. BRCA1 expression restores radiation resistance in BRCA1-defective cancer cells through enhancement of transcription-coupled DNA repair. J Biol Chem, 1999; 274(26): 18808–18812.

32. Moynahan ME, Cui TY, and Jasin M. Homology-directed DNA repair, mitomycin-c resistance, and chromosome stability is restored with correction of a Brca1 mutation. Cancer Res, 2001; 61: 4842–4850.

33. Wang H et al. Nonhomologous end-joining of ionizing radiation-induced DNA doulbe-stranded breaks in human tumor cells deficient in BRCA1 and BRCA2. Cancer Res., 2001; 61: 270–277.


10. Catteau A et al. Methylation of the BRCA1 promoter region in sporadic breast

and ovarian cancer: correlation with disease characteristics. Oncogene, 1999; 18: 1957–1965.

11. Esteller M et al. Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors. J Natl Cancer Inst, 2000; 5: 564–569.

36. Ongusaha PP et al. BRCA1 shifts p53-mediated cellular outcomes towards irreversible growth arrest. Oncogene, 2003; 22: 3749–3758.

37. Ouchi T et al. Collaboration of signal transducer and activator of transcription 1 (STAT1) and BRCA1 in differential regulation of IFN-gamma target genes. Proc Natl Acad Sci USA, 2000; 97(10): 5208–5213.

38. Houvras Y et al. BRCA1 physically and functionally interacts with ATF1. J Biol Chem, 2000; 275(46): 36230–36237.

39. Zheng L et al. Sequence-specific transcriptional corepressor function for BRCA1

40. Fan S et al. Role of direct interaction in BRCA1 inhibition of estrogen receptor activity. Oncogene, 2001; 20(1): 77–87.

41. 1(AD1) through a coiled-coil-mediated interaction. Genes & Dev, 2002; 16: 1509–1517.

42. Neish AS et al. Factors associated with the mammalian RNA polymerase II holoenzyme. Nucleic Acids Res, 1998; 26(3): 847–53.

43. Yarden RI and Brody LC. BRCA1 interacts with components of the histone deacetylase complex. Proc Natl Acad Sci USA, 1999; 96(9): 4983–4988.

44. Pao GM et al. CBP/p300 interact with and function as transcriptional coactivators of BRCA1. Proc Natl Acad Sci USA, 2000; 97(3): 1020–1025.

45. Bochar DA et al. BRCA1 is associated with a human SWI/SNF-related complex: linking chromatin remodeling to breast cancer. Cell, 2000; 102: 257–265.

46. Scully R et al. BRCA1 is a component of the RNA polymerase II holoenzyme.

47. Anderson SF et al. BRCA1 protein is linked to the RNA polymerase II holoenzyme complex via RNA helicase A. Nat Genet, 1998; 19: 254–256.

48. Krum SA et al. BRCA1 associates with processive RNA polymerase II. J Biol Chem, 2003; 278: 52012–52020.

49. Somasundaram K et al. Arrest of the cell cycle by the tumour-suppressor BRCA1 requires the CDK-inhibitor p21WAF1/CiP1. Nature, 1997; 389(6647): 187–190.

50. Ouchi T et al. BRCA1 regulates p53-dependent gene expression. Proc Natl Acad Sci USA, 1998; 95(5): 2302–2306.

51. Harkin DP et al. Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of BRCA1. Cell, 1999; 97: 575–586.

52. MacLachlan TK et al. BRCA1 effects on the cell cycle and DNA damage response are linked to altered gene expression. J Biol Chem, 2000; 275: 2777–2785.

53. Aprelikova O et al. BRCA1 is a selective coactivator of 14-3-3s gene transcription in mouse embryonic stem cells. J Biol Chem, 2001; 276: 25647–25650.

54. Wang RH, Yu H, and Deng CX. A requirement for breast-cancer-associated gene 1 (BRCA1) in the spindle checkpoint. Proc Natl Acad Sci USA, 2004; 101: 17108–17113.

55. Zheng L et al. BRCA1 mediates ligand-independent transcriptional repression of the estrogen receptor. Proc Natl Acad Sci USA, 2001; 98: 9587–9592.

56. Furuta S et al. Removal of BRCA1/CtIP/ZBRK1 repressor complex on ANG1 promoter leads to accelerated mammary tumor growth contributed by prominent vasculature. Cancer Cell, 2006; 10: 13–24.

Proc Natl Acad Sci USA, 1997; 94(11): 5605–5610.

through a novel zinc finger protein, ZBRK1. Mol. Cell, 2000; 6: 757–768.


Hu Y-F. and Li R. JunB Potentiates function of BRCA1 Activation Domain

34. Zhong Q et al. Deficient nonhomologous end-joining activity in cell-free

extracts from brca1-null fibroblasts. Cancer Res, 2002; 62: 3966–3970. 35. Zhang H et al. BRCA1 physically associated with p53 and stimulates its

transcriptional activity. Oncogene, 1998; 16: 1713–1721.

61. Hakem R et al. The Tumor Suppressor Gene Brca1 Is Required for Embryonic Cellular Proliferation in the Mouse. Cell, 1996; 85: 1009–1023.

62. Hakem R et al. Partial rescue of Brca1 (5-6) early embryonic lethality by p53 or p21 null mutation. Nat Genet, 1997; 16: 298–302.

63. Cao L et al. ATM-Chk2-p53 activation prevents tumorigenesis at an expense of organ homeostasis upon Brca1 deficiency. EMBO J, 2006; 25: 2167–2177.

64. Schuyer M and Berns EM. Is TP53 dysfunction required for BRCA1-associated carcinogenesis? Mol Cell Endocrinol, 1999; 155: 143–152.

65. Elledge SJ and Amon A. The BRCA1 suppressor hypothesis: an explanation for the tissue-specific tumor development in BRCA1 patients. Cancer Cell, 2002; 1: 129–132.

66. Monteiro ANA. BRCA1: the enigma of tissue-specific tumor development. Trends Genet, 2003; 19: 312–315.

67. Hennighausen L and GW Robinson. Think globally, act locally: the making of a mouse mammary gland. Genes & Dev, 1998; 12: 449–455.

68. Nilsson S et al. Mechanisms of estrogen action. Physiol Rev, 2001; 81: 1535–1565.

69. Hall JM, JF Couse, and KS Korach. The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem, 2001; 276: 36869–36872.

70. Anzick SL et al. AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science, 1997; 277: 965–968.

71. Khan SA et al. Estrogen receptor expression in benign breast epithelium and breast cancer risk. J Natl Cancer Inst, 1998; 90: 37–42.

72. Fan S et al. BRCA1 inhibition of estrogen receptor signaling in transfected cells. Science, 1999; 284(5418): 1354–1356.

73. Xu J, Fan S, and Rosen EM. Regulation of the estrogen-inducible gene expression profile by the breast cancer susceptibility gene BRCA1. Endocrinology, 2005; 146: 2031–2047.

74. oncology Clinics of North America, 2001; 13(2): 1–23.

75. Nilsson S and Gustafsson J-A. Basic aspects of estrogen action. Breast Cancer Res, 2000; 2: 360–366.

76. Persson I. Estrogens in the causation of breast, endometrial, and ovarian cancers-evidence and hypotheses from epidemiological findings. Steroid Biochem. & Mol Biol, 2000; 74: 357–364.

77. Ali S and Coombes RC. Endocrine-responsive breast cancer and strategies for combating resistance. Nature Reviews Cancer, 2002; 2: 101–112.

78. Dumitrescu RG and Cotarla I. Understanding breast cancer risk - where do we stand in 2005? J Cell Mol Med, 2005; 9: 208–221.

79. Wooster R and Weber BL. Breast and ovarian cancer. N Engl J Med, 2003; 348: 2339–2347.

80. Simpson ER and Davis SR. Minireview: aromatase and the regulation of estrogen biosynthesis-some new perspectives. Endocrinol, 2001; 142: 4589–4594.

81. Hu Y-F et al. Modulation of aromatase expression by BRCA1: a possible link to tissue-specific tumor suppression. Oncogene, 2005; 24: 8343–8348.


Nass SJ and Davidson NE. Advance in Breast Cancer Therapy. Hematology/

57. Ohta T and Fukuda M. Ubiquitin and breast cancer. Oncogene, 2004; 23: 2079–2088.

targeted degradation of RNA polymerase II. Genes Dev, 2005; 15: 1227–1237. 58. Kleiman FE et al. BRCA1/BARD1 inhibition of mRNA 3’ processing involves

59. Starita LM et al. BRCA1/BARD1 ubiquitinate phosphorylated RNA polymerase II. J Biol Chem, 2005; 280: 24498–24505.

60. Starita LM et al. BRCA1-dependent ubiquitination of gamma-tubulin regulates centrosome number. Mol Cell Biol, 2004; 24: 8457–8466.

85. Sasano H and Harada N. Intratumoral aromatase in human breast, endometrial, and ovarian malignacies. Endocrine Rev, 1998; 19: 583–607.

86. Purohit A and Reed MJ. Regulation of estrogen synthesis in postmenopausal women. Steroids, 2002; 67: 979–983.

87. Bulun SE et al. A link between breast cancer and local estrogen biosynthesis suggested by quantification of breast adipose tissue aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after reverse transcription. J Clin Endocrinol Metab, 1993; 77: 1622–1628.

88. Harada N. Aberrant expression of aromatase in breast cancer tissues. J Steroid Biochem Mol Biol, 1997; 61: 175–184.

89. Zhao Y et al. Estrogen biosynthesis proximal to a breast tumor is stimulated by PGE2 via cyclic AMP, leading to activation of promoter II of the CYP19 (aromatase) gene. Endocrinology, 1996; 137: 5739–5742.

90. Morales L, Neven and R Paridaens, Choosing between an aromatase inhibitor and tamoxifen in the adjuvant setting. Curr Opin Oncol, 2005; 17: 559–565.

91. Ghosh S et al. Tumor Suppressor BRCA1 Inhibits a Breast Cancer-Associated Promoter of the Aromatase Gene (Cyp19) in Human Adipose Stromal Cells. Am J Physiol Endocrinol Metab, 2006. [Epub ahead of print].

92. Lu M et al. BRCA1 negatively regulates the cancer-associated aromatase promoters I.3 and II in breast adipose fibroblasts and malignant epithelial cells. J Clin Endocrinol Metab, 2006. [Epub ahead of print].

93. Russo J et al. Estrogen and its metabolites are carcinogenic agents in human breast epithelial cells. J Steroid Biochem Mol Biol, 2003; 87: 1–25.

94. Perou CM et al. Molecular portraits of human breast tumors. Nature, 2000; 406: 747–752.

95. breast cancer. Nature, 2002; 415: 530–536.

96. Kauff ND et al., Risk-reducing salpingo-oophorectomy in women with a

97. Rebbeck TR et al. Prophylactic oophorectomy in carriers of BRCA1 or BRCA2 mutations. New Engl J Med, 2002; 346: 1616–1622.

98. Bachelier R et al. Effect of bilateral oophorectomy on mammary tumor formation in BRCA1 mutant mice. Oncol Rep, 2005; 14: 1117–1120.

99. Narod SA et al. Tamoxifen and risk of contralateral breast cancer in BRCA1 and BRCA2 mutation carriers: a case-control study. Hereditary Breast Cancer Clinical Study Grou Lancet, 2000; 356: 1876–1881.

100. Xu X et al. Conditional mutation of Brca1 in mammary epithelial cells results in blunted ductal morphogenesis and tumour formation. Nature Genetics, 1999; 22: 37–43.

101. Albiges L et al. Spectrum of breast cancer metastasis in BRCA1 mutation carriers: highly increased incidence of brain metastases. Ann Oncol., 2005; 16: 1846–1847.

102. Bissell MJ and Radisky D. Putting tumors in context. Nature Reviews Cancer, 2001; 1: 46–54.

103. Bissell MJ et al. The organizing principle: microenvironmental influences in the normal and malignant breast. Differentiation, 2002; 70: 537–546.

van ’t Veer LJ et al. Gene expression profiling predicts clinical outcome of

BRCA1 or BRCA2 mutation. N. Engl. J. Med., 2002. 346: 1609–1615.

453. BRCA1 in initiation, invasion, and metastasis

82. Chodankar R et al. Cell-nonautonomous induction of ovarian and uterine serous cystadenomas in mice lacking a functional Brca1 in ovarian granulosa cells. Curr Biol, 2005; 15: 561–565.

83. Bulun SE et al. The human CYP19 (aromatase P450) gene: update on physio-logic roles and genomic organization of promoters. J Steroid Biochem Mol Biol, 2003; 86: 219–224.

Rev, 2005; 26: 332–330. 84. Simpson ER et al. Estrogen–the good, the bad, and the unexpected. Endocr

106. Weber F et al. Total-genome analysis of BRCA1/2-related invasive carcinomas of the breast identifies tumor stroma as potential landscaper for neoplastic initiation. Am J Hum Genet., 2006; 78: 961–972.

107. Kiaris H et al. Evidence for nonautonomous effect of p53 tumor suppressor in carcinogenesis. Cancer Res, 2005; 65: 1627–1630.

108. Hill R et al. Selective evolution of stromal mesenchyme with p53 loss in response to epithelial tumorigenesis. Cell, 2005; 123: 1001–1011.

109. Jacobsen BM et al. Spontaneous fusion with, and transformation of mouse stroma by, malignant human breast cancer epithelium. Cancer Res, 2006; 66: 8274–8279.


104. Furuta S et al. Depletion of BRCA1 impairs differentiation but enhances proliferation of mammary epithelial cells. Proc Natl Acad Sci USA, 2005; 102: 9176–9181.

105. Kawai H et al. Direct interaction between BRCA1 and the estrogen receptor regulates vascular endothelial growth factor (VEGF) transcription and secretion in breast cancer cells. Oncogene, 2002; 21: 7730–7739.

Chapter 4

CELL MOTILITY AND BREAST CANCER METASTASIS

Marc E. Bracke1, Daan De Maeseneer1, Veerle Van Marck1, 1 1 1

2

1Laboratory of Experimental Cancer Research, Department of Experimental Cancer Research, Radiotherapy and Nuclear Medicine, Department of Gynaecological Oncology, University Hospital, De Pintelaan 185, B-9000 Gent, Belgium

Abstract: Motility and invasion of breast cancer cells are the result of the concerted

filaments and microtubules), establishment and disruption of cell-matrix and homotypic/heterotypic cell-cell adhesions, and extracellular proteolysis. Metastasis formation is not only related to cancer cell motility, but also necessitates the collaboration of other, coined “host” cells. Newly discovered ligand-receptor interactions between cancer cells and these host elements offer a molecular explanation for Paget’s “seed and soil” hypothesis, and indicate new targets for possible anti-metastatic therapeutic agents

motility, metastasis, breast cancer, actin, microtubles, cytoskeleton, extra-ellular matrix, collagen, laminin, hyaluronate, cadherin, CXCL12/CXCR4 interaction, integrin, CD44, proteinases

1. INTRODUCTION

The relation between cancer cell motility and the development of meta-stases was historically first suggested by Rudolf Virchow, who situated the onset of cancer at the primary tumour. Here, normal cells have

© 2007 Springer.

47

Herman T. Depypere Lara Derycke , Barbara Vanhoecke , Olivier De Wever , and

by the dynamic organisation of cytoskeletal components (actin micro-action of a number of cell activities: directional migration underpinned

Keywords:

transformed into cancer cells, and from this site the cancer cells can

2

disseminate towards locoregional and distant organs to found metastases. Bridging the distance between a primary tumour and its metastases is


48 Bracke et al.

become highly motile when isolated and brought in culture, but do not metastasize when reinoculated into the host organism. Normal cells some-times do “metastasize”, like leukocytes which can migrate from the bone marrow towards their homing or inflammation sites, or like trophoblast cells towards the lungs of certain rodents during pregnancy (1). The common denominator in the motility by these normal cells is the restrict-tion in time and space: sensitivity to contact inhibition and the switching off by an “internal clock” appear to be mechanisms that are deficient in cancer cells. Again, this should not be taken too strictly, since cancer cell motility is a transient phenomenon, which can be switched off spon-taneously and often temporarily in cells once they have established a metastasis. Aware of this complexity, we should consider motility as a necessary, but not as a sufficient condition for metastasis, and thus not conceive it as a functional marker of metastatic capability.

The content of this chapter is strictly related to the contribution of breast cancer motility to metastasis. Cell motility covers a number of aspects we will deal with separately for didactic reasons (Figure 1). First, breast cancer cells dispose of highly dynamic structures like actin fila-ments and a cytoplasmic microtubular complex, for which the traditional term “cytoskeleton” probably is a misnomer because it is too static (2). This intrinsic motility machinery can be considered as both the engine and the steering wheel of the cell, since it allows directional migration. This implicates that motility is not random, but that moving breast cancer cells are always on their way to form metastases. Second, motility is only one prerequisite for invasion, and is influenced by transient adhesive inter-actions with the cell’s microenvironment. So, homotypic (between cells of the same type) cell–cell adhesions usually keep the cells in contact with each other, and serve an invasion suppressive aim. Heterotypic (between cells of different types) cell–cell adhesions, however, can help the invading cancer cell to use neighbouring stromal cells as a “grip”. For cell–matrix adhesions the role in invasion is dual: some extracellular matrix structures, such as the basement membrane, can act as barriers or anchors for the cancer cells, while others rather offer tracks for the moving cell, such as interstitial type I collagen fibres. Third, extracellular proteases continuously help to remodel the cancer cell’s microenvironment by disrupting cell–cell and cell–matrix adhesion proteins temporarily, and

by dissolving extracellular matrix structures to facilitate cell displacement. For these reasons extracellular proteolysis is an important activity in invasion, not at least because it can generate chemotactic and angiogenic peptide fragments (3).

routes. Yet, the relation between motility and metastasis formation is not always straightforward. Normal cells with a stable tissue position, can

possible only if cancer cells can move actively and passively along certain

4. Cell motility and breast cancer metastasis 49

Figure 1. Schematic overview of the different aspects implicated in cancer cell motility and the first steps of metastasis formation. Normal epithelial cells (EP) transform into cancer cells (CA), where different components of the cytoskeleton (CS) organize to provide the machinery for directional migration (DM, dotted arrow). Integrins (INT) on normal and cancer cells can interact with components of the extracellular matrix (ECM), such as laminin in the basement membrane (BM). Cadherins (CAD) are implicated in cell–cell adhesion, while proteolysis (PL) by proteinases dissolves the matrix. Host cells have a molecular cross-talk with normal and cancer cells (full arrows). Chemotactic ligands (L) and receptors (R) guide the cancer cells towards the vessels for intravasation (EC: endothelial cell).

The old “seed and soil” hypothesis by Paget (4), stating that organ-specific metastasis from the primary tumour depends on the right combi-nation of tumour cell and host organ factors, is still valid. For motility and breast cancer metastasis we will rephrase this hypothesis in terms of ligands and receptors as much as possible. Not only can these lead to a more complete picture of molecular interactions in metastasis, they may also indicate new targets for anti-metastatic therapeutic strategies in onco-logy. The latter concern is inspired by recent statistics telling that meta-

50 Bracke et al.

In this chapter we will apply three major restrictions. First, the data will only relate to breast cancer cells, which does not exclude that they may be relevant for other cancer types as well. Second, only cell motility information is included in order to avoid overlap with the other chapters of this book. Third, the motility data have been related to breast cancer invasion and metastasis, and are relevant to the general theme of this book.

2. THE DYNAMICS OF THE CYTOSKELETON IN BREAST CANCER CELLS: THE DRIVING FORCE OF THE CANCER CELL ON ITS WAY TO METASTASIS

The dynamic assembly/disassembly of the cytoplasmic actin micro-filament complex is based on the rapid and reversible polymerisation of monomeric globular G-actin monomers into polymeric filamentous F-actin. Actin microfilament formation is typically observed in membrane protrusions coined lamellipodia and invadopodia. Analyses based on micro-arrays and proteomics have shown that the dynamics of actin poly-merisation/depolymerisation reactions are controlled by actin-binding proteins. In cancer cells the expression of these proteins can be aberrant: some are downregulated like gelsolin (5,6), while others like fascin are upregulated (7,8). More recent data have modified our thinking of how actin-binding proteins regulate cancer cell motility: their localisation in-side the cell appears to be more important than their gross general concentration. Some of them, like the LIM-and-SH3 protein (LASP1), are phosphorylated upon stimulation by external signals, relocate in the cytoplasm of the leading edge of the cancer cell and locally associate with actin to build up focal adhesion complexes in a dynamic way (9).

Remarkably, actin and actin-binding proteins also co-exist in the nucleus. How this finding relates to cell motility is not always clear, but for the actin-capping protein CapG for example, it was shown that transport from the cytoplasm to the nucleus stimulates cell invasion (10). If this phenol-menon proves to be clinically relevant, the import receptor importin β, which is responsible for this nuclear relocation of CapG, may become an interesting therapeutic target for anti-invasive and anti-metastatic agents.

An underestimated actin-binding protein with relation to metastasis is probably myosin (11). In breast cancer cells in vitro non-muscle myosin II A and B are localised both in the rear end of the moving cell, where they are crucial for the retraction of the posterior cell part, and in the

stases are by far the major cause of death in cancer patients, including breast cancer.


and its expression has been related with the metastatic phenotype repeatedly (12–14). We have shown that a non-invasive cell line variant, derived from a human breast cancer, did only weakly express the heavy chains of non-muscle myosins II A and B, as compared to an invasive variant from the same tumour, expressing high levels of these molecules. Attempts in our laboratory to downregulate the expression of non-muscle myosin II A and B in invasive and metastatic breast cancer cells are currently in progress, and try to confirm our hypothesis that these myosins are targets for anti-invasive agents.

While actin and non-muscle myosin II are the motor of the cell, the function of the cytoplasmic microtubular complex has been referred to as a steering wheel, because it is instrumental for direction–finding during cell movement. One strong indication for this idea was provided by experiments in vitro with cancer cells on glass, in which different classes of microtubule inhibitors all blocked directional migration, but not random motility nor cell ruffling. Treated cells lost their elongated shape, and became flattened and disclike with intense membrane ruffling all over the perimeter of the cell. Moreover, this inhibition was sufficient to block invasion in different assays in cell and organ culture (15), and offered an explanation for the anti-metastatic effect of chemotherapy regimens containing microtubule inhibitors, such as vinca-alkaloids (16). Indeed, the microtubule inhibitors not only block the cancer cell cycle in the M-phase, but also prevent dissemination to locoregional tissues and distant organs.

Rho, Rac, and Cdc42, three small Rho GTPases, control signal trans-duction pathways linking membrane receptor signals to the assembly and disassembly of the actin cytoskeleton. Rho regulates stress fiber and focal adhesion assembly, Rac regulates the formation of lamellipodia

and membrane ruffles and Cdc42 triggers filopodial extensions at the cell periphery. These observations have led to the suggestion that, wherever filamentous actin is used to drive a cellular process, e.g., cell movement, axon guidance, phagocytosis, or cytokinesis, the Rho GTPases may play an important regulatory role. Furthermore, Rho, Rac, and Cdc42 have been reported to control other cellular activities, including regulation of the JNK and p38 MAP kinase cascades. So, all three GTPases have been implicated in growth control and although mutations at the gene loci of these molecules have not been found in human cancers, experiments suggest that Rac in particular might play an important role in invasion and metastasis (17). As a rule, activation of Rac and Cdc42 and inactiva-tion of Rho lead to increased motility, invasion and metastasis. Yet, some considerations have to be added to this generalisation. First, the family of

leading edge, where they associate with S100A4 (also known as metastasin-1). The A4 isoform of S100 is a motogenic molecule, that can induce the epithelioid-to-mesenchymal transition (EMT) in cancer cells,

52 Bracke et al.

invasion (20,21). RhoC overexpression is positively correlated with breast cancer metastasis formation (22). Moreover, the balance between Rho GTPases and guanine nucleotide dissociation inhibitors also appears to regulate the metastatic capability of breast cancer cells (23). Second, the effects of Rho on motility, invasion, and metastasis may be influenced by the cellular context: effects of Tiam-1 on cell motility for instance depend on the cell type under study (24).

3. EXTRACELLULAR PROTEINASES AND THEIR INHIBITORS IN MOTILITY, INVASION, AND METASTASIS: NOT ONLY A MATTER OF EXTRACELLULAR MATRIX BREAKDOWN

In the three-step hypothesis of Liotta (3), proteolysis is conceived as extracellular matrix (ECM) breakdown which creates a virtual gap to be continuously filled up by the leading edge of the moving cancer cell. Different classes of extracellular proteases have been associated with this phenomenon, of which matrix metalloproteinases (MMPs), the serine proteinases coined plasminogen activators (PAs) and the aspartic protei-nase cathepsine D have been studied in depth in breast cancer. One timely aspect of this type of motility regulation is the recent insight that synthesis and secretion of these proteinases is controlled via different path-ways as a response to extracellular stimuli or inhibitors. Known examples of MMP upregulation relevant to metastasis are: MMP-7 (matrilysin-1) upregulation as a result of Wnt pathway stimulation or cyclo-oxygenase-2 (COX-2) overexpression (25), and MMP-9 (gelatinase B) upregulation by integrin αvβ3 (26) or Src homology phosphatase (Shp-2) (27). Better under-standing of these regulatory pathways will help to identify new and old anti-invasive and anti-metastatic agents, such as bisphosphonates, vanillin,

while calcitonin was found to inhibit the expression (32). Importantly,

motility/invasion and metastasis, respectively (33).

u-PA has received major interest, and expression was found to be induced and COX-2 inhibitors (28,29). Among PAs, the expression control of

these up- and downregulations correlated with stimulated or inhibited

by, for example, hypoxia, osteopontin, and the tyrosine kinase Syk (30,31),

Rho GTPases is continuously increasing with new members (e.g., Tiam-1 (18) and Deleted in Liver Cancer-1 (19), both motogenic factors) and isoforms. These new members add a new level of complexity to our general rules. So, Rac1 is associated with lamellipodia formation, while Rac3 is not, but activation of either isoform increases motility and


however, appears to be a bona fide inhibitor of motility (36). A third level of regulation relates to the localisation of the proteinases with respect to the surface of the invading cancer cell. Some MMP’s are anchored to the plasma membrane (the so-called membrane-type MMP’s and the “a disintegrin and metalloproteinase” (ADAMs)) (37), while for u-PA a typical surface receptor (u-PAR) can be expressed. A fourth level

fibroblasts and myofibroblasts) present in the tumour’s microenviron-ment. Taken together, the role of extracellular proteinases in cancer progression can hardly be inferred from their simple presence as such, but needs confirmation by signs of their in situ activity. We are con-vinced that ultrastructural visualisation of typical ECM breakdown products (like collagen type I fragments) at the invading edge of cancer cells is the most relevant technique to identify proteolysis in situ.

Breakdown of ECM is probably only one important aspect of extra-cellular proteinase activity. Cleavage of ectodomains of surface receptors and cell–cell adhesion molecules can also influence motility, invasion, and metastasis, described in more detail in section 4.

4. CELL–MATRIX AND CELL–CELL

AND AS RECEPTORS

Integrins are the sensors of the cancer cells for their surrounding ECM, and participate in the molecular translation of the “seed and soil” hypothesis by Paget. As mentioned before, the integrin-basement mem-brane interaction can help in maintaining positional stability in normal epithelia, but in carcinoma many clues indicate that integrins promote motility, invasion, and hence, metastasis. Many data from the recent literature point toward the relation between (over)expression of certain types of integrins, cell motility, and breast cancer metastasis (38). Examples of integrin subunits related to motility and metastasis are α3

and α4 (both inducing MMP-9 secretion) (39,40), α5, α6 (via the “nuclear factor of activated T-cells” mediators, abbreviated as NFAT1 and NFAT5) (41), α8 (interacting with tenascin V, an ECM molecule thought

of regulation is brought in by factors from other cell types (e.g.,

ADHESION: CADHERINS AS LIGANDS

The expression of extracellular proteinases is only one level of regula-tion of enzymatic activity. Of key importance is also the balance between active enzymes and their natural inhibitors: tissue inhibitors of metallo-proteinase (TIMPs) for MMPs and plasminogen activator inhibitors

1 and PAI-3 inhibit u-PA, but finally stimulate motility of breast cancer cells as a result of an intrinsic motogenic activity (35). TIMP-2,

(PAIs) for PAs (34). Here, the interpretation becomes tricky when PAI-

54 Bracke et al.

by the cancer cells is not explaining all aspects of motility and metastasis formation. Also important appears to be the composition of the ECM in the tumor microenvironment, and a seminal paper by Barsky et al. (48) revealed ECM differences between normal breast and cancer. More recent papers seem to confirm that subtle ECM differences can have an impact on cancer cell motility and invasion. This was shown for type I collagen (telopeptide-free, invasion-permissive versus normal, invasion-resistant) (49), fibronectin (involuting breast-derived, invasion-permissive versus nulliparous breast-derived, invasion-resistant) (50), laminin (catechin- pretreated, invasion resistant versus native, invasion-permissive) (51) and hyaluronate (hyaluronate synthase 2 antisense-treated, invasion, resistant versus parental, invasion-permissive) (52).

One important cytoplasmic signalling molecule transducing inhibitory integrin signals to the effector machineries of breast cancer cell motility, invasion, and metastasis, is integrin-linked kinase (ILK) (53). Eliminating chromosome 11, where the ILK gene locus is situated, induces invasion and metastasis, and has led to consider ILK as a metastasis suppressor. Another metastasis suppressor gene Kiss-1, coding for kisspeptin (54) (metastin), was shown to exert its inhibitory effect on breast cancer metastasis via increased adhesion to collagen type IV, which is a consti-tuent of basement membrane (54,55).

Homotypic cell–cell interaction mediated by homophilic cadherin recog-

recognition, histidine–alanine–valine (HAV) amino acid sequence in their extracellular part. However, this does not imply an underestimation of the role of other cadherins, such as cadherin-11, in breast cancer motility. The three cadherins of interest here are epithelial (E), neural (N), and placental (P) cadherin, which consist of a highly conserved cytoplasmic, a membrane-spanning part, and an ectodomain composed of five calcium-binding protomers, which marks the identity of each cadherin type. The role of these cadherins in breast cancer motility and invasion is not entirely elucidated, but available data indicate that E-cadherin is a motility/invasion suppressor, while the effects of N- and P-cadherin are in line with motility/invasion promotion.

In this chapter the discussion will be restricted to type I (also called nitions is a crucial regulator of cell motility, invasion, and metastasis.

“classical”) cadherins, which are characterised by a typical homophilic

to be involved in motogenic and invadogenic effects by the ECM) (42), αv (often related to breast cancer metastasis) (26,43,44), β1 (activated by phosphatidyl inositol-3 kinase, abbreviated as PI3K (45), and Akt2 (46)), and β4. One indication of the importance of integrins in metastasis is the

peptidomimetics S137 and S246, and provide a proof of principle of the integrin implication (47). Yet and again, the integrin repertoire expressed

for the integrins. Some of these drugs are referred to as the RGD anti-metastatic effect of drugs designed to act as inactivating ligands


level/pattern and pathology grade: experimental manipulation of E-cadherin expression via antisense technology has confirmed the invasion suppressor function of this molecule in mammary cells in vitro (58), and subsequently in transgenic rat pancreas in vivo (59), and the metastasis suppressor function in drosophila larva eye disk (60). Hence, one important question in oncology is: what are the downregulating mechanisms of E-cadherin in breast cancer, and can targets for therapy be discovered among them? Mutations in the E-cadherin gene are not a frequent cause of human cancers. They occur incidentally in the germ-line of some families in New Zealand and Portugal, and lead to the development of breast and other cancers with an early age onset (61). Somatic mutations of E-cadherin appear to be a frequent phenomenon in lobular breast carcinoma, but a rare one in ductal carcinoma. These mutations are detectable early at the very stage of carcinoma in situ, and often create premature stop codons giving rise to truncated versions of the molecule (62,63). Theoretical considerations predict that many of these truncated forms lack the membrane-spanning part, and are not anchored in the cell membrane. So, if they are secreted, they may diffuse into the extracellular fluids and in blood, and can be developed as a circulating tumor marker for lobular breast carcinoma patients. While mutations are rare, downregulation of E-cadherin expression can often be traced back to promoter silencing. The list of negative transcription factors for E-cadherin is increasing steadily: slug, snail, twist, SIP1, δEF1, E47 (64,65). Another mechanism of silencing may be the result of methylation of promoter DNA bases. Post-translational modifications of the E-cadherin molecule have been shown to be crucial in the regulation of epithelial cell–cell adhesion, motility, and invasion as well. These modifications such as extracellular domain N-glycosylation and cytoplasmic tail serine/threonine/tyrosine phosphorylation have to be described within a broader intracellular signalling context. The cytoplasmic tail of E-cadherin associates in a non-covalent way with a group of catenin molecules (α-, β-, γ-catenin, –the latter one being identical to plakoglobin–, and p120 catenin), which form a link with the actin cytoskeleton.

E-cadherin is present in all normal epithelia, promotes cell–cell adhesion in the adherens junctions and plays a role as master molecule during the organisation of the other epithelial cell junction types. In epi-thelial layers E-cadherin is responsible for contact inhibition of motility. When the expression of this molecule is downregulated in an epithelioid background, such as in certain embryonic stages or in invasive carcino-mas, the cells become more motile and start to occupy the neighbouring stromal tissues (56,57). Evidence that this downregulation is indeed causally related to invasion not only stems from correlation studies in breast cancer between the immunohistochemical E-cadherin expression

56 Bracke et al.

cancer cell motility and metastasis here are the receptor families for epidermal growth factor (EGF), insulin-like growth factor I (IGF-1) (66), and nerve growth factor (NGF) (67). Heregulin, a ligand of the EGF receptor-3, was shown to increase E-cadherin-mediated breast cancer

improving the function of the E-cadherin/catenin complex. Our team has gathered indications that these ligands trigger rapid (within 10 minutes) exocytosis of a pool of subcortically stored E-cadherin in human MCF-7/6 breast adenocarcinoma cells (69). The E-cadherin/catenin complex is also amenable to modulation by estrogens and anti-estrogens. The selec-tive estrogen receptor modulator (SERM) tamoxifen, which has been administered successfully to breast carcinoma patients as an adjuvant therapy for three decades, was shown to activate the complex and to inhibit motility and invasion. A potent natural phyto-estrogen from hops coined 8-prenylnaringenin (8-PN) or hopein, was also shown to increase E-cadherin-mediated cell-cell adhesion between MCF-7/6 cells (70). For a number of empirical stimulators of E-cadherin cell–cell adhesion no

was described for: the citrus methoxyflavone tangeretin (71), the hops prenylated chalcone xanthohumol (72), the vitamin A analog retinoic acid (73) and a number of related polyphenols. A large group of closely related congeners of these polyphenols were tested for potentially anti-invasive effects on MCF-7/6 cells in confronting cultures with embryo-nic chick heart fragments (74). The degree of anti-invasive activity of these compounds was related to their three-dimensional features by means of the QSAR software, and predictions on the characteristics of optimally anti-invasive compounds are expected to be available soon (75).

N-cadherin is functionally the opponent of E-cadherin in many aspects (76). While E-cadherin is a suppressor of epithelioid motility and

in vitro, a stimulation of cell–cell adhesion and an inhibition of invasion molecular target has been found yet. Using MCF-7/6 cells as a model

cell–cell adhesion, and to inhibit their invasion (68). Similarly, IGF-I and insulin were anti-invasive in organotypic confronting cultures by

While serine/threonine phosphorylation of E-cadherin and β-catenin are implicated in the regulation of cell–cell adhesion, it is the tyrosine phosphorylation of β-catenin that has gained major attention. The latter phenomenon has been related to a dissociation of β-catenin from the E-cadherin/catenin complex, and to inactivation of the cell–cell adhesion structures. Moreover, tyrosine-phosphorylated β-catenin becomes resistant to proteasome degradation, and diffuses into the nucleus to activate pro-

illustration of how an invasion suppressor molecule, provided it is integrated in the E-cadherin/catenin complex, can adopt the role of an oncogene and invasion promoter after the proper post-translational modifications have occurred. Tyrosine phosphorylation of β-catenin can result from activation of cell surface peptide receptors. Relevant to breast

tein transcription of, among others, MMP-7 and myc genes. This is an

Upregulation of N-cadherin leads to enzymatic cleavage of the ecto-domain close to the plasma membrane (Figure 2). Several proteinases have been shown to perform this cleavage: while ADAM10 appears to be the main actor, other enzymes such as plasmin and the membrane-type matrix metalloproteinases MT1-MMP and MT5-MMP can be implicated as well. All this results in shedding of a 90 kD soluble N-cadherin fragment (sN-CAD) into the extracellular fluid. This sN-CAD was shown to stimulate cancer cell motility and angiogenesis, and future experiments will show whether or not this fragment contributes to meta-stasis formation in vivo (83). Using an ELISA assay, we were able to detect sN-CAD in a number of biological fluids, such as blood and semen (84). For several tumour types, including breast cancer, the patient’s serum con-centration of sN-CAD was higher than in a reference population with no evidence of disease (median value 584 versus 99 ng/ml respectively). We are currently evaluating the potential value of serum sN-CAD as a tumour marker in cancer patients.

For P-cadherin, data on its role in motility and invasion of breast cancer

expressed in the normal human breast by the myoepithelial cells. It is implicated in growth and differentiation, as evidenced by knockout mice displaying precocious differentiation of the mammary gland, and is aber-rantly expressed in mammary carcinomas of high histological grade and poor prognosis. It has been suggested that suppression of the P-cadherin gene is lost during carcinogenesis, but the nature of this mechanism and the biological role of the newly acquired P-cadherin still remain interesting areas of research. In one study, blocking the estrogen receptor

cycle gatekeeper is no longer implicated in proliferation and apoptosis only, but has to be considered an important EMT suppressor and hence a major motility and metastasis suppressor (82).


are still sparse, but the list is growing steadily (Table 1). P-cadherin is

invasion, N-cadherin is an activator of both activities, and hence a factor of bad prognosis (77). Simultaneous expression of E- and N-cadherin in human breast cancer cells showed that the function of N-cadherin dominates the one of E-cadherin with respect to cell motility, invasion, and metastasis formation (78). In malignant tumours, including breast cancers, downregulation of E-cadherin is accompanied or followed by upregulation of N-cadherin. Aberrant expression studies of the transcript-tion factors snail and SIP1 show that these phenomena may be elements of a broader dedifferentiation program observed in a number, but not in all cancers, namely the epithelial-to-mesenchymal transition (EMT) (79,80). In EMT epithelial cells not only resemble mesenchymal fibroblasts mor-phologically, but also express mesenchymal markers (e.g., N-cadherin and vimentin) (81). Recent data indicate that EMT is a reversible process (as sometimes observed at metastatic sites), and that the maintenance of the epithelial state is one of the multiple functions of p53. So, this cell

Figure 2. Schematic overview of the enzymatic cleavage of the N-cadherin ectodomain (sN-CAD). Epithelial cadherin (E-CAD) is normally present on the breast epithelia (EP) while the cancer cell (CA) often express Neural cadherin (N-CAD). N-cadherin is also present on stromal cells (SC), like myofibroblast and endothelial cells (EC). Plasmin, ADAM10, MT1-MMP, and MT5-MMP are responsible for the proteolysis (PL) of N-cadherin. sN-CAD can be present in the extracellular matrix (ECM) but also in the blood.

Bracke et al.58

invasive capacity and the loss of cell–cell adhesion in vitro. Retroviral transduction in MCF-7/AZ cells confirmed the pro-invasive activity of P-cadherin, which required the juxtamembrane, p120 catenin-binding do-main of its cytoplasmic tail. The effect of P-cadherin on cell–cell adhesion, motility, and invasion are clearly dependent on the cell context, since opposite effects were obtained in melanoma cells (85). Here, P-cadherin expression suppresses invasion in different assays, and may be a target for future gene therapy in this tumour.

with the pure antagonist ICI 182,780 induced the expression of P-cadherin in MCF-7/AZ cells, which coincided with the acquisition of

5. THE INTERACTION OF THE BREASTCANCER CELL WITH SOLUBLE AND

In accordance with the Paget hypothesis, breast cancer cells express different types of receptors, which make them sensitive to extracellular signals. Some of these signals are secreted, others are cell-bound, but all of them contribute to the organ-specific nature of metastasis. The chemo-kine with C-X-C motif ligand 12 (CXCL12) is secreted by hepatocytes and is recognized by the CXC receptor 4 (CXCR4) on circulating breast cancer cells (105–107). The receptor is absent on normal breast epithelial cells, but is expressed on ductal cells at very early stages of tumori-genesis such as in atypical hyperplasia or carcinoma in situ (108), and this receptor is upregulated by heregulin and by adenosine in the hypoxic tumor micro-environment (109). The molecular interaction between the ligand and the receptor triggers a number of signalling pathways in the cancer cell. The G-protein coupled CXCR4 receptor recrutes the α-, β-, and γ-subunits (110), and activates the following pathways: (a) phos-phatidyl inositide-3 kinase (PI3K), Akt1, and focal adhesion kinase (FAK), (b) β-arrestin and erk (111), and (c) epidermal growth factor receptor (EGFR) (112). These signals result in effects that eventually prepare the cancer cell for extravasation into the organ of metastasis: apoptosis is inhibited, endothelial transmigration is promoted, and motility/invasion is induced through an reorganisation of the actin cytoskeleton. The CXC example has at least two merits. First, it translates the Paget hypothesis on seed-and-soil into CXCR4 and CXCL12 respectively, and, second, it appears to be a useful target for anti-invasive treatments, as will be outlined in section 6. Recently, an opposite ligand/receptor interaction effect was published on cancer cell motility and metastasis: the tetra-spanin KAI1/CD82 on cancer cells interacts with the Duffy antigen receptor for cytokines (DARC). The interaction suppresses metastasis by inhibiting extravasation of circulating cancer cells (113).

The CXCR4/CXCL12 system is only one example of molecular inter-actions between breast cancer cells and distant organs that trigger motility and determine organ-specific metastasis formation. Table 2 lists a number of interactions affecting both breast cancer motility and metastasis.


WITHIN A MICRO-ECOSYSTEM CELL-ASSOCIATED SIGNALS: INVASION

Tabl

e 1.

Stu

dies

con

cern

ing

the

expr

essi

on a

nd fu

nctio

n of

P-c

adhe

rin in

neo

plas

tic b

reas

t tis

sues

and

thei

r phy

siol

ogic

al c

ount

erpa

rts

Aut

hor

Met

hods

Fi

ndin

gs1

Pala

cios

et a

l., 1

995

(86)

IH

C

Nor

mal

: P-C

AD

++

in m

yo-e

pith

elia

l cel

ls, E

-CA

D +

in lu

min

al e

pith

elia

l cel

ls.

IDC

: P-C

AD

+ 2

0% (9

/45)

, E-C

AD

↓ 6

6% (3

0/45

) am

ong

whi

ch a

ll P-

CA

D +

, ILC

: 0%

E- a

nd P

-CA

D +

(0

/9).

P-C

AD

+ a

nd E

-CA

D ↓

~ h

ighe

r his

tolo

gica

l gra

de, E

R- e

n PR

-neg

ativ

ity. N

o co

rrel

atio

n w

ith

tum

our s

ize

and

lym

ph n

ode

met

asta

sis.

P-C

AD

+ in

IDC

doe

s not

cor

rela

te w

ith m

yoep

ithel

ial m

arke

rs (S

100,

smoo

th m

uscl

e ac

tin, c

ytok

erat

in

34βE

12)

Rad

ice

et a

l., 1

997

(87)

W

este

rn B

lot,

IHC

M

utan

t P-C

AD

(Kno

ckou

t mic

e): p

rem

atur

e di

ffer

entia

tion

of b

reas

t gla

nd a

lveo

li, fo

cal e

pith

eliu

m

hype

rpla

sia

and

dysp

lasi

a, a

nd in

crea

sed

lym

phoc

ytic

infil

tratio

n.

P-C

AD

inhi

bits

gro

wth

and

diff

eren

tiatio

n of

bre

ast g

land

epi

thel

ium

Deu

gnie

r et a

l., 1

999

(88)

IC

C, W

este

rn

Blo

t, N

orth

ern

Blo

t

Bre

ast e

pith

elia

l cel

l lin

e H

C11

(>C

OM

MA

-1D

) clo

nes:

P-C

AD

↑ a

nd k

erat

in 1

4 (K

14) ↑

in E

GF−

gr

owth

med

ium

, upr

egul

atio

n on

RN

A le

vel.

BC

20

and

BC

44

(P-C

AD

and

K14

stro

ngly

+ c

lone

s of

HC

11):

P-C

AD

and

K14

↓ in

EG

F+ g

row

th m

ediu

m, r

ever

sal t

o ep

ithel

ioid

phe

noty

pe. P

-CA

D R

NA

↑

whe

n α6

and

β1

inte

grin

s are

blo

cked

, and

by

inte

ract

ion

with

EC

M (l

amin

and

fibr

onec

tin)

1 CA

D: c

adhe

rin, C

TN: c

aten

in, I

DC

: inv

asiv

e du

ctal

car

cino

ma,

DC

IS: d

ucta

l car

cino

ma

in s

itu, I

LC: i

nvas

ive

lobu

lar

carc

inom

a, E

CM

: ext

race

llula

r m

atrix

, ~:

cor

rela

tes

with

, ↑:

inc

reas

ed, ↓:

dec

reas

ed,

>:or

igin

atin

g fr

om,

+: e

xpre

ssio

n, +

+: s

trong

exp

ress

ion,

−:

no e

xpre

ssio

n, I

HC

: im

mun

o-hi

stoc

hem

istry

, IP:

imm

unop

reci

pita

tion,

ICC

: im

mun

ocyt

oche

mis

try, (

RT-

)PC

R: (

reve

rse

trans

crip

tase

) pol

ymer

ase

chai

n re

actio

n, R

R: r

elat

ive

ratio

, O

R: o

dd’s

ratio

, ER

: est

roge

n re

cept

or, P

R: p

roge

ster

on re

cept

or.

Bracke et al.60

Pera

lta S

oler

et a

l. , 1

999

(89)

IH

C

IDC

: 52%

P-C

AD

+ (9

5/18

3), i

ndep

ende

nt o

f tum

our s

ize

and

lym

ph n

ode

met

asta

sis,

ILC

: 0%

P-C

AD

+ (0

/18)

P-C

AD

exp

ress

ion

corr

elat

es w

ith in

crea

sed

mor

talit

y, E

R a

nd P

R n

egat

ivity

. P-

CA

D is

a b

ette

r pro

gnos

is in

dica

tor t

han

E-, N

-CA

D o

r β-C

TN

Aut

hor

Met

hods

Fi

ndin

gs1

Knu

dsen

et a

l., 2

000

(90)

W

este

rn B

lot

sP-C

AD

+ (s

E-C

AD

+) in

car

cino

ma

and

beni

gn b

reas

t les

ions

(sam

e le

vels

), no

cor

rela

tion

with

P-C

AD

ex

pres

sion

in b

reas

t can

cer

Mad

hava

n et

al.,

200

1 (9

1)

IHC

B

reas

t car

cino

ma

(N=5

1):

E-C

AD

↓ ~

lym

ph n

ode

met

asta

sis,

high

his

tolo

gica

l gra

de

P-C

AD↓

~ ly

mph

nod

e m

etas

tasi

s (si

gnifi

cant

ly)

Gam

allo

et a

l., 2

001

(92)

IH

C

IDC

: 35%

P-C

AD

+ (7

4/21

0), ~

tum

our s

ize,

hig

h hi

stol

ogic

al g

rade

, lym

ph n

ode

met

asta

sis,

ER a

nd P

R

nega

tivity

, dec

reas

ed E

-CA

D e

xpre

ssio

n (a

fter m

ultiv

aria

te a

naly

sis)

Kov

ács e

t al.,

200

2 (9

3)

IHC

M

yoep

ithel

ial c

ells

: 100

% P

-CA

D+

(10/

10)

IDC

: 43.

5% P

-CA

D+

(30/

69),

~ hi

gh h

isto

logi

cal g

rade

(Gr I

II)

Pare

des e

t al.,

200

2 (9

4)

IHC

D

CIS

: P-C

AD

+ (2

3/69

) (E-

CA

D+

59/6

6, N

-CA

D+

9/63

) P-

CA

D e

xpre

ssio

n co

rrel

ates

with

ER

neg

ativ

ity a

nd h

igh

hist

olog

ical

(DC

IS) t

umou

r gra

de, h

ighe

r pr

olife

ratio

n an

d ex

pres

sion

of c

-erB

2

Sole

r et a

l., 2

002

(95)

IH

C, W

este

rn

Blo

t La

ctat

ing

brea

st g

land

tiss

ue: P

-CA

D++

, shi

ft of

myo

epith

elia

l cel

ls (m

embr

anou

s) →

lum

inal

epi

thel

ial

cells

(cyt

opla

sm) a

nd lu

min

al se

cret

ion

fluid

. Hum

an m

ilk: s

P-C

AD

(80k

D)

Kov

ács e

t al .,

200

3 (9

6)

IHC

ID

C (N

=100

): P-

CA

D +

40%

, N-C

AD

+ 3

0% (c

ytop

lasm

) and

E-C

AD

+ 8

1%

P-C

AD

exp

ress

ion

~ hi

gher

his

tolo

gica

l gra

de, E

R n

egat

ivity

and

EG

FR e

xpre

ssio

n (n

ot tu

mou

r siz

e or

ly

mph

nod

e m

etas

tasi

s)

Kov

ács a

nd W

alke

r, 20

03 (9

7)

IHC

H

igh

sens

itivi

ty o

f P-C

AD

to d

istin

guis

h be

twee

n m

yoep

ithel

ial c

ells

( +) a

nd m

yofib

robl

astic

cel

ls ( −

). P-

CA

D c

an b

e us

ed in

the

diff

eren

tial d

iagn

osis

of s

cler

osin

g ad

enos

is (b

enig

n) a

nd in

vasi

ve c

arci

nom

a


Aut

hor

Met

hods

Fi

ndin

gs1

Pala

cios

et a

l., 2

003

(98)

IH

C

P-C

AD

− :

BR

CA

1: 1

5/19

; BR

CA

2: 1

4/15

; non

-BR

CA

1/2:

29/

29

Rad

ice

et a

l., 2

003

(99)

IH

C, W

este

rn

Blo

t Tr

ansg

enic

mic

e w

ith h

uman

(h)P

-CA

D e

xpre

ssio

n co

nstra

ined

to m

amm

ary

glan

d ep

ithel

ium

: via

ble,

no

diff

eren

ce c

ompa

red

to n

orm

al p

heno

type

, no

spon

tane

ous t

umou

r gro

wth

. MM

TV/n

eu in

duce

d tu

mou

rs:

loos

e hP

-CA

D e

xpre

ssio

n

Pare

des e

t al.,

200

4 (1

00)

Wes

tern

Blo

t, Fu

nctio

nal

Ass

ays

Bre

ast c

ance

r cel

l lin

es: A

nti-e

stro

gen

ICI 1

82,7

80: P

-CA

D ↑

in E

Rα

posi

tive

cells

, red

uces

cel

l-cel

l ad

hesi

on a

nd in

crea

ses i

nvas

ion

in M

CF-

7/A

Z ce

lls.

De

novo

P-C

AD

-exp

ress

ion

(MC

F-7/

AZ)

: inc

reas

es in

vasi

on b

ut n

o di

ffer

ence

in c

ell-c

ell a

dhes

ion,

(H

EK c

ells

): in

crea

sed

inva

sion

med

iate

d vi

a ju

xtam

embr

anou

s dom

ain

Arn

es e

t al.,

200

5 (1

01)

IHC

In

vasi

ve b

reas

t car

cino

ma

(in A

shke

nazi

jew

s): 8

0/26

1 (3

1%) P

-CA

D +

, cor

rela

ted

with

bas

al e

pith

elia

l ph

enot

ype

and

BR

CA

1 m

utat

ion.

P-C

AD

pos

itivi

ty ~

RR

=2.9

(sig

nific

antly

) to

die

in 1

0 ye

ars.

P-C

AD

po

sitiv

ity (m

ultiv

aria

te a

naly

sis)

hig

hly

pred

ictiv

e of

poo

r sur

viva

l in

smal

l, ly

mph

nod

e ne

gativ

e ca

ncer

s

Col

lett

et a

l., 2

005

(102

) IH

C

Fast

gro

win

g br

east

can

cer (

inte

rval

can

cer s

): ~

basa

l epi

thel

ial p

heno

type

~ P

-CA

D p

ositi

vity

(OR

=2.5

)

Jacq

uem

ier e

t al. ,

200

5 (1

03)

IHC

Ty

pica

l Med

ulla

r bre

ast c

ance

r : ~

P-C

AD

pos

itivi

ty (R

R=2

.29)

Pare

des e

t al.,

200

5 (1

04)

IHC

, W

este

rn B

lot,

RT-

PCR

Inva

sive

bre

ast c

arci

nom

a (N

=150

): P-

CA

D e

xpre

ssio

n ~

high

his

tolo

gica

l gra

de, i

ncre

ased

pro

lifer

atio

n,

c-er

b2 a

nd p

53 e

xpre

ssio

n, E

R n

egat

ivity

and

poo

r pat

ient

surv

ival

. G

ene

prom

oter

met

hyla

tion

treat

men

t in

crea

sed

P-C

AD

mR

NA

and

pro

tein

leve

ls (M

CF-

7/A

Z ce

lls).

Inva

sive

car

cino

ma:

P-C

AD

neg

ativ

ity ~

CD

H3

prom

oter

met

hyla

tion

(71%

) and

P-C

AD

pos

itivi

ty ~

un

met

hyla

ted

CD

H3

prom

oter

(65%

) N

orm

al P

-CA

D n

egat

ive

brea

st e

pith

elia

l cel

ls: 1

00%

CD

H3

prom

oter

met

hyla

tion

Bracke et al.62

Tabl

e 2.

Lig

and/

rece

ptor

inte

ract

ions

regu

latin

g br

east

can

cer c

ell m

otili

ty a

nd m

etas

tasi

s

Liga

nd

Rec

epto

r Si

gnal

ling

Effe

ct o

n m

otili

ty

and

met

asta

sis

Ref

eren

ce

Aut

ocrin

e M

otili

ty F

acto

r (A

MF)

A

MF

rece

ptor

+ 11

4 B

one

Sial

opro

tein

In

tegr

in α

Vβ 5

43

, 115

C

D24

P-

sele

ctin

α

4β1, α

3β1

+ 11

6–11

8 C

XC

L12

CX

CR

4 Se

e te

xt

+ 11

9 Ep

ider

mal

Gro

wth

Fac

tor (

EGF)

EG

F re

cept

or

Sphi

ngos

ine

kina

se 1

+

Fibr

obla

st G

row

th F

acto

r (FG

F)

FGF

rece

ptor

+ 12

4, 1

25

Hep

atoc

yte

Gro

wth

Fac

tor

c-m

et

CX

CR

4, C

D44

+

126,

127

Her

egul

in

EGF

rece

ptor

+ 12

8

Hya

luro

nate

C

D44

(v3)

52, 1

29–1

32

Insu

line-

like

Gro

wth

Fac

tor I

(I

GF-

I)

IGF-

1 re

cept

or

CX

CR

4, in

sulin

rece

ptor

su

bstra

te (I

RS-

2), P

I3K

, M

EK 1

/2

+ 13

3-13

7

Inte

rleuk

in-1

(IL-

1α)

IL-1

rece

ptor

+ 13

8 K

AI/C

D82

D

uffy

Ant

igen

Rec

epto

r fo

r Cyt

okin

es (D

AR

C)

-

113

Muc

-1

Inte

rcel

lula

r Adh

esio

n M

olec

ule

1 (I

CA

M-1

)

+ 13

9, 1

40

Ost

eone

ctin

C

D44

C

hem

otax

is

+ 14

1 Si

alyl

Lew

isx

P-se

lect

in

+

142

Trom

bin

PAR

-2

β-ar

rest

in, e

rk

phos

phor

ylat

ion

+ 14

3–14

5


(HG

F)/S

catte

r Fac

tor (

SF)

120–

123

+ or

–

6. BREAST CANCER CELL MOTILITY: MOLECULAR TARGETS FOR POSSIBLE ANTI-METASTATIC AGENTS

Insight into the molecular mechanisms of breast cancer cell motility has revealed a number of possible targets for the development of anti-metastatic drugs. Some of these targets were already indicated earlier in the text. In animal models for breast cancer metastasis, neutralizing antibodies against CXCR4 reduce the number of metastases (146). Furthermore, CXCR4 has been studied intensively in AIDS research, and CXCR4 antagonists from this research area such as T140, have shown to be capable of inhibiting cancer cell motility and pulmonary metastasis formation (147). In another study the synthetic inhibitor TN1 4003 was shown to reduce the number of metastasis in laboratory animals (148). Together with the RGD peptidomimetics already mentioned before, the CXCR4 antagonists are candidate molecules for anti-metastatic treat-ment. Cyclooxygenase-2 (COX-2) inhibitors have regained interest in oncology as well, since it was shown convincingly that they can inhibit cancer cell motility and invasion, and presumably metastasis formation by a mechanism that encompasses integrin adhesion and MMP pro-duction (29,149,150).

Other agents are currently being used in treatment and prevention of cancer, and, through a better knowledge of their action mechanisms, appear to reduce breast cancer motility and metastasis. Examples of such molecules are: the bisphosphonate zoledronic acid (151–153), the soy phytoestrogens genistein and daidzein (via NFκB) (154), green tea poly-phenols (via uPA secretion) (155), modified citrus pectin (via galectin-3) (156) and the phytosterol β-sitosterol (via cell–matrix adhesion) (157). Still other molecules, like γ-linoleic acid, are promising, because they inhibit cancer cell motility and angiogenesis (158).

7. CONCLUSION

In breast cancer metastasis the old “seed and soil” hypothesis has started to become elucidated at the molecular level. Cancer cell motility is a necessary “seed” factor, but it is neither a marker for metastatic capa-bility, due to its transient occurrence, nor sufficient on its own. Agents that interfere with the different aspects of cancer cell motility (directional migration, adhesion, and proteolysis) are also candidates for anti-metastatic

Bracke et al.64

approaches. Critical interactions with the “soil”, as exemplified by the CXCL12/CXCR4 ligand/receptor recognition, are expected to become useful targets for therapy as well. This insight leads to the concept that the cancer cells are not the only targets for treatment, but that “host” cells can also be central players in future anti-metastatic strategies.

We thank Georges De Bruyne and Jean Roels-Van Kerckvoorde for their help with the preparation of the manuscript, and the Foundation against Cancer, the FWO-Vlaanderen and the Foundation Emmanuel van der Schueren (Vlaams Liga tegen Kanker), Brussels, Belgium and the Centrum voor Gezwelziekten, Ghent, Belgium.

REFERENCES

Billington WD, Weir BJ. Deportation of trophoblast in the chinchilla. J Reprod Fertil. 1967; 13: 593–595 Insall RH, G. E. Jones GE. Moving matters: signals and mechanisms in directed cell migration. Nat Cell Biol. 2006; 8: 776–779 Liotta LA. Tumor invasion and metastases - Role of the extracellular matrix: Rhoads Memorial Award Lecture., Cancer Res. 1986; 46: 1–7 Paget P. The distribution of secondary growths in cancer of the breast. Lancet 1889; 1: 571–573 Winston JS, Asch HL, Zhang P, Edge SB, Hyland A, Asch BB. Downregulation of gelsolin correlates with the progression to breast carcinoma. Breast Cancer Res. Treat. 2001; 65: 11–21 De Corte V, Bruyneel E, Boucherie C, Mareel C, Vandekerckhove J, Gettemans J. Gelsolin-induced epithelial cell invasion is dependent on Ras-Rac signalling. EMBO J. 2002; 21: 6781–6790 Hu W, McCrea PD, Deavers M, Kavanagh JJ, Kudelka AP, Verschraegen CF. Increased expression of fascin, motility associated protein, in cell cultures derived from ovarian cancer and in borderline and carcinomatous ovarian tumors, Clin Exp Metastasis 2000; 18: 83–88

Hicks DG. The expression of fascin, an actin-bundling motility protein, correlates with hormone receptor-negative breast cancer and a more aggressive clinical

Lin YH, Park ZY, Lin D, Brahmbhatt AA, Rio MC, Yates JR 3rd, Klemke RL. Regulation of cell migration and survival by focal adhesion targeting of Lasp-1,

Van Impe K, De Corte V, Eichinger L, Bruyneel E, Mareel M, Vandekerckhove J, Gettemans J. The nucleo-cytoplasmic actin binding protein CapG lacks a nuclear export sequence present in structurally related proteins, J Biol Chem. 2003; 278: 17945–17952


ACKNOWLEDGMENTS

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

Yoder BJ, Tso E, Skacel M, Pettay J, Tarr S, Budd S, Tubbs RR, Adams JC,

J Cell Biol. 2004; 165: 421–432

course. Clin Cancer Res. 2005; 11: 186–192

Jenkinson SR, Barraclough R, West CR, Rudland PS. S100A4 regulates cell motility and invasion in an in vitro model for breast cancer metastasis. Br J Cancer 2004; 90: 253–262 Wang G, Zhang S, Fernig DG, Martin-Fernandez M, Rudland PS, Barraclough R. Mutually antagonistic actions of S100A4 and S100A1 on normal and metastatic phenotypes, Oncogene 2005; 24: 1445–1454 Zhang S, Wang G, Liu D, Bao Z, Fernig DG, Rudland PS, Barraclough R. The C-terminal region of S100A4 is important for its metastasis-inducing properties. Oncogene 2005; 24: 4401–4411 Mareel MMK, De Brabander MJ. Effect of microtubule inhibitors on malignant

Atassi G, Dumont P, Vandendris M. Investigation of the in vivo anti-invasive and anti-metastatic effect of desacetyl vinblastine amide sulphate or vindesine, Invasion Metastasis 1982; 2: 217–231 Hall A. Ras-related GTPases and the cytoskeleton. Mol Biol Cell. 1992; 3: 475–479 Minard ME, Kim LS, Price JE, Gallick GE. The role of the guanine nucleotide exchange factor Tiam1 in cellular migration, invasion, adhesion and tumor pro-gression. Breast Cancer Res Treat. 2004; 84: 21–32

cells. Cancer Res. 2005; 65: 6042–6053 Chan AY, Coniglio SJ, Chuang YY, Michaelson D, Knaus UG, Philips M R, Symons M. Roles of the Rac1 and Rac3 GTPases in human tumor cell invasion. Oncogene 2005; 24: 7821–7829 Baugher PJ, Krishnamoorthy L, Price JE, Dharmawardhane SF. Rac1 and Rac3 isoform activation is involved in the invasive and metastatic phenotype of human breast cancer cells. Breast Cancer Res. 2005; 7: R965–R974 Kleer CG, van Golen KL, Zhang Y, Wu ZF, Rubin MA, Merajver SD. Characteri-zation of RhoC expression in benign and malignant breast disease: a potential new marker for small breast carcinomas with metastatic ability. Am J Pathol. 2002; 160: 579–584 Jiang WG, Watkins G, Lane J, Cunnick GH, Douglas-Jones A, Mokbel K, Mansel RE. Prognostic value of rho GTPases and rho guanine nucleotide disso-ciation inhibitors in human breast cancers. Clin Cancer Res.2003; 9: 6432–6440 Hordijk PL, ten Klooster JP, van der Kammen RA, Michiels F, Oomen LCJM, Collard JG. Inhibition of invasion of epithelial cells by Tiam1-Rac signalling. Science 1997; 278: 1464–1466

enhances cellular invasiveness and pro-gelatinase activation. Cancer Lett. 2006; 236: 292–301

alphavbeta3 cooperates with metalloproteinase MMP-9 in regulating migration of metastatic breast cancer cells. Proc Natl Acad Sci USA 2003; 100: 9482–9487

Bracke et al.66

12.

13.

14.

15.

16.

17. 18.

19.

20.

21.

22.

23.

24.

25.

26.

Betapudi V, Licate LS, Egelhoff TT. Distinct roles of nonmuscle myosin II isoforms in the regulation of MDA-MB-231 breast cancer cell spreading and

11.

migration, Cancer Res. 2006; 66: 4725–4733

invasion in vitro. J Natl Cancer Inst. 1978; 61: 787–792

Goodison S, Yuan J, Sloan D, Kim R, Li C, Popescu N C, Urquidi V. The RhoGAP protein D LC-1 functions as a metastasis suppressor in breast cancer

Wang F, Reierstad S, Fishman DA. Matrilysin over-expression in MCF-7 cells

Rolli M, Fransvea E, Pilch J, Saven A, Felding-Habermann B. Activated integrin

Wang FM, Liu HQ, Liu SR, Tang SP, Yang L, Feng GS. SHP-2 promoting migration and metastasis of MCF-7 with loss of E-cadherin, dephosphorylation of FAK and secretion of MMP-9 induced by IL-1beta in vivo and in vitro, Breast Cancer Res Treat. 2005; 89: 5–14

27.

Lirdprapamongkol K, Sakurai H, Kawasaki N, Choo MK, Saitoh Y, Aozuka Y, Singhirunnusorn P, Ruchirawat S, Svasti J, Saiki I. Vanillin suppresses in vitro invasion and in vivo metastasis of mouse breast cancer cells. Eur J Pharm Sci. 2005; 25: 57–65 Larkins TL, Nowell M, Singh S, Sanford GL. Inhibition of cyclooxygenase-2 decreases breast cancer cell motility, invasion and matrix metalloproteinase expres-sion. BMC Cancer 2006; 6: 18 Mahabeleshwar GH. Kundu GC. Syk, a protein-tyrosine kinase, suppresses the cell motility and nuclear factor kappa B-mediated secretion of urokinase type plasminogen activator by inhibiting the phosphatidylinositol 3’-kinase activity in

cells and regulated by Syk protein-tyrosine kinase. Int J Cancer 2005; 117: 14–20 Han B, Nakamura M, Zhou G, Ishii A, Nakamura A, Bai Y, Mori I, Kakudo K. Calcitonin inhibits invasion of breast cancer cells: involvement of urokinase-type plasminogen activator (uPA) and uPA receptor. Int J Oncol. 2006; 28: 807–814 Subramanian R, Gondi CS, Lakka SS, Jutla A, Rao JS. siRNA-mediated simul-taneous downregulation of uPA and its receptor inhibits angiogenesis and invasive-ness triggering apoptosis in breast cancer cells, Int J Oncol. 2006; 28: 831–839 Fauquette W, Dong-Le Bourhis X, Delannoy-Courdent A, Boilly B, and Desbiens X. Characterization of morphogenetic and invasive abilities of human mammary epithelial cells: correlation with variations of urokinase-type plasminogen activator activity and type-1 plasminogen activator inhibitor level. Biol Cell. 1997; 89: 453–465 Palmieri D, Lee JW, Juliano RL, Church FC. Plasminogen activator inhibitor-1 and -3 increase cell adhesion and motility of MDA-MB-435 breast cancer cells. J Biol Chem. 2002; 277: 40950–40957 Ahn SM, Jeong SJ, Kim YS, Sohn Y, Moon A. Retroviral delivery of TIMP-2 inhibits H-ras-induced migration and invasion in MCF10A human breast epithelial cells. Cancer Lett. 2004; 207: 49–57 Jiang WG, DaviesG, Martin TA, Parr C, Watkins G, Mason MD, Mansel RE. Expression of membrane type-1 matrix metalloproteinase, MT1-MMP in human breast cancer and its impact on invasiveness of breast cancer cells, Int J Mol Med. 2006; 17: 583–590 Felding-Habermann B, O’Toole TE, Smith JW, Fransvea E, Ruggeri ZM, Ginsberg MH, Hughes PE, Pampori N, Shattil SJ, Saven A, Mueller BM. Integrin activation controls metastasis in human breast cancer, Proc Natl Acad Sci USA 2001; 98: 1853–1858 Morini M, Mottolese M, Ferrari N, Ghiorzo F, Buglioni S, Mortarini R, Noonan DM, Natali PG, Albini A. The alpha 3 beta 1 integrin is associated with mammary carcinoma cell metastasis, invasion, and gelatinase B (MMP-9) activity. Int J Cancer. 2000; 87: 336–342 Wang HS, Hung Y, Su CH, Peng ST, Guo YJ , Lai MC, Liu CY, Hsu JW. CD44 cross-linking induces integrin-mediated adhesion and transendothelial migration in breast cancer cell line by up-regulation of LFA-1 (alpha L beta2) and VLA-4 (alpha4beta1). Exp Cell Res. 2005; 304: 116–126


28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

Li J and Sidell N Growth-related oncogene produced in human breast cancer breast cancer cells. J Biol Chem. 2003; 278: 6209–6221

Jauliac S, Lopez-Rodriguez C, Shaw LM, Brown LF, Rao A, Toker A. The role of NFAT transcription factors in integrin-mediated carcinoma invasion. Nat Cell Biol. 2002; 4: 540–544 Scherberich A, Tucker RP, Degen M, Brown-Luedi M, Andres AC, Chiquet-Ehrismann R. Tenascin-W is found in malignant mammary tumors, promotes

41.

42.

Sung V, Stubbs JT 3rd, Fisher L, Aaron AD, Thompson EW. Bone sialoprotein supports breast cancer cell adhesion proliferation and migration through differential usage of the alpha(v)beta3 and alpha(v)beta5 integrins, J Cell Physiol. 1998; 176: 482–494 Pecheur I, Peyruchaud O, Serre, Guglielmi J, Voland C, Bourre F, Margue C, Cohen-Solal M, Buffet A, Kieffer N, Clezardin P. Integrin alpha(v)beta3 expression confers on tumor cells a greater propensity to metastasize to bone. FASEB J. 2002; 16: 1266–1268 Sturge J, Hamelin J, Jones GE. N-WASP activation by a beta1-integrin-dependent mechanism supports PI3K-independent chemotaxis stimulated by urokinase-type plasminogen activator. J Cell Sci. 2002; 115: 699–711 Arboleda MJ, Lyons JF, Kabbinavar FF, Bray MR, Snow BE, Ayala R, Danino M, Karlan BY, Slamon DJ. Overexpression of AKT2/protein kinase Bbeta leads to up-regulation of beta1 integrins, increased invasion, and metastasis of human breast and ovarian cancer cells. Cancer Res. 2003; 63: 196–206 Shannon KE, Keene JL, Settle SL, Duffin TD, Nickols MA, Westlin M, Schroeter S, Ruminski PG, Griggs DW. Anti-metastatic properties of RGD-peptidomimetic agents S137 and S247. Clin Exp Metastasis 2004; 21: 129–138 Barsky SH, Rao CN, Grotendorst GR, Liotta LA. Increased content of Type V Collagen in desmoplasia of human breast carcinoma. Am J Pathol. 1982; 108: 276–283

Boucher Y. Lack of telopeptides in fibrillar collagen I promotes the invasion of a

Bemis LT, Schedin P. Reproductive state of rat mammary gland stroma modulates human breast cancer cell migration and invasion. Cancer Res. 2000; 60: 3414–3418 Bracke ME, Castronovo V, Van Cauwenberge RM-L, Vakaet L Jr, Strojny P, Foidart J-M, Mareel MM. The anti-invasive flavonoid (+)-catechin binds to laminin and abrogates the effect of laminin on cell morphology and adhesion. Exp Cell Res. 1987; 173: 193–205 Udabage L, Brownlee GR, Waltham M, Blick T, Walker EC, Heldin P, Nilsson SK, Thompson EW, Brown TJ. Antisense-mediated suppression of hyaluronan synthase 2 inhibits the tumorigenesis and progression of breast cancer. Cancer

Chen P, Shen WZ, Karnik P. Suppression of malignant growth of human breast cancer cells by ectopic expression of integrin-linked kinase. Int J Cancer 2004; 111: 881–891 Lee J-H, Welch DR. Suppression of metastasis in human breast carcinoma MDA-MB-435 cells after transfection wih the metastasis suppressor gene, KiSS-1. Cancer Res. 1997; 57: 2384–2387 Navenot JM, Wang Z, Chopin M, Fujii N, Peiper SC. Kisspeptin-10-induced signaling of GPR54 negatively regulates chemotactic responses mediated by CXCR4: a potential mechanism for the metastasis suppressor activity of kisspeptins. Cancer Res. 2005; 65: 10450–10456

Bracke et al.68

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

metastatic breast tumor cell line, Cancer Res. 2005; 65: 5674–5682

Demou ZN, Awad M, McKee T, Perentes JY, Wang X, Munn LL, Jain RK,

Res. 2005; 65: 6139–6150

Takeichi M. Cadherins: a molecular family important in selective cell-cell adhesion. Annu Rev Biochem. 1990; 59: 237–252

56.

alpha8 integrin-dependent motility and requires p38MAPK activity for BMP-2 and TNF-alpha induced expression in vitro. Oncogene 2005; 24: 1525–1532

Bracke ME, Van Roy FM, Mareel MM. The E-cadherin/catenin complex in invasion and metastasis, in: Attempts to Understand Metastasis Formation I, edited by U. Günthert and W. Birchmeier (Springer, Berlin, 1996) pp. 123–161. Vleminckx K, Vakaet LJr, Mareel M, Fiers W, Van Roy F. Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role. Cell 1991; 66: 107–119 Perl A-K, Wilgenbus P, Dahl U, Semb H, Christofori G. A causal role for E-

Pagliarini RA, Xu T. A genetic screen in Drosophila for metastatic behaviour. Science. 2003; 302: 1227–1231 Suriano G, Mulholland D, De Wever O, Ferreira P, Mateus AR, Bruyneel E, Nelson CC, Mareel MM, Yokota J, Huntsman D, Seruca R. The intracellular E-cadherin germline mutation V832M lacks the ability to mediate cell-cell adhesion and to suppress invasion. Oncogene 2003; 22: 5716–5719 Sarrio D, Perez-Mies B, Hardisson D, Moreno-Bueno G, Suarez A, Cano A, Martin-Perez J, Gamallo C, Palacios J. Cytoplasmic localization of p120ctn and E-cadherin loss characterize lobular breast carcinoma from preinvasive to meta-static lesions. Oncogene. 2004; 23: 3272–3283 Berx G, Cleton-Jansen A-M, Strumane K, de Leeuw WJF, Nollet F, Van Roy F, Cornelisse C. E-cadherin is inactivated in a majority of invasive human lobular breast cancers by truncation mutations throughout its extracellular domain. Onco-gene 1996; 13: 1919–1925 Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, Savagner P, Gitelman I, Richardson A, Weinberg RA. Twist, a master regulator of morpho-genesis, plays an essential role in tumor metastasis.Cell 2004; 117: 927–939 Comijn J, Berx G, Vermassen P, VerschuerenK, van Grunsven L, Bruyneel E, Mareel M, Huylebroeck D, van Roy F. The two-handed E box binding zinc finger

1267–1278 Bracke ME, Vyncke BM, Bruyneel EA, Vermeulen SJ, De Bruyne GK, Van Larebeke NA, Vleminckx K, Van Roy FM, Mareel MM. Insulin-like growth factor I activates the invasion suppressor function of E-cadherin in MCF-7 human mammary carcinoma cells in vitro. Br J Cancer 1993; 68: 282–289 Dollé L, Oliveira M-J, Bruyneel E, Hondermarck H, Bracke M. Nerve growth factor mediates its pro-invasive effect in parallel with the release of a soluble E-

Stove C, Boterberg T, Van Marck V, Mareel M, Bracke M. Bowes melanoma cells secrete heregulin, which can promote aggregation and counteract invasion of human mammary cancer cells. Int J Cancer 2005; 114: 572–578 Boterberg T, Vennekens KM, Thienpont M, Mareel MM, Bracke ME. Inter-nalization of the E-cadherin/catenin complex and scattering of human mammary carcinoma cells MCF-7/AZ after treatment with conditioned medium from human skin squamous carcinoma cells COLO 16. Cell Adhesion Commun. 2000; 7: 299–310 Rong H, Boterberg T, Maubach J, Stove C, Depypere H, Van Slambrouck S, Serreyn R, De Keukeleire D, Mareel M, Bracke M. 8-Prenylnaringenin, the phyto-estrogen in hops and beer, upregulates the function of the E-cadherin/catenin complex in human mammary carcinoma cells. Eur J Cell Biol. 2001; 80: 580–585


57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

cadherin in the transition from adenoma to carcinoma. Nature 1998; 392: 190–193

protein SIP1 downregulates E-cadherin and induces invasion. Mol Cell 2001; 7:

cadherin fragment from breast cancer MCF-7/AZ cells. J Dairy Res. 2005; 72: 20–26

Van Slambrouck S, Parmar VS, Sharma SK, De Bondt B, Foré F, Coopman P,

Tangeretin inhibits extacellular-signal-regulated kinase (ERK) phosphorylation. FEBS Lett. 2005; 579: 1665–1669

71. Vanhoecke BW, Boterberg T, Depypere HT, Leclercq G, MBracke ME.

Vanhoecke B, Derycke L, Van Marck V, Depypere H, De Keukeleire D, Bracke M. Antiinvasive effect of xanthohumol, a prenylated chalcone present in hops

Vermeulen SJ, Bruyneel EA, Van Roy FM, Mareel MM, Bracke ME. Activation of the E-cadherin/catenin complex in human MCF-7 breast cancer cells by all-trans-retinoic acid. Br J Cancer 1995; 72: 1447–1453 Vanhoecke BW, Depypere HT, De Beyter A, Sharma SK, Parmar VS, De Keukeleire D, Bracke ME. New anti-invasive compounds: results from the Indo-Belgian screening program. Pure Appl Chem. 2005; 77: 65–74 Katritzky AL, Kuanar M, Dobchev DA, Vanhoecke BWA, Karelson M, Parmar VS, Stevens CV, Bracke ME. QSAR modeling of anti-invasive activity of organic compounds using structural descriptors. Bioorg Med Chem. 2006; 14: 6933–6939 Derycke LDM, BrackeME. N-cadherin in the spotlight of cell-cell adhesion,

463–476 Hazan RB, Qiao R, Keren R, Badano I, Suyama K. Cadherin switch in tumor pro-gression. Ann NY Acad Sci. 2004; 1014: 155–163 Hazan RB, Phillips GR, Fang Qiao R, Norton L, Aaronson SA. Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis. J Cell Biol. 2000; 148: 779–790 Vandewalle C, Comijn J, De Craene B, Vermassen P, Bruyneel E, Andersen H, Tulchinsky E, Van Roy F, Berx G. SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell-cell junctions. Nucleic Acids Res. 2005; 33: 6566–6578 De Craene B, Gilbert B, Stove C, Bruyneel E, van Roy F, Berx G. The trans-cription factor Snail induces tumor cell invasion through modulation of the epithelial cell differentiation program. Cancer Res. 2005; 65: 6237–6244 Van Marck VL, Bracke ME. Epithelial-mesenchymal transitions in human cancer, in: Rise and Fall of Epithelial Phenotype. Concepts of Epithelial-Mesenchymal Transition, edited by P. Savagner (Landes Bioscience, Georgetown, Texas, USA, 2005), Chapter 9, pp. 135–159. Marine JC, Francoz S, Maetens M, Wahl G, Toledo F, Lozano G. Keeping p53 in check: essential and synergistic functions of Mdm2 and Mdm4. Cell Death Differ. 2006; 13: 927–234 Derycke L, Morbidelli L, Ziche M, De Wever O, Bracke M, Van Aken E. Soluble N-cadherin fragment promotes angiogenesis. Clin Exp Metastasis 2006, In Press.

Bracke M. Soluble N-cadherin in human biological fluids. Int J Cancer 2006 In Press Van Marck V, Stove C, Van Den Bossche K, Stove V, Paredes J, Vander Haeghen Y, Bracke M. P-cadherin promotes cell-cell adhesion and counteracts invasion in human melanoma. Cancer Res. 2005; 65: 8774–8783 Palacios J, Benito N, Pizarro A, Suarez A, Espada J, Cano A, Gamallo C. Anomalous expression of P-cadherin in breast carcinoma. Am J Pathol. 1995; 146: 605–612

Bracke et al.70

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

differentiation, embryogenesis, invasion and signalling. Int J Dev Biol. 2004; 48:

Derycke L, De Wever O, Stove V, Vanhoecke B, Delanghe J, Depypere H,

Radice GL, Ferreira-Cornwell MC, Robinson SD, Rayburn H, Chodosh LA, Takeichi M, and Hynes RO. Precocious mammary gland development in P-cadherin-deficient mice. J Cell Biol. 1997; 139: 1025–1032 Deugnier M-A, Faraldo MM, Rousselle P, Thiery JP, and Glukhova MA. Cell-extracellular matrix interactions and EGF are important regulators of the basal mammary epithelial cell phenotype. J Cell Sci. 1999; 112: 1035–1044

87.

88.

(Humulus lupulus L.) and beer. Int J Cancer 2005; 117: 889–895

Peralta Soler A, Knudsen KA, Salazar H, Han AC, and Keshgegian AA. P-Cadherin expression in breast carcinoma indicates poor survival. Cancer 1999; 86: 1263–1272 Knudsen KA, Lin CY, Johnson KR, Wheelock MJ, Keshgegian AA, and Soler AP. Lack of correlation between serum levels of E- and P-cadherin fragments and the presence of breast cancer. Hum Pathol. 2000; 31: 961–965 Madhavan M, Srinivas P, Abraham E, Ahmed I, Mathew A, Vijayalekshmi NR, Balaram P. Cadherins as predictive markers of nodal metastasis in breast cancer. Mod Pathol. 2001; 14: 423–427 Gamallo C, Moreno-Bueno G, Sarrio D, Calero F, Hardisson D, Palacios J. The prognostic significance of P-cadherin in infiltrating ductal breast carcinoma. Mod Pathol. 2001; 14: 650–654 Kovacs A, Walker RA, Nagy A, Gomba S, Jones L, Dearing S. Immuno-histochemical study of P-cadherin in breast cancer. Orv Hetil. 2002; 143: 405–409 Paredes J, Milanezi F, Reis-Filho JS, Leitão D, Athanazio D, Schmitt F. Aberrant P-cadherin expression: is it associated with estrogen-independent growth in breast cancer? Pathol Res Pract. 2002; 198: 795–801

adhesion protein found in human milk. J Cell Biochem. 2002; 85: 180–184

Kovács A, Walker RA. P-cadherin as a marker in the differential diagnosis of

Palacios J, Honrado E, Osorio A, Cazorla A, Sarrió D, Barroso A, Rodríguez S, Cigudosa JC, Diez O, Alonso C, Lerma E, Sánchez L, Rivas C, Benítez J. Immuno-histochemical characteristics defined by tissue microarray of hereditary breast cancer not attributable to BRCA1 or BRCA2 mutations: differences from breast carcinomas arising in BRCA1 and BRCA2 mutation carriers. Clin Cancer Res. 2003; 9: 3606–3614 Radice GL, Sauer CL, Kostetskii I, Peralta Soler A, Knudsen KA. Inappropriate P-cadherin expression in the mouse mammary epithelium is compatible with normal mammary gland function. Differentiation 2003; 71: 361–373 Paredes J, Stove C, Stove V, Milanezi F, Van Marck V, Derycke L, Mareel M, Bracke M, Schmitt F. P-cadherin is up-regulated by the antiestrogen ICI 182,780 and promotes invasion of human breast cancer cells. Cancer Res. 2004; 64: 8309–8317 Arnes JB, Brunet J-S, Stefansson I, Bégin LR, Wong N, Chappuis PO, Akslen LA, Foulkes WD. Placental cadherin and the basal epithelial phenotype of BRCA1-related breast cancer. Clin Cancer Res. 2005; 11: 4003–4011 Collett K, Stefansson IM, Eide J, Braaten A, Wang H, Eide GE, Thoresen S Ø, Foulkes WD, Akslen LA. A basal epithelial phenotype is more frequent in interval breast cancers compared with screen detected tumors. Cancer Epidemiol. Biomarkers Prev. 2005; 14: 1108–1112


89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

99.

100.

101.

102.

Soler AP, Russo J, Russo IH, Knudsen KA. Soluble fragment of P-cadherin

Kovacs A, Dhillon J, Walker RA. Expression of P-cadherin, but not E-cadherin or N-cadherin, relates to pathological and functional differentiation of breast

breast lesions. J Clin Pathol. 2003; 56: 139–141

carcinomas. J Clin Pathol.: Mol Pathol. 2003; 56: 318–322

Jacquemier J, Padovani L, Rabayrol L, Lakhani SR, Penault-Llorca F, Denoux Y, Fiche M, Figueiro P, Maisongrosse V, Ledoussal V, Martinez Penuela J, Udvarhely N, El Makdissi G, Ginestier C, Geneix J, Charafe-Jauffret E, Xerri L, Eisinger F, Birnbaum D, Sobol H. The European Working Group for Breast Screening Pathology, The Breast Cancer Linkage Consortium and Hagay Sobol, Typical medullary breast carcinomas have a basal/myoepithelial phenotype. J Pathol. 2005; 207: 260–268

103.

Paredes J, Albergaria A, Oliveira JT, Jeronimo C, Milanezi F, Schmitt FC. P-cadherin overexpression is an indicator of clinical outcome in invasive breast carcinomas and is associated with CDH3 promoter hypomethylation. Clin Cancer Res. 2005; 11: 5869–5877 Müller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, McClanahan T, Murphy E, Yuan W, Wagner SN, Barrera JL, Mohar A, Verástegui E, Zlotnik A. Involvement of chemokine receptors in breast cancer metastasis. Nature 2001; 410: 50–56 Darash-Yahana M, Pikarsky E, Abramovitch R, Zeira E, Pal B, Karplus R, Beider K, Avniel S, Kasem S, Galun E, Peled A. Role of high expression levels of CXCR4 in tumor growth, vascularization, and metastasis., FASEB J. 2004; 18: 1240–1242

Schmid BC, Rudas M, Rezniczek GA, Leodolter S, Zeillinger R. CXCR4 is expressed in ductal carcinoma in situ of the breast and in atypical ductal hyperplasia. Breast Cancer Res Treat. 2004; 84: 247–250 Richard CL, Tan EY, Blay J. Adenosine upregulates CXCR4 and enhances the pro-liferative and migratory responses of human carcinoma cells to CXCL12/SDF-1α. Int J Cancer 2006; 119: 2044–2053 Holland JD, Kochetkova M, Akekawatchai C, Dottore M, Lopez A, SMcColl SR. Differential functional activation of chemokine receptor CXCR4 is mediated by G proteins in breast cancer cells. Cancer Res. 2006; 66: 4117–4124 Sun Y, Cheng Z, Ma L, Pei G. Beta-arrestin2 is critically involved in CXCR4-mediated chemotaxis, and this is mediated by its enhancement of p38 MAPK activation. J Biol Chem. 2003; 277: 49212–49219 Fernandis AZ, Prasad A, Band H, Klosel R, Ganju RK. Regulation of CXCR4-mediated chemotaxis and chemoinvasion of breast cancer cells. Oncogene 2004; 23: 157–167

mediated metastasis suppression in the vascular tunnel. Cancer Cell 2006; 10: 177–178 Jiang WG, Raz A, Douglas-Jones A, Mansel RE. Expression of autocrine motility factor (AMF) and its receptor, AMFR, in human breast cancer. J. Histochem Cytochem. 2006; 54: 231–241 Bellahcène A, Castronovo V. Expression of bone matrix proteins in human breast cancer: potential roles in microcalcification formation and in the genesis of bone metastases. Bull Cancer 1997; 84: 17–24 Aigner S, Ramos CL, Hafezi-Moghadam A, Lawrence MB, Friederichs J, Altevogt P, Ley K. CD24 mediates rolling of breast carcinoma cells on P-selectin. FASEB J. 1998; 12: 1241–1251 Baumann P, Cremers N, Kroese F, Orend G, Chiquet-Ehrismann T, Uede T, Yagita H, Sleeman JP. CD24 expression causes the acquisition of multiple cellular

10783–10793

Bracke et al.72

104.

105.

106.

107.

108.

109.

110.

111.

112.

113. Zijlstra A, Quigley JP. The DARC side of metastasis: shining a light on KAI1-

114.

115.

116.

117.

Kang H, Mansel RE, Jiang WG. Genetic manipulation of stromal cell-derivedfactor-1 attests the pivotal role of the autocrine SDF-1-CXCR4 pathway in theaggressiveness of breast cancer cells. Int J Oncol. 2005; 26: 14291–434

properties associated with tumor growth and metastasis. Cancer Res. 2005; 65:

Schabath H, Runz S, Joumaa S, Altevogt P. CD24 affects CXCR4 function in pre-B lymphocytes and breast carcinoma cells. J Cell Sci. 2006; 119: 314–325 Burger JA, Kipps TJ. CXCR4: a key receptor in the crosstalk between tumor cells and their microenvironment. Blood 2006; 107: 1761–1767 Dittmar T, Husemann A, Schewe Y, Nofer JR, Niggemann B, Zanker KS, Brandt BH. Induction of cancer cell migration by epidermal growth factor is initiated by

118.

119.

120.

specific phosphorylation of tyrosine 1248 of c-erbB-2 receptor via EGFR. FASEB J. 2002; 16: 1823–1825 Wang SJ, Saadi W, Lin F, Minh-Canh Nguyen C, Li Jeon N. Differential effects of EGF gradient profiles on MDA-MB-231 breast cancer cell chemotaxis. Exp Cell Res. 2004; 300: 180–189 Hart S, Fischer OM, Prenzel N, Zwick-Wallasch E, Schneider M, Hennighausen L, Ullrich A. GPCR-induced migration of breast carcinoma cells depends on both EGFR signal transactivation and EGFR-independent pathways. Biol Chem. 2005; 386: 845–855

Sphingosine kinase 1 is required for migration, proliferation and survival of MCF-7 human breast cancer cells. FEBS Lett. 2005; 579: 5313–5317 Korah R, Choi L, Barrios J, Wieder R. Expression of FGF-2 alters focal adhesion dynamics in migration-restricted MDA-MB-231 breast cancer cells. Breast Cancer Res Treat. 2004; 88: 17–28 Stadler CR, Knyazev P, Bange J, Ullrich A. FGFR4 GLY388 isotype suppresses motility of MDA-MB-231 breast cancer cells by EDG-2 gene repression. Cell

Mine S, Fujisaki T, Kawahara C, Tabata T, Iida T, Yasuda M, Yoneda T, Tanaka Y. Hepatocyte growth factor enhances adhesion of breast cancer cells to endo-thelial cells in vitro through up-regulation of CD44. Exp Cell Res. 2003; 288: 189–197 Matteucci E, Locati M, Desiderio MA. Hepatocyte growth factor enhances CXCR4 expression favoring breast cancer cell invasiveness. Exp Cell Res.2005; 310: 176–185 Stove C, Bracke M. Roles for neuregulins in human cancer. Clin Exp Metastasis 2004; 21: 665–684 Kalish ED, Iida N, Moffat FL, Bourguignon LY. A new CD44V3-containing isoform is involved in tumor cell growth and migration during human breast carcinoma progression. Front Biosci. 1999; 4: A1–A8 Bourguignon LY, Singleton PA, Zhu H, Zhou B. Hyaluronan promotes signaling interaction between CD44 and the transforming growth factor beta receptor I in metastatic breast tumor cells. J Biol Chem. 2002; 277: 39703–39712 Lopez JI, Camenisch TD, Stevens MV, Sands BJ, McDonald J, JSchroeder JA. CD44 attenuates metastatic invasion during breast cancer progression. Cancer Res. 2005; 65: 6755–6763 Tzircotis G, Thorne RF, Isacke CM. Chemotaxis towards hyaluronan is dependent on CD44 expression and modulated by cell type variation in CD44-hyaluronan binding. J Cell Sci. 2005; 118: 5119–5128 Mauro L, Salerno M, Morelli C, Boterberg T, Bracke ME, Surmacz E. Role of the IGF-I receptor in the regulation of cell-cell adhesion: implications in cancer development and progression. J Cell Physiol. 2002; 194: 108–116


121.

122.

123. Sarkar S, Maceyka M, Hait NC, Paugh SW, Sankala H, Milstien S, Spiegel S.

124.

125.

126.

127.

128.

129.

130.

131.

132.

133.

Signal 2006; 18: 783–794

Jackson JG, Zhang X, Yoneda T, Yee D. Regulation of breast cancer cell motility by insulin receptor substrate-2 (IRS-2) in metastatic variants of human breast cancer cell lines. Oncogene 2001; 20: 7318–7325 Guvakova MA, Adams JC, Boettiger D. Functional role of alpha-actinin, PI 3-kinase and MEK1/2 in insulin-like growth factor I receptor kinase regulated

insulin-like growth factor receptor inhibits metastasis of human cancer cells. J Biol Chem. 2004; 79: 5017–5024

134.

135.

136. motility of human breast carcinoma cells. J Cell Sci. 2002; 115: 4149–4165 Sachdev D, Hartell JS, Lee AV, Zhang X, Yee D. A dominant negative type I

Akekawatchai C, Holland JD, Kochetkova M, Wallace JC, McCollSR. Transactivation of CXCR4 by the insulin-like growth factor-1 receptor (IGF-1R) in human MDA-MB-231 breast cancer epithelial cells. J Biol Chem. 2005; 280: 39701–39708 Nozaki S, Sledge GW Jr, Nakshatri H. Cancer cell-derived interleukin 1alpha contributes to autocrine and paracrine induction of pro-metastatic genes in breast cancer. Biochem Biophys Res Commun. 2000; 275: 60–62 Rahn JJ, Shen Q, Mah BK, Hugh JC. MUC1 initiates a calcium signal after ligation by intercellular adhesion molecule-1. J Biol Chem. 2004; 279: 29386–29390 Rahn JJ, Chow JW, Horne GJ, Mah BK, Emerman JT, Hoffman P, Hugh JC. MUC1 mediates transendothelial migration in vitro by ligating endothelial cell ICAM-1. Clin Exp Metastasis 2005; 22: 475–483 Campo McKnight DA, Sosnoski DM, Koblinski JE, Gay CV. Roles of osteonectin in the migration of breast cancer cells into bone. J Cell Biochem. 2006; 97: 288–302

sialyl Lewis(x) and sialyl Lewis(a) in lesions of breast carcinoma. Int J Cancer 1997; 74: 296–300 Nguyen Q-D, De Wever O, Bruyneel E, Hendrix A, Xie W-Z, Lombet A, Leibl M, Mareel M, Gieseler F, Bracke M, Gespach C. Commutators of PAR-1 signaling in cancer cell invasion reveal an essential role of the Rho-Rho kinase axis and tumor microenvironment. Oncogene 2005; 24: 8240–8251

receptor in breast cancer invasiveness. Br J Cancer 1999; 79: 401–406 Hjortoe GM, Petersen LC, Albrektsen T, Sorensen BB, Norby PL, Mandal SK, Pendurthi UR, Rao LV. Tissue factor-factor VIIa-specific up-regulation of IL-8 expression in MDA-MB-231 cells is mediated by PAR-2 and results in increased cell migration. Blood 2004; 103: 3029–3037 Hatse S, Princen K, Bridger G, De Clercq E, Schols D. Chemokine receptor inhibition by AMD3100 is strictly confined to CXCR4. FEBS Lett. 2002; 527: 255–262 Tamamura H, Hori A, Kanzaki N, Hiramatsu K, Mizumot Mo, Nakashima H, Yamamoto N, Otak Aa, Fujii N. T140 analogs as CXCR4 antagonists identified as anti-metastatic agents in the treatment of breast cancer. FEBS Lett. 2003; 550: 79–83 Liang Z, Wu T, Lou H, Yu X, Taichman RS, Lau SK, Nie S, Umbreit J, Shim H. Inhibition of breast cancer metastasis by selective synthetic polypeptide against CXCR4. Cancer Res. 2004; 64: 4302–4308 Rozic JG, Chakraborty C, Lala PK. Cyclooxygenase inhibitors retard murine mammary tumor progression by reducing tumor cell migration, invasiveness and angiogenesis. Int J Cancer 2001; 93: 497–506

Bracke et al.74

137.

138.

139.

140.

141.

142.

143.

144.

145.

146.

147.

148.

149.

Renkonen J, Paavonen T, Renkonen R. Endothelial epithelial expression of

Henrikson KP, Salazar SL, Fenton JW 2nd, Pentecost BT. Role of thrombin

Singh B, Berry JA, Shoher A, Ramakrishnan V, Lucci A. COX-2 overexpression increases motility and invasion of breast cancer cells. Int J Oncol. 2005; 26: 1393–1399 Boissier S, Ferreras M, Peyruchaud O, Magnetto S, Ebetino FH, Colombel M, Delmas P, Delaissé J-M, Clézardin P. Bisphosphonates inhibit breast and prostate carcinoma cell invasion, an early event in the formation of bone metastases. Cancer Res. 2000; 60: 2949–2954 Denoyelle C, Hong L, Vannier JP, Soria J, Soria C. New insights into the actions of bisphosphonate zoledronic acid in breast cancer cells by dual RhoA-dependent and -independent effects. Br J Cancer 2003; 88: 1631–1640

150.

151.

152.

visceral metastases in the 4T1/luc mouse breast cancer model. Clin Cancer Res. 2004; 10: 4559–4567 (2004). Valachovicova T, Slivova V, Bergman H, Shuherk J, Sliva D. Soy isoflavones suppress invasiveness of breast cancer cells by the inhibition of NF-kappaB/AP-1-dependent and -independent pathways. Int J Oncol. 2004; 25: 1389–1395 Slivova V, Zaloga G, DeMichele SJ, Mukerji P, Huang YS, Siddiqui R, Harvey K, Valachovicova T, Sliva D. Green tea polyphenols modulate secretion of urokinase plasminogen activator (uPA) and inhibit invasive behavior of breast cancer cells. Nutr Cancer 2005; 52: 66–73 Nangia-Makker P, Hogan V, Honjo Y, Baccarini S, Tait L, Bresalier R, Raz A. Inhibition of human cancer cell growth and metastasis in nude mice by oral intake of modified citrus pectin. J Natl Cancer Inst. 2002; 94: 1854–1862 Awad AB, Williams H, Fink CS. Phytosterols reduce in vitro metastatic ability of MDA-MB-231 human breast cancer cells. Nutr Cancer 2001; 40: 157–164 Cai J, Jiang WG, Mansel RE. Inhibition of angiogenic factor- and tumour-induced angiogenesis by gamma linolenic acid. Prostaglandins Leukot Essent Fatty Acids 1999; 60: 21–29


153. Hiraga T, Williams PJ, Ueda A, Tamura D, Yoneda T. Zoledronic acid inhibits

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Chapter 5

TIGHT JUNCTIONS AND METASTASIS OF BREAST CANCER

Tracey A. Martin Metastasis and Angiogenesis Research Group, School of Medicine, Cardiff University,Heath Park, Cardiff, CF14 4XN, UK

Abstract: TJs are the apical most structure between epithelial and endothelial cells. Although well known as functioning as a control for paracellular diffusion of ions and certain molecules, it has recently become apparent that the TJ has a vital role in maintaining cell integrity and that loss of cohesion of the TJ structure can lead to invasion and thus metastasis of breast cancer cells.

Keywords: TJ, metastasis, breast cancer, occludin

1. INTRODUCTION

Tight Junctions (TJs) govern the permeability of epithelial and endo-thelial cells and are the most topical structures of these cell types (1–3). It is a region where the plasma membrane of adjacent cells forms a series of contacts that appear to completely occlude the extracellular space thus creating an intercellular barrier and intramembrane diffusion fence (4).

TJs in endothelial cells function as a barrier through which molecules and inflammatory cells can pass. In epithelial cells the TJ functions in an adhesive manner and can prevent cell dissociation (5). An important step in the formation of cancer metastases is interaction and penetration of the vascular endothelium by dissociated cancer cells. TJs are therefore the first barrier that cancer cells must overcome in order to metastasize. We have previously demonstrated that TJs of vascular endothelium in vivo function as a barrier between blood and tissues against metastatic cancer cells (6).

© 2007 Springer.

77 R.E. Mansel et al. (eds.), Metastasis of Breast Cancer, 77–110.

78 Martin

Changes in expression of TJ proteins may be due to regulatory mecha-nisms or promoter methylation. Regulatory mechanisms may be via the suggested pathway of the epithelial-mesenchymal-transition (EMT) as the process of acquisition of an invasive phenotype by tumours of epi-thelial origin can be regarded as a pathological version EMT (17–18). TJ determine epithelial cell polarity and disappear during EMT. Snail and Slug are factors thought to be responsible for this loss (19). Regulation also occurs via the Rho GTPase family, which is able to regulate TJ assembly (20). Thus the TJ can be regulated in response to physiological and tissue-specific requirements (4). TJs are able to rapidly change their permeability and functional properties in response to stimuli, permiting dymanic fluxes of ions and solutes in addition to the passage of whole cells (21).

This chapter will overview the recent progress in elucidating the role of TJs in the invasion and metastasis of breast cancer via changes in expression of TJ proteins and alterations in the structure of the TJ itself.

Figure 1. A schematic illustrating the structures between endothelial and epithelial cells. The TJ is located at the most apical membrane between adjacent cells.

Early studies demonstrated a correlation between the reduction of TJs

years that their possible role in tumorigenesis has been studied and to date most of the work has been concentrated on cell lines and to a limited

out on breast cancer which have concentrated on Claudin-1 (SEMP-1), Claudin-7, ZO-1 and ZO-2 expression (9-16) of which, more detail later.

degree on colorectal and pancreatic cancers, with a few studies carried

and tumour differentiation and experimental evidence has emerged to place TJs in the frontline as the structure that cancer cells must overcome in order to metastasize (6–9) (Figure 1). Although a considerable body of work exists on TJs and their role in a number of diseases, it is only in the last few

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2. TJ MOLECULAR STRUCTURE

The TJ has a characteristic structure, appearing as discrete sites of fusion between the outer plasma membrane of adjacent cells when viewed

fracture, they appear as continuous intramembrane particle strands in the protoplasmic face with complimentary grooves in the extracellular face (22). These completely circumscribe the apices of the cells as a network of intramembrane fibrils (4) appearing as a series of “kissing” points (Figure 2).

Figure 2. Distribution of TJs at the apical membranes (left) forming a series of “kissing” points (Right).

This ultrastructure is representative of the conglomerate of molecules that constitute, associate with or regulate TJs (23), Figure 3. Although a number of proteins were identified in the mid-1980s, the list of additional molecules has expanded considerably over recent years (Table 1). The molecular components of the TJ have been extensively investigated (3, 24) and it became apparent that the junctions could be reasonably separated into three regions: (i) the integral transmembrane proteins- occludin, clau-

5. Tight junctions and metastasis of breast cancer

in ultrathin section electron microscopy. When visualised using freeze-

dins, nectins, and junctional adhesion molecules (JAM), together with

etc.; and (iii) TJ-associated/regulatory proteins- α-catenin, cingulin, etc. often containing PDZ motifs- zonula occludens (ZO)-1, -2, -3, MAGI-1,other CTX family members; (ii) the peripheral or plaque anchoring protein,

80 Martin

involved in TJ structure and function (see text).

The integral transmembrane proteins are the essential adhesion pro-teins responsible for correct assembly of the TJ structure and controlling TJ functions via homotypic and heterotypic interactions. Successful assembly and maintenance of the TJ is accomplished by anchorage of the transmembrane proteins by the peripheral or plaque proteins such as ZO-1 which act as a scaffold to bind the raft of TJ molecules together and provide the link to the actin cytoskeleton and the signalling mechanism

Table 1. Proteins involved in TJ structure, function and regulation

Integral transmembrane proteins Peripheral plaque proteins Associated proteins Occludin Claudins 1–24 Junctional Adhesion Molecules (A–C, 4) and other CTX proteins such as Coxsackie Adenovirus Receptor (CAR) Nectins 1–4 Nectin-like 1–5

ZO-2 ZO-3 MAGI-1, -2, -3 MUPP-1 PAR-3/ASIP, PAR-6 AF-6/s-afadin CASK CAROM

Cingulin, 7H6, Symplekin, ZONAB Rab-13, 19B1, Ponsin Rab 3B, PKC, l-afadin c-src, Gαi-2, Gαi-12, α-catenin, Pals, PATJ

ZAK, SCRIB, ITCH, Rho-GTPases, WNK4, Vinculin

Figure 3. Schematic representation of the interactions suggested between proteins

of the cell. This is in conjunction with the associated/regulatory proteins.

Zonula occludens-1(ZO-1)

PKA, JEAP, Pilt, PTEN,

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2.1.1 Zonular occludens and PDZ proteins

ZO-1 was the first molecule to be identified in TJs, as a phosphor-protein of 210-225 kDa in size (25–26) localised in the immediate vicinity of the plasma membrane of both endothelial and epithelial cells (22). ZO-1 is concentrated at the TJs although it is also found within the adherens junction, the nucleus and in cells that do not have distinct TJ structure (23). It is phosphorylated on serine residues under normal condi-tions but becomes phosphorylated on tyrosine residues after stimulatiuon. ZO-1 is a member of the MAGUK protein family (membrane associated and having the presence of a guanylate kinase or GUK domain) of which the members share the common domains of SH3, homologous to GUK, and have one or more PDZ domains (27–28). The PDZ domains mediate a reversible and regulated protein–protein interaction through contact with other PDZ domains or bu recognition of sequence motifs at the C-termini of integral proteins (27). The SH3 domain acts as a protein–protein interaction molecule binding to the PXXP motif. The UGK domain is also involved in protein to protein interactions.

ZO-2 and ZO-3 are also members of this MAGUK family of 160 kDa and 130 kDa, respectively. ZO-2 is a phosphoprotein present at the TJs of epithelia and endothelia and at the adherens junctions of non-TJ con-taining cells. ZO-2 and ZO-1 co-precipitate as heterodimers through PDZ-2/ PDZ-2 interactions. Soluble ZO-1, -2 and –3 are found as independent ZO-1, ZO-2 and ZO-3 complexes. The N-terminal domain of ZO-2 directly binds to claudins, occludin, and alpha-catenin, while the C-terminal domain co-localises with actin filaments and interacts with the actin-binding protein 4.1 (28). ZO-3 interacts with both ZO-1 and ZO-2, sharing a high sequence homology with both (27).

ZO proteins also contain some unique motifs not shared by other MAGUK family members, including nuclear localisation and nuclear export signals and a leucine zipper-like sequence. Nuclear ZO-2 directly

factor-B) (30). ZO-2 associates with SAF-B via its PDZ-1 domain, linking to the C-domain of SAF-B. SFA-B does not associate with ZO-1, supporting the idea that junctional MAGUK’s serve non-redundant functions. ZO-3 directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. Increased nuclear staining of ZO-2 is observed in epithelial cells subjected to environmental stress conditions.

Sequence analysis of ZO-1 and ZO-2 revealed them to be homologous to members of the lethal discs large-1 (Dlg), PSD-95/SAP90 and p55 protein family indicating a role in signal transduction (27, 22). Evidence


interacts with the DNA-binding protein SAF-B (scaffold attachment

TJ molecules 2.1

82 Martin suggests that ZO-1 may well act as a tumour suppressor in mammals as mutations in the Dlg cause neoplastic overgrowth of imaginal discs in Drosophila (31).

MUPP-1, like ZO-1 may also function as a cross-linker between Claudin-based TJ strands and JAM oligomers in TJs. The difference is in the 13 PDZ domains in tandem repeat within a single MUPP-1 molecule (32). This may indicate that other integral membrane proteins can be recruited to the Claudin-based TJ through MUPP-1. It is interesting to note, that viral oncogene products bind to MUPP-1, and one can speculate that MUPP-1 is involved in the formation of macromolecular complexes beneath the plasma membranes at TJs which may play an important role in the regulation of the growth and/or differentiation of epithelial cells (32). Claudins generally have a valine residue at their COOH termini, suggesting that they strongly attract PDZ-containing proteins, such as

partner for claudins at the COOH termini (32). MUPP1 is not well characterised, but is exclusively concentrated at TJs of epithelial cells via its binding to claudins and JAM. It thus may play a role as a multivalent scaffold protein recruiting various proteins to the TJ (32).

The subfamily of MAGUKs termed MAGIs (MAGUKs with inverted domain structure) are also located at TJs. Two of the three known MAGI isoforms, MAGI-1 and MAGI-3 are present in the TJs of cultured epi-thelial cells (33); indeed, MAGI-1 is expressed in the TJs of all epithelial cell types examined. Human MAGI-1 transcripts are alternatively spliced at three sites, and two forms are expressed only in non-epithelial tissues, mainly the brain, although all are localised at the TJ. The major form expressed in colon cancer epithelial cell cultures contains an extended carboxy terminus encoding potential nuclear targeting signals. MAGI-1, ZO-1 and ZO-2 all col-ocalise in non-polarised epithelial cells, suggesting a pre-assembled structure incorporated into the TJ structure at polarisation.

Zonulin may participate in the physiological regulation of intercellular TJs throughout a wide range of extraintestinal epithelia, as well as vascular endothelium, including the blood-brain barrier (34–35). Such disregula-tion may contribute to disordered intercellular communication, including inflammation, malignant transformation, and metastasis. Indeed, human brain plasma membrane contains a zonulin protein receptor of 45 kDa, which is a glycoprotein containing multiple sialic acid residues (36). This receptor has a striking similarity to MRP-8, a calcium-binding protein.

Membrane-associated guanykate kinase with inverted orientation (MAGI)-1/brain angiogenesis inhibitor 1-associated protein (BAP1) inter-acts with many transmembrane proteins, including receptors and channels through these domains (37). MAGI-1/BAP1 is ubiquitously expressed and localised at TJs in epithelial cells and is an isoform of the neurone-specific

ZO-1, -2, and -3. MUPP1 (multi-PDZ domain protein 1) is also a binding

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synaptic scaffolding molecule (S-SCAM), known to interact with NMDA erceptors and neuro-ligins. S-SCAm also interacts with a signalling molecule, a GDP/GTP exchange protein (GEP) that is specific for Rap1 small G protein, Rap GEP. MAGI-1/BAP1 serves as a scaffolding molecule for Rap GEP at TJs in epithelial cells (37).

CASK (originally identified as a neurexin-interacting protein, a human homologue of Lin-2, (38) is a membrane-associated guanylate kinases of epithelial TJ. CASK is localised along the lateral membranes. Carom has a coiled-coil diamin in the middle region and two src homology 3 domains and a PSD-95/Dlg-A/ZO-1 (PDZ)-binding motif in the C-terminal region (39). Carom binds to the fifth PDZ domain of MAGI-1 and the calmodulin kinase domain of CASK in vitro. MAGI-1 and CASK bind to distinct sequences in the C-terminal region of Carom, but still compete with each other for Carom binding. MDCK cells expressing GFP-Carom revealed that Carom was partially over-lapped by MAGI-1 in MDCK cells which have not yet established mature cell junctions, but became separated from MAGI-1 and co-localised with CASK in polarised cells. Carom was highly resistant to Triton X-100 extractions and recruited CASK to the Triton X-100-insoluble structures. Carom is a binding partner for CASK, which interacts with CASK in polarised epithelial cells, and may link it to the cytoskeleton. CASK also interacts with syndecans, JAM-A, protein 4.1, hDLA, (40). CASK may be important for the proper targeting of junctional components and link them to the cytoskeleton (41). Moreover, CASK has been shown to be translocated to the nucleus and may be involved in the regulation of gene transcription (42).

2.1.2 Occludin

The first transmembrane TJ protein identified was Occludin, 60–65 (82) kDa (43–44). It bears four transmembrane domains in its N-terminal half, with both the N- and C- termini located in the cytoplasm; the C-terminal (approximately, 150 amino acids) binding to ZO-1 (1, 22). The cytoplasmin domain (domain E) also interacts with both ZO-1 and ZO-2. The topology of occludin predicts two extracellular loops pro-jecting into the paracellular space which interact with loops originating from occludin in the neighbouring cell or unidentified molecules to pro-mote interaction and sealing of the paracellular space (45). The C-terminal of occludin is sufficient to mediate endocytosis, as the C-terminal governs intracellular transport of occludin (46). Occludin is a functional com-ponent of the TJ and widely expressed in both endothelial and epithelial cells, but not in cells and tissues without TJs (3).

The extracellular surface of occludin was found to be directly involved in cell-cell adhesion and the ability to confer adhesiveness correlated with the ability to co-localise with ZO-1 (47). The discrepancy in the size


84 Martin

within the TJ whereas the smaller less phosphrylated forms are found in the basolateral membrane and cytosol (45, 48). Thus its phosphorylation is directly related to its function. Small differences in the electrophoretic mobility of occludin were found to be distinct phosphorylated variants with altered membrane localisation, indicating that phosphorylation of occludin in an important step in TJ assembly (48–50). In endothelial cells it has been shown that selective proteolytic cleavage of occludin by metallo-proteinases after inhibition of protein tyrosine phosphatases raises para-cellular permeability (51).

Little is known about occludin kinases. However, a recombinant C-terminal fragment of occludin is a substrate for a kinase in crude extracts

An alternatively spliced form of occludin, occludin 1B was identified in MDCK cells and cultured T84 human colon cancer cells (54). There are two gene products, the larger, predominant product corresponded to the canonical occludin (TM4+), whilst the smaller product exhibited a 162bp deletion encoding the entire TM4 and immediate C-terminal flanking region (TM4-) (55). The deleted section corresponded to exon 4, suggesting that TM4- is an alternatively spliced isoform. TM4- was also found in monkey epithelial cells, but not murine or canine. Staining of occludin in Caco-2 cells with a C-terminal occludin antibody revealed weak, discontinuous staining restricted to the periphery of subconfluent islands. A weak band at 58kDa (smaller than the predominant band at 65 kDa) corresponded to the predicted mass after blotting. The authors suggest that the TM4- isoform is upregulated in subconfluent cells, and that it is translated at low levels in specific conditions and may contribute to the regulation of occludin function; i.e., if occludin has no C-terminus, it cannot bind to ZO-1.

Occludin’s function in the TJ is poorly defined (56). Suppression of

of brain. CK2 is a candidate kinase for regulation of occludin phosphorylation in vivo (52). Phosphorylation of serine residues on occludinwill increase the formation of the TJs (53). Occludin is a Ca2+ -indepen-dent intercellular adhesion molecule that confers adhesiveness in proportion to the level of occludin expressed (24).

occludin is associated with a decrease in claudin-1 and claudin-7 and anincrease in claudin-3 and claudin-4. It is indicated that occludin transducesexternal (apoptotic cells) and intramembrane (rapid cholesterol depletion)signals via a Rho signalling pathway that, in turn, elicits reorganizationof the actin cytoskeleton. Impaired signalling in the absence of occludinmay also alter the dynamic behaviour of TJ strands, as reflected byan increase in permeability to large organic cations; the permeability ofion pores formed of claudins, however, is less affected.

of occludin protein is a result of differential serine and threonine phos-phorylation. The larger phosphorylated form of occludin is found localised

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structurally related, are 23 kDa in size and have four membrane-spanning regions, although they share no homology with occludin and the other transmembrane proteins located within the TJ. These are found in cells with and without TJs, but are highly expressed in those that do. There are currently 25 claudins described, which often have distinct tissue-specific distributions, although this is debatable as previously claudins that were thougth only to be expressed in certain cells types have been found

the primary seal-forming elements of the paracellular space when


expressed–albeit at low levels–in disparate cells and tissues. Claudins are

occludin is absent (24). They mediate calcium-independent cell–cell adhesion (22).

2.1.3 The Claudin family

Claudins are a family of integral trasnmembrane proteins located in the TJ (22). Claudin-1 and claudin-2 were the first described and are

Mutations in claudin genes give rise to a number of human hereditary diseases. Claudin-14 defects suffer autosomal deafness (62); Mutations in claudin-16 (paracellin-1) lead to hypomagnesemia syndrome (63). Claudin-16 was originally thought to be uniquely expressed in kidney tissues, but has been found to be expressed in low levels in normal breast tissues (15). Claudin-1 originally named senescence-associated epithelial membrane protein 1 (SEMP-1) was the first to be described and was found to be expressed in most tissue types (64). Moreover, it was the first TJ protein to be indicated as a tumour suppressor in human mammary epithelial cells

expressed in endothelial cells, it has subsequently been detected in human epithelial cells also, albeit at low levels (6). It is believed that claudins are

(64). Although claudin-5 was originally described as being specifically

The claudin protein structure is predicted to consist of cytoplasmic

loops via which interactions with claudins on adjacent cells occur (57). These interactions can be homo- or heterotypic. The sealing function of claudins is mediated in part by phosphorylation events on the cytoplasmic C-terminus (58–59). In addition, the cytoplasmic C-terminal domain con-tains a PDZ-binging motif and thus claudins are able to bind to ZO-1,

N- and C-termini, four transmembrane domains, and two extracellular

ZO-2, and ZO-3. Claudins are also able to bind to the other PDZ contain-ing proteins such as PAR3 and PAR6. Claudins are also then, involvedin cytoskeletal and cell signalling events via regulation of protein localisa-tion in addition to their adhesive functions (61). In contrast to the othermain constituents of the TJ, claudins have become an active area ofresearch attempting to understand carcinogeneis and progression to meta-stasis as many claudins exhibit altered expression in cancer, which wasnoted shortly after their discovery (61). Claudins are usually over or underexpressed in cancers.

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2.1.4 Junctional Adhesion Molecules (JAM)/CTX molecules (CAR)

JAMs are members of the immunoglogulin superfamily of protein and are expressed in most cell types, including epithelial, endothelial cells, leukocytes, and platelets. The members of this family are approximately 40 kDa in size and are located at TJs in a similar distribution to ZO-1 (66). There are four members of the JAM protein family, which have recently been renamed; JAM-A, JAM-B, JAM-C, and JAML (67-68). JAM-A and JAM-C localise to the TJ in epithelia and JAM-B to the lateral membrane (69). JAMs have the structural and sequence conserva-tion features of IgSF molecules with two extracellular Ig-like domains and sites for N-glycosylation (70). They are thus unlike occludin and the claudins in having a single transmembrane domain (71). The extra-

JAM-B, or VE-JAM, was originally believed to be a vascular molecule participating in interendothelial junctional complexes (69, 78). JAM-C is highly expressed during embryogenesis, in lymph nodes, stains darkly in endothelial venules, vascular structures in the kidney and in lymphatic vessels in lymphoid organs (69). JAM-B binds in a homotypic manner to

cellular domains of JAM-A, -B and -C contain dimerisation motifs that play a role in their interactions (72). JAMs interact in both homo- andheterotypic fashion, as well as with integrins (73).

JAMs regulate both paracellular permeability and leukocyte trans-migration via homphilic interaction (74–75). JAM has been suggested to play an important role in the regulation of TJ assembly in epithelia, and JAM-mediated effects may occur by direct or indirect interactions with occludin (76), as JAM is associated with occludin and not ZO-1 in re-assembling the TJ structure. JAM’s associate through their extracellular domains with the leukocyte beta2 integrins LFA-1 and Mac-1 as well as with the beta1 integrin alpha4beta1. All three integrins are involved in the regulation of leukocyte–endothelial cell interactions (77). Through their cytoplasmic domain JAMs directly associate with ZO-1, AF6, MUPP-1, and the cell polarity protein PAR-3. PAR-3 is part of a ternary protein complex containing PAR-3, atypical protein kinase C and PAR-6. This complex is highly conserved throughout evolution. This may suggest a dual function for JAMs; they appear to regulate leukocyte–platelet–

JAM-B, but also has a receptor in JAM-C (73, 79) within numerous cell

the paracellular channels which have selectivity for specific ions and play central roles in the regulation of paracellular permeability; the diversity of claudin expression contributes to the physiological homeostasis in response to a particular tissues requirement (65).

types, including endothelial cells. JAM-B adheres to T cells through

in epithelial and endothelial cells during the acquisition of cell polarity (77). endothelial cell interactions in the immune system, as well as TJ formation

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prostate adenocarcinoma, and colonic carcinoma (73). JAM-1 is also an adhesion molecule for T-cell lines and some circulating lymphocytes and

into the TJ structure via its binding to the PDZ2 domain of ZO-1 (claudins bind to its PDZ1 domain). JAM also recruits PAR-3 (ASIP), a determinant of asymmetric cell division and polarised cell growth to TJs through binding to its COOH terminus (81). AF6, a PDZ domain protein in also an intracellular binding partner of JAM-1 via its C-terminus, which has a classical type II PDZ domain-binding motif (82). JAM also binds to the PDZ domains 2 and 3 of ZO-1.

JAML, a novel MAGI-1-binding protein co-localises with ZO-1 in

whereas JAM-A did not bind to MAGI-1. They also found that MAGI-1, AO-1 and occludin were recruited to JAML-based cell contacts. JAML appeared to reduce the permeability of CHO cell monolayers. It is suggested that JAML and MAGI-1 provide an adhesion machinery at TJs, which may regulate the permeability of kidney glomeruli and small intestinal epithelial cells.

The Coxsackie-Adenovirus Receptor (CAR) is a 46 kDa transmembrane protein enabling the attachment of virus via the interaction of the adenovirus finger-knob (84). It is expressed ubiquitously in most benign epithelial tissues and although its role is poorly understood has been suggested to be associated with the TJ structure in normal cells (84) and loss of CAR expression can reduce infectivity. Earlier studies have reported a frequent reduction in CAR expression in highly malignant bladder and prostate tumours (85–88). CAR has been much studied due to its importance as a means of entry to cancer cells regarding adenovirus-based cancer therapies. The expression is reported to be often low in a number of cancer types, including ovarian, colorectal, lung, prostate, head and neck tumours, and breast (89–95). CAR expression may correlate inversely with tumour progression (96).

There is a downregulation of CAR gene expression in invasive tran-sitional cell carcinoma in bladder cancer (97). This low expression may have an impact on developing adenoviral-based gene therapies, and they proposed that loss of CAR expression could decrease rigid cell adhesion,


in vitro studies revealed that JAML bound to MAGI-1 but not to ZO-2, kidney glomeruli and in intestinal epithelial cells (83). Biochemical

heterotypic interactions with JAM-C. The engagement of α4β1 by JAM-B is only enabled following prior adhesion of JAM-B with JAM-C (80).

There is a preferential expression of JAM-B mRNA in the endothelium in and around tumours and at sites of inflammation at tumour types such as breast, pulmonary squamous cell, pulmonary adenocarcinoma,

possibly increasing migratory potential. Loss of CAR correlates with invasive bladder cancer.

dendritic cells. JAM-2 and JAM-3 are an interacting pair in the A33/JAM family of adhesion molecules. JAM is thought to be integrated

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The Raf-MEK-ERK pathway is suggested to be involved in regulating ZO-1 expression at the cell surface (99); ZO-1 is restored after inhibition of MEK. CAR expression in pancreatic and colorectal cancer cell lines is

protein at the cell surface (100). They conclude that CAR expression loss in cancer cells is at least in part mediated through the Raf-MEK-ERK signal transduction pathway.

2.1.5 The Nectin family

Nectins, Ca(2+)-independent immunoglobulin-like cell adhesion mole-cules (CAMs), first form cell–cell adhesion where cadherins are recruited, forming adherens junctions in epithelial cells and fibroblasts. In addition,

of which are actin filament-binding proteins. The nectin-1-based cell–cell adhesion is formed and maintained irrespective of the presence and absence of the actin filament-disrupting agents, such as cytochalasin D and latrunculin A (101). In the presence of these agents, only afadin remains at the nectin-1-based cell–cell adhesion sites, whereas E-cadherin and other PMPs at adherens junctions, α-catenin, β-catenin, vinculin, α-actinin, ADIP, and LMO7, are not concentrated there. Claudin-1, occludin, and JAM-A, or the PMPs at TJs, ZO-1, and MAGI-1, are not

other CAMs and PMPs at adherens and TJs. Although nectin was initially thought to be only localised at adherens

junctions, recent studies have suggested that a role in the formation or organisation of TJs may be found. Nectin-3 (PRR3) interacts with afadin by interaction of their C-terminal to the PDZ domain of afadin (102). The nectin-afadin system is able to recruit ZO-1 to the nectin-based cell–cell adhesion sites in non-epithelial calls that have no TJs (103).

There is a nectin trans-hetero-interaction network; nectin-3 binds to nectin-1, nectin-2, and PVR (poliovirus receptor); nectin-1 also binds to nectin-4 (104). Nectin-1/nectin-3 and nectin-1/nectin-4 trans-hetero-

upregulated by inhibition of MEK, accompanied by increased CAR

cancer cell lines, with disruption of cell–cell contacts increasing adenoviral gene transfer into human cancer cells (98). Moreover, TNF alpha increases CAR expression in HeLa and ovarian cancer cells, but decreases CAR expression in U87MG glioblastoma cells. Dexamethasone downregulates CAR expression in both cell types.

There is localisation of CAR at cell–cell adhesions in several human

interactions are mediated through trans- V – V domain interactions, whereas C domains contribute to increase the affinity of the interaction. Nectin-3 and

nectins recruit claudins, occludin, and JAMs to the apical side of adherensjunctions, forming TJs in epithelial cells. Nectins are associated withthese CAMs through peripheral membrane proteins (PMPs), many

skeleton is required for the association of the nectin-afadin unit with concentrated there, either. These results indicated that the actin cyto-

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over recent years. They are too numerous to detail fully here and the reader is directed to the numerous reviews available (65, 106–110).

3. TJ FUNCTIONS

Five main functions are ascribed to the TJ:


The formation of cis-dimers is necessary for the formation of nectintrans-dimers. The authors noted that the first Ig-like loop of nectin-3 isessential and sufficient for the formation of trans-dimers with nectin-1,but that the second Ig-like loop of nectin-3 was furthermore necessaryfor its cell–cell adhesion activity.

TJ-associated molecules 2.2

An increasing number of TJ-associated molecules has been revealed

All four nectin family members have one extracellular region with three Ig-like loops, one transmembrane segment, and one cytoplasmic tail (106).

nectin-4 share a common binding region in the nectin-1 V domain: (i)

binding to nectin-1 was reduced by monoclonal antibodies directed towards the nectin-1 V domain, (iii) the glycoprotein D of HSV-1 that binds to the V domain of nectin-1 reduced nectin-3 and nectin-4 binding.

ling, thus playing a role in the processes of polarity, cell differen-tiation, cell growth, and proliferation.

Cell adhesion to adjacent cells and the extracellular matrix is key not only

cytoskeletal all of which are organised into multimolecular complexes and

(4) TJ proteins act as cell–cell adhesion molecules. (5) The TJ functions as a barrier to cell migration.

to the organisation of epithelium into a tissue but also to the regulation of cellular processes such as gene expression, differentiation, motility, and growth (111). Cell adhesion molecules, transmembrane receptors, and

the activation of signalling pathways, mediate these regulatory functions.

nectin-3 strongly competed with nectin-4 binding, (ii) nectin-3 and nectin-4

(3) TJ molecules act as intermediates and transducers in cell signal-

(1) The TJ seals the intercellular space and is responsible for the separation of apical and basolateral fluid compartments of epithelia and endothelia. Macromolecules of radii ≥15 angstroms cannot pass. However, such barriers regulate the passage of small ions etc.

(2) The TJ functions as a diffusion barrier to plasma membrane lipids and proteins, which helps to define apical and basolateral mem-brane domains of these polarised epithelial and endothelial cells. Therefore the TJ is crucial for the epithelium to generate chemical and electrical gradients across the cell monolayer that is necessary for vectorial transport processes such as absorption and secretion.

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structure with roles in other cellular processes such as cell polarity, proliferation, and differentiation has been recognised (68). Moreover, it is becoming increasingly clear that the development of human cancer is frequently associated with the failure of epithelial cells to form TJs and to establish correct apicobasal polarity (112).

4. TJ AND BREAST CANCER METASTASIS

Role of TJs in Breast Cancer Metastasis 4.1

Studies suggest that some of the cell adhesion and cytoskeletal proteins may subserve an additional and important function, namely, suppression of the malignant phenotype of cells in tumorigenesis (111). Whilst the barrier and fence functions of TJs have been well appreciated, it is only recently that concept of the TJ as a complex, multiprotein

Cancer metastasis proceeds by a series of steps, among which the capacity of cancer cells to invade surrounding normal tissues is of central importance in the dissemination of disease (113). The interaction between cancer cells and mesothelial cells lining the cavity is crucial for achieving the complex sequence of cancer cell dissemination into the body cavity. In the process of submesothelial invasion of cancer cells, TJs of mesothelial cells may function as a defence against the invasion of cancer cells, because the TJs are known to work as a barrier to the paracellular passage of cells and substances between epithelial or endothelial cells (113).

Metastasis is the primary cause of fatality in breast cancer patients. Although there are believed to be numerous events contributing to the process of metastasis, it is widely accepted that the loss of cell–cell adhesion in neoplastic epithelium is necessary for invasion of surrounding stromal elements and subsequent metastatic events (10). Regulation of vascular permeability is one of the most important functions of endothelial cells, and endothelial cells from different organ sites show different degrees of permeability (114). Tumour blood vessels are more permeable on macro-molecular diffusion than normal tissue vessels. However, the

organization of TJs in mammary gland biology can be found in (65).

Expression of TJ Proteins in Breast Cancer 4.2

Most cancers, including breast cancer, originate from epithelial tissues and are characterised by aberrant growth control, and loss of differentiation and tissue architecture. It is a fundamental property of cancer cells that their mutual adhesiveness is significantly weaker than that of normal cells.

endothelial cell permeability irreversibly. A timely discussion of the

cause and mechanism of hyperpermeability of human vessels had notbeen clear (114). Although, tumour-cell-conditioned medium increases

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4.2.1 Transmembrane protein expression in breast cancer

Claudin-1 expression has been observed in human mammary epi-thelial cells, but was observed to be at low or undetectable levels in a number of breast tumours and breast cancer cell lines (11). This points to a possible tumour suppressor function. Kramer et al. found that in sporadic and hereditary breast cancer, there were no genetic changes, implying that regulatory or epigenetic factors may be involved in the downregulation of the claudin-1 gene during breast cancer development.

Loss of claudin-7 correlates with histological grade in both ductal carcinoma in situ and invasive ductal carcinoma of the breast (10). The expression of claudin-7 is lost in both pre-neoplastic and invasive ductal carcinoma of the breast occurring predominately in high grade lesions. Expression is also frequently lost in LCIS correlating with the increased cellular discohesion observed in LCIS. Additionally, the majority of IDC cases displaying a low claudin-7 expression have a positive lymph node status. Such findings suggest that the loss of claudin-7 may aid in tumour cell dissemination and augment metastatic potential. Moreover, silencing of claudin-7 expression correlated with promoter hypermethylation in 3/3 breast cancer cell lines but not in invasive ductal carcinomas (0/5). In addition, HGF treatment results in disassociation of MCF-7 and T47D


Recent studies have shown that several TJ components are, directly or indirectly, involved in breast cancer progression including ZO-1, ZO-2,claudin-7, claudin-1, and occludin.

cells in culture, and a loss of claudin-7 expression within 24 hours.

Claudin-1 (SEMP-1) is normally expressed in mammary gland-derived epithelial cells, but is absent in most human breast cancer cell lines. Claudin-1 expression was not detectable in subconfluent MDA-MB-435 and MDA-MB-361 breast cancer cells (9). Neither of these cell lines express occludin protein, and MDA-MB-435 do not express ZO-1 protein. Claudin-1 retroviral transduced breast cancer cells showed expression of Claudin-1 at the usual cell–cell contact sites, suggesting that other proteins may be able to target claudin-1 to the TJ in the absence of occludin and ZO-1. Moreover, paracellular permeability was reduced in these transduced cells. The authors suggest that Claudin-1 gene transfer may be in itself enough to exert TJ-mediated gate function in metastatic breast cancer cells even in the absence of other TJ associated proteins such as occludin.

Reduced cell–cell interaction allows cancer cells to disobey the social order, resulting in destruction of overall tissue architecture, the morpho-logical hallmark of malignancy. Loss of contact inhibition, which reflects disorder in the signal transduction pathways that connect cell–cell interactions are typical of both early (loss of cell polarity and growth control) and late (invasion and metastasis) stages of tumour progression.

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Tokes et al. (115) compared levels of protein and mRNA expression of three members of the claudin family in malignant breast tumours and benign lesions. Altogether, 56 sections from 52 surgically resected breast

by immunohistochemistry and mRNA was also analysed using real-time PCR. Claudins were rarely observed exclusively at TJ structures. Claudin-1 was present in the membrane of normal duct cells and in some of the cell membranes from ductal carcinoma in situ, and was frequently observed in eight out of nine areas of apocrine metaplasia, whereas invasive tumours were negative for claudin-1 or it was present in a scat-tered distribution among such tumour cells (in 36/39 malignant tumours). Claudin-3 was present in 49 of the 56 sections and calsuin-4 was present in all 56 tissue sections. However, claudin-4 was highly positive in normal epithelial cells and was decreased or absent in 17 out of 21 ductal carcinoma grade 1, in special types of breast carcinoma (mucinous, papillary, tubular) and in areas of apocrine metaplasia. Claudin-1 mRNA was downregulated by 12-fold in the tumour group. Claudin-3 and claudin-4 mRNA exhibited no difference in expression between invasive tumours and surrounding tissue. The significant loss of claudin-1 protein in breast cancer cells suggests that this protein may play a role in invasion and metastasis. The loss of claudin-4 expression in areas of apocrine metaplasia and in the majority of grade 1 invasive carcinomas also suggests a particular role for this protein in mammary glandular cell differentiation and carcinogenesis.

mammary cases), and compared the results with those of other neoplastic skin lesions, including actinic keratoses, basal cell carcinomas, and malignant melanomas. To compare claudin expression in Paget’s disease and breast neoplasia, it was also studied in a large set of breast carcino-mas. Membrane-bound claudin-3 and -4 expression was seen in all cases

seen in most of them, suggesting an inverse expression of these claudins between Paget’s disease and epidermal and nevocytic lesions. Claudin expression in breast carcinomas was claudin-2 in 52%, claudin-3 in 93%,

found more often in ductal carcinomas than in lobular carcinomas. Expression of claudins were frequently associated with each other. They were not associated with estrogen or progesterone receptor status or with tumour grade. No significant differences were found between claudin

specimens were analysed for claudin-1, claudin-3, and claudin-4 expression

Soini (116), evaluated the expression of claudin-2, claudin-3, claudin-4, and claudin-5 in 20 cases of Paget’s disease (13 mammary and 7 extra-

of Paget’s disease, whereas claudin-5 was seen in 50% of cases and claudin-2 was seen in 32% of cases. In contrast, claudin-3, claudin-4, and claudin-5 were not seen in the other skin lesions, and claudin 2 was

claudin-4 in 92%, and claudin-5 in 47%. Claudins-2 and claudin-5 were

expression in Paget’s disease and breast carcinomas. The results demon-strate that claudins could be useful in diagnosing Paget’s disease and

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actinic keratoses, basal cell carcinomas, and nevocytic lesions. The lack

are reduced with poor prognosis of patients with breast cancer, however JAM-2 does not show differences in expression (117). The levels of transcripts of claudin-16 and vinculin were significantly lower in patients that had poor prognosis (with metastasis, recurrence, or mortality), compared with those that remained healthy after a median follow-up of 72.2 months Immunohistochemistry confirmed these results, as there is decreased levels in staining for claudin-16 and AF6. In normal tissue, staining was confined to the intercellular regions whereas in the tumour tissues the staining was diffuse and cytosolic. The conclusion was that low levels of TJ molecules claudin-16 and vinculin in breast cancer are associated with poor prognosis in patients, underscoring the idea that regulation of TJs could be of fundamental importance in the prevention of metastasis of breast cancer cells.

Martin and Jiang (118) investigated the expression of occludin in human breast cancer tissues and cell lines. Tissues and breast cancer cell lines were amplified for functional regions of occludin. 6/6 tumour tissues showed truncated and/or variant signals for N-terminal and first trans-membrane loop of occludin; 4/6 tumour tissues did not express the C-terminal region of Occludin. Paired background tissues showed similar expression profiles. None of the breast tissues showed methylation of the

express the N-terminal, 6 expressed 2 or more variants; 3 did expressed a truncated message for the first trans-membrane loop, 5 expressed the correct message, MDA-MB-231 cells did not express this region; the C-terminal region was expressed correctly in 3 cell lines, 4 expressed variants, and 4 were missing this region. Overall, only 3/10 breast cancer

probed with 3 antibodies specific for the N-terminus, first membrane loop and C-terminus. These variants did not fit the expected occludin signals for changes in phosphorylation status of the protein. Immuno-staining showed similarly disparate levels of expression, with more invasive cell lines showing reduced cell junction location. This study showed for the first time that occludin is differentially expressed in breast tumour tissues and in human breast cancer cell lines. The changes in occludin message indicate that variants are expressed in tumour tissue. The loss of or truncation of the N-terminus indicates reduced assembly


tumours suggests that changes in the phenotype of claudin-2, claudin-3, claudin-4, and claudin-5 are not necessary for epidermal invasion.

occludin promotor. Of the 10 human breast cancer cell lines, 3 did not

in differentiating these lesions from other epidermal lesions, such as

observed. Western blotting also demonstrated variants of occludin when the more invasive phenotype. Methylation of the promotor was not cell lines expressed full length occludin; interestingly, these were of

Claudin-16 (Paracellin-1), Ponsin, ZO-2, AF6, Vinculin, and Nectin

of difference in claudin expression between Paget’s disease and breast

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to the basolateral membrane and binding to ZO-1, resulting in reduced TJ anchoring, assembly, and cell–cell adhesion. This has clear repercus-sions as to the importance of occludin in maintaining TJ integrity in breast tissues. Such inappropriate expression may play a part in breast cancer development.

4.2.2 Peripheral Plaque protein expression in breast cancer

ZO-2 can be expressed in two isoforms, ZO-2A and ZO-2C, in normal epithelia. ZO-2A is absent in pancreatic adenocarcinoma of the ductal type, with none of the common mechanisms of gene inactivation res-ponsible (13). Analysis of the ZO-2 promotors (PA and PC) showed that lack of expression of ZO-A in neoplastic pancreatic cells is caused by inactivation of the downstream promoter PA, probably due to structural or functional alterations in the regulatory elements localised outside the analysed promoter region as hypermethylation was not a convincing reason in early cancers. However, methylation of PA is responsible for the inactivation of the suppressed promoter at the late stages of tumour development (111). ZO-2 was found to be deregulated in breast adeno-carcinoma, but not in colon or prostate adenocarcinoma, both of which are considered to be of acinar rather than ductal type.

MAGUKs may play a vital role in cellular functions preventing tumori-genesis as indicated by neoplastic phenotypes in Drosophila; Normal breast tissues have shown the expected intense staining at cell–cell junc-tions; however, ZO-1 staining is found to be reduced or lost in 69% of breast cancers analysed using immunohistochemistry (12). Normal tissue showed intense staining for ZO-1 at the position of the epithelial TJs, but

of tumours. The ZO-1 gene tjp-1 was mapped relative to other markers flanking the gene. There was a loss of heterozygosity in 23% of informative tumours. Loss of a tjp-1-linked marker suggests that genetic loss may, in some cases, be responsible for a reduction in ZO-1 in breast cancer.

In 18 breast cancer cell lines, the most poorly differentiated, fibro-blastic cell lines were ZO-1 negative, and were highly invasive (119).

Martin et al. (16) investigated the expression of Zonula Occludens (ZO) proteins ZO-1, ZO-2, and ZO-3, and MUPP-1 in patients with pri-mary breast cancer (Figure 4). Breast cancer primary tumours. Standar-

differentiated, in 83% of moderately differentiated and in 93% of poorly

this was lost or reduced in 69% of breast cancers analysed. In infiltrat-ing ductal carcinomas there was a reduction in staining in 42% of well-

differentiated tumours. ZO-1 was positively correlated with tumour differentiation, and more specifically with the glandular differentiation

of TJ structure and reduced maintenance of barrier function. Loss of C-terminal expression suggests reduced intracellular trafficking of occludin

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these results, with decreased levels in ZO-1 staining. For both ZO-1 and ZO-3, staining was confined to the intercellular regions in normal tissue, whereas in tumour tissues staining was diffuse and cytosolic. Q-PCR revealed a reduction in the levels of ZO-1 and MUPP-1 in patients with disease recurrence. Prognostic indicators of breast cancer were also inversely correlated with ZO-1 expression. It was concluded that low levels of TJ plaque molecules, such as ZO-1 and MUPP-1, in breast cancer are associated with poor patients prognosis.


Figure 4. Panel shows the differential expression of peripheral/plaque proteins in represen-tative sections from patients with breast cancer. (A) Immunohistochemical staining (×100) of ZO-1, ZO-2, and ZO-3 in human breast cancer tissues. Clear staining is shown in normal tissue (left), reduced staining for ZO-1 and ZO-3 shown in the right. (B) Western blotting of paired normal and tumour tissues and densimetric analysis. Total levels of ZO-1 and ZO-3 were seen in tumours (8/10). (C) Comparison of grade and RNA trans-cript level of plaque proteins. All four were reduced with increasing tumour grade. (D) Comparison of histology of primary tumours and RNA transcript level of plaque proteins. ZO-1 and ZO-2 were increased in lobular carcinoma compared to other types. ZO-3 was significantly reduced. MUPP-1 was significantly reduced in ductal carcinoma.

dised transcript levels of ZO-1 and MUPP-1 were significantly lower in patients with metastatic disease compared with those remaining disease-free (median follow-up 72.2 months). Immunohistochemistry confirmed

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were analysed by IHC: ZO-1 expression did not correlate with HER-

overexpression should be sought. Interestingly, the authors report that ZO-1 IHC stained DCIS were positive for ZO-1 in 18/20 cases, with 4/18 negative for ZO-1 in the invasive tumour.

Interestingly, the nectin family has been little studied as regards TJs in cancer, being originally described as molecules involved in adherens junctions only. Recently however, it has become apparent the nectins are also involved in recruitment and maintenance of proteins within the TJ. Studies have shown that nectin-3 expression showed clear changes in distribution between normal and cancerous cells (120). However, there was little difference in overall expression when analysed by Q-PCR. Breast cancer cell lines screened showed aberrant expression for nectin-3. nectin-3 transformed cells showed retarded invasion, even when treated

cells were significantly less motile and more resistance to HGF-induced reduced TJ functionality. As anticipated, breast cancer cells with endo-genous nectin-3 knocked out using ribozyme technology showed both increased invasiveness and motility. The staining pattern in human breast cancer tissues indicated that the distribution of the molecule is more crucial than the level of expression. The introduction of nectin-3 into human breast cancer cells results in breast cancer cells with reduced invasive phenotype and increased TJ function; conversely, breast cancer cells with nectin-3 knockout showed increased invasion and motility. This, together with the reported aberrant expression of other nectins in human cancer, indicates that nectin-3 may be a key component in the formation of cell–cell junctions and be a putative suppressor molecule to the invasion of breast cancer cells (120).

Application of all transretinoic acid correlates well with paracellular barrier function of endothelial cells, significantly reducing the rate by which tumour cells transmigrate across the endothelial cell monolayers (8, 113). Such experiments (6) suggest that TJs of vascular endothelium in vivo function as a barrier between blood and tissues against metastatic cancer cells. Dexamethasone induces “normal-like” differentiated pro-perty of TJ formation, and suppresses growth of the rat Con8 mammary epithelial tumour cell line (121).

2/neu expression in breast carcinomas, and so other causes of HER-2/neu

Regulation of TJs in Breast Cancer 4.3

repressor of the Her-2/neu gene promoter (14). ZO-1 was examined in a series of breast cancers: one group contained those invasive cancers scoring for HER-2/neu status (negative (12), 2+ (13) and 3+ (10)) and

ZO-1 can upregulate HER-2/neu expression in vitro by sequestering a

with HGF. Invasion was significantly different between these cells and thewild type and when treated with HGF. Moreover, these transformed

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were related to the proliferation and detachment of tumour cells from the primary site in the initial stage of tumour metastasis (7).

In Con8 rat mammary epithelial tumour cells, dexamethasone stimulates TER, promotes remodelling of apical junctions, and downregulates the level of fascin, an actin-bundling protein that can bind beta-catenin. It has been shown that TGFα ablates dexamethsone-induced remodelling of the apical junction and stimulation of TER (122). This response was polarised in that basolateral but not apical exposure reversed fascin production and TJ formation. The authors propose the regulation of fascin protein levels as a target of cross-talk between the Ras-dependent growth factor signalling and dexamethasone (glucocorticoid) signalling pathways that control TJ dynamics in mammary epithelial tumour cells.

Hyperactivation of the insulin-like growth factor I receptor (IGF-IR) contributes to primary breast cancer development, but its role in meta-stasis is unclear. IGF-IR overexpression markedly stimulates aggregation in E-cadherin positive MCF-7 cells, but not in E-cadherin negative MDA 231 cells (123). IGF-IR-dependent cell–cell adhesion of MCF-7 cells coincided with the upregulation of ZI-1. ZO-1 expression (mRNA and protein) was induced by IGF-I and was blocked in MCF-7 cells with a tyrosine kinase-defective IGF-IR mutant. ZO-1 associates with the E-cadherin complex (immunoprecipitation) and IGF-IR. High levels of ZO-1 coincide with increased IGF-IR/alpha-catenin/ZO-1-binding and

anti-ZO-1 RNA inhibited IGF-IR-dependent cell-cell adhesion. The results are suggestive of a mechanism by which activated IGF-IR regulated E-cadherin-mediated cell–cell adhesion by over-expression of ZO-1 and the resulting stronger connections between the E-cadherin complex and the actin cytoskeleton. IGF-IR may thus provide an anti-metastatic effect in E-cadherin positive breast cancer cells.

It has been demonstrated that involvement of the Ras-MEK-ERK pathway is likely not involved in the dysregulated TJ formation in breast tumour cells and indicates that elevated activity of Ras might not be of general importance for the disruption of TJ structures in breast tumours (124). Constitutive activation of Ras of Ras-mediated signalling path-

neoplastic transformation. Clostridium perfringens enterotoxin (CPE), induces cytolysis very


improved ZO-1/actin association, whereas downregulation of ZO-1 by

ways is one of the initial steps during tumorogenesis that promotes

rapidly through binding to its receptors, the TJ proteins claudin-3 and -4 (125). In primary human breast cancers (21) claudin-3 and claudin-4

An early paper looked at metastatic, weakly metastatic, and parent clones spontaneously developed from rat mammary carcinoma (7). EMs were used to look at TJ formation. When highly and weakly metastatic clones were co-cultured with normal fibroblasts, TJ structures were ob-served only in the weakly metastatic clones. Ultrastructural differences

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necrosis after CPE treatment. Necrotic reaction was also observed in fresh resected primary breast carcinoma samples treated with CPE for 12

Although most malignant tumours are epithelia-derived carcinomas, methods for specific and effective delivery of anti-tumour agents to car-cinomas have not been developed. Recent reports indicate that epithelia overexpress claudin-3 and claudin-4, which are integral membrane pro-teins of epithelial TJs. This suggests that claudins can be targeted for tumour therapy, but there is not currently a method for delivering drugs to claudin-expressing cells. It was evaluated whether a potent claudin- 4-binding C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) would allow targeting to claudin-4-expressing cells (126). C-CPE was fused to the protein synthesis inhibitory factor (PSIF), which lacks the cell-binding domain of Pseudomonas exotoxin. This fusion protein, C-CPE-PSIF, was cytotoxic to MCF-7 human breast cancer cells, which express endogenous claudin-4, but it was not toxic to mouse fibroblast L cells, which lack endogenous claudin-4. The cytotoxicity of C-CPE-PSIF was attenuated by pretreating the MCF-7 cells with C-CPE but not bovine serum albumin. Also, deletion of the claudin-4-binding region of C-CPE reduced the cytotoxicity of C-CPE-PSIF. Finally, it was found that C-CPE-PSIF is toxic to L cells expressing claudin-4 but not to normal L cells or cells expressing claudin-1, claudin-2, or claudin-5. These results indicate that use of the C-CPE peptide may provide a novel way to target drugs to claudin-expressing cells (126).

HGF, a cytokine secreted by stromal cells, is capable of modulating expression and function of TJ molecules in human breast cancer cell

and immunohistochemistry also showed modulation of expression of the TJ molecule, occludin. It is suggested that HGF disrupts TJ function in human breast cancer cells by effecting changes in the expression of TJ molecules at both the mRNA and protein levels. The conclusion was that regulation of TJs could be of fundamental importance in the prevention of metastasis of breast cancer cells (Figure 5).

hours, while isolated primary breast cancers underwent rapid and complete cytolysis within 1 hour. Thus, expression of claudin-3 and claudin-4sensitises breast carcinomas to CPE-mediated cytolysis and emphasises the potential of CPE in breast cancer therapy.

TJ molecule (occludin, claudin-1 and claudin-5, JAM-1 and JAM-2) mRNA transcripts in MDA-MB-231 and MCF-7 cells. Western blotting

were both detected and compared to normal mammary epithelium, were

breast cancer cell lines with CPE resulted in a dose-dependent cytolysis

demonstrated a significant reduction on volume with accompanying

over-expressed in approximately 62% – 26%, respectively. Treatment of

exclusively in cells expressing claudin-3 and claudin-4. In vivo models

lines (127). HGF decreases transepithelial resistance and increases para-cellular permeability of human breast cancer cell lines, MDA-MB-231and MCF-7. Q-PCR shows that HGF modulates the levels of several

99

Figure 5. Effect of HGF on expression of TJ molecules in human breast cancer cells. (A) Reduction of transepithelial resistance by HGF. Changes effected by HGF could be

assessed using Quantitative-PCR. Results are expressed as transcript copy/50 ng/RNA. (C) Western blots of TJ proteins after HGF treatment. (D) HGF and the increased phosphorylation status of ZO-1 in human breast cancer cell lines. This suggests de-activation of ZO-1 by HGF. (E) Immunostaining of human breast cancer cell lines treated with HGF for 1 hour. Cells were stained with ZO-1 or Occludin. MCF-7 cells, but not

hour. HGF reduced staining of both by 1 hour. Both cell lines show increased cytosolic staining and relocation of Occludin and ZO-1 to ruffled membrane areas.

Ye et al. (128) sought to determine the role of oestrogen in the regulation of TJs and expression of molecules making TJs in endothelial cells. Human endothelial cell, HECV, which express ER-beta but not ER-alpha was used. 17beta estradiol induced a concentration- and time-dependent biphasic effect on TJ. At 10(-9) and 10(-6) M, it decreased the level of occludin and increased in paracellular permeability of HECV cells, but at 10(-12) M it decreased in paracellular permeability and increased the level of occludin. The transendothelial electrical resistance (TER), however, was reduced by 17beta estradiol at lower concentra-tions (as low as 10(-12) M). Furthermore, the time-dependent biphasic effect was observed over a period of 4 days, with the first reduction of TER seen within 15 minutes and the second drop occurring 48 hours after 17beta estradiol treatment. It was further revealed that protein and mRNA levels of occludin, but not claudin-1 and -5, and ZO-1, were reduced by 17beta estradiol, in line with changes of TER. This study shows that 17beta estradiol can induce concentration- and time-related biphasic effects on TJ functions expression of occludin in endothelial cells and that this perturbation of TJ functions may have implications in the etiology of mastalgia and the vascular spread of breast cancer.


inhibited by NK4, the HGF antagonist. (B) Modulation of TJ molecules by HGF as

MDA-MB-231 cells showed typical TJ pattern staining for ZO-1 and Occludin at 0

100 Martin

Glucocorticoid hormones stimulate adherens and TJ formation in Con8

the membrane organisation of structural apical junction proteins and TJ sealing is controlled by specific signal transduction components. Dexa-methasone stimulation of apical junction formation requires downregula-tion of the small GTPase RhoA. Rnd3/RhoE, a GTPase-deficient Rho family member and RhoA antagonist was defined as a key regulator of apical junction dynamics. Exogenously expressed Rnd3/RhoE co-localised with actin at the cell periphery and induced the localisation of the adherens junction protein β-catenin and the TJ protein ZO-1 to sites of cell–cell contact, and led to the formation of highly sealed TJs. Treat-ment with glucocorticoids was not required to achieve complete apical junction remodeling. Consistent with Rnd3/RhoE acting as an antagonist of RhoA, expression of Rnd3/RhoE rescued the disruptive effects of constitutively active RhoA on apical junction organisation. Therse results demonstrate a new role for the Rho family member Rnd3/RhoE in regulat-ing the assembly of the apical junction complex and TJ sealing (129).

Transforming growth factor beta (TGF-beta) facilitates metastasis during the advanced stages of cancer. Smad6, Smad7, and c-Ski block signaling by the TGF-beta superfamily proteins through different modes of action. Expression of Smad7 in JygMC(A) cells was associated with increased expression of major components of adherens and TJs, including E-cadherin, decreased expression of N-cadherin, and decreases in the migratory and invasive abilities of the JygMC(A) cells. Smad7 inhibits metastasis, possibly by regulating cell–cell adhesion. Systemic expression of Smad7 may be a novel strategy for the prevention of metastasis of advanced cancers (130).

Claudin-1 and Slug transcripts were observed in breast cancer cell lines. E-box elements in the Claudin-1 promoter were found to play a critical negative regulatory role in breast cancer cell lines that expressed low levels of Claudin-1 transcript. Significantly, in invasive human breast tumours, high levels of Snail and Slug correlated with low levels of

5. PROMISING NEW TARGETS FOR BREAST CANCER DIAGNOSIS AND THERAPY

The work carried out investigating TJs over recent years indicates that this is an area of great interest as targets for cancer diagnosis and

mammary epithelial tumour cells through a multi-step process in which

Snail and Slug bind to the E-box motifs present in the human Claudin-1promoter (131). Moreover, an inverse correlation in the levels of

Claudin-1 expression. Taken together, these results support the hypo-thesis that Claudin-1 is a direct downstream target gene of Snail familyfactors in epithelial cells.

101

Agents that inhibit the effects of cytokines and growth factors such as TNF-α, TGF-β, VEGf, and HGF, all of which are able to decrease tran-

be useful tools in the fight against breast cancer metastasis. It has been shown that the HGF variant NK4 is able to successfully inhibit HGF induced decrease in both epithelial and endothelial cell TJ function (133). Moreover, other less likely substances appear to have profound effects on the inhibition of TJ disruptive elements such as estrogen. Disruption of TJs in endothelial and epithelial cells can lead to leaky vas-cular bed and potentially to oedema and swelling of tissues, the aetiology of mastalgia, and a potential means of escape for tumour cells from the primary tumour. A recent study aimed to determine whether the function of TJs in endothelial cells can be strengthened by gamma linolenic acid (GLA), selenium (Se), and iodine (I) in the presence of 17beta estradiol (17beta estradiol), which causes leakage of endothelial cells by disrupt-tion of TJs in endothelium (134). GLA, I, and Se individually increased transendothelial resistance. The combination of all three agents also had a significant effect. Addition of GLA/Se/I reduced paracellular permeabi-lity of the endothelial cells. Treatment with GLA/Se/I reversed the effect of 17beta estradiol in reducing resistance and increasing permeability. Immunofluorescence revealed that after treatment with Se/I/GLA over 24 hours there was increasing relocation to endothelial cell-cell junctions of the TJs proteins claudin-5, occludin, and ZO-1. Interestingly, this relocation was particularly evident with treatments containing I when probing with claudin-5 and those containing Se for occludin. There was a small increase in overall protein levels after treatment with GLA/Se/I when probed with claudin-5 and occludin. GLA, I, and Se alone, or in combination are able to strengthen the function of TJs in human endo-thelial cells, by way of regulating the distribution of claudin-5, occludin, and ZO-1. Interestingly, this combination was also able to completely reverse the effect of 17beta estradiol in these cells.


that changes in TJ molecule expression, such as occludin, claudin-1,

ZO-1, ZO-2, and MUPP-1. These provide potential prognostic indicators for breast tumours. The claudin family has caused considerable interest as an emerging target for cancer therapy (61); however, it remains to be seen how much of this potential can be translated into real treatments. Interestingly, levels of CAR have been found to be significantly correlated with long-term survival of patients with breast cancer with total CAR levels being elevated in primary breast cancers (132). This may have a

claudin-2, claudin-3, claudin-4, claudin-5, claudin-7, and MAGUK proteins

potential therapeutics. From the work outlined in section 4, it can be seen

sepithelial/transendothelial resistance and increase paracellular perme-ability, as well as promote cell–cell dissociation, invasion, and spread could

bearing on its use as means of delivery for gene therapy.

102 Martin 6. CONCLUSIONS

TJs have been increasingly shown to be deregulated in cancer cells, as a consequence of epigenetic changes, downregulation at mRNA or protein level, aberrant expression (truncated), and the activity of regulatory hormones. Many of these processes begin early in cancer progression

of breast cancer. Studies have demonstrated a correlation between the reduction of TJs

and tumour differentiation, where lower levels of TJs correlated with poorer differentiation of tumours (3). It is evident that increasing data

metastasis. TJs have emerged as the frontline structure that cancer cells must overcome in order to metastasize.

1. Tsukita S, Furuse M. Occludin and claudins in tight-junction strands: leading or supporting players? Trends Cell Biol. 1999; 9(7):268–273.

2. Jiang WG, Martin TA, Matsumoto K, Nakamura T, Mansel RE. Hepatocyte growth factor/scatter factor decreases the expression of occludin and trans-endothelial resistance (TER) and increases paracellular permeability in human vascular endothelial cells. J Cell Physiol. 1999; 181(2):319–329.

3. Jiang WG, Bryce RP, Horrobin DF, Mansel RE. Regulation of tight junction permeability and occludin expression by polyunsaturated fatty acids. Biochem Biophys Res Commun. 1998; 244(2):414–420.

4. Wong V, Gumbiner BM. A synthetic peptide corresponding to the extracellular domain of occludin perturbs the tight junction permeability barrier. J Cell Biol. 1997; 136(2):399–409.

5. Hollande F, Blanc EM, Bali JP, Whitehead RH, Pelegrin A, Baldwin GS, Choquet A. HGF regulates tight junctions in new nontumorigenic gastric epithelial cell line. Am J Physiol Gastrointest Liver Physiol. 2001;280(5):G910–921.

6. Martin TA, Mansel RE, Jiang WG. Antagonistic effect of NK4 on HGF/SF induced changes in the transendothelial resistance (TER) and paracellular per-meability of human vascular endothelial cells. J Cell Physiol. 2002; 192(3): 268–275.

7. Ren J, Hamada J, Takeichi N, Fujikawa S, Kobayashi H. Ultrastructural differences in junctional intercellular communication between highly and weakly metastatic clones derived from rat mammary carcinoma. Cancer Res. 1990; 50(2):358–362.

8. Satoh H, Zhong Y, Isomura H, Saitoh M, Enomoto K, Sawada N, Mori M. Localization of 7H6 tight junction-associated antigen along the cell border of vascular endothelial cells correlates with paracellular barrier function against ions, large molecules, and cancer cells. Exp Cell Res. 1996; 222(2):269–274.

9. Hoevel T, Macek R, Mundigl O, Swisshelm K, Kubbies M. Expression and targeting of the tight junction protein CLDN1 in CLDN1-negative human breast tumor cells. J Cell Physiol. 2002; 191(1):60–68.

show that TJs have a vital role to play in the prevention of cancer

and as such are interesting targets for diagnosis, prognosis, and treatment

REFERENCES

103

11.

12. Hoover KB, Liao SY, Bryant PJ. Loss of the tight junction MAGUK ZO-1 in

13. Chlenski A, Ketels KV, Korovaitseva GI, Talamonti MS, Oyasu R, Scarpelli DG. Organization and expression of the human zo-2 gene (tjp-2) in normal and neoplastic tissues. Biochim Biophys Acta. 2000; 1493(3):319–324.

14. Bell J, Walsh S, Nusrat A, Cohen C. Zonula occludens-1 and Her-2/neu expression in invasive breast carcinoma. Appl Immunohistochem Mol Morphol. 2003; 11(2):125–129.

15. Martin TA, Watkins G, Mansel RE, Jiang WG. Hepatocyte growth factor disrupts tight junctions in human breast cancer cells. Cell Biol Int. 2004 28(5):361–371.

16. Martin TA, Watkins G, Mansel RE, Jiang WG. Loss of tight junction plaque molecules in breast cancer tissues is associated with a poor prognosis in patients with breast cancer. Eur J Cancer. 2004; 40(18):2717–2725.

17. Ikenouchi J, Matsuda M, Furuse M, Tsukita S. Regulation of tight junctions during the epithelium-mesenchyme transition: direct repression of the gene expression of claudins/occludin by Snail. J Cell Sci. 2003; 116(Pt 10):1959–1967.

18. Martin TA, Goyal A, Watkins G, Jiang WG. Expression of the transcription factors snail, slug, and twist and their clinical significance in human breast

19. Ohkubo T, Ozawa M. The transcription factor Snail downregulates the tight junction components independently of E-cadherin downregulation. J Cell Sci. 2004; 117(Pt 9):1675–1685.

20. Gopalakrishnan S, Raman N, Atkinson SJ, Marrs JA. Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion. Am J Physiol. 1998; 275(3 Pt 1):C798–809.

21. Chen Y, Merzdorf C, Paul DL, Goodenough DA. COOH terminus of occludin is required for tight junction barrier function in early Xenopus embryos. J Cell Biol. 1997; 138(4):891–899.

22. Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol. 1998; 141(7):1539–1550.

23. Martin TA, Jiang WG. Tight junctions and their role in cancer metastasis. Histol Histopathol. 2001; 16(4):1183–1195.

24. Fanning AS, Mitic LL, Anderson JM. Transmembrane proteins in the tight junction barrier. J Am Soc Nephrol. 1999; 10(6):1337–1345.

25. Stevenson BR, Siliciano JD, Mooseker MS, Goodenough DA. Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol. 1986; 103(3):755–766.

26. Anderson JM, Stevenson BR, Jesaitis LA, Goodenough DA, Mooseker MS. Characterization of ZO-1, a protein component of the tight junction from mouse

27. Itoh M, Morita K, Tsukita S. Characterization of ZO-2 as a MAGUK family member associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin. J Biol Chem. 1999; 274(9):5981–5986.


correlates with histological grade in both ductal carcinoma in situ and invasive ductal carcinoma of the breast. Oncogene 2003; 22(13):2021–2033.

cancer. Ann Surg Oncol. 2005; 12(6):488–496.

liver and Madin-Darby canine kidney cells. J Cell Biol. 1988; 106(4):1141–1149.

10. Kominsky SL, Argani P, Korz D, Evron E, Raman V, Garrett E, Rein A, Sauter G, Kallioniemi OP, Sukumar S. Loss of the tight junction protein claudin-7

breast cancer: relationship to glandular differentiation and loss of heterozygosity.Am J Pathol. 1998; 153(6):1767–1773.

zation of claudin-1 and its assessment in hereditary and sporadic breast cancer.Hum Genet. 2000; 107(3):249–256.

Kramer F, White K, Kubbies M, Swisshelm K, Weber BH. Genomic organi-

104 Martin

29. Mattagajasingh SN, Huang SC, Hartenstein JS, Benz EJ Jr. Characterization of the interaction between protein 4.1R and ZO-2. A possible link between the tight junction and the actin cytoskeleton. J Biol Chem. 2000; 275(39):30573–30585.

30. Traweger A, Fang D, Liu YC, Stelzhammer W, Krizbai IA, Fresser F, Bauer HC, Bauer H. The tight junction-specific protein occludin is a functional target of the E3 ubiquitin-protein ligase itch. J Biol Chem. 2002; 277(12):10201–10208.

31. Chlenski A, Ketels KV, Tsao MS, Talamonti MS, Anderson MR, Oyasu R, Scarpelli DG. Tight junction protein ZO-2 is differentially expressed in normal pancreatic ducts compared to human pancreatic adenocarcinoma. Int J Cancer. 1999; 82(1):137–144.

32. Hamazaki Y, Itoh M, Sasaki H, Furuse M, Tsukita S. Multi-PDZ domain protein 1 (MUPP1) is concentrated at tight junctions through its possible interaction with claudin-1 and junctional adhesion molecule. J Biol Chem. 2002; 277(1):455–461.

33. Laura RP, Ross S, Koeppen H, Lasky LA. MAGI-1: a widely expressed, alternatively spliced tight junction protein. Exp Cell Res. 2002; 275(2):155–170.

34. Fasano A. Regulation of intercellular tight junctions by zonula occludens toxin and its eukaryotic analogue zonulin. Ann N Y Acad Sci. 2000; 915:214–222.

35. Wang WL, Lu RL, DiPierro M, Fasano A. Zonula occludin toxin, a microtubule binding protein. World J Gastroenterol. 2000; 6(3):330–334.

36. Di Pierro M, Lu R, Uzzau S, Wang W, Margaretten K, Pazzani C, Maimone F, Fasano A. Zonula occludens toxin structure-function analysis. Identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain. J Biol Chem. 2001; 276(22):19160–19165.

37. Mino A, Ohtsuka T, Inoue E, Takai Y. Membrane-associated guanylate kinase with inverted orientation (MAGI)-1/brain angiogenesis inhibitor 1-associated protein (BAP1) as a scaffolding molecule for Rap small G protein GDP/GTP exchange protein at tight junctions. Genes Cells. 2000; 5(12):1009–1016.

38. Dimitratos SD, Woods DF, Bryant PJ. Camguk, Lin-2, and CASK: novel membrane-associated guanylate kinase homologs that also contain CaM kinase domains. Mech Dev. 1997;63(1):127–130.

39. Ohno H, Hirabayashi S, Kansaku A, Yao I, Tajima M, Nishimura W, Ohnishi H, Mashima H, Fujita T, Omata M, Hata Y. Carom: a novel membrane-associated guanylate kinase-interacting protein with two SH3 domains. Oncogene 2003; 22(52):8422–8431.

40. Tabuchi K, Biederer T, Butz S, Sudhof TC. CASK participates in alternative tripartite complexes in which Mint 1 competes for binding with caskin 1, a novel CASK-binding protein. J Neurosci. 2002; 22(11):4264–4273.

41. Biederer T, Sudhof TC. CASK and protein 4.1 support F-actin nucleation on neurexins. J Biol Chem. 2001; 276(51):47869–47876.

42. Hsueh YP, Wang TF, Yang FC, Sheng M. Nuclear translocation and transcription regulation by the membrane-associated guanylate kinase CASK/LIN-2. Nature 2000; 404(6775):298–302.

43. Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S. Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol. 1993; 123(6 Pt 2):1777–1788.

44. Itoh M, Morita K, Tsukita S. Characterization of ZO-2 as a MAGUK family member associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin. J Biol Chem. 1999; 274(9):5981–5986.

28. Balda MS, Matter K. The tight junction protein ZO-1 and an interacting trans-cription factor regulate ErbB-2 expression. EMBO J. 2000; 19(9):2024–2033.

105

48. Sakakibara A, Furuse M, Saitou M, Ando-Akatsuka Y, Tsukita S. Possible involvement of phosphorylation of occludin in tight junction formation. J Cell Biol. 1997; 137(6):1393–1401.

49. Wong V. Phosphorylation of occludin correlates with occludin localization and function at the tight junction. Am J Physiol. 1997; 273(6 Pt 1):C1859–1867.

50. Farshori P, Kachar B. Redistribution and phosphorylation of occludin during opening and resealing of tight junctions in cultured epithelial cells. J Membr Biol. 1999; 170(2):147–156.

51. Wachtel M, Frei K, Ehler E, Fontana A, Winterhalter K, Gloor SM. Occludin proteolysis and increased permeability in endothelial cells through tyrosine phosphatase inhibition. J Cell Sci. 1999; 112( Pt 23):4347–4356.

52. Smales C, Ellis M, Baumber R, Hussain N, Desmond H, Staddon JM. Occludin phosphorylation: identification of an occludin kinase in brain and cell extracts as CK2. FEBS Lett. 2003; 545(2-3):161–166.

53. Jiang WG, Bryce RP, Horrobin DF, Mansel RE. Regulation of tight junction permeability and occludin expression by polyunsaturated fatty acids. Biochem Biophys Res Commun. 1998; 244(2):414–420.

54. Muresan Z, Paul DL, Goodenough DA. Occludin 1B, a variant of the tight junction protein occludin. Mol Biol Cell. 2000; 11(2):627–634.

55. Ghassemifar MR, Sheth B, Papenbrock T, Leese HJ, Houghton FD, Fleming TP. Occludin TM4(-): an isoform of the tight junction protein present in primates lacking the fourth transmembrane domain. J Cell Sci. 2002; 115(Pt 15):3171–3180.

56. Yu AS, McCarthy KM, Francis SA, McCormack JM, Lai J, Rogers RA, Lynch RD, Schneeberger EE. Knockdown of occludin expression leads to diverse phenol-typic alterations in epithelial cells. Am J Physiol Cell Physiol. 2005; 288(6):

57. Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol. 1998; 141(7):1539–1550.

58. Tanaka M, Kamata R, Sakai R. EphA2 phosphorylates the cytoplasmic tail of Claudin-4 and mediates paracellular permeability. J Biol Chem. 2005; 280(51): 42375–42382.

59. D’Souza T, Agarwal R, Morin PJ. Phosphorylation of claudin-3 at threonine 192 by cAMP-dependent protein kinase regulates tight junction barrier function in ovarian cancer cells. J Biol Chem. 2005; 280(28):26233–26240.

60. Katoh M. Epithelial-mesenchymal transition in gastric cancer (Review). Int J Oncol. 2005; 27(6):1677–1683.

61. Kominsky SL. Claudins: emerging targets for cancer therapy. Expert Rev Mol Med. 2006; 8(18):1–11.

62. Wilcox ER, Burton QL, Naz S, Riazuddin S, Smith TN, Ploplis B, Belyantseva I, Ben-Yosef T, Liburd NA, Morell RJ, Kachar B, Wu DK, Griffith AJ, Riazuddin S, Friedman TB. Mutations in the gene encoding tight junction claudin-14 cause autosomal recessive deafness DFNB29. Cell 2001; 104(1):165–172.

63. Simon DB, Lu Y, Choate KA, Velazquez H, Al-Sabban E, Praga M, Casari G, Bettinelli A, Colussi G, Rodriguez-Soriano J, McCredie D, Milford D, Sanjad S, Lifton RP. Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption. Science 1999; 285(5424):103–106.


C1231–1241.

45. Denker BM, Nigam SK. Molecular structure and assembly of the tight junction. Am J Physiol. 1998; 274(1 Pt 2):F19.

46. Matter K, Balda MS. Biogenesis of tight junctions: the C-terminal domain of occludin mediates basolateral targeting. J Cell Sci. 1998; 111 ( Pt 4):511–519.

47. Van Itallie CM, Anderson JM. Occludin confers adhesiveness when expressed in fibroblasts. J Cell Sci. 1997; 110 ( Pt 9):1113–1121.

106 Martin

65. Itoh M, Bissell MJ. The organization of tight junctions in epithelia: implications for mammary gland biology and breast tumorigenesis. J Mammary Gland Biol Neoplasia. 2003; 8(4):449–462.

66. Martin-Padura I, Lostaglio S, Schneemann M, Williams L, Romano M, Fruscella P, Panzeri C, Stoppacciaro A, Ruco L, Villa A, Simmons D, Dejana E. Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration. J Cell Biol. 1998; 142(1):117–127.

67. Mandell KJ, Parkos CA. The JAM family of proteins. Adv Drug Deliv Rev. 2005; 57(6):857–867.

68. Shin K, Fogg VC, Margolis B. Tight junctions and cell polarity. Annu Rev Cell Dev Biol. 2006; 22:207–235.

69. Aurrand-Lions M, Duncan L, Ballestrem C, Imhof BA. JAM-2, a novel immunoglobulin superfamily molecule, expressed by endothelial and lymphatic cells. J Biol Chem. 2001; 276(4):2733–2741.

70. Williams LA, Martin-Padura I, Dejana E, Hogg N, Simmons DL. Identification and characterisation of human Junctional Adhesion Molecule (JAM). Mol Immunol. 1999; 36(17):1175–1188.

71.

structure of junctional adhesion molecule: structural basis for homophilic adhesion via a novel dimerization motif. EMBO J. 2001; 20(16):4391–4398.

72. Mandell KJ, Babbin BA, Nusrat A, Parkos CA. Junctional adhesion molecule 1 regulates epithelial cell morphology through effects on beta1 integrins and Rap1 activity. J Biol Chem. 2005; 280(12):11665–11674.

73. Liang TW, Chiu HH, Gurney A, Sidle A, Tumas DB, Schow P, Foster J, Klassen T, Dennis K, DeMarco RA, Pham T, Frantz G, Fong S. Vascular endothelial-junctional adhesion molecule (VE-JAM)/JAM 2 interacts with T, NK, and dendritic cells through JAM 3. J Immunol. 2002; 168(4):1618–1626.

74. Chavakis T, Preissner KT, Santoso S. Leukocyte trans-endothelial migration: JAMs add new pieces to the puzzle. Thromb Haemost. 2003;89(1):13–17.

75. Bazzoni G, Martinez-Estrada OM, Orsenigo F, Cordenonsi M, Citi S, Dejana E. Interaction of junctional adhesion molecule with the tight junction components ZO-1, cingulin, and occludin. J Biol Chem. 2000; 275(27):20520–20526.

76. Liu Y, Nusrat A, Schnell FJ, Reaves TA, Walsh S, Pochet M, Parkos CA. Human junction adhesion molecule regulates tight junction resealing in

77. Ebnet K, Suzuki A, Ohno S, Vestweber D. Junctional adhesion molecules (JAMs): more molecules with dual functions? J Cell Sci. 2004; 117(Pt 1):19–29.

78. Palmeri D, van Zante A, Huang CC, Hemmerich S, Rosen SD. Vascular endothelial junction-associated molecule, a novel member of the immuno-globulin superfamily, is localized to intercellular boundaries of endothelial cells. J Biol Chem. 2000; 275(25):19139–19145.

79. Arrate MP, Rodriguez JM, Tran TM, Brock TA, Cunningham SA. Cloning of human junctional adhesion molecule 3 (JAM3) and its identification as the JAM2 counter-receptor. J Biol Chem. 2001; 276(49):45826–45832.

80. Cunningham SA, Arrate MP, Rodriguez JM, Bjercke RJ, Vanderslice P, Morris AP, Brock TA. A novel protein with homology to the junctional adhesion molecule. Characterization of leukocyte interactions. J Biol Chem. 2000; 275(44): 34750–34756.

Kostrewa D, Brockhaus M, D’Arcy A, Dale GE, Nelboeck P, Schmid G, Mueller, Bazzoni G, Dejana E, Bartfai T, Winkler FK, Hennig M. X-ray

epithelia. J Cell Sci. 2000; 113( Pt 13):2363–2374.

64. Swisshelm K, Machl A, Planitzer S, Robertson R, Kubbies M, Hosier S. SEMP1, a senescence-associated cDNA isolated from human mammary epithelial cells, is a member of an epithelial membrane protein superfamily. Gene 1999; 226(2):285–295.

107

82. Ebnet K, Schulz CU, Meyer Zu Brickwedde MK, Pendl GG, Vestweber D. Junctional adhesion molecule interacts with the PDZ domain-containing proteins AF-6 and ZO-1. J Biol Chem. 2000; Sep 8;275(36):27979–27988.

83. Hirabayashi S, Tajima M, Yao I, Nishimura W, Mori H, Hata Y. JAM4, a junctional cell adhesion molecule interacting with a tight junction protein,

84. Cohen CJ, Shieh JT, Pickles RJ, Okegawa T, Hsieh JT, Bergelson JM. The coxsackievirus and adenovirus receptor is a transmembrane component of the

85. Li Y, Pong RC, Bergelson JM, Hall MC, Sagalowsky AI, Tseng CP, Wang Z, Hsieh JT. Loss of adenoviral receptor expression in human bladder cancer cells: a potential impact on the efficacy of gene therapy. Cancer Res. 1999; 59(2): 325–330.

86. Okegawa T, Li Y, Pong RC, Bergelson JM, Zhou J, Hsieh JT. The dual impact of coxsackie and adenovirus receptor expression on human prostate cancer gene therapy. Cancer Res. 2000; 60(18):5031–5036.

87. Rauen KA, Sudilovsky D, Le JL, Chew KL, Hann B, Weinberg V, Schmitt LD, McCormick F. Expression of the coxsackie adenovirus receptor in normal prostate and in primary and metastatic prostate carcinoma: potential relevance to gene therapy. Cancer Res. 2002; 62(13):3812–3818.

88. Rein DT, Breidenbach M, Curiel DT. Current developments in adenovirus–based cancer gene therapy. Future Oncol. 2006; 2(1):137–143.

89. Hemminki A, Zinn KR, Liu B, Chaudhuri TR, Desmond RA, Rogers BE, Barnes MN, Alvarez RD, Curiel DT. In vivo molecular chemotherapy and noninvasive imaging with an infectivity-enhanced adenovirus. J Natl Cancer Inst. 2002; 94(10):741–749

90. Kanerva A, Wang M, Bauerschmitz GJ, Lam JT, Desmond RA, Bhoola SM, Barnes MN, Alvarez RD, Siegal GP, Curiel DT, Hemminki A. Gene transfer to ovarian cancer versus normal tissues with fiber-modified adenoviruses. Mol Ther. 2002;5(6):695–704.

91. Miller CR, Buchsbaum DJ, Reynolds PN, Douglas JT, Gillespie GY, Mayo MS, Raben D, Curiel DT. Differential susceptibility of primary and established human glioma cells to adenovirus infection: targeting via the epidermal growth factor receptor achieves fiber receptor-independent gene transfer. Cancer Res. 1998;58(24):5738–5748.

92. Shayakhmetov DM, Li ZY, Ni S, Lieber A. Analysis of adenovirus sequestration in the liver, transduction of hepatic cells, and innate toxicity after injection of fiber-modified vectors. J Virol. 2004; 78(10):5368–5381.

93. Dmitriev I, Krasnykh V, Miller CR, Wang M, Kashentseva E, Mikheeva G, Belousova N, Curiel DT. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adeno-virus receptor-independent cell entry mechanism. J Virol. 1998; 72(12):9706–9713.

94. Kasono K, Blackwell JL, Douglas JT, Dmitriev I, Strong TV, Reynolds P, Kropf DA, Carroll WR, Peters GE, Bucy RP, Curiel DT, Krasnykh V.Selective gene delivery to head and neck cancer cells via an integrin targeted adenoviral vector. Clin Cancer Res. 1999; 5(9):2571–2579.

95. Jee YS, Lee SG, Lee JC, Kim MJ, Lee JJ, Kim DY, Park SW, Sung MW, Heo DS. Reduced expression of coxsackievirus and adenovirus receptor (CAR) in tumor tissue compared to normal epithelium in head and neck squamous cell carcinoma patients. Anticancer Res. 2002; 22(5):2629–2634.


MAGI-1. Mol Cell Biol. 2003; 23(12):4267–4282.

tight junction. Proc Natl Acad Sci USA 2001; 98(26):15191–15196.

81. Itoh M, Sasaki H, Furuse M, Ozaki H, Kita T, Tsukita S. Junctional adhesion molecule (JAM) binds to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight junctions. J Cell Biol. 2001; 154(3):491–497.

108 Martin

97. Sachs MD, Rauen KA, Ramamurthy M, Dodson JL, De Marzo AM, Putzi MJ, Schoenberg MP, Rodriguez R. Integrin alpha(v) and coxsackie adenovirus receptor expression in clinical bladder cancer. Urology 2002; 60(3):531–536.

98. Bruning A, Runnebaum IB. CAR is a cell-cell adhesion protein in human cancer cells and is expressionally modulated by dexamethasone, TNFalpha, and TGFbeta. Gene Ther. 2003; 10(3):198–205.

99. Li D, Mrsny RJ. Oncogenic Raf-1 disrupts epithelial tight junctions via downregulation of occludin. J Cell Biol. 2000; 148(4):791–800.

100. Anders M, Christian C, McMahon M, McCormick F, Korn WM. Inhibition of the Raf/MEK/ERK pathway up-regulates expression of the coxsackievirus and adenovirus receptor in cancer cells. Cancer Res. 2003; 63(9):2088–2095.

101. Yamada A, Irie K, Fukuhara A, Ooshio T, Takai Y. Requirement of the actin cytoskeleton for the association of nectins with other cell adhesion molecules at

102. Reymond N, Borg JP, Lecocq E, Adelaide J, Campadelli-Fiume G, Dubreuil P, Lopez M. Human nectin3/PRR3: a novel member of the PVR/PRR/nectin family that interacts with afadin. Gene 2000; 255(2):347–355.

103. Inagaki M, Irie K, Deguchi-Tawarada M, Ikeda W, Ohtsuka T, Takeuchi M, Takai Y. Nectin-dependent localization of ZO-1 at puncta adhaerentia junctions between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of adult mouse hippocampus. J Comp Neurol. 2003; 460(4):514–524.

104. Fabre S, Reymond N, Cocchi F, Menotti L, Dubreuil P, Campadelli-Fiume G, Lopez M. Prominent role of the Ig-like V domain in trans-interactions of nectins.

V domain. J Biol Chem. 2002; 277(30):27006–27013. 105. Yasumi M, Shimizu K, Honda T, Takeuchi M, Takai Y. Role of each immuno-

globulin-like loop of nectin for its cell-cell adhesion activity. Biochem Biophys Res Commun. 2003; 302(1):61–66.

106. Aijaz S, Balda MS, Matter K. Tight junctions: molecular architecture and function. Int Rev Cytol. 2006; 248:261–298.

107. Morin PJ. Claudin proteins in human cancer: promising new targets for diagnosis

108. Kohler K, Zahraoui A. Tight junction: a co-ordinator of cell signalling and membrane trafficking. Biol Cell. 2005; 97(8):659–665.

109. Van Itallie CM, Anderson JM. The molecular physiology of tight junction pores. Physiology (Bethesda). 2004; 19:331–338.

110. Schneeberger EE, Lynch RD. The tight junction: a multifunctional complex.

111. Chlenski A, Ketels KV, Tsao MS, Talamonti MS, Anderson MR, Oyasu R, Scarpelli DG. Tight junction protein ZO-2 is differentially expressed in normal pancreatic ducts compared to human pancreatic adenocarcinoma. Int J Cancer. 1999; 82(1):137–144.

112. Latorre IJ, Roh MH, Frese KK, Weiss RS, Margolis B, Javier RT.Viral oncoprotein-induced mislocalization of select PDZ proteins disrupts tight junctions and causes polarity defects in epithelial cells. J Cell Sci. 2005; 118(Pt 18):4283–4293.

113. Tobioka H, Sawada N, Zhong Y, Mori M. Enhanced paracellular barrier function of rat mesothelial cells partially protects against cancer cell penetration. Br J Cancer. 1996; 74(3):439–445.

114. Utoguchi N, Mizuguchi H, Dantakean A, Makimoto H, Wakai Y, Tsutsumi Y, Nakagawa S, Mayumi T. Effect of tumour cell-conditioned medium on

adherens and tight junctions in MDCK cells. Genes Cells 2004; 9(9):843–855.

Nectin3 and nectin 4 bind to the predicted C-C’-C”-D beta-strands of the nectin1

and therapy. Cancer Res. 2005; 65(21):9603–9606.

Am J Physiol Cell Physiol. 2004; 286(6):C1213–1228.

96. Okegawa T, Pong RC, Li Y, Bergelson JM, Sagalowsky AI, Hsieh JT. The mechanism of the growth-inhibitory effect of coxsackie and adenovirus receptor (CAR) on human bladder cancer: a functional analysis of car protein structure. Cancer Res. 2001; 61(17):6592–6600.

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116. Soini Y. Expression of claudins 1, 2, 3, 4, 5 and 7 in various types of tumours. Histopathology 2005; 46(5):551–560.

117. Martin TA, Watkins G, Mansel RE, Jiang WG. Reduction of levels of paracellin-1 and viculin aare associated with poor clincoal outcome in patients with breast cancer. Proceedings of American Association for Cancer Research, 2003; 44:511.

118. Martin TA, Jiang WG. Occludin is aberrantly expressed in human breast cancer.

119. Sommers CL, Byers SW, Thompson EW, Torri JA, Gelmann EP. Differen-tiation state and invasiveness of human breast cancer cell lines. Breast Cancer Res Treat. 1994; 31(2-3):325–335.

120. Martin TA, Watkins G, Mansel RE, Jiang WG. Loss of tight junction plaque molecules in breast cancer tissues is associated with a poor prognosis in patients with breast cancer. Eur J Cancer. 2004; 40(18):2717–2725.

121. Buse P, Woo PL, Alexander DB, Reza A, Firestone GL. Glucocorticoid-induced functional polarity of growth factor responsiveness regulates tight junction dynamics in transformed mammary epithelial tumor cells. J Biol Chem. 1995; 270(47):28223–28227.

122. Guan Y, Woo PL, Rubenstein NM, Firestone GL. Transforming growth factor-alpha abrogates the glucocorticoid stimulation of tight junction formation and reverses the steroid-induced down-regulation of fascin in rat mammary epithelial tumor cells by a Ras-dependent pathway. Exp Cell Res. 2002; 273(1):1–11.

123. Mauro L, Bartucci M, Morelli C, Ando S, Surmacz E. IGF-I receptor-induced cell-cell adhesion of MCF-7 breast cancer cells requires the expression of

124. Macek R, Swisshelm K, Kubbies M. Expression and function of tight junction associated molecules in human breast tumor cells is not affected by the Ras-MEK1 pathway. Cell Mol Biol (Noisy-le-grand). 2003; 49(1):1–11.

125. Kominsky SL, Vali M, Korz D, Gabig TG, Weitzman SA, Argani P, Sukumar S. Clostridium perfringens enterotoxin elicits rapid and specific cytolysis of breast carcinoma cells mediated through tight junction proteins claudin 3 and 4. Am J Pathol. 2004; 164(5):1627–1633.

126. Ebihara C, Kondoh M, Hasuike N, Harada M, Mizuguchi H, Horiguchi Y, Fujii M, Watanabe Y. Preparation of a claudin-targeting molecule using a C-terminal fragment of Clostridium perfringens enterotoxin. J Pharmacol Exp Ther. 2006; 316(1):255–260.

127. Martin TA, Watkins G, Mansel RE, Jiang WG. Hepatocyte growth factor disrupts tight junctions in human breast cancer cells. Cell Biol Int. 2004; 28(5):361–371.

128. Ye L, Martin TA, Parr C, Harrison GM, Mansel RE, Jiang WG. Biphasic effects of 17-beta-estradiol on expression of occludin and transendothelial resistance and paracellular permeability in human vascular endothelial cells. J Cell Physiol. 2003; 196(2):362–369.

129. Rubenstein NM, Chan JF, Kim JY, Hansen SH, Firestone GL. Rnd3/RhoE induces tight junction formation in mammary epithelial tumor cells. Exp Cell Res. 2005; 305(1):74–82.


and malignant breast lesions: a research study. Breast Cancer Res. 2005; 7(2):R296–305.

Breast Cancer Res Treat. 2005; 94(1):2096.

junction protein ZO-1. J Biol Chem. 2001; 276(43):39892–39897.

endothelial macromolecular permeability and its correlation with collagen. Br J Cancer. 1996; 73(1):24–28.

115. Tokes AM, Kulka J, Paku S, Szik A, Paska C, Novak PK, Szilak L, Kiss A, Bogi K, Schaff Z. Claudin-1, -3 and -4 proteins and mRNA expression in benign

110 Martin

131. Martinez-Estrada OM, Culleres A, Soriano FX, Peinado H, Bolos V, Martinez FO, Reina M, Cano A, Fabre M, Vilaro S. The transcription factors Slug and Snail act as repressors of Claudin-1 expression in epithelial cells. Biochem J. 2006; 394(Pt 2):449–457.

132. Martin TA, Watkins G, Jiang WG. The Coxsackie-adenovirus receptor has

133. Jiang WG, Martin TA, Parr C, Davies G, Matsumoto K, Nakamura T. Hepatocyte growth factor, its receptor, and their potential value in cancer therapies. Crit Rev Oncol Hematol. 2005; 53(1):35–69.

134. Martin TA, Das T, Mansel RE, Jiang WG. Synergistic regulation of endothelial tight junctions by antioxidant (Se) and polyunsaturated lipid (GLA) via Claudin-5 modulation. J Cell Biochem. 2006; 98(5):1308.

elevated expression in human breast cancer. Clin Exp Med. 2005; 5(3):122–128.

130. Azuma H, Ehata S, Miyazaki H, Watabe T, Maruyama O, Imamura T, Sakamoto T, Kiyama S, Kiyama Y, Ubai T, Inamoto T, Takahara S, Itoh Y, Otsuki Y, Katsuoka Y, Miyazono K, Horie S. Effect of Smad7 expression on metastasis of mouse mammary carcinoma JygMC(A) cells. J Natl Cancer Inst. 2005; 97(23):1734–1746.

Chapter 6

CELL ADHESION MOLECULES IN BREAST CANCER INVASION AND METASTASIS

Lalita A. Shevde1,2 and Judy A. King2,3,4 1Mitchell Cancer Institute, 2Department of Pathology, 3Department of Pharmacology, Center for Lung Biology, College of Medicine, University of South Alabama, Mobile,

AL 36688-0002, USA

Abstract: Metastasis occurs through a series of sequential steps, all of which a metastatic cell must successfully complete in order to establish growth at the secondary site. Cell adhesion molecules including the cadherins, immunoglobulin superfamily, selectins, and integrins play important roles in tumor metastasis. Mucins can also be involved in tumor cell adhesion. In this chapter we review the current knowledge of these groups of cell adhesion molecules in breast cancer.

Keywords: adhesion, cadherin, selectin, immunoglobulin, integrin, metastasis

Metastasis occurs through a series of sequential steps, all of which a metastatic cell must successfully complete in order to establish growth at the secondary site. Upon establishment of a blood supply to support its metabolic needs, the new blood vessels provide an escape route for the tumor cells to enter directly into the vasculature (intravasation). The tumor cells eventually end up in blood circulation via the lymphatics as well. The tumor cells need to survive in the circulation until they arrest in a new organ and extravasate. Multiple fates await tumor cells once they land at secondary sites: (a) they may be destroyed by immune or non-immune defenses; (b) they may lie dormant in the tissue for years, reactivating later with appropriate stimuli (immune suppression); or (c) they may proliferate either intravascularly or in tissues following extravasation (1). Among the many changes in gene expression and protein function that occur during tumor progression, alterations in cell-cell and cell-matrix adhesion seem to have a central role in facilitating

© 2007 Springer.

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tumor cell migration, invasion, and metastatic dissemination (2, 3). At


112 Shevde and King

tumor cell acquires a profile that is distinct from the primary tumor. In

molecules in breast cancer and discuss their potential applications in the management of breast cancer.

tumor cells change from a highly differentiated, epithelial morphology to

1. CELL ADHESION MOLECULES

2. CADHERINS

More than 80 cadherins have now been described. The name “cadherin” comes from “calcium (Ca)-dependent adhesion.” Cadherins are often invol-ved in homotypic adhesion between the same cell type, and are not involved in the attachment of cells to the extracellular matrix. Cadherins are located in adherens junctions and desmosomes, and intercellular adhe-sion depends on the interaction of the cadherins with the cytoskeleton (7).

Breast E-cadherin (epithelial cadherin), the prototype member of the cadherin family of calcium-dependent cell–cell adhesion molecules, is expressed in normal adults in luminal epithelial cells (8), and is lost

2.1 E-cadherin

every step of the process of tumor transformation and progression, the profile of the surface cell adhesion molecules changes and ultimately the

a migratory and invasive mesenchymal phenotype (4). During this process of epithelial–mesenchymal transition (EMT), cells progressively redistri-bute or downregulate their apical and basolateral epithelial-specific tight

The most apparent morphological change that occurs during the

re–express mesenchymal molecules (including vimentin and N-cadherin)

transition from benign tumor to a malignant and metastatic one is that

(4–6). These changes lead to the loss of cell–cell contacts and the gain

this review we summarize the involvement of various cell adhesion

of cell motility; changes that are necessary for invasion.

characteristics and include the following: cadherins, immunoglobulin-cell

include mucins (7). Each of these families of adhesion proteins has dis-

Adhesion molecules are divided into four major groups based upon their

tinctive characteristics. In this chapter we will examine the current know-

adhesion molecules, selectins, and integrins (7). Other adhesion molecules

ledge of these cell adhesion molecules and their roles in breast cancer.

and adherens junction proteins (including E-cadherin and cytokeratins) and

concomitantly with tumor progression in breast cancers (9–14). This is due to irreversible and reversible mechanisms and is related to the

6. Adhesion molecules in invasion and metastasis 113

carcinoma in situ frequently lacks E-cadherin (19). Thus, inactivation of E-cadherin expression may play an important role in the development and progression of these cancers. This results from loss of heterozygosity (LOH) at 16q22.1, involving the E-cadherin gene CDH1 (approximately

silencing of the remaining CDH1 allele (14, 20–27). The E-cadherin promoter is frequently repressed by specific transcriptional repressors, including Snail, Slug, SIP1, δEF1, Twist, and E12/E47 (28–34). The zinc

promoter and repress transcription (31, 35, 36). E-cadherin can also be

phorylation of E-cadherin and catenins, resulting in their ubiquitylation

(37–39). Finally, secreted proteases, such as MMPs can cleave E-cadherin

level can be reduced and its cellular localization abnormal, that is, not restricted to sites of cell–cell interaction (11). Both E-cadherin positive

50%) (11), frequently in combination with mutation (50%) or epigenetic

histological subtype. E-cadherin expression is irreversibly lost in >85% of invasive lobular breast cancers (8, 14–18). Loss of E-cadherin appears to be an early event in these tumors, since even noninvasive lobular

The status of the estrogen receptor (ER) can also have regulatory effects

by the E3 lipase Hakai, and subsequent endocytosis and degradation

metastasis-associated protein, MTA3, which plays a role in chromatin re-

1R, FGF receptors (FGFRs), and the non-RTK c-Src can induce phos-

complex normally represses Snail, which in turn represses E-cadherin.

on E-cadherin (41). Absence of the ER results in decreased levels of a

downregulated at the protein level. RTKs, such as EGFR, c-Met, IGF-

finger transcription factors bind to three E-box elements in the CDH1

correlates with ER negativity, supporting this as one possible mechanism

and disrupt cadherin-mediated cell–cell contacts (40).

ErbB2 and TGF-β negatively regulate E-cadherin expression (11).

and E-cadherin-negative metastatic lesions have been reported. In general, while E-cadherin expression correlates inversely with histological grade,

modeling as part of a larger repressive complex, Mi-2/NuRD (42). This

and thus differentiation, its expression is not well correlated with survival.

for E-cadherin loss in some breast cancers. Lastly, growth factors including

In some studies reduced E-cadherin correlates with shorter metastasis-free

In contrast to lobular breast cancers, ductal carcinomas, which represent

periods and poor prognosis in node negative patients, while other reports

increase and subsequent repression of E-cadherin (42). Loss of E-cadherin

indicate that heterogeneous staining of the tumor for E-cadherin is a poorindicator. In contrast, other studies suggested that E-cadherin presence

the predominant form of breast cancer, express E-cadherin. However, the

was actually a marker of poor survival. Clearly, evaluating E-cadherin

Loss of estrogen signaling reverses the repression of Snail, resulting in its

expression alone in breast cancers is more useful for distinguishinglobular from ductal carcinomas than predicting clinical outcome.

114 Shevde and King

What are the tumor-invasion-promoting signals elicited by the loss of E-cadherin function?

An alternative mechanism for inactivating the adhesive function of E-cadherin in tumor cells is to disrupt the connection between cadherin and the cytoskeleton (43). The catenins tether E-cadherin to the actin cytoskeleton (9). Catenins α-, β-plakoglobin, and p120 form a complex with E-cadherin in normal mammary epithelial cells. In general, the expression and cellular localization of catenins in breast cancers appear to correspond to the presence or loss of E-cadherin. In the absence of a cadherin for them to bind to, α-, β-catenins, and plakoglobin, but not p120, are degraded in most cells (44–48). Consistent with this observation, in lobular carcinoma, which is E-cadherin-negative, β-catenins are typically reduced or absent (49). On the other hand, p120 is present in the cytoplasm and nucleus, consistent with its stability in the absence of E-cadherin. In ductal carcinoma, which is E-cadherin-positive, p120 is mostly at the plasma membrane, presumably bound to E-cadherin (49, 50). Abnormal cytosolic localization of α-catenin has been correlated with high histologic grade, advanced stage, and poor survival in the case of ductal carcinomas. In addition, abnormal β-catenin staining has been correlated with advanced stage and lymph node metastasis. In general, alterations in catenin expression or localization are correlated with invasive breast cancers. The absence or presence of E-cadherin may affect the levels of β-catenin and therefore potentially its signaling activity; however, there are no compelling data to confirm a primary role for the Wnt signaling pathway in human breast cancers (43, 51). No activating mutations for β-catenin or other members of the Wnt signaling pathway have been reported. The loss of β-catenin with E-cadherin downregulation may indicate that degradation of β-catenin, and thus regulation of its signaling activity, is very efficient in mammary epithelial cells, perhaps indicating the importance of tightly regulating the Wnt pathway in the mammary gland. However, in a mouse model, stabilized β-catenin and increased β-catenin/TCF signaling induces mammary carcinomas, so it remains possible that this pathway plays a role in some human breast cancers (11, 12, 44, 52).

First, E-cadherin loss disrupts adhesion junctions between neighboring cells and thereby supports detachment of malignant cells from the epithelial-cell layer. Second, loss of E-cadherin has direct effects on signaling path-ways involved in tumor-cell migration and tumor growth, including the canonical Wnt signaling pathway and Rho family GTPase-mediated modu-lation of the actin cytoskeleton (9, 10, 23, 51–55). However, as part of EMT, the loss of E-cadherin is frequently contrasted by the gain of expression of mesenchymal cadherins, such as N-cadherin, which enhance tumor-cell

the gain of N-cadherin (a.k.a. the cadherin switch) may make a critical motility and migration (56). Hence, in addition to the loss of E-cadherin,


Normal epithelial cells express E-cadherin. However, tumor cells that have undergone an EMT begin to inappropriately express N-cadherin (neural cadherin). Expression of N-cadherin in mammary tumor cell lines leads to increased cell migration and invasion, regardless of E-cadherin expression (56, 57, 62). It has also been suggested that N-cadherin pro-motes breast cancer metastasis by reestablishing homophilic cell–cell adhesion in metastasis (57). In highly invasive breast tumors, N-cadherin was shown to replace E-cadherin at cell–cell contacts, and it has been proposed that N-cadherin mediates carcinoma cell interaction with mammary stromal and endothelial cells. Moreover, intravenous injection of MCF7 cells engineered to overexpress N-cadherin into nude mice results in increased metastasis, compared to parent MCF7 cells lacking N-cadherin. N-cadherin expression influences downstream signaling from the FGFR. Suyama et al., 2002, have implicated a direct interaction between N-cadherin and FGFR, resulting in receptor stabilization and prolonged signaling by FGF (59). N-cadherin-expressing breast carc-inoma cells were specifically sensitized to FGF-2-induced invasion and upregulation of the proteolytic enzyme MMP-9. However, the findings are consistent with the observation that, although breast carcinoma cells expressing N-cadherin are more motile and invasive (57, 58) and many human breast cancers express N-cadherin, its presence does not correlate with poor survival (12, 63, 64). It is possible that additional events besides N-cadherin misexpression, such as overexpression of FGF or its receptor, decrease in E-cadherin expression, or increased levels of metal-loproteinases, are required to act in concert with N-cadherin to promote mammary tumor cell invasion and metastasis in vivo (11).

While P-cadherin (placental cadherin) is expressed in the myo-epithelium in the normal, nonlactating mammary gland, many ductal (but not lobular) carcinomas, express P-cadherin, even though they are thought to be of epithelial origin. In ductal carcinoma in situ as well, a high grade is associated with increased P-cadherin expression. P-cadherin expression correlates with increased tumor aggressiveness, high pro-liferation rate, and histologic grade, absence of ER/PR, high c-ErbB-2,

2.2 N-cadherin

2.3 P-cadherin

contribution to tumor invasion and metastatic dissemination, not only by changing the adhesive repertoire of a tumor cell, but also by modulating various signaling pathways and transcriptional responses (56–61).

and poor prognosis (65). The causal role of P-cadherin in aggressive tumor

116 Shevde and King

role of desmosomes in breast cancer metastasis is enigmatic. The loss of desmoplakin in breast cancers correlates with amplified proliferation and increased tumor size, suggesting that desmosomal proteins might be important in suppressing breast cancer progression. Desmoplakin levels are generally lower in metastases compared to primary tumors (67). Downregulation of DSC3 in breast cancer was first reported by Klus (68). Desmocollin 3 (DSC3), a p53 responsive gene, is expressed in normal breast while its expression is downregulated in both primary breast tumors and breast tumor cell lines (69). Decreased expression of DSC3 is partly due to cytosine hypermethylation and histone deacetyla-tion (70). Therefore, the loss of DSC3 expression in the cell lines appears to be due to both epigenetic and genetic changes. Hence, loss of desmo-somes might play a role in progression of tumor cells from the well to poorly differentiated phenotype. Clearly, the role of desmosomes in breast cancer is an area that needs more attention.

VE-cadherin (vascular endothelial cadherin) is localized at inter-endothelial cell adherens junctions and has an important role in maintaining endothelial permeability (71). It gets rapidly redistributed upon interaction with breast cancer cells, possibly due to the increase in tyrosine phosphorylation of members of the VE-cadherin/catenin adhesion complex. This, in turn, may increase vascular endothelial permeability and facilitate the transendothelial migration of tumor cells during extravasation (72–77). Previous studies support a role of VE-cadherin in angiogenesis and tumor growth when there is active vessel growth (78). Antibodies directed toward VE-cadherin inhibit angiogenesis and modulate endothelial permeability (78–80). This is complemented by a study showing that dominant-negative mutants of VE-cadherin inhibit endothelial growth (81). Recent results demonstrated an enhanced expres-sion of VE-cadherin as disease progresses suggesting a role for VE-cadherin in angiogenesis as opposed to vasculogenesis (82). Moreover, in patients with a poor prognosis determined by high Nottingham Prognostic Index, tumor samples stained intensely for VE-cadherin (83).

2.5 VE-cadherin

Desmosomes are multifaceted intracellular junctions that participate in cell adhesion and maintenance of normal tissue structure. Desmosomes connect epithelial cells, myoepithelial cells, and the two cell types to each other. Despite the strong adhesion that desmosomes provide, the

2.4 Desmosomes in breast cancer

cell behavior is yet to be determined since transgenic mice overexpressing P-cadherin do not develop spontaneous mammary tumors (66).


3.1 ALCAM

ALCAM (activated leukocyte cell adhesion molecule, CD166, human melanoma metastasis clone D [MEMD], HB2) is a glycoprotein of the immunoglobulin superfamily that is involved in both homotypic/homo-philic adhesion and heterotypic/heterophilic (to CD6) adhesion (85, 86). In a study of 120 primary breast carcinomas, levels, of ALCAM RNA transcripts (by real–time PCR) were analyzed in relation to clinical data from a 6-year follow-up period (87). Decreased levels of ALCAM cor-related with nodal involvement, higher grade, higher TNM (tumor, node, metastasis) stage, worse NPI (Nottingham Prognostic Index), and clinical outcome (local recurrence and death due to breast cancer). Burkhardt et al. (88) performed an immunohistochemical study of 162 primary breast carcinomas and correlated the staining pattern with the clinical findings (Figure 1). There was a mean follow-up period of 53 months. Both intraductal and invasive breast carcinomas had higher ALCAM expression than normal breast. High cytoplasmic ALCAM expression was associated with shortened patient disease-free survival.

Jezierska et al. (89) used laser scanning cytometry and confocal micro-scopy to evaluate 56 breast cancer specimens. The results were correlated to clinical and pathologic data from the cases. High levels of ALCAM correlated with small tumor diameter, low tumor grade, presence of pro-gesterone receptor, and presence of estrogen receptors. Lower levels of ALCAM were associated with HER2/neu gene amplification (but the numbers were not statistically significant). Small tumors and those with low tumor grade had higher ALCAM/MMP-2 ratios. In a separate report

between breast cancer cells is important for survival in the primary tumor. Loss of ALCAM is associated with programmed cell death, both apoptosis and autophagy.

3.2 VCAM-1

VCAM-1 (vascular cell adhesion molecule-1) is involved in hetero-typic adhesion. VCAM-1 is increased in the tumor cytosol and sera of

the same research group (90) found that ALCAM–ALCAM interactions

3. IMMUNOGLOBULIN SUPERFAMILY OF CELL ADHESION MOLECULES

Members of the immunoglobulin superfamily of cell adhesion mole-cules (Ig-CAM) have 1–7 extracellular immunoglobulin-like domains. They are attached to the plasma membrane by a single, hydrophobic transmembrane sequence and have a cytoplasmic tail (7). They can be involved in both homotypic and heterotypic adhesion (84).

118 Shevde and King

tories) (1A and 1B) and N-cadherin (1:80 dilution; Calbiochem-Novabiochem Corp.) (1C and 1D). Normal breast ducts and acini exhibit staining for ALCAM (1A) in a membranous and cytoplasmic distribution, and staining for N-cadherin (1C) in a cytoplasmic distribution with some nuclear staining. Invasive breast carcinoma exhibits strong staining for ALCAM (1B) in a membranous and cytoplasmic distribution, and less intense staining for N-cadherin (1D) in a cytoplasmic distribution.

3.3 ICAM-1

ICAM-1 (intercellular adhesion molecule-1) is involved in heterotypic adhesion. Rosette et al. (94) studied five breast cancer cell lines and found that ICAM-1 expression on the cell surface positively correlated with metastatic potential. Breast tumors had increased ICAM-1 mRNA levels

Immunohistochemical staining for ALCAM (1:40 dilution; Novocastra Labora-Figure 1.

patients with breast cancer (91). Prognostic value could not be established, however (91). In a study of 92 patients with breast cancer and 31 age-

that serum levels of VCAM-1 were elevated in patients with Stage 4 disease compared with controls. In addition, elevated serum levels of VCAM-1 in patients with Stage 2 disease were predictive of decreased survival, even when corrected for T and N status. In an immuno-

(downregulation) is an independent predictor of nodal metastasis.

matched controls with benign breast disease O’Hanlon et al. (92) found

histochemical study, Madhavan et al. (93) found that VCAM-1 level

compared to normal tissue (94). Lynch et al. (95) found that ICAM-1


3.4 CEACAM1

CEACAM1 (biliary glycoprotein, BGP, CD66a, cell-CAM, C-CAM-1) is a cell adhesion glycoprotein that belongs to the carcinoembryonic antigen (CEA) family and the immunoglobulin superfamily (97). Human BGP has four isoforms (98), but only three of the four isoforms are present in normal and malignant breast (99). BGP is involved in homophilic and heterophilic binding and requires calcium for adhesion (100). BGP is expressed in normal, premalignant, and malignant breast (99, 101), but the subcellular localization is different (99, 101). BGP is expressed on the apical surface of normal ductal and lobular epithelial cells, and is located in the cytoplasm or uniformly over the entire membrane in invasive carcinoma (99, 101). C-CAM-1 has been found to suppress breast cancer tumors (growth suppression) (102). BGP is downregulated at the mRNA and protein levels in 30% of breast cancers (99). Using immuno-

breast carcinomas there was downregulation or loss of BGP expression. Using immunohistochemistry Riethdorf et al. (101) found that 80% of ductal carcinomas and 68% of lobular carcinomas had staining for BGP. There was no relationship between the expression of BGP in the invasive breast carcinomas and grade, age, tumor size, menopausal status, or hormone receptor status (101). Well- differentiated invasive ductal carcinomas (including papillary carcinoma and tubular carcinoma) had strong apical membrane staining for BGP rather than uniform membrane staining as seen in the majority of other carcinomas (101).

3.5 NCAM

NCAM (neural cell adhesion molecule, CD56, Leu19, NKH1) belongs to the immunoglobulin superfamily, is involved in homotypic and hetero-

histochemistry Riethdorf et al. (101) showed that in a portion of invasive

serum levels were increased in patients with breast carcinoma. ICAM-1 is increased in the tumor cytosol and sera of patients with breast cancer, however, prognostic value could not be established (91). An immuno-histochemical study of 274 patients with invasive breast carcinoma revealed that ICAM-1 was expressed in 50.3% of cases (96). ICAM-1 expression was negatively correlated to tumor size, lymph node meta-stasis, tumor infiltration, nuclear pleomorphism, and nuclear grade (96). There was improved relapse-free and overall survival in patients with ICAM-1 positive tumors (96). The findings in that study suggested a

ICAM is downregulated in node positive breast cancer (compared to node negative cases).

tumor suppressor role for ICAM-1 (96). Madhavan et al. (93) found that

typic adhesion (103, 104), and is expressed by neural, neuroendocrine,

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3.7 HepaCAM

HepaCAM is a cell adhesion molecule that is a member of the immunoglobulin superfamily (112). It was originally identified in liver. Moh et al. transfected HepaCAM into MCF-7 breast cancer cells (112). In nonconfluent cells hepaCAM was located in cell protrusions; in confluent cells it was located at cell-cell borders; and in polarized cells it was localized to the lateral and basal membranes. HepaCAM is thought to be important in cell–matrix interaction and cell motility (112).

3.8 PECAM

PECAM (platelet endothelial cell adhesion molecule, PECAM-1, CD31, endocam) is an adhesion molecule that is expressed by endo-thelial cells, leukocytes, and platelets and is involved in homotypic and heterotypic interactions (113). PECAM is expressed in the endothelium of normal breast (100%) and tumor-associated vessels (100%) (114), and antibodies to PECAM have been used to study the vascularization of tumors. CD31 is not seen in normal or hyperplastic breast epithelium (115). A few studies have found that PECAM can be expressed in

Mel-CAM negative breast cancer cells produced a cohesive cell growth pattern and inhibition of tumor growth (109) (compared to mock transfectants). Therefore, Mel-CAM is considered to be a tumor suppressorin breast cancer (111).

NCAM is localized to the cell membrane in breast cancer, but can sometimes be cytoplasmic (107). Breast cancer cells (MDA-MB-231) transfected with NCAM produced tumors with slower growth rates (longer latency) than NCAM negative cells (108).

3.6 Mel-CAM

Mel-CAM (CD146, MUC18, MCAM, A32 antigen, S-Endo-1) is a member of the immunoglobulin superfamily and is involved in Ca2+-independent heterophilic adhesion (109–111). Using a new mouse mono-clonal antibody (MN-4) Shih et al. (110) found positive immuno-histochemical staining in normal epithelial and myoepithelial cells of the breast, and in 2 of 11 infiltrating breast carcinomas. In a different study by the same group Mel-CAM was identified in 100% of normal breast and benign proliferating breast epithelium, but only seen (focally) in 17% of breast cancers (109). Transfection of Mel-CAM cDNA into

and some biphasic tumors including breast phyllodes tumors (105, 106).

neoplastic breast epithelium. Fox et al. (114) performed an immuno-histochemical study of 64 invasive breast carcinomas and found one case


E-selectin, also known as endothelial leukocyte adhesion molecule-1 (ELAM-1) is present on endothelial cells adjacent to tumor cells (118, 119). E-selectin is involved in mediating the adhesion of breast carcinoma cells to the endothelium and inhibition reduces adherence (120, 121). The highest levels were observed in patients with hepatic metastases and previous studies have suggested that E-selectin expression is a risk factor for the development of metastases (122, 123). Breast cancer patients have high circulating levels of E-selectin (in serum) (124, 125). Breast cancer cell lines induce the expression of E-selectin on vascular endothelium. E-selectin has been found to enhance ICAM-1 expression in human tumor cell lines and a positive correlation between ICAM and E-selectin has also been reported in breast cancers (92, 93).

P-selectin (CD62P) is a member of the selectin family of cell adhesion molecules. It is a presynthesized protein stored in the Weibel-Palade bodies of endothelial cells and the α-granules of platelets. Upon inflam-matory and thrombogenic challenges, it translocates from these cellular granules to the cell surfaces of endothelial cells and platelets by exo-cytosis in seconds (116, 117). Furthermore, it can be upregulated by

4.1 E-selectin

4.2 P-selectin

where the malignant epithelial cells expressed PECAM. Sapino et al. (115) studied 32 cases of high nuclear grade ductal carcinoma in situ (>/= 2 cm) and found CD31 positivity in 11 cases. In that study the associated poorly differentiated invasive ductal carcinomas were CD31 and CD44 positive. The authors suggested that CD31 expression correlates with tumor cell spreading within the ductal system (including

4. SELECTINS

The selectins are a group of cell surface lectins that mediate the adhesion between leukocytes, platelets, and endothelial cells under blood flow (116, 117). Selectin mediated adhesion ensures that leukocytes roll in the direction of flow, which is a prerequisite for recruitment of leukocytes to areas of injury and inflammation. The selectin family is small, consisting of three closely related proteins - L-selectin, E-selectin, and P-selectin—expressed by both platelets and vascular endothelium.

Paget’s cells at the nipple) (115).

interacts with P-selectin glycoprotein ligand-1 (PSGL-1) (CD162), a de novo synthesis in the stimulated endothelial cells in hours. P-selectin

122 Shevde and King

5. INTEGRINS

Integrins are ubiquitously transmembrane glycoproteins expressed as

subunits, α and β, which form distinct integrin subtypes. Their extracellular

homodimeric mucin-like protein expressed on a majority of leukocytes (126). Sulfation of P-selectin is essential for binding to ZR-75-30 cells, a cell line derived from a human breast carcinoma (127). P-selectin has been shown to bind to several human cancers and human cancer-derived cell lines, including breast cancer (128, 129). Increased levels of P-selectin are found in the sera of breast cancer patients, although its implications are unclear (130).

While there are no studies that report a role for L-selectin in the metastasis of breast cancer, in colon cancer, L-selectin serves as a mole-cular link between recruitment of inflammatory leukocytes to the sites of tumor cell emboli in microvasculature and their potential to facilitate metastasis (131). Cancer metastasis is also known to be impaired in L-selectin-deficient mice (132). In melanoma, L-selectin and ICAM-1 contri-bute cooperatively to the antitumor reaction by regulating lymphocyte infiltration to the tumor (133, 134). In another melanoma model of cancer L-selectin–mediated NK cell recruitment plays a crucial role in the control of tumor metastasis into secondary lymphoid organs (135).

4.3 L-selectin

Importantly, binding to these ECM components activates integrins, which, in turn, induce intracellular signaling cascades that modulate cell proliferation, survival, polarity, motility, and differentiation (140). There

Integrins are pivotal in controlling cell attachment, cell migration, cell cycle progression, and apoptosis (136–138). It has been reported that integrinsfunction in signaling by two mechanisms, ‘‘inside out’’ signaling and

domains link extracellular matrix (ECM) ligands, such as fibronectin,

connect directly or indirectly via linker proteins to the actin cytoskeleton.

alters the adhesive state of its integrin receptors, allowing it to bind to other

vitronectin, laminin, and collagen, whereas their intracellular domains

proteins that can modulate the integrin activity state. Outside in signaling

‘‘outside in’’ signaling. Inside out signaling is the process by which a cell

transmits signals from the ECM after integrin ligation, which can influence

heterodimeric cell-surface receptors that consist of two transmembrane

vital cellular processes, such as gene transcription (139).

are 18 α and 8 β subunits, which dimerize to yield at least 24 molecular permutations, each with distinct ligand-binding and signaling properties. The β1, β3, β4, β5, and β6 integrins have all been identified in breast cancer, where their expression may influence the metastatic process (141).


depend on changes not only in cell–cell, but also in cell–matrix, inter-action. The loss of α2β1 integrins by oncogenes is evident as changes in the tissue integrity, implicating this integrin in the initiation of metastatic spread of breast cancer (164). Additionally the loss of integrins, α2β1, α3β1, α6β1, αvβ1, and αvβ5 show reduced expression in breast cancers correlated to their positive lymph node involvement (165, 166).

Malignant human mammary epithelial cells no longer depend on liga-tion and activation of β1 integrins for survival in culture (143, 167, 168). In fact, as the nontransformed cells in this series progress toward malig-nancy, they gradually lose their dependency upon β1 integrin for survival (168). Concomitant with the loss of β1 integrin dependency for survival is a dramatic increase in the expression and activity of EGFR (2) which is essential to maintain the malignant phenotype of the tumor cells and repress their anchorage independence for growth and survival.

Recent reports document the implication of the loss of β4 signaling on mammary tumor onset and invasive growth (169). The β4 integrin comp-lexes with ErbB2 and enhances activation of the transcription factors STAT3 and c-Jun. While STAT3 contributes to disruption of epithelial adhesion and polarity, c-Jun enhances proliferation. Finally, deletion of

Integrins act to promote the growth, and retard the death, of both normal and tumorigenic cells. While the α2β1 integrin (collagen/laminin receptor) is abundantly expressed on the epithelium of ductules of normal breast tissues (142–146), its expression is lost concomitant with the loss of estrogen receptor on poorly differentiated breast adeno-carcinoma cells (141). The fibronectin receptor, α5β1 and the vitronectin receptor, αvβ3 also show similar changes in these mammary tumors (147, 148). Highly differentiated adenocarcinoma cells show intermediate expression levels of these integrins. The expression of αvβ3 is directly associated with the ability of cells in culture to adhere and migrate, correlating with the metastatic potential (149–153). Various breast cancer cell lines also show expression of αvβ5 and αvβ1 integrins (154). The expression of α6β1 integrin gives the expressing breast cancer cells a survival advantage and also regulates metastatic potential (155–158). The α6β1 integrin may also contribute to tumor cell growth by inhibiting erbB2 signals by inducing proteasome-dependent proteolytic cleavage of the erbB2 cytoplasmic domain (159). Tumor-specific αvβ3 contributes to spontaneous metastasis of breast tumors to bone suggesting a critical role for this receptor in mediating chemotactic and haptotactic migration towards bone factors (160–163). To detach and migrate, tumor cells

the β4 signaling domain enhances the efficacy of ErbB2-targeted therapy (170). Thus β4 integrin promotes tumor progression by amplifying ErbB2 signaling. Additionally, ErbB2-mediated transcriptional upregulation of the α5β1 integrin fibronectin receptor promotes mammary adenocarci-noma cell survival under adverse conditions such as hypoxia and serum

124 Shevde and King

6. OTHER CELL ADHESION MOLECULES

EpCAM (epithelial cell adhesion molecule, EGP40, GA733-2, ESA, KSA, 17-1A antigen) is a 40kD glycoprotein on human epithelium that is involved in Ca++-independent homophilic intercellular adhesion and is not likely involved in cell-substrate adhesion (177). EpCAM is not thought to be related to the major families of cell adhesion molecules (cadherins, integrins, selectins, and the immunoglobulin superfamily) (178). It is localized to the lateral domain of polarized epithelial cells, but cells in suspension exhibit it on the entire cell surface (177). EpCAM is overexpressed 100- to 1,000-fold in primary and metastatic breast cancer (179). In a study of 205 patients Gastl et al. (180) found that 35.6% of invasive breast cancers had overexpression of Ep-CAM by immuno-histochemical staining, and there was an association with poor disease-free and overall survival (independent of tumor size, nodal status, histo-logical grade, and hormone receptor expression). A more recent study of

6.1 EpCAM

withdrawal (171). In serum-depleted breast cancer cells, integrin α6β4 upregulates ErbB2 through translational control followed by phos-phorylation of EGFR and activation of Ras, substantiating the role of α6β4 in carcinoma invasion (172). Furthermore, the α6β4 integrin pro-motes tumor formation by regulating tumor cell survival in a VEGF-dependent manner (169). Moreover, α6β4 integrin-mediated activation of PI-3K-Akt is amplified by integrin-stimulated VEGF expression providing yet another mechanism for α6β4 in carcinoma progression (173). The E2F family of transcription factors promote H-ras mediated invasion by upregulating the expression of the β4 integrin, culminating in an enhanced α6β4-dependent invasion (174). In MDA-MB-435 breast carci-

expression of the autocrine motility factor autotoxin which enhances chemotaxis (175). Also, adhesion independent clustering of α6β4 integrin, known to be important in mediating tumor cell motility, is driven by phosphatidylinositol 3-kinase (PI3K) but does not require activation of the PI3K-Akt pathway (176). These data collectively demonstrate a role of the integrins and their altered expression in predisposing breast cancer cells to metastasize.

noma cells, the α6β4 integrins leads to increased NFAT1-dependent

1715 patients (181) showed high levels of Ep-CAM (by immuno-histochemical staining) in 41.7% of invasive breast carcinomas. In that study the expression of Ep-CAM was predictive of poor overall survival, but was not an independent prognostic marker. Ep-CAM was a marker of poor prognosis in node-positive invasive breast carcinoma (181).


7. SUMMARY AND PERSPECTIVES

Women in the USA and most of the Western world have a 12% lifetime risk of developing breast cancer, which rivals lung cancer in being the most common cause of cancer-related deaths. Approximately 25% of women diagnosed with breast cancer die of the disease. There is a need for better prognostic markers for accurately predicting clinical outcome. As summarized above, adhesion molecules regulate several mechanisms that control tumor cell survival, proliferation, migration, inva-sion, and the ability to survive in various microenvironments. Therapeutics targeting various cellular invasive and migratory activities might be useful in treating pathologies that are associated with these cell phenotypes, such as metastasis and angiogenesis. Over the past several years, research has led to the development of integrin and protease inhibitors that are now being tested in clinical trials. As the underlying mechanisms and relevant key molecules become progressively identified, there are pos-

functional upregulation of E-cadherin in breast cancers. Further research sibilities to develop antitumor and antiinvasion strategies aimed at

Silencing EpCAM using short interfering RNA (siRNA) resulted in decreased breast cancer cell proliferation (35%–80%), decreased cell migration (91.8%), and decreased cell invasion (96.4%) (179).

6.2 MUC1

MUC1 (CA 15-3, episialin, epithelial membrane antigen, human milk fat globule membrane antigen) is a membrane-bound epithelial mucin/ glycoprotein found on the apical aspect of normal breast duct cells (182, 183). Its ligand is ICAM-1 (184, 185). In breast carcinoma MUC1 is overexpressed, aberrantly glycosylated (183), and present over the entire surface (186). Nearly 90% of breast cancers express MUC1 (187). It has been proposed that the overexpression by breast carcinoma protects the cells from immune response and prevents cell adhesion (blocks E-cadherin) (183, 188–191). The glycoprotein in cancer, however, is abnormally glycosylated so it acts as a self-antigen and an immune response occurs (183). MUC1 expression correlates to adhesion and invasion of MDA-MB-231 cells (192). Serum MUC1 is a prognostic marker for breast cancer independent of tumor size and nodal status (193).

on other possible factors that affect the N-cadherin switch, on the

perspective of N-cadherin as a potential marker of invasion, is needed. In the future, we expect the generation and testing of various known, and as yet unknown, molecules that interfere with EMT. Continuing research

signaling pathways initiated in N-cadherin-mediated invasion and on the

126 Shevde and King

10. Cowin P, Rowlands TM, and Hatsell SJ. Cadherins and catenins in breast cancer. Curr Opin Cell Biol, 2005; 17: 499–508.

11. Knudsen KA. and Wheelock MJ. Cadherins and the mammary gland. J Cell Biochem, 2005; 95: 488–496.

12. Knudsen KA. and Soler AP. Cadherin-mediated cell–cell interactions. Methods Mol Biol, 2000; 137: 409–440.

13. Wheelock MJ. and Knudsen KA. Cadherins and associated proteins. In Vivo, 1991; 5: 505–513.

14. Vos CB, Cleton-Jansen AM, Berx G, de Leeuw W J, ter Haar NT, van Roy F, Cornelisse CJ, Peterse JL, and van de Vijver M J. E-cadherin inactivation in lobular carcinoma in situ of the breast: an early event in tumorigenesis. Br J Cancer, 1997; 76: 1131–1133.

15. Gamallo C, Palacios J, Suarez A, Pizarro A, Navarro P, Quintanilla M, and Cano A. Correlation of E-cadherin expression with differentiation grade and histological type in breast carcinoma. Am J Pathol, 1993; 142: 987–993.

16. Moll R, Mitze M, Frixen UH, and Birchmeier W. Differential loss of E-cadherin expression in infiltrating ductal and lobular breast carcinomas. Am J Pathol, 1993; 143: 1731–1742.

REFERENCES

1. Chambers AF, Groom AC, and MacDonald IC. Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer, 2002; 2: 563–572.

2. Chrenek MA, Wong P, and Weaver VM. Tumour-stromal interactions. Integrins and cell adhesions as modulators of mammary cell survival and transformation. Breast Cancer Res, 2001; 3: 224–229.

3. Nguyen TH. Mechanisms of metastasis. Clin Dermatol, 22: 209–216, 2004. 4. Christofori G. New signals from the invasive front. Nature, 2006; 441: 444–450. 5. Christofori G. and Semb, H. The role of the cell-adhesion molecule E-cadherin

as a tumour-suppressor gene. Trends Biochem Sci, 1999; 24: 73–76. 6. Cavallaro U, Schaffhauser B, and Christofori G. Cadherins and the tumour

progression: is it all in a switch? Cancer Lett, 2002; 176: 123–128. 7. Pollard T D. and Earnshaw W C. Cellular Adhesion. In: Cell Biology, Phila-

8. Rasbridge SA, Gillett CE, Sampson, S. A, Walsh, F. S, and Millis, R. R. Epithelial (E-) and placental (P-) cadherin cell adhesion molecule expression in breast carcinoma. J Pathol, 1993; 169: 245–250.

9. Berx G and Van Roy F. The E-cadherin/catenin complex: an important gate-keeper in breast cancer tumorigenesis and malignant progression. Breast Cancer Res, 2001; 3: 289–293.

delphia: Saunders, 2002; pp.507–524.

into the pathways that are intrinsic to the invasive and migratory phenotype of metastatic breast cancer holds promise for the development of new, more effective cancer therapeutics.

18. Droufakou S, Deshmane V, Roylance R, Hanby A, Tomlinson I, and Hart IR. Multiple ways of silencing E-cadherin gene expression in lobular carcinoma of the breast. Int J Cancer, 2001; 92: 404–408.

19. Frixen UH, Behrens J, Sachs M, Eberle G, Voss B, Warda A, Lochner D, and Birchmeier W. E-cadherin-mediated cell-cell adhesion prevents invasiveness of human carcinoma cells. J Cell Biol, 1991; 113: 173–185.

17. Huiping C, Sigurgeirsdottir JR, Jonasson JG, Eiriksdottir G, Johannsdottir JT, Egilsson V, and Ingvarsson S. Chromosome alterations and E-cadherin gene mutations in human lobular breast cancer. Br J Cancer, 1999; 81: 1103–1110.

20. Mielnicki LM, Asch HL, and Asch BB. Genes, chromatin, and breast cancer: an epigenetic tale. J Mammary Gland Biol Neoplasia, 2001; 6: 169–182.

21. Berx G, Cleton-Jansen AM, Strumane K, de Leeuw WJ, Nollet F, van Roy F, and Cornelisse C. E-cadherin is inactivated in a majority of invasive human lobular breast cancers by truncation mutations throughout its extracellular domain. Oncogene, 1996; 13: 1919–1925.

22. Berx G, Becker KF, Hofler H, and van Roy F. Mutations of the human E-cadherin (CDH1) gene. Hum Mutat, 1998; 12: 226–237.

23. Berx G, Nollet F, and van Roy F. Dysregulation of the E-cadherin/catenin complex by irreversible mutations in human carcinomas. Cell Adhes Commun, 1998; 6: 171–184.

24. Cleton-Jansen AM. E-cadherin and loss of heterozygosity at chromosome 16 in breast carcinogenesis: different genetic pathways in ductal and lobular breast cancer? Breast Cancer Res, 2002; 4: 5–8.

25. Cleton-Jansen AM, Timmerman MC, van de Vijver MJ, van Asperen CJ, Kroon HM, Eilers PH, and Hogendoorn PC. A distinct phenotype characterizes tumors from a putative genetic trait involving chondrosarcoma and breast cancer occurring in the same patient. Lab Invest, 2004; 84: 191–202.

26. Lombaerts M, van Wezel T, Philippo K, Dierssen JW, Zimmerman RM, Oosting J, van Eijk R, Eilers PH, van de Water B, Cornelisse CJ, and Cleton-Jansen AM. E-cadherin transcriptional downregulation by promoter methylation but not mutation is related to epithelial-to-mesenchymal transition in breast cancer cell lines. Br J Cancer, 2006; 94: 661–671.

27. Berx G, Cleton-Jansen AM, Nollet F, de Leeuw WJ, van de Vijver M, Cornelisse C, and van Roy F. E-cadherin is a tumour/invasion suppressor gene mutated in human lobular breast cancers. Embo J, 1995; 14: 6107–6115.

28. Vandewalle C, Comijn J, De Craene B, Vermassen P, Bruyneel E, Andersen H, Tulchinsky E, Van Roy F, and Berx G. SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell-cell junctions. Nucleic Acids Res, 2005; 33: 6566–6578.

29. De Craene B, Gilbert B, Stove C, Bruyneel E, van Roy F, and Berx G. The transcription factor snail induces tumor cell invasion through modulation of the epithelial cell differentiation program. Cancer Res, 2005; 65: 6237–6244.

30. Kang Y. and Massague J. Epithelial-mesenchymal transitions: twist in develop-ment and metastasis. Cell, 2004; 118: 277–279.

31. Bolos V, Peinado H, Perez-Moreno MA, Fraga MF, Esteller M, and Cano A. The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors. J Cell Sci, 2003; 116: 499–511.

32. Peinado H, Marin F, Cubillo E, Stark HJ, Fusenig N, Nieto MA, and Cano A. Snail and E47 repressors of E-cadherin induce distinct invasive and angiogenic properties in vivo. J Cell Sci, 2004; 117: 2827–2839.

33. Peinado H, Ballestar E, Esteller M, and Cano A. Snail mediates E-cadherin repression by the recruitment of the Sin3A/histone deacetylase 1 (HDAC1)/ HDAC2 complex. Mol Cell Biol,2004; 24: 306–319.

34. Strathdee G. Epigenetic versus genetic alterations in the inactivation of E-cadherin. Semin Cancer Biol, 2002; 12: 373–379.

35. Batlle E, Sancho E, Franci C, Dominguez D, Monfar M, Baulida J, and Garcia De Herreros A. The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol, 2000; 2: 84–89.

36. Perez-Moreno MA, Locascio A, Rodrigo I, Dhondt G, Portillo F, Nieto MA, and Cano A. A new role for E12/E47 in the repression of E-cadherin expression and epithelial-mesenchymal transitions. J Biol Chem, 2001; 276: 27424–27431.


37. Fujita Y, Krause G, Scheffner M, Zechner D, Leddy HE, Behrens J, Sommer T, and Birchmeier W. Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex. Nat Cell Biol, 2002; 4: 222–231.

38. Hogan C, Serpente N, Cogram P, Hosking CR, Bialucha CU, Feller SM, Braga V. M, Birchmeier W, and Fujita Y. Rap1 regulates the formation of E-cadherin-based cell-cell contacts. Mol Cell Biol, 2004; 24: 6690–6700.

39. Fujita Y, Hogan C, and Braga VM. Regulation of cell-cell adhesion by rap1. Methods Enzymol, 2005; 407: 359–372.

40. Munshi HG and Stack MS. Reciprocal interactions between adhesion receptor signaling and MMP regulation. Cancer Metastasis Rev, 2006; 25: 45–56.

41. crucial regulators of structure and function in reproductive tissues. Rev Reprod, 2000; 5: 53–61.

42. Fujita N, Jaye DL, Kajita M, Geigerman C, Moreno CS, and Wade PA. MTA3, a Mi-2/NuRD complex subunit, regulates an invasive growth pathway in breast cancer. Cell, 2003; 113: 207–219.

50. Dillon D. A, D’Aquila T, Reynolds AB, Fearon ER, and Rimm DL. The expression of p120ctn protein in breast cancer is independent of alpha- and beta-catenin and E-cadherin. Am J Pathol, 1998; 152: 75–82.

51. Conacci-Sorrell M, Zhurinsky J, and Ben-Ze’ev A. The cadherin-catenin adhesion system in signaling and cancer. J Clin Invest, 2002; 109: 987–991.

52. Nelson WJ. and Nusse R. Convergence of Wnt, beta-catenin, and cadherin pathways. Science, 2004; 303: 1483–1487.

43. mammary development and cancer. J Mammary Gland Biol Neoplasia, 2003; 8: 145–158.

44. Anastasiadis PZ. and Reynolds AB. The p120 catenin family: complex roles in adhesion, signaling and cancer. J Cell Sci, 2000; 113 ( Pt 8): 1319–1334.

45.

46. Davis MA, Ireton RC, and Reynolds AB. A core function for p120-catenin in cadherin turnover. J Cell Biol, 2003; 163: 525–534.

47. Reynolds AB and Roczniak-Ferguson A. Emerging roles for p120-catenin in cell adhesion and cancer. Oncogene, 2004; 23: 7947–7956.

48. Kowalczyk AP and Reynolds AB. Protecting your tail: regulation of cadherin degradation by p120-catenin. Curr Opin Cell Biol, 2004; 16: 522–527.

49. Sarrio D, Perez-MiesB, HardissonD, Moreno-BuenoG, SuarezA, CanoA, Martin-Perez J, Gamallo C, and Palacios J. Cytoplasmic localization of p120ctn and E-cadherin loss characterize lobular breast carcinoma from preinvasive to metastatic lesions. Oncogene, 2004; 23: 3272–3283.

53. Pecina-Slaus N. Tumor suppressor gene E-cadherin and its role in normal and malignant cells. Cancer Cell Int, 2003; 3: 17

54. De Leeuw WJ, Berx G, Vos CB, Peterse JL, Van de Vijver MJ, Litvinov S, Van Roy F, Cornelisse CJ, and Cleton-Jansen AM. Simultaneous loss of E-cadherin and catenins in invasive lobular breast cancer and lobular carcinoma in situ. J Pathol, 1997; 183: 404–411.

55. Nollet F, Berx G, and van Roy F. The role of the E-cadherin/catenin adhesion complex in the development and progression of cancer. Mol Cell Biol Res Commun, 1999; 2: 77–85.

56. Nieman MT, Prudoff RS, Johnson KR, and Wheelock MJ. N-cadherin promotes motility in human breast cancer cells regardless of their E-cadherin expression. J Cell Biol, 1999; 147: 631–644.

57. Hazan RB, Kang L, Whooley BP, and Borgen PI. N-cadherin promotes adhesion between invasive breast cancer cells and the stroma. Cell Adhes Commun, 1997; 4: 399–411.

Hatsell S, Rowlands T, Hiremath M, and Cowin P. Beta-catenin and Tcfs in

Thoreson MA and Reynolds AB. Altered expression of the catenin p120 in human cancer: implications for tumor progression. Differentiation, 2002; 70: 583–589.

Rowlands TM, Symonds JM, Farookhi R, and Blaschuk OW. Cadherins:

128 Shevde and King

58. Hazan RB, Phillips GR, Qiao RF, Norton L, and Aaronson SA. Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis. J Cell Biol, 2000; 148: 779–790.

59. Suyama K, Shapiro I, Guttman M, and Hazan RB. A signaling pathway leading to metastasis is controlled by N-cadherin and the FGF receptor. Cancer Cell, 2002; 2: 301–314.

60. Nagi C, Guttman M, Jaffer S, Qiao R, Keren R, Triana A, Li M, Godbold J, Bleiweiss IJ, and Hazan RB. N-cadherin expression in breast cancer:

61. Pishvaian MJ, Feltes CM, Thompson P, Bussemakers MJ, Schalken JA, and Byers SW. Cadherin-11 is expressed in invasive breast cancer cell lines. Cancer Res, 1999; 59: 947–952.

carcinoma. Breast Cancer Res Treat, 2005; 94: 225–235. correlation with an aggressive histologic variant–invasive micropapillary

69. Oshiro MM, Watts GS, Wozniak RJ, Junk DJ, Munoz-Rodriguez JL, Domann FE, and Futscher B. W. Mutant p53 and aberrant cytosine methylation cooperate to silence gene expression. Oncogene, 2003; 22: 3624–3634.

70. Oshiro MM, Kim CJ, Wozniak RJ, Junk DJ, Munoz-Rodriguez JL, Burr JA, Fitzgerald M, Pawar SC, Cress AE, Domann FE, and Futscher BW. Epigenetic silencing of DSC3 is a common event in human breast cancer. Breast Cancer Res, 2005; 7: R669–680.

62. Cavallaro U. N-cadherin as an invasion promoter: a novel target for antitumor

63. Knudsen KA, Lin CY, Johnson KR, Wheelock MJ, Keshgegian AA, and Soler AP. Lack of correlation between serum levels of E- and P-cadherin fragments and the presence of breast cancer. Hum Pathol, 2000; 31: 961–965.

64. Wheelock MJ, Soler AP, and Knudsen KA. Cadherin junctions in mammary tumors. J Mammary Gland Biol Neoplasia, 2001; 6: 275–285.

65. Peralta Soler A, Knudsen KA, Salazar H, Han AC, and Keshgegian AA. P-cadherin expression in breast carcinoma indicates poor survival. Cancer, 1999; 86: 1263–1272.

66. Radice GL, Sauer CL, Kostetskii I, Peralta Soler A, and Knudsen KA.

67. Davies EL, Gee JM, Cochrane RA, Jiang WG, Sharma AK, Nicholson RI, and Mansel RE. The immunohistochemical expression of desmoplakin and its role in vivo in the progression and metastasis of breast cancer. Eur J Cancer, 1999; 35: 902–907.

68. Klus GT, Rokaeus N, Bittner ML, Chen Y, Korz DM, Sukumar S, Schick A, and Szallasi Z. Down-regulation of the desmosomal cadherin desmocollin 3 in human breast cancer. Int J Oncol, 2001; 19: 169–174.

Inappropriate P-cadherin expression in the mouse mammary epithelium is compatible

therapy? Curr Opin Investig Drugs, 2004; 5: 1274–1278.

with normal mammary gland function. Differentiation, 2003; 71: 361–373.

71. Navarro P, Ruco L, and Dejana E. Differential localization of VE- and

N-cadherins in human endothelial cells: VE-cadherin competes with N-cadherin for junctional localization. J Cell Biol, 1998; 140: 1475–1484.

72. Lampugnani MG, Corada M, Caveda L, Breviario F, Ayalon O, Geiger B, and Dejana E. The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, beta-catenin, and alpha-catenin with vascular endothelial cadherin (VE-cadherin). J Cell Biol, 1995; 129: 203–217.

73. Navarro P, Caveda L, Breviario F, Mandoteanu I, Lampugnani M, and Dejana E. Catenin-dependent and -independent functions of vascular endothelial cadherin. J Biol Chem, 1995; 270: 30965–30972.

74. Lampugnani MG, Caveda L, Breviario F, Del Maschio A, and Dejana E. Endothelial cell-to-cell junctions. Structural characteristics and functional role in the regulation of vascular permeability and leukocyte extravasation. Baillieres Clin Haematol, 1993; 6: 539–558.


75. Esser S, Lampugnani MG, Corada M, Dejana E, and Risau W. Vascular endothelial growth factor induces VE-cadherin tyrosine phosphorylation in endothelial cells. J Cell Sci, 1998; 111 ( Pt 13): 1853–1865.

76. Dejana E, Bazzoni G, and Lampugnani MG. The role of endothelial cell-to-cell junctions in vascular morphogenesis. Thromb Haemost, 1999; 82: 755–761.

77. Dejana E, Lampugnani MG, Martinez-Estrada O, and Bazzoni G. The molecular organization of endothelial junctions and their functional role in vascular morphogenesis and permeability. Int J Dev Biol, 2000; 44: 743–748.

78. Corada M, Zanetta L, Orsenigo F, Breviario F, Lampugnani MG, Bernasconi S, Liao F, Hicklin DJ, Bohlen P, and Dejana E. A monoclonal antibody to vascular endothelial-cadherin inhibits tumor angiogenesis without side effects on endothelial permeability. Blood, 2002; 100: 905–911.

85. Swart GW. Activated leukocyte cell adhesion molecule (CD166/ALCAM): developmental and mechanistic aspects of cell clustering and cell migration. Eur J Cell Biol, 2002; 81: 313–321.

79. Corada M, Liao F, Lindgren M, Lampugnani MG, Breviario F, Frank R, Muller W. A, Hicklin D. J, Bohlen P, and Dejana E. Monoclonal antibodies directed to different regions of vascular endothelial cadherin extracellular domain affect adhesion and clustering of the protein and modulate endothelial

80. Liao F, Li Y, O’Connor W, Zanetta L, Bassi R, Santiago A, Overholser J, Hooper, A, Mignatti P, Dejana E, Hicklin D. J, and Bohlen P. Monoclonal antibody to vascular endothelial-cadherin is a potent inhibitor of angiogenesis, tumor growth, and metastasis. Cancer Res, 2000; 60: 6805–6810.

81. Venkiteswaran K, Xiao K, Summers S, Calkins CC, Vincent PA, Pumiglia K, and Kowalczyk AP. Regulation of endothelial barrier function and growth by VE-cadherin, plakoglobin, and beta-catenin. Am J Physiol Cell Physiol, 2002; 283: C811–821.

82. Parker BS, Argani P, Cook BP, Liangfeng H, Chartrand SD, Zhang M, Saha S, Bardelli A, Jiang Y, St Martin TB, Nacht M, Teicher BA, Klinger KW, Sukumar S, and Madden SL. Alterations in vascular gene expression in invasive breast carcinoma. Cancer Res, 2004; 64: 7857–7866.

83. Martin T A, Watkins G, Lane J, and Jiang WG. Assessing microvessels and angiogenesis in human breast cancer, using VE-cadherin. Histopathology, 2005; 46: 422–430.

84. Epstein RJ. Adhesion molecules and the extracellular matrix. In: Human

permeability. Blood, 2001; 97: 1679–1684.

Molecular Biology, Cambridge, UK: Cambridge University Press, 2003; 209–234.

86. van Kempen LC, Nelissen JM, Degen WG, Torensma R, Weidle UH,

Bloemers H. P, Figdor CG, and Swart GW. Molecular basis for the homophilic activated leukocyte cell adhesion molecule (ALCAM)-ALCAM interaction. J Biol Chem, 2001; 276: 25783–25790.

87. King JA, Ofori-Acquah SF, Stevens T, Al-Mehdi AB, Fodstad O, and Jiang WG. Activated leukocyte cell adhesion molecule in breast cancer: prognostic indicator. Breast Cancer Res, 2006; 6: R478–487.

88. Burkhardt M, Mayordomo E, Winzer KJ, Fritzsche F, Gansukh T, Pahl S, Weichert W, Denkert C, Guski H, Dietel M, and Kristiansen G. Cytoplasmic overexpression of ALCAM is prognostic of disease progression in breast cancer. J Clin Pathol, 2006; 59: 403–409.

89. Jezierska A, Olszewski WP, Pietruszkiewicz J, Olszewski W, Matysiak W, and Motyl T. Activated Leukocyte Cell Adhesion Molecule (ALCAM) is associated with suppression of breast cancer cells invasion. Med Sci Monit, 2006; 12: BR245–256.

90. Jezierska A, Matysiak W, and Motyl T. ALCAM/CD166 protects breast cancer cells against apoptosis and autophagy. Med Sci Monit, 2006; 12: BR263–273.

130 Shevde and King

91. Regidor PA, Callies R, Regidor M, and Schindler AE. Expression of the cell adhesion molecules ICAM-1 and VCAM-1 in the cytosol of breast cancer tissue, benign breast tissue and corresponding sera. Eur J Gynaecol Oncol, 1998; 19: 377–383.

92. Soluble adhesion molecules (E-selectin, ICAM-1 and VCAM-1) in breast carcinoma. Eur J Cancer, 2002; 38: 2252–2257.

93. Madhavan M, Srinivas P, Abraham E, Ahmed I, Vijayalekshmi NR, and Balaram P. Down regulation of endothelial adhesion molecules in node positive breast cancer: possible failure of host defence mechanism. Pathol Oncol Res, 2002; 8: 125–128.

O’Hanlon DM, Fitzsimons H, Lynch J, Tormey S, Malone C, and Given HF.

94. Rosette C, Roth RB, Oeth P, Braun A, Kammerer S, Ekblom J, and Denissenko M. F. Role of ICAM1 in invasion of human breast cancer cells. Carcinogenesis, 2005; 26: 943–950.

95. Lynch DF, Jr, Hassen W, Clements MA, Schellhammer PF, and Wright GL, Jr. Serum levels of endothelial and neural cell adhesion molecules in prostate cancer. Prostate, 1997; 32: 214–220.

96. Ogawa Y, Hirakawa K, Nakata B, Fujihara T, Sawada T, Kato Y, Yoshikawa K, and Sowa M. Expression of intercellular adhesion molecule-1 in invasive breast cancer reflects low growth potential, negative lymph node involvement, and good prognosis. Clin Cancer Res, 1998; 4: 31–36.

97. Thompson JA, Grunert F, and Zimmermann W. Carcinoembryonic antigen gene family: molecular biology and clinical perspectives. J Clin Lab Anal, 1991; 5: 344–366.

98. Barnett TR, Drake L, and Pickle W. 2nd Human biliary glycoprotein gene: characterization of a family of novel alternatively spliced RNAs and their expressed proteins. Mol Cell Biol, 1993; 13: 1273–1282.

99. Huang J, Simpson JF, Glackin C, Riethorf L, Wagener C, and Shively JE. Expression of biliary glycoprotein (CD66a) in normal and malignant breast epithelial cells. Anticancer Res, 1998; 18: 3203–3212.

100. Rojas M, Fuks A, and Stanners CP. Biliary glycoprotein, a member of the immunoglobulin supergene family, functions in vitro as a Ca2(+)-dependent intercellular adhesion molecule. Cell Growth Differ, 1990; 1: 527–533.

101. Riethdorf L, Lisboa BW, Henkel U, Naumann M, Wagener C, and Loning T. Differential expression of CD66a (BGP), a cell adhesion molecule of the carcino-embryonic antigen family, in benign, premalignant, and malignant lesions of the human mammary gland. J Histochem Cytochem, 1997; 45: 957–963.

102. Luo W, Wood CG, Earley K, Hung MC, and Lin SH. Suppression of tumori-genicity of breast cancer cells by an epithelial cell adhesion molecule (C-CAM1): the adhesion and growth suppression are mediated by different domains. Oncogene, 1997; 14: 1697–1704.

103. Cole GJ. and Glaser L. A heparin-binding domain from N-CAM is involved in neural cell-substratum adhesion. J Cell Biol, 1986; 102: 403–412.

104. Probstmeier R, Kuhn K, and Schachner M. Binding properties of the neural cell adhesion molecule to different components of the extracellular matrix. J Neurochem, 1989; 53: 1794–1801.

105.

106. Miettinen M. and Cupo W. Neural cell adhesion molecule distribution in soft tissue tumors. Hum Pathol, 1993; 24: 62–66.

107. Zoltowska A, Stepinski J, Lewko B, Serkies K, Zamorska B, Roszkiewicz A, Izycka-Swieszewska E, and Kruszewski WJ. Neural cell adhesion molecule in breast, colon and lung carcinomas. Arch Immunol Ther Exp (Warsz), 2001; 49: 171–174.

Mechtersheimer G. Towards the phenotyping of soft tissue tumours by cell surface molecules. Virchows Arch A Pathol Anat Histopathol, 1991; 419: 7–28.


108. Edvardsen K, Bock E, Jirus S, Frandsen TL, Holst-Hansen C, Moser C, Spang-Thomsen M, Pedersen N, Walsh FS, Vindelov LL, and Brunner N. Effect of NCAM-transfection on growth and invasion of a human cancer cell line. Apmis, 1997; 105: 919–930.

109. Shih LM, Hsu MY, Palazzo JP, and Herlyn M. The cell-cell adhesion receptor Mel-CAM acts as a tumor suppressor in breast carcinoma. Am J Pathol, 1997; 151: 745–751.

110. Shih IM, Nesbit M, Herlyn M, and Kurman RJ. A new Mel-CAM (CD146)-specific monoclonal antibody, MN-4, on paraffin-embedded tissue. Mod Pathol, 1998; 11: 1098–1106.

112. Moh MC, Zhang C, Luo C, Lee L, and Shen S. Structural and functional analyses of a novel ig-like cell adhesion molecule, hepaCAM, in the human

113. Newman PJ. The biology of PECAM-1. J Clin Invest, 1997; 100: S25–S29. 114. Fox S, Turner G D, Gatter KC, and Harris AL. The increased expression of

adhesion molecules ICAM-3, E- and P-selectins on breast cancer endothelium. J Pathol, 1995; 177: 369–376.

115. Sapino A, Bongiovanni M, Cassoni P, Righi L, Arisio R, Deaglio S, and Malavasi F. Expression of CD31 by cells of extensive ductal in situ and invasive carcinomas of the breast. J Pathol, 2001; 194: 254–261.

116. Butcher EC and Picker LJ. Lymphocyte homing and homeostasis. Science, 1996; 272: 60–66.

117. Springer TA. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu Rev Physiol, 1995; 57: 827–872.

118. Steinbach F, Tanabe K, Alexander J, Edinger M, Tubbs R, Brenner W, Stockle M, Novick AC, and Klein EA. The influence of cytokines on the adhesion of renal cancer cells to endothelium. J Urol, 1996; 155: 743–748.

119. Daneker GW, Lund SA, Caughman SW, Staley CA, and Wood WC. Anti-metastatic prostacyclins inhibit the adhesion of colon carcinoma to endothelial cells by blocking E-selectin expression. Clin Exp Metastasis, 1996; 14: 230–238.

120. Narita T, Kawakami-Kimura N, Matsuura N, Hosono J, and Kannagi R. Corticosteroids and medroxyprogesterone acetate inhibit the induction of E-selectin on the vascular endothelium by MDA-MB-231 breast cancer cells. Anticancer Res, 1995; 15: 2523–2527.

breast carcinoma MCF7 cells. J Biol Chem, 2005; 280: 27366–27374.

111. Shih IM. The role of CD146 (Mel-CAM) in biology and pathology. J Pathol, 1999; 189: 4–11.

122. Mitsuoka C. and Kannagi R. [Clinical significance of circulating soluble E-selectin (ELAM-1) in patients with cancers]. Nippon Rinsho, 1995; 53: 1770–1775.

123. Zhang GJ and Adachi I. Serum levels of soluble intercellular adhesion molecule-1 and E-selectin in metastatic breast carcinoma: correlations with clinicopathological features and prognosis. Int J Oncol, 1999; 14: 71–77.

124. Matsuura N, Narita T, Mitsuoka C, Kimura N, Kannagi R, Imai T, Funahashi H, and Takagi H. Increased level of circulating adhesion molecules in the sera of breast cancer patients with distant metastases. Jpn J Clin Oncol, 1997; 27: 135–139.

125. Matsuura N, Narita T, Mitsuoka C, Kimura N, Kannagi R, Imai T, Funahashi H, and Takagi H. Increased concentration of soluble E-selectin in the sera of breast cancer patients. Anticancer Res, 1997; 17: 1367–1372.

126. McEver RP, Moore KL, and Cummings RD. Leukocyte trafficking mediated by selectin-carbohydrate interactions. J Biol Chem, 1995; 270: 11025–11028.

121. Narita T, Kawasaki-Kimura N, Matsuura N, Funahashi H, and Kannagi R. Adhesion of Human Breast Cancer Cells to Vascular Endothelium Mediated by Sialyl Lewis &supx; /E-selectin. Breast Cancer, 1996; 3: 19–23.

132 Shevde and King

127. Ma YQ. and Geng JG. Obligatory requirement of sulfation for P-selectin binding to human salivary gland carcinoma Acc-M cells and breast carcinoma ZR-75-30 cells. J Immunol, 2002; 168: 1690–1696.

128. Aigner S, Ramos CL, Hafezi-Moghadam A, Lawrence MB, Friederichs J, Altevogt P, and Ley K. CD24 mediates rolling of breast carcinoma cells on P-selectin. Faseb J, 1998; 12: 1241–1251.

129. Aruffo A, Dietsch MT, Wan H, Hellstrom KE, and Hellstrom I. Granule membrane protein 140 (GMP140) binds to carcinomas and carcinoma-derived cell lines. Proc Natl Acad Sci U S A, 1992; 89: 2292–2296.

130. Blann AD, Gurney D, Wadley M, Bareford D, Stonelake P, and Lip GY. Increased soluble P-selectin in patients with haematological and breast cancer: a comparison with fibrinogen, plasminogen activator inhibitor and von Willebrand factor. Blood Coagul Fibrinolysis, 2001; 12: 43–50.

131. Laubli H, Stevenson JL, Varki A, Varki NM, and Borsig L. L-selectin facilitation of metastasis involves temporal induction of Fut7-dependent ligands at sites of tumor cell arrest. Cancer Res, 2006; 66: 1536–1542.

132. Borsig L, Wong R, Hynes RO, Varki NM, and Varki A. Synergistic effects of L- and P-selectin in facilitating tumor metastasis can involve non-mucin ligands and implicate leukocytes as enhancers of metastasis. Proc Natl Acad Sci U S A, 2002; 99: 2193–2198.

133. Yamada M, Yanaba K, Hasegawa M, Matsushita Y, Horikawa M, Komura K, Matsushita T, Kawasuji A, Fujita T, Takehara K, Steeber DA, Tedder TF, and Sato S. Regulation of local and metastatic host-mediated anti-tumour mechanisms by L-selectin and intercellular adhesion molecule-1. Clin Exp Immunol, 2006; 143: 216–227.

134. Yamada M, Yanaba K, Takehara K, and Sato S. Clinical significance of serum levels of soluble intercellular adhesion molecule-1 and soluble L-selectin in malignant melanoma. Arch Dermatol Res, 2005; 297: 256–260.

135. Chen S, Kawashima H, Lowe JB, Lanier LL, and Fukuda M. Suppression of tumor formation in lymph nodes by L-selectin-mediated natural killer cell recruitment. J Exp Med, 2005; 202: 1679–1689.

136. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell, 1992; 69: 11–25.

137. Albelda SM and Buck CA. Integrins and other cell adhesion molecules. Faseb J, 1990; 4: 2868–2880.

141. Mizejewski GJ. Role of integrins in cancer: survey of expression patterns. Proc Soc Exp Biol Med, 1999; 222: 124–138.

142. Koukoulis GK, Howeedy AA, Korhonen M, Virtanen I, and Gould VE. Distribution of tenascin, cellular fibronectins and integrins in the normal, hyperplastic and neoplastic breast. J Submicrosc Cytol Pathol, 1993; 25: 285–295.

143. Howlett AR, Bailey N, Damsky C, Petersen OW, and Bissell M J. Cellular growth and survival are mediated by beta 1 integrins in normal human breast epithelium but not in breast carcinoma. J Cell Sci, 1995; 108 ( Pt 5): 1945–1957.

144. Damjanovich L, Fulop B, Adany R, and Nemes Z. Integrin expression on normal and neoplastic human breast epithelium. Acta Chir Hung, 1997; 36: 69–71.

138. Nair KS, Naidoo R, and Chetty R. Expression of cell adhesion molecules in oesophageal carcinoma and its prognostic value. J Clin Pathol, 2005; 58: 343–351.

139. Hood JD. and Cheresh DA. Role of integrins in cell invasion and migration. Nat Rev Cancer, 2002; 2: 91–100.

140. Rust WL, Carper SW, and Plopper GE. The Promise of Integrins as Effective Targets for Anticancer Agents. J Biomed Biotechnol, 2002; 2: 124–130.


145. Zutter MM, Krigman HR, and Santoro SA. Altered integrin expression in adenocarcinoma of the breast. Analysis by in situ hybridization. Am J Pathol, 1993; 142: 1439–1448.

146. Zutter MM, Sun H, and Santoro SA. Altered integrin expression and the malignant phenotype: the contribution of multiple integrated integrin receptors. J Mammary Gland Biol Neoplasia, 1998; 3: 191–200.

147. Hata H, Matsuzaki H, Takeya M, Yoshida M, Sonoki T, Nagasaki A, Kuribayashi N, Kawano F, and Takatsuki K. Expression of Fas/Apo-1 (CD95) and apoptosis in tumor cells from patients with plasma cell disorders. Blood, 1995; 86: 1939–1945.

148. Green, L. J, Mould, A. P, and Humphries, M. J. The integrin beta subunit. Int J Biochem Cell Biol, 1998; 30: 179–184.

149. Wong NC, Mueller BM, Barbas CF, Ruminski P, Quaranta V, Lin EC, and Smith JW. Alphav integrins mediate adhesion and migration of breast carcinoma cell lines. Clin Exp Metastasis, 1998; 16: 50–61.

150. Deryugina EI, Bourdon MA, Jungwirth K, Smith JW, and Strongin AY. Functional activation of integrin alpha V beta 3 in tumor cells expressing membrane-type 1 matrix metalloproteinase. Int J Cancer, 2000; 86: 15–23.

151. Tuck AB, Elliott BE, Hota C, Tremblay E, and Chambers AF. Osteopontin-induced, integrin-dependent migration of human mammary epithelial cells involves activation of the hepatocyte growth factor receptor (Met). J Cell Biochem, 2000; 78: 465–475.

152. Bartsch JE, Staren ED, and Appert HE. Adhesion and migration of extracellular matrix-stimulated breast cancer. J Surg Res, 2003; 110: 287–294.

153. Rolli M, Fransvea E, Pilch J, Saven A, and Felding-Habermann B. Activated integrin alphavbeta3 cooperates with metalloproteinase MMP-9 in regulating

100: 9482–9487. 154. Meyer T, Marshall JF, and Hart IR. Expression of alphav integrins and vitronectin

receptor identity in breast cancer cells. Br J Cancer, 1998; 77: 530–536. 155. Wewer UM, Shaw LM, Albrechtsen R, and Mercurio AM. The integrin alpha 6

beta 1 promotes the survival of metastatic human breast carcinoma cells in

156. Chung J. and Mercurio AM. Contributions of the alpha6 integrins to breast carcinoma survival and progression. Mol Cells, 2004; 17: 203–209.

migration of metastatic breast cancer cells. Proc Natl Acad Sci USA, 2003;

mice. Am J Pathol, 1997; 151: 1191–1198.

160. Sloan EK, Pouliot N, Stanley KL, Chia J, Moseley JM, Hards DK, and Anderson R. L. Tumor-specific expression of alphavbeta3 integrin promotes spontaneous metastasis of breast cancer to bone. Breast Cancer Res, 2006; 8: R20.

161. Takayama S, Ishii S, Ikeda T, Masamura S, Doi M, and Kitajima M. The relationship between bone metastasis from human breast cancer and integrin alpha(v)beta3 expression. Anticancer Res, 2005; 25: 79–83.

162. Karadag A, Ogbureke KU, Fedarko NS, and Fisher LW. Bone sialoprotein, matrix metalloproteinase 2, and alpha(v)beta3 integrin in osteotropic cancer cell invasion. J Natl Cancer Inst, 2004; 96: 956–965.

158. Shaw LM. Integrin function in breast carcinoma progression. J Mammary Gland Biol Neoplasia, 1999; 4: 367–376.

159. Shimizu H, Seiki T, Asada M, Yoshimatsu K, and Koyama N. Alpha6beta1 integrin induces proteasome-mediated cleavage of erbB2 in breast cancer cells. Oncogene, 2003; 22: 831–839.

157. Chung J, Yoon S, Datta K, Bachelder RE, and Mercurio AM. Hypoxia-induced vascular endothelial growth factor transcription and protection from apoptosis are dependent on alpha6beta1 integrin in breast carcinoma cells. Cancer Res, 2004; 64: 4711–4716.

134 Shevde and King

163. Harms JF, Welch DR, Samant RS, Shevde LA, Miele ME, Babu GR, Goldberg SF, Gilman VR, Sosnowski DM, Campo DA, Gay CV, Budgeon LR, Mercer R, Jewell J, Mastro AM, Donahue HJ, Erin N, Debies MT, Meehan WJ, Jones AL, Mbalaviele G, Nickols A, Christensen ND, Melly R, Beck LN, Kent J, Rader RK, Kotyk JJ, Pagel MD, Westlin WF, and Griggs DW. A small molecule antagonist of the alpha(v)beta3 integrin suppresses MDA-MB-435 skeletal metastasis. Clin Exp Metastasis, 2004; 21: 119–128.

164. Alford D, Pitha-Rowe P, and Taylor-Papadimitriou J. Adhesion molecules in breast cancer: role of alpha 2 beta 1 integrin. Biochem Soc Symp, 1998; 63: 245–259.

165. Gui GP, Wells CA, Browne PD, Yeomans P, Jordan S Puddefoot JR, Vinson GP, and Carpenter R. Integrin expression in primary breast cancer and its

166. Gui GP, Wells CA, Yeomans P, Jordan SE, Vinson GP, and Carpenter R. Integrin expression in breast cancer cytology: a novel predictor of axillary metastasis. Eur J Surg Oncol, 1996; 22: 254–258.

167. Weaver VM, Petersen OW, Wang F, Larabell CA, Briand P, Damsky C, and Bissell MJ. Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies. J Cell Biol, 1997; 137: 231–245.

168. Weaver VM. and Bissell MJ. Functional culture models to study mechanisms governing apoptosis in normal and malignant mammary epithelial cells. J Mammary Gland Biol Neoplasia, 1999; 4: 193–201.

169. Lipscomb EA, Simpson KJ, Lyle SR, Ring JE, Dugan AS, and Mercurio AM. The alpha6beta4 integrin maintains the survival of human breast carcinoma cells in vivo. Cancer Res, 2005; 65: 10970–10976.

170. Guo W, Pylayeva Y, Pepe A, Yoshioka T, Muller WJ, Inghirami G, and Giancotti FG. Beta 4 integrin amplifies ErbB2 signaling to promote mammary tumorigenesis. Cell, 2006; 126: 489–502.

relation to axillary nodal status. Surgery, 1995; 117: 102–108.

171. Spangenberg C, Lausch EU, Trost TM, Prawitt D, May A, Keppler R, Fees SA, Reutzel D, Bell C, Schmitt S, Schiffer IB, Weber A, Brenner W, Hermes M, Sahin U, Tureci O, Koelbl H, Hengstler JG, and Zabel BU. ERBB2-mediated transcriptional up-regulation of the alpha5beta1 integrin fibronectin receptor promotes tumor cell survival under adverse conditions. Cancer Res, 2006; 66: 3715–3725.

176. Gilcrease MZ, Zhou X, and Welch K. Adhesion-independent alpha6beta4 integrin clustering is mediated by phosphatidylinositol 3-kinase. Cancer Res, 2004; 64: 7395–7398.

177. Litvinov SV, Bakker HA, Gourevitch MM, Velders MP, and Warnaar SO. Evidence for a role of the epithelial glycoprotein 40 (Ep-CAM) in epithelial cell-cell adhesion. Cell Adhes Commun, 1994; 2: 417–428.

172. Yoon SO, Shin S, and Lipscomb EA. A novel mechanism for integrin-mediated ras activation in breast carcinoma cells: the alpha6beta4 integrin regulates ErbB2 translation and transactivates epidermal growth factor receptor/ErbB2 signaling. Cancer Res, 2006; 66: 2732–2739.

173. Chung J, Bachelder RE, Lipscomb EA, Shaw LM, and Mercurio AM. Integrin (alpha 6 beta 4) regulation of eIF-4E activity and VEGF translation: a survival mechanism for carcinoma cells. J Cell Biol, 2002; 158: 165–174.

174. Yoon SO, Shin S, and Mercurio AM. Ras stimulation of E2F activity and a consequent E2F regulation of integrin alpha6beta4 promote the invasion of breast carcinoma cells. Cancer Res, 2006; 66: 6288–6295.

175. Chen M and O’Connor KL. Integrin alpha6beta4 promotes expression of autotaxin/ENPP2 autocrine motility factor in breast carcinoma cells. Oncogene, 2005; 24: 5125–5130.


178. Litvinov SV, Balzar M, Winter MJ, Bakker HA, Briaire-de Bruijn IH, Prins F, Fleuren GJ, and Warnaar SO. Epithelial cell adhesion molecule (Ep-CAM) modulates cell-cell interactions mediated by classic cadherins. J Cell Biol, 1997; 139: 1337–1348.

179. Osta WA, Chen Y, Mikhitarian K, Mitas M, Salem M, Hannun YA, Cole DJ, and Gillanders WE. EpCAM is overexpressed in breast cancer and is a potential target for breast cancer gene therapy. Cancer Res, 2004; 64: 5818–5824.

180. Gastl G, Spizzo G, Obrist P, Dunser M, and Mikuz G. Ep-CAM overexpression in breast cancer as a predictor of survival. Lancet, 2000; 356: 1981–1982.

181. Spizzo G, Went P, Dirnhofer S, Obrist P, Simon R, Spichtin H, Maurer R, Metzger U, von Castelberg B, Bart R, Stopatschinskaya S, Kochli OR, Haas P, Mross F, Zuber M, Dietrich H, Bischoff S, Mirlacher M, Sauter G, and Gastl G. High Ep-CAM expression is associated with poor prognosis in node-positive breast cancer. Breast Cancer Res Treat, 2004; 86: 207–213.

182. Gendler SJ. MUC1, the renaissance molecule. J Mammary Gland Biol Neoplasia, 2001; 6: 339–353.

183. von Mensdorff-Pouilly S, Snijdewint FG, Verstraeten AA, Verheijen RH, and Kenemans P. Human MUC1 mucin: a multifaceted glycoprotein. Int J Biol Markers, 2000; 15: 343–356.

184. Rahn JJ, Shen Q, Mah BK, and Hugh JC. MUC1 initiates a calcium signal after ligation by intercellular adhesion molecule-1. J Biol Chem, 2004; 279: 29386–29390.

185. Regimbald LH, Pilarski LM, Longenecker BM, Reddish MA, Zimmermann G, and Hugh JC. The breast mucin MUCI as a novel adhesion ligand for endothelial intercellular adhesion molecule 1 in breast cancer. Cancer Res, 1996; 56: 4244–4249.

186. Wesseling J, van der Valk SW, Vos HL, Sonnenberg A, and Hilkens J. Episialin (MUC1) overexpression inhibits integrin-mediated cell adhesion to extracellular matrix components. J Cell Biol, 1995; 129: 255–265.

187. Berry N, Jones DB, Smallwood J, Taylor I, Kirkham N, and Taylor-Papadimitriou J. The prognostic value of the monoclonal antibodies HMFG1 and HMFG2 in breast cancer. Br J Cancer, 1985; 51: 179–186.

188. Ligtenberg MJ, Buijs F, Vos HL, and Hilkens J. Suppression of cellular aggregation by high levels of episialin. Cancer Res, 1992; 52: 2318–2324.

189. Hilkens J, Vos HL, Wesseling J, Boer M, Storm J, van der Valk S, Calafat J, and Patriarca C. Is episialin/MUC1 involved in breast cancer progression? Cancer Lett, 1995; 90: 27–33.

190. Kondo K, Kohno N, Yokoyama A, and Hiwada K. Decreased MUC1 expression induces E-cadherin-mediated cell adhesion of breast cancer cell lines. Cancer Res, 1998; 58: 2014–2019.

191. Walsh MD, Luckie SM, Cummings MC, Antalis TM, and McGuckin MA. Heterogeneity of MUC1 expression by human breast carcinoma cell lines in vivo and in vitro. Breast Cancer Res Treat, 1999; 58: 255–266.

192. Lu L, Deng HY, and Fan WK. [Correlation of MUC1 expression to adhesion of breast cancer cell line MDA-MB-231.]. Ai Zheng, 2004; 23: 1294–1296.

193. Duffy MJ, Shering S, Sherry F, McDermott E, and O'Higgins N. CA 15-3: a prognostic marker in breast cancer. Int J Biol Markers, 2000; 15: 330–333.

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Chapter 7

ENDOCRINE RESISTANCE AND BREAST CANCER INVASION

Stephen Hiscox, Julia Gee, and Robert I. Nicholson Tenovus Centre for Cancer Research, Welsh School of Pharmacy, Cardiff University,

Despite the initial success of endocrine therapies, a significant proportion of women will acquire resistance to such treatments. Furthermore, clinical relapse during anti-hormonal therapy has been linked to tumours that have gained an aggressive phenotype and enhanced metastatic capacity and is frequently associated with a poorer outlook for the patient. Recently, we have demonstrated that the acquisition of an endocrine resistant state in breast cancer cells is accompanied by a profound increase in invasive capacity. Tumour cell invasion is fundamental to the subsequent develop-ment of metastasis, the most significant factor that affects the survival of patients with cancer. Despite this, past therapeutic approaches have paid relatively little attention to these important issues; thus a greater under-standing of this process will lead to the identification of potential targets for anti-invasive intervention for such patients. To this end, we are currently addressing potential mechanisms which may underlie such processes in acquired anti-hormone resistance and have identified several molecular elements through the study of cell models of acquired endocrine resistance.

1. INTRODUCTION

Steroid hormones are of central importance in directing the growth and development of breast tumours; as such, endocrine therapies which seek to perturb the steroid hormone environment of the tumour cells can promote extensive remissions in established tumours and furthermore provide significant patient survival benefits (1). Since hormone mani-

© 2007 Springer.


breast cancer; endocrine-resistance; invasion; EGFR; Src kinase; c-Met

Abstract:

Keywords:

Heath Park, Cardiff, CF10 3XF, UK

as the preferred therapeutic choice in the management of breast cancer patients and are now routinely used as an adjuvant to surgery to treat micro-metastatic deposits. Unfortunately, however, despite the initial suc-cess of endocrine therapies, data from clinical applications of such treat-ments has revealed that their beneficial actions are limited and can eventually be counteracted by the capacity of breast cancer cells to circum-vent the need for steroid hormones, allowing them to grow and progress despite such therapy. Thus, at presentation of these cancers, current endocrine therapies are not effective in all patients (de novo endocrine resistance) and initially responsive tumours will invariably progress despite such treatments (acquired resistance) resulting in patient relapse associated with reduced survival (2). Clinical relapse during anti-hormonal therapy has been linked to tumours that have gained an aggressive phenotype and enhanced metastatic capacity and is frequently associated with a poorer outlook for the patient. However, little is known about the mechanism(s) that underlie such disease progression and spread and whether they are induced by drug treatment.

Importantly, recent evidence is emerging which reveals that the acquisi-tion of resistance to endocrine therapies is also accompanied by a signi-ficant enhancement of the cells’ migratory and invasive potential in vitro (3–5). Clearly, these in vitro observations suggest that endocrine-resistant tumours possess aggressive characteristics which, in vivo, are likely to favour the dissemination of tumour cells from the primary tumour and thus promote disease spread. Despite the significance of such findings, little is known about the molecular changes which precipitate an aggres-sive phenotype during the acquisition of endocrine resistance. In light of this, this chapter highlights several key mechanisms recently identified by our laboratory through which the invasive phenotype of breast cancer cells may be augmented following the acquisition of endocrine resistance. Understanding such mechanisms will ultimately aid in the development of therapies which may prove central to the successful treatment of aggressive disease associated with relapse on endocrine therapies and improve prognosis as a consequence.

2. ALTERED GROWTH FACTOR SIGNALLING CONTRIBUTES TO AN INVASIVE PHENOTYPE

In comparison with endocrine-sensitive breast cancer cells, anti-hormone-resistant variants display a significantly enhanced invasive and

Hiscox, Gee, and Nicholson138

pulations are relatively non-toxic when compared to other major therapies (notably cytotoxic chemotherapy), they have become widely established

at least, a transition towards a more mesenchymal phenotype. Such characteristics indicative of an epithelial-to-mesenchymal transition (EMT) have been commonly reported in cancer cells and tissues where they act to promote an aggressive, invasive phenotype. To this end, we are currently addressing potential mechanisms which may underlie such processes in acquired anti-hormone resistance and have identified several molecular signaling elements through the study of cell models of acquired endocrine resistance.

2.1 EGFR signalling in ER-positive, acquired endocrine-resistant breast cancer cells

There is now substantial in vitro and in vivo experimental evidence revealing that the control of endocrine-resistant breast cancer growth is a multifaceted event, involving signalling through many different growth factor receptor tyrosine kinases which provide a complex network of interacting signal transduction pathways impinging on tumour proli-feration and cell survival parameters (6, 7, and references therein). For example, several studies have established that the intracellular signalling pathways associated with oestrogen-receptor (ER) and IGF-1R action are highly interactive. As such, anti-hormonal drugs can exert their anti-oestrogenic activity through disruption of oestrogen/IGF-1R signalling cross-talk (6) in addition to their more classical effects of blockade of ER/oestrogen response element (ERE) signalling. It follows that the growth inhibitory properties of such drugs are thus a combination of anti-oestrogenic and anti-growth factor activities (8–10). Similarly, members of the EGFR family of receptors have a well-established role in acquired endocrine resistance: oestrogens suppress the transcription of

models in vitro (13,14) and, as might be predicted, anti-hormones such as tamoxifen are able to promote the expression of EGFR and HER2. This, in turn, can lead to the mitogen-activated protein kinase (MAPK)/ AKT-mediated activation of the ER and, as a consequence, increased production of key ER-regulated EGFR ligands such as transforming growth factor alpha (TGFα) and amphiregulin (15–17), thereby completing an autocrine signalling loop. These events subsequently provide an effi-cient mechanism to drive anti-hormone-resistant growth (18). Significantly, EGFR expression, kinase activity, and reactivation of ER incrementally increase during treatment, culminating in emergence of EGFR-mediated, ER-positive, acquired tamoxifen-resistant growth (15,19).

7. Endocrine resistance and invasion 139

both the EGFR and HER2 (7, 11, 12) in ER-positive breast cancer cell

migratory capacity. Additionally, such cells commonly show a more angular, de-differentiated morphology with numerous lamellipodia and membrane ruffling in addition to growing as loose, disorganised colonies in which cells appear to have partially dissociated cell-cell contacts promoting cell scattering (3, 4). These events appear to reflect, behaviourally

tumours (7). Furthermore, elevated levels of EGFR correlate with increased invasiveness and metastasis and are associated with a poor clinical

increased in tumour tissue, it is likely that its activation state has a greater bearing on prognosis than expression of the protein alone. Constitutive activation of the EGFR may arise from autocrine production of EGFR ligands such as TGFα. Indeed, co-expression of EGFR and TGFα has been reported in non-small-cell lung cancers (22), prostate cancer (23), gastrointestinal tumours (24), and in invasive breast carci-nomas, where expression is significantly correlated with poor patient prognosis (25). Signalling through the EGFR subsequently causes the simultaneous activation of multiple, functionally interlinked signalling pathways (which include the Grb2/Ras/MAPK pathway, phospholipid metabolism involving PLD, PLCγ and PI3K and activation of the cytosolic Src family kinases (26, and references therein), ultimately

aggressive cell phenotype (27–29). The critical role that the EGFR plays in malignant transformation and cancer progression has thus identified it as a promising therapeutic target. Current strategies that exist to target this molecule include various EGFR tyrosine kinase inhibitors such as gefitinib (30).

Our data has demonstrated that signalling through EGFR/HER2 also contributes to the increased migratory and invasive capacity of ER-

with TGFα and amphiregulin (19). Inhibition of EGFR-mediated signal-ling in tamoxifen-resistant cells with gefitinib results in a reduction of the cells’ migratory and invasive capacity in vitro (3). Furthermore, abrogation of HER2 function through use of Herceptin is also able to partially suppress these cells’ aggressive phenotype (S. Hiscox, un-published observations). Interestingly, time-lapse analysis of TAM-R cell movement has revealed that it occurs in a directional, rather than random, fashion (3). This phenomenon is reported to be controlled by localised EGFR signalling in other cell types including keratinocytes (31), fibroblasts (32), and human mammary epithelial cells (33) as a consequence of the asymmetrical activation of motility-promoting signal-ling pathways. Interestingly, expression of the EGFR on TAMR-R cells is observed to be predominantly located to the areas of the cells that


promoting chemotaxis, migration, invasion, and the development of an

positive, acquired tamoxifen-resistant (TAM-R) breast cancer cellsin vitro. Such resistant cells highly express the EGFR and HER2 together

prognosis (11, 20, 21). Although expression of the EGFR protein may be

Tumour progression and spread requires a cell phenotype that displays altered biological activities other than simply deregulated proliferation, such as invasiveness and motility, and it has been speculated that the EGFR may play a role in this process. High levels of EGFR have been demonstrated in a number of aggressive tumour types including head and neck cancer, non-small-cell lung cancer, colorectal cancer, and ovarian

Moreover, it demonstrates the potential of EGFR signalling inhibitors as a means of controlling this adverse phenotype.

2.2 Src kinase

Although therapies targeting the EGFR/HER2 pathway such as those mentioned above can eliminate the agonistic effect of tamoxifen and

cells’ invasive capacity. Thus these data suggest that an EGFR/HER2-driven input contributes to, but is not essential for, their invasive in vitro phenotype. Significantly, these anti-growth factor therapies are not spared the problem of resistance; chronic exposure of tamoxifen-resistant breast cancer cells to anti-growth factor monotherapies such as gefitinib, results in the development of a further resistant state, with these now “dually resistant” cells (insensitive to both anti-hormone and anti-growth factor) utilizing additional growth factor receptor pathways such as the IGF1R signalling pathway and having an even greater invasive capacity

due to the tumour cells’ ability to “switch” between growth factor receptor pathways and circumvent these inhibitors, resulting in further resistant phenotypes. However, our recent signalling studies have revealed that, in parallel with their increased migratory and invasive capacity, many of our models of tamoxifen-, faslodex-, oestrogen withdrawal, and anti-growth factor resistance share significantly elevated activity of the non-receptor tyrosine kinase Src, known to play a central role in promoting invasion and motility in cancer.

Src is an important element in many growth factor receptor pathways,

expression of members of such receptors have been demonstrated to possess constitutively activated Src (39) where it may potentiate EGF-dependent tumour formation and growth in animal models (40, 41). Furthermore, elevated levels of both Src and EGFR in breast cancer cell lines correlates with an enhanced tumorigenicity in vivo (42). Src has been identified as playing a central role in tumour invasion and motility and the process of EMT, with Src-deficient cells showing defects in


metastatic phenotype in acquired tamoxifen-resistant breast cancer cells.

restore its anti-tumour activity (12, 35), they only partially reduce the

than their tamoxifen-resistant counterparts (36). Thus it is likely that thera-pies targeting individual growth factor receptors will prove unsuccessful

including that of the EGFR (37, 38) and tumours that exhibit elevated

displayed membrane activity (ruffles, lamellapodia) (3). Subsequently, gefitinib-treatment of TamR cells reduced the numbers of cells dis-playing membrane protrusions. Such observations have also been reported by others who have demonstrated the ability to block asymmetric EGFR expression using the EGFR inhibitor, PD158780 (31) and the invasion-suppressive effects of gefitinib in other cancer cell lines (34).

Modulation of EGFR activity using gefitinib has thus enabled us to establish the EGFR as a key player in the development of an enhanced

prognosis in some cancer types (50–53). Together, these observations suggest a role for Src in tumour development and progression.

In anti-hormone resistant cells, Src activity is elevated up to 20-fold greater compared to their endocrine-sensitive counterparts, an effect independent of expression levels of Src gene or protein (5). It is known that Src, along with focal adhesion kinase (FAK), cooperate to regulate cell attachment to the substratum and their subsequent migration. As such, we have demonstrated that elevated Src activity in tamoxifen-resistant cells alters the phosphorylation state of FAK and thus the turnover of focal adhesions leading to increased migratory and invasive behaviours. Furthermore, our immunohistochemical profiling of Src expression and activation in clinical breast cancer samples has similarly observed a correlation between increased Src activity, presence of distant metastasis, and shortened survival with tamoxifen therapy in ER-positive patients. Significantly, targeted inhibition of Src kinase activity in tamoxifen-resistant breast cancer cells using the dual Src/Abl inhibitor, AZD0530 (54), is clearly accompanied by an efficient reduction of their

activity promotes the elongation of focal adhesion structures and enhance-ment of focal adhesion “strength”, preventing focal adhesion turnover

tude of which is unachievable with anti-EGFR strategies. Interestingly, it is noteworthy that the anti-growth factors described above as only partially affecting tumour cell invasion are equally only modest inhibitors of Src activity (3). Moreover, AZD0530-treated TamR cells demonstrate impaired spreading over matrix components and grow as tightly packed colonies with very few membrane projections, similar to the morphology of the parental cells (5). These data confirm an import-ance for Src in the regulation of anti-hormone-resistant tumour cell motility and invasion, suggesting considerable therapeutic potential for Src inhibitors. Clearly there are further upstream regulators of Src activity in resistant cells that require deciphering. Intriguingly, Src inhibition as a monotherapy can itself result in a resistant state and suggests that therapies individually targeting either growth factor


invasive and migratory behaviour (5, 55). Furthermore, inhibition of Src

(5, 56). This leads to a reduction in migration and invasion, the magni-

cytoskeletal organisation (43) and spreading (44). Src is also a key element in the regulation of integrin-dependent attachment, acting in conjunction with focal adhesion kinase (FAK) to regulate focal adhesion

in tumour cells may lead to an aberrant intrinsic migratory capacity. In addition to modification of cell–matrix interactions, Src may also pro-mote loss of epithelial adhesion and cell scattering through modulation of cell-cell adhesions (47–49). Many studies now report elevation of Src protein and/or activity in a variety of tumours and Src appears to be an emerging independent indicator of disease stage and/or poor clinical

turnover and cell migration (45, 46). Thus elevated levels of Src activity

β-catenin, an element reported to interact with E-cadherin and the actin cytoskeleton, is considerably modified (4). Using integrated microarray and signalling studies we have revealed that β-catenin expression is increased at the mRNA/protein level whilst its phosphorylation status is significantly modified (elevated tyrosine phosphorylation, decreased serine/threonine phosphorylation). This deregulation is associated with PI3K/AKT-induced inactivation of GSK3β in TAM-R cells resulting in reduced association of β-catenin with E-cadherin. As a consequence, a disruption of cell-cell contacts and elevated migration and invasion is seen. Further evidence for an impaired adherens junction system has come from studies in which E-cadherin function has been neutralised using the calcium chelator, EGTA, or the HECD-1 antibody; these have only a modest impact on TAM-R invasion in marked contrast to the promotion of this feature in parental MCF-7 cells. Furthermore, failure of GSK3β/ubiquitin-mediated degradation of β-catenin in TAM-R cells results in elevated intracellular levels of β-catenin, promoting its nuclear translocation and interaction with the TCF/LEF-1 transcription factor. This triggers increased transcription of β-catenin/TCF/LEF-1 target genes, including CD44 which has, in turn, been linked to invasive cellular responses and EMT (4).

4. PROLONGED GROWTH FACTOR SIGNALLING CONTRIBUTES TO THE INVASIVE PHENOTYPE OF ER-NEGATIVE BREAST CANCER CELLS

At its greatest extremes, it is believed that aberrant growth factor

it may also drive the very highly invasive phenotype associated with ER-


dysfunction of components of the E-cadherin adhesion system since

signalling may promote ER loss (58,59) and we now have evidence that

receptors or Src kinases will be unable to effectively compromise both the growth and invasive properties of cancer cells and that combination therapy should provide a superior approach (57).

3. INTERCELLULAR ADHESION DEFICIENCIES

Although loss of E-cadherin is well associated with a more aggressive cell phenotype, its expression is not altered in our TAM-R cells. However, consistent with the observed poor cell-cell adhesion and the increased invasiveness of these cells, TAM-R cells display evidence of

partially methylated in ER-negative, FAS-R cells, an event that may contribute to their increased invasiveness. Interestingly, conventional RT-PCR analysis has also revealed further changes in TIMP and MMP expression, notably increased MMP2. This correlates with ability of broad-spectrum MMP inhibitors to suppress the invasive behaviour of FAS-R cells (62).

Using microarray analysis, we have also highlighted pro-invasive genes that are induced in FAS-R cells potentially arising as a con-sequence of chronically elevated growth factor signalling. Among these is the extracellular matrix protein vitronectin that is paralleled by sub-stantially increased αvβ3 integrin, and also the cell surface receptor CD44, a glycoprotein involved in cell-cell and cell-matrix interactions

such cells including CD44v3, which, interestingly, appears to have a close, inverse correlation with ER expression and provides a marker for

overexpression of CD44 has been linked to the growth and spread of a range of different types of malignancies, particularly lymphomas.

Evidence suggests that antisence and antibody-mediated targeting of CD44 markedly reduces the malignant activities of tumours in vivo thus suggesting the therapeutic potential of anti-CD44 agents. Furthermore, because alternative splicing and post-translational modifications which generate the many different CD44 variants, some of which may be only associated with tumours, the production of anti-CD44 tumour-specific agents may be a realistic therapeutic approach.


and demonstrated that TIMP3 (tissue inhibitor of metalloproteinase 3) is

poor prognosis in clinical breast cancer (65). Along with its role as a

(63, 64). A number of splice variants of this molecule are expressed in

responses. Furthermore, of cell motility and activator of cell survivalmediator of cellular adhesion, CD44 has been identified as an inducer

substantial in de novo ER-negative breast cancer models such as MDA-MB-231 cells that exhibit extensive aberrant growth factor signalling. Moreover, our studies in ER-negative faslodex-resistant (FAS-R) cells also reveal that chronic exposure to more modest increases in EGFR/kinase/NFκB signalling during prolonged faslodex treatment can associate with morphological features characteristic of an epithelial-to-mesenchymal transition (EMT), together with very high levels of migratory and invasive activity in vitro alongside adaptive silencing of

growth factor signalling that promotes ER negativity may culminate in parallel silencing of ER-regulated genes which play a central role in suppressing cellular invasion. In such cells, we are exploring whether there is a role for transcriptional silencing of any key anti-invasive genes

for ER-negative clinical breast cancer (7, 9,18,60,61), and invasiveness is

ER (4,59). In view of these observations, it is feasible that the chronic

ER-negative cells. Certainly, a poor prognosis has invariably been reported

has been demonstrated by a modest suppression of invasion following siRNA-mediated knockdown of Met activity (66). These in vitro data suggest that the development of an anti-hormone mediated, ER-negative, endocrine-resistant state in vivo may confer a metastatic advantage to the cells by allowing their migratory and invasive behaviour to be augmented by surrounding stromal cells. As such, the potential application of a num-ber of c-Met signalling inhibitors currently under development (77–79) may provide a new option for the suppression of the adverse disease phenotype associated with endocrine resistance.

6. CONCLUSIONS

We have highlighted here how the acquisition of endocrine resistance is often associated with gain of aggressive tumour features (epithelial to mesenchymal transition, increased cell motility, and invasive capacity). The underlying biology of such adverse properties in drug-resistant cells has been largely understudied to date and we have described here several cellular mechanisms whereby these poor prognostic features may be promoted in such cells. Importantly, while studies have shown that invasiveness in the presence of exogenous growth factors can be fully blocked by individual anti-growth factor therapies, basal invasiveness of our various endocrine-resistant models is only partially reduced by such


with its expression being a stronger prognostic indicator than HER2 and tumours correlate with a significantly reduced survival rate (69,71–74)

c-Met in the intrinsic, basal invasive capacity of faslodex-resistant cells EGFR (75, 76). As well as sensitising these cells to HGF/SF, a role for

5. SENSITISATION TO STROMAL-DERIVED GROWTH FACTORS THROUGH C-MET OVEREXPRESSION OCCURS IN ENDOCRINE-RESISTANT CELLS

Interestingly, array analysis of ER-negative, faslodex-resistant MCF7 cells has revealed significantly elevated levels of the HGF/SF receptor gene, c-Met. Furthermore, this is reflected at protein level and results in cells which are highly sensitive to exogenous HGF/SF ligand (66). Activation of the receptor tyrosine kinase, c-Met, promotes a diverse array of cellular responses resulting in cell ‘scattering’ and increased

activated in an autocrine manner or by ligand secreted by cells of the in vivo. In this context, c-Met expressed on epithelial tumour cells may be

surrounding stroma (69,70). Furthermore, high levels of c-Met in breast

c-Met signalling as a promoter of tumour progression and metastasisinvasion (67, 68). These in vitro observations have suggested a role for

2. Nicholson RI, Robertson JFR, Hayes DF. Endocrine management of Breast Cancer, Taylor & Francis, London., RI Nicholson, JFR Robertson, and DF Hayes. eds., 2002; pp. 1 296.

3. Hiscox S, Morgan L, Barrow D, Dutkowski C, Wakeling A, Nicholson RI. Tamoxifen resistance in breast cancer cells is accompanied by an enhanced motile and invasive phenotype: inhibition by gefitinib (‘Iressa’, ZD1839). Clin.

4. Hiscox S, Jiang WG, Obermeier K, Taylor K, Morgan L, Burmi R, Barrow D, Nicholson RI. Tamoxifen-resistance in MCF7 cells promotes EMT-like behaviour and involves modulation of β-catenin phosphorylation. Int J Cancer 2006; 118:290–301.

5. Hiscox S, Morgan L, Green T, Barrow D, Gee JM, Nicholson RI. Elevated Src activity promotes cellular invasion and motility in tamoxifen-resistant breast cancer cells. Breast Cancer Res Treat 2006; 97:263–274.

6.

7. Nicholson RI, Gee JMW, Harper ME. EGFR and cancer prognosis. Eur J Cancer 2001; 37:9–15.

8. Gee J, Rubini M, Robertson JF, Ellis IO, Gutteridge E, Nicholson RI. Type 1 insulin-like growth factor receptor expression and activation in clinical breast cancer. Breast Cancer Res Treat 2003; 82:S102.

9. Gee JM, Robertson JF, Gutteridge E, Ellis IO, Pinder SE, Rubini M, Nicholson RI. Epidermal growth factor receptor/HER2/insulin-like growth factor receptor signalling and oestrogen receptor activity in clinical breast cancer. Endocr Relat Cancer 2005; 12:99–111.

10. Hiscox SE, Barrow D, Gee JM. Non-endocrine pathways and endocrine resis-tance: observations with antiestrogens and signal transduction inhibitors in combination. Clin Cancer Res 2004; 10:346–354.


Exp Met 2004; 21:201–212.

approaches, suggesting other contributory regulatory elements. Moreover, the targeting of intracellular kinases such as Src that represent the con-vergence points for multiple growth factor signalling pathways to promote aggressive behavior may provide valuable anti-invasive targets. The detailed understanding of these events will lead to novel combination strategies with which to best control tumour cell growth and invasiveness in breast cancer patients.

REFERENCES

1. Nicholson RI, Johnston SR. Endocrine therapy-current benefits and limitations. Breast Cancer Res Treat 2005; 93:3–10.

ACKNOWLEDGMENTS

11. McClelland RA, Jones HE, Wakeling AE, Gee JM. Modulation of epidermal

Nicholson RI, Hutcheson IR, Knowlden JM, Jones HE, Harper ME, Jordan N,

Nicholson RI, Hutcheson IR, Harper ME, Knowlden JM, Barrow D,

The authors would like to thank the staff of the Tenovus tissue culture

sented here.and immunohistochemisty units for their contribution to the work repre-

sitivity and acquired resistance in breast cancer. Br J Cancer 2000; 82:501–513. Nicholson RI, Gee JM. Oestrogen and growth factor cross-talk and endocrine insen-

19. Knowlden JM, Hutcheson IR, Jones HE, Madden T, Gee JM, Harper ME,

20. Verbeek BS, Adriaansen-Slot SS, Vroom TM, Beckers T, Rijksen G. Over-expression of EGFR and c-erbB2 causes enhanced cell migration in human breast cancer cells and NIH3T3 fibroblasts. FEBS Lett 1998; 425:145–150.

21. Kaufmann AM, Lichtner RB, Schirrmacher V, Khazaie K. Induction of apoptosis by EGF receptor in rat mammary adenocarcinoma cells coincides with enhanced spontaneous tumour metastasis. Oncogene 1996; 13:2349–2358.

22. Hsieh ET, Shepherd FA, Tsao MS. Co-expression of epidermal growth factor receptor and transforming growth factor-alpha is independent of ras mutations in lung adenocarcinoma. Lung Cancer 2002; 29:151–157.

23. Seth D, Shaw K, Jazayeri J, Leedman PJ. Complex post-transcriptional regulation of EGF-receptor expression by EGF and TGF-alpha in human prostate cancer cells. Br J Cancer 1999; 80:657–669.

24. Cai YC, Jiang Z, Vittimberga F, Xu X, Savas L, Woda B, Callery M, Banner B. Expression of transforming growth factor-alpha and epidermal growth factor receptor in gastrointestinal stromal tumours. Virchows Arch 1999; 435:112–115.

25. Umekita Y, Ohi Y, Sagara Y, Yoshida H. Co-expression of epidermal growth factor receptor and transforming growth factor-alpha predicts worse prognosis in breast-cancer patients. Int J Cancer 2000; 89:484–487.

26. Jorissen RN, Walker F, Pouliot N, Garrett TP, Ward CW, Burgess AW. Epidermal growth factor receptor: mechanisms of activation and signalling. Exp Cell Res 2003; 284:31–53.


growth factor receptor in endocrine-resistant, oestrogen receptor-positive breast cancer. Endocr Relat Cancer 2001; 8:175–182.

12. McClelland RA, Jones HE, Wakeling AE, Gee JM. Modulation of epidermal growth factor receptor in endocrine-resistant, estrogen-receptor-positive breast cancer. Ann NY Acad Sci 2002; 963:104–115.

13. Yarden RI, Wilson MA, Chrysogelos SA. Estrogen suppression of EGFR expression in breast cancer cells: A possible mechanism to modulate growth. J Cell Biochem 2001; 81:232–246.

14. Wilson MA, Chrysogelos SA. Identification and characterization of a negative regulatory element within the epidermal growth factor receptor gene first intron in hormone-dependent breast cancer cells. J Cell Biochem 2002; 85:601–614.

15. Britton DJ, Hutcheson IR, Knowlden JM, Barrow D, Giles M, McClelland RA,

signalling pathways regulates tamoxifen–resistant growth, Breast Cancer Res Treat 2005; in press.

16. Hutcheson IR, Knowlden JM, Barrow D, Jones HE, Nicholson RI. The novel ER down-regulator, Faslodex, modulates EGFR/MAPK activity in tamoxifen-resistant MCF-7 breast cancer cells. Clin Cancer Res 2001; 7:A622.

17. Hutcheson IR, Knowlden JM, Madden TA, Barrow D, Gee JM, Wakeling AE,

18. Gee J, Madden TA, Robertson JFR, Nicholson RI. Clinical response and resistance to SERMS, in: Endocrine Therapy in Breast Cancer, Robertson JFR, Nicholson RI, Hayes DF, eds., Martin Dunitz Ltd, London, 2002; pp. 155–190.

Gee JMW, Nicholson RI. Bidirectional cross talk between ERα and EGFR

Nicholson RI. Oestrogen receptor-mediated modulation of the EGFR/MAPK pathway in tamoxifen-resistant MCF-7 cells. Breast Cancer Res Treat 2003; 81:81–93.

Barrow D, Wakeling AE, Nicholson RI. Elevated levels of epidermal growth factor receptor/c-erbB2 heterodimers mediate an autocrine growth regulatory pathwayin tamoxifen-resistant MCF-7 cells. Endocrinology 2003; 144:1032–1044.

27. Schafer B, Gschwind A, Ullrich A. Multiple G-protein-coupled receptor signals converge on the epidermal growth factor receptor to promote migration and invasion. Oncogene 2004; 23:991–999.

Nicholson RI, Hutcheson IR, Harper ME, Knowlden JM, Barrow D,

37.

38. Leu TH, Maa MC. Functional implication of the interaction between EGF receptor and c-Src. Front Biosci 2003; 8:S28–38.

39. Muthuswamy SK, Siegel PM, Dankort DL, Webster MA, Muller WJ. Mammary tumours expressing the neu proto-oncogene possess elevated c-Src tyrosine kinase activity. Mol Cell Biol 1994; 14:735–743.

40. Tice DA, Biscardi JS, Nickles AL, Parsons SJ. Mechanism of biological synergy between cellular Src and epidermal growth factor receptor. Proc Natl

41.

42. Biscardi JS, Belsches AP, Parsons SJ. Characterization of human epidermal growth factor receptor and c-Src interactions in human breast tumour cells. Mol Carcinog 1998; 21:261–272.

43. Schwartzberg PL, Xing L, Hoffmann O, Lowell CA, Garrett L, Boyce BF, Varmus HE. Rescue of osteoclast function by transgenic expression of kinase-deficient Src in src-/- mutant mice.Genes Dev 1997; 11:2835–2844.


Acad Sci USA 1999; 96:1415–1420.

28. El-Obeid A, Bongcam-Rudloff E, Sorby M, Ostman A, Nister M, Westermark B. Cell scattering and migration induced by autocrine transforming growth factor alpha in human glioma cells in vitro. Cancer Res 1997; 57:5598–5604.

29. McCawley LJ, O’Brien P, Hudson LG. Overexpression of the epidermal growth factor receptor contributes to enhanced ligand-mediated motility in keratinocyte cell lines. Endocrinology 1997; 138:121–127.

30. Wakeling AE, Guy SP, Woodburn JR Ashton SE, Curry BJ, Barker AJ, Gibson KH. ZD1839 (Iressa): an orally active inhibitor of epidermal growth factor signaling with potential for cancer therapy. Cancer Res 2002; 62:5749–5754.

31. Fang KS, Ionides E, Oster G, Nuccitelli R, Isseroff RR. Epidermal growth factor receptor relocalization and kinase activity are necessary for directional migration of keratinocytes in DC electric fields. J Cell Sci 1999; 112:1967–1978.

32. Ware JL. Growth factor network disruption in prostate cancer progression. Cancer Metastasis Rev 1998; 17:443–447.

33. Maheshwari G, Wiley HS, Lauffenburger DA. Autocrine epidermal growth factor signaling stimulates directionally persistent mammary epithelial cell migration. J Cell Biol 2001; 155:1123–1128.

34. Kumar R. Suppression of epidermal growth factor receptor, mitogen-activated protein kinase, and pak1 pathways and invasiveness of human cutaneous squamous cancer cells by the tyrosine kinase inhibitor ZD1839 (Iressa). Mol Cancer Ther 2003; 2:345–351.

35. Witters L, Engle L, Lipton A. Restoration of estrogen responsiveness by blocking the HER-2/neu pathway. Oncol Rep 2002; 9:1163–1166.

36. Jones HE, Goddard L, Gee JMW, Hiscox S, Rubini M, Barrow D, Williams S, Wakeling AE, Nicholson RI. Insulin-like growth factor –1 receptor signalling and resistance to gefitinib (ZD1839; IRESSATM) in human breast and prostate cancer cells. Endocrine–Related Cancer 2004; 1:793–814.

Barnes CJ, Bagheri-Yarmand R, Mandal M, Yang Z, Clayman GL, Hong WK,

Biscardi JS, Maa MC, Tice DA, Cox ME, Leu TH, Parsons SJ. c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function. J Biol Chem 1999; 274:8335–8343.

Maa MC, Leu TH, McCarley DJ, Schatzman RC, Parsons SJ. Potentiation of epi-dermal growth factor receptor-mediated oncogenesis by c-Src: implications for theetiology of multiple human cancers. Proc Natl Acad Sci USA 1995; 92: 6981–6985.

44. Kaplan KB, Swedlow JR, Morgan DO, Varmus HE. c-Src enhances the spreading of src-/- fibroblasts on fibronectin by a kinase-independent mechanism. Genes Dev 1995; 9:1505–1517.

45. Fincham VJ, Frame MC. The catalytic activity of Src is dispensable for translocation to focal adhesions but controls the turnover of these structures during cell motility. Embo J 1998; 17:81–92.

55. Morgan LD, Hiscox S, Nicholson RI. Elevated Src kinase activity accompanies endocrine resistance in breast cancer and promotes an aggressive cell pheno-type. Proc Adv Breast Cancer Res: Gen Biol Clin Appl 2005; B54.

56. Hiscox SE, Barrow D, Green T, Nicholson RI. Adhesion independent focal adhesion kinase activation involves Src and promotes cell adhesion and motility in tamoxifen-resistant MCF-7 cells and is inhibited by the Src/Abl kinase inhibitor, AZD0530. Proc Am Ass Cancer Res 2005; 46:A266.

57.

2006; in press. 58. McClelland RA, Barrow D, Madden TA, Dutkowski CM, Pamment J,

Knowlden J, Gee JM, Nicholson RI. Enhanced epidermal growth factor receptor signaling in MCF7 breast cancer cells after long-term culture in the presence of the pure antiestrogen ICI 182,780 (Faslodex). Endocrinology 2001; 142:2776–2788.

59. Giles MG, Fiegl H, Widschwendter M, Gee JM, Wakeling AE, Nicholson RI. Irreversible loss of oestrogen receptor expression in MCF-7 cells following long-term exposure to fulvestrant. Breast Cancer Res Treat 2006; in press.


46. Jones RJ, Avizienyte E, Wyke AW, Owens DW, Brunton VG, Frame MC. Elevated c-Src is linked to altered cell-matrix adhesionn rather than proliferation in KM12C human colorectal cancer cells. Br J Cancer 2002; 87:1128–1135.

47. Irby RB, Yeatman TJ. Increased Src activity disrupts cadherin/catenin-mediated homotypic adhesion in human colon cancer and transformed rodent cells. Cancer Res 2004; 62:2669–2674.

48. Boyer B, Roche S, Denoyelle M, Thiery JP. Src and Ras are involved in separate pathways in epithelial cell scattering. Embo J 1997; 16:5904–5913.

49. Boyer B, Bourgeois Y, Poupon MF.Src kinase contributes to the metastatic spread of carcinoma cells. Oncogene 2002; 21:2347–2356.

50. Masaki T, Okada M, Shiratori Y, Rengifo W, Matsumoto K, Maeda S, Kato N,

51. Masaki T, Igarashi K, Tokuda M, Yukimasa S, Han F, Jin YJ, Li JQ, Yoneyama H, Uchida N, Fujita J, Yoshiji H, Watanabe S, Kurokohchi K, Kuriyama S.

52. Wiener JR, Windham TC, Estrella VC, Parikh NU, Thall PF, Deavers MT,

53. Summy JM, Gallick GE. Src family kinases in tumour progression and metastasis. Cancer Metastasis Rev 2003; 22:337–358.

54. Hennequin LF, Allen J, Curwen J, Fennell M, Green TP, Lambert-van der Brempt C, Morgentin R, Olivier R, Plé PA, Warin N, Costello G. The discovery of N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine (AZD0530) a novel highly selective, orally available, dual-specific Src/abl kinase inhibitor. J Med Chem 2006; in press.

Kanai F, Komatsu Y, Nishioka M, Omata M. pp60c-src activation in hepato-cellular carcinoma of humans and LEC rats. Hepatology 1998; 27:1257–1264.

pp60c-src activation in lung adenocarcinoma. Eur J Cancer 2003; 39:1447–1455.

anti-hormone/anti-growth factor-resistant breast cancer. Endocr Rel Cancer Hiscox S, Morgan L, Green TP, Nicholson RI. Src as a therapeutic target in

Bast RC, Mills GB, Gallick GE. Activated SRC protein tyrosine kinase is overexpressed in late-stage human ovarian cancers. Gynecol Oncol 2003; 88:73–79.

60. Cheung KL, Nicholson RI, Blamey RW, Robertson JF. Selection of primary breast cancer patients for adjuvant endocrine therapy-is oestrogen receptor alone adequate? Breast Cancer Res Treat 2001; 65:155–162.

61. Rampaul RS, Pinder SE, Nicholson RI, Gullick WJ, Robertson JF, Ellis IO. Clinical value of epidermal growth factor receptor expression in primary breast cancer. Adv Anat Pathol 2005; 12:271–273.

62. Khirwadkar Y, Jordan NJ, Hiscox SE, Nicholson RI. Increased matrix metallo-proteinase (MMP) expression in anti-oestrogen resistant breast carcinoma cell lines. Eur J Cancer 2005; 3:P19.

73. Ghoussoub RA, Dillon DA, D’Aquila T, Rimm EB, Fearon ER, Rimm DL. Expression of c-met is a strong independent prognostic factor in breast carcinoma. Cancer 1998; 82:1513–1520.

74. Lee WY, Chen HH, Chow NH, Su WC, Lin PW, Guo HR. Prognostic signi-ficance of co-expression of RON and MET receptors in node-negative breast cancer patients. Clin Cancer Res 2005; 11:2222–2228.

75. Lengyel E, Prechtel D, Resau JH, Gauger K, Welk A, Lindemann K, Salanti G, Richter T, Knudsen B, Vande Woude GF, Hardeck D. C-Met overexpression in node-positive breast cancer identifies patients with poor clinical outcome independent of Her2/neu. Int J Cancer 2005; 113:678–682.

76. microarray-based studies of patients with lymph node negative breast carcinoma show that met expression is associated with worse outcome but is not correlated with epidermal growth factor family receptors. Cancer 2003; 97:1841–1848.

77. Matsumoto K, Nakamura T. NK4 (HGF-antagonist/angiogenesis inhibitor) in cancer biology and therapeutics. Cancer Sci 2003; 94:321–327.

78. Cassinelli G, Lanzi C, Petrangolini G, Tortoreto M, Pratesi G, Cuccuru G, Laccabue D, Supino R, Belluco S, Favini E, Poletti A, Zunino F. Inhibition of c-Met and prevention of spontaneous metastatic spreading by the 2-indolinone

79. Wang SY, Chen B, Zhan YQ, Xu WX, Li CY, Yang RF, Zheng H, Yue PB, Larsen SH, Sun HB, Yang X. SU5416 is a potent inhibitor of hepatocyte growth factor receptor (c-Met) and blocks HGF-induced invasiveness of human HepG2 hepatoma cells. J Hepatol 2004; 41:267–273.


Tolgay Ocal I, Dolled-Filhart M, D’Aquila TG, Camp RL, Rimm DL. Tissue

63. Burmi RS, McClelland RA, Nicholson RI, Barrow D, Ellis IO, Robertson JF, Gee JM. Microarray studies reveal novel genes associated with antiestrogen resistance in breast cancer. Proc Adv Breast Cancer Res: Genetics, Biology and Clin Appl 2005; A22.

64. Gee J, Shaw V, Burmi R, McClelland R, Morgan H, Harper M, Hiscox S, Barrow D, Lewis P, Nicholson RI. Array profiling of survival and resistance genes in anti-hormone-treated breast cancer cells. Intl J Mol Med 2004; 14:S81.

65. Harper ME, Smith C, Nicholson RI. Upregulation of CD44s and variants in anti-hormone resistant breast cancer cells. Eur J Cancer 2005; 3:A25.

66. Hiscox S, Jordan NJ, Jiang W, Harper M, McClelland R, Smith C, Nicholson RI. Chronic exposure to fulvestrant promotes overexpression of the c-Met receptor in breast cancer cells: implications for tumour-stroma interactions. Endocr Rel Cancer 2006; in press.

67. Jiang WG, Hiscox S. Hepatocyte growth factor/scatter factor, a cytokine playing multiple and converse roles. Histol Histopathol 1997; 12:537–555.

68. Parr C, Jiang WG. Hepatocyte growth factor activators, inhibitors and antagonists and their implication in cancer intervention. Histol Histopathol 2001; 16:251–268.

69. Edakuni G, Sasatomi E, Satoh T, Tokunaga O, Miyazaki K. Expression of the hepatocyte growth factor/c-Met pathway is increased at the cancer front in breast carcinoma. Pathol Int 2001; 51:172–178.

70. Parr C, Jiang W G. Expression of hepatocyte growth factor/scatter factor, its activator, inhibitors and the c-Met receptor in human cancer cells. Int J Oncol 2001; 19:857–863.

71. Camp RL, Rimm EB, Rimm DL. Met expression is associated with poor outcome in patients with axillary lymph node negative breast carcinoma. Cancer

72. Elliott BE, Hung WL, Boag AH, Tuck AB. The role of hepatocyte growth factor

Physiol Pharmacol 2002; 80:91–102.

86:2259–2265.

(scatter factor) in epithelial-mesenchymal transition and breast cancer. Can J

RPI-1. Mol Cancer Ther 2006; 5:2388–2397.

Chapter 8

THE ROLE OF AROMATASE AND OTHER

IN MAMMARY CARCINOGENESIS Mohamed Salhab and Kefah Mokbel

There is a large and compelling body of epidemiological and experimental evidence that oestrogens are the fuel behind the aetiology of breast cancer. The local biosynthesis of oestrogens especially in postmenopausal women as a result of the interactions of various enzymes is believed to play a very important role in the pathogenesis and development of hormone-dependent breast carcinoma. The over-expression of such enzymes seems to be asso-ciated with the development of a more aggressive disease and associated with poor outcome and increased local and distant recurrences. In this chapter we shed light on CYP19 gene expression, aromatase enzyme activity

ducing enzymes such as 17beta hydroxysteroid dehydrogenase 1, 2 and steroid sulphatase and their role in breast cancer development are

these enzymes is crucial to the development of new endocrine preventa-tive and therapeutic strategies in postmenopausal females with hormone- dependant breast cancer. Currently, the third generation of aromatase

cancer. However, the important role of both STS and 17beta HSD type 1

therapy. Such endocrine therapy is currently being explored and the development of STS inhibitors and 17beta HSD 1 inhibitors is underway with promising initial results.

breast cancer, estrogen, postmenopausal, aromatase, 17beta HSD type 1,

and its role in mammary carcinogenesis. In addition, other oestrogen pro-

inhibitors has revolutionised the treatment of oestrogen-dependant breast

in local oestrogen production provides novel potential targets for endocrine

discussed in details. The understanding of the mechanisms that regulate

St. George’s Hospital, London, SW17 0QT, UK

steroid sulphatase, carcinogenesis

OESTROGEN - PRODUCING ENZYMES

Abstract:

Keywords:

© 2007 Springer.


1. INTRODUCTION

There is a large and compelling body of epidemiological and experi-mental evidence that oestrogens are the fuel behind the aetiology of

152 Salhab and Mokbel breast cancer and actually some of breast carcinomas require oestrogen for continued growth and progression (1, 2).

The progression from proliferative disease without atypia (PDWA) to

be a possible model for the development of human breast invasive ductal carcinoma (3, 4). In the early stages of this proposed cascade of breast cancer development, oestrogens, especially oestradiol (E2), have been considered as one of the most important factors (5).

Animal studies demonstrated that oestrogens can induce and promote mammary tumours in rodents and the removal of animals’ ovaries or administration of anti-oestrogenic drugs had the opposite effect (6). Additionally, oestrogens induce the expression of peptide growth factors which are responsible for the proliferative responses of cancer cells (7, 8). Furthermore, oestrogen has been shown to upregulate oncogenes such as c-myc through binding to its receptor, and through the Src/ p21ras/mitogen-activated protein kinase pathway of c-fos and c-jun,

In women, oestradiol originates from different sources before and after menopause. In premenopausal women, the ovary or membrana granulosa of dominant follicles is the main source of circulating oestrogens (11, 12). Oestrogens are produced, secreted, and transported through the circu-lation, and act on their target tissues where their specific receptors are expressed. This system is known as the endocrine system. In classical endocrine systems, only a small amount of hormone is generally utilized in the target tissues, and the great majority is metabolized or converted to inactive forms. However, most oestrogen after the menopause is syn-thesised in peripheral tissues from abundantly present circulating precursor steroids (13) where the enzymes involved in the formation of androgens and oestrogens are expressed. Several epidemiological studies indicate that plasma oestradiol, adrenal androgens, and testosterone levels are higher in women who develop breast cancer over a period of several years than in those who do not (14, 15, 16).

In postmenopausal women, oestrogens act in an autocrine fashion where oestrogen is synthesised in tumour epithelial cells. Moreover, neighbouring stromal cells can produce oestrogen which is transported to the tumour cells without release to the circulation. This is known as the paracrine mechanism. Furthermore, locally produced bioactive androgens and/or oestrogens exert their action in the cells where synthesis occurs without release into the extracellular space. This phenomenon is different from the autocrine, paracrine, and classical endocrine action, and is called ‘intracrine’. Oestrogens are biosynthesised in peripheral tissues through the conversion of circulating inactive steroids (17). Androgens such as androsteonedione of both adrenal and ovarian origin, especially

in situ (DCIS), and from DCIS to invasive carcinoma has been proposed to atypical ductal hyperplasia (ADH), from ADH to ductal carcinoma

leading to increased breast cancer cell proliferation (9, 10).

8. Aromatase in breast cancer 153

tissues, including skin, muscle (19), fat (20), and bone (21). The intracrine system requires minimal amounts of biologically active hormones to exert their maximum effects. Therefore, the intracrine pathway is an efficient mode of hormone action and plays important roles especially in the development of hormone-dependent neoplasms. It is also important to note that, in an intracrine system, serum concentrations of hormones do not necessarily reflect the local hormonal activity in the target tissues.

The relative contribution of any of the above-mentioned mechanisms is likely to vary with the physiological status of the female and possibly also with the local and systemic changes occurring during breast tumori-genesis and progression. Experimental evidence supports the potential of

tumorigenesis (22). Higher levels of estradiol were seen in breast cancer tissue when

compared with areas considered as morphologically normal (23). In addition, it has been observed that in postmenopausal patients with breast cancer, oestrogens levels in specimens were found to be several-

decline sharply after menopause, it has been reported that in some breast

bution to the oestrogen content of breast cancer cells (26, 27). Moreover, experimental evidence using xenograft models provides a direct proof that locally produced oestrogen can stimulate the growth of oestrogen-

produced locally in tumours arising from these xenografted cells may exceed the amount taken up from plasma. Furthermore, oestrogen influences the clinical outcome of breast cancer patients by stimulating

Intratumoral oestradiol levels were not observed to be significantly different between premenopausal and postmenopausal breast cancer

higher in postmenopausal than in premenopausal breast cancers (28).

steroid sulfatase (STS) hydrolyzes oestrone sulphate (E1S) to oestrone (Figure 1). Oestrone is subsequently converted to oestradiol (E2) by

acts on breast cancer cells through ER. Therefore, it is important to

cells (22).

The local production of oestrogens is mediated by a number of enzymes; aromatase catalyzes androstenedione into oestrone (E1), while

patients, but the intratumoral oestradiol/oestrone ratio was significantly

17beta hydroxysteroid dehydrogenase type 1 (17beta HSD1), and locally

the zona reticularis of adrenal cortex (18) and oestrone sulphate, are considered major precursor substrates of local oestrogen production. The conversion of androgen to oestrone occurs principally in peripheral

examine these enzymes in human breast carcinomas in order to understand

the proliferation of oestrogen receptor (ER) positive tumour epithelial

fold higher than those of plasma (24, 25). Although oestrogens levels

dependent MCF-7 human breast tumours to a greater extent than

tumours, in situ formation of oestrogens can make an important contri-

can oestrogen delivered via an endocrine mechanism (10). Oestrogen

each mechanism to contribute to oestrogen synthesis and influence breast

Figu

re 1

. The

orig

in o

f oes

troge

nic

ster

oids

in p

ostm

enop

ausa

l wom

en w

ith h

orm

one-

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ance

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SD (h

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Salhab and Mokbel 154

2. AROMATASE

The human CYP19 (P450 arom) gene is localized at chromosome 15q21.21. It belongs to the cytochrome P-450 superfamily compromising over 460 members in 74 families, of which cytochrome P450 arom is the sole member of the family 19 (29). CYP19 encodes aromatase that is the key enzyme for oestrogen biosynthesis (30, 31) which is achieved by sequential hydroxylation, oxidation, and removal of the C-19 carbon and aromatization of the A ring of the steroid.

women, and in the peripheral adipose tissues of postmenopausal women and men (10, 32, 33).

The CYP19 gene is found between markers stSG12786 and stSG47530 with the 3′-end of the gene centromeric to the 5′-end of the gene, showing the direction of transcription as from telomere to centromere. It spans about 123 Kb. Only the 30 kb (exon II-exon X) 3′-region encodes aromatase, whereas the large 93 kb 5′-flanking region serves as the regu-latory unit of the gene. The unusually large regulatory region contains 10 tissue-specific promoters that are alternatively used in various cell types.

Further upstream of exon II, there are a number of alternative first-exons which are differently spliced into distinct 5′-untranslated regions (34, 35, 36). In addition, up to nine different transcriptional start sides with individual promoters permitting tissue-specific regulation of expression have been described. However, even though each tissue expresses a unique first-exon 5′-untranslated region by splicing into a highly promiscuous splice acceptor site (AG-GACT) of the exon II, coding regions and translated products are identical in all tissue sites of expression (34, 36). This means that although transcripts in different

therefore the proteins expressed in these tissues remain the same. The recently published Human Genome Project Data allowed us for

the first time to precisely locate all known promoters and elucidate the extraordinarily complex organization of the entire human CYP19 gene. Each promoter is regulated by a distinct set of regulatory sequences in DNA and transcription factors that bind to these specific sequences.


CYP19 gene expression 2.1.

in the ovaries of premenopausal women, in the placenta of pregnant It is well established that the highest levels of aromatase are present

tissues have different 5′-termini, the coding region is the same and

oestrogen producing enzymes and their role in breast cancer development. In this chapter we will shed light on aromatase enzyme and other

how they play an essential part in the local production of oestrogens and subsequently their role in mammary carcinogenesis.

156 Salhab and Mokbel

The in vivo cellular distribution and physiologic roles of promoter I.7 in healthy tissues, however, are not known.

It is now known that the aromatase gene expression is regulated in a tissue-specific manner by the use of alternative promoters (37). Normal breast adipose tissue maintains low levels of aromatase expression pri-marily via promoter I.4 that lies 73 kb upstream of the common coding region. Promoters I.3 and II are used only minimally in normal breast adipose tissue. By performing primer-specific RT-PCR analyses (38, 39, 40), it was revealed that the two major exons (I.3 and PII) are present in aromatase mRNAs isolated from breast tumours. These results suggest that promoters I.3 and II are the major promoters directing aromatase expression in breast cancer and surrounding stromal cells and fibroblasts. It appears that the prototype oestrogen-dependent malignancy breast cancer takes advantage of four promoters (II, I.3, I.7, and I.4) for aro-matase expression. The sum of P450arom mRNA species arising from these four promoters markedly increases the total P450arom mRNA levels in breast cancer compared with the normal breast that uses almost exclusively promoter I.4.

Many studies showed that a switch from an adipose-specific exon 1

ovary-specific exon 1 (exon 1c or exon I.2) occurred in breast cancer tissue (41, 42).

activity in the stromal rather than the epithelial component of breast tumours (44). Furthermore, measurements of aromatase activity in fibro-blasts derived from breast tumours or MCF-7 cells have demonstrated a much higher level of aromatase activity in fibroblasts (45).

possible to induce arornatase activity with dexamethasone, breast cyst fluid (BCF) and breast tumour cytosol were found to stimulate aromatase activity (46, 47, 48). It was revealed that IL-6 could stimulate aromatase activity in stromal cells derived from subcutaneous adipose tissue (49). This stimulation requires the IL-6 soluble receptor (IL-6sR) which is

Enzyme activity 2.2.

(17, 43). Biochemical studies, however, have revealed higher a aromatase epithelial and stromal location for the aromatase enzyme complex

The promoter I.7 was cloned by analyzing P450arom mRNA in breast cancer tissue levels. P450arom mRNA with exon I.7 expression was significantly increased in breast cancer tissues and adipose tissue adjacent to tumours (37). This TATA-less promoter accounts for the transcription of 29–54% of P450arom mRNAs in breast cancer tissues.

been identified. Using breast tumour-derived fibroblasts, in which it is

Immunohistochemical studies have provided evidence for both an

Several factors which can stimulate aromatase activity have now

(exon 1b or exon I.4) promoter used in non-tumour breast tissues to the


gene expression is regulated by PII and PI.3 to a greater extent than PI.4

Prostaglandin E2 (PGE2) is able to cause promoter switching from I.4 to II in adipose stromal cells, and can increase aromatase activity (53). It has also been found to be the most potent factor stimulating aromatase expression via promoter II. A correlation between COX-2 and CYP19 mRNA levels has been demonstrated in human breast cancer specimens using semi-quantitative RT-PCR. It has been also found that PGE2 may act by stimulating IL-6 production in fibroblasts derived from normal and malignant breast tissues (54).

The regulation of aromatase activity in malignant tissues is highly

of a tumour within the breast influenced aromatase activity in the quadrant in which the tumour was located. This finding, which was subsequently confirmed at the expression and activity levels, suggested that either tumours developed in an area of high aromatase activity within the breast or tumours were capable of producing factors that stimulated aromatase activity in adjacent tissues. Bulun et al. (56) reported that CYP19 mRNA levels were highest in tumour bearing quadrants. CYP19 mRNA levels were observed to be significantly higher

in non-malignant breast tissue (57). Using quantitative polymerase chain reaction (PCR) analysis, it was

confirmed that adipose stromal cells surrounding the cancer cells contained higher levels of CYP19 mRNA than adipose stromal cells in

Cell line experiments have confirmed the role of aromatase in stimu-lating the growth of breast cancer cells (24, 59, 60, 61). Additionally, aromatase overexpression has been reported to be associated with a poor clinical outcome in women with breast cancer (62) (Figure 2). Such a relationship was not seen with the clinicopathological parameters of other tumour characteristics. The lack of correlation between aromatase

Role in mammary carcinogenesis 2.3.

(38, 52).

complex. Miller and O’Neill (55) were the first to show that the location

in tumour bearing quadrants than in those regions distal to the tumour or

adjacent to the tumour or in normal breast fat. in vitro aromatase activity was higher in breast tumours than in the fat non-cancerous areas (56) Furthermore, James et al. (58) reported that

produced by breast tumour-derived fibroblasts and acts synergistically with IL-6 to stimulate aromatase activity in these cells (50). In addition

inhibitory factor and insulin-like growth factor Type I are known to stimulate aromatase activity (49, 51). Cytokines, in the presence of glucocorticoids, regulate aromatase gene expression via the PI.4. In malignant breast tissue promoter switching occurs resulting in aromatase

to IL-6, other cytokines such as TNF α, IL-Il, oncostatin M, leukaemia


The increasing evidence that aromatase inhibitors are superior to tamoxifen in postmenopausal women with ER positive early and advanced breast cancer is in keeping with our observation that higher

Figure 2. Kaplan-Meier analysis of disease-free survival of breast cancer patients depending

(cut-off point: 10 000)).

DEHYDROGENASE TYPE 1 AND 2

Estradiol (E2), a biologically potent estrogen, contributes greatly to

0 50 100 150 200

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aromatase expression correlates with poor clinical outcome (67, 68, 69).

on the expression of Aromatase mRNA (P = 0.0105). (0 = Low levels; 1.00 = High levels

3. 17BETA HYDROXYSTEROID

the growth and development of breast carcinoma cells. 17beta HSD type 1, which is associated with a high specificity for C18 steroids, primarily

expression and these clinicopathological factors including age, tumour size, axillary lymph node involvement, grade, and histological type was previously reported (63, 64, 65).

Interestingly, Brodie et al. (66) found that tumours with a relatively

observed a significant trend towards an association between aromatase activity and the presence of ERα, alt ugh tumours expressing active aromatase included both ERα positive and negative tumours.

high aromatase activity tended to be ER-positive. Miller et al. (63) also


(IGF-1) and an albumin-like molecule isolated from breast tumour cytosol, were also found to regulate the conversions of oestrone to oestradiol (77).

Miller et al. (78) and Perel et al. (79) demonstrated that human breast and its neoplasms could produce 17beta estradiol in vitro. 17beta HSD type 1 was immunolocalized in the cytoplasm of carcinoma cells in 60% of invasive ductal carcinomas (80) whereas 17beta HSD type 2 immuno-reactivity was not detected in all cases examined.

A few immunohistochemical studies of 17beta HSD type 1 in human breast carcinoma have been reported and no clear relation to prognosis

however, have shown that 17beta HSD type 1 positive carcinoma cells of mammary epithelial proliferative lesions tend to be positive for ER (83) and 17beta HSD type 1 can be an independent prognostic marker in breast cancer patients (84). It was suggested that 17beta HSD type 1 played an important role in hormone-dependent breast carcinomas (85). In a study conducted by Gunnarson et al. (86), the authors found that a high level of 17beta HSD 1 indicated an increased risk of developing a late relapse of breast cancer. The authors suggested that abnormal expres-sion of 17beta HSD isoforms had prognostic significance in breast cancer and that altered expression of these enzymes could have importance in breast cancer progression. Feigelson et al. (87) found that a polymorphism in the gene for 17beta HSD type 1 could be used to identify women at an increased risk of developing advanced breast cancer. In principle, our recent study (62) supports these findings and highlights the significant relationship between poor survival and high expression of 17beta HSD 1 in breast cancer patients (Figure 3).

Based on the above reports, inhibition of intratumoral 17beta HSD type 1 activity or expression should be considered as a potential novel endocrine therapy and can contribute greatly to the suppression of oestrogen-dependent proliferation of tumour cells.

The development of potent inhibitors of 17beta HSD type 1 has been attempted by many researchers; Fischer et al. (88) have recently reported the potential of E-ring modified steroids as a useful template for the

and clinical parameters has been found (26, 81, 82). Recent studies,

The gene coding for 17beta HSD type 1 is located at 17q12-21 (72). 17beta HSD type 2, on the other hand, is an enzyme that converts E2 to E1 (73, 74) and plays important roles in the peripheral inactivation of

IL-6 and TNF-α as have been demonstrated to stimulate the activity of 17beta HSD type 1(75. 76). In addition, insulin-like growth factor type I

converts the inactive C18 steroid, oestrone, to the biologically active oestradiol (70, 71).

in target tissues. androgens and oestrogens, thus determining the steady oestrogen levels

design of specific inhibitors of 17beta HSD type 1. It is important


1.00 = High levels (cut-off point: 1000)).

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Figure 3. Kaplan-Meier analysis of disease-free survival of breast cancer patients

regulated and no correlations between these two enzymes have been reported in patients with breast cancer (80). Therefore inhibition of 17beta HSD type 1 might be a much more efficacious therapy than aromatase

HSD type 1 but not aromatase. Furthermore, it may become possible to employ future 17beta HSD

type 1 inhibitors as third or later lines of endocrine therapy after develop-ment of resistance against conventional endocrine therapy including ER antagonists or aromatase inhibitors in patients with intratumoral over-expression of 17beta HSD type 1.

inhibition in breast cancer patients whose tumours over-express 17beta

however to point out that aromatase and 17beta HSD type 1 are differently

(89, 90). The gene coding for human STS is located on the distal short

4.

arm of the X-chromosome and maps to Xp22.3-Xpter. STS gene is pseudo-

STEROID SULPHATASE (STS)

autosomal and escapes X-inactivation. It has been cloned, characterized,

STS is a member of a superfamily of 12 different mammalian sulfatases

and sequenced (91). On the Y-chromosome, there is a pseudogene forSTS, which is transcriptionally inactive as the promoter, and several exons

depending on the expression of 17beta HSD type 1 mRNA (P = 0.0182). (0 = Low levels


(R-5020) (95). In contrast, it was observed that exposure of MCF-7 and MDA-MB-231 breast cancer cells to the progestagen; medroxypro-gesterone acetate, stimulated STS activity in these cells (93).

Immunohistochemistry and STS mRNA expression of laser-captured micro-dissected samples were also used to examine the location of STS within breast tumours (82). STS immunoreactivity was detected in the cytoplasm of cancer cells with STS mRNA expression being detected in micro-dissected carcinoma cells but not in stromal cells.

STS hydrolyzes circulating oestron sulphate (E1-S) to E1 in various human tissues (96, 97, 98, 99, 100, 101) and acts on DHEAS which is considered the most abundant steroid secreted by the adrenal cortex reducing it to DHEA by the removal of the sulfate group. DHEA in turn can undergo reduction to Adiol (102) which is known to have affinity for

verted to an oestrogen in order to stimulate tumour growth. Further studies have revealed that DHEA and Adiol can directly activate the ER and stimulate the proliferation of breast cancer cells (104). Moreover, recent research has shown that DHEAS, DHEA, and Adiol can stimulate

tumours in vivo (105) and their ability to do so is blocked by the ER antagonist nafoxidene, but not by aromatase inhibitors. These results provide strong evidence that the stimulation of cell growth by DHEAS

blocked by an STS inhibitor.

consistent with the higher STS enzymatic activity that has been detected

STS mRNA expression was found to be an independent prognostic in malignant breast tissue (22, 107).

The STS mRNA expression in malignant breast tissue seems tobe significantly higher than in normal tissue (106). This finding is

ER and can stimulate the growth of ER positive breast cancer cells

Information about the molecular regulation of STS is still limited. It was observed that both basic fibroblast growth factor and IGF-I increase STS activity in a dose- and time-dependent manner in MCF-7 and MDA-MB- 231 breast cancer cells (93). Moreover, both cytokines TNFα and IL-6 upregulate STS enzyme activity in MCF-7 breast cancer cells.

rather than occurring via any changes in gene transcription or mRNA stability (94). Interestingly, STS mRNA levels decreased when MCF-7

However, this upregulation appears to be post-translationally mediated

have been deleted. The gene consists of 10 exons and spans 146 kb, with

breast cancer cells were treated with the progestagen; Promegestone

the intron sizes ranging from 102 bp up to 35 kb (92).

indicator in predicting relapse-free survival, with high levels of expres-

the proliferation of breast cancer cells in vitro and induce mammary

sion being associated with larger tumour size, lymph node metastasis,

in vitro (75, 103). This finding shows that Adiol does not need to be con-

increased risk of recurrence, and poor prognosis (28, 82, 107, 108).

occurs via an aromatase-independent pathway that can be potentially


inhibitors (111). However, accurate determination of STS and ER levels in tumour specimens is required in order to achieve the maximum potential benefits from STS inhibitors. Phase III clinical trials will determine the usefulness of such drugs.

Figure 4. Kaplan-Meier analysis of over all survival of breast cancer patients depending on the expression of STS mRNA (p = 0.0452). (0 = Low levels; 1.00 = High levels).

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demonstrated a significant correlation between High levels of STS mRNA and poor survival (Figure 4). In addition, STS mRNA levels were correlated with aromatase mRNA levels (109). Interestingly, high STS mRNA expression was observed to be associated with a poor prognosis in both pre- and postmenopausal women. This finding led to

synthesis may play an important role in the growth of breast tumours. Finally, both steroidal and non-steroidal STS inhibitors have been

recently developed and seem to be effective in depressing the prolife-ration of oestrogen-dependent MCF-7 cells (110). Since oestrogen formation from E1S and DHEAS (STS pathway) cannot be blocked by aromatase inhibitors, STS is thought to be a new molecular target for the treatment of oestrogen-dependent tumour post-SERM and/or aromatase

and prognosis applied only to ER positive tumours. Recently, our group

the suggestion that even in premenopausal women, intratumoral oestrogen

It was also reported that the association between STS mRNA expression


women (112). However, the important role of both STS and 17beta HSD type 1 in local estrogen production, provides novel potential targets for endocrine therapy. The inhibition of STS and 17beta HSD 1, in addition to aromatase inhibition, is believed to be very important in stopping the local production of estrogen and therefore the inhibition of development and recurrence of breast carcinoma. Such new strategies are currently being explored and the development of STS inhibitors and 17beta HSD 1 inhibitors is underway and the initial results are promising.

REFERENCES

1. Henderson IC, Canellos GP. Cancer of the breast: The past decade. N Engl J Med 1990; 302:17–30.

2. The Richard and Hinda Rosenthal Foundation award Lecture. Cancer Research 1988; 48:246.

3. Page DL, Dupont WD, Rogers LW, Rados MS. Atypical hyperplastic lesions of the female breast; a longterm follow-up study. Cancer 1985; 55:2698–2708.

4. London SJ, Connolly JL, Schnitt SJ, Colditz GA. A prospective study of benign breast disease and the risk of breast cancer. JAMA. 1992; 267:941–944.

5. Shekhar PVM, Werdell J, Barsrur VS. Environmental estrogen stimulation of growth and estrogen receptor function in preneoplastic and cancerous human breast cell lines. J Natl Cancer Inst 1997; 89:1774–1782.

6. Dao TL. The role of the ovarian steroid hormones in mammary carcinogenesis.

7. Pietras RJ, Marquez DC, Chen HW, Tsai E, Weinberg O, Fishbein M. Estrogen and growth factor receptor interactions in human breast and non-small cell lung cancer cells. Steroids. 2005; 70:372–381.

Henderson HS, Ross RK, Bernstein L. Oestrogens as a cause of human Cancer.

In Pike, MC, Siiteri PK, and Welsch, CW (eds) Hormones and breast cancer. (anbury repport no.8). Cold Spring Harbor Laboratory Press, Cold Spring Harbor,

5. SUMMARY

The local biosynthesis of oestrogens especially in postmenopausal women as a result of the interactions of various enzymes is believed to play a very important role in the pathogenesis and development of hor-mone dependent breast carcinoma. The overexpression of such enzymes seems to be associated with the development of a more aggressive disease and associated with poor outcome and increased local and distant recurrences. The understanding of the mechanisms that regulate these

Currently the third generation of aromatase inhibitors has revolutionised

and therapeutic strategies in postmenopausal females with hormone-dependant breast cancer.

enzymes is crucial to the development of new endocrine preventative

the treatment of oestrogen dependant breast cancer in postmenopausal

NY, 1981; pp. 281–295.


14. Berrino F, Muti P, Micheli A, Bolelli G, Krogh V, Sciajno R, Pisani P, Panico S,

Secreto G. Serum sex hormone levels after menopause and subsequent breast cancer. J Natl Cancer Inst 1996; 88:291–296.

15. Lipworth L, Adami HO, Trichopoulos D, Carlstrom K, Mantzoros C. Serum steroid hormone levels, sex hormone-binding globulin, and body mass index in the etiology of postmenopausal breast cancer. Epidemiology 1996; 7:96–100.

16. Dorgan JF, Stanczyk FZ, Longcope C, Stephenson Jr HE, Chang L, Miller R, Franz C, Falk RT, Kahle L. Relationship of serum dehydroepiandrosterone (DHEA), DHEA sulfate, and 5-androstene-3 beta, 17 beta-diol to risk of breast cancer in postmenopausal women. Cancer Epidemiol Biomarkers Prev 1997; 6:177–181.

17. Sasano H, Harada N. Intratumoral aromatase in human breast, endometrial, and ovarian malignancies. Endocrine Reviews 1998; 19:593–607.

18. Miller WR. Aromatase activity in breast tissue. J Steroid Biochem Mol Biol 1991; 39:783–790.

19. Schweikert HU, Milewich L, Wilson JD. Aromatization of androstenedione by cultured human fibroblasts. J Clin Endocrinol Metab 1976; 43:785–795.

20. Longcope C, Pratt JH, Schneider SN, Fineberg SE. Aromatization of androgens

152. 21. Sasano H, Uzuki M, Sawai T, Nagura H, Matsunaga G, Kashimoto O, Harada N.

Aromatase in human bone tissue. J Bone Miner Res 1997; 12:1416–1423. 22. Santner SJ, Chen S, Zhou D, Korsunsky Z, Martel J, Santen RJ. Effect of

androstenedione on growth of untransfected and aromatase-transfected MCF-7 cells in culture. J Steroid Biochem Mol Biol 1993; 44:611–616.

23. Chetrite GS, Cortes-Prieto J, Philippe JC, Wright F, Pasqualini JR. Comparison of estrogen concentrations, estrone sulfatase and aromatase activities in normal,

24. Pasqualini JR, Chetrite G, Blacker C, Feinstein MC, Delalonde L, Talbi M, 23–27. and in cancerous, human breast tissues. J Steroid Biochem Mol Bio 2000; 72:

8. Garvin S, Nilsson UW, Dabrosin C. Effects of oestradiol and tamoxifen on VEGF, soluble VEGFR-1, and VEGFR-2 in breast cancer and endothelial cells. Br J Cancer 2005; 93:1005–1010.

9. Redeuilh G, Attia A, Mester J, Sabbah M. Transcriptional activation by the oestrogen receptor alpha is modulated through inhibition of cyclin-dependent kinases. Oncogene 2002; 21:5773–5782.

10. Yue W, Wang JP, Hamilton CJ, Demers LM, Santen RJ. In situ aromatization enhances breast tumor estradiol levels and cellular proliferation. Cancer Res 1998; 58:927–932.

11. Silverberg SG. Immunolocalization of aromatase, 17 alpha-hydroxylase and side-chain-cleavage cytochromes P-450 in the human ovary. Journal of Reproduction & Fertility 1989; 85:163–169.

12.

13. Labrie F, Luu-The V, Labrie C, Belanger A, Simard J, Lin SX, Pelletier G. Endocrine and intracrine sources of androgens in women: inhibition of breast cancer and other roles of androgens and their precursor dehydroepiandrosterone. Endocrine Reviews 2003; 24:152–182.

Sasano H. Okamoto M, Mason JI, Simpson ER, Mendelson CR, Sasano N,

81–89. cycling ovary and steroidproducing neoplasms. Endocrine Pathology 1994; 5:Sasano H. Functional pathology of human ovarian steroidogenesis: Normal

Maloche C. Concentrations of estrone, estradiol, and estrone sulfate and evaluation

by muscle and adipose tissue in vivo. J Clin Endocrinol Metab 1978; 46:146–

of sulfatase and aromatase activities in pre- and postmenopausal breast cancer patients. Clin Endocr Metab 1996; 81:1460–1464.


31.

Graham-Lorence S, Amarneh B, Ito Y, Fisher CR, Michael MD, Mendelson CR, Bulun SE. Aromatase cytochrome P450, the enzyme responsible for estrogen

32. Simpson ER. Sources of estrogen and their importance. J Steroid Biochem Mol Biol 2003; 86:225–230.

33.

34. Simpson ER, Davis SR. Minireview. Aromatase and the regulation of estrogen biosynthesis- some new perspectives. Endocrinology 2001; 142:4589–4594.

35. Simpson ER, Mahendroo MS, Means GD, Kilgore MW, Corbin CJ, Mendelson

36. Sebastian S, Bulun SE. A highly complex organization of the regulatory region of

37.

38.

Commun 1993; 189:1001–1007. 39.

aromatase messenger RNA by reverse transcription-polymerase chain reaction

40. Bulun SE, Noble LS, Takayama K, Michael MD, Agarwal V, Fisher C, Zhao Y, Hinshelwood MM, Ito Y, Simpson ER. Endocrine disorders associated with

61: 133–139. 41. Simpson ER, Michael MD, Agarwal VR, Hinshelwood MM, Bulun SE, Zhao Y.

Cytochromes P450 11: expression of the CYP19 (aromatase) gene: an unusual case of alternative promoter usage. FASEB J 1997; 11:29–36.

42. Harada N, Utsumi T, Takagi Y. Tissue-specific expression of the human aromatase cytochrome P-450 gene by alternative use of multiple exons 1 and

biosynthesis. Endocr Rev 1994; 15:342–355.

2001; 45:116–124.

upregulated in breast cancer tissue Mol Endocrinol 2002; 16:2243–2254.

of tissue-specific exons 1 in human skin fibroblasts. Biochem Biophys Res Harada N. A unique aromatase (P-450arom) mRNA formed by alternative use

(RT-PCR). J Steroid Biochem Mol Biol 1996; 59:163–171.

inappropriately high.aromatase expression. J Steroid Biochem Mol Biol 1997;

25. Pasqualini JR. The selective estrogen enzyme modulators in breast cancer: a

26. Reed MJ, Owen AM, Lai LC, Coldham NG, Ghilchik MW, Shaikh NA, James VHT. In situ oestrone synthesis in normal breast and breast tumour tissues: effect

27. Reed MJ, Purohit A, Howarth NM, Potter BVL. Steroid sulphatase inhibitors: a

28. Miyoshi Y, Ando A, Hasegawa S, Ishitobi M, Taguchi T, Tamaki Y, Noguchi S. High expression of steroid sulfatase mRNA predicts poor prognosis in patients with estrogen receptor-positive breast cancer. Clin Cancer Res 2003; 9:2288–2293

29. Nelson DR, Koymans L, Kamataki T, stegeman JJ, Feyereisen R, Waxman DJ, Waterman MR, Gotoh O, Coon MJ, Estabrook RW, Gunsalus IC, Nebert DW. P450 superfamily: Update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 1996; 6:1–42.

30. Chen SA, Besman MJ, Sparkes RS, Zollman S, Klisak I, Mohandas T, Hall PF,

assignment of the gene to chromosome 15. DNA 1988; 7:27–38.

review. Biochimica et Biophysica Acta 2004; 1654:123–143.

new endocrine therapy. Drugs Future 1994; 19:673–680.

of treatment with 4-hydroxyandrostenedione. Int J Cancer 1989; 44:233–237.

Shively JE. Human aromatase: cDNA cloning, Southern blot analysis, and

the human CYP 19 (Aromatase) gene revealed by the Human Genome Project. J Clin Endocrinol Metab 2001; 86:4600–4602.

a novel endothelial promoter of the human CYP19 (aromatase P450) gene that is

CR. Tissue specific promoters regulate aromatase cytochrome P450 expression.

Sebastian S, Takayama K, Shozu M, Bulun SE. Cloning and characterization of

Simpson ER, Mahendroo MS, Means GD, Kilgore MW, Hinshelwood MM,

gene expression and its exon I usage in human breast tumors. Detection of

J Steroid Biochem Mol Biol 1993; 44:321–330.

Zhou C, Zhou D, Esteban J, Murai J, Siiteri PK, Wilczynski S, Chen S. Aromatase

Nelson LR, Bulun SE. Estrogen production and action. J Am Acad Dermatol


49. Zhao Y, Nichols JE, Bulnn SE, Mendelson CR, Simpson ER. Aromatase P450

50. Singh A, Purohit A, Wang DY, Duncan LJ, Ghilchik MW, Reed MJ. IL-6sR: release from MCF-7 breast cancer cells and role in regulating peripheral

51. Stimulation of aromatase activity in breast fibroblasts by tumour necrosis factor α Molec Cell Endocr 1994; 106:17–21.

52. Agarwal V, Bulun SE, Leitch M, Rohrich R, Simpson ER. Use of alternative promoters to express the aromatase P450 (CYP19) gene in breast adipose tissues of cancer-free and breast cancer patients. J Clin Endocrinol Metab 1996;

53. Zhao Y, Agarwal VR, Mendelsohn CR, Simpson ER. Transcriptional regulation of CYP19 (aromatase) gene expression in adipose stromal cells in primary culture. J Steroid Biochem Mol Biol 1997; 61 (3-6) 203–210.

54. Singh A, Purohit A, Ghilchik MW, Reed MJ. The regulation of aromatase

55. breast. Steroids 1987; 50:537–547.

56.

breast adipose tissue aromatase cytochrome P450 transcripts using competitive

1993; 77:1622–1628. 57. Utsumi T, Harada N, Maruta M, Takagi Y. Presence of alternatively spliced

transcripts of aromatase gene in human breast cancer. J Clin Endocrinol Metab 1996; 81:2344–2349.

58.

59. mammalian cells - A useful system for aromatase inhibitor screening. Cancer Res

60.

gene expression in human adipose tissue. J Biol Chem 1995, 270:16449–16457.

oestrogen synthesis. J Endocrinol 1995; 147:R9–R12.

81:3843–3849.

Endocr-Relat Cancer 1999; 6:139–147.

1990; 50:6949–6954.

promoters, and switching of tissuespecific exons 1 in carcinogenesis. Proc Natl

43. Esteban JM, Warsi Z, Haniu M, Hall P, Shively JE, Chen S. Detection of intratumoral aromatase in breast carcinomas. Am J Pathol 1992; 140:337–343.

44. Purohit A, Ghilchik MW, Duncan LJ, Wang DY, Singh A, Walker MM, Reed MJ. Aromatase activity and interleukin 6 production by normal and malignant

45. aromatase in breast tumours: the role of the immune system. J Steroid Biochem Mol Biol 1997; 61:185–192.

46. Reed MJ, Coldham NG, Patel SR, Ghilchik MW, James VHT. Interleukin-1 and interleukin-6 in breast cyst fluid: their role in regulating aromatase activity in breast cancer cells. J Endocrinol 1992, 132:R5–R8.

47. Reed MJ, Purohit A. Sulphatase inhibitors: the rationale for the development of a

48.

Acad Sci USA 1993; 90:11312–11316.

breast tissues. J Clin Endocrinol Metab 1995; 80:3052–3058.

Miller WR, Mullen P. Factors influencing aromatase activity in the breast. J Steroid

Singh A, Purohit A, Duncan LJ, Mokbel K, Ghilchik MW, Reed MJ. Control of

Biochem Molec Biol 1993; 44:597–604.

new endocrine therapy. Rev Endocr Rel Cancer 1993; 45:51–62.

Macdiarmid F, Wang DY, Duncan LJ, Purohit A, Ghilchik MW, Reed MJ.

James VHT, McNeill JM, Lai LC, Newton CJ, Ghilchik MW, Reed MJ. Aromatase

Miller WR, O’Neill JS. The importance of local synthesis of estrogen within the

activity in normal breast and breast tumor tissues: in vivo and in vitro studies.

activity in breast fibroblasts: the role of interleukin- 6 and prostaglandin E2.

Steroids 1987; 50:269–279. Zhou D, Pompon D, Chen S. Stable expression of human aromatase cDNA in

breast cancer and local estrogen biosynthesis suggested by quantification of

Yue W, Zhou D, Chen S, Brodie A. A new nude mouse model for postmeno-

Bulun SE, Price TM, Aitken J, Mahendroo MS, Simpson ER. A link between

pausal breast cancer using MCF-7 cells transfected with the htmmn aromatase

polymerase chain reaction after reverse transcription. J Clin Endocrinol Metab

gene. Cancer Res 1994; 54:5092–5095.


67. The ATAC (Arimidex Tamoxifen alone or in Combination) Trialists’ Group, Anastrozole alone or in combination with tamoxifen versus tamoxifen alone for adjuvant treatment of postmenopausal women with early breast cancer: first results of the ATAC randomised trial Lancet 2002; 359:2131–2139.

68. 217–22.

69.

70. Luu-The V, Labrie C, Zhao HF, Couet J, Lachance Y, Simard J, Leblanc G, Cote J,

17beta-dehydrogenase and assignment of the gene to chromosome 17: evidence of two mRNA species with distinct 50 -termini in human placenta. Molecular Endocrinology 1989; 3:1301–1309.

71. Miettinen MM, Mustonen MVJ, Poutanen MH, Isomaa VV, Vihko RK. Human 17b-hydroxysteroid dehydrogenase type 1 and type 2 isozymes have opposite activities in cultured cells and characteristic celland tissue-specific expression. Biochem J 1996; 314:839–845.

72. Gunnarsson C, Ahnstrom M, Kirschner K, Olsson B, Nordenskjold B, Rutqvist LE, Skoog L, Stal O. Amplification of HSD17B1 and ERBB2 in primary breast

73. Takeyama J, Sasano H, Suzuki T, Iinuma K, Nagura H, Andersson S. 17Beta-

74. Takeyama J, Suzuki T, Hirasawa G, Muramatsu Y, Nagura H, Iinuma K, Nakamura J, Kimura KI, Yoshihama M, Harada N, Andersson S, Sasano H. 17beta-hydroxysteroid dehydrogenase type 1 and 2 expression in the human

75. Adams EF, Rafferty B, White MC. Interleukin-6 is secreted by breast fibroblasts and stimulates 17beta oxidoreductase activity in MCF-7 breast cancer cells: possible paracrine regulation of 17beta oestradiol levels. Int J Cancer 1991; 49:118–121.

76. Duncan LJ, Coldham NG, Reed MJ. The interaction of cytokines in regulating oestradiol 17beta hydroxysteroid dehydrogenase activity in MCF-7 cells. J Steroid Biochem Mol Biol 1994; 49:63–68.

Cunnick GH, Mokbel K. Aromatase inhibitors. Curr Med Res Opin 2001; 17:

Mokbel K. The evolving role of aromatase inhibitors in breast cancer. Int J Clin Oncol 2002; 7:279–283.

Berube D, Gagne R, Labrie F. Characterization of cDNAs for human estradiol

cancer. Oncogene 2003; 22:34–40.

hydroxysteroid dehydrogenase types 1 and 2 in human placenta: an immunohistochemical study with correlation to placental development. J Clin EndocrinMetabol 1998; 83:3710–3715.

fetus. J Clin Endocrinol Metabol 2000; 85:410–416.

61. Biological effects of stable overexpression of aromatase in human hormone-dependent breast cancer ceils. Br J Cancer 1994; 69:77–83.

62. Salhab M, Reed MJ, Al Sarakbi W, Jiang WG, Mokbel K. The role of aromatase and 17-beta hydroxysteroid dehydrogenase type 1 mRNA expression in predicting the clinical outcome of human breast cancer. Breast Cancer Res Treat

63.

64.

65. Zhang Z, Yamashita H, Toyama T, Hara Y, Omoto Y, Sugiura H, Kobayashi S,

Treat 2002; 74:47–53. 66.

Miller WR, Anderson TJ, Lack WJL. Relationship between tumor aromatase

Biol 1990; 37:1055–1059.

of aromatase and other steroidogenic enzymes in human breast disorders.Hure Pathol 1994; 25:530–535.

Sasano H, Nagura H, Harada N, Goukon Y, Kimyra M. Immunolocalization

Macaulay VM, Nicholls JE, Gledhi UJ, Rowlands MG, Dowsett M, Ashworth A.

2006; 99(2):155–162. Epub 2006 Mar 16.

Harada N, Iwase H. Semi quantitative immunohistochemical analysis of aromatase

activity, tumor characteristics and response to therapy. Steroid Biochem Molec

expression in ductal carcinoma in situ of the breast. Breast Cancer Res Treat

J Steroid Biochem Mol Biol 1997; 61:281– 286. Brodie A, Lu Q, Nakamura J. Aromatase in the normal breast and breast cancer.


84. Oduwole OO, Li Y, Isomaa VV, Mantyniemi A, Pulkka AE, Soini Y, Vihko PT. 17beta-hydroxysteroid dehydrogenase type 1 is an independent prognostic marker in breast cancer. Cancer Res 2004; 64:7604–7609.

85. Zhang Z, Yamashita H, Toyama T, Omoto Y, Sugiura H, Hara Y, Wu X, Kobayashi S, Iwase H. Quantitative determination, by real-time reverse transcription polymerase chain reaction, of aromatase mRNA in invasive ductal carcinoma of the breast. Breast Cancer Res 2003; 5:250–256.

86. Gunnarsson C, Olsson BM, Stal O. Southeast Sweden Breast Cancer GroupAbnormal expression of 17beta-hydroxysteroid dehydrogenases in breast

87. Feigelson, SH, McKean-Cowdin, R, Coetzee, GA, Stram, DO, Kolonel, LN, and Henderson, BE. Building a multigenic model of breast cancer susceptibility: CYP 17 and HSD 17 beta 1 are two important candidates. Cancer Res 2001; 61:785–789.

88. Fischer DS, Allan GM, Bubert C, Vicker N, Smith A, Tutill HJ, Purohit A, Wood L, Packham G, Mahon MF, Reed MJ, Potter BV. E-ring modified steroids as novel

89. Bond CS, Clements PR, Ashby SJ, Collyer CA, Harrop SJ, Hopwood JJ, Guss

90. Ferrante P, Messali S, Meroni G, Ballabio A. Molecular and biochemical characterisation of a novel sulphatase gene: aryl sulphatase G (ARSG). Eur J

91. Yen PH, Marsh B, Allen E, Tsai SP, Ellison J, Connoly L, Neiswanger K, Shapiro LJ. The human X-linked steroid sulfatase gene and a Y-encoded pseudogene: evidence for an inversion of the Y chromosome during primate

92. Reed MJ, Purohit A, Woo LW, Newman SP, Potter BV. Steroid sulfatase: molecular biology, regulation, and inhibition. Endocr Rev 2005; 26(2):171–202.

93. Purohit A, Reed MJ. Oestrogen sulphatase activity in hormone-dependent and hormone-independent breast cancer cells: modulation by steroidal and non-

and intraductal proliferative lesions of the human breast. Anticancer Res 2000; 20:1101–1108.

cancer predicts late recurrence. Cancer Res. 2001; 61:8448–8451.

potent inhibitors of 17 beta-hydroxysteroid dehydrogenase type 1. J Med Chem 2005; 48:5749–5770.

Hum Genet 2002; 10:813–818.

evolution. Cell 1988; 55:1123–1135.

steroidal therapeutic agents. Int J Cancer 1992; 50:901–905.

JM. Structure of a human lysosomal sulfatase. Structure 1997; 5:277–289.

77. Singh A, Blench I, Morris HR, Savoy L-A, Reed MJ. Synergistic interaction of growth factors and albumin in regulating estrogen synthesis in breast cancer cells. Mol Cell Endocrinol 1992; 85:165–173.

78. Miller WR, Hawkins RA, Forrest APM. Significance of aromatase activity in human breast cancer. Cancer Res 1982; 42:3365–3368.

79. Perel E, Wilkins D, Killinger DW. The conversion of androstenedione to estrone,

80. Suzuki T, Moriya T, Ariga N, Kaneko C, Kanazawa M, Sasano H. 17Beta-hydroxysteroid dehydrogenase type 1 and type 2 in human breast carcinoma: a

81. Sasano H, Frost AR, Saitoh R, Harada N, Poutanen M, Vihko R, Buhm SE, Silverberg SG, Nagura H. Aromatase and 17beta hydroxysteroid dehydrogenase

82. Suzuki T, Nakata T, Miki Y, Kaneko C, Moriya T, Ishida T, Akinaga S, Hirakawa H, Kimura M, Sasano H. Estrogen sulfotransferase and steroid

83. beta-Hydroxysteroid dehydrogenase type 1 and type 2 in ductal carcinoma in situ

correlation to clinicopathological parameters. Br J Cancer 2000; 82:518–523.

type 1 in human breast carcinoma. J Clin Endocrinol Metab 1996; 11:4042–4046.

sulfatase in human breast carcinoma. Cancer Res 2003; 63:2762–2770. Ariga N, Moriya T, Suzuki T, Kimura M, Ohuchi N, Satomi S, Sasano H. 17

estradiol and testosterone in breast tissue. J Steroid Biochem, 1980; 13:89–94.


101. X-linked ichthyosis. Effects on the structure and function of the steroid sulfatase

102. Poortman J, Andriesse R, Agema A, Douker GH, Schwarz F, Thijssen JHH. Adrenal androgen secretion and metabolism in postmenopausal women. In: Genazzani AR, Thijssen JHH, Siiteri PK, eds. Adrenal androgens. Raven Press,

103. Poulin R, Labrie F. Stimulation of cell proliferation and estrogenic response by adrenal C19- delta 5-steroids in the ZR-75–1 human breast cancer cell line.

104. Maggiolini M, Donze O, Jeannin E, Ando S, Picard D. Adrenal androgens stimulate the proliferation of breast cancer cells as direct activators of estrogen

105. Morris KT, Toth-Fejel SE, Schmidt J, Fletcher WS, Pommier RF. High dehydroepiandrosterone-sulfate predicts breast cancer progression during new aromatase inhibitor therapy and stimulates breast cancer cell growth in tissue

106. Utsumi T, Yoshimura N, Takeuchi S, Maruta M, Maeda K, Harada N. Elevated steroid sulfatase expression in breast cancer. J Steroid Biochem Mol Biol 2000;

107. Evans TR, Rowlands MG, Law M, Coombes RC. Intratumoral oestrone sulphatase activity as a prognostic marker in human breast carcinoma. Br J Cancer 1994; 69:555–561.

108. Utsumi T, Yoshimura N, Takeuchi S, Ando J, Maruta M, Maeda K, Harada N. Steroid sulfatase expression is an independent predictor of recurrence in human breast cancer. Cancer Res 1999; 59:377–381.

109. Al Sarakbi W, Mokbel R, Salhab M, Jiang WG, Reed MJ, Mokbel K. The Role of STS and OATP-B mRNA Expression in Predicting the Clinical Outcome in

110. Selcer KW, Hegde PV, Li PK. Inhibition of estrone sulfatase and proliferation of

New York, 1980; 219–240.

Cancer Res 1986; 46:4933–4937.

receptor alpha. Cancer Res 1999; 59:4864–4869.

culture: a renewed role for adrenalectomy. Surgery 2001; 130:947–953.

73:141–145.

Human Breast Cancer. Anticancer Research 2006; 26 (In Press)

human breast cancer cells by nonsteroidal (p-O-sulfamoyl)-N-alkanoyl tyra-mines. Cancer Res 1997; 57:702–707.

Alperin ES, Shapiro LJ. Characterization of point mutations in patients with

protein. J Biol Chem 1997; 272:20756–20763.

94. Newman SP, Purohit A, Ghilchik MW, Potter BVL, Reed MJ. Regulation of steroid sulphatase expression and activity in breast cancer. J Steroid Biochem

95. Pasqualini JR, Maloche C, Maroni M, Chetrite G. Effect of the progestagen Promegestone (R-5020) on mRNA of the oestrone sulphatase in the MCF-7

96. Reed MJ. The role of aromatase in breast tumors. Breast Cancer Res Treat 1994;

97.

98. Reed MJ, Topping L, Coldham NG, Purohit A, Ghilchik MW, James VHT. Control of aromatase activity in breast cancer cells: the role of cytokines and

99. Reed MJ, Purohit A. Inhibition of steroid sulphatases. In: Sandler M, Smith HJ, eds. Design of enzyme inhibitors as drugs, vol. 2. Oxford: Oxford University

100. Basler E, Grompe M, Parenti G, Yates J, Ballabio A. Identification of point mutations in the steroid sulfatase gene of three patients with X-linked ichthyosis.

Mol Biol 2000; 75:259–264.

human mammary cancer cells. Anticancer Res 1994; 14:1589–1594.

30:7–17.

sulphatase inhibitors. Endocr Rel Cancer 1996; 3:9–23.

Press 1994; 481–494.

Reed MJ, Purohit A, Woo LWL, Potter BVL. The development of steroid

growth factors. J Steroid Biochem Mol Biol 1993; 44:589–596.

Am J Hum Genet 1992; 50:483–491.

111. Nakata T, Takashima S, Shiotsu Y, Murakata C, Ishida H, Akinaga S, Li PK, Sasano H, Suzuki T, Saeki T. Role of steroid sulfatase in local formation of estrogen in post-menopausal breast cancer patients. J Steroid Biochem Mol Biol 2003; 86:455–460.

112. Mokbel R, Karat I, Mokbel K. Adjuvant endocrine therapy for postmenopausal breast cancer in the era of aromatase inhibitors: an update. Int Semin Surg Oncol 2006; 3:31.

Salhab and Mokbel170

Chapter 9

THE ROLE OF THE HGF REGULATORY FACTORS IN BREAST CANCER

Christian Parr and Wen G Jiang Metastasis and Angiogenesis Research Group, School of Medicine, Cardiff University,

Hepatocyte growth factor (HGF) plays a pivotal role in the invasion and motility of breast cancer cells, and is also a key angiogenic and lym-phangiogenic factor. The cytokine, which is primarily synthesised as inactive pro-HGF by stromal fibroblasts in breast tumours, requires activation to function as a biologically active factor. A number of pro-HGF activators have been identified in recent years, together with some naturally occurring activation inhibitors. This chapter discusses the impact of the HGF activators and activation inhibitors in the development and metastasis of breast cancer, and discusses their potential therapeutic value.

breast cancer, hepatocyte growth factor, HGF activator (HGFA), HAI-1,

1. INTRODUCTION

Breast cancer is one of the leading causes of cancer death worldwide (1). It is by far the commonest form of cancer in women, and was responsible for 27.4% of all new cancer cases, and 17.4% of all cancer-related death of European women in 2004 (2). Cancer metastasis is the single most important factor influencing cancer patient mortality. Controlling the metastatic spread of tumours remains a crucial target for the successful treatment of cancer.

The metastatic cascade is a complex multistep process which is influenced by a number of factors, including (i) the genetic events of cancer cells, (ii) extrinsic aspects such as cytokines and paracrine factors, (iii) immune cells and (iv) the micro-environment of the primary tumour and the host organ, for example the bone micro-environment (as dis-cussed in chapter 12). Amongst these factors, interactions between tumour cells and the surrounding environment are thought to be essential in the

© 2007 Springer.


HAI-2, matriptase, c-MET

Heath Park, Cardiff, CF14 4XN, UK

Abstract:

Keywords:

172 Parr and Jiang

produced by stromal cells are vital to the development of metastasis. Stromal cells, mainly fibroblasts in mammary tumours, produce a rich array of cytokines. One of the most documented stromal-derived factors is a cytokine known as hepatocyte growth factor (HGF). HGF plays a plethora of roles in tumour growth and metastasis. This cytokine demon-strates the ability to stimulate proliferation, dissociation, migration, and invasion in a wide variety of tumour cells, and is also a potent angio-genic and lymphangiogenic factor. The investigation of HGF, and the factors that govern the influence of HGF, may therefore lead to exciting new strategies to combat the metastatic spread of tumours.

HGF was originally identified in 1984, from the serum of partially hepatectomised animals, as a protein that was able to stimulate DNA synthesis and growth of hepatocytes (3–5). Soon after the initial dis-covery of HGF, a protein termed scatter factor (SF) was isolated by a separate group working within a different field. Scatter factor was identified as a fibroblast-derived protein that demonstrated the ability to scatter tightly packed colonies of epithelial cells (6). However, subse-quent structural and functional studies revealed HGF and SF to be identical proteins (7–10).

HGF is synthesised as a single chain peptide of 728 amino acid resi-dues. This biologically inactive form is known as pro-HGF and requires enzymatic processing to generate the active, heterodimeric form of HGF (11–13). The mature active form of HGF is composed of a 69kDa α-chain and a 34kDa serine protease-like β-chain (14) (Figure 1). The α-chain contains the N-terminal hairpin domain and four kringle domains that are essential for the correct biological functioning of the molecule (15, 16). HGF’s domain structure and proteolytic mechanism of active-tion are similar to that of the serine protease known as plasminogen, although HGF is devoid of protease activity. Interestingly, HGF is thought to have evolved from the same ancestral gene as plasminogen and hepatocyte growth factor-like/macrophage stimulating protein (17). Under normal conditions, the active form of HGF plays a role in the development of the liver, placenta, skeletal muscle, and is also involved in the tissue regeneration process (18, 19).

Tumour-stromal interactions are known to facilitate the metastatic spread of cancer. In the last 15 years HGF has attracted considerable

AND ITS RECEPTOR, C-MET 2. HEPATOCYTE GROWTH FACTOR

early development of tumours. Beyond the classical cell–matrix inter-actions (as already discussed in chapter 4), cytokines and growth factors

attention as a stromal-derived mediator of tumour-stromal interactions, particularly due to its key involvement in cancer invasion and metastasis.

9. HGF regulators in metastasis 173

detected in a variety of carcinoma tissues, including breast, thereby sug-

The various cellular responses to HGF are mediated through a cell surface receptor specific to HGF (Figure 2). This receptor is a protein encoded by the c-MET proto-oncogene, known as c-Met. c-Met is a receptor tyrosine kinase and is the prototype of a distinct subfamily, which also includes Ron and Sea, and was originally identified as an activated oncogene in an osteosarcoma cell line (27). The expression of c-Met by tumour cells has since been shown to be associated with tumour progression (26, 28–34).

Figure 1. Hepatocyte growth factor. Schematic representation of single chain inactive pro-HGF and the mature heterodimeric form of biologically active HGF.

The receptor protein arises from a single polypeptide precursor, which undergoes co- and post-translational glycosylation and endopro-

may not be the case in all tumours, as HGF production has also been

gesting an autocrine mechanism of stimulation within tumours (22–26).

Originally, HGF was considered to be produced by cells of mesenchymal origin and act on epithelial cells through a paracrine mechanism of stimulation (20, 21). However, increasing evidence suggests that this

teolytic cleavage (35). c-Met is a 190kDa heterodimer composed of two disulphide-linked chains, an extracellular 50kDa α-chain and a trans-membrane 145kDa β-chain, both of which are necessary for c-Met bio-logical activity. The α-chain is exposed at the cell surface whilst the

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dimerisation, followed by trans-phosphorylation of regulatory tyrosines, which is critical for receptor activation (38). The C-terminal domain is responsible for the biological activity and, upon phosphorylation of specific tyrosine residues, provides a docking site for multiple signal transducers and adaptors (39). The bulk of receptor signalling activity is funneled through this multifunctional docking site made of the tandemly arranged degenerate sequence YVH/NV. The SH2 domains of the PI 3 kinase (phosphatidylinositol 3-kinase), phospholipase C-γ, c-AktShc, and pp60c-Src bind with quick association and dissociation rates to either of the phosphotyrosines in the sequence Y1349VHVNATY1356VNV, where both residues can be phosphorylated simultaneously (39–44). The Grb2-associated receptor (Gab 1) has been identified as a multisubstrate adapter protein that associates with c-Met to mediate epithelial morphogenesis (45), and also acts as an inhibitor to HGF signalling pathways, down-stream of PI3K, for cell survival and DNA repair (46, 47).

The c-Met receptor is expressed by a wide variety of epithelial cells, whereas its ligand, HGF, is normally produced by the stromal tissues. Interestingly, possible autocrine signalling mechanisms have also been

Figure 2. The c-Met receptor. The receptor is composed of two disulphide linked chains; a 50 kDa α-chain and a 145 kDa β-chain. The β-chain contains the tyrosine kinase domain and a docking site which interacts with signalling molecules upon HGF complexing.

β-chain spans the cell membrane and possesses an intracellular tyrosine kinase domain (36, 37). HGF binds to the β-chain and induces receptor

demonstrated in human carcinomas of the breast, lung, colon, and pro-state through the co-expression of HGF and c-Met in the tumour tissue (26, 28, 48–52).

9. HGF regulators in metastasis 175 3. HGF, C-MET, AND METASTASIS

The presence of metastatic disease in cancer patients is the most significant factor affecting their survival (53). Many studies have demon-strated that tumour cell stimulation with HGF results in an enhancement of cellular functions that are central to the process of metastasis. HGF, upon complexing with its specific receptor, c-Met, evokes an array of biological actions within cancer cells, such as enhanced cell migration, matrix degradation, invasion, and induction of angiogenesis. The signi-ficance of HGF activity in cancer development and progression has also been confirmed through clinical studies; where the level of HGF and its receptor correlated with disease progression and prognosis of cancer patients. The significance of the HGF-Met complex in cancer has been reviewed recently (54, 55).

Our studies, and others, have shown that in contrast to what occurs in normal epithelium, HGF and Met are frequently overexpressed in a wide variety of cancers, including invasive human breast carcinomas (26, 50, 56–59). The forced expression of HGF within mouse mammary epithet-lium led to the formation of metastatic carcinomas (60), however, the mechanism behind HGF expression in breast carcinoma cells is currently unclear. A recent study suggests it may be in response to an activating function of c-Src and Stat3 on HGF transcription (61). Reports demon-strate that patients with breast cancer have elevated serum HGF levels; however, following removal of the malignant breast tumours serum HGF levels decrease (62–65). Elevated HGF expression levels correlate with disease progression, with levels rising in cases of recurrence (66–68). One study even reports that the immunoreactive level of HGF was a stronger independent predictor of recurrence and survival than that of lymph node involvement (69). We also report that HGF levels are elevated in breast cancer patients with an overall poor prognosis, in comparison with patients who remained disease-free (26). These observations suggest that establishment of an autocrine HGF loop and sustained activation of the Met-signalling pathway in carcinoma cells may promote progression to more aggressive cancers.

The status of the c-Met receptor has also been evaluated in a wide

by many tumours, including tumours of the breast, thyroid, ovary, pan-

c-Met will result in the tumour being more sensitive to the influence of

variety of cancers. These reports demonstrate that c-Met is overexpressed

creas, prostate, and gastrointestinal tract (70–75). An elevated level of

HGF. Reports reveal that c-Met overexpression is associated with breast cancer progression and poor outcome in breast cancer patients (48, 76–79). Interestingly, c-Met has also demonstrated the ability to act as an independent prognostic factor for breast cancer patients, when compared against traditional breast markers in a multivariate analysis comparison (80). Collectively, these studies have strongly indicated that

176 Parr and Jiang

In the current chapter, we focus on the biological and clinical aspects of the hepatocyte growth factor regulators in breast cancer.

4. THE HGF REGULATORY FACTORS

Interactions between tumour cells and their surrounding stromal envi-ronment play a key role in modulating the aggressive nature of tumour invasion and metastasis (82–84). HGF is synthesised and released by stromal fibroblasts as an inactive single chain precursor, known as pro-HGF, and requires site-specific cleavage to function as a biologically active cytokine (85, 86). A number of proteases have been proposed as possessing HGF-converting properties, however, the initial factor reported to convert inactive pro-HGF to active HGF was a serine protease known as HGF activator (HGFA) (87). Additional factors which possess pro-HGF converting ability include the pro-metastatic factors known as matriptase, hepsin, and uPA (88–90). It is the activation of HGF that forms a key step in governing the influence of HGF in cancer metastasis. Recent studies have also described two serine protease inhibitors with

Figure 3. The HGFA and matriptase serine proteases convert inactive HGF into the active form of HGF. HAI-1 and HAI-2 are two Kunitz-type inhibitors that suppress the proteolytic activation of HGF, through inhibition of HGFA and matriptase action.

HGF and its receptor are potential therapeutic targets in cancer treat-ment. This aspect has been documented in recent articles (54, 55, 81).

the ability to bind to the HGF activators, and block the pro-HGF con-version properties. These inhibitors were termed HGFA inhibitor type-1 and type-2 (HAI-1 and HAI-2) (91, 92). HAI-1 and HAI-2 are regulators of HGF action, and may therefore limit the pro-metastatic effects of HGF on tumour cells (Figure 3). We report that the degree to which HGF,c-Met, HGFA, matriptase, HAI-1 and HAI-2 are expressed within breast


4.1 The Activators of HGF

HGF is secreted as the inactive single chain form of pro-HGF, and is unable to exert any biological influence until it has been activated, via site-specific proteolytic cleavage, into the biologically active form of HGF (11, 85, 86). Activation occurs through the extracellular hydrolysis of the Arg494-Val495 peptide bond of pro-HGF. This cleavage generates the active 2-chain form of mature HGF. Once converted to this hetero-dimeric form, HGF is able to stimulate numerous responses, via c-Met stimulation in the target cells. HGF activator (HGFA) was originally thought to be the main serine protease responsible for the active HGF conversion (93, 94); however, several other factors have since demon-strated pro-HGF converting abilities. These enzymes include the hepato-cyte growth factor-converting enzyme (95), blood coagulation factor XIIa (96) and, albeit weakly, both types of plasminogen activator, uPA (urokinase-type) and tPA (tissue-type) (89, 97). Although, it appears HGFA appears to be a far more potent converter of pro-HGF to HGF than these enzymes (88). In recent years several other proteases have revealed a more potent pro-HGF converting ability; these include the proteases known as matriptase and hepsin (88, 90). However, the predo-minant converters of pro-HGF in the breast tissues are HGFA and matrip-tase, as these serine proteases process pro-HGF at a similar rate (98).

HGFA is a blood coagulation factor XII-like serine protease, respon-sible for the activation of HGF in tumours and injured tissues. Shimomura et al. (99) first reported the purification of a HGF-converting enzyme present in fetal bovine serum. Subsequently, a protease was purified from human serum with the ability to convert pro-HGF into the active form of HGF in vitro, and was thus termed HGF Activator (HGFA) (87). HGFA has since been shown to be the key mediator of the localised activation of HGF in injured tissue (94).

Many serine proteases are generated from their precursors, via limited proteolysis, upon the initiation of blood coagulation. HGFA appears to follow this trend, as HGFA also exists as a precursor form in the plasma.

4.1.1 HGFA

cancer tissues determines the biological activity of HGF (26). Therefore, these factors have direct bearing on the metastatic spread of cancer cells.

The HGFA precursor is made up of a single polypeptide chain, consist-ing of 655 amino acids, has a molecular weight of around 96kDa, and has no HGF converting ability (100). The cDNA sequence for this novel serine protease revealed that the active form of HGFA is derived from the COOH-terminal region of a precursor protein, and is composed of multiple domains. The chromosomal location of the HGFA gene has been determined as 4p16 (101).

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HGFA precursor. They identified thrombin as the most effective protease for cleavage of the precursor. This cleavage occurred, via limited proteoly-sis, at the bond between Arg407 and Ile408, in vitro. Thrombin therefore, links HGFA to the blood coagulation cascade, as upon initiation of blood coagulation, the serine protease thrombin is generated from its precursor, pro-thrombin. The HGFA precursor circulates in the plasma in this inactive form and does not have the ability to bind to heparin. However, in the active form HGFA does possess the ability to bind to heparin-like molecules, thereby associating with the cell surface to ensure localised HGFA action. This binding enables a more efficient pro-HGF conversion as the pro-HGF molecule also binds to heparin-like molecules on the cell surface awaiting activation (12, 36, 94). Cleavage of the HGFA precursor results in the generation of two major fragments of 66kDa and 34kDa in size. The 66kDa fragment represents the inactive NH2-terminal region of the precursor, which may have been involved in the binding of the precursor to the cell surface for activation by thrombin (102). Whereas, the 34kDa fragment represents the active form of HGFA and is com-posed of the COOH-terminal region (Figure 4).

HGF activation by HGFA occurs mainly in the extracellular environ-ment and is the limiting step in the HGF signalling pathway. HGFA was initially detected in the liver, through northern blot analysis, and it has since been established as the main source of HGF in the body (87). HGFA has also been detected in white matter astrocytes of brain tissue, glioma cells, and in colorectal carcinoma (103–105). Our studies have demonstrated in recent years that HGFA is expressed by a wide variety

tissues (26, 106).

More recently, a member of the transmembrane type II serine protease family, matriptase, has demonstrated pro-HGF converting properties (88). Matriptase was initially discovered and purified as a matrix degrading

of cancer cell lines, and is also overexpressed in human breast cancer

4.1.2 Matriptase

The HGFA precursor is inactive in plasma and requires activation to fulfill the function of HGF activator. It was observed that human serum revealed a high degree of HGF converting activity, and Shimomura et al. (100), examined the ability of various serine proteases, from the blood coagulation and fibrinolysis mechanisms, to act as activators of the

protease from breast cancer cell lines and human breast milk (107–109). Interestingly, matriptase has been separately identified by four different groups, subsequent cloning revealed matriptase to be identical to MT-SP1, TADG-15, epithin, and ST14 (110–113).

Matriptase is an 80kDa – 90kDa protease that consists of multiple domains, including a short cytoplasmic domain at the NH2 terminus followed by a putative transmembrane domain; a sperm protein, enteroki-


studied to date, and incompletely understood (114). Matriptase requires proteolytic processing at Gly-149 in the SEA domain of the protease, glycosylation of the first CUB domain and the first serine protease domain, and intact LDL receptor class A domains. It was suggested that activation of matriptase required the presence of its cognate inhibitor, HAI-1 (115). A recent study reports that after its activation, matriptase is rapidly bound to HAI-1. Subsequently, the matriptase-HAI-1 complex is shed into the extracellular milieu (116). These observations indicate that activation and HAI-1-mediated inhibition of matriptase are well organised and controlled in human mammary epithelial cells.

In addition to generating active HGF, matriptase can also create active forms of urokinase-type plasminogen activator and protease-activated receptor 2 (PAR2) (88, 113). Furthermore, purified matriptase was also found to activate one of the important matrix metalloproteases, stromely-sin (MMP-3) (117). The normal physiological role of matriptase may be in epithelial biology, as matriptase is reported to be an essential com-ponent of the profilagrin-processing pathway in keratinocytes, a crucial

follicle growth (116, 118). In addition, transgenic knockout studies have shown that matriptase elimination results in a malfunction in epidermal barrier formation, the cellular immune system, and reduces post-natal survival in mice (119). Matriptase also demonstrates the ability to degrade extracellular matrix proteins, such as gelatin, fibronectin and laminin (109, 120). Therefore, matriptase may contribute to the remodeling of the ECM and aid tumour cell invasion (121). The fact that matriptase is synthesised as a transmembrane form may also prove to aid the pericellular activation of HGF.

4.2 Inhibitors of HGF Activation

The two main factors responsible for HGF activation are HGFA and matriptase. HGFA and matriptase action is regulated by two novel Kunitz-

Similarly to HGFA, matriptase requires activation via cleavage at its canonical activation motif to convert the single-chain zymogen to a two-chain active protease. However, the activation process of the matriptase zymogen is extraordinarily complex, unique among all serine proteases

nase, and agrin

domain; two tandem C1r/C1s, urchin embryonic growth factor, and bone morphogenetic protein-1 (CUB) domains; four tandem

low-density lipoprotein (LDL) receptor class A domains; and a trypsin-like serine protease domain at its COOH terminus (109) (Figure 4).

regulator of epidermal terminal differentiation, and also critical for hair

type serine protease inhibitors termed hepatocyte growth factor activator inhibitor type 1 (HAI-1) and hepatocyte growth factor activator inhibitor type 2 (HAI-2). However, the roles of HAI-1 and HAI-2 in the body are still unclear; these inhibitors may play multiple roles in the body, and have been linked to a variety of physiological processes. Very little is

180 Parr and Jiang

Figure 4. Domain structures of the factors in the HGF regulatory system.

known about the regulation of HGF activity, and the interaction between the HGF activators (HGFA and matriptase) and the HGF activation inhibitors (HAI-1 and HAI-2).


HGFA is found as an inactive precursor in human plasma, however, in human serum HGFA is detected in its active form. This suggested that a factor responsible for inhibiting HGFA action would not be present in the serum. Therefore, it seemed reasonable to assume that an inhibitor to HGFA may be produced by the tissues. Shimomura et al. (91), decided to examine human cell lines for an inhibitory factor against HGFA action. This group identified an inhibitory protein in the conditioned media of a variety of cell lines. The protein was then purified from a human MKN45 stomach carcinoma cell line and cloned to reveal a novel Kunitz-type serine protease inhibitor. The newly discovered HGFA inhibitor was designated HGF activator inhibitor (HAI). However, soon after the discovery of HAI, a second inhibitor of HGFA action was identified from the conditioned media of the same stomach carcinoma cell line (92). This newly discovered HGFA inhibitor was purified, cloned, and found to be another new Kunitz-type serine protease inhibitor. To distinguish between these two very similar HGFA inhibitors, they were designated HAI-1 and HAI-2.

HAI-1 and HAI-2 are a unique class of serine protease inhibitors as they are synthesised as transmembrane glycoproteins rather than secreted forms. They are type 1 transmembrane proteins, and have two Kunitz-type serine protease domains, the first of which is thought to be respon-sible for the HGFA-inhibitory action (122). Presently the target protease/s for the second Kunitz domain is unknown. HAI-1 and HAI-2 are synthe-sised on the cell surface and appear to be secreted by ectodomain shed-ding through proteolytic cleavage at the juxtamembrane part of the protein, this release of the inhibitors could decide the function of HAI-1 and HAI-2.

HAIs have emerged to play roles in mediating a diverse range of cellular functions through their ability to control the biological activation of proteins. HAI-1 is now reported to inhibit the action of HGFA, matriptase, hepsin, plasmin, and trypsin; whereas, HAI-2 inhibits HGFA, hepsin, trypsin, plasmin, tissue kallikreins, and factor XIa activity.

HAI-1 is classed as a Kunitz-type serine protease inhibitor due to the similarity it shares with this family of inhibitors in the Kunitz domains residue regions 250–300 and 375–425. The primary HAI-1 translation

4.2.2 Structure and function of HAI’s

HAI-1

4.2.1 Discovery of the HAI’s

product is 66 kDa, made up of 513 amino acid residues, and is composed of an NH2-terminal putative signal peptide (1–35 residues), Kunitz domain 1 (250–300 residues), and Kunitz domain 2 (375–25 residues). The region between these Kunitz domains (319–353 residues) shares a high similarity to the low-density lipoprotein (LDL) receptors binding

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Shimomura et al. (123) identified several new soluble forms of HAI-1. These soluble forms differ in form depending upon their site of cleavage, as the primary translation product contains multiple sites for proteolytic processing, resulting in two different sizes of HAI-1. Importantly, these forms of HAI-1 also possess the ability to inhibit the activity of other serine proteases, such as plasmin and matriptase (109). HAI-1 also plays role in plasminogen activator cascade through its regulation of matrip-tase action (124). The presence of a hydrophobic sequence in the COOH-terminal of the primary translation products, of both HAI-1 and HAI-2, suggests that they are produced in a membrane-associated form, and then proteolytically cleaved into a truncated form and secreted into the extracellular environment (92). This transmembrane associated form reveals multiple sites for proteolytic processing, which has resulted in production of two major secreted forms of 40 and 58kDa (123). The 58kDa form contains both Kunitz domains, whereas the 40kDa from only contains the first Kunitz domain (105, 122). There are at least two proteases, one of which is a metalloproteinase, which can cleave the sites to release these soluble forms of HAI-1, however, presently the factors responsible for shedding of the HAI’s is unclear.

HAI-1 is very potent inhibitor of matriptase, HGFA, and trypsin, specifically due to the action of the first Kunitz domain (98, 108, 125). While Kunitz domain 1 is known to possess inhibitory action against

As with HAI-1, the originally identified form of HAI-2 was the proteolytically truncated version, rather than the membrane form. This shed version of HAI-2 was much smaller then HAI-1, with a molecular

HAI-2 has similar structural domains to HAI-1, but does not have the

between the two Kunitz domains (Figure 4). The primary translation pro-duct of HAI-2 is composed of 252 amino acid residues. The NH2-terminal

inhibition of HGFA, due to the fact that the HGFA precursor has ahigh affinity for negatively charged substances. There is also a trans-membrane domain and a hydrophobic region at the COOH-terminal end(91, 104) (Figure 4).

same length NH2-terminal, and there is no LDL ligand-binding domain

HAI-2

domain. This LDL receptor-like domain, absent in HAI-2, contains a

it may aid in formation of a protease inhibitor complex during thenegatively charged domain, the reason for which is unknown, although

is composed of the putative signal peptide (1–27 residues), Kunitz domain 1 (38–88 residues), Kunitz domain 2 (133–183 residues), and a hydrophobic region (198–221 residues) at the COOH-terminal end (92).

HGFA and matriptase, the inhibitory targets of the second Kunitz domainare unknown at present.

mass of about 14kDa. Full size HAI-2 has a molecular mass of 30kDa.


new HAI-2 variant in mice. This HAI-2 variant, although not present in human tissue, was shorter than human HAI-2 due to the fact that it lacked the first Kunitz domain. HAI-2 has been found to be identical to a protein called placental bikunin and another protein overexpressed in pancreatic cancer known as KOP (127, 128). Placental bikunin contains

HAI-1 and HAI-2 bind to HGFA and matriptase, to prevent these serine proteases from attaching to and cleaving pro-HGF into the active form of HGF. It also appears that the different forms of the HAI’s inhibit HGF activation with varying potency. However, it is unclear if HAI-2 possesses the ability to inhibit matriptase action. A recent study revealed that both HAI-1 and HAI-2 demonstrate that ability to inhibit the action of hepsin, another factor with pro-HGF converting properties (130).

Matriptase and HAI-1 are reported co-localize on the cell periphery of breast cancer cells and form stable complexes in the extracellular milieu, suggesting that the inhibitor serves to prevent undesired proteolysis in these cells. Subsequently, the matriptase-HAI-1 complex is shed into the extracellular milieu (116). Another study reports that HAI-1 acts not only as an inhibitor to matriptase, but plays a role in all aspects of matriptase functionality (124). HAI-1 was shown to be responsible for controlling inappropriate matriptase synthesis, trafficking, activation, and inhibition. This constant monitoring of matriptase by HAI-1 may be necessary to ensure that this potentially hazardous enzyme functions properly, thus avoiding its harmful effects.

While in the transmembrane form, only HAI-1 can bind to HGFA. This is a reversible reaction and may act as a means of pooling the

released to activate HGF accordingly (131). HAI-2 was not capable of forming this complex with HGFA; this may be due to the absence of the LDL receptor-like domain, although the secreted form of HAI-2 is a highly potent inhibitor of HGFA action. Therefore, it appears to be the shed forms of HAI-1 and HAI-2 that inhibit HGFA and matriptase action

4.2.3 Inhibition of HGFA and Matriptase by HAI-1 and HAI-2

Structural differences occur between HAI-1 and HAI-2, as HAI-1 was adsorbed by a hydrophobic column, whereas, HAI-2 was not. This indi-cates that HAI-2 is more hydrophilic than HAI-1. As only the presence of Kunitz domain 1 is required for HGFA suppression, then maybe Kunitz domain 2 has an alternative responsibility. Itoh et al. (126) detected a

tors is unknown. It has been suggested that the TACE-like (tumour necro-sis factor-α-converting enzyme) proteases of the ADAM (a disintegrin

the Kunitz domain 2, and it seems possible that this domain is responsible for inhibiting trypsin, tissue kallikrein, plasma kallikrein, and plasmin (129).

and metalloprotease) family may be involved in the ectodomain shedding of membrane proteins.

available HGFA on the cell surface at a desired site to ensure a con-centrated pericellular supply of HGFA activity, whereupon it may be

most effectively. However, the process behind shedding of these inhibi-

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against HGFA, and may be due to other physiological events. Their normal roles in the body may be governed by whether they are in their trans-membrane form or their secreted form, if they are composed of one Kunitz-domain or two, and to what their target protease is in a particular situation.

HAI-1 is generally expressed in the simple columnar epithelial cells of ducts, tubules, and mucosal surface of various organs. HAI-1 mRNA has been detected in adult breast, placenta, brain, pancreas, kidney, prostate, small intestine, and colon (26, 104, 106, 131, 132). Interestingly, HAI-1 was not detected in hepatocytes, endocrine cells, stromal mesenchymal cells, and inflammatory cells (133).

HAI-2 is expressed by a wide variety of human normal and cancer

identical to HAI-2, was also reported to be highly expressed in placenta and pancreatic tissue, but was undetectable in heart, lung, brain, liver, and skeletal muscle (128).

Patterns of HAI-1 and HAI-2 expression have been assessed, and it appears that they generally share similar distribution within the majority of human tissues examined. There are exceptions however, as observed in human testis, where HAI-2 is strongly expressed and HAI-1 was hardly detected at all, therefore HAI-2 may pay a role in spermatogenesis (92). Interestingly, the examination of HAI-2 mRNA from adult testes revealed a shorter HAI-2 transcript than normally found in the body (134). The HAI’s may play also different physiological roles in the body as HAI-1 expression was unregulated in response to tissue injury and inflammation (135), whereas HAI-2 was not. Currently, very little is known about induction of HAI-1 and HAI-2 gene expression.

HGF is known to be a key factor in liver and lung regeneration, therefore, requires the influence of HGFA for the conversion of pro-HGF to HGF at these sites. HGFA is secreted from the liver, thus this organ represents a rich source of HGFA. HAI expression in human liver and lung is low (91, 92), thereby intensifying the potency of matriptase and HGFA. We also report that human fibroblasts are a good source of HGF in the body, and express high levels of HGF, HGFA, and matriptase,

Although HAI-1 and HAI-2 possess similar structure, immuno-histochemical staining and other reports suggest they have a variety of different functions in the body, which may be unrelated to their action

4.2.4 Expression of HAI-1 and HAI-2

however, HAI expression in human fibroblasts is low or absent (126). Certain conditions reveal an upregulation of HAI-1, as during hepatitis scattered hepatocytes reveal the presence of HAI-1. In addition, HAI-1 expression was also observed during regeneration of kidney tubule epithelial cells following infarction (133).

cells of ducts, tubules, and mucosal surfaces (26, 106). KOP, which is cells, and like HAI-1 is found within the simple columnar epithelial


vascularisation of tumours. Matriptase also activates latent urokinase-type plasminogen activator, which subsequently activates plasmin (88). This cascade results in the activation of plasmin, and the coactivation of matrix metalloproteases, which leads to the degradation of ECM components and further enhances tumour cell invasion, extravasation and metastasis (136). Therefore, increased HGFA and matriptase activity may therefore correlate with enhanced metastatic potential of tumours.

A balance between protease activity and protease inhibition is crucial for maintaining normal cell function. The balance between proteases and their inhibitors is disrupted in cancer cells, and this shift in regulation can lead to the progression of tumour cells (137, 138). Therefore, the balance between HGF activation and HGF activation suppression is the crucial step controlling the metastatic influence of HGF. Overexpression of HGFA and/or matriptase may disrupt the activator/inhibitor ratio in favour of increased HGF activation, resulting in an increase in HGF activity, and subsequently enhancing the metastatic stimulus.

5.1 The Role of HGFA in Cancer

We have previously reported that HGFA is expressed in a number of human cancer cell lines, including breast, colon, prostate, lung, and liver (106). In addition, we have also demonstrated that the expression of HGFA is upregulated in human breast cancer tissues compared with normal background breast tissues, whereas the levels of the HGFA inhibitors are reduced in breast cancer tissues (26) (Figure 5). This observation is also reported in colorectal carcinomas, where an upregulation of HGFA is accompanied by a downregulation of HAI-1 (105). Plasminogen acti-vator, which shows significant homology to the HGFA precursor, also displays enhanced expression in breast cancer cells and correlates with tumour progression (139). In addition to the changes of HGFA in tumour and tumour cells, recent studies also reveal that serum HGFA was elevated in patients with advanced stage prostate cancer (140). The study, although in small scale, is interesting and indicated that a systemic rise of the

5. HGF ACTIVATORS ENHANCE THE SPREAD OF TUMOURS

HGFA and matriptase are responsible for the activation of HGF in tumours, which suggests that these proteases will aid the growth and motility of cancer cells, particularly carcinomas, and further enhance

HGFA may be a feature in these patients. A large-scale study would be important. The forced expression of HGFA within human glioblastoma cell lines resulted in significantly enhanced tumour growth with increased vascular density when these cells were implanted in nude mouse brain (140, 141). These elevated HGFA levels lead to enhanced HGF activity and in turn may promote tumour metastasis.

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tative analysis of the HGF system. Graphs show the mean number of respective transcripts

chemical staining of breast specimens (Top = background breast tissue from breast cancer patient; bottom = tumour tissue from breast cancer patient). HGF (A,B), c-Met (C,D), and HGFA (E,F) all displayed a low degree of staining in the background specimens, however, the level of these influential factors in the breast cancer tissues was dramatically increased. In contrast, HAI-1 (G,H) and HAI-2 (I,J) showed intense staining in the normal tissue, but the breast cancer tissues displayed far lower levels of these

These reports suggest that the HGFA serine protease is a target for inhibition. Serine protease inhibitors have previously been employed in an attempt to examine the possible methods of decreasing HGFA activity. The serine protease inhibitors examined were naturally present in human serum and included antithrombin III, c-1 inhibitor and α2-antiplasmin. The HGF-converting ability of Factor XIIa and HGFA was

5.2 The Role of Matriptase in Cancer

The type II transmembrane serine protease known as matriptase has the potential to mediate the dissolution of extracellular matrix components surrounding tumour cells, catalyse the degradation of intercellular cohesive structures that allows shedding of tumour cells into the extracellular environment, and activate growth and angiogenic factors during tumour progression, and has also recently demonstrated the ability to promote carcinogenesis (142).

Figure 5. HGF, c-MET and HGF regulators in human breast cancer. Top panel - Quanti-

comparing normal background tissue and tumour tissues. Bottom panel - Immunohisto-

inhibitory factors. (Adapted from Parr et al., 2004. Clin Cancer Res; 10: 202–11).

examined in the presence of these protease inhibitors. The inhibitors suppressed the influence of Factor XIIa, but none of these inhibitors couldprevent HGFA converting pro-HGF to HGF (96). This suggests that theHAI inhibitors may be crucial to controlling the pro-metastatic influenceof HGF in breast cancer.


convert matriptase

from a well-regulated cell junctional protease in mammary epithelial cells to a dysregulated invasion protease at the leading edges of breast cancer cells (143). Thus, matriptase may play an important role in the progression of breast carcinomas. Matriptase is overexpressed in a wide variety of malignant tumours including breast, prostate, ovarian, renal, uterine, colon, oesophageal, epithelial-type meso-thelioma, and cervical cell carcinoma and often correlates with advanced

inhibition of matriptase expression led to suppression of both prostate and ovarian primary tumour growth and metastasis in murine models (108, 112, 155, 156); whereas, overexpression of matriptase-1 was found

enhance epidermal tumour formation in transgenic mice (142). It has recently been shown that matriptase also possesses a strong oncogenic potential, as its overexpression in the skin of transgenic mice caused 100% of the mice to develop tumours, 70% of which progressed into carcinomas (142). Importantly, increased expression of HAI-1 com-pletely negated the oncogenic effects of matriptase overexpression (142).

These data strongly suggest that a shift in the balance between matriptase to HAI-1 action causes malignant transformation to occur at a high frequency. There is mounting evidence that demonstrates that an increase in matriptase expression is also accompanied by the concomi-tant downregulation of HAI-1 expression (26, 144, 146, 157, 158).

stage clinicopathological parameters (144–154). Studies report that the

IN THE REGULATION OF METASTASIS 6. EMERGING ROLE OF HAI-1 AND HAI-2

The significance of HGF in human cancer metastasis is well established. However, the role of the HAI inhibitors is less clear. The fact that HGF plays such a pivotal role, suggests that the HGF activators (HGFA and matriptase), and HGF activation inhibitors (HAI-1 and HAI-2), are important factors that can influence the metastatic spread of tumours. Recently, a number of studies demonstrate that the ratio between HAI-1/2 and matriptase/HGFA expression is a crucial factor governing the malignant progression of tumour cells in a variety of human cancers including breast, prostate, renal, and colorectal (26, 144, 146, 157, 159, 160). These reports reveal a shift in the balance between HGF activation and inhibition; and demonstrate the malignant progresssion of tumour cells may be a consequence of protease and/or inhibitor dysregulation.

We have shown that both HAI-1 and HAI-2 are downregulated in breast cancer tissues in comparison with normal mammary tissue from a cohort of 120 breast cancer patient samples (26) (Figure 5). In addition we also reveal that HGFA expression is elevated, thus, activation of HGF becomes deregulated and results in enhanced HGF activity. Furthermore,

Breast cancer cells constitutively activate matriptase and concentrate the activated protease at membrane ruffles, a relocalisation that may

188 Parr and Jiang

Our most recent studies reinforce the opinion that both HAI-1 and HAI-2 play an important role governing the metastatic nature of tumour cells (160). We employed a ribozyme transgene system to knock down the expression of HAI-1 and HAI-2 in a human breast cancer cell line. Loss of either HAI-1 or HAI-2 resulted in a significantly more aggressive cancer cell phenotype (Figure 6). This study revealed that breast cancer cells with experimentally reduced HAI levels down became dramatically more invasive, and also revealed enhanced motile and proliferative pro-perties. These breast cancer cells expressed such proteolytic enzymes as matriptase, HGFA and hepsin. Therefore, the lack of the HAI-1 or HAI-2 expression leads to dysregulated protease activity, which may subsequently promote cancer progression and metastasis.

Overall, the importance of the HGF regulatory system in cancer metastasis is yet to be fully appreciated. The two Kunitz domains of both HAI-1 and HAI-2 are the key to their inhibitory aspect against a variety of serine proteases. As yet the full list of protease targets for these inhi-bitors is unclear, however, understanding the interaction of these HAI’s with a variety of other serine proteases such as HGFA, matriptase, hepsin, plasma kallikrein, and trypsin will help elucidate the roles and the true value of HAI-1 and HAI-2 in cancer metastasis. We believe that HAI-1 and HAI-2 play a crucial role in breast cancer progression and may have prognostic value. Importantly, an increasing number of studies are suggesting that the HAI inhibitors also display the potential for use in future as anti-cancer agents or biomarkers of cancer progression (26, 98, 157, 158, 160, 163, 164).

7. THERAPEUTIC POTENTIAL OF HAI-1

The last decade has witnessed the rapid increase of knowledge available on the role of HGF and c-Met in human cancer. HGF stimulates, through c-Met coupling, the metastatic spread and angiogenesis of tumours. Therefore, the blockade of HGF signalling has become a strategy to

AND HAI-2 AS ANTI-CANCER AGENTS

(55, 81, 165). An increasing number of reports support this theory, as shown by recent NK4 studies.

inhibit tumour invasion and metastasis, as indicated in recent articles

(26). We reveal that HAI-1/2 levels are both significantly reduced in poorly differentiated Grade 3 breast tumour, and that low levels of HAI-1/2 are associated with advanced stage tumours and may possess prognostic value. Several other studies also report that HAI expression inversely correlates with patient prognosis in ovarian, gastrointestinal, glioblastoma, colorectal, and prostate tumours (158, 161–163).

our studies have shown that low levels of HAI expression within breast tumours are associated with a poor prognosis for the breast cancer patient


Figure 6. (A) Ribozyme transgenes were used to inhibit HAI-1 and HAI-2 expression in MDA-MB-231 breast cancer cells (Wild type = control; HAI-1 suppressed = HAI-1 KO; HAI-2 inhibited = HAI-2 KO). HAI-1 KO and HAI-2 KO breast cancer cells revealed a dramatically more aggressive nature compared to the control group. (B) Crystal Violet Staining of Invaded Breast Cancer Cells. (i) Wild type breast cancer cell control group following 72 hour incubation. (ii) The elimination HAI-1 resulted in a significantly higher degree of breast cancer cell invasion. (iii) Suppression of HAI-2 expression dramatically influenced the nature of these cells, resulting in enhanced tumour cell invasion. (C) Wound Closure Migration Assay. The knockdown of HAI-1 or HAI-2 expression significantly increased the migratory nature of the breast cancer cells. (D) Recombinant HAI’s were used to potently reduce, MRC5 fibroblast-induced, breast cancer cell invasion. (E) Retroviral Expression of HAIs within human MRC5 fibroblasts inhibited the ability of these fibroblasts to induce breast cancer cell invasion. (Adapted from Parr et al., 2006. Int J Cancer; 119: 1176–1183).

190 Parr and Jiang

NK4 is a variant of HGF that competitively blocks HGF binding to the c-Met receptor, thereby reducing HGF-related pro-metastatic effects. NK4 acts as an antagonist of HGF-Met coupling, and has demonstrated significant potential as a novel anti-cancer agent (166–174). Similarly, small molecule inhibitors to the HGF receptor have also been recently reported, e.g., PHA-665752 and SU5416 (175, 176).

Suppression of HGF activity is due to a shift in the balance between the HGF-converting proteases and the HAI inhibitors. This shift between HGF activation and HGF activation suppression is the crucial step con-trolling the metastatic influence of HGF, and may represent a method of limiting tumour progression. The presence of our HAI-1 or HAI-2 proteins altered the balance to favour suppression of HGF activity. Therefore, the ability of HGFA, matriptase, and hepsin to enhance the potent effects of HGF on tumour cells, has been quenched through the presence of HAI-1 and HAI-2. Importantly, the most effective suppression of breast cancer cell invasion was observed when rHAI-1 and rHAI-2 were used in combination. These inhibitors appear to possess additional and individual inhibitory properties (98, 113, 122, 180, 181), and may therefore benefit

The HAI inhibitors address the issue of HGF suppression from a dif-ferent angle to NK4, in that HAI inhibitors prevent pro-HGF being con-verted into the active form in the first instance. Pro-HGF is ineffective as a pro-metastatic factor prior to its interaction and subsequent activation via the HGFA or matriptase proteases (94, 105, 177, 178). The conversion of pro-HGF to the biologically active HGF is the critical limiting step in the HGF regulatory system and will play a key role in the control of metastatic events. If no active HGF is available in the tumour micro-environment, tumour cells, or tumour-infiltrated endothelial cells will not receive pro-invasive signals even if their c-Met levels ensure high ligand sensitivity.

The importance of suppressing pro-HGF processing was recently highlighted in an interesting study that described, through a single amino acid substitution in the proteolytic site, how an uncleavable form of pro-HGF suppressed tumour growth and dissemination in a mouse model (179). HAI-1 and HAI-2 are two novel Kunitz-type serine protease inhi-bitors that inhibit the influence of a range of proteases, most notably HGFA and matriptase (91, 92, 125). Our most recent studies have generated recombinant HAI-1 and HAI-2 proteins to further assess the function and anti-cancer properties of these protease inhibitors. Crucially, these HAI studies further implicated the potential value of the HAI-1 and HAI-2 as anti-cancer agents (160). Addition of either rHAI-1 or rHAI-2 to cultured fibroblasts significantly reduced the production of biologically active HGF (Figure 6). These HAI-1 and HAI-2 proteins also dramatically reduced fibroblast-mediated breast cancer cell invasion and migration (160). These exciting results may be due to the ability of the HAI’s to interact and inhibit the pro-invasive function of matriptase, HGFA and hepsin. Therefore, the natural ability of fibroblasts to facilitate cancer cell invasion had been suppressed by the addition of recombinant HAI proteins.

9. HGF regulators in metastasis 191 from being deployed in tandem. However, it has to be pointed out that the development of the HGF activation inhibitors is in the early stages. The true clinical value of using HAIs as therapeutic modalities requires substantial work.

8. CONCLUDING REMARKS

HGF and its partner c-Met play a definitive role in tumour-stromal interactions, leading to particularly invasive and metastatic cancers. The invasion and subsequent establishment of metastasis are devastating events for patients with cancer. Therapeutic strategies targeting the activa-tion of HGF warrant investigation for their potential value in combating the spread of tumours. HAI-1 and HAI-2 are serine protease inhibitors that display unique therapeutic potential due to their ability inhibit HGFA and matriptase action, and thus prevent the generation of biologically active HGF. These inhibitors play important roles in controlling the aggres-sive nature and spread of cancer. Further progress will undoubtedly lead to the application of these advances in the generation of future therapies to prevent the spread of breast cancer.

REFERENCES

1. Althuis MD, Dozier JM, Anderson WF, Devesa SS, Brinton LA. Global trends in breast cancer incidence and mortality 1973–1997. Int J Epidemiol 2005; 34: 405–412.

2. Boyle P, Ferlay J Cancer incidence and mortality in Europe, 2004. Ann Oncol 2005; 16: 481–488.

3. Nakamura T, Nawa K, Ichihara A. Partial purification and characterization of hepatocyte growth factor from serum of hepatomized rats. Biochem. Biophys. Res. Commun. 1984; 122: 1450–1459.

4. Michalopoulos G, Houck KA, Dolan ML, Leutteke NC. Control of hepatocyte replication by two serum factors. Cancer Res. 1984; 44: 4414–4419.

5. Russel WE, McGowen JA, and Butcher NL. Partial characterization of an hepato-cyte growth factor from rat platelets. J. Cell Physiol. 1984; 119: 183–192.

6. Stoker M and Perryman M. An epithelial scatter factor released by embryo fibroblasts. J. Cell Sci. 1985; 77: 209–223.

7. Gherardi E and Stoker M. Hepatocyte and scatter factor. Nature 1990; 346: 228 8. Weidner KM, Behrens J, Vandekerckhove J, and Birchmeier W. Scatter factor:

molecular characteristics and effect on the invasiveness of epithelial cells. J. Cell Biol. 1990; 111: 2097–2108.

9. Weidner KM, Arakaki N, Hartmann G, Vandekerckhove J, Weingart S, Rieder H, Fonatsch C, Tsubouchi H, Hishida T, Daikuhara Y, and Birchmeier W. Evidence for the identity of human scatter factor and human hepatocyte growth factor. Proc. Natl. Acad. Sci. USA. 1991; 88: 7001–7005.

10. Furlong RA, Takehara T, Taylor WG, Nakamura T, and Rubin JS. Comparison of biological and immunochemical properties indicates that scatter factor and hepatocyte growth factor are indistinguishable. J. Cell Sci. 1991; 100: 173–177.

192 Parr and Jiang

cleavage by urokinase is required for activation of hepatocyte growth factor/ scatter factor. Embo J. 1992; 11: 4825–4833.

13. Naldini L, Vigna E, Bardelli A, Follenzi A, Galimi F, and Comoglio PM. Biological activation of pro-HGF (hepatocyte growth factor) by urokinase is controlled by a stoichiometric reaction. J. Biol. Chem. 1995; 270: 603–611.

14. Gohda E, Matsunaga T, Kataoka H, Takebe T, and Yamamoto I. Induction of hepatocyte growth-factor in human skin fibroblasts by epidermal growth-factor, platelet-derived growth-factor and fibroblast growth-factor. Cytokine 1994; 6: 633–640.

15. Nakamura T, Nishizawa T, Hagiya M, Seki T, Shimonishi M, Sugimura A, Tashiro K, and Shimizu S. Molecular cloning and expression of human hepato-cyte growth factor. Nature 1989; 342: 440–443.

16. Matsumoto K, Takehara T, Inoue H, Hagiya M, Shimizu S, and Nakamura T. Deletion of kringle domains or the N-terminal hairpin structure in hepatocyte growth factor results in marked decreases in related biological activities. Biochem. Biophys. Res. Commun. 1991; 18: 691–699.

17. Donate LE, Gherardi E, Srinivasan N, Sowdhamini R, Aparicio S, and Blundell TL. Molecular evolution and domain structure of plasminogen-related growth factors (HGF/SF and HGF1/MSP). Protein Sci. 1994; 3: 2378–2394.

18. clinical applications. Clin Chim Acta 2003; 327: 1–2.

19. Birchmeier C, Birchmeier W, Gherardi E, and Vande Woude GF. Met,

20. Rosen EM, Nigam SK, and Goldberg ID. Scatter factor and the c-Met receptor: a paradigm for mesenchymal/epithelial interaction. J. Cell Biol. 1994; 127: 1783–1787.

21. Birchmeier C, Birchmeier W, and Brand-Saberi B. Epithelial-mesenchymal transitions in cancer progression. Acta Anat. 1996; 156: 217–226.

22. the met protooncogene and hepatocyte growth factor in human pancreatic

23. Joseph A, Weiss GH, Jin L, Fuchs A, Chowdhury S, O’Shaugnessy P, Goldberg

24. Lamszuz K, Schmidt NO, Jin L, Laterra J, Zagzag D, Way D, Witte M, Weinland M, Goldberg ID, Westphal M, and Rosen EM. Scatter factor promotes motility of human glioma and neuromicrovascular endothelial cells. Int. J. Cancer 1998; 75: 19–28.

25. Watanabe M, Fukutome K, Kato H, Murata M, Kawamura J, Shiraishi T, and Yatani R. Progression-linked overexpression of c-Met in prostatic intraepithelial neoplasia and latent as well as clinical prostate cancers. Cancer Lett. 1990; 141: 173–177.

26. Parr C, Watkins G, Mansel RE, and Jiang WG. The hepatocyte growth factor regulatory factors and human breast cancer. Clin Cancer Res 2004; 10: 202–211.

27. Cooper CS, Park M, Blair DG, Tainsky MA, Heubner K, Croce CM, and Vande Woude GF. Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature 1984; 311: 29–33.

28. Kammula US, Kuntz EJ, Francone TD, Zeng Z, Shia J, Landmann RG, Paty PB, and Weiser MR. Molecular co-expression of the c-Met oncogene and hepato-cyte growth factor in primary colon cancer predicts tumor stage and clinical

Funakoshi H and Nakamura T. Hepatocyte growth factor: from diagnosis to

metastasis, motility and more. Nat Rev Mol Cell Biol 2003; 4: 915–925.

J. cancer. Cancer Res. 1994; 54: 5775–5778.

ID, and Rosen EM. Expression of scatter factor in human bladder carcinoma.

Eberg M, Yokoyama M, Friess H, Bulcher MW, and Korc M. Co-expression of

outcome. Cancer Lett. 2006 (in press).

J. Natl. Cancer Inst. 1995; 87: 372–377.

11. Nakamura T. Structure and function of hepatocyte growth factor. Prog. Growth Factor Res. 1990; 3: 67–85.

12. Naldini L, Tamagnone L, Vigna E, Sachs M, Hartmann G, Birchmeier W, Daikuhara Y, Tsubouchi H, Blasi F, and Comoglio PM. Extracellular proteolytic


31. Peghini PL, Iwamoto M, Raffeld M, Chen YJ, Goebel SU, Serrano J, and Jensen RT. Overexpression of epidermal growth factor and hepatocyte growth factor receptors in a proportion of gastrinomas correlates with aggressive

32. Qian CN, Guo X, Cao B, Kort EJ, Lee CC, Chen J, Wang LM, Mai WY, Min HQ, Hong MH, Vande Woude GF, Resau JH, and Teh BT. Met protein expression level correlates with survival in patients with late-stage nasopharyn-

33. Prat M, Narisham R, Crepaldi T, Nicotra MR, Natali PG, and Comoglio PM. The receptor encoded by the human c-MET oncogene is expressed in hepato-cytes, epithelial cells and solid tumours. Int. J. Cancer 1991; 49: 323–328.

34. Di Renzo MF, Narsiman RP, Olivero M, Bretti S, Giolano S, Medico E, Gaglia P, Zara P, and Comoglio PM. Expression of the MET/HGF receptor in normal and neoplastic human tissues. Oncogene 1991; 6: 1997–2003.

35. Giordano S, Di Renzo MF, Narsimhan RP, Cooper CS, Rosa C, and Comoglio PM. Biosynthesis of the protein encoded by the c-met proto-oncogene. Oncogene 1989; 4: 1383–1388.

36. Naldini L, Vigna E, Narsimhan RP, Gaudino G, Zarnegar R, Michalopoulos GK, and Comoglio PM. Hepatocyte growth factor (HGF) stimulates the tyrosine kinase activity of the receptor encoded by the proto-oncogene c-MET. Oncogene 1991; 6: 501–504.

37. Bottaro DP, Rubin JS, Faletto DL, Chan AM, Kmiecik TE, Vande Woude GF, and Aaronson SA. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 1991; 251: 802–804.

38. Faletto DL, Kaplan DR, Halverson DO, Rosen EM, and Vande Woude GF. Signal transduction in c-met mediated motogenesis. Experientia supplimentum

39. Ponzetto C, Bardelli A, Zhen Z, Maina F, dalla Zonca P, Giordano S, Graziani A, Panayotou G, and Comoglio PM. A multifunctional docking site mediates signalling and transformation by the hepatocyte growth factor/scatter factor receptor family. Cell 1994; 77: 261–271.

40. Ponzetto C, Bardelli A, Maina F, Longati P, Panayotou G, Dhand R, Waterfield Md, and Comoglio PM. A novel recognition motif for phosphatidylinositol 3-kinase binding mediates its association with the hepatocyte growth- factor scatter factor-receptor. Mol. Cell Biology. 1993; 13: 4600–4608.

41. Graziani A, Gramaglia D, Cantley LC, and Comoglio PM. The tyrosine-phosphorylated HGF/SF receptor associates with phosphatidyllinositol 3-kinase. J. Biol. Chem. 1991; 266: 22087–22090.

42. Bardelli A, Maina F, Gout I, Fry MJ, Waterfield MD, Comoglio PM, and Ponzetto C. Autophosphorylation promotes complex-formation of recombinant HGF receptor with cytoplasmic effectors containing SH2 domains. Oncogene 1992; 7: 1973–1978.

43. Bowers DC, Fan S, Walter K, Abounader JA, Rosen EM, and Laterra J. Scatter factor/hepatocyte growth factor activates AKT and protects against cytotoxic death in human gliomblasoma cell via PI3-kinase-dependant pathways. Cancer Res. 2000; 60: 4277–4283.

44. Fan S, Ma YX, Wang JA, Yuan RQ, Meng Q, Laterra JJ, Goldberg ID, and Rosen EM. The cytokine scatter factor inhibits apoptosis and enhances DNA

growth and lower curability. Clin Cancer Res. 2002; 8: 2273–2285.

geal carcinoma. Cancer Res. 2002; 62: 589–596.

1993; 65: 107–130.

29. Hansel DE, Rahman A, House M, Ashfaq R, Berg K, Yeo CJ, and Maitra A. Met proto-oncogene and insulin-like growth factor binding protein 3 over-expression correlates with metastatic ability in well-differentiated pancreatic endocrine neoplasms. Clin Cancer Res. 2004; 10: 6152–6158.

30. Knudsen BS, Gmyrek GA, Inra J, Scherr DS, Vaughan ED, Nanus DM, Kattan MW, Gerald WL, and Vande Woude GF. High expression of the Met receptor in prostate cancer metastasis to bone. Urology. 2002; 60: 1113–1117.

194 Parr and Jiang

c-Met signalling for cell survival and DNA repair. Mol. Cell Biol. 2001; 21: 4968–4984.

48. Edakuni G, Sasatomi E, Satoh T, Tokunaga O, and Miyazaki K. Expression of the hepatocyte growth factor/c-Met pathway is increased at the cancer front in breast carcinoma. Pathol Int. 2001; 51: 172–178.

49. Harvey P, Warn A, Newman P, Perry LJ, Ball RY, and Warn RM. Immunore-activity for hepatocyte growth factor/scatter factor and its receptor, met, in human lung carcinomas and malignant mesotheliomas. J. Path. 1996; 180: 389–394.

50. Tuck AB, Park M, Sterns EE, Boag A, and Elliott BE. Coexpression of hepatocyte growth factor and receptor (met) in human breast carcinoma. Am J Pathol 1996; 148: 225–232.

51. Trusolino L, Serini G, Cecchini G, Besati C, Ambesi-Impiombato FS, Marchisio PC, and De Filippi R. J. Cell Biol. 1998; 142: 1145–1156.

52. Tam NNC, Chung SSM, Lee DTW, and Wong YC. Aberrant expression of hepatocyte growth factor and its receptor, c-Met, during hormone-induced

53. Stracke ML and Liotta LA. The molecular basis of Cancer (Mendelson J, Howley PM, Israel MA and Liotta LA eds.), WB Saunders Company, Philadelphia. 1995; 233–247.

54. Jiang WG, Martin TA, Parr C, Davies G, Matsumoto K, Nakamura T. Jiang WG, Hiscox S, Matsumoto K, and Nakamura T. Hepatocyte growth factor, its receptor, and their potential value in cancer therapies. Crit Rev Oncol Hematol 2005; 53: 35–69.

55. Matsumoto K, Nakamura T. Hepatocyte growth factor and the Met system as a mediator of tumor-stromal interactions. Int J Cancer. 2006; 119: 477–483.

56. Wang Y, Selden AC, Morgan N, Stamp GW, and Hodgson HJ. Hepatocyte growth factor/scatter factor expression in human mammary epithelium. Am J Pathol. 1994; 144: 675–682.

57. Bussolino F, DiRenzo MF, and Ziche M. Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J. Cell Biol. 1992; 119: 629–641.

58. Olivero M, Rizzo M, Madeddu R, Casadio C, Pennacchietti S, Nicotra MR, Prat M, Maggi G, Arena N, Natali PG, Comoglio PM, and Di Renzo MF. Over-expression and activation of hepatocyte growth factor/scatter factor in human non-small-cell lung carcinomas. Br J Cancer. 1996; 74: 1862–1868.

59. Trovato M, Vitarelli E, Grosso M, Alesci S, Benvenga S, Trimarchi F, and Barresi G. Immunohistochemical expression of HGF, c-MET and transcription factor STAT3 in colorectal tumors. Eur J Histochem. 2004; 48: 291–297.

60. Gallego MI, Bierie B, and Hennighausen L. Targeted expression of HGF/SF in mouse mammary epithelium leads to metastatic adenosquamous carcinomas through the activation of multiple signal transduction pathways. Oncogene. 2003; 22: 8498–8508.

61. Wojcik EJ, Sharifpoor S, Miller NA, Wright TG, Watering R, Tremblay EA, Swan K, Mueller CR, and Elliott BE. A novel activating function of c-Src and

prostatic carcinogenesis in the Noble rat. Carcinogenesis 2000; 21: 2183–2191.

repair by a common mechanism involving signalling through phosphatidyl-inositol 3’ kinase. Oncogene 2000; 19: 2212–2223.

45. Holgado-Madruga M, Emlet DR, Moscatello DK, Godwin AK, and Wong AJ. A Grb2-associated docking protein in EGF- and insulin-receptor signalling. Nature 1996; 379: 560–564.

46. Korhonen JM, Said FA, Wong AJ, and Kaplan DR. Gab 1 mediates neurite outgrowth, DNA synthesis, and survival in PC12 cells. J. Biol. Chem. 1999; 274: 37307–37314.

47. Fan S, Ma YX, Gao M, Yuan RQ, Meng Q, Goldberg ID, and Rosen EM. The multisubstrate adapter Gab1 regulates hepatocyte growth factor (scatter factor)-


65. Elliot BE, Hung WL, Boag AH, and Tuck AB. The role of hepatocyte growth factor (scatter factor) in epithelial-mesenchymal transition in breast cancer. Can J Physiol Pharmacol 2002; 80: 91–102.

66. Sheen-Chen SM, Liu YW, Eng HL, and Chou FF. Serum levels of hepatocyte growth factor in patients with breast cancer.Cancer Epidemiol Biomarkers Prev. 2005; 14: 715–717.

67. Narita T, Toi M, Sekiguchi K, Iwanari H, Matsuura N, Kimura N, Mitsuoka C, and Kannagi R. Hepatocyte growth factor in the sera of patients with gastrointestinal and breast cancer. Gan To Kagaku Ryoho 1997; 24: 2159–2162.

68. Fukuura T, Mik, C, Inoue T, Matsumoto K, and Suzuki H. Serum hepatocyte growth factor as an index of disease status of patients with colorectal carcinoma. Br J Cancer 1998; 78: 454–459.

69. Yamashita J, Ogawa M, Yamashita S, Nomura K, Kuramoto M, Saishoji T, and Shin S. Immunoreactive hepatocyte growth-factor is a strong and independent predictor of recurrence and survival in human breast-cancer. Cancer Res 1994; 54: 1630–1633.

70. Soman NR, Correa P, Ruiz BA, and Wogan G. The TPR-met oncogenic rearrangement is present and expressed in human gastric carcinoma and precursor lesions. Proc. Natl. Acad. Sci. USA, 1991; 88: 4892–4896.

71. Costantino A, Vigneri R, Pierotti MA, and Comoglio PM. Overexpression of the c-MET/HGF receptor gene in human thyroid carcinomas. Oncogene, 1992; 7: 2549–2553.

72. Di Renzo MF, Olivero M, and Katsaros D. Overexpression of the met/HGF receptor in ovarian cancer. Int. J. Cancer 1994; 58: 658–662.

73. growth factor receptor in human pancreatic cancer. Cancer Res 1995; 55: 1129–1138.

74. Watanabe M, Fukutome K, Kato H, Murata M, Kawamura J, Shiraishi T, and Yatani R. Progression-linked overexpression of c-Met in prostatic intraepithelial neoplasia and latent as well as clinical prostate cancers. Cancer Lett. 1999; 141: 173–177.

75. Ruco LP, Stoppacciaro A, Ballarini F, Prat M, and Scarpino S. Met protein and hepatocyte growth factor (HGF) in papillary carcinoma of the thyroid: evidence for a pathogenic role in tumorigenesis. J. Pathol. 2001; 194: 4–8.

76. Lengyel E, Prechtel D, Resau JH, Gauger K, Welk A, Lindemann K, Salanti G,

node-positive breast cancer identifies patients with poor clinical outcome independent of Her2/neu. Int J Cancer. 2005; 113: 678–682.

77. Tsarfaty I, Alvord WG, Resau JH, Altstock RT, Lidereau R, Bieche I, Bertrand F, Horev J, Klabansky RL, Keydar I, Vande Woude GF. Alteration of Met protooncogene product expression and prognosis in breast carcinomas. Anal Quant Cytol Histol 1999; 21: 397–408.

78. Ocal IT, Dolled-Filhart M, D’Aquilla TG, Camp RL, and Rimm DL. Tissue microarray-based studies of patients with lymph node negative breast carcinoma

Di Renzo MF, Olivero M, Ferro S, Prat M, Bongarzone I, Pilotti S, Belfiore A,

Di Renzo MF, Poulsom R, and Olivero M. Expression of the met/hepatocyte

Richter T, Knudsen B, Vande Woude GF, Harbeck NC. Met overexpression in

Stat3 on HGF transcription in mammary carcinoma cells. Oncogene. 2006; 25: 2773–2784.

62. Toi M, Taniguchi T, Ueno T, Asano M, Funata N, Sekiguchi K, Iwanari H, and Tominaga T. Significance of circulating hepatocyte growth factor level as a

63. Taniguchi T, Toi M, Inada K, Imazawa T, Yamamoto Y, Tominaga T. Serum concentrations of hepatocyte growth factor in breast cancer patients. Clin Cancer Res 1995; 1: 1031–1034.

64. Woodbury RL, Varnum SM, and Zangar RC. Elevated HGF levels in sera from breast cancer patients detected using a protein microarray ELISA. J Proteome Res 2002; 1: 233–237.

prognostic indicator in primary breast cancer. Clin Cancer Res 1998; 4: 659–664.

196 Parr and Jiang

82. Grey AM, Schlor AM, Rushton G, Ellis I, and Schlor SL. Purification of the migration stimulating factor produced by fetal and breast cancer patients fibroblasts. Proc Natl Acad Sci USA 1989; 86: 2438–2442.

83. Camps JL, Chang SM, Hsu TC, Freeman MR, Hong SJ, Zhau HE, von Eschenbach AC, and Chung LW. Fibroblast-mediated acceleration of human epithelial tumour growth in vivo. Proc Natl Acad Sci USA 1990; 87: 75–79.

84. Ohuchida K, Mizumoto K, Murakami M, Qian LW, Sato N, Nagai E, Matsumoto K, Nakamura T, and Tanaka M. Radiation to stromal fibroblasts increases invasiveness of pancreatic cancer cells through tumor-stromal inter-actions. Cancer Res 2004; 64: 3215–3222.

85. Hartmann G, Naldini L, Weidner KM, Sachs M, Vigna E, Comoglio PM, and Birchmeier W. A functional domain in the heavy-chain of scatter factor hepatocyte growth-factor binds the c-met receptor and induces cell-dissociation but not mitogenesis. Proc Natl Acad Sci USA 1992; 89: 11574–11578.

86. Gak E, Taylor WG, Chan AM, and Rubin JS. Processing of hepatocyte growth factor to the heterodimeric form is required for biological activity. FEBS Lett 1992; 311: 17–21.

87. Miyazawa K, Shimomura T, Kitamura A, Kondo J, Morimoto Y, and Kitamura N. Molecular-cloning and sequence-analysis of the cDNA for a human serine protease responsible for activation of hepatocyte growth-factor - structural similarity of the protease precursor to blood-coagulation factor-xii. J Biol Chem 1993; 268: 10024–10028.

88. Lee S, Dickson RB, and Lin CY. Activation of Hepatocyte growth factor and urokinase/plasminogen activator by matriptase, an epithelial membrane serine

89. Naldini L, Vigna E, Bardelli A, Follenzi A, Galimi F, and Comoglio PM. Biological activation of pro-HGF (hepatocyte growth factor) by urokinase is controlled by a stoichiometric reaction. J Biol Chem 1995; 270: 603–611.

90. Herter S, Piper DE, Aaron W, Gabriele T, Cutler G, Cao P, Bhatt AS, Choe Y, Craik CS, Walker N, Meininger D, Hoey T, and Austin RJ. Hepatocyte growth factor is a preferred in vitro substrate for human hepsin, a membrane-anchored serine protease implicated in prostate and ovarian cancers. Biochem J 2005;

91. Shimomura T, Denda K, Kitamura A, Kawaguchi T, Kito M, Kondo J, Kagaya S, Qin L, Takata H, Miyazawa K, and Kitamura N. Hepatocyte growth factor activator inhibitor, a novel Kunitz-type serine protease inhibitor. J Biol Chem 1997; 272: 6370–6376.

92. Kawaguchi T, Qin L, Shimomura T, Kondo J, Matsumoto K, Denda K, and Kitamura N. Purification and cloning of hepatocyte growth factor activator inhibitor type 2, a Kunitz-type serine protease inhibitor. J Biol Chem 1997; 272: 27558–27564.

93. Miyazawa K, Shimomura T, Daiji N, and Kitamura N. Proteolytic activation of hepatocyte growth factor in response to tissue injury. J. Biol. Chem. 1994; 269: 8966–8970.

protease. J Biol Chem 2000; 275: 36720–36725.

390: 125–136.

show that met expression is associated with worse outcome but is not correlated

79. Kang JY, Dolled-Filhart M, Ocal IT, Singh B, Lin C-Y, Dickson RB, Rimm DL, and Camp RL. Tissue microarray analysis of hepatocyte growth factor/met pathway components reveals a role for met, matriptase, and hepatocyte growth factor activator inhibitor 1 in the progression of node-negative breast cancer. Cancer Res 2003; 63: 1101–1105.

80. Expression of c-met is a strong independent prognostic factor in breast carcinoma. Cancer 1998; 82: 1513–1520.

81. Jiang WG. HGF and its receptor as potential therapeutic targets in cancer. Current Oncology 2007 (in press).

Ghoussoub RAD, Dillon DA, D’Aquila T, Rimm EB, Fearon ER, and Rimm DL.

with epidermal growth factor family receptors. Cancer 2003; 97: 1841–1848.


98. Kirchhofer D, Peek M, Li W, Stamos J, Eigenbrot C, Kadkhodayan S, Elliott JM, Corpuz RT, Lazarus RA, and Moran P. Tissue expression, protease specificity, and Kunitz domain functions of hepatocyte growth factor activator inhibitor-1B (HAI-1B), a new splice variant of HAI-1. J Biol Chem 2003; 278: 36341–36349.

99. Shimomura T, Ochiai M, Kondo J, and Morimoto Y. A novel protease obtained from FBS-containing culture supernatant, that processes single chain form hepatocyte growth factor to 2 chain form in serum-free culture. Cytotechnology 1992; 8: 219–229.

100. Shimomura T, Kondo J, Ochiai M, Naka D, Miyazawa K, Morimoto Y, and Kitamura N. Activation of the zymogen of hepatocyte growth factor by thrombin. J. Biol. Chem. 1993; 268: 22927–22932.

101. Miyazawa K, Wang Y, Minoshima S, Shimuzu N, and Kitamura N. Structural organisation and chromosomal localization of the human hepatocyte growth factor activator gene. Phylogenetic and functional relationship with blood coagulation factor XII, urokinase, and tissue-type plasminogen activator. Eur. J. Biochem. 1998; 258: 355–361.

102. Furie B and Furie BC. The molecular basis of blood coagulation. Cell 1988; 53: 505–518.

103. Moriyama T, Kataoka H, Tsubouchi H, and Koono M. Concomitant expression of hepatocyte growth factor (HGF), HGF activator and c-met genes in human glioma cells in vitro. FEBS Lett. 1995; 372: 78–82.

104. Yamada T, Tsujioka Y, Taguchi J, Takahashi M, Tsuboi Y, and Shimomura T. White matter astrocytes produce hepatocyte growth factor activator inhibitor in human brain tissues. Exp. Neurology. 1998; 153: 60–64.

105. Kataoka H, Hamasuna R, Itoh H, and Kitamura Nand Koono M. Activation of hepatocyte growth factor/scatter factor in colorectal carcinoma. Cancer Res. 2000; 60: 6148–6159.

106. Parr C, and Jiang WG. Expression of hepatocyte growth factor/scatter factor, its activator, inhibitors and the c-Met receptor in human cancer cells. Int J Oncology 2001; 19: 857–863.

107. Lin CY, Wang JK, Torri J, Dou L, Sang A, and Dickson RB. Characterisation of a novel membrane bound 80 kDa matrix degrading protease from human breast cancer cells. J. Biol. Chem. 1997; 272: 9147–9152.

108.

109. Lin CY, Anders J, Johnson M, Sang QA, and Dickson RB. Molecular cloning of cDNA for matriptase, a matrix-degrading serine protease with trypsin-like activity. J Biol Chem. 1999; 274: 18231–18236.

110. Tanimoto H et al. Ovarian tumor cells express a transmembrane serine protease: a potential candidate for early diagnosis and therapeutic intervention. Tumour Biol. 2001; 22: 104–114.

Lin CY, Anders J, Johnson M, and Dickson RB. Purification and characterisation of a complex containing matripase and a kunitz-type serine proteaseinhibitor from human milk. J. Biol. Chem. 1999; 274: 18237–18242.

94. Miyazawa K, Shimomura T, and Kitamura N. Activation of hepatocyte growth factor, in the injured tissues, is mediated by hepatocyte growth factor activator. J. Biol. Chem. 1996; 271: 3615–3618.

95. Mizuno K, Tanoue Y, Okano I, Harano T, Takada K, and Nakamura T. Purification and characterization of hepatocyte growth-factor (HGF)-converting enzyme - activation of pro-HGF. Biochem. Biophys. Res. Commun. 1994; 198: 1161–1169.

96. Shimomura T, Miyazawa K, Komiyama Y, Hiraoka H, Naka D, Morimoto Y, and Kitamura N. Activation of hepatocyte growth factor by two homologous proteases, blood-coagulation factor XIIa and hepatocyte growth factor activator.

97. Mars WM, Kim TH, Stolz DB, Liu ML, and Michalopoulos GK. Presence of urokinase in serum-free primary rat hepatocyte cultures and its role in activating hepatocyte growth factor. Cancer Res. 1996; 56: 2837–2843.

Eur. J. Biochem. 1995; 229: 257–261.

198 Parr and Jiang

115. Oberst MD, Williams CA, Dickson RB, Johnson MD, and Lin CY. The activation of matriptase requires its noncatalytic domains, serine protease domain, and its cognate inhibitor. J Biol Chem. 2003; 278: 26773–26779.

116. Lee MS, Kiyomiya K, Benaud C, Dickson RB, and Lin CY. Simultaneous activation and hepatocyte growth factor activator inhibitor 1-mediated inhibition of matriptase induced at activation foci in human mammary epithelial cells. Am J Physiol Cell Physiol. 2005; 288: 932–941.

117. Jin X, Yagi M, Akiyama N, Hirosaki T, Higashi S, Lin CY, Dickson RB, Kitamura H, and Miyazaki K. Matriptase activates stromelysin (MMP-3) and promotes tumor growth and angiogenesis. Cancer Science 2000; 97: 1327–1334.

118. List K, Bugge TH, Szabo R. Matriptase: potent proteolysis on the cell surface. Mol Med. 2006; 12: 1–7.

119. List K, Haudenschild CC, Szabo R, Chen W, Wahl SM, Swaim W, Engelholm LH, Behrendt N, and Bugge TH. Matriptase/MT-SP1 is required for postnatal survival, epidermal barrier function, hair follicle development, and thymic homeostasis. Oncogene. 2002; 21: 3765–3779.

120. Satomi S, Yamasaki Y, Tsuzuki S, Hitomi Y, Iwanaga T, and Fushiki T. A role for membrane-type serine protease (MT-SP1) in intestinal epithelial turnover. Biochem Biophys Res Commun. 2001; 287: 995–1002.

121. Ihara S, Miyoshi E, Ko JH, Murata K, Nakahara S, Honke K, Dickson RB, Lin CY, and Taniguchi N. Prometastatic effect of N-acetylglucosaminyltransferase V is due to modification and stabilization of active matriptase by adding beta 1-6 GlcNAc branching. J Biol Chem. 2002; 277: 16960–16967.

122. Denda, K., Shimomura, T., Kawaguchi, T., Miyazawa, K. and Kitamura, N. Functional characterisation of kunitz domains in hepatocyte growth factor activator inhibtor type-1. J. Biol. Chem. 2002; 277: 14053–14059.

123. Shimomura T, Denda K, Kawaguchi T, Matsumoto K, Miyazawa K, Kitamura N. Multiple sites of proteolytic cleavage to release soluble forms of hepatocyte growth factor activator inhibitor type 1 from a transmembrane form. J Biochem. 1999; 126: 821–828.

124. Oberst MD, Chen LY, Kiyomiya K, Williams CA, Lee MS, Johnson MD, Dickson RB, and Lin CY. HAI-1 regulates activation and expression of matrip-tase, a membrane-bound serine protease. Am J Physiol Cell Physiol. 2005; 289:

125. Benaud C, Dickson RB, and Lin CY. Regulation of the activity of matriptase on epithelial cell surfaces by a blood-derived factor. Eur J Biochem. 2001; 268:

126. Itoh H, Kataoka H, Hamasuna R, Kitamura K, and Koono M. Hepatocyte growth factor activator inhibitor type 2 (HAI-2) lacking the first Kunitz-type serine proteinase inhibitor domain is a predominant product in mouse but not in human, Biochem. Biophys. Res. Commun. 1999; 255: 740–748.

462–470.

1439–1447.

111. Kim MG et al. Cloning and chromosomal mapping of a gene isolated from thymic stromal cells encoding a new mouse type II membrane serine protease, epithin, containing four LDL receptor modules and two CUB domains. Immuno-genetics. 1999; 49: 420–428.

112. Takeuchi T, Shuman MA, and Craik CS. Reverse biochemistry: use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue. Proc Natl Acad Sci USA. 1999; 96: 11054–11061.

113. Takeuchi T, Harris JL, Huang W, Yan KW, Coughlin SR, and Craik CS. Cellular localization of membrane-type serine protease 1 and identification of protease-activated receptor-2 and single-chain urokinase-type plasminogen acti-vator as substrates. J Biol Chem. 2000; 275: 26333–26342.

114. List K, Szabo R, Molinolo A, Nielsen BS, and Bugge TH. Delineation of matriptase protein expression by enzymatic gene trapping suggests diverging roles in barrier function, hair formation, and squamous cell carcinogenesis. Am J Pathol. 2006; 168: 1513–1525.


(HGFA) and regulates HGFA activity in the pericellular microenviroenvironment. J. Biol. Chem., 2000 275: 40453–40462.

132. Kataoka H, Uchino H, Denda K, Kitamura N, Itoh H, Tsubouchi H, Nabeshima K, and Koono M. Evaluation of hepatocyte growth factor activator inhibitor expression in normal and malignant colonic mucosa. Cancer Lett. 1998; 128: 219–227.

133. Kataoka H, Itoh H, Kitamura N, Nabeshima K, and Koono M. Distribution of hepatocyte growth factor activator inhibitor type 1 (HAI-1) in human tissues: Cellular surface localisation of HAI-1 in simple columnar epithelium and its modulated expression in injured and regenerative tissues. J. Histochem. Cytochem. 1999; 47: 673–682.

134. Yamauchi M, Itoh H, Naganuma S, Koono M, Hasui Y, Osada Y, and Kataoka H. Expression of hepatocyte growth factor activator inhibitor type 2 (HAI-2) in human testis: identification of a distinct transcription start site for the HAI-2 gene in testis. Biol Chem. 2002; 383: 1953–1957.

135. Itoh H, Yamauchi M, Kataoka H, Hamasuna R, Kitamura N, and Koono M. Genomic structure and chromosomal mapping of the human hepatocyte growth factor activator inhibitor type 1 and 2 genes. Eur. J. Biochem. 2000; 267: 3351–3359.

136. Andreasen PA, Kjoller L, Christensen L, and Duffy MJ. The urokinase-type plasminogen activator system in cancer metastasis: a review. Int J Cancer. 1997; 72: 1–22.

137. use in human cancer. Eur. J. Cancer 1994; 14: 2170–2180.

138. Takada S, Tsuchida T, Kobayashi M, and Koike K. Disruption of the function of tumor-suppressor gene p53 by the hepatitis-b virus x-protein and hepatocarcinogenesis J Cancer Res. Clin. Oncol. 1995; 121: 593–601.

139. Spyratos F, Martin P-M, Hacene K, Romain S, Andrieu C, Ferrero-Pous M,

prognostic evaluation of biological factors in primary breast cancer. J. Natl. Cancer. Inst. 1992; 84: 1266–1272.

140. Nagakawa O, Yamagishi T, Fujiuchi Y, Junicho A, Akashi T, Nagaike K, and Fuse H. Serum hepatocyte growth factor activator (HGFA) in benign prostatic hyperplasia and prostate cancer. Eur Urol. 2005; 48: 686–690.

141.

growth of human glioblastoma cells in vivo. Int J Cancer. 2006; 118: 583–592.

Declerk YA, and Imren S. Protease inhibitors: role and potential therapeutic

Deytieux S, Doussal VL, Tubiana-Hulin M, and Brunet M. Multiparametric

Uchinokura S, Miyata S, Fukushima T, Itoh H, Nakano S, Wakisaka S, and Kataoka H. Role of hepatocyte growth factor activator (HGF activator) in invasive

127. Qin L, Denda K, Shimomura T, Kawaguchi T, and Kitamura N. Functional characterization of Kunitz domains in hepatocyte growth factor activator inhibitor type 2. FEBS Lett. 1998; 436: 111–114.

128. Muller-Pillasch F, Wallrapp C, Bartels K, Varga G, Friess H, Buchler M, Adler G, and Gress TM. Cloning of a new Kunitz-type protease inhibitor with a putative transmembrane domain overexpressed in pancreatic cancer. Biochim. Biophys. Acta 1998; 1395: 88–95.

129. Delaria KA, Muller DK, Marlor CW, Brown JE, Das RC, Roczniak SO, and Tamburini PP. Characterisation of placental bikunin, a novel human serine protease inhibitor. J. Biol. Chem. 1997; 272: 12209–12214.

130. Kirchhofer D, Peek M, Lipari MT, Billeci K, Fan B, and Moran P. Hepsin activates pro-hepatocyte growth factor and is inhibited by hepatocyte growth factor activator inhibitor-1B (HAI-1B) and HAI-2. FEBS Lett. 2005; 579:

131. Kataoka H, Shimomura T, Kawaguchi T, Hamasuna R, Itoh H, Kitamura, N, Miyazawa K, and Koono M. Hepatocyte growth factor activator inhibitor type 1 is a specific cell surface binding protein of hepatocyte growth factor activator

1945–1950.

200 Parr and Jiang

148. Jin JS, Chen A, Hsieh DS, Yao CW, Cheng MF, and Lin YF. Expression of serine protease matriptase in renal cell carcinoma: correlation of tissue microarray immunohistochemical expression analysis results with clinicopathological para-meters. Int J Surg Pathol. 2006; 14: 65–72.

149. Tanimoto H, Shigemasa K, Tian X, Gu L, Beard JB, Sawasaki T, and O’brien TJ. Transmembrane serine protease TADG-15 (ST14/Matriptase/MT-SP1): expression and prognostic value in ovarian cancer. Br J Cancer. 2004; 91: 1834–1841.

150. Oberst M, Anders J, Xie B, Singh B, Ossandon M, Johnson M, Dickson RB, and Lin CY. Matriptase and HAI-1 are expressed by normal and malignant epithelial cells in vitro and in vivo. Am J Pathol. 2001; 158: 1301–1311.

151. Hoang CD, D’Cunha J, Kratzke MG, Casmey CE, Frizelle SP, Maddaus MA, and Kratzke RA. Gene expression profiling identifies matriptase overexpression in malignant mesothelioma. Chest. 2004; 125: 1843–1852.

152. Santin AD, Zhan FH, Bellone S, Palmieri M, Cane S, Bignotti E, Anfossi S, Gokden M, Dunn D, Roman JJ, O’Brien TJ, Tian EM, Cannon MJ, Shaughnessy J, and Pecorelli S. Gene expression profiles in primary ovarian serous papillary tumors and normal ovarian epithelium: Identification of candidate molecular

153. S, Roman JJ, O’Brien T, and Pecorelli S. The novel serine protease tumor-associated differentially expressed gene-15 (matriptase/MT-SP1) is highly overexpressed in cervical carcinoma. Cancer. 2003; 98: 1898–1904.

154. Riddick ACP, Shukla CJ, Pennington CJ, Bass R, Nuttall RK, Hogan A, Sethia KK, Ellis V, Collins AT, Maitland NJ, Ball RY, and Edwards DR. Identification of degradome components associated with prostate cancer progression by expres-sion analysis of human prostatic tissues. Br J Cancer. 2005; 92: 2171–2180.

155. Suzuki M, Kobayashi H, Kanayama N, et al. Inhibition of tumor invasion by genomic down-regulation of matriptase through suppression of activation of

156. Galkin AV, Mullen L, Fox WD, et al. CVS-3983, a selective matriptase inhibitor, suppresses the growth of androgen independent prostate tumor xenografts. Prostate 2004; 61: 228–235.

markers for ovarian cancer diagnosis and therapy. Int J Cancer. 2004; 112: 14–25. Santin AD, Cane S, Bellone S, Bignotti E, Palmieri M, Las Casas LE, Anfossi

receptor-bound pro-urokinase. J Biol Chem 2004; 279: 14899–14908.

142. List K, Szabo R, Molinolo A, et al. Deregulated matriptase causes ras-independent multistage carcinogenesis and promotes ras-mediated malignant transformation. Genes Dev 2005; 19: 1934–1950.

143. Benaud CM, Oberst M, Dickson RB, and Lin CY. Deregulated activation of matriptase in breast cancer cells. Clin Exp Metastasis. 2002; 19: 639–649.

144. Kang JY, Dolled-Filhart M, Ocal IT, Singh B, Lin CY, Dickson RB, Rimm DL, and Camp RL. Tissue microarray analysis of hepatocyte growth factor/Met pathway components reveals a role for Met, matriptase, and hepatocyte growth factor activator inhibitor 1 in the progression of node-negative breast cancer. Cancer Res. 2003; 63: 1101–1105.

145. Cheng MF, Tzao C, Tsai WC, Lee WH, Chen A, Chiang H, Sheu LF, and Jin JS. Expression of EMMPRIN and matriptase in esophageal squamous cell carcinoma: Correlation with clinicopathological parameters. Dis Esophagus. 2006; 19: 482–486.

146. Vogel LK, Saebo M, Skjelbred CF, Abell K, Pedersen ED, Vogel U, and Kure EH.The ratio of Matriptase/HAI-1 mRNA is higher in colorectal cancer adenomas and carcinomas than corresponding tissue from control individuals. BMC Cancer. 2006; 6: 176.

147. Lee JW, Yong SS, Choi JJ, Lee SJ, Kim BG, Park CS, Lee JH, Lin CY, Dickson RB, and Bae DS. Increased expression of matriptase is associated with histopathologic grades of cervical neoplasia. Hum Pathol. 2005; 36: 626–633.


163. Nagakawa O, Yamagishi T, Akashi T, Nagaike K, and Fuse H. Serum hepato-cyte growth factor activator inhibitor type I (HAI-I) and type 2 (HAI-2) in prostate cancer. Prostate. 2006; 66: 447–452.

164. Miyata S, Uchinokura S, Fukushima T, Hamasuna R, Itoh H, Akiyama Y, Nakano S, Wakisaka S, and Kataoka H. Diverse roles of hepatocyte growth factor activator inhibitor type 1 (HAI-1) in the growth of glioblastoma cells in vivo. Cancer Lett. 2005; 227: 83–93.

165. Jiang WG. HGF antagonists in cancer treatment. Expert Reviews in Oncology, 2007, (in press).

166. Ueda K, Iwahashi M, Matsuura I, Nakamori M, Nakamura M, Ojima T, Naka T, Ishida K, Matsumoto K, Nakamura T, and Yamaue H. Adenoviral-mediated Gene transduction of the hepatocyte growth factor (HGF) antagonist, NK4, suppresses peritoneal metastases of gastric cancer in nude mice. Eur J Cancer 2004; 40: 2135–2142.

167. Kubota T, Fujiwara H, Amaike H, Takashima K, Inada S, Atsuji K, Yoshimura M, Matsumoto K, Nakamura T, Yamagishi H. Reduced HGF expression in sub-cutaneous CT26 tumor genetically modified to secrete NK4 and its possible relation with antitumor effects. Cancer Sci 2004; 95: 321–327.

168. Wen J, Matsumoto K, Taniura N, Tomioka D, and Nakamura T. Hepatic gene expression of NK4, an HGF-antagonist/angiogenesis inhibitor, suppresses liver metastasis and invasive growth of colon cancer in mice. Cancer Gene Ther

169. Brockmann MA, Papadimitriou A, Brandt M, Fillbrandt R, Westphal M, and Lamszus K. Inhibition of intracerebral glioblastoma growth by local treatment with the scatter factor/hepatocyte growth factor-antagonist NK4. Clin Cancer Res 2003; 9: 4578–4585.

170. Davies G, Mason MD, Martin TA, Parr C, Watkins G, Lane J, Matsumoto K, Nakamura T, and Jiang WG. The HGF/SF antagonist NK4 reverses fibroblast- and HGF-induced prostate tumor growth and angiogenesis in vivo. Int J Cancer 2003; 106: 348–354.

type-2/placental bikunin (HAI-2/PB) in human glioblastomas: implication for anti-invasive role of HAI-2/PB in glioblastoma cells. Int J Cancer. 2001; 93: 339–345.

2004; 11: 419–430.

157. Saleem M, Adhami VM, Zhong W, Longley BJ, Lin CY, Dickson RB, Reagan-Shaw S, Jarrard DF, and Mukhtar H. A novel biomarker for staging human prostate adenocarcinoma: overexpression of matriptase with concomitant loss of its inhibitor, hepatocyte growth factor activator inhibitor-1. Cancer Epidemiol Biomarkers Prev. 2006; 15: 217–227.

158. Oberst MD, Johnson MD, Dickson RB, Lin CY, Singh B, Stewart M, Williams A, al-Nafussi A, Smyth JF, Gabra H, and Sellar GC. Expression of the serine protease matriptase and its inhibitor HAI-1 in epithelial ovarian cancer: correlation with clinical outcome and tumor clinicopathological parameters. Clin Cancer Res. 2002; 8: 1101–1107.

159. Yamauchi M, Kataoka H, Itoh H, Seguchi T, Hasui Y, and Osada Y. Hepatocyte growth factor activator inhibitor types 1 and 2 are expressed by tubular epithelium in kidney and down-regulated in renal cell carcinoma. J. Urology. 2004; 171: 890–896.

160. Parr C and Jiang WG. Hepatocyte growth factor activation inhibitors (HAI-1 and HAI-2) regulate HGF-induced invasion of human breast cancer cells. Int J Cancer 2006; 119: 1176–1183.

161. Zeng L, Cao J, and Zhang X. Expression of serine protease SNC19/matriptase and its inhibitor hepatocyte growth factor activator inhibitor type 1 in normal and malignant tissues of gastrointestinal tract. World J Gastroenterol. 2005; 11: 6202–6207.

162. Hamasuna R, Kataoka H, Meng JY, Itoh H, Moriyama T, Wakisaka S, and Koono M. Reduced expression of hepatocyte growth factor activator inhibitor

202 Parr and Jiang

178. van Adelsberg J, Sehgal S, Kukes A, Brady C, Barasch J, Yang J, and Huan Y. Activation of hepatocyte growth factor (HGF) by endogenous HGF activator is required for metanephric kidney morphogenesis in vitro. J Biol Chem 2001; 18: 15099–15106.

179. Mazzone M, Basilico C, Cavassa S, Pennacchietti S, Risio M, Naldini L, Comoglio PM, and Michieli P. An uncleavable form of pro-scatter factor suppresses tumor growth and dissemination in mice. J Clin Invest 2004; 114: 1418–1432.

180. Suzuki M, Kobayashi H, Tanaka Y, Hirashima Y, Kanayama N, Takei Y, Saga Y, Suzuki M, Itoh H, and Terao T. Suppression of invasion and peritoneal carcinomatosis of ovarian cancer cell line by overexpression of bikunin. Int J Cancer 2003; 104: 289–302.

181. Kobayashi H, Suzuki M, Kanayama N, Takashi N, Takigawa M, and Terao T. Suppression of urokinase receptor expression by bikunin is associated with inhibition of upstream targets of extracellular signal-regulated kinase-dependant cascade. Eur J Biochem 2002; 269: 3945–3957.

development of the rat gastrointestinal tract. Biochem Biophys Res Commun 1998; 253: 477–484.

171. Martin TA, Parr C, Davies G, Watkins G, Lane J, Matsumoto K, Nakamura T, Mansel RE, and Jiang WG. Growth and angiogenesis of human breast cancer in a nude mouse tumour model is reduced by NK4, a HGF/SF antagonist. Carcinogenesis 2003; 24: 1317–1323.

172. Parr C, Davies G, Nakamura T, Matsumoto K, Mason MD, and Jiang WG. The HGF/SF-induced phosphorylation of paxillin, matrix adhesion, and invasion of prostate cancer cells were suppressed by NK4, an HGF/SF variant. Biochem Biophys Res Co 2001; 285: 1330–1337.

173. Parr C, Hiscox S, Nakamura T, Matsumoto K, and Jiang WG. Nk4, a new HGF/SF variant, is an antagonist to the influence of HGF/SF on the motility and invasion of colon cancer cells. Int J Cancer. 2000; 85: 563–570.

174. Jiang WG, Hiscox SE, Parr C, Martin TA, Matsumoto K, Nakamura T, and Mansel RE. Antagonistic effect of NK4, a novel hepatocyte growth factor variant, on in vitro angiogenesis of human vascular endothelial cells. Clin Cancer Res 1999; 5: 3695–3703.

175. Wang SY, Chen B, Zhan YQ, Xu WX, Li CY, Yang RF, Zheng H, Yue PB, Larsen SH, Sun HB, and Yang X. SU5416 is a potent inhibitor of hepatocyte growth factor receptor (c-Met) and blocks HGF-induced invasiveness of human HepG2 hepatoma cells. J Hepatol. 2004; 41: 267–273.

176. Hov H, Holt RU, Ro TB, Fagerli UM, Hjorth-Hansen H, Baykov V, Christensen JG, Waage A, Sundan A, and Borset M. A selective c-met inhibitor blocks an autocrine hepatocyte growth factor growth loop in ANBL-6 cells and prevents migration and adhesion of myeloma cells. Clin Cancer Res. 2004; 10: 6686–6694.

177. Matsubara Y, Ichinose M, Yahagi N, Tsukada S, Oka M, Miki K, Kimura S, Omata M, Shiokawa K, Kitamura N, Kaneko Y, and Fukamachi H. Hepatocyte Growth Factor Activator: A possible regulator of morphogenesis during fetal

Chapter 10

LIGAND IN BREAST CANCER MANAGEMENT

Department of Breast and Endocrine Surgery, St. George’s Hospital, London, SW17 0QT, UK

The insulin-like growth factor-1 (IGF-1) system plays an important role in normal human development and is also a potent mitogen which can stimulate the development and progression of breast cancer cells. This review aims at looks at how measuring IGF-1 levels may be used in the clinical management of breast cancer patients. Many studies have shown that IGF-1 acts synergistically with oestrogen to stimulate breast cancer cells. Case-control studies have also shown that premenopausal women with high levels of serum IGF-1 have a high risk of developing breast cancer later in life which does not apply to postmenopausal women with correspondingly high serum levels. Serum IGF-1 levels can therefore potentially be used as biomarkers for predicting breast cancer risk while some studies have started using serum IGF-1 levels as a response bio-marker for chemopreventive drug trials. Measuring IGF-1 ligand expression in breast cancer tissue is not consistently associated with better or worst prognosis features. Identifying the IGF-1 gene polymorphism can potentially be used in predicting breast cancer risk and 17beta HSD 1 inhibitors is underway with promising initial results.

IGF-1, IGFBP, biomarker, clinicopathological relevance, survival, breast cancer

1. INTRODUCTION

The association between the insulin-like growth factor-1 (IGF-1) system and breast cancer has been extensively research over the past two decades. Despite its original role as a mediator of normal human growth

THE INSULIN-LIKE GROWTH FACTOR-1

© 2007 Springer. R.E. Mansel et al. (eds.), Metastasis of Breast Cancer, 203–217.

203

Abstract:

Keywords:

Yoon M. Chong, Ash A. Subramanian, and Kefah Mokbel

204

consists of the IGF-1 ligand, the receptor, binding proteins, and proteaseswhich interact in dynamic equilibrium to regulate the effects of IGF-1 (1). This chapter aims to elaborate on the various laboratory and clinical findings regarding IGF-1 and discusses how measuring IGF-1 could potentially be used in a clinical setting.

THE INSULIN-LIKE GROWTH FACTOR-1 (IGF-1) SYSTEM

2.1 In Normal Physiology

The IGF-1 system plays an important role in normal growth and development. It is particularly important for growth of specific organs such as the nervous system in which IGF-1 signaling regulates neuronal proliferation, apoptosis, and cell survival (2). IGF-1 acts as a mediator for growth hormone (GH) which is produced by the anterior pituitary and is fundamental to linear growth. GH stimulates production of the IGF-1 ligand in almost all tissue types especially the liver which serves as the main source of circulating IGF-1 ligand (1). A negative feedback loop exists in which serum IGF-1 suppresses the secretion of GH (3) (Figure 1). In normal development growth, serum IGF-1 is expressed at low levels during embryonic growth, increases post natally from birth to puberty, surges in puberty, and then declines with age thereafter its

2.2 In Cancer Pathology

Initial evidence that the GH/IGF-1 axis contributed to breast cancer progression was provided 30 years ago when hypophysectomy was shown to favourably improve outcome in metastatic breast cancer patients (5). Much of IGF-1 research over the past two decades has focused on the role it plays in the development and progression in numerous varied cancer types. IGF-1 acts as a potent mitogen which can stimulate both normal breast and breast cancer cells. The IGF-1 system has been shown

tenance of the malignant phenotype, increased metastatic potential (8, 9), resistance to apoptosis and cytotoxic drugs (10–12), multi-drug resistance (13, 14) and hormone independence (15–17). These are all features of more aggressive and resistant phenotypes which would eventually translate into poorer prognosis for patients with breast cancers which show inc-

Chong, Subramanian, and Mokbel

2.

levels being affected by nutrition (4).

to promote malignant transformation of normal breast cells (6, 7), main-

and development, the IGF-1 system has now been heavily implicated in the development and progression of breast cancer. The IGF-1 system

205

This chapter will focus on the role of the IGF-1 ligand in the development and progression of breast cancer and how its measurement can potentially be used to manage breast cancer patients.

Figure 1. The Growth Hormone Axis.

3. THE INSULIN-LIKE GROWTH FACTOR-1 LIGAND

The IGF-1 gene is located on chromosome 12q22 and codes for A and B domains which are homologous to the A and B chains of the insulin hormone. The liver produces the largest amount of IGF-1 as a result of

(18). Many studies have also shown that stromal cells adjacent to breast cancer cells can produce IGF-1 locally to stimulate tumour cells via an paracrine or autocrine mechanism (19–23). In fact, several studies have

10. IGF-1 ligand in breast cancer management

GH stimulation and is the main contributor to serum IGF-1. Serum IGF-1can stimulate normal breast cells and promote malignant transfor-mation as well as breast cancer progression via an endocrine fashion

HYPOTHALAMUS

GHRH

PITUITARYGLAND

End organ

GROWTH HORMONE

-ve feedback

LIVER

IGF-1

IGF-1

• IGF-1 is important for mammalian development

• produced by almost all tissue types but main producer of circulatory IGF-1 is liver

• IGF-1 serum levels highest during post-natal period and puberty

• IGF-1 is also produced IGF-1 by normal tissue and is important for

organ development (1)

In normal human physiology• IGF-1 mediates the effects of Growth Hormone

components of the IGF-1 system and looked at their ability to predict the risk of developing breast cancer whilst others aimed to correlate IGF-1 levels with prognosis in breast cancer patients.

reased IGF-1 activity. Several clinical studies have investigated the

206

4. THE ASSOCIATION BETWEEN THE IGF-1 SYSTEM AND OESTROGEN

Research has suggested that IGF-1 and oestrogen act synergistically to stimulate breast cancers and that IGF-1 may have little effect on pro-liferation in the absence of oestrogen (28). Oestrogen stimulation is thought to induce the expression of IGF-1R (29). Cell line studies from various normal and malignant human tissues have established that oestrogen sensitizes cells to the mitogenic effect of IGF-1 through several mecha-nisms such as increasing the expression and binding of IGF-1R signaling components (30, 31), promotion of cell cycle progression while decreasing cell cycle suppressors (32), increasing the expression intracellular signal-ing molecules including IRS-1, IRS-2, (33–35) and increasing the activity of PI3K and Ras-Ref-MAPK intracellular pathways (36, 37).

(38–40) and IGF-1 has been also shown to increase the expression of ER in breast tissue (41). A study in our unit has shown that there may be a reciprocal and cross-stimulatory relationship between IGF-1 ligand and oestrogen production (42). Some studies suggests also that oestrogen can itself stimulate proliferation of breast cancer cells directly or indirectly by elaborating a number of growth factors (43). In other oestrogen-sensitive cancers such as endometriomas, the growth promoting effects of oestrogen are mediated by the induction of IGF-1 (44, 45). Such findings, suggest that IGF-1 and oestrogen are important cofactors of the same pathway which may lead to the development and progression of breast cancers.

5. THE ASSOCIATION BETWEEN SERUM IGF-1 AND BREAST CANCER RISK

Many studies support the role of IGF-1 in malignant transformation of breast epithelia. Animal studies have shown that transgenic mice which over-express growth hormone and IGF-1 exhibit an increased rate of developing mammary tumours (46, 47). Likewise, liver-IGF-1-deficient mice showed a 75% reduction of circulating IGF-1 compared to control mice which also correlated with a significant reduction in risk of mammary tumour development (48) while treatment of primates with

Chong, Subramanian, and Mokbel

Anti-oestrogens like tamoxifen can reduce plasma IGF-1 by 25%–30%

suggested that local tissue production could be an important source of IGF-1 which may play a role in growth of normal tissue (24) as well breast cancer development and progression (20, 25–27).

207

controls. The results showed that when looking at the overall group, there was no relationship between serum IGF-1 and risk of developing breast cancer. However, on sub-analysis based on menopausal status, there was a significant association between elevated serum IGF-1 and breast cancer risk in women who were premenopausal at the time of blood collection (50). A subsequent larger case-control study by Schernhammer et al. involving 800 breast cancer patients and 1,129 age-matched controls also showed that serum IGF-1 levels were modestly associated with an increased breast cancer risk among premenopausal women only (45, 51). Other studies have confirmed that this risk was not present in postmenopausal women with high serum IGF-1 levels (52–58). One study which examined postmenopausal women alone did show a significant association between high serum IGF-1 levels and a risk of breast cancer but this was not significant once the hormone replacement therapy users were removed from the series (59). A meta-analytical study by Shi et al. involving 16 similar studies has concluded that there is a nearly 40% increase in breast cancer risk among premenopausal women with higher IGF-1 in circulation (53, 60). These findings reinforce the understanding that oestrogen may act as a cofactor in promoting the effects of IGF-1 on normal breast cells and may lead to malignant transformation.

6. THE ASSOCIATION BETWEEN SERUM IGF-1 LEVELS WITH CLINICOPATHOLOGICAL FACTORS, DISEASE-FREE SURVIVAL, AND OVERALL SURVIVAL

Many studies have confirmed that premenopausal women generally have higher levels of serum IGF-1 levels compared to postmenopausal women (61–63). Some studies have shown that breast cancer patients in general have higher plasma IGF-1 levels at the time of diagnosis compared to normal (control) subjects (64, 65).


growth hormone and IGF-1 led to mammary gland hyperplasia (49). Animal studies suggested that high levels of circulating IGF-1 could be responsible for an increased risk of breast cancer in humans and this hypothesis prompted studies looking at the relationship between serum

The first prospective study on this relationship was performed by Hankinson et al. (18) who carried out a case-control study by retrospec-tively measuring serum IGF-1 on blood samples collected from 397 women who subsequently developed breast cancer against 620 age-matched

IGF-1 and risk of breast cancer in human subjects.

sample size and cancer cases were not matched to an equal number of controls (67). Holdaway et al. looked at serum IGF-1 levels at baseline and at 1 week post commencement of chemotherapy in patients with early and advanced breast cancer. In this study, there was no significant relationship between basal serum IGF-1 level and survival. Serum IGF-1 levels did not change with chemotherapy in the overall group. Contrary to serum IGFBP-3 levels, the fall in serum IGF-1 also did not seem to have any association with overall survival (58).

Measuring local breast tissue IGF-1 expression seems logical considering that most studies conclude that serum IGF-1 level falls after onset of menopause and would not appear to contribute to late postmenopausal breast cancer leading to the hypothesis that local IGF-1 production may contribute to postmenopausal breast cancer. However, studies correlating local breast tissue IGF-1 expression with clinico-pathological feature and prognosis are also limited. Yu et al. showed tissue expression of IGF-1 in 135 tumour tissue cytosols using radio-immunoassay did not show significant correlation between IGF-1 expression with ER, PR, or any other biochemical markers of poor prognosis such as p53, HER-1, HER-2 protein, S-phase fraction or DNA ploidy (59). An earlier study by Mizukami et al. who used immuno-histochemistry also failed to show any correlation between IGF-1 expression, histological features and prognosis but did show a positive correlation between tumour IGF-1 expression and ER content (60). Al-Sarakbi showed that IGF-1 mRNA levels in breast tissue adjacent to breast tumour correlated with the number of metastatic lymph nodes only, but not with any other pathological prognostic factor (61). Toropainen et al. measured IGF-1 expression in tumour and breast stromal tissue using immunohistochemistry in a series of 211 breast cancer cases and showed that IGF-1 immunostaining in tumour areas tended to be higher in cases with axillary lymph node negativity as compared to those with

receptor status, nodal status, tumour size, and histological grading are lacking. Vadgama et al. measured the serum IGF-1 in breast cancer patients after primary treatment and found that it correlated only to tumour size and progesterone receptor (PR) immunostaining, without any association with age, nodal status, or oestrogen receptor status. His study also showed that patients who received adjuvant Tamoxifen had lower serum IGF-1 levels and this corresponded with a lower probability of recurrent

that serum IGF-1 levels were higher in patients with metastases com-pared with those without or ‘normal’ controls. There were no differences found between ER+ve and ER-ve metastatic groups or between the non-metastatic and control groups. However, this study involved only a small

breast cancer and longer overall survival (56, 66). Coskun et al. showed

Information looking at the relationship between serum IGF-1, clinical outcome and clinicopathological prognostic factors such as oestrogen

208 Chong, Subramanian, and Mokbel

7. USING SERUM IGF-1 AS A SURROGATE

PRIMARY AND SECONDARY BREAST CANCER END-POINT BIOMARKER OF DEVELOPING

probability compared to negative tumour-IGF-1 staining cases but no effect on recurrence-free survival. IGF-1 immunostaining intensity in stromal tissue adjacent to breast tumours correlated with tumour size, nuclear pleomorphism, DNA diploidy and increased likelihoods of meta-stasis at time of diagnosis but did not have any association with recurrence-free survival or overall survival (62). Eppler et al. measured IGF-1 using radioimmunoassay and found that IGF-1 expression was significantly lower in grade 3 tumours compared to grade 1 and 2 tumours. In all histopathological grades, IGF-1 immunoreactivity increased along with ER and PR levels but was inversely related to S-phase fraction. In low grade tumours, tumour IGF-1 levels was associated with longer survival

axillary metastasis and also higher in cases with low S-phase fraction compared to higher S-phase fraction. In all cases, patients with positive tumour-IGF-1 staining cases had significantly longer overall survival


The strong association between breast cancer risk and serum IGF-1 has prompted clinical drug trials to use serum IGF-1 as a surrogate end-point biomarker for predicting risk of developing primary breast carcinogenesis. In this way, circulating IGF-1 could be a cofactor in the

that lead to carcinogenesis. As mentioned previously, several case-

could potentially be used to predict risk of developing breast cancer later in life. In the clinical setting, serum IGF-1 could be used as a risk biomarker that could allow evaluation of risk of breast cancer in the general population or at least in groups of patients with high risk of developing early breast cancer such as BRCA1 and BRCA-2 gene mutation carriers or patients on exogenous oestrogen treatment.

time (63). Overall, most studies suggests that IGF-1 expression is asso-

However, more studies are needed to validate these findings.

development of breast cancer or it may be a by product of other processes

control studies have shown that serum IGF-1 in premenopausal women

inhibit or reverse the process of carcinogenesis. It aims to treat premalig-nant cells thereby disrupting the series of events involved in carcino-genesis which would promote their progression to neoplastic disease.Traditionally, many drug chemoprevention trials recruited early-stage

as the prevention of cancer by the use of pharmacological agents that

Another use of serum IGF-1 is its use as a response biomarker in testing chemopreventive drugs. Chemoprevention has been defined

ciated with favourable histopathological features and better prognosis.

(64). Response biomarkers allow breast cancer risk to be evaluated before the incident occurs which can play a very important role in chemo-prevention trials. In addition to this, once the biomarker has been validated to be a consistent predictor of risk, it can also be utilised outside trial settings to help patients and physicians make decisions regarding initia-tion or continuation of chemoprevention drug treatment. To date serum IGF-1 and serum IGFBP-3 has been shown been one of the most widely used biomarkers of response used in chemoprevention trials in addition to Ki-67, breast intraepithelial neoplasia morphology by FNA, nipple aspiration or biopsy, and mammographic density (65).

Many early studies have shown that adjuvant tamoxifen treatment of breast cancer patients led to a reduction in serum IGF-1 (66–68) which suggests that oestrogen stimulation may be required in order to produce IGF-1 in circulation. The National Surgical Adjuvant Breast Project (NSABP) trial showed that women with a high Gail-risk of 1.7% or higher who were randomised to a 5-year treatment of tamoxifen enjoyed a 50% reduction in breast cancer risk incidence relative to those who received placebo (69). Likewise, chemopreventive trials involving Tamoxifen treatment of normal women showed a reduction of levels of biomarkers including serum IGF-1 (70). However, whether tamoxifen lowers breast cancer risk directly or via modulation of serum IGF-1 levels still remains to be validated and further drug trial studies are required before we can confidently use serums IGF-1 as a response biomarker.

This effect was investigated further in a phase III drug trial using fenretinide (a synthetic retinoid). The trial looked at whether the administration of the drug could reduce the risk of contralateral and recurrent ipsilateral breast cancer in treated breast cancer patients bet-ween ages 30 and 70. Fenretinide which inhibits cell growth and induces apoptosis was shown to reduce the risk of secondary breast malignancy in premenopausal women by 35%. Incidentally, this reduction in risk corresponded to a reduction in circulating IGF-1 which was observed one year after drug administration only in premenopausal women but not in postmenopausal women (51). The observed modulation of IGF-1 by Fenretinide together with its clinical effects of secondary cancer risk suggests that a decline in IGF-1 levels may at least partially account for its chemopreventive activity. A 2 × 2 randomised trial of fenretinide and low dose tamoxifen and another randomised trial involving fenretinide and women on hormone replacement therapy are currently underway and aim to measure the change in serums IGF-1 levels to clarify the role of IGF-1 as a response biomarker of carcinogenesis (70).

and then prospectively looked at their risk of developing either contra-lateral or ipsilateral breast cancer in another quadrant as an end-point


breast cancer patients who had completed breast cancer treatment,

8. THE RELATIONSHIP BETWEEN IGF-1 PHENOTYPE, SERUM IGF-1, AND RISK

There is evidence that serum IGF-1 levels vary considerably between healthy adults. Twin studies show that 50% of the inter-individual varia-bility of circulating IGF-1 is genetically determined (71, 72). Some studies have suggested that this variablility may be due to an inheritable poly-morphism of the IGF-1 gene which in turn may be due to the alleic variations upstream to the IGF-1 gene that lead to changes in the pro-

variation in the frequency of the 19-repeat allele between ethnic groups. The absence of a common 19-repeat allele in the IGF-1 gene is associated with high levels of serum IGF-1 during oral contraceptive (OC) use in nulliparous women (75). The risk of early-onset breast cancer after teenage OC use also varies considerably between ethnic groups, and this appears to correlate with the relative frequencies of the absence of this 19-repeat allele (76). Jernstrom et al. found that the absence of the IGF-1 19-repeat allele was more common in premenopausal women with breast cancer than those without breast cancer. Even though this IGF-1 polymorphism did not have any effect on serum IGF-1 in nulliparous non-OC users,

OF EARLY BREAST CANCER

moter region (73). The promoter region in the IGF-1 gene contains a CA- repeat sequence which ranges from 12 to 23 repeats (74). There is wide

women with absent 19-repeat alleles demonstrated higher levels of IGF-1 during OC use. This study suggested that there was an increased risk of breast cancer after hormonal exposure especially in teenage OC use or pregnancy in women who lack the 19-repeat allele (75). In addition, this study showed the absence of the 19-repeat allele was more common in BRCA1 mutation carriers than other women, and that these women were more prone to develop early-onset breast cancer than BRCA1 carriers who expressed the 19-repeat allele. As with circulating IGF-1, the IGF-1

Several studies have suggested an association between breast

densities using computer-assisted analysis of mammograms has shown a consistent association between high breast density and breast cancer risk.

women who underwent hormonal breast augmentation, only women who

volumes/density and risk of breast cancer (77). Measurement of breast

with larger breast volumes (78, 79). Hartmann et al. showed that in IGF-1 stimulates cell proliferation, reduces apoptosis, and is associated

lacked the 19-repeat allele demonstrated a substantial increase in breast

that OC users with absent 19-repeat alleles had larger body-weight

volume (81) whilst other studies studies noted that larger breasts may be associated with higher risk of breast cancer (82). Jernstrom et al. showed


genotype did not seem to affect the risk of breast cancer in postmeno-pausal women (75).

adjusted breast volumes than those with at least one copy of the 19-

endogenous and exogenous oestrogen. A consequence to this interaction may be an effect on proliferation of normal breast epithelia which leads to larger breasts and also an increased risk of breast cancer. In future, gene testing on the presence or absence of the 19-repeat allele may be useful in determining risk of breast cancer in high risk premenopausal women such as those with family history breast cancer and those on oral contraceptives.

9. HOW CAN IGF-1 MEASUREMENT BE USED

Of all the components in the IGF-1 system, serum IGF-1 (and IGFBP-3- not elaborated in this review) has shown promise in the clinical manage-ment of breast cancer (53). Measuring serum IGF-1 may be used to predict breast cancer risk in premenopausal women which is especially useful in high-risk women such as those with strong familial breast cancer histories, young oral contraceptive users, and gene mutation carriers. Even though many studies have shown a strong association between serum IGF-1 and the risk of recurrent new primary or contra-lateral breast cancers, further studies are needed to validate this. We look

repeat allele (80). These findings suggest that the IGF-1 genotype may

IN THE FUTURE?

play an important role in early breast cancer and its effect on serum IGF-1and breast cancer risk may rely on the availability of high levels of

forward to results of chemopreventive drug trials which use serum IGF-1 as a response biomarker and further reinforce serum IGF-1 as a useful biomarker of measuring drug efficacy rather than using just clinical outcome as the end-point. So far, studies looking at IGF-1 expression in normal and malignant breast tissue and its prognostic value in breast cancer patients have been inconsistent but this may be limited by the relatively few studies performed on this subject. Consistently, research has shown that the IGF-1 system and oestrogen hormone system interact substantially in stimulating breast cancer and that IGF-1 may be the key step between oestrogen stimulation and breast cancer carcinogenesis. If these results are borne out by further studies, inhibiting the action of IGF-1 systemically or locally at breast tissue level by growth factor-targeted therapy may be the next step in IGF-1 research.


REFERENCES

1. Moschos SJ, Mantzoros CS. The role of the IGF system in cancer: from basic

2. LeRoith D, Roberts CT, Jr. The insulin-like growth factor system and cancer. Cancer Lett 2003; 195(2):127–137.

3. Chapman IM, Hartman ML, Pieper KS, Skiles EH, Pezzoli SS, Hintz RL, et al. Recovery of growth hormone release from suppression by exogenous insulin-like growth factor I (IGF-I): evidence for a suppressive action of free rather than bound IGF-I. J Clin Endocrinol Metab 1998; 83(8):2836–2842.

4. Thissen JP, Ketelslegers JM, Underwood LE. Nutritional regulation of the insulin-like growth factors. Endocr Rev 1994; 15(1):80–101.

5. PEARSON OH, RAY BS. Results of hypophysectomy in the treatment of metastatic mammary carcinoma. Cancer 1959; 12(1):85–92.

6. Wu Y, Cui K, Miyoshi K, Hennighausen L, Green JE, Setser J, et al. Reduced circulating insulin-like growth factor I levels delay the onset of chemically and genetically induced mammary tumors. Cancer Res 2003; 63(15):4384–4388.

7. Baserga R, Hongo A, Rubini M, Prisco M, Valentinis B. The IGF-I receptor in

1332(3):F105–F126. 8. Long L, Rubin R, Baserga R, Brodt P. Loss of the metastatic phenotype in

murine carcinoma cells expressing an antisense RNA to the insulin-like growth factor receptor. Cancer Res 1995; 55(5):1006–1009.

9. Guerra FK, Eijan AM, Puricelli L, Alonso DF, Bal de Kier JE, Kornblihgtt AR, et al. Varying patterns of expression of insulin-like growth factors I and II and their receptors in murine mammary adenocarcinomas of different metastasizing ability. Int J Cancer 1996; 65(6):812–820.

10. Adams TE, Epa VC, Garrett TP, Ward CW. Structure and function of the type 1 insulin-like growth factor receptor. Cell Mol Life Sci 2000; 57(7):1050–1093.

11. Resnicoff M, Baserga R. The role of the insulin-like growth factor I receptor in transformation and apoptosis. Ann N Y Acad Sci 1998; 842:76–81.

12. Pollak MN. Endocrine effects of IGF-I on normal and transformed breast epithelial cells: potential relevance to strategies for breast cancer treatment and prevention. 2. Breast Cancer Res Treat 1998; 47(3):209–217.

to clinical studies and clinical applications. Oncology 2002; 63(4):317–332.

cell growth, transformation and apoptosis. Biochem Biophys Acta 1997;

13. Guo YS, Jin GF, Houston CW, Thompson JC, Townsend CM, Jr. Insulin-like growth factor-I promotes multidrug resistance in MCLM colon cancer cells. J Cell Physiol 1998; 175(2):141–148.

14. Geier A, Beery R, Haimsohn M, Karasik A. Insulin-like growth factor-1 inhibits cell death induced by anticancer drugs in the MCF-7 cells: involvement of growth factors in drug resistance. Cancer Invest 1995; 13(5):480–486.

15. Nicholson RI, Hutcheson IR, Knowlden JM, Jones HE, Harper ME, Jordan N, et al. Nonendocrine pathways and endocrine resistance: observations with anti-estrogens and signal transduction inhibitors in combination 8. Clin Cancer Res 2004; 10(1 Pt 2):346S–354S.

16. limitations. Breast Cancer Res Treat 2005; 93 Suppl 1:S3–10.

17. Nicholson RI, Staka C, Boyns F, Hutcheson IR, Gee JM. Growth factor-driven mechanisms associated with resistance to estrogen deprivation in breast cancer: new opportunities for therapy. Endocr Relat Cancer 2004; 11(4):623–641.

Nicholson RI, Johnston SR. Endocrine therapy–current benefits and

18. Hankinson SE, Willett WC, Colditz GA, Hunter DJ, Michaud DS, Deroo B, et al. Circulating concentrations of insulin-like growth factor-I and risk of breast cancer. Lancet 1998; 351(9113):1393–1396.


19. Yee D, Paik S, Lebovic GS, Marcus RR, Favoni RE, Cullen KJ, et al. Analysis of insulin-like growth factor I gene expression in malignancy: evidence for a

20. Ellis MJ, Jenkins S, Hanfelt J, Redington ME, Taylor M, Leek R, et al. Insulin-like growth factors in human breast cancer. Breast Cancer Res Treat 1998; 52(1-3):175–184.

21. growth factor (IGF)-system mRNA quantities in normal and tumor breast tissue of women with sporadic and familial breast cancer risk. Breast Cancer Res Treat 2004; 84(3):225–233.

22. Gebauer G, Jager W, Lang N. mRNA expression of components of the insulin-like growth factor system in breast cancer cell lines, tissues, and metastatic breast cancer cells. Anticancer Res 1998; 18(2A):1191–1195.

23. Chong YM, Williams SL, Elkak A, Sharma AK, Mokbel K. Insulin-like growth factor 1 (IGF-1) and its receptor mRNA levels in breast cancer and adjacent non-neoplastic tissue. Anticancer Res 2006; 26(1A):167–173.

24. Sjogren K, Liu JL, Blad K, Skrtic S, Vidal O, Wallenius V, et al. Liver-derived insulin-like growth factor I (IGF-I) is the principal source of IGF-I in blood but

1999; 96(12):7088–7092. 25. Umayahara Y, Kawamori R, Watada H, Imano E, Iwama N, Morishima T, et al.

Estrogen regulation of the insulin-like growth factor I gene transcription involves an AP-1 enhancer. J Biol Chem 1994; 269(23):16433–16442.

26. Richards RG, Walker MP, Sebastian J, DiAugustine RP. Insulin-like growth factor-1 (IGF-1) receptor-insulin receptor substrate complexes in the uterus. Altered signaling response to estradiol in the IGF-1(m/m) mouse. J Biol Chem

27. Stewart AJ, Johnson MD, May FE, Westley BR. Role of insulin-like growth factors and the type I insulin-like growth factor receptor in the estrogen-stimulated proliferation of human breast cancer cells. J Biol Chem 1990; 265(34):21172–21178.

28. Dupont J, Karas M, LeRoith D. The potentiation of estrogen on insulin-like growth factor I action in MCF-7 human breast cancer cells includes cell cycle components. J Biol Chem 2000; 275(46):35893–35901.

29. Ruan W, Catanese V, Wieczorek R, Feldman M, Kleinberg DL. Estradiol enhances the stimulatory effect of insulin-like growth factor-I (IGF-I) on

paracrine role in human breast cancer. Mol Endocrinol 1989; 3(3):509–517.

Voskuil DW, Bosma A, Vrieling A, Rookus MA, ’t Veer LJ. Insulin-like

is not required for postnatal body growth in mice. Proc Natl Acad Sci USA

1998; 273(19):11962–11969.

30. Guvakova MA, Surmacz E. Overexpressed IGF-I receptors reduce estrogen growth requirements, enhance survival, and promote E-cadherin-mediated cell-cell adhesion in human breast cancer cells. Exp Cell Res 1997; 231(1):149–162.

31. Kleinman D, Karas M, Roberts CT, Jr., LeRoith D, Phillip M, Segev Y, et al. Modulation of insulin-like growth factor I (IGF-I) receptors and membrane-associated IGF-binding proteins in endometrial cancer cells by estradiol. Endocrinology 1995; 136(6):2531–2537.

32. Teruel T, Valverde AM, Benito M, Lorenzo M. Insulin-like growth factor I and insulin induce adipogenic-related gene expression in fetal brown adipocyte

33. Valverde AM, Lorenzo M, Navarro P, Benito M. Phosphatidylinositol 3-kinase is a requirement for insulin-like growth factor I-induced differentiation, but not for mitogenesis, in fetal brown adipocytes. Mol Endocrinol 1997; 11(5): 595–607.

34. Pollak M, Costantino J, Polychronakos C, Blauer SA, Guyda H, Redmond C, et al. Effect of tamoxifen on serum insulinlike growth factor I levels in stage I breast cancer patients. J Natl Cancer Inst 1990; 82(21):1693–1697.

primary cultures. Biochem J 1996; 319 ( Pt 2):627–632.

mammary development and growth hormone-induced IGF-I messenger ribonucleic acid. Endocrinology 1995; 136(3):1296–1302.


35. Lonning PE, Hall K, Aakvaag A, Lien EA. Influence of tamoxifen on plasma levels of insulin-like growth factor I and insulin-like growth factor binding protein I in breast cancer patients. Cancer Res 1992; 52(17):4719–4723.

36. Colletti RB, Roberts JD, Devlin JT, Copeland KC. Effect of tamoxifen on plasma insulin-like growth factor I in patients with breast cancer. Cancer Res 1989; 49(7):1882–1884.

37. Yu H, Rohan T. Role of the insulin-like growth factor family in cancer development and progression. J Natl Cancer Inst 2000; 92(18):1472–1489.

38. Chong YM, Colston K, Jiang WG, Sharma AK, Mokbel K. The relationship between the insulin-like growth factor-1 system and the oestrogen metabolising enzymes in breast cancer tissue and its adjacent non-cancerous tissue. Breast Cancer Res Treat 2006 Jun 5.

39. Bulun SE, Lin Z, Imir G, Amin S, Demura M, Yilmaz B, et al. Regulation of aromatase expression in estrogen-responsive breast and uterine disease: from bench to treatment. Pharmacol Rev 2005; 57(3):359–383.

40. Giudice LC, Dsupin BA, Gargosky SE, Rosenfeld RG, Irwin JC. The insulin-like growth factor system in human peritoneal fluid: its effects on endometrial stromal cells and its potential relevance to endometriosis. J Clin Endocrinol Metab 1994; 79(5):1284–1293.

41. Kudoh M, Susaki Y, Ideyama Y, Nanya T, Mori M, Shikama H. Inhibitory effects of a novel aromatase inhibitor, YM511, on growth of endometrial explants and insulin-like growth factor-I gene expression in rats with experimental endometriosis. J Steroid Biochem Mol Biol 1997; 63(1-3):75–80.

42. Wennbo H, Tornell J. The role of prolactin and growth hormone in breast

43. Hadsell DL, Murphy KL, Bonnette SG, Reece N, Laucirica R, Rosen JM. Cooperative interaction between mutant p53 and des(1-3)IGF-I accelerates mammary tumorigenesis. Oncogene 2000; 19(7):889–898.

44. Ng ST, Zhou J, Adesanya OO, Wang J, LeRoith D, Bondy CA. Growth hormone treatment induces mammary gland hyperplasia in aging primates. Nat Med 1997; 3(10):1141–1144.

45. Schernhammer ES, Holly JM, Pollak MN, Hankinson SE. Circulating levels of insulin-like growth factors, their binding proteins, and breast cancer risk. Cancer Epidemiol Biomarkers Prev 2005; 14(3):699–704.

46. Allen NE, Roddam AW, Allen DS, Fentiman IS, Dos SS, I, Peto J, et al. A prospective study of serum insulin-like growth factor-I (IGF-I), IGF-II, IGF-binding protein-3 and breast cancer risk. Br J Cancer 2005; 92(7):1283–1287.

cancer. Oncogene 2000; 19(8):1072–1076.

48. Toniolo P, Bruning PF, Akhmedkhanov A, Bonfrer JM, Koenig KL, Lukanova A, et al. Serum insulin-like growth factor-I and breast cancer. Int J Cancer 2000; 88(5):828–832.

49. Li BD, Khosravi MJ, Berkel HJ, Diamandi A, Dayton MA, Smith M, et al. Free insulin-like growth factor-I and breast cancer risk. Int J Cancer 2001; 91(5):736–739.

50. Keinan-Boker L, Bueno De Mesquita HB, Kaaks R, van Gils CH, Van Noord PA, Rinaldi S, et al. Circulating levels of insulin-like growth factor I, its binding proteins -1,-2, -3, C-peptide and risk of postmenopausal breast cancer. Int J Cancer 2003; 106(1):90–95.

51. Rollison DE, Newschaffer CJ, Tao Y, Pollak M, Helzlsouer KJ. Premenopausal levels of circulating insulin-like growth factor I and the risk of postmenopausal breast cancer. Int J Cancer 2006; 118(5):1279–1284.

47. Krajcik RA, Borofsky ND, Massardo S, Orentreich N. Insulin-like growth factor I (IGF-I), IGF-binding proteins, and breast cancer. Cancer Epidemiol Biomarkers Prev 2002; 11(12):1566–1573.


52. Kaaks R, Lundin E, Rinaldi S, Manjer J, Biessy C, Soderberg S, et al. Prospective study of IGF-I, IGF-binding proteins, and breast cancer risk, in northern and

53. Shi R, Yu H, McLarty J, Glass J. IGF-I and breast cancer: a meta-analysis. Int J Cancer 2004; 111(3):418–423.

54. Muti P, Quattrin T, Grant BJ, Krogh V, Micheli A, Schunemann HJ, et al. Fasting glucose is a risk factor for breast cancer: a prospective study. Cancer Epidemiol

55. Peyrat JP, Bonneterre J, Hecquet B, Vennin P, Louchez MM, Fournier C, et al. Plasma insulin-like growth factor-1 (IGF-1) concentrations in human breast cancer. Eur J Cancer 1993; 29A(4):492–497.

56. Vadgama JV, Wu Y, Datta G, Khan H, Chillar R. Plasma insulin-like growth factor-I and serum IGF-binding protein 3 can be associated with the pro-gression of breast cancer, and predict the risk of recurrence and the probability of survival in African-American and Hispanic women. Oncology 1999; 57(4): 330–340.

57. Coskun U, Gunel N, Sancak B, Gunel U, Onuk E, Bayram O, et al. Significance of serum vascular endothelial growth factor, insulin-like growth factor-I levels and nitric oxide activity in breast cancer patients. Breast 2003 ; 12(2):104–110.

58.

protein-3 following chemotherapy for advanced breast cancer. ANZ J Surg 2003; 73(11):905–908.

59. Yu H, Levesque MA, Khosravi MJ, Papanastasiou-Diamandi A, Clark GM, Diamandis EP. Associations between insulin-like growth factors and their binding proteins and other prognostic indicators in breast cancer. Br J Cancer 1996; 74(8):1242–1247.

60. Mizukami Y, Nonomura A, Yamada T, Kurumaya H, Hayashi M, Koyasaki N, et al. Immunohistochemical demonstration of growth factors, TGF-alpha, TGF-beta, IGF-I and neu oncogene product in benign and malignant human breast tissues. Anticancer Res 1990; 10(5A):1115–1126.

61. expression of IGF-1 and IGF-1R in human breast cancer: association with clinico-pathological parameters. J Carcinog 2006; 5:16.

62. Toropainen E, Lipponen P, Syrjanen K. Expression of insulin-like growth factor I (IGF-I) in female breast cancer as related to established prognostic factors and long-term prognosis. Eur J Cancer 1995; 31A(9):1443–1448.

63. Eppler E, Zapf J, Bailer N, Falkmer UG, Falkmer S, Reinecke M. IGF-I in human breast cancer: low differentiation stage is associated with decreased IGF-I content. Eur J Endocrinol 2002; 146(6):813–821.

southern Sweden. Cancer Causes Control 2002; 13(4):307–316.

Biomarkers Prev 2002; 11(11):1361–1368.

et al. Serum insulin-like growth factor-I and insulin-like growth factor binding Holdaway IM, Mason BH, Lethaby AE, Singh V, Harvey VJ, Thompson PI,

W Al-Sarakbi, Chong Y, Williams S, Sharma A, Mokbel K. The mRNA

66. Friedl A, Jordan VC, Pollak M. Suppression of serum insulin-like growth factor-1 levels in breast cancer patients during adjuvant tamoxifen therapy. Eur J Cancer 1993; 29A(10):1368–1372.

67. Gail MH, Brinton LA, Byar DP, Corle DK, Green SB, Schairer C, et al. Project-ing individualized probabilities of developing breast cancer for white females who are being examined annually. J Natl Cancer Inst 1989; 81(24):1879–1886.

68. Gail MH, Costantino JP, Bryant J, Croyle R, Freedman L, Helzlsouer K, et al. Weighing the risks and benefits of tamoxifen treatment for preventing breast cancer. J Natl Cancer Inst 1999; 91(21):1829–1846.

64. Torrisi R, Decensi A, Formelli F, Camerini T, De PG. Chemoprevention of breast cancer with fenretinide. Drugs 2001; 61(7):909–918.

65. Fabian CJ, Kimler BF. Chemoprevention for high-risk women: tamoxifen and beyond. Breast J 2001; 7(5):311–320.


70. Bonanni B, Ramazzoto F, Franchi D, et al. A randomised trial of fenretinide in

Treat. 2000; A152. 71. Kao PC, Matheny AP, Jr., Lang CA. Insulin-like growth factor-I comparisons

in healthy twin children. J Clin Endocrinol Metab 1994; 78(2):310–312. 72. Harrela M, Koistinen H, Kaprio J, Lehtovirta M, Tuomilehto J, Eriksson J, et al.

Genetic and environmental components of interindividual variation in circulating levels of IGF-I, IGF-II, IGFBP-1, and IGFBP-3. J Clin Invest 1996; 98(11): 2612–2615.

73. Comuzzie AG, Blangero J, Mahaney MC, Haffner SM, Mitchell BD, Stern MP, et al. Genetic and environmental correlations among hormone levels and measures of body fat accumulation and topography. J Clin Endocrinol Metab 1996; 81(2):597–600.

74. Rosen CJ, Kurland ES, Vereault D, Adler RA, Rackoff PJ, Craig WY, et al. Association between serum insulin growth factor-I (IGF-I) and a simple sequence repeat in IGF-I gene: implications for genetic studies of bone mineral density. J Clin Endocrinol Metab 1998; 83(7):2286–2290.

75. Jernstrom H, Deal C, Wilkin F, Chu W, Tao Y, Majeed N, et al. Genetic and nongenetic factors associated with variation of plasma levels of insulin-like growth factor-I and insulin-like growth factor-binding protein-3 in healthy premenopausal women. Cancer Epidemiol Biomarkers Prev 2001; 10(4):377–384.

76. Jernstrom H, Chu W, Vesprini D, Tao Y, Majeed N, Deal C, et al. Genetic factors related to racial variation in plasma levels of insulin-like growth factor-1: implications for premenopausal breast cancer risk. Mol Genet Metab 2001; 72(2):144–154.

77. Jernstrom H, Sandberg T, Bageman E, Borg A, Olsson H. Insulin-like growth factor-1 (IGF1) genotype predicts breast volume after pregnancy and hormonal contraception and is associated with circulating IGF-1 levels: implications for risk of early-onset breast cancer in young women from hereditary breast cancer families. Br J Cancer 2005; 92(5):857–866.

78. Missmer SA, Haiman CA, Hunter DJ, Willett WC, Colditz GA, Speizer FE, et al. A sequence repeat in the insulin-like growth factor-1 gene and risk of breast

79. Maskarinec G, Williams AE, Kaaks R. A cross-sectional investigation of breast density and insulin-like growth factor I. Int J Cancer 2003; 107(6):991–996.

80. Hartmann BW, Laml T, Kirchengast S, Albrecht AE, Huber JC. Hormonal breast augmentation: prognostic relevance of insulin-like growth factor-I. Gynecol Endocrinol 1998; 12(2):123–127.

HRT users using IGf-1 as a surrogate biomarker. (abstract). Breast Cancer Res

cancer. Int J Cancer 2002; 100(3):332–336.

82. Jernstrom H, Olsson H. Breast size in relation to endogenous hormone levels, body constitution, and oral contraceptive use in healthy nulligravid women aged 19-25 years. Am J Epidemiol 1997; 145(7):571–580.

81. Dupont WD, Page DL. Breast cancer risk associated with proliferative disease, age at first birth, and a family history of breast cancer. Am J Epidemiol 1987; 125(5):769–779.


69. Decensi A, Bonanni B, Guerrieri-Gonzaga A, Gandini S, Robertson C, Johansson H, et al. Biologic activity of tamoxifen at low doses in healthy women. J Natl Cancer Inst 1998; 90(19):1461–1467.

Chapter 11

LYMPHANGIOGENESIS AND METASTATIC SPREAD OF BREAST CANCER

Mahir A. Al-Rawi and Wen G. Jiang Metastasis and Angiogenesis Research Group, School of Medicine, Cardiff University,

Abstract: Lymphangiogenesis, the growth and formation of new lymphatic vessels, has been extensively studied in recent years. With the identification of new lymphangiogenic factors and new lymphatic markers, the role of lymphangiogenesis in the progression of breast cancer and in the lymphatic spread of breast cancer cells have been recognized. The current chapter overviews the progress in this area.

Keywords:

1. INTRODUCTION

Lymphangiogenesis (growth and formation of new lymphatic vessels) occurs in both normal developing tissues and in pathological processes like inflammation, wound healing, lymphoedema, and most importantly in cancer lymphatic spread. Recently, there has been an increasing interest in lymphangiogenesis due to the discovery of molecular markers including; podoplanin, prox-1, and LYVE-1 that are specific to the lymphatic endothelium. Although the molecular mechanisms of lymphan-giogenesis are still not very clear, it is through that the production of growth factors like the vascular endothelial growth factors – C and D (VEGF-C and VEGF-D) within tumours could induce the endothelial cells within tumour tissues to grow and generate new lymphatics that could establish a connection to the peri-tumoral lymphatics and eventually tumours cells could metastasise to the regional lymph nodes. Formation of lymphatic vessels occurs early during fetal development by sprouting of endothelial cells. There are two main theories behind

© 2007 Springer.

lymphangiogenesis, breast cancer, VEGF-D, lymphatic markers

Heath park, Cardiff, CF14 4XN, UK

219R.E. Mansel et al. (eds.), Metastasis of Breast Cancer, 219–240.

220 Al-Rawi and Jiang

system from isolated primitive lymph sacs exclusively by sprouting of endothelial cells into the surrounding tissues and organs (1–3). Most recent data favours this theory, including expression studies of lymphatic specific markers (4–5). The second theory of lymphatic development, the “centripetal sprouting”, was proposed by Huntington and McClure (6). Huntington and McClure proposed a vasculogenic mechanism for the development of the peripheral lymphatic system. In this theory lymphatic spaces would arise independently from the veins, fusing into a primitive lymphatic network, and subsequently spread centripetally and connect to the venous system. The centripetally sprouting lymphatics would either integrate or replace the embryonic lymph sacs.

The lymphatic system is an excellent pathway for malignant cells dissemination, because the initial lymphatics are much larger than the blood capillaries and have incomplete basement membrane. Additionally, flow velocity of lymph is much slower than blood flow and has similar consistency to that of the interstitial fluid enabling cell viability (7–9). Conversely bloodstream is a highly aggressive medium for neoplastic cells due to serum toxicity, high shear stresses and mechanical deformation (9, 10). Additionally, haematogeneous metastasis has low efficiency because a significant number of neoplastic cells are either quiescent or apoptotic (11, 12). Furthermore, cancer cells may pass to bloodstream via lympho-venous shunts, high endothelial venules inside lymph nodes, or may be drained through the thoracic duct (7–9).

One of the major limitations of research on lymphatic vessels was the lack of histological, ultrastructural, and immunohistochemical markers to accurately discriminate between the lymphatic and blood endothelial cells. Lymphatic capillaries are identified by the fact that they are lined by a single layer of endothelial cells, which are characterised by having poorly developed junctions with frequent large gaps between cells. These loose junctions readily permit the passage of large biological macromole-

capillaries is only slightly higher than the interstitium, lumen potency is

blood capillaries, lymphatic capillaries lack a continuous basement membrane, and they are devoid of pericytes (14). However, it should be noted that the latter is not true for larger collecting lymphatic ducts, which are supported by a thin connective tissue coat and higher up the lymphatic drainage tree by an additional smooth muscle wall. Although the initial lymphatics have no valves, the larger collecting ducts do (14). However these anatomical differences do not provide a practical

cules, pathogens, and migrating cells. Because pressure within lymphatic

the embryonic endothelial cell sprouting. Sabin proposed the “centrifugal sprouting” theory; that is, the development of the peripheral lymphatic

of endothelial cells to the perivascular extracellular matrix (7, 13). Unlike

way in the differentiation between blood and lymphatic vessels, parti-cularly in regard to studies involving lymphatics.

maintained by anchoring filaments that connect the abluminal surfaces

221 2. LYMPHATIC MARKERS

Specific markers for lymphatic endothelial cells have been traditionally lacking. However, the past few years have seen the identification of increasing number of molecules (markers) that appear to be specific to lymphatic endothelial cells, and therefore are thought to be highly useful in identification of lymphatic vessels.

2.1. Podoplanin

on normal rat kidney podocytes, but not on podocytes in kidneys with a puromycin aminonucleoside nephrosis (PAN), a model for human

has a single membrane spanning domain, two phosphorylation sites and six O-glycosylation sites in the large ectodomain. Originally, podoplanin was first cloned as OTS-8 in TPA-treated osteoplastic cells (16) and as the antigen recognised by the E11 antibody, which binds to osteoblast and osteocytes and is a marker for cells of the late osteogenic lineage (17). The identical sequence was reported by Rishi et al. (18) as T1α, a protein expressed on alveolar epithelial type 1 cells. The lung is a major site of podoplannin expression in the adult (17, 18). Intravenous injection

shape of podocyte foot processes (19, 20). Podoplanin is also expressed on epithelial cells of the choroids plexus cells and on lymphatic

demonstrate the specificity of podoplanin expression on lymphatic but

co-localise with VEGFR-3 (21, 22). These data suggest that podoplanin

2.2. Prox-1

Another lymphatic marker is Prox-1, the homologue of the Drosophila

during early development (23). Prox-1 gene spans more than 40 kb,

minimal change nephropathy (15). It consists of 163 amino acids and

11. Lymphangiogenesis in metastatic

podoplanin is involved in maintaining lamellar permeability and the

is a very promising marker for differentiating between lymphatic and blood vascular endothelium. To date, the exact function of podoplanin is still

of antibodies against podoplanin caused proteinurea and flattening of podocytes, typical of the pathology seen in PAN suggesting that

unknown. However podoplanin may be involved in regulating the perme-ability of lymphatic vessels, or perhaps in maintaining their integrity (8).

consists of at least five exons and four introns and encodes an 83 kDa protein. Prox-1 gene is mapped to human chromosome 1q32.2–q32.3.

Podoplanin, a specific lymphatic marker, is a 43 kDa surface glyco-protein that was recently cloned as a cell surface protein expressed

homeobox gene prospero, is a marker for the subpopulation of endo-thelial cells that bud and sprout to give rise to the lymphatic system

endothelial cells (17). Light and electron microscopic immunohistology

not blood vasculature endothelia in the skin (21). Furthermore, podo-planin was found to be expressed on PAL-E-negative vessels and to


pancreas (24). Mouse Prox-1 expression was detected in the young neurons of the subventricular region of the CNS as well as the developing lens and the pancreas (25). Targeted deletion of the Prox-1 gene does not affect development of the blood vascular system, but the budding and sprouting of the developing lymphatics is ablated, suggesting that prox-1 plays a key role in lymphatic system development (26). These data point

2.3. LYVE-1

towards a possible exclusive expression of prox-1 in lymphatic endothelium.

Recently, it has demonstrated that LYVE-1 receptor is a type I integral membrane polypeptide expressed on the cell surface as a 60 kDa protein, which is reduced to approximately 40 kDa by glycosidase

fetal liver, less abundant in appendix, bone marrow, placenta, muscle, and adult liver, and absent in peripheral blood lymphocytes, thymus, brain, kidney, and pancreas. Expression of LYVE-1 is largely restricted to endothelial cells lining lymphatic vessels and splenic sinusoidal endothelial cells (27). LYVE-1 may be involved in hyaluronan metabolism in the lymphatic system (8, 28, 29). The co-localisation of LYVE-1 and hyaluronan on the luminal surface of lymphatic vessels suggests that HA may coat the lumen of lymphatic vessels through binding to LYVE-1 allowing hyaluronan-binding cells to adhere and migrate (27). The central core of the LYVE-1 Link module (C2-C3) is 57% identical to that of the human CD44 HA receptor, the only other Link superfamily HA receptor described to date with the closest homologue to LYVE-1. Nevertheless, there are distinct differences between LYVE-1 and CD44 suggesting that the two homologues differ either in the mode of HA

restricted to lymph vessel endothelial cells, while CD44 is almost completely absent (27). While the highest concentration of LYVE-1 expression was found in submucosal lymph vessels underlying smooth muscle in the colon, and the lacteal vessels of intestinal villi that transport dietary lipid absorbed

endothelial cells of the spleen and placental syncytiotrophoblasts (8). The development of antibodies against LYVE-1 has made detection of lymphatics within tumours possible. For example, proliferating intra-tumoral lymph vessels have been identified in head and neck cancer (31).

treatment (27). LYVE-1 is abundant in spleen, lymph node, heart, lung, and

Chicken Prox-1 is highly expressed in the developing lens, retina, and

Studies on LYVE-1 as a lymphatic marker was also helped in detecting

binding or in its regulation. LYVE-1 receptor is almost exclusively

from the small intestine. CD44 is

phatic vessels (30). However, LYVE-1 is also expressed on sinusoidalexpressed abundantly in blood vessels and largely absent from lym-

lymphatics in primary malignant melanoma (32). Furthermore, the pre-sence of LYVE-1 in tumours can indeed promote lymph node metastasis.

223

2.4. VEGF receptors

While, VEGFR-1 and -2 are expressed almost exclusively on vascular endothelial cells, VEGFR-3 is restricted to lymphatic endothelium (36–38).

40). VEGFR-3, a tyrosine kinase receptor, has been shown to control the development and growth of the lymphatic system. The importance of VEGFR-3 for the development of the lymphatic vasculature has been further strengthened by the fact that early onset primary lymphoedema is linked to the VEGFR-3 locus in distal chromosome 5q (41–43). However, in the early embryonic development, VEGFR-3 is essential in the forma-tion of the primary cardiovascular network before the emergence of the lymphatic vessels, as VEGFR-3 knockout embryos die early in develop-ment because of cardiovascular failure (44). In humans, two isoforms of the VEGFR-3 protein occur: VEGFR-3S (short) and VEGFR-3L (long). The difference between the two lies in their carboxyl termini as a result of alternative mRNA splicing (45, 46). VEGFR-3L is the predominant isoform in the tissues. It contains three additional tyrosyl residues, of which Tyr1337 serves as an important autophosphorylation site in the receptor (45, 47). The long isoform was able to mediate anchorage inde-pendent growth in soft agar and tumorigenicity in nude mice (47–49). Stimulation of VEGFR-3, using the specific ligand, induces a rapid tyro-sine phosphorylation of Shc and activation of MAPK pathway results in an increased cell motility, actin reorganisation and proliferation (50–51). In a human erythroleukaemia cell line which expresses high levels of the VEGFR-3, VEGF-C stimulation induced activation of the signalling molecules Shc, Grb2 and SOS which lead to cell growth response (52). In these cells VEGF-C also induced tyrosine phosphorylation of the cytoskeletal protein paxillin by RAFTK, a member of the focal adhesion kinase family. The binding of VEGFR-3 to Grb2 is mediated by the Grb2 SH2 domain. The PTB domain of Shc is required for Shc tyrosine

However, VEGFR-3 can also be upregulated on tumour blood vessels (39,

rylation sites increased VEGFR-3 transforming activity in the soft

vessels and increased subsequent metastasis of tumour to lymph nodes.

phosphorylation by VEGFR-3 (47, 48, 53). Mutations in Shc phospho-


identified as novel targets for the VEGFRs, suggesting that they may be involved in the regulation of endothelial function. Stat proteins are also involved in other cytokines signalling suggesting that the regulation of VEGFR-3 signalling might be controlled by other cytokines. VEGFR-3

Overexpression of VEGF-C in orthotopically transplanted MDA MB-435or MCF-7 breast carcinoma (33, 34) or RIP1/Tag2-RIP1/VEGF-C trans-genic mice (35), promoted proliferation of LYVE-1-positive lymph

a strong activator of Stat-3 and Stat-5. Stat proteins were therefore

agar assay, suggesting that Shc has an inhibitory role in VEGFR-3mediated growth response. Recently, VEGFR-3 has been found to be


pathological tissue samples (54) and has been used to demonstrate an apparent lymphatic origin of Kaposi’s sarcoma cells (55). However, although VEGFR-3 stains PAL-E-negative capillaries (54, 55), recent data show that VEGFR-3 can also be expressed in blood vessel endothelia (57). It is also expressed in blood capillaries during the neovasculari-sation of tumours and in chronic inflammatory wounds (39, 56, 58–60). A mutation in VEGFR-3 has recently been linked to hereditary lympho-edema (41). The mutation, which converts proline 1114 to leucine, occurs in the VEGFR-3 tyrosine kinase domain, indicating that a disturbance in VEGFR-3 signalling may play a part in the development of this disease.

3. LYMPHANGIOGENIC FACTORS

3.1. VEGFs

molecular biology. The detection of the vascular endothelial growth

Since other vascular growth factors were identified and the VEGF family

(36, 63–65). There are three VEGF tyrosine kinase receptors identified

differ from other VEGF family members by the presence of long N- and C-terminal propeptides flanking the VEGF homology domain (63–65, 71–74). The fully processed or mature forms of VEGF-C and VEGF-D consist of the VHD, which acts as a ligand not only for VEGFR-3, but

prominently expressed in regions where the lymphatic vessels undergo sprouting from embryonic veins, such as in the perimetanephric, axillary,

also for VEGFR-2 (73–75). In mid-gestation embryos, VEGF-C is

has been employed as a marker for lymphatic vessels in normal and

and jugular areas, and in the developing mesenterium (5). In adults, VEGF-C is expressed in the heart, small intestine, placenta, ovary, and

the thyroid gland. VEGF-C stimulates mitosis and migration of endo-thelial cells and it increases vascular permeability. VEGF-C has beenshown to induce lymphangiogenesis in transgenic mouse skin and inmature chick chorioallantoic membrane (76, 77). However, recombinant

factors (VEGFs) started with the discovery of VEGF in 1989 (61, 62).

are ligands for receptors VEGFR-1 and VEGFR-2, and considered to

The last 20 years of angiogenesis research have been dominated by

revealed that VEGF family members are expressed in a variety of human tumours in different ways and tumour cells have been reportedto be able to secrete VEGF-A, VEGF-B, VEGF-C, and VEGF-D (67–69). However, the angiogenic switch is thought to be carefully regulated, and

is currently consists mainly of VEGF-A, VEGF-B, VEGF-C, and VEGF-D

at least some specific genetic events in tumour progression correlate

so far, VEGFR-1, VEGFR-2, and VEGFR-3. VEGF-A and VEGFR-B

mechanism is also a distinct possibility (70). VEGF-C and VEGF-D

play an important role in tumour angiogenesis (66). It has been recently

with lymphatic metastasis, suggesting that a “lymphangiogenic switch”

VEGF-C also promotes angiogenesis when applied to early chorioallantoic

225 membrane of chicks, to mouse cornea or to ischaemic hindlimbs of rabbits (50, 78). Therefore, VEGF-C is likely to play a dual role both as an angiogenic and a lymphangiogenic growth factor. If VEGF-C induces

VEGF-D is structurally 48% identical to VEGF-C (90, 91). It contains the eight conserved cysteine residues characteristic of the VEGF family and has a cysteine-rich COOH terminal extension similar to that of VEGF-C. In midgestation mouse embryos, VEGF-D expression is particularly abundant in the developing lung. VEGF-D is expressed in many adult tissues including the vascular endothelium, heart, skeletal muscle, lung, small and large bowel. VEGF-D is mitogenic for endothelial cells. Like VEGF-C, VEGF-D is proteolytically processed after secretion, and it binds

controversy remains as it has been shown that transgenic overexpression of VEGF-D led to lymphatic hyperplasia but not angiogenesis (92).

The secretion of VEGF-C and VEGF-D by some tumours could induce the activation of their receptor, VEGFR-3 on the vascular endothelium and thereby inducing the formation of new lymphatic vessels (Fig. 2). However, little is currently known about the factors that make dome tumours secret these lymphangiogenic factors. Like angiogenesis (formation of new blood vessels), factors such as hypoxia, other growth factors, cytokines and hormones have been studied (93). Regulation by other cytokines and growth factors seems to be promising as it has been recently found that VEGF-C and VEGF-D could indeed be regulated by

talks and interactions between signalling pathways of these cytokines do exist. Although signalling via VEGFR-3 involves complex molecular

IL-1β (94) and IL-7 (95) respectively. It is well established that cross-


lymphangiogenesis, is it sufficient enough to increase the rate of meta-

pathways, but it mainly involves the MAPK and PI3-K pathways. Recent studies have indicated the presence of cross-talks between the MAPK and the PI3-K pathways as phosphorylation of Raf by Akt resulted in inhibition of the Raf-MEK (MAP kinase) – ERK pathway (96). PI3-Kinase activation is known to mediate signalling transduction of many several cytokines and growth factors. The PI3-K pathway is linked to mitogenesis, but several studies subsequently have shown that this pathway has an important function in regulating cell survival by the

stasis to the lymph nodes? It has recently been reported that lymphaticssurrounding a VEGF-C overexpressing tumour are enlarged, and it hasbeen suggested that the increase in lymphatic diameter may be sufficientto increase metastasis (7). Clinical studies correlating the levels of VECF-Cin tumours and their metastatic potential have revealed controversialresults. However, a significant correlation between VEGF-C expressionand lymph node metastasis have been observed in a variety of carcinomasincluding breast (79), oesophageal (80), gastric (81, 82), colorectal (83),thyroid (84, 85), head and neck (86), prostate (87), and lung (88,89).

binds also VEGFR-2 has made it to be possibly angiogenic. However, the to and activates both VEGFR-2 and -3 (65, 73, 90). The fact that VEGF-D

226 Al-Rawi and Jiang activation of the serine-threonine kinase Akt (protein kinase B). The cross talk between MAPK and PI3-K pathways leads to increased cell survival by stimulating the transcription of the pro-survival gene(s) and by post-translational modification and inactivation of components of the cell death machinery. VEGFR-3 can also strongly activates Stat-5 (97), also activated and phosphorylated by IL-7 (98) suggesting that Stat-5 activation is involved in the regulation of lymphatic endothelium.

3.2. Interleukin-7

IL-7 is a proliferative and trophic cytokine that induces the development and proliferation of haematopoietic cells and malignancies (98–110). The intracellular mechanisms mediating signalling for the various effects of IL-7 are not clearly established. However, engagement of IL-7R with its ligand, IL-7, leads to series of intracellular phosphorylation events mediated by signalling molecules including the Janus kinases (Jak-1 and Jak-3), stat-5 (signal transducers and activators of transcription) (98). IL-7 induces the activation of PI3-K (111). PI3-K activation is involved in transducing proliferative signals (112, 113). IL-7-induced PI3-K activation is mediated by tyrosine phosphorylation of the PI3-K p85 subunit and occurs in the absence of SRc family kinase activity (114). Furthermore, Jak-3 is associated with the p85 subunit of PI3-K and regulate its activation (114). Recently, several publications indicated the expression of IL-7 receptor in non-haematopoietic neoplasms (110, 115). The expression of

IL-7 by some human solid tumours including colon and other cancers suggest a possible impact on the process of tumorigenesis and lymphangio-genesis. This is supported by the detection of a functional IL-7R in human solid malignancies. IL-7 mRNA was expressed in colorectal (116, 117),

(120) as well as Warthin’s tumour of parotid gland (121) molecule involved in the downstream signalling pathway of IL-7, in invasive breast cancers than those from benign and normal breast tissue (122),

We have recently studied the expression of IL-7 and IL-7R in breast

with the more invasive and aggressive breast cancer, particularly within tumours that have metastasised to the regional lymph nodes (123). There is increasing evidence suggesting that lymphangiogenesis is higher in

podoplanin, and Prox-1 in endothelial cells, and it induces the formation

breast cancer for example in the node positive tumours compared to

possibility for an autocrine growth pathway for IL-7. The production of

node negative tumours (124). Recently, IL-7 has been identified as a

IL-7 mRNA in some non-haematopoietic malignancies suggest the

oesophageal (118), renal (119), head and neck squamous cell carcinoma

suggesting a possible enhanced IL-7 signalling in invasive breast cancer.

strong lymphangiogenic factor in endothelial cells (125). IL-7 specifically

cancer and quantitative RT-PCR revealed a significant positive correlation

increases the expression of lymphatic markers, including LYVE-1,

227

that has multiple functions over a number of cell types (127). In epithelial cells and epithelium-derived malignant cells, HGF was able to stimulate the migration, morphogenesis, and in some cases proliferation, thus has been widely referred to as motogen, morphogen, and mitogen (128). Inter-

stimulating the migration, morphogenesis, cell adhesion, events central to angiogenesis (129–132). Recently reported that HGF is able to induce formation of lymphatics both in vitro and in vivo breast tumour model (133). The same has been demonstrated in a prostate tumour model (134).

a dual role in tumour endothelium, by acting as an angiogenic factor, and also as a lymphangiogenic factor.

4. LYMPHANGIOGESIS AND LYMPHATIC SPREAD OF BREAST CANCER

4.1. Lymphangiogenesis and metastatic spread of cancer cells

The dissemination of malignant cells to the regional lymph nodes is an early step in the progression of many solid tumours and is an important determinant of prognosis. Recently, some tumours are thought to be lymph-angiogenic, i.e., they have the ability to generate their own lymphatics and thereby provide direct conduit to metastasise to the regional lymph nodes.

These observations have provide tantalising evidence that HGF may play


Although the molecular regulation of lymphangiogenesis is still unclear, the discovery of the vascular endothelial growth factors and receptors has made a real progress in this field. Understanding the molecular signalling pathways in lymphangiogenesis might help to develop new therapeutic strategies against cancer lymphatic spread.

Tumour cell dissemination is mediated by mechanisms including local tissue invasion, lymphatic and blood spread, or direct seeding of body cavities (135). Regional lymph nodes are often the first sites to develop metastases (32, 136), either draining via pre-existing afferent lymphatic

of lymphatic vessels in vivo (126). This effect of IL-7 was mainly mediated via the induction of VEGF-D, both in breast cancer cells and endothelial cells, thus suggesting a paracrine and autocrine regulation. Our recent work has further demonstrated that these effects of IL-7 on breast cancer cells and endothelial cells are via a Wortmannin sensitive

important, as aberrant expression of IL-7, IL-7 receptor and its signalling complex in human breast cancer has been recently demonstrated (123).

of these patients. This aberrant expression was strongly linked to the nodal involvement

IL-7 acts as a powerful lymphangiogenic factor. This is particularly pathway (126). These studies have established, for the first time, that

Hepatocyte growth factor, also known as scatter factor, is a cytokine

estingly, HGF also act on endothelial cells and induces angiogenesis by


basis of the sentinel lymph node biopsy and indicates the particular importance in surgical management of cancers including breast, melanoma, and others. However, not all tumours metastasise to the regional lymph nodes first. Furthermore, the presence of a metastasis in a lymph node does not necessarily mean that the tumour cells have been arrived via the lymphatic vessels (137). Tumour cells may pass directly into the blood vascular system through veno-lymphatic communications. The mechanisms determining whether regional lymph nodes or other sites first develop metastases remain poorly understood. In fact, most disseminated tumour

cells remain dormant in the host tissues, only a few develop into clinicallydetectable micrometastases. However, identification of those occult tumours cells, and prevention of their re-growth would be of great clinical significance.

Tumorigenesis in humans is a multistep process, and these steps reflect the genetic alterations that drive the progressive transformation to cancer. Contrary to normal cells, cancer cells have defective regulatory circuits that control normal proliferation and homeostasis. While normal cells require mitogenic signals to proliferate, malignant cells are self-sufficient for the growth signals and insensitive to the growth-inhibitory signals. Therefore, tumour cells are independent in generating their own growth signals. It has been well established that a complex series of cellular interactions between several types of cells like fibroblasts, immune cells, and endothelial cells as well as malignant cells within the tumour tissues could lead to cancer cells growth and metaststasis (138). In addition to the ability to synthesise their own growth factors leading to an autocrine stimulation, cancer cells could indeed induce the stimulation of other cells like endothelial cells via a paracrine mechanism, thus

Although the significance of pre-existing peritumoral lymphatics as conduits for tumour cell dissemination has been well recognised (139), lymphatic vessels have been thought to be absent from tumours themselves (140). Until recently, it has remained unclear whether tumours can stimu-late lymphangiogenesis or tumour metastasis stimulates molecular activa-tion of the lymphatic system. Previous studies have failed to detect

cells have a limited lifespan in bloodstream. While many surviving cancer

generating neovascularization in the local tumour micro-environment.

vessels and/or via newly formed lymphatic capillaries. This is indeed the

intratumoral functional lymphatics and therefore it was thought that lymphangiogenesis might not play a role in tumour metastasis (141–143). There the initial concept of lymphatic spread of tumours was that tumour cells metastasise solely by the invasion of pre-existing lymphatics surround-ing the tumour margin, i.e., tumours are not lymphangiogenic. However, the absence of intratumoral lymphatics may simply reflect the collapse of

stress generated by the proliferating cancer cells (144). The detection of lymphatics within tumours due to the increased pressure and mechanical

dilated and engorged lymphatics in the peritumoral stroma was not sufficient evidence to claim that they are newly formed, although they were

229

4.2.

Early metastasis to lymph nodes is a frequent complication in human breast cancer. However, the extent to which this depends on lymphangio-genesis or on invasion of existing lymph vessels remains ill-defined. It has been suggested that breast carcinomas invade and destroy lymph

postulated that lymphangiogenesis does not appear to be a feature of invasive breast carcinomas (151). However, the same study revealed that a proportion of the peritumoral lymphatics contained tumour emboli associated with hyaluronan, indicating a possible role for LYVE-1/ hyaluronan interactions in lymphatic invasion or metastasis (151). Intra-tumoral lymphatic vessels have been demonstrated immunohistochemi-cally in breast cancer (152). Using a quantitative approach, the level of expression of a range of lymphangiogenic markers was analysed in a cohort of human breast cancer and compared with the clinical parameters and outcome demonstrated that a high transcript level of LYVE-1 in breast tissues compared with matched normal tissues (124). LYVE-1 level of expression was found to be higher in tumours that had spread to the regional lymph nodes (153). The increased lymphangiogenic markers were also seen together with an increase in the transcript levels of lymphangiogenic factor, VEGF-C and VEGF-D, in the same mammary tumours (154). While it is possible that this increase in lymphangiogenic markers in tumour tissues reflect merely as the presence of pre-existing

of clinical breast cancer Lymphatic markers, lymphangiogenesis and spread


lymphatic vessels by invading tumour cells (140, 141, 143, 144), the possibility may also strongly exists that the quantitation approach also sensitively detected minutes lymphatics with the tumour that cannot be seen by routine immunohistochemistry.

It has been recently reported that interleukin-7 is a lymphangiogenic factor both in vitro and in vivo (125, 126). It was also demonstrated that interleukin-7 and its receptor are aberrantly expressed in human breast cancer (123). The signalling complex molecules of IL-7R, including PI3-K, Jak-3, and Stat-5 are also aberrant in tumour tissues. Aggressive tumours particularly node positive tumours are seen with some most obvious changes.

Increased lymphangiogenesis was correlated to VEGF-C over-expression in metastatic breast cancer (34). This was associated with profound lung

linked to the growth factors produced by tumour cells (144). Therefore the existense of intratumoral lymphatic vessels was rather a disputable issue (7, 140, 142–147). However, most of these studies are indirect and per-formed using tracers or perfusion models, in which no lymphatics could be observed inside tumours. However, during the last 2 years, several studies have demonstrated the existence of intratumoral lymphatics using experimental xenotransplanted tumour models (34, 35, 148–150).

can proceed via pre-existing lymphatics (151). In another study, it was vessels rather than promoting their proliferation and nodal metastasis

230 Al-Rawi and Jiang metastasis and enlargement of the peritumoral lymphatics (34, 155). The rate of lung metastases was directly correlated with the extent of lymphatic microvascular density inside the tumour mass (34). A recent study found that VEGF-C expression was only detectable in node positive breast

and node negative tumours (79). However, other studies claim that although VEGF-C is present, it is not always sufficient to induce the formation of functional lymphatic vessels (144). It has been recently demonstrated that HRG-beta 1 stimulated up-regulation of VEGF-C mRNA and protein of

that this upregulation was de novo RNA synthesis-dependent (156). The

As tumours need neovascularization to grow and metastasise, micro-vascular density has been used as a measure of tumour angiogenesis which is correlated to prognosis (157–162). However, lymphatic microvessel density (LMVD) was rarely assessed because of the lack of a reliable lymphatic marker that is suitable for paraffin sections. Recently, antibodies against VEGFR-3 (80, 163) and LYVE-1 (164) that work on paraffin embedded tissue sections were used to evaluate the presence of intratumoral lymphatics and LMVD as a prognostic factor in several neoplasms. So far, most studies on LMVD have used VEGFR-3 as a

specific marker for normal adult lymphatic vessels, its upregulation in some tumour angiogenesis has made the role of LMVD as a prognostic factor unclear. Therefore, there is currently little conclusive evidence as to the influence of LMVD on patients’ survival. In ovarian cancer for example, the LMVD has no influence on the progression of the disease and in cervical cancer an increased amount of LMVD may even be associated with a better prognosis (166, 167). It has been recently shown that increased flt-4-positive vessel density was correlated with lymph

lymphatic marker (8, 80, 163, 165). Although VEGFR-3 is a highly

cancers, whereas expression of VEGF-A detected in both node positive

HRG-beta 1-induced increase in VEGF-C expression was effectively reduced

human breast cancer cells in a dosage- and time-dependent manner and

by treatment with Herceptin, an antibody specifically against HER2 (156).

The current targeting technologies make it possible to develop drugs into a targeted compound, thereby increasing the potency of the drug at the intended target tissue while reducing side effects elsewhere in the body (169–171). Inhibition of angiogenesis for example, is already considered a promising area in cancer therapy.

As stated above, tumours with a higher incidence of lymph node positivity express high levels of VEGF-C and VEGF-D, inhibition of VEGFR-3 signalling might be an attractive approach to inhibit cancer lymphatic metastasis. In transgenic mice with targeted expression of a soluble form of VEGFR-3 in the skin, lymphatic vessels initially formed normally, but the onset of the transgene expression led to regression of

node metastasis and VEGF-D expression (168). High flt-4-positive vessel

survival in breast cancer (168). density may be a significant unfavourable prognostic factor for long-term

231 lymphatic vessels in embryos (172). Furthermore, a soluble VEGFR-3

in a transplantable human breast carcinoma model using MCF-7 cell line

quent collapse of large tumour vessels was also reported in mice injected

5. PERSPECTIVE

It has been recognised that lymphangiogenesis occurs inside tumours and is associated with nodal and distal metastasis. There is now evidence to suggest that there is significant correlation between the expression of these molecules and several clinicopathologiocal parameters in several human cancers. This might be of particular importance in determining patients’ prognosis and survival. Although tumours can secrete lymphangiogenic growth factors like VEGF-C and VEGF-D and can


system, was recently linked to mutations in the VEGFR-3 tyrosine kinase

protein produced via an adenovirus vector could inhibit lymphangiogenesis

lymphoedema, a rare autosomal dominant disorder of the lymphatic

antibodies against VEGF-C and VEGF-D might also be an area of interest.

domain (173). Interruption of VEGFR-3 signalling results in lymphatic

VEGF-D decreases the number of lymphatic metastases of the VEGF-D-293tumours in the mammary fat pads of SCID/NOD mice (150). Therefore,

with blocking monoclonal antibodies against VEGFR-3 (60). Primary

the association of lymphangiogeneic factors with increased lymphatic

It was recently revealed that the use of neutralizing antibodies against

growth and metastasis of cancers (34, 35, 148, 150) has made them an

in SCID mice (171). In another study, microhaemorrahge and the subse-

attractive target for an additional therapeutic modality against cancer.

unanswered. For example, why different tumours have heterogeneity in

the intrinsic or extrinsic factors that regulate VEGFR-3 signalling? Further work is required to clarify whether these growth factors could also induce pre-existing lymphatic vessels formation? Does interrupting VEGFR-3 signalling have any impact on lymphatic spread and cancer metastasis? The elucidation of molecular components of VEGFR-3 sig-nalling could be beneficial both in terms of diagnosis and therapy by selective targeting of this pathway. Angiogenesis that occurs only in

described (174–176). Does tumour-specific lymphangiogenesis exists? Are there potential markers to distinguish the existing lymphactic vessels from the newly derived ones? Finally, the early research results have tenta-

tumour, also known as tumour-specific angiogenesis, has been recently

regards to the expression and secretion of these growth factors? What are

induce the growth of new lymphatic vessels, several questions remain

lymphatic function during embryonic development (155, 1 73). Neutralisinghypoplasia, underlining the importance of VEGFR-3 in the maintenance of

tively suggested that the degree of lymphangiogenesis have prognostic importance in solid tumours. They also pointed a strong possibility that tar-geting tumour-associated lymphatics may have potential therapeutic value.


1 Clark AH. On the fate of the jugular lymph sacs and the development of lymph channels in the neck of the pig. Am J Anat 1912; 14: 47–62.

2 Sabin FR. On the origin of the lymphatic system from the veins and the development of lymph hearts and thoracic duct in the pig. Am J Anat 1902; 1: 367–391.

3 Sabin FR. On the development of the superficial lymphatics in the skin of pig. Am J Anat 1904; 9: 43–91.

4 Kaipainen A, Korhonen J, Mustonen T et al. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci USA 1995; 92: 3566–3570.

5 Kukk E, Lymboussaki A, Taira S et al. VEGF-C receptor binding and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development. Development 1996; 122: 3829–3837.

6 Hunttington GS, McClure CFW. The anatomy and developmentof the jugular lymph sac in the domestic cat (Felis domestica). Am J Anat 1910; 10: 177–311.

7 Cancer Research, 2001; 7: 462–468.

8 endothelium: In search of the holy grail? Microscopy Research and Technique 2001; 55: 61–69.

9 Swartz MA, Skobe M. Lymphatic function, lymphangiogenesis, and cancer metastasis. Microscopy Research and Technique 2001; 55: 92–99.

10 Cancer Metastasis Rev 2000; 19: I-XI, 193–383.

11 Mehes G, Witt A, Kubista E, Ambros PF. Circulating breast cancer cells are frequently apoptotic. Am J Pathol 2001; 159: 17–20.

12 as a possible source of tumour dormancy? Semin Cancer Biol 2001; 11: 271–276.

13 Biology 1968; 36: 129–149.

REFERENCES

Pepper MS. Lymphangiogenesis and tumor metastasis: Myth or reality? Clinical

Sleeman JP, Krishnan J, Kirkin V, Baumann P. Markers for the lymphatic

Weiss L. Metastasis of cancer: a conceptual history from antiquity to the 1990s.

Naumov GN, MacDonald IC, Chambers AF, Groom AC. Solitary cancer cells

Leak L BJ. Ultrastructural studies on the lymphatic ancchoring filaments. Biol

14

15 Breiteneder-Geleff S, Matsui K, Soleiman A et al. Podoplanin, novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis. Am J Pathol 1997; 151: 1141–1152.

16 promoting 12-O-tetradecanoylphorbol-13-acetate in mouse osteoblastic cells (MC3T3-E1) and expressed constitutively in ras-transformed cells. Cell Growth Differ 1990; 511–518.

17 Wetterwald A, Hoffstetter W, Cecchini M et al. Characterization and cloning of the E11 antigen, a marker expressed by rat osteoblasts and osteocytes. Bone

18 Rishi A, Joyce-Brady M, Fisher J et al. Cloning, characterization, and development expression of a rat lung alveolar type I cell gene inembryonic endodermal and

19 Podoplanin, a novel 43-kDa membrane protein, controls the shape of podocytes. Nephrology Dialysis Transplantation 1999; 14: 9–11.

20 the 43-kD glomerular membrane protein podoplanin cause proteinuria and rapid

Nose K, Saito H, Kuroki T. Isolation of a gene sequence induced later by tumor-

1996; 125–132.

neural derivatives. Dev Biol 1995; 294–306. Matsui K, Breiteneder-Geleff S, Soleiman A, Kowalski H, Kerjaschki D.

Matsui K, Breiteneder-Geleff S, Kerjaschki D. Epitope-specific antibodies to

flattening of podocytes. J Am Soc Nephrol 1998; 2013–2026.

Aukland KRR. Interstitial-lymphatic mechanisms in the control of extracellularfluid volume. physiol Rev 1993; 73: 1–78.

233

Podoplanin as a specific marker for lymphatic endothelium. American Journal of Pathology 1999; 154: 385–394.

22 Weninger W, Partanen TA, Breiteneder-Geleff S et al. Expression of vascular endothelial growth factor receptor-3 and podoplanin suggests a lymphatic

1999; 79: 243–251. 23 Wigle JT OG. Prox-1 function is required for the development of the murine

lymphatic system. Cell 1999; 98: 769–778. 24 Tomarev SI, Sundin O, BanerjeeBasu S, Duncan MK, Yang JM, Piatigorsky J.

Chicken homeobox gene Prox 1 related to Drosophila prospero is expressed in the developing lens and retina. Developmental Dynamics 1996; 206: 354–367.

25 Prospero-Related Homeobox Gene Expressed During Mouse Development. Mechanisms of Development 1993; 44: 3–16.

26 Wigle J, Oliver G. Prox-1 function is required for the development of the murine lymphatic system. Cell 1999; 98: 769–778.

27 Banerji S, Ni J, Wang SX et al. LYVE-1, a new homologue of the CD44

28 Fraser JR, Laurent TC. Turnover and metabolism of hyaluronan. Ciba Found Symp 1989; 143: 41–53; discussion 53–49, 281–285.

29 degradation of hyaluronan in lymphatic tissue. Biochem J 1988; 256: 153–158.

30

31 Beasley NJ, Prevo R, Banerji S et al. Intratumoral lymphangiogenesis and lymph node metastasis in head and neck cancer. Cancer Res 2002; 62: 1315–1320.

32 Oliver G, Detmar M. The rediscovery of the lymphatic system: old and new insights into the development and biological function of the lymphatic vasculature. Genes Dev 2002; 16: 773–783.

express mixed endothelial phenotypes of blood and lymphatic capillaries:

endothelial cell origin of Kaposi’s sarcoma tumor cells. Laboratory Investigation

Oliver G, Sosapineda B, Geisendorf S, Spana EP, Doe CQ, Gruss P. Prox-1, a

glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol 1999; 144: 789–801.

Fraser JR, Kimpton WG, Laurent TC, Cahill RN, Vakakis N. Uptake and


21

33 VEGF-C induced lymphangiogenesis is associated with lymph node metastasis in orthotopic MCF-7 tumors. Int J Cancer 2002; 98: 946–951.

34 Skobe M, Hawighorst T, Jackson DG et al. Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nature Medicine 2001; 7: 192–198.

35 Mandriota SJ, Jussila L, Jeltsch M et al. Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis. Embo Journal 2001; 20: 672–682.

36 VEGF-C. Curr Opin Biotechnol 1999; 10: 528–535.

37 angiogenesis via vascular endothelial growth factor receptors. Cancer Research 2000; 60: 203–212.

38 novel vascular endothelial growth factors. Curr Top Microbiol Immunol 1999; 237: 41–57.

39 Valtola R, Salven P, Heikkila P et al. VEGFR-3 and its ligand VEGF-C are associated with angiogenesis in breast cancer. American Journal of Pathology 1999; 154: 1381–1390.

Mattila MM, Ruohola JK, Karpanen T, Jackson DG, Alitalo K, Harkonen PL.

Eriksson U, Alitalo K. Structure, expression and receptor-binding properties of

Olofsson B, Jeltsch M, Eriksson U, Alitalo K. Current biology of VEGF-B and

Veikkola T, Karkkainen M, Claesson-Welsh L, Alitalo K. Regulation of

Breiteneder-Geleff S AS, Kowalski H, Horvat R, Amman G, Kriehuber E,Diem K, Weninger W, Tschachler E, Alitalo K, Kerjaschki D. Angiosarcomas

Picker LJ, Nakache M, Butcher EC. Monoclonal antibodies to human lympho-cyte homing receptors define a novel class of adhesion molecules on diversecell types. J Cell Biol 1989; 109: 927–937.


42 in familial Milroy lymphedema. Lymphology 1998; 31: 145–155.

43 Evans AL, Brice G, Sotirova V et al. Mapping of primary congenital lymphedema to the 5q35.3 region. Am J Hum Genet 1999; 64: 547–555.

44 Dumont D, Jussila L, Taipale J et al. Cardiovascular failure in mouse embryos

45 Pajusola K, Aprelikova O, Armstrong E, Morris S, Alitalo K. Two human FLT4 receptor tyrosine kinase isoforms with distinct carboxy terminal tails are produced by alternative processing of primary transcripts. Oncogene 1993; 8: 2931–2937.

46 Galland F, Karamysheva A, Pebusque MJ et al. The FLT4 gene encodes a transmembrane tyrosine kinase related to the vascular endothelial growth factor receptor. Oncogene 1993; 8: 1233–1240.

47 Fournier E, Dubreuil P, Birnbaum D, Borg JP. Mutation at tyrosine residue 1337 abrogates ligand-dependent transforming capacity of the FLT4 receptor. Oncogene 1995; 11: 921–931.

48 Pajusola K, Aprelikova O, Pelicci G, Weich H, Claesson-Welsh L, Alitalo K. Signalling properties of FLT4, a proteolytically processed receptor tyrosine kinase related to two VEGF receptors. Oncogene 1994; 9: 3545–3555.

49 Borg JP, deLapeyriere O, Noguchi T, Rottapel R, Dubreuil P, Birnbaum D. Biochemical characterization of two isoforms of FLT4, a VEGF receptor-related tyrosine kinase. Oncogene 1995; 10: 973–984.

50 Cao Y, Linden P, Farnebo J et al. Vascular endothelial growth factor C induces angiogenesis in vivo. Proc Natl Acad Sci USA 1998; 95: 14389–14394.

51 Joukov V, Kumar V, Sorsa T et al. A recombinant mutant vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding, activation, and vascular permeability activities. J Biol Chem 1998; 273: 6599–6602.

52 transduction in human hematopoietic cells by vascular endothelial growth factor related protein, a novel ligand for the FLT4 receptor. Blood 1997; 90: 3507–3515.

deficient in VEGF receptor-3. Science 1998; 946–949.

Wang JF, Ganju RK, Liu ZY, Avraham H, Avraham S, Groopman JE. Signal

Witte MH, Erickson R, Bernas M et al. Phenotypic and genotypic heterogeneity

40 Partanen TA, Arola J, Saaristo A et al. VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissues. Faseb J 2000; 14: 2087–2096.

41 Ferrell RE, Levinson KL, Esman JH et al. Hereditary lymphedema: evidence for linkage and genetic heterogeneity. Hum Mol Genet 1998; 7: 2073–2078.

53 tyrosine residues and protein interaction domains of SHC adaptor in VEGF receptor 3 signaling. Oncogene 1999; 18: 507–514.

54 growth factor (VEGF) and VEGF-C show overlapping binding sites in embryonic endothelia and distinct sites in differentiated adult endothelia. Circulation Research 1998; 85: 992–999.

55 Jussila L VR, Partanen TA, Salven P, Heikkila P, Matikainen, MT RR, Kaipainen A, Detmar M, Tschachler E, Alitalo R, KA. Lymphatic endothelium and Kaposi’s sarcoma spindle cells detected by antibodies against the vascular

56 Paavonen K, Puolakkainen P, Jussila L, Jahkola T, Alitalo K. Vascular endothelial growth factor receptor-3 in lymphangiogenesis in wound healing. Am J Pathol 2000; 156: 1499–1504.

57 Partanen TA AJ, Saaristo A, Jussila L, Ora A, Miettinen M, Stacker SA, Achen

their receptor, VEGFR-3, in fenestrated blood vessels in human tissues. FASEB J 2000; 2087–2096.

Fournier E, Blaikie P, Rosnet O, Margolis B, Birnbaum D, Borg JP. Role of

Lymboussaki A, Olofsson B, Eriksson U, Alitalo K. Vascular endothelial

endothelial growth factor receptor-3. Cancer Res 1998; 1599–1604.

MG, Alitalo K. VEGF-C and VEGF-D expression in neuroendocrine cells and

235

60 Kubo H, Fujiwara T, Jussila L et al. Involvement of vascular endothelial growth factor receptor-3 in maintenance of integrity of endothelial cell lining during

61 Keck PJ, Hauser SD, Krivi G et al. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 1989; 246: 1309–1312.

62 Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989; 246: 1306–1309.

63 growth factor-related protein: a ligand and specific activator of the tyrosine

64 Joukov V PK, Kaipainen A, Chilov D, Lahtinen I, Kukk E, Saksela O, Kalkkinen N, Alitalo K. A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. EMBO J 1996; 290–298 [erratum EMBO J 1996;1915:1751].

65 Orlandini M, Marconcini L, Ferruzzi R, Oliviero S. Identification of a c-fos-induced gene that is related to the platelet-derived growth factor/vascular endothelial growth factor family. Proc Natl Acad Sci USA 1996; 93: 11675–11680.

66 Shweiki D, Neeman M, Itin A, Keshet E. Induction of vascular endothelial growth factor expression by hypoxia and by glucose deficiency in multicell

92: 768–772. 67 Achen MG, Williams RA, Minekus MP et al. Localization of vascular

endothelial growth factor-D in malignant melanoma suggests a role in tumour angiogenesis. J Pathol 2001; 193: 147–154.

68 Endocr Rev 1997; 18: 4–25.

69 Salven P, Lymboussaki A, Heikkila P et al. Vascular endothelial growth factors VEGF-B and VEGF-C are expressed in human tumors. Am J Pathol 1998; 153: 103–108.

tumor angiogenesis. Blood 2000; 546–553.

Lee J GA, Yuan J, Luoh SM, Avraham H, Wood WI. Vascular endothelial

spheroids: implications for tumor angiogenesis. Proc Natl Acad Sci USA 1995;

Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor.


kinase receptor Flt4. Proc Natl Acad Sci USA 1996; 1988–1992.

58 Kubo H FT, Jussila L, Hashi H, Ogawa M, Shimizu K, Awane M, Sakai Y, Takabayashi A, Alitalo K, Yamaoka Y, Nishikawa SI. Involvement of vascular endothelial growth factor receptor-3 in maintenance of integrity of endothelial

59 Partanen T, Alitalo K, Miettinen M. Lack of lymphatic vascular specificity of vascular endothelial growth factor receptor 3 in 185 vascular tumors. Cancer Res 1999; 2406–2412.

70 Jussila L, Alitalo K. Vascular growth factors and lymphangiogenesis. Physiol Rev 2002; 82: 673–700.

71 Joukov V, Pajusola K, Kaipainen A et al. A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2)

72 Lee J, Gray A, Yuan J, Luoh S, Avraham H, Wood W. Vascular endothelial growth factor-related protein: a ligand and specific activator of the tyrosine kinase receptor Flt4. Proc Natl Acad Sci USA 1996; 1988–1992.

73 Achen M, Jeltsch M, Kukk E et al. Vascular endothelial growth factor-D (VEGF-D) is a ligand for tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flk4). Proc Natl Acad Sci USA 1998; 95: 548–553.

74 Achen MG JM, Kukk E, Makinen T, Vitali A, Wilks AF, Alitalo K, Stacker SA. Vascular endothelial growth factor-D (VEGF-D) is a ligand for tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flk4). Proc Natl Acad Sci USA 1998; 95: 548–553.

receptor tyrosine kinases. EMBO J 1996: 290–298 [erratum EMBO J 1996;1915: 1751].

cell lining during tumor angiogenesis. Blood 2000; 546–553.


79 Kurebayashi J, Otsuki T, Kunisue H et al. Expression of vascular endothelial growth factor (VEGF) family members in breast cancer. Japanese J Cancer Res 1999; 90: 977–981.

80 Kitadai Y, Amioka T, Haruma K et al. Clinicopathological significance of vascular endothelial growth factor (VEGF)-C in human esophageal squamous cell carcinomas. Int J Cancer 2001; 93: 662–666.

81 the expression of vascular endothelial growth factor (VEGF) and VEGF-C in gastric carcinoma. J Surg Oncol 2001; 78: 132–137.

82 Yonemura Y, Endo Y, Fujita H et al. Role of vascular endothelial growth factor C expression in the development of lymph node metastasis in gastric cancer. Clin Cancer Res 1999; 5: 1823–1829.

83 Akagi K, Ikeda Y, Miyazaki M et al. Vascular endothelial growth factor-C (VEGF-C) expression in human colorectal cancer tissues. British Journal of Cancer 2000; 83: 887–891.

84 Bunone G, Vigneri P, Mariani L et al. Expression of angiogenesis stimulators

85

1056–1061. 86 O-charoenrat P, Rhys-Evans P, Eccles SA. Expression of vascular endothelial

growth factor family members in head and neck squamous cell carcinoma correlates with lymph node metastasis. Cancer 2001; 92: 556–568.

87 Tsurusaki T, Kanda S, Sakai H et al. Vascular endothelial growth factor-C expression in human prostatic carcinoma and its relationship to lymph node metastasis. Br J Cancer 1999; 80: 309–313.

88 Niki T, Iba S, Tokunou M, Yamada T, Matsuno Y, Hirohashi S. Expression of vascular endothelial growth factors A, B, C, and D and their relationships to

Ichikura T, Tomimatsu S, Ohkura E, Mochizuki H. Prognostic significance of

Fellmer PT, Sato K, Tanaka R et al. Vascular endothelial growth factor-C gene expression in papillary and follicular thyroid carcinomas. Surgery 1999; 126:

75 Joukov V, Sorsa T, Kumar V et al. Proteolytic processing regulates receptor specificity and activity of VEGF-C. Embo Journal 1997; 16: 3898–3911.

76 Jeltsch M, Kaipainen A, Joukov V et al. Hyperplasia of lymphatic vessels in

77 Oh S, Jeltsch M, Birkenhager R et al. VEGF and VEGF-C: specific induction of angiogenesis and lymphangiogenesis in the differentiated avian chorioallantoic

78 Witzenbichler B, Asahara T, Murohara T et al. Vascular endothelial growth factor-C (VEGF-C/VEGF-2) promotes angiogenesis in the setting of tissue ischemia. Am J Pathol 1998; 153: 381–394.

membrane. Dev Biol 1997; 96–109.

92 Marconcini L, Marchio S, Morbidelli L et al. c-fos-induced growth factor/ vascular endothelial growth factor D induces angiogenesis in vivo and in vitro. Proc Natl Acad Sci USA 1999; 96: 9671–9676.

89 Ohta Y, Nozawa H, Tanaka Y, Oda M, Watanabe Y. Increased vascular endothelial growth factor and vascular endothelial growth factor-C and decreased NM23 expression associated with microdissemination in the lymph

90 vascular endothelial growth factor, VEGF-D. Genomics 1997; 42: 483–488.

91 Achen MG, Jeltsch M, Kukk E et al. Vascular endothelial growth factor-D (VEGF-D) is a ligand for tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flk4). Proc Natl Acad Sci USA 1998; 95: 548–553.

Yamada Y, Nezu J, Shimane M, Hirata Y. Molecular cloning of a novel

lymph node status in lung adenocarcinoma. Clinical Cancer Research 2000; 6: 2431–2439.

VEGF-C transgenic mice. Science 1997; 1423–1425 [erratum Science 1997;1277: 1463].

and inhibitors in human thyroid tumors and correlation with clinical patho-logical features. Am J Pathol 1999; 155: 1967–1976.

nodes in stage I non-small cell lung cancer. Journal of Thoracic and Cardio-vascular Surgery 2000; 119: 804–813.

237

causing venous malformations signals a distinct STAT activation response. Oncogene 1999; 18: 1–8.

98 Foxwell BMJ, Beadling C, Guschin D, Kerr I, Cantrell D. Interleukin-7 Can Induce the Activation of Jak-1, Jak-3 and Stat-5 Proteins in Murine T-Cells. Eur J Immunol 1995; 25: 3041–3046.

99 Touw I, Pouwels K, van Agthoven T et al. Interleukin-7 is a growth factor of

100 Eder M, Ottmann OG, Hansen-Hagge TE et al. In vitro culture of common acute lymphoblastic leukemia blasts: effects of interleukin-3, interleukin-7, and accessory cells. Blood 1992; 79: 3274–3284.

101 Digel W, Schmid M, Heil G, Conrad P, Gillis S, Porzsolt F. Human interleukin-7 induces proliferation of neoplastic cells from chronic lymphocytic leukemia and acute leukemias. Blood 1991; 78: 753–759.

102 Dalloul A, Laroche L, Bagot M et al. Interleukin-7 is a growth factor for Sezary lymphoma cells. J Clin Invest 1992; 90: 1054–1060.

103 Qin JZ, Zhang CL, Kamarashev J, Dummer R, Burg G, Dobbeling U. Interleukin-7 and interleukin-15 regulate the expression of the bcl-2 and c-myb genes in cutaneous T-cell lymphoma cells. Blood 2001; 98: 2778–2783.

104 Foss HD, Hummel M, Gottstein S et al. Frequent expression of IL-7 gene

33–39. 105

Lymphocytic-Leukemia. J Exper Medi 1993; 177: 955–964. 106

B-Cell Il-7 - Human B-Cell Lines Constitutively Secrete Il-7 and Express Il-7 Receptors. J Immunol 1994; 152: 4749–4757.

precursor B and T acute lymphoblastic leukemia. Blood 1990; 75: 2097–2101.

transcripts in tumor cells of classical Hodgkin’s disease. Am J Pathol 1995; 146:

Benjamin D, Sharma V, Knobloch TJ, Armitage RJ, Dayton MA, Goodwin RG.


Are Transcribed in Leukemic-Cell Subsets of Individuals with ChronicFrishman J, Long B, Knospe W, Gregory S, Plate J. Genes for Interleukin-7

93 Bellomo D, Headrick JP, Silins GU et al. Mice lacking the vascular endothelial growth factor-B gene (Vegfb) have smaller hearts, dysfunctional coronary vasculature, and impaired recovery from cardiac ischemia. Circ Res 2000; 86: E29–35.

94 Akagi Y, Liu W, Xie K, Zebrowski B, Shaheen RM, Ellis LM. Regulation of vascular endothelial growth factor expression in human colon cancer by interleukin-1beta. Br J Cancer 1999; 80: 1506–1511.

95 Al-Rawi MA, Mansel RE, Jiang WG. Interleukin-7 induced lymphangiogenesis is mediated by autocrine secretion of vascular endothelial growth factor-D (VEGF-D) in breast cancer. Breast Cancer Res Treat 2003; 82 (Supp 1): S134.

96 Zimmermann S, Moelling K. Phosphorylation and regulation of Raf by Akt (protein kinase B). Science 1999; 286: 1741–1744.

97 Korpelainen EI, Karkkainen M, Gunji Y, Vikkula M, Alitalo K. Endothelial receptor tyrosine kinases activate the STAT signaling pathway: mutant Tie-2

111 ligation of the interleukin-7 receptor is dependent on protein tyrosine kinase activity. Blood 1994; 84: 1579–1586.

Dadi H, Ke S, Roifman CM. Activation of phosphatidylinositol-3 kinase by

107 Trumper L, Jung W, Dahl G, Diehl V, Gause A, Pfreundschuh M. Interleukin-7, interleukin-8, soluble TNF receptor, and p53 protein levels are elevated in the

108 lymphoproliferation and lymphomas in interleukin 7 transgenic mice. J Exp Med 1993; 177: 305–316.

109

110 Yamada K, Shimaoka M, Nagayama K, Hiroi T, Kiyono H, Honda T. Bacterial invasion induces interleukin-7 receptor expression in colonic epithelial cell line,

Rich BE, Campos-Torres J, Tepper RI, Moreadith RW, Leder P. Cutaneous serum of patients with Hodgkin’s disease. Ann Oncol 1994; 5 (Suppl 1): 93–96.

T84. Eur J Immunol 1997; 3456–3460.

Takakuwa T, Nomura S, Matsuzuka F, Inoue H, Aozasa K. Expression of inter-leukin-7 and its receptor in thyroid lymphoma. Lab Invest 2000; 80: 1483–1490.

117 Maeurer MJ, Walter W, Martin D et al. Interleukin-7 (IL-7) in colorectal cancer: IL-7 is produced by tissues from colorectal cancer and promotes preferential

118 Oka M, Hirose K, Iizuka N et al. Cytokine mRNA expression patterns in human esophageal cancer cell lines. J Interferon Cytokine Res 1995; 15: 1005–1009.

119 Trinder P, Seitzer U, Gerdes J, Seliger B, Maeurer M: Constitutive and IFN-gamma regulated expression of IL-7 and IL-15 in human renal cell cancer. Int J

120

121 Takeuchi T, Yamanouchi H, Yue Q, Ohtsuki Y. Epithelial component of

122 Watson CJ, Miller WR. Elevated levels of members of the STAT family of transcription factors in breast carcinoma nuclear extracts. Br J Cancer 1995; 71: 840–844.

123 Al-Rawi MA, Rmali K, Watkins G, Mansel RE, Jiang WG. Aberrant expression of interleukin-7 (IL-7) and its signalling complex in human breast cancer. Eur J Cancer 2004; 40: 494–502.

124 Cunnick GH, Jiang WG, Gomez KF, Mansel RE. Lymphangiogenesis quantification using quantitative PCR and breast cancer as a model. Biochem Biophys Res Commun 2001; 288: 1043–1046.

125

126 Al-Rawi MA, Mansel RE, Jiang W. Interleukin-7 (IL-7) induces lymphangio-genesis in vivo by stimulating endothelial cell migration. Br J Surg 2005; 92: 305–310.

Oncol 1999; 14: 23–31.

Al-Rawi M, Mansel R, Jiang W. Interleukin-7 is a putative lymphangiogenic

expansion of tumour infiltrating lymphocytes. Scand J Immunol 1997; 45: 182–192.

112

113 Zvelebil MJ, MacDougall L, Leevers S et al. Structural and functional diversity

of London Series B-Biological Sciences 1996; 351: 217–223. 114 Sharfe N, Dadi HK, Roifman CM. Jak3 Protein-Tyrosine Kinase Mediates

Interleukin-7-Induced Activation of Phosphatidylinositol-3' Kinase. Blood 1995; 86: 2077–2085.

115 Saito S, Harada N, Ishii N et al. Functional expression on human trophoblasts of interleukin 4 and interleukin 7 receptor complexes with a common gamma chain. Biochem Biophys Res Commun 1997; 231: 429–434.

116 Intestinal Epithelial-Cells and Regulates the Proliferation of Intestinal Mucosal Lymphocytes. J Clin Investi 1995; 95: 2945–2953.

Watanabe M, Ueno Y, Yajima T et al. Interleukin-7 Is Produced by Human

127 Jiang W, Hiscox S, Matsumoto K, Nakamura T. Hepatocyte growth factor/scatter factor, its molecular, cellular and clinical implications in cancer. Crit Rev Oncol Hematol 1999; 29: 209–248.

128 liver regeneration and cancer metastasis. Br J Surg 1993; 80: 1368–1373.

129 Grant DS, Kleinman HK, Goldberg ID et al. Scatter factor induces blood vessel formation in vivo. Proc Natl Acad Sci USA 1993; 90: 1937–1941.

130 Jiang WG, Hiscox SE, Parr C et al. Antagonistic effect of NK4, a novel hepatocyte growth factor variant, on in vitro angiogenesis of human vascular endothelial cells. Clin Cancer Res 1999; 5: 3695–3703.

131 Jiang WG, Martin TA, Matsumoto K, Nakamura T, Mansel RE. Hepatocyte

M11–M16.

lymphoid stroma-rich Warthin’s tumour expresses interleukin (IL)-7. Histo-pathology 1998; 32: 383–384.

Carpenter CL, Cantley LC. Phosphoinositide 3-kinase and the regulation of

Paleri V, Pulimood A, Davies GR, Birchall MA. Interleukins 7 and 12 are expres-sed in head and neck squamous cancer. Clin Otolaryngol 2001; 26: 302–306.

cell growth. Biochimica Et Biophysica Acta-Reviews on Cancer 1996; 1288:

vascular endothelial cells. J Cell Physiol 1999; 181: 319–329.

of phosphoinositide 3-kinases. Philosophical Transactions of the Royal Society


growth factor/scatter factor decreases the expression of occludin and trans-

Jiang WG, Hallett MB, Puntis MC. Hepatocyte growth factor/scatter factor,

endothelial resistance (TER) and increases paracellular permeability in human

factor in endothelial cells. Breast Cancer Res Treat. 2002; 76 (Supp 1): S166.

239

138 Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100: 57–70. 139 Fisher B FE. Role of the lymphatic system in dissemination of tumour. Lymph

and lymphatic system 1968: 324. 140 Folkman J. Angiogenesis and tumour growth. New Englang journal of Medicine

1996; 334: 921. 141 Carmeliet P, Jain R. Angiogenisis in cancer and other diseases. Nature Medicine

2000; 407: 249–257. 142 Jain R. Transport of molecules in the tumour interstitium: a review. Cancer

Research 1987; 47: 3039–3051. 143 Tanigawa N, Kanazawa T, Satomura K et al. Experimental-Study on Lymphatic

Vascular Changes in the Development of Cancer. Lymphology 1981; 14: 149–154. 144

lymphatics within a murine sarcoma: A molecular and functional evaluation. Cancer Research 2000; 60: 4324–4327.

145 of Heterogeneous Perfusion and Lymphatics. Microvascular Research 1990; 40: 246–263.

146 Jackson DG, Prevo R, Clasper S, Banerji S. LYVE-1, the lymphatic system and tumor lymphangiogenesis. Trends in Immunology 2001; 22: 317–321.

147 Witte MH, Witte CL. On tumor (and other) lymphangiogenesis. Lymphology 1997; 30: 1–2.

148 Karpanen T, Egeblad M, Karkkainen MJ et al. Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer Research 2001; 61: 1786–1790.

149 Skobe M, Hawighorst T, Alitalo K, Jackson D, Detmar M. Lymphangiogenesis in melanoma, squamous cell carcinoma and breast carcinoma. J Investi Dermatol 2001; 117: 017.

Leu AJ, Berk DA, Lymboussaki A, Alitalo K, Jain RK. Absence of functional


132 Martin TA, Matsumot K, Nakamura T, Mansel RE, Jiang WG. NK4 reduced HGF/SF and fibroblast induced breast tumour growth. Carcinogenesis 2003.

133 Jiang WG, Parr C, Martin TA, Davies G, Watkins G, Mansel RE. Is hepatocyte growth factor/scatter factor (HGF/SF) lymphangiogenic? Breast Cancer Res Treat 2003; 82: 272.

134 Jiang WG, Davies G, Parr C, Martin TA, Mason MD. The potential lymphan-giogenic effects of hepatocyte growth factor/scatter factor in vitro and in vivo.

135 Jiang WG, Puntis MC, Hallett MB. Molecular and cellular basis of cancer invasion and metastasis: implications for treatment. Br J Surg 1994; 81: 1576–1590.

136 Alitalo K, Carmeliet P. Molecular mechanisms of lymphangiogenesis in health and disease. Cancer Cell 2002; 1: 219–227.

137 Van Trappen PO, Pepper MS. Lymphangiogenesis and lymph node micro-dissemination. Gynecologic Oncology 2001; 82: 1–3.

Int J Mol Medicine. 2005; 16: 723–728.

150

151 Williams CS, Leek RD, Robson AM et al. Absence of lymphangiogenesis and intratumoural lymph vessels in human metastatic breast cancer. J Pathol 2003; 200: 195–206.

152 Witte MH WD, Witte CL, Bernas M. Lymphangiogenesis. mechanisms, signi- 153

Correlation between the rate of lymphangiogenesis and lymph node metastasis in breast cancer. Eur J Cancer 2002; 38: 57.

154 Expression of angiogenic and lymphangiogenic factors and markers in breast cancer. Br J Surg 2002; 89 (Suppl 1): 48.

ficance and clinical implications. EXS 1997; 65–112.

Baxter LT, Jain RK. Transport of Fluid and Macromolecules in Tumors. 2. Role

Stacker S, Caeser C, Baldwin M et al. VEGF-D promots the metastatic spreadof tumour cells via the lymphatics. Nat Med 2001; 7: 186–191.

Cunnick GH, Jiang WG, Douglas-Jones A, Watkins G, Gomez K, Mansel RE.

Cunnick GH, Jiang WG, Douglas-Jones A, Watkins G, Gomez K, Mansel RE.


161 Fox SB. Tumour angiogenesis and prognosis. Histopathology 1997; 30: 294–301. 162 Marinho A, Soares R, Ferro J, Lacerda M, Schmitt FC. Angiogenesis in breast

cancer is related to age but not to other prognostic parameters. Pathol Res Pract 1997; 193: 267–273.

163 Clarijs R, Schalkwijk L, Ruiter DJ, de Waal RMW. Lack of lymphangiogenesis despite coexpression of VEGF-C and its receptor Flt-4 in uveal melanoma. Investigative Ophthalmology & Visual Science 2001; 42: 1422–1428.

164 Carreira CM, Nasser SM, di Tomaso E et al. LYVE-1 is not restricted to the lymph vessels: expression in normal liver blood sinusoids and down-regulation in human liver cancer and cirrhosis. Cancer Res 2001; 61: 8079–8084.

165 factors and receptors in humans. Microscopy Research and Technique 2001; 55: 108–121.

166 Lymphatic microvessel density as a novel prognostic factor in early-stage invasive cervical cancer. Int J Cancer 2001; 95: 29–33.

167 Birner P, Schindl M, Obermair A et al. Lymphatic microvessel density in epithelial ovarian cancer: its impact on prognosis. Anticancer Res 2000; 20: 2981–2985.

168 Nakamura Y, Yasuoka H, Tsujimoto M et al. Flt-4-positive vessel density correlates with vascular endothelial growth factor-d expression, nodal status, and prognosis in breast cancer. Clin Cancer Res 2003; 9: 5313–5317.

169 Arap W, Pasqualini R, Ruoslahti E. Chemotherapy targeted to tumor vasculature. Curr Opin Oncol 1998; 10: 560–565.

170 Ruoslahti E. Targeting tumor vasculature with homing peptides from phage display. Semin Cancer Biol 2000; 10: 435–442.

Partanen TA, Paavonen K. Lymphatic versus blood vascular endothelial growth

Birner P, Schindl M, Obermair A, Breitenecker G, Kowalski H, Oberhuber G.

156 Tsai PW, Shiah SG, Lin MT, Wu CW, Kuo ML. Up-regulation of vascular endothelial growth factor C in breast cancer cells by heregulin-beta 1. A critical role of p38/nuclear factor-kappa B signaling pathway. J Biol Chem 2003; 278: 5750–5759.

157 Weidner N. Intratumor microvessel density as a prognostic factor in cancer. Am J Pathol 1995; 147: 9–19.

158 Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and

1–8. 159 Folkman J, Shing Y. Angiogenesis. Journal of Biological Chemistry 1992; 267:

10931–10934. 160 Weidner N. Tumor angiogenesis: review of current applications in tumor

prognostication. Semin Diagn Pathol 1993; 10: 302–313.

metastasis–correlation in invasive breast carcinoma. N Engl J Med 1991; 324:

171 Karpanen T, Alitalo K. Lymphatic vessels as targets of tumor therapy? Journal of Experimental Medicine 2001; 194: F37–F42.

172 Makinen T, Jussila L, Veikkola T et al. Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3.

173 Karkkainen MJ, Ferrell RE, Lawrence EC et al. Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema. Nat Genet 2000; 25: 153–159.

174 St Croix B, Rago C, Velculescu V et al. Genes expressed in human tumor endothelium. Science 2000; 289: 1197–1202.

175 Carson-Walter EB, Watkins DN, Nanda A, Vogelstein B, Kinzler KW, St Croix B. Cell surface tumor endothelial markers are conserved in mice and humans. Cancer Res 2001; 61: 6649–6655.

176 Davies G, Mason MD, Mansel RE, Jiang WG. Expression of tumour endothelial

Nature Medicine 2001; 7: 199–205.

markers in human bresat cancer. Clin Exp Metastasis 2004; 21: 31–37.

155 Makinen T, Jussila L, Veikkola T et al. Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3. Nat Med 2001; 7: 199–205.

Chapter 12

BREAST CANCER SECRETED FACTORS ALTER THE BONE MICROENVIRONMENT Potential New Targets for Bone Metastasis Treatment

Valerie A. Siclari, Theresa A. Guise, and John M. Chirgwin Division of Endocrinology, University of Virginia, PO Box 801401, Charlottesville, VA22908-1401, USA

Abstract: Bone is the most common site of breast cancer metastasis. Over eighty percent of patients with advanced breast cancer develop bone metastases. Once breast cancer has spread to bone, the cancer is incurable and patients develop mostly osteolytic, but also osteoblastic, or mixed bone lesions and suffer from extreme bone pain, skeletal fractures, hypercalcemia, and nerve compression. Current treatment is the use of antiresorptive bisphos-phonates, which reduces bone pain and skeletal fractures but does not improve overall survival. Mouse models of bone metastasis have led to an understanding of the complex interactions that occur within bone that contribute to the incurability of the disease. Once breast cancer cells enter bone, a “vicious cycle” develops between breast cancer cells and the other cells within bone. Breast cancer cells secrete factors that stimulate bone cells, causing them in turn to secrete factors back onto the cancer cells. Inhibiting the actions of cancer-secreted factors may break this vicious cycle. The list of tumor-secreted factors is long, but they can be divided into three groups: (1) bone-resorbing, (2) bone-forming, and (3) metastasis-opposing factors. These factors may share upstream regulatory pathways. Such central pathways could provide new targets for more effective treatment of bone metastasis. The TGFβ and hypoxia-induced Hif1α path-ways are two leading targets for such adjuvant treatments.

breast cancer, bone metastasis, tumor-secreted factors

1.

Breast cancer is one of several cancers, including lung and prostate cancer that displays osteotropism or a preferential growth in bone (1).

© 2007 Springer. R.E. Mansel et al. (eds.), Metastasis of Breast Cancer, 241–258.

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Keywords:

INTRODUCTION

patients with advanced breast cancer will develop bone lesions and suffer from skeletal fractures, hypercalcemia, bone pain, or nerve compression (2). Bone metastases are currently incurable (2). The approved treatment, antiresorptive bisphosphonates, is only palliative (2). Median survival from time of diagnosis of bone metastases is about two years (2). Therefore, new treatments need to be identified to cure this disease. Understanding why breast cancer spreads to bone and aspects of both the breast cancer cell and the bone microenvironment may reveal new targets. This chapter focuses on breast cancer-secreted factors with the goal of identifying molecular targets for improved treatment.

2. THE “SEED AND SOIL” HYPOTHESIS: AN EXPLANATION FOR THE PREFERENTIAL SPREAD OF CANCER CELLS

The “Seed and Soil Hypothesis” was proposed by Stephen Paget in

states: “when a plant goes to seed, its seeds are carried in all directions;but they can only grow if they fall on congenial soil” (4). The “seed” is the breast cancer cell, which can only grow or form metastases in particular, compatible parts of the body or “soils” (3). Aspects of both the seed and the soil contribute to the successful formation of a metastasis (3). Not every seed can grow in every soil (3). In the case of breast cancer, bone serves as a fertile “soil” for the breast cancer “seed” to grow.

3. BONE: A FERTILE SOIL FOR THE BREAST CANCER SEED

The mineralized matrix of the bone is a rich store of growth factors and calcium that are released during bone resorption (5). The released growth factors contribute to the growth of breast cancer cells in bone (6). Insulin-like growth factors (IGFs) I and II and transforming growth factor β (TGFβ) are the most abundant growth factors in bone (5). A role of bone matrix IGF I and II in bone metastasis has not been completely demonstrated. Currently, only TGFβ has been shown to be actively released from the bone matrix by osteoclast resorption (7). Expression of a dominant negative TGFβ receptor subunit in MDA-MB-231 breast cancer cells blocked

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Bone is the most common site of breast cancer metastasis, and over 80% of

1889 to explain the preferential spread of breast cancer to bone (3, 4). It

Siclari, Guise, and Chirgwin

12. Breast cancer secreted factors and BM microenvironment 243 responsiveness to TGFβ and decreased bone metastases in mice (8). TGFβ inhibitors are effective in preclinical models to block bone metastases (9–12).

Actions of the two main bone cell types are coupled. The bone-forming osteoblast and the bone-resorbing osteoclast maintain bone homeostasis by a process of remodeling (13). Osteoclasts resorb bone, leaving a pit within which osteoblasts then form new bone (13). Osteoclast formation is regulated by cells of the osteoblast lineage that express macrophage-colony stimulating factor (M-CSF) and receptor activator of NFkappaB ligand (RANKL) (14). M-CSF induces monocyte/macrophage cell pre-cursors to express the receptor activator of NFkappaB (RANK) (14). Binding of RANKL to RANK stimulates the differentiation of the pre-cursor cells into osteoclasts and increases osteoclast activation and survival (14). Imbalances in the activities of osteoblasts and osteoclasts can lead to increased bone loss or bone formation. Breast cancer cells in bone cause such imbalances, producing predominantly osteolytic (bone destructive), but also osteoblastic (bone forming) and mixed bone lesions (14).

4.

Only the murine mammary carcinoma 4T1 model spontaneously forms metastases to the bone, but it also spreads to the liver, lungs, and brain (15). Standard bone metastasis models are produced by injecting cancer cells into the left cardiac ventricle of immunocompromised mice (6). Within this model, MDA-MB-231 human breast cancer cells produce osteolytic bone lesions within a month after tumor cell inoculation (6). MDA-MB-435s and BT549 breast cancer cell lines also produce osteolytic lesions (6). Other breast cancer cell lines produce osteoblastic (T47D, MCF-7,

bone metastasis mouse models have led to an understanding of the com-plex interactions that develop between breast cancer cells and the bone microenvironment that lead to lesion formation and the incurability of the disease (6), although they lack important regulators of cancer progresssion such as T lymphocytes. Data from these models provide evidence that a “vicious cycle” develops between breast cancer cells and the other cells within bone (6). Once breast cancer cells have entered bone, they secrete various factors that act on bone cells and other cells within the bone, causing them to secrete factors back onto the breast cancer cells, driving a “vicious cycle” that renders the disease incurable. Inhibiting the secreted factors may

AND THE VICIOUS CYCLE BONE METASTASIS MOUSE MODELS

ZR75.1) or mixed (BT483) bone lesions within this model (6, 16). These

interfere with the vicious cycle and lead to a cure for breast cancer bone metastasis (6).

5.

Breast cancer cells secrete many factors that in combination contribute to bone metastases (17). They can be broken down into two groups: (i) bone-resorbing and (ii) bone-forming factors (14). Osteolytic breast cancer-secreted factors include: PTHrP, IL-11, IL-6, VEGF, IL-8, CSFs, EGF, oxygen-derived free radicals, PDGF, prostaglandins, PTH, TNFs, TGFs, and IL-1 (14,18). Potential osteoblastic factors include: ET-1, stanniocalcins, AM, many of the six CCN proteins, BMPs, PTHrP fragments generated by PSA proteolysis, BDGF, FGFs, IGFs, PDGF,

group of breast cancer-secreted factors may oppose the development of bone metastases (14). These factors are often downregulated in breast cancer cells or upregulated as an anti-tumor host response. They include IL-18, IL-4, IL-12, OPG, BMP antagonists such as noggin, and Wnt signaling antagonists (DKKs and soluble frizzled related proteins) (14).

The large list of breast cancer-secreted factors makes the task of identifying the best targets daunting. Some of these factors play roles in other diseases, for which drugs/inhibitors have already been developed and tested. Understanding the role of these particular factors in breast cancer bone metastasis provides the opportunity to translate existing drugs into the clinic for improved treatment of metastases. The rest of this chapter focuses on tumor-secreted factors with established roles in breast cancer bone metastasis; potential new treatment targets will be highlighted.

5.1 Bone-Resorbing Breast Cancer-Secreted Factors

Breast-cancer secreted factors induce bone resorption by both indirect and direct actions on the osteoclast. Parathyroid hormone-related protein (PTHrP) is the most studied breast cancer-secreted factor. It indirectly activates osteoclastic bone resorption by stimulating osteoblasts and stromal cells to express RANKL, which in turn activates osteoclasts (20). PTHrP was first identified as a causal factor in humoral hypercalcemia of mali-gnancy and was later shown to be a major factor in promoting osteolytic metastases (14). Breast cancer cells that have metastasized to bone

Inhibiting PTHrP with neutralizing antibodies decreased osteolytic bone

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TUMOR-SECRETED FACTORS

express higher PTHrP mRNA levels than in soft tissue sites (21, 22).


prostaglandins, TGFβ, TNFs, and urokinase (uPA) (14, 18, 19). A third

12. Breast cancer secreted factors and BM microenvironment 245 metastases formed by MDA-MB-231 breast cancer cells in mice (23). A humanized PTHrP neutralizing antibody is currently in clinical trial for the treatment of breast cancer bone metastasis. Paradoxically, higher PTHrP expression in the primary breast tumor is correlated with a better prognosis and is not associated with the presence of bone metastases (24). Therefore, the role of PTHrP in bone lesion formation is local, and factor expression may be increased subsequent to the arrival of the metastatic tumor cells in bone.

Other secreted factors also act indirectly on osteoclasts via the RANKL pathway, including vascular endothelial growth factor (VEGF), interleukin-11 (IL-11), and interleukin-6 (IL-6) (2). Primary breast tumors express the pro-angiogenic factor VEGF and its receptors (VEGFRs) (25–27). Increased VEGF expression is correlated with increased tumor size and

expressed by breast cancer bone metastases, and VEGFRs are expressed by breast cancer bone metastases, osteoclasts, and osteoclast precursors

ment in combination with RANKL, similarly to M-CSF in combination with RANKL, stimulates osteoclast differentiation and bone resorption

bone metastases may promote osteoclastic bone resorption and promote lytic bone lesions. Anti-VEGF therapies have been developed for anti-angiogenic therapy, including VEGF antibodies, soluble VEGFRs, VEGFR antibodies, and small-molecule receptor kinase inhibitors (31). Anti-VEGFR-2 and anti-VEGFR-3 antibody combination therapy decreased lymph node and lung metastases in an orthotopic spontaneous breast cancer metastasis model (32). Currently, anti-VEGF therapy has only been shown to improve survival in combination with chemotherapy in clinical trials in patients with metastatic colorectal cancer and not in

bone resorption, anti-VEGF therapy may reduce osteolytic breast cancer bone metastases.

Interleukin-11 (IL-11) also indirectly activates osteoclasts via the RANKL pathway (2). IL-11 induced bone resorption in calvarial organ culture assays, and this effect was inhibited by Cox inhibitors (34). IL-11

combination were identified to contribute to the development of bone metastasis (17). IL-11 expression was higher in highly bone metastatic MDA-MB-231 subpopulations compared to parental cells. Combined overexpression of IL-11 and osteopontin, but not overexpression of IL-11 alone, increased bone metastases formed by MDA-MB-231 cells (17).

Interleukin-8 (IL-8) is a breast cancer-secreted factor that induces bone resorption in a PTHrP/RANKL-independent manner by acting directly on

grade (27, 28). Vascular endothelial growth factor (VEGF) is also highly

breast cancer (31, 33). However, since VEGF stimulates osteoclastic

is expressed by breast cancer cells (17, 35). It is one of five factors that in

the IL-8 receptor (CXCR1) on osteoclasts and osteoclast precursors (36, 37).

(27, 29). Therefore, the high VEGF expression found in breast cancer

(27, 29, 30). VEGF is also a monocyte chemoattractant (27, 30). VEGF treat-

The chemokine is expressed by breast cancer cell lines, and higher expression is associated with greater osteolytic potential (37). Patients with breast cancer have elevated IL-8 serum concentrations compared to normal controls, with the highest levels found in patients with advanced disease (38). MDA-MET breast cancer cells are highly metastatic to bone and differ from parental MDA-MB-231 cells by having increased IL-8 expression and no PTHrP expression, suggesting that IL-8 can drive osteolytic metastases to bone (39). An IL-8-specific neutralizing antibody inhibited osteoclast formation induced by MDA-MET conditioned media (37). Combined treatment of mice injected subcutaneously with MDA-MB-231 cells with a human IL-8 antibody and an epidermal growth factor receptor antibody increased overall survival, decreased metastatic spread, and decreased tumor size (40).

5.2 Bone-Forming Breast Cancer-Secreted Factors

About 15% of breast cancer bone metastases are osteoblastic (6). Endothelin-1 (ET-1) is a tumor-secreted peptide with a role in osteoblastic bone metastases (16). ET-1 stimulates osteoblast activity and new bone formation (41). It is secreted by breast cancers and cell lines that produce

and ETA receptor than nonneoplastic tissue (42). Patients with breast cancer and lymph node metastases possess higher ET-1 serum levels than patients without lymph node metastases (42). Selective inhibition of the endothelin A receptor decreased osteoblastic metastases formed by ET-1-secreting ZR-75-1 breast cancer cells (16). An ETA receptor antago-nist is currently in Phase III clinical trials in men with advanced prostate cancer. Adrenomedullin (AM) is another secreted peptide that may play a role in osteoblastic breast cancer bone metastases. AM is expressed by human breast cancers and breast cancer cell lines (43). Higher levels of AM tumor peptide expression and AM plasma levels were found in patients with axillary lymph node metastasis compared to patients without axillary lymph

(unpublished data). Overexpression of AM increased lesion formation in a prostate cancer mouse model, while decreasing AM expression decreased bone lesion formation in a lung cancer bone metastasis mouse model (unpublished data). Small molecule inhibitors of AM have been developed (46) that inhibit AM-induced new bone formation in neonatal mouse calvariae (unpublished data). Such agents may reduce breast cancer bone metastases. Like ET-1, AM is a potent stimulator of pain (47). Both

246

osteoblastic and mixed bone lesions in mouse models e.g., T47D, MCF-7,ZR75.1, and BT483 (16). Invasive breast tumors express higher ET-1

in vivo (44, 45) and induces new bone formation in neonatal mouse calvariae node metastasis (43). AM stimulates osteoblast proliferation in vitro and


12. Breast cancer secreted factors and BM microenvironment 247 peptides may contribute to bone metastasis-associated bone pain, which is a major complication of skeletal metastases. Tumor-secreted platelet derived growth factor-BB (PDGF-BB) may contribute to osteoblastic metastases. PDGFs are multifunctional cytokines that stimulate both osteoclasts and osteoblasts (48). Breast cancer cells

plasma and tumor tissue levels are associated with a poorer prognosis for breast cancer, including increased metastases, lower chemotherapeutic

breast cancer cells that overexpress the neu oncogene decreased osteo-blastic bone metastases in nude mice (48). Overexpression of PDGF-BB in MDA-MB-231 breast cancer cells, which normally produce osteolytic lesions, produced osteoblastic lesions (48). Gleevac, a selective inhibitor of PDGF receptor tyrosine kinase activity, decreased growth of breast cancer cells injected into the tibia of mice (49). Such inhibitors could reduce osteoblastic breast cancer bone metastases. The pro-angiogenic factor connective tissue growth factor (CTGF) is a member of the cysteine-rich CCN protein family and is another breast cancer-secreted factor that stimulates new bone formation (52). Recom-binant CTGF increases bone formation and angiogenesis when injected into the femoral marrow cavity of rats (52). CTGF is expressed by breast

compared to normal tissues (54). Low CTGF levels are associated with a poor prognosis, metastasis, local recurrence, and mortality (54). However,CTGF expression at sites of bone metastases has not been reported. CTGF is a member of the bone metastatic gene profile identified by Kang et al. in 2003 (17). Overexpressing CTGF alone did not increase

the rate and incidence of bone metastases (17). CTGF neutralizing antibodies decreased osteolytic lesions formed by MDA-MB-231 cells in mice (55). Thus, CTGF appears to play an important role in bone meta-stases. The bone microenvironment may induce an increase in CTGF expression.

Another member of the CCN family that stimulates osteoblasts, cysteine-

Breast cancer tumor tissues expressed higher Cyr61 levels than normal breast tissues (54). High Cyr61 levels were associated with poor prognosis, nodal involvement, and metastatic disease in breast cancer patients (54). It was recently found that a bone-metastatic variant of MDA-MB-231 cells showed increased expression of Cyr61, CTGF, and ET-1, as well as the osteolytic factors IL-11 and IL-8 (58).

response, and lower survival (50, 51). Reduction of PDGF-BB in MCF-7

secrete PDGFs and express the PDGF receptor (48, 49). High PDGF

rich protein 61 (Cyr61), may also play a role in bone metastases (56, 57).

cancer cells (17, 53). Lower levels of CTGF were detected in breast tumors

expressing IL-11, osteopontin, and CTGF together significantly increased bone metastases formed by MDA-MB-231 in mice. However, over-

PTHrP also may play a role in osteoblastic metastases (59). PTHrP expression is commonly found in prostate cancer cells that produce osteoblastic metastases (60). PTHrP can be cleaved at residue 23 by the serine proteinase prostate-specific antigen (PSA) (59) that is commonly

not activate the PTH/PTHrP receptor. PTHrP fragments 1–16 and 1–23 stimulate new bone formation in ex vivo calvarial organ cultures, and this stimulation was blocked by an ETAR antagonist, ABT-627, suggesting that PTHrP fragments may stimulate new bone formation through the endothelin A receptor (59). However, Langlois et al. (2005) were unable to show binding of PTHrP 1–16 and 1–23 to the ETA or ETB receptor (60). Proteolysis may convert PTHrP from an osteolytic to an osteoblastic factor. Therefore, neutralizing PTHrP may also be beneficial for osteo-blastic bone metastases, while ETA receptor antagonists may be effective against tumors that make PTHrP fragments but are ET-1-negative.

The bone morphogenetic proteins (BMPs) are a family of growth factors that stimulate bone formation and are part of the TGFβ super-family (63). Breast cancer cells express BMPs and BMP receptors (64). Different BMPs may have both growth inhibitory and stimulatory effects

genetic protein receptor IB is associated with increased tumor grade, proliferation, cytogenetic instability, and poor prognosis of estrogen receptor-positive breast carcinomas (67). Overexpression of BMP-2 in

model (68). Overexpression of the BMP antagonist, noggin, in PC3 and LAPC-9 prostate cancer cells decreased osteolytic and osteoblastic lesions, respectively, produced by the prostate cancer cells after injection

5.3 Secreted Factors that Can Oppose Bone Metastasis Formation

Breast cancer cells can secrete factors that oppose bone metastasis formation (14). These factors are often decreased in breast cancer cells or increased as a host anti-tumor defense response. Increasing these factors in breast cancer patients might be another means to treat breast cancer bone metastases. Osteoprotegerin (OPG) is a secreted decoy receptor for RANKL (71). OPG is expressed by breast cancer cells, osteoblasts, and bone stromal cells (72). Binding of OPG to RANKL prevents RANKL from binding to its receptor RANK on osteoclast precursor cells and osteoclasts, preventing the formation and activation of osteoclasts (71). Therefore, OPG is a potent inhibitor of osteoclast formation and bone resorption. Breast cancer cells

248

expressed by breast cancers (61, 62). The resulting PTHrP fragment does

on breast cancer cells (65, 66). Increased expression of the bone morpho-


in vitro and enhanced estrogen-independent growth in a xenograft mouse MCF-7 breast cancer cells increased the invasive ability of these cells

into the tibia of SCID mice (69, 70).

12. Breast cancer secreted factors and BM microenvironment 249 may reduce OPG and increase RANKL expression in the bone to increase osteolysis (72). Inhibiting RANKL signaling with OPG may inhibit the

and VEGF) that induce osteolysis through the RANKL pathway and therefore may be more effective than inhibiting one of these factors alone. Recombinant OPG treatment reduced osteolytic lesion formation, skeletal tumor burden, and tumor-associated osteoclasts formed by MDA-MB-231 breast cancer cells after intracardiac injection in nude mice (73). A recombinant OPG construct (AMGN-0007) decreased bone resorption without significant adverse effects in a phase I trial using 26 patients with breast carcinoma and established lytic bone lesions (74). However, OPG constructs have not succeeded through clinical trials so far. Small molecule stimulators of OPG expression have also been developed (75). The small molecule OPG stimulator (Cmpd 5) decreeased lytic bone lesions formed by 13762 rat mammary carcinoma cells after intracardiac injection of Fischer-344 rats (75). However, overexpressing OPG in breast cancer cells increased tumor growth in the tibiae of mice (71), contraindicating the use of small molecule OPG stimulators. Anti-RANKL antibodies have been more successful. The humanized anti-RANKL antibody, denosumab, reduced bone resorption and was well tolerated in patients with multiple myeloma and breast cancer bone metastases (76).

Interleukin-18 (IL-18) enhances the anti-tumor immune response and inhibits osteoclast formation and bone resorption via a mechanism involv-ing granulocyte/macrophage colony-stimulating factor (77–79). IL-18 upre-gulates OPG expression by osteoblastic and stromal cells (80). Patients with breast cancer have higher serum IL-18 levels than patients without breast cancer (78). Higher IL-18 levels were also found in metastatic patients compared to nonmetastatic with the highest levels found in

reduced osteolytic bone metastases formed by intracardiac injection of MDA-MB-231 breast cancer cells but had no effect on subcutaneous tumor growth (82). Systemic administration of recombinant IL-18 in humans could reduce breast cancer bone metastases.

Soluble frizzled related protein 1 (Sfrp1) is a breast cancer secreted protein that inhibits the Wnt signaling pathway (83). The Wnt signaling pathway has a known role in osteogenesis and oncogenesis (84). Wnt sig-naling activates osteoblasts and Wnt signaling inhibitors like Sfrp1 and dickkopf-1 (DKK-1) inhibit this activation (84). Activation of the Wnt

regulation of repressors of Wnt signaling, Sfrp1 and the transcription factor TCF-4, was identified in a subset of breast cancers (83). Deletion of the chromosomal region containing Sfrp1 is often detected in breast cancer

actions of multiple bone-resorbing tumor factors (e.g., PTHrP, IL-11,

patients with bone metastasis (78, 81). IL-18 injections into nude mice

signaling pathway also promotes mammary carcinogenesis (83, 85). Down-

(86). Aberrant hypermethylation (gene-silencing) of Sfrp1 was also associ-ated with an unfavorable prognosis for breast cancer (86). Increasing Wnt activity by knocking down DKK-1 expression with DKK-1 short hairpin RNA caused osteolytic PC3 prostate cancer cells to induce

phenotype to an osteolytic phenotype (87). Wnt signaling contributes to prostate cancer osteoblastic bone metastasis formation (87) and may in the same way contribute to breast cancer bone metastasis. Suppression of the Wnt signaling pathway may reduce osteoblastic bone metastasis. A green tea compound (-)-epigallocatchin 3-gallate (EGCG) inhibits Wnt signaling and reduces breast cancer cell proliferation and invasiveness (88). Green tea consumption has been correlated with reduced recurrence of breast cancers in Japanese women. Oral administration of EGCG reduced breast cancer tumor progression in animal models (88). EGCG may reduce osteoblastic bone metastases. However, Wnt signaling inhibition has also been suggested to be one of the mechanisms that multiple myeloma

myeloma cells and multiple myeloma patients with advanced osteolytic lesions secreted the Wnt inhibitor, secreted frizzled related protein-2 (Sfrp-2) and Sfrp-2 inhibits bone formation (89). Further research is needed to test the role of the Wnt signaling inhibitors in breast cancer bone metastasis.

6. CURRENT PROBLEMS AND POSSIBLE FUTURE TREATMENT DIRECTIONS: IDENTIFYING UPSTREAM REGULATORS TO TARGET MULTIPLE FACTORS INVOLVED IN BREAST CANCER BONE METASTASIS

The approved treatment for breast cancer bone metastases is anti-resorptive bisphosphonates (2). Bisphosphonates bind to bone matrix and reduce osteoclastic bone resorption (14). They promote osteoclast apopto-sis, while their effects in vivo on osteoblasts and tumor growth remain con-troversial (14). Bisphosphonates reduce bone pain and skeletal fractures but do not improve overall survival (2). Additional classes of antiresorptive agents include anti-RANKL antibodies and cathepsin K inhibitors. These are in clinical development but are not yet approved for patient use. Anti-RANKL antibodies prevent interaction of RANKL with RANK, interfering with formation and activation of osteoclasts (76). Cathepsin K inhibitors inhibit one of the proteolytic enzymes secreted by osteoclasts, cathepsin K,

250

osteoblast activity (87). Decreasing Wnt activity by overexpressing DKK-1converts prostate cancer cells with a mixed osteolytic/osteoblastic


induces bone destruction by inhibiting bone formation (89, 90). Multiple

12. Breast cancer secreted factors and BM microenvironment 251 that is necessary for bone resorption (91). Out of the three groups, Cathepsin K inhibitors are the only agents that do not prevent osteoclast formation or induce osteoclast death. If osteoclasts have other functions in bone beyond osteolysis, drugs that allow osteoclast formation, but block their bone resorptive activity, may have fewer side effects. Current treatment flaws leave the need for the development of more effective therapies. This chapter has demonstrated a method of targeting tumor-secreted factors such as PTHrP to treat breast cancer bone metastases. Many additional factors are involved in breast cancer bone metastases. The important question is: How to find the best target(s) out of the long list of factors to effectively cure breast cancer bone metastases? The best strategy may be to target multiple tumor-secreted factors. Kang et al. (2003) demonstrated that not one, but a combination of four to five factors were necessary for bone metastasis formation (17). They identified a bone metastatic gene profile consisting of 43 genes with varying functions, among which included the bone-resorbing factor IL-11 and the bone-forming, angiogenic factor CTGF (17). These genes only in combi-nation enhanced bone metastasis formation produced by poorly meta-static MDA-MB-231 cells (17). Therefore, multiple factors are important in bone metastasis formation and targeting multiple factors may be more effective in treating breast cancer bone metastases than targeting one factor alone. Indeed, breast cancers secrete multiple factors from the lists of both bone-resorbing and bone-forming proteins (6). Therefore, a more effective treatment may be to target an upstream regulator of multiple factors. Many of the known tumor-secreted factors, both osteolytic and osteoblastic, are regulated by the hypoxia-induced Hif-1α pathway and

bone microenvironment and are important targets for treatment of bone metastases. TGFβ inhibitors have been effective in blocking bone meta-stases in preclinical models (9–12). Additional upstream regulators need to be identified and may prove to be more effective treatment targets for breast cancer bone metastasis treatment. Combining this approach of targeting tumor-secreted factors with other therapies (bisphosphonates and chemotherapeutics) may improve treatment (93). Inhibitors of tumor-secreted factors may be important adjuvant therapies for breast cancer bone metastasis.

ACKNOWLEDGMENTS

The authors would like to acknowledge support from the University of Virginia, the US Army, and the NIH. VA Siclari was supported by a US

the TGFβ signaling pathway (8, 17, 92). Both pathways are active in the

Army Breast Cancer Predoctoral Traineeship Award BC051563. JM Chirgwin and TA Guise were supported by grants from the NIH.

1. Coleman RE. Skeletal complications of malignancy. Cancer 1997; 80(8 Suppl): 1588–1594

2. Kozlow W, Guise TA. Breast cancer metastasis to bone: mechanisms of osteolysis and implications for therapy. J Mammary Gland Biol Neoplasia 2005; 10(2): 169–180

3. Fidler IJ. The pathogenesis of cancer metastasis: the “seed and soil” hypothesis revisited. Nat Rev Cancer 2003; 3(6): 453–458

4. Paget S. The distribution of secondary growths in cancer of the breast. Cancer Metastasis Rev 1989; 8(2): 98–101

5. Hauschka PV, Mavrakos AE, Iafrati MD, Doleman SE, Klagsbrun M. Growth factors in bone matrix. Isolation of multiple types by affinity chromatography on heparin-Sepharose. J Biol Chem 1986; 261(27): 12665–12674

6. Guise TA, Kozlow WM, Heras-Herzig A, Padalecki SS, Yin JJ, Chirgwin JM. Molecular mechanisms of breast cancer metastases to bone. Clin Breast Cancer 2005; 5 Suppl(2): S46–53

7. Dallas SL, Rosser JL, Mundy GR, Bonewald LF. Proteolysis of latent transforming growth factor-beta (TGF-beta)-binding protein-1 by osteoclasts. A cellular mechanism for release of TGF-beta from bone matrix. J Biol Chem 2002; 277(24): 21352–21360

8. Yin JJ, Selander K, Chirgwin JM, Dallas M, Grubbs BG, Wieser R, Massague J, Mundy GR, Guise TA. TGF-beta signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development. J Clin Invest 1999; 103(2): 197–206

9. Muraoka RS, Dumont N, Ritter CA, Dugger TC, Brantley DM, Chen J, Easterly E, Roebuck LR, Ryan S, Gotwals PJ, Koteliansky V, Arteaga CL. Blockade of TGF-beta inhibits mammary tumor cell viability, migration, and metastases. J Clin Invest 2002; 109(12): 1551–1559

10. Yang YA, Dukhanina O, Tang B, Mamura M, Letterio JJ, MacGregor J, Patel SC, Khozin S, Liu ZY, Green J, Anver MR, Merlino G, Wakefield LM. Lifetime exposure to a soluble TGF-beta antagonist protects mice against metastasis without adverse side effects. J Clin Invest 2002; 109(12): 1607–1615

11. Bandyopadhyay A, Agyin JK, Wang L, Tang Y, Lei X, Story BM, Cornell JE, Pollock BH, Mundy GR, Sun LZ. Inhibition of pulmonary and skeletal metastasis by a transforming growth factor-beta type I receptor kinase inhibitor. Cancer Res 2006; 66(13): 6714–6721

12. Ge R, Rajeev V, Ray P, Lattime E, Rittling S, Medicherla S, Protter A, Murphy A, Chakravarty J, Dugar S, Schreiner G, Barnard N, Reiss M. Inhibition of growth and metastasis of mouse mammary carcinoma by selective inhibitor of transfor-ming growth factor-beta type I receptor kinase in vivo. Clin Cancer Res 2006; 12(14 Pt 1): 4315–4330

13. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular Biology

252

REFERENCES

of THE CELL: 4th edn. (Garland Science, NY, 2002)


12. Breast cancer secreted factors and BM microenvironment 253 14. Clines GA, Guise TA. Hypercalcaemia of malignancy and basic research on

mechanisms responsible for osteolytic and osteoblastic metastasis to bone. Endocr Relat Cancer 2005; 12(3): 549–583

15. Heppner GH, Miller FR, Shekhar PM. Nontransgenic models of breast cancer. Breast Cancer Res 2000; 2(5): 331–334

16. Yin JJ, Mohammad KS, Kakonen SM, Harris S, Wu-Wong JR, Wessale JL, Padley RJ, Garrett IR, Chirgwin JM, Guise TA. A causal role for endothelin-1 in the pathogenesis of osteoblastic bone metastases. Proc Natl Acad Sci USA

17. Kang Y, Siegel PM, Shu W, Drobnjak M, Kakonen SM, Cordon-Cardo C, Guise TA, Massague J. A multigenic program mediating breast cancer metastasis to bone. Cancer Cell 2003; 3(6): 537–549

18. Boyce BF, Yoneda T, Guise TA. Factors regulating the growth of metastatic cancer in bone. Endocr Relat Cancer 1999; 6(3): 333–347

19. Chirgwin JM, Mohammad KS, Guise TA. Tumor-bone cellular interactions in skeletal metastases. J Musculoskelet Neuronal Interact 2004; 4(3): 308–318

20. Thomas RJ, Guise TA, Yin JJ, Elliott J, Horwood NJ, Martin TJ, Gillespie MT. Breast cancer cells interact with osteoblasts to support osteoclast formation. Endocrinology 1999; 140(10): 4451–4458

21. van der Pluijm G, Sijmons B, Vloedgraven H, Deckers M, Papapoulos S, Lowik C. Monitoring metastatic behavior of human tumor cells in mice with species-specific polymerase chain reaction: elevated expression of angiogenesis and bone resorption stimulators by breast cancer in bone metastases. J Bone Miner Res 2001; 16(6): 1077–1091

22. Powell GJ, Southby J, Danks JA, Stillwell RG, Hayman JA, Henderson MA, Bennett RC, Martin TJ. Localization of parathyroid hormone-related protein in breast cancer metastases: increased incidence in bone compared with other sites. Cancer Res 1991; 51(11): 3059–3061

23. Guise TA, Yin JJ, Taylor SD, Kumagai Y, Dallas M, Boyce BF, Yoneda T, Mundy GR. Evidence for a causal role of parathyroid hormone-related protein in the pathogenesis of human breast cancer-mediated osteolysis. J Clin Invest 1996; 98(7): 1544–1549

24. Henderson M, Danks J, Moseley J, Slavin J, Harris T, McKinlay M, Hopper J, Martin T. Parathyroid hormone-related protein production by breast cancers, improved survival, and reduced bone metastases. J Natl Cancer Inst 2001; 93(3): 234–237

25. Yoshiji H, Gomez DE, Shibuya M, Thorgeirsson UP. Expression of vascular endothelial growth factor, its receptor, and other angiogenic factors in human breast cancer. Cancer Res 1996; 56(9): 2013–2016

26. Dales JP, Garcia S, Carpentier S, Andrac L, Ramuz O, Lavaut MN, Allasia C, Bonnier P, Taranger-Charpin C. Prediction of metastasis risk (11-year follow-up) using VEGF-R1, VEGF-R2, Tie-2/Tek and CD105 expression in breast cancer (n=905). Br J Cancer 2004; 90(6): 1216–1221

27. Aldridge SE, Lennard TW, Williams JR, Birch MA. Vascular endothelial growth factor acts as an osteolytic factor in breast cancer metastases to bone. Br J Cancer 2005; 92(8): 1531–1537

28. Linderholm B, Tavelin B, Grankvist K, Henriksson R. Vascular endothelial growth factor is of high prognostic value in node-negative breast carcinoma. J Clin Oncol 1998; 16(9): 3121–3128

2003; 100(19): 10954–10959

29. Niida S, Kaku M, Amano H, Yoshida H, Kataoka H, Nishikawa S, Tanne K,

Maeda N, Nishikawa S, Kodama H. Vascular endothelial growth factor can substitute for macrophage colony-stimulating factor in the support of osteoclastic bone resorption. J Exp Med 1999; 190(2): 293–298

30. Clauss M, Weich H, Breier G, Knies U, Rockl W, Waltenberger J, Risau W. The vascular endothelial growth factor receptor Flt-1 mediates biological activities. Implications for a functional role of placenta growth factor in monocyte activation and chemotaxis. J Biol Chem 1996; 271(30): 17629–17634

31. Rosen LS. VEGF-targeted therapy: therapeutic potential and recent advances. Oncologist 2005; 10(6): 382–391

32. Roberts N, Kloos B, Cassella M, Podgrabinska S, Persaud K, Wu Y, Pytowski B, Skobe M. Inhibition of VEGFR-3 activation with the antagonistic antibody more potently suppresses lymph node and distant metastases than inactivation of VEGFR-2. Cancer Res 2006; 66(5): 2650–2657

33. Miller KD, Trigo JM, Wheeler C, Barge A, Rowbottom J, Sledge G, Baselga J. A multicenter phase II trial of ZD6474, a vascular endothelial growth factor receptor-2 and epidermal growth factor receptor tyrosine kinase inhibitor, in patients with previously treated metastatic breast cancer. Clin Cancer Res 2005; 11(9): 3369–3376

34. Morinaga Y, Fujita N, Ohishi K, Zhang Y, Tsuruo T. Suppression of interleukin-11-mediated bone resorption by cyclooxygenases inhibitors. J Cell Physiol 1998; 175(3): 247–254

35. Singh B, Berry JA, Shoher A, Lucci A. COX-2 induces IL-11 production in human breast cancer cells. J Surg Res 2006; 131(2): 267–275

36. Bendre M, Gaddy D, Nicholas RW, Suva LJ. Breast cancer metastasis to bone: it is not all about PTHrP. Clin Orthop Relat Res 2003; 415 (Suppl): S39–45

37. Bendre MS, Margulies AG, Walser B, Akel NS, Bhattacharrya S, Skinner RA, Swain F, Ramani V, Mohammad KS, Wessner LL, Martinez A, Guise TA, Chirgwin JM, Gaddy D, Suva LJ. Tumor-derived interleukin-8 stimulates osteolysis independent of the receptor activator of nuclear factor-kappaB ligand pathway. Cancer Res 2005; 65(23): 11001–11009

38. Benoy IH, Salgado R, Van Dam P, Geboers K, Van Marck E, Scharpe S, Vermeulen PB, Dirix LY. Increased serum interleukin-8 in patients with early and metastatic breast cancer correlates with early dissemination and survival. Clin Cancer Res 2004; 10(21): 7157–7162

39. Bendre MS, Gaddy-Kurten D, Mon-Foote T, Akel NS, Skinner RA, Nicholas RW, Suva LJ. Expression of interleukin 8 and not parathyroid hormone-related protein by human breast cancer cells correlates with bone metastasis in vivo. Cancer Res 2002; 62(19): 5571–5579

40. Salcedo R, Martins-Green M, Gertz B, Oppenheim JJ, Murphy WJ. Combined administration of antibodies to human interleukin 8 and epidermal growth factor receptor results in increased antimetastatic effects on human breast carcinoma xenografts. Clin Cancer Res 2002; 8(8): 2655–2665

41. Guise TA, Yin JJ, Mohammad KS. Role of endothelin-1 in osteoblastic bone metastases. Cancer 2003; 97(3 Suppl): 779–784

42. Hagemann T, Binder C, Binder L, Pukrop T, Trumper L, Grimshaw MJ. Expression of endothelins and their receptors promotes an invasive phenotype of breast tumor cells but is insufficient to induce invasion in benign cells. DNA Cell Biol 2005; 24(11): 766–776

254 Siclari, Guise, and Chirgwin

12. Breast cancer secreted factors and BM microenvironment 255 43. Oehler MK, Fischer DC, Orlowska-Volk M, Herrle F, Kieback DG, Rees MC,

Bicknell R. Tissue and plasma expression of the angiogenic peptide adrenome-dullin in breast cancer. Br J Cancer 2003; 89(10): 1927–1933

44. Cornish J, Callon KE, Coy DH, Jiang NY, Xiao L, Cooper GJ, Reid IR. Adrenomedullin is a potent stimulator of osteoblastic activity in vitro and in vivo. Am J Physiol 1997; 273(6 Pt 1): E1113–1120

45. Cornish J, Grey A, Callon KE, Naot D, Hill BL, Lin CQ, Balchin LM, Reid IR. Shared pathways of osteoblast mitogenesis induced by amylin, adrenomedullin, and IGF-1. Biochem Biophys Res Commun 2004; 318(1): 240–246

46. Martinez A, Julian M, Bregonzio C, Notari L, Moody TW, Cuttitta F. Identification of vasoactive nonpeptidic positive and negative modulators of adrenomedullin using a neutralizing antibody-based screening strategy. Endocrinology 2004; 145(8): 3858–3865

47. Ma W, Chabot JG, Quirion R. A role for adrenomedullin as a pain-related peptide in the rat. Proc Natl Acad Sci U S A 2006; 103(43): 16027–16032

48. Yi B, Williams PJ, Niewolna M, Wang Y, Yoneda T. Tumor-derived platelet-derived growth factor-BB plays a critical role in osteosclerotic bone metastasis

49. Lev DC, Kim SJ, Onn A, Stone V, Nam DH, Yazici S, Fidler IJ, Price JE. Inhibition of platelet-derived growth factor receptor signaling restricts the growth of human breast cancer in the bone of nude mice. Clin Cancer Res 2005; 11(1): 306–314

50. Seymour L, Bezwoda WR. Positive immunostaining for platelet derived growth factor (PDGF) is an adverse prognostic factor in patients with advanced breast cancer. Breast Cancer Res Treat 1994; 32(2): 229–233

51. Seymour L, Dajee D, Bezwoda WR. Tissue platelet derived-growth factor (PDGF) predicts for shortened survival and treatment failure in advanced breast cancer. Breast Cancer Res Treat 1993; 26(3): 247–252

52. Safadi FF, Xu J, Smock SL, Kanaan RA, Selim AH, Odgren PR, Marks SC, Jr., Owen TA, Popoff SN. Expression of connective tissue growth factor in bone: its role in osteoblast proliferation and differentiation in vitro and bone formation in vivo. J Cell Physiol 2003; 196(1): 51–62

53. Shimo T, Kubota S, Kondo S, Nakanishi T, Sasaki A, Mese H, Matsumura T, Takigawa M. Connective tissue growth factor as a major angiogenic agent that is induced by hypoxia in a human breast cancer cell line. Cancer Lett 2001; 174(1): 57–64

54. Jiang WG, Watkins G, Fodstad O, Douglas-Jones A, Mokbel K, Mansel RE. Differential expression of the CCN family members Cyr61, CTGF and Nov in human breast cancer. Endocr Relat Cancer 2004; 11(4): 781–791

55. Shimo T, Kubota S, Yoshioka N, Ibaragi S, Isowa S, Eguchi T, Sasaki A, Takigawa M. Pathogenic role of connective tissue growth factor (CTGF/CCN2) in osteolytic metastasis of breast cancer. J Bone Miner Res 2006; 21(7): 1045–1059

56. Schutze N, Kunzi-Rapp K, Wagemanns R, Noth U, Jatzke S, Jakob F. Expression, purification, and functional testing of recombinant CYR61/CCN1. Protein Expr Purif 2005; 42(1): 219–225

57. Bartholin L, Wessner LL, Chirgwin JM, Guise TA. The human Cyr61 gene is a transcriptional target of transforming growth factor beta in cancer cells. Cancer Lett (2006)

58. Bellahcene A, Bachelier R, Detry C, Lidereau R, Clezardin P, Castronovo V. Transcriptome analysis reveals an osteoblast-like phenotype for human osteotropic breast cancer cells. Breast Cancer Res Treat (2006)

in an animal model of human breast cancer. Cancer Res 2002; 62(3): 917–923

59. Chirgwin JM, Mohammad KS, Guise TA. Parathyroid hormone-related protein

(PTHrP) fragments are potent agonists of the endothelin A receptor (Abstract). Book of the Eighth International Conference on Endothelin 29 (2003)

60. Langlois C, Letourneau M, Turcotte K, Detheux M, Fournier A. PTHrP fragments 1-16 and 1-23 do not bind to either the ETA or the ETB endothelin receptors. Peptides 2005; 26(8): 1436–1440

61. Narita D, Raica M, Suciu C, Cimpean A, Anghel A. Immunohistochemical expression of androgen receptor and prostate-specific antigen in breast cancer. Folia Histochem Cytobiol 2006; 44(3): 165–172

62. Narita D, Cimpean AM, Anghel A, Raica M. Prostate-specific antigen value as a marker in breast cancer. Neoplasma 2006; 53(2): 161–167

63. Wozney JM. The bone morphogenetic protein family and osteogenesis. Mol Reprod Dev 1992; 32(2): 160–167

64. Arnold SF, Tims E, McGrath BE. Identification of bone morphogenetic proteins and their receptors in human breast cancer cell lines: importance of BMP2. Cytokine 1999; 11(12): 1031–1037

65. Pouliot F, Blais A, Labrie C. Overexpression of a dominant negative type II bone morphogenetic protein receptor inhibits the growth of human breast cancer cells. Cancer Res 2003; 63(2): 277–281

66. Ghosh-Choudhury N, Ghosh-Choudhury G, Celeste A, Ghosh PM, Moyer M, Abboud SL, Kreisberg J. Bone morphogenetic protein-2 induces cyclin kinase inhibitor p21 and hypophosphorylation of retinoblastoma protein in estradiol-treated MCF-7 human breast cancer cells. Biochim Biophys Acta 2000; 1497(2): 186–196

67. Helms MW, Packeisen J, August C, Schittek B, Boecker W, Brandt BH, Buerger H. First evidence supporting a potential role for the BMP/SMAD pathway in the progression of oestrogen receptor-positive breast cancer. J Pathol 2005; 206(3): 366–376

68. Clement JH, Raida M, Sanger J, Bicknell R, Liu J, Naumann A, Geyer A, Waldau A, Hortschansky P, Schmidt A, Hoffken K, Wolft S, Harris AL. Bone morphogenetic protein 2 (BMP-2) induces in vitro invasion and in vivo hormone independent growth of breast carcinoma cells. Int J Oncol 2005; 27(2): 401–407

69. Feeley BT, Gamradt SC, Hsu WK, Liu N, Krenek L, Robbins P, Huard J, Lieberman JR. Influence of BMPs on the formation of osteoblastic lesions in metastatic prostate cancer. J Bone Miner Res 2005; 20(12): 2189–2199

70. Feeley BT, Krenek L, Liu N, Hsu WK, Gamradt SC, Schwarz EM, Huard J, Lieberman JR. Overexpression of noggin inhibits BMP-mediated growth of osteolytic prostate cancer lesions. Bone 2006; 38(2): 154–166

71. Fisher JL, Thomas-Mudge RJ, Elliott J, Hards DK, Sims NA, Slavin J, Martin TJ, Gillespie MT. Osteoprotegerin overexpression by breast cancer cells enhances orthotopic and osseous tumor growth and contrasts with that delivered therapeutically. Cancer Res 2006; 66(7): 3620–3628

72. Park HR, Min SK, Cho HD, Kim DH, Shin HS, Park YE. Expression of osteoprotegerin and RANK ligand in breast cancer bone metastasis. J Korean Med Sci 2003; 18(4): 541–546

73. Morony S, Capparelli C, Sarosi I, Lacey DL, Dunstan CR, Kostenuik PJ. Osteoprotegerin inhibits osteolysis and decreases skeletal tumor burden in syngeneic and nude mouse models of experimental bone metastasis. Cancer Res 2001; 61(11): 4432–4436

74. Body JJ, Greipp P, Coleman RE, Facon T, Geurs F, Fermand JP, Harousseau JL, Lipton A, Mariette X, Williams CD, Nakanishi A, Holloway D, Martin SW,


12. Breast cancer secreted factors and BM microenvironment 257

Dunstan CR, Bekker PJ. A phase I study of AMGN-0007, a recombinant osteopro-tegerin construct, in patients with multiple myeloma or breast carcinoma related bone metastases. Cancer 2003; 97(3 Suppl): 887–892

75. Onyia JE, Galvin RJ, Ma YL, Halladay DL, Miles RR, Yang X, Fuson T, Cain RL, Zeng QQ, Chandrasekhar S, Emkey R, Xu Y, Thirunavukkarasu K, Bryant HU, Martin TJ. Novel and selective small molecule stimulators of osteoprotegerin expression inhibit bone resorption. J Pharmacol Exp Ther 2004; 309(1): 369–379

76. Body JJ, Facon T, Coleman R, Lipton A, Geurs F, Fan M, Holloway D, Peterson MC, Bekker P. A study of the biological receptor activator of nuclear factor-kB ligand inhibitor, Denosumab, in patients with multiple myeloma or bone metastases from breast cancer. Clin Cancer Res 2006; 12(4): 1221–1228

77. Yamada N, Niwa S, Tsujimura T, Iwasaki T, Sugihara A, Futani H, Hayashi S, Okamura H, Akedo H, Terada N. Interleukin-18 and interleukin-12 synergistically inhibit osteoclastic bone-resorbing activity. Bone 2002; 30(6): 901–908

78. Gunel N, Coskun U, Sancak B, Gunel U, Hasdemir O, Bozkurt S. Clinical importance of serum interleukin-18 and nitric oxide activities in breast carcinoma patients. Cancer 2002; 95(3): 663–667

79. Udagawa N, Horwood NJ, Elliott J, Mackay A, Owens J, Okamura H, Kurimoto M, Chambers TJ, Martin TJ, Gillespie MT. Interleukin-18 (interferon-gamma-inducing factor) is produced by osteoblasts and acts via granulocyte/macrophage colony-stimulating factor and not via interferon-gamma to inhibit osteoclast formation. J Exp Med 1997; 185(6): 1005–1012

80. Makiishi-Shimobayashi C, Tsujimura T, Iwasaki T, Yamada N, Sugihara A, Okamura H, Hayashi S, Terada N. Interleukin-18 up-regulates osteoprotegerin expression in stromal/osteoblastic cells. Biochem Biophys Res Commun 2001; 281(2): 361–366

81. Soheir AL, Eissa MD, Samar A, Zaki MD, Shereen M, El-Maghraby MD, Dalia Y, Kadry MD. Importance of serum IL-18 and RANTES as markers for breast carcinoma progression. J Egyptian Nat. Cancer Inst. 2005; 17(1): 51–55

82. Nakata A, Tsujimura T, Sugihara A, Okamura H, Iwasaki T, Shinkai K, Iwata N, Kakishita E, Akedo H, Terada N. Inhibition by interleukin 18 of osteolytic bone metastasis by human breast cancer cells. Anticancer Res 1999; 19(5B): 4131–4138

83. Shulewitz M, Soloviev I, Wu T, Koeppen H, Polakis P, Sakanaka C. Repressor roles for TCF-4 and Sfrp1 in Wnt signaling in breast cancer. Oncogene 2006; 25(31): 4361–4369

84. Hall CL, Kang S, MacDougald OA, Keller ET. Role of Wnts in prostate cancer bone metastases. J Cell Biochem 2006; 97(4): 661–672

85. Michaelson JS, Leder P. beta-catenin is a downstream effector of Wnt-mediated tumorigenesis in the mammary gland. Oncogene 2001; 20(37): 5093–5099

86. Veeck J, Niederacher D, An H, Klopocki E, Wiesmann F, Betz B, Galm O, Camara O, Durst M, Kristiansen G, Huszka C, Knuchel R, Dahl E. Aberrant methylation of the Wnt antagonist SFRP1 in breast cancer is associated with unfavourable prognosis. Oncogene 2006; 25(24): 3479–3488

87. Hall CL, Bafico A, Dai J, Aaronson SA, Keller ET. Prostate cancer cells promote osteoblastic bone metastases through Wnts, Cancer Res 2005; 65(17): 7554–7560

88. Kim J, Zhang X, Rieger-Christ KM, Summerhayes IC, Wazer DE, Paulson KE, Yee AS. Suppression of Wnt signaling by the green tea compound (-)-epigallocatechin 3-gallate (EGCG) in invasive breast cancer cells. Requirement of the transcript-tional repressor HBP1. J Biol Chem 2006; 281(16): 10865–10875

89. Oshima T, Abe M, Asano J, Hara T, Kitazoe K, Sekimoto E, Tanaka Y, Shibata

H, Hashimoto T, Ozaki S, Kido S, Inoue D, Matsumoto T. Myeloma cells suppress bone formation by secreting a soluble Wnt inhibitor, sFRP-2. Blood 2005; 106(9): 3160–3165

90. Heider U, Hofbauer LC, Zavrski I, Kaiser M, Jakob C, Sezer O. Novel aspects of osteoclast activation and osteoblast inhibition in myeloma bone disease. Biochem Biophys Res Commun 2005; 338(2): 687–693

91. Shinozuka T, Shimada K, Matsui S, Yamane T, Ama M, Fukuda T, Taki M, Takeda Y, Otsuka E, Yamato M, Mochizuki S, Ohhata K, Naito S. Potent and selective cathepsin K inhibitors. Bioorg Med Chem 2006; 14(20): 6789–6806

92. Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003; 3(10): 721–732

93. Kim SJ, Uehara H, Yazici S, He J, Langley RR, Mathew P, Fan D, Fidler IJ. Modulation of bone microenvironment with zoledronate enhances the therapeutic effects of STI571 and paclitaxel against experimental bone metastasis of human prostate cancer. Cancer Res 2005; 65(9): 3707–3715


Chapter 13

CYCLOOXYGENASE-2 AND BREAST CANCER Gurpreet Singh-Ranger and Kefah Mokbel Department of Breast and Endocrine Surgery, St. George’s Hospital, London, SW17 0QT, UK

Recent research suggests that the cyclooxygenase-2 (COX-2), an isoenzyme of the COX enzyme system has a fundamental role in breast cancer pathogenesis and metastasis. COX-2 appears to be expressed by a large proportion of invasive breast cancers and by DCIS, and levels seem to correlate with those of angiogenic factors such as VEGF, and with tumour-promoting systems such as aromatase. This is particularly important when we consider that COX-2 is amenable to suppression by simple medications such as aspirin and selective COX-2 inhibitors. This chapter reviews the research exploring the role of COX-2 in breast cancer, and studies investigating COX-2 inhibition.

cyclooxygenase-2, breast cancer, ductal carcinoma in situ, prostaglandins, non-steroidal anti-inflammatory drugs (NSAIDs)

1. INTRODUCTION

Breast cancer affects around 1 in 12 women, and is the leading cause of death in females between the ages of 40 and 50 in the West world (1).

In the last decade a wealth of studies have indicated a link between the pathogenesis of breast cancer and the expression of cyclooxygenases, particularly cyclooxygenase-2 (COX-2).

Non-steroidal anti-inflammatory drugs (NSAIDs), well-tolerated, accessible, and inexpensive medications, suppress COX activity, leading

© 2007 Springer.


Abstract:

Keywords:

to speculation of a role for NSAIDs in breast cancer treatment and prevention. Not surprisingly, the activity and implications of expression

Singh-Ranger and Mokbel

(DCIS) are now under intense scrutiny.

2.

The cyclooxygenase enzyme system, also known as prostaglandin H synthetase (PGHS), is composed of two distinct isoenzymes, cyclooxy-genase-1 (COX-1) and cyclooxygenase-2 (COX-2). These enzymes form part of the prostaglandin synthetase complex of enzymes, which plays a key role in the conversion of arachidonic acid into prostaglandin G2 (PGG2). This molecule is subsequently transformed into individual prostaglandins by tissue-specific components of the synthetase complex, such as hydroperoxidase (Figure 1).

Both enzymes are both homodimeric, haem-containing, glycosylated proteins.

Although approximately 60% identical, studies of crystal structure have revealed COX-2 has a larger active site. This is consistent with what is termed “substrate promiscuity” of COX-2, a property which ena-bles the enzyme to metabolise molecules structurally similar to prostag-landins, such as linoleic acid and anandamide (2, 3).

of the COX enzyme system in breast cancer and ductal carcinoma in situ

260

COX-1 and COX-2 are encoded by genes which are tightly regulated,

and located on different chromosomes (4). The isoenzymes differ substan-tially in patterns of expression and biology.

The COX-1 gene is essentially a “housekeeping gene”, expressed at a constant level throughout the cell cycle, and by almost all tissues. It has therefore been termed “the constitutive isoenzyme”.

In contrast, the COX-2 gene is an “immediate to early gene” (5, 6). COX-2 is induced rapidly in response to growth factors, tumour

promoters, hormones, bacterial endotoxin, cytokines, and shear stress (7), and has a number of inducible enhancer elements. COX-2 is often termed “the inducible isoenzyme” (8), but this is an oversimplification, as the gene is expressed constitutively in brain, testes, and trachea (9–12).

3. CELLULAR LOCALISATION OF COX-1 AND COX-2 AND PROBABLE BIOLOGICAL ROLES

Immunoelectron microscopy reveals both enzymes are found on the luminal surface of the endoplasmic reticulum (ER), and in the nuclear

the inner and outer nuclear membranes. envelope of human cells (13). They are present in similar proportions in

STRUCTURE AND EXPRESSION OF COX-1 AND COX-2

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Laboratory studies using COX-1 and COX-2 deficient mice, and COX-specific inhibitors suggest there are some biological events in which distinct COX isoenzymes are involved (Table 1), some in which they act together, and others where one isoenzyme can compensate if the other is lacking. There are also likely to be unique prostanoid synthetic pathways for COX-1 and COX-2, via designated coupling to various downstream prostaglandin synthases (14).

A specific PGE synthase has been identified which is induced with COX-2 and may function preferentially with it rather than with COX-1 (15, 16).

Some insight into the biological roles of COX-2 has been gained from animal studies.

COX-2 null mice are infertile, and although COX-2 deficient mice undergo follicular development, they demonstrate a marked reduction in ovulation, and in the release of fertilised eggs (17, 18). This may be caused by a deficiency of ovarian PGE2, as exogenous supplementation of this prostaglandin restores ovulatory function (19). COX-2 deficiency also retards implantation of the blastocyst (18), and disrupts renal develop-ment. Affected mice develop severe renal disease, which has a different pathology from NSAID-induced renal toxicity (17, 20).

13. COX-2 in breast cancer

Figure 1. Role of cyclooxygenase (COX) in prostaglandin synthesis.


COX-2 has been implicated in development of the cardiovascular

of birth due to a patent ductus arteriosus. Both COX isoenzymes also subserve critical roles during T-cell

development in the developing thymus, and COX-2 may have a specific influence on CD4 cell differentiation (22).

COX isoenzymes also activate cellular signalling reactions which involve electron transfer (reduction/oxidation or “redox”) reactions. This is via an intrinsic, highly active peroxidase (POX) activity (23, 24). The COX and POX activities are physically and functionally separate (4).

A variety of substrates are oxidised by the POX component, some are carcinogenic and lead to the production of more mutagens (25).

Significantly, POX activity is not necessarily blocked by NSAIDs (4), and selective COX-2 inhibitors also have little effect against POX activity. Steroids, however, due to their effects on transcription, downregulate total cellular COX-2 protein content, and cause a fall in both activities (3).

Table 1. important biological roles of the COX isoenzymes

Isoenzyme Biological roles COX-1 Parturition, platelet aggregation COX-2

remodelling of the ductus arteriosus. Compensatory effects COX-1 can compensate for COX-2 deficiency in parturition

and closure of the ductus arteriosus Both T-cell development, protection against gastric ulceration.

4. EXPERIMENTAL EVIDENCE OF A ROLE FOR COX-2 IN BREAST CANCER

4.1 COX-2 Immunoreactivity

Experimental studies of COX-2 expression in breast cancer specimens have produced varying and sometimes conflicting results. The general consensus of opinion however, seems to be that:

• COX-2 is expressed by invasive ductal and lobular carcinoma, •

• Where specifically investigated, COX-2 expression correlates to poor prognostic parameters, such as hormone receptor negativity,

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system (21). Nearly 35% of COX-2 null mice die within 48 hours of birth

The proportion of immunohistochemically identified COX-2 positive tumours varies between studies (range between 4.5% and 85%, Table 2, 26–32).

Ovulation, implantation, perinatal renal development,

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HER2 positivity, increased size of tumour, high grade, develop-ment of distant metastases, and reduced survival.

• COX-2 expression correlates with aromatase expression.

Variations in findings for COX-2 protein expression between studies could partly be attributable to different scoring systems and cut-offs used for COX-2 immunoreactivity. For example, Kelly et al. reported even weak COX-2 staining as positive immunoreactivity (31), whilst Boland et al. reported COX-2 staining as positive only if there was moderate staining in the specimens (30).

Ristimaki et al. found COX-2 expression was significantly associated with hormone receptor negativity, large tumour size, high histological grade, HER2 overexpression, and high Ki67 proliferative rate (28).

Contrary to this, Half et al. did not find any significant relationships between COX-2 expression and clinicopathological factors (29). These results were supported by Kelly et al. (31). Our own work indicates a significant correlation between COX-2 expression and development of distant metastases on follow-up (32). These findings suggests that COX-2 expression facilitates metastases of breast cancer. This may occur by induction of angiogenic factors such as vascular endothelial growth factor (VEGF). Table 2. Immunochemical studies of COX-2 expression in breast cancer

Study Total No. of cancers studied

COX-2 positive (%)

Pathological correlates

Clinical correlates

1998 (26) 44 2 (4.5%) Not studied Not studied

2000 (27) 17 7 (42%) Not studied Not studied

2002, (28) 1576 589 (37.4%) ER/PR negativity

HER2 expression High grade

Reduced disease free survival

(29) 42 18 (43%) None found None found

2004 (30)

65 41 (63%) ER negativity HER2 expression

Not studied

2003 (31). 106 90 (85%) None found

None found

Singh Ranger 29 11 (37.9%, high COX-2 expression)

None found Development of distant metastases


Hwang et al.,

Ristimaki et al.,

Half et al., 2002

Boland et al.,

Kelly et al.,

et al., 2004 (32)

Soslow et al.,

sivity and clinicopathological factors, and again, there is variability in Few studies have examined the correlations between COX-2 expres-

findings (Table 2).

Singh-Ranger and Mokbel 4.2 COX-2 mRNA Expression

COX-2 mRNA-expression in breast cancers varies between 50% and 100% in the literature (Table 3; 29, 33–38, 19–23). There is a parallel relationship between COX-2 immunoreactivity and mRNA expression in tumours (26).

Few studies have examined correlations of COX-2 mRNA with other clinical parameters. Increased transcription has been reported in hormone receptor-positive breast cancer (36), a finding confirmed by our own work demonstrating a significant relationship between COX-2 mRNA expression and PR expression (38). These findings seem to conflict with the results of most immunochemical studies which indicate COX-2 expression is associated with hormone receptor negativity.

There could be a number of explanations for this.

4.3 Post-transcriptional processing of COX-2 mRNA

Messenger RNA once transcribed from DNA, undergoes post-transcriptional processing prior to leaving the nucleus and directing protein synthesis via translation.

It is possible that COX-2 mRNA may be synthesised, but not proceed to formal protein synthesis if destabilized in the cell. Therefore, the association between COX-2 and hormone receptors at mRNA level, may not manifest when COX-2 protein product is investigated. The genomic structure of human COX-2 has been characterised (39, 40), and part of the gene, the 3’ untranslated region (UTR) has an important role in post-transcriptional regulation (41). Several factors have been shown to influence the stability of COX-2 mRNA once transcribed. Interleukin-1 can stabilise the highly unstable COX-2 mRNA transcript (42, 43), and steroids may encourage destabilisation (44). There seem to be two major transcript isoforms – COX-24.6 (4.6kb) and COX-22.8 (2.8kb). In response to steroids, the shorter COX-22.8 isoform decays with a longer half-life than the COX-24.6 isoform (45).

Clearly further work is needed to clarify the basis of these findings - concurrent reverse transcriptase-PCR and immunostaining of cancer sections using antibodies directed against COX-2 and relevant hormone receptors could go some way towards answering these questions, but to our knowledge, has not yet been explored in the literature.

4.4 Subsets of breast cancer which are hormone receptor positive and COX-2 positive

Breast cancer can be classified broadly into ER-positive and ER-negative groups, but it is likely that subgroups exist which are genetically

264

265

(28) – positive immunoreactivity for COX-2 significantly predicted decrea-

in tumours with a low proliferation rate (identified by Ki67, p = 0.001). This seems to suggest that there are subgroups of tumours within the hormone receptor positive group that express COX-2, with significantly poorer survival. Cell line work indicates that enhanced COX-2 levels result in increased production of prostaglandins, such as PGE2, which subsequently increased aromatase activity in breast stromal cells (47). Expression of aromatase leads to oestrogen production, and from cell line studies, we know that hormone receptor expression can be induced

basis for the correlations observed between COX-2 and hormone receptor expression.

Study Total Number of.

cancers studied positive cancers correlates Clinical correlates

Half et al., 2002, (29)

9 9 (100%) Not studied Not studied

Kirkpatrick et al., 2001 (33)


Watanabe et al., 2003 (34)

7 7 (100%) None found None found

Yoshimura et al., 2003 (35)


Zhao et al., 2003 (36)

30 27 (90%) ER-positivity Not studied

Guo et al., 2003 (37)


Singh Ranger 18 18 (100%) PR Not studied

4.5 Animal and Cell Line Studies COX-2 protein has been immunolocalised to malignant epithelial

cells within breast tumours induced in a rat model by various carcinogens (52, 53). Transgenic mice overexpressing breast-targeted oncogenes such as Wnt-1 develop mammary tumours which contain significant amounts of COX-2 (54).

A recent study used transgenic mice to examine the effects of COX-2 overexpression in the breast (55). Invasive breast tumours occur with

sion of COX-2 was dissimilar in different hormonal subgroups of patients Ristimaki et al. found that the prognostic impact of elevated expres-

sed survival in hormone receptor positive patients ( p < 0.0001), and also

by sex steroid hormones (48–51). These findings provide a speculative

Table 3. Studies of COX-2 mRNA expression in breast cancer

et al., 2003 (38)


heterogeneous (46). Within such subgroups, it is possible that there are tumours in which COX-2 is expressed and associated with both hormone receptor positivity and negativity.

COX-2 mRNA Pathological


but no tumour formation. This suggests a high level of COX-2 expression is required to allow carcinoma development, probably in addition to other sustained insults to the genome. Although the effects of targeted disruption of COX-2 genes upon mammary carcinogenesis has not yet been studied, it is informative that in a rodent colorectal cancer model, this strategy leads to a reduction in intestinal adenoma incidence of 86% in COX-2 null mice, and 66% in heterozygous mice (56).

4.6 COX-2 Expression In Ductal Carcinoma In Situ (DCIS)

Ductal carcinoma in situ (DCIS) of the breast is characterised by the proliferation of abnormal epithelial cells with morphological features of malignancy within the basement membrane of the mammary ductal system, without the presence of stromal invasion. It is clear that DCIS is a lesion capable of progressing to form an invasive malignancy. In an effort to understand the biology of the disease, and factors which may predict conversion into invasive ductal cancer, much attention has focused

have addressed the issue of COX-2 expression in DCIS, with varying conclusions (Table 4).

The general consensus of opinion appears to be that:

• COX-2 expression occurs in DCIS. •

•

•

Variations in findings might again, be explained by differences in experimental methodology. In addition, DCIS likely comprises a vast heterogeneous spectrum of disease, composed of subsets of lesion with differing biological potential.

The presence of COX-2 experssion in preinvasive breast cancer is an important finding. It suggests that COX-2 may play a role in the develop-ment of these lesions, or even in their transition into invasive cancers.

266

case with invasive cancers (67–100%).

high frequency after successive rounds of pregnancy in these mice, and are prone to metastasis. The study revealed only pregnancy or lactation resulted in exaggerated induction of COX-2, and tumorogenesis occurred only in multiparous mice. A low level of COX-2 expression occurred in virgin mice, which displayed precocious mammary gland development,

A larger proportion of lesions express COX-2 compared to the

Where specifically analysed, COX-2 expression in DCIS appears

COX-2 expression tends to correlate with increasing grade of lesion – but this is contentious.

may indicate the existence of a subgroup of DCIS lesions with more Correlations with HER2 positivity and hormone receptor negativity

to correlate with HER2 expression, and hormone receptor

on clarifying patterns of protein expression. Surprisingly few studies

negativity.

267

Table 4. Findings of various studies examining COX-2 expression in DCIS Immunochemical study cases

Clinicopathological correlates

Half et al. (29) 16 100 None found Shim et al. (57) 46 85 None found Boland et al. (30) 187 67 ER negativity

HER2 expression Tan et al. (58) 51 80 High grade Perrone et al. (59) 49 87.8 HER2 expression

VEGF expression

5.

In order for COX-2 expression to be categorically implicated in the pathogenesis of breast cancer:

• the gene must first be induced • the consequences of its expression must favour the development

of a malignant state

5.1 Mechanisms of COX-2 upregulation

COX-2 expression is regulated at transcriptional and post-transcrip-tional levels, and also by factors influencing the rate of protein synthesis and degradation.

The human COX-2 gene contains multiple transcription factor binding sites (60), for example, for cAMP (cAMP response element, CRE), inter-leukin-6 (IL6), and nuclear factor κB (NF-κB).

The chemical environment of malignancy is an ideal medium for COX-2 transcription. Dysregulated oncogenes, cytokines, growth factors and hormones, have all been shown to cause induction of COX-2 expression (61–66). In addition, loss of function of tumour suppressor genes may be an explanation for overexpression of COX-2. Mouse fibroblast cell lines engineered to express p53 demonstrate a large reduction in activity of the

Once the gene is expressed, potential factors then come into play which can encourage the development and potentiation of a malignancy.


No. of DCIS % COX-2 expression

OF TUMORIGENESIS COX-2: MECHANISMS

aggressive biological potential. Studies with large sample numbers and adequate follow-up are required to clarify why there is disparity between studies, and to give an insight into the mechanisms of this disease process.

ment favouring increased transcription of the gene itself. tively (67). As we shall see, COX-2 expression can lead to an environ-COX-2 promoter compared to cells which lack p53 expression constitu-


5.2 Prostaglandin-dependent mechanisms

COX-2 expression leads to the synthesis of prostaglandins. These mole-

alter the activity of both the cells in which they are synthesised and that of adjacent cells. There is clear evidence of a link between high prostag-landin levels and cancer. These molecules stimulate cell proliferation (68, 69), and in particular have been shown to induce mitogenesis of mammary epithelial cells (70). Conversely, they are also able to suppress proliferation of immune cells, and alter antigen processing by dendritic cells, which may account in part for the ability of malignant cells to evade immunosurveillance (71, 72). Prostaglandin production is higher in lesions associated with the presence of neoplastic cells in tumour

Figure 2. COX-2 and mechanisms of carcinogenesis.

268

For ease of description, these can be classified as prostaglandin-dependent, and prostaglandin-independent mechanisms of carcinogenesis (Figure 2).

cules are local hormones which help regulate essential cellular physio-logical processes. They have short half-lives, often only minutes, and can

lymphatics, blood vessels, and axillary nodes. Prostaglandin levels appearto be greater in sites of nodal metastases compared to primary tumourareas, and are elevated in tumours of moderate-high grade. Notably,

lesions expressing steroid receptors (73). steroid receptor-negative tumours may produce more prostaglandin than

269

Furthermore, increased prostaglandin levels lead to a rise in cellular cyclic AMP (cAMP). This can directly result in reduced apoptosis and increased cell survival. The aromatase gene CYP19, is responsible for local oestrogen biosynthesis in breast cancer, and therefore, is an impor-tant influence in the development and growth of hormone-dependent tumours (74).

CYP19 from promoter I.4 to promoter II in adipose stromal cells,

PGE2 may itself induce COX-2 expression by binding to the PGE receptor. Cell line work has shown incubation of a mouse osteoblastic cell line with TNF-alpha leads to a biphasic increase in COX-2 production. The second phase of COX-2 expression is considered to be the result of induction by accumulated PGE2 (77).

5.3 Prostaglandin-Independent Mechanisms

Elevated expression of COX-2 will deplete levels of arachidonic acid, its natural substrate. There is some evidence that depletion of arachidonic acid can by itself, lead to reduced cellular apoptosis (78). Furthermore, as mentioned earlier, COX-2 is a potent oxidiser, and it is possible that adjacent substrates can be “co-oxidised” by the enzyme, in some cases producing molecules capable of damaging DNA (78). Substrate promi-scuity of COX-2 may allow the formation of carcinogenic molecules from a wide variety of dietary and environmental agents (79)

There is experimental evidence that COX-2 expression leads to the induction of angiogenic factors, such as VEGF, bFGF, TGF-1, PDGF, and endothelin (80, 81). We recently reported a significant correlation between COX-2 and VEGF-189 mRNA copy numbers in invasive breast cancer specimens (82), which supports these observations. Moreover, tumour growth is markedly attenuated in COX-2 null mice compared to wild-type mice, and these tumours have reduced vascular density (83). COX-2-mediated induction of angiogenesis would provide a basis for tumour spread and metastasis.

Malondialdehyde (MDA) is a carcinogenic molecule, which can form

substitutions (84). MDA can be formed by the isomerisation of prostag-landin H2 (PGH2), cellular levels of which are increased due to over-expression of COX-2.

Members of the matrix metalloproteinase family of enzymes have been implicated in a wide variety of disorders thought to have their basis in aberrant degradation of the extracellular matrix. Transfection of the


adducts with deoxynucleosides, inducing frame-shifts, and base pair

Prostaglandin E2 (PGE2) facilitates switching of expression of

thereby leading to a three- to four-fold increase in activity (75, 76). In support of linkage between the two enzyme systems, there is a signi-ficant correlation between COX-2 and CYP19 mRNA levels in breastcancer (75).


to an increase in the expression and activity of matrix metalloproteinase-2 (MMP-2), resulting in increasingly invasive behaviour of the cells (85). Co-expression of COX-2 and MMP-2 has been found in the atherosclerotic lesions of human aortic aneurysms (86), and MMP-2 immunoreactivity has also been associated with neoplastic cells in human breast cancer specimens (87). Induction of MMPs could facilitate degradation of the basement membrane thereby encouraging tumour growth and spread. This hypothesis needs further exploration in breast cancer, and particularly in DCIS, where breach of the basement membrane defines evolution into cancer. To our knowledge, this has not yet been addressed in the literature.

6.

Non-steroidal anti-inflammatory drugs (NSAIDs) can suppress the COX system non-selectively or selectively. Therefore, long-term usage could theoretically reduce breast cancer risk.

A number of epidemiological studies have investigated if long-term NSAID reduces breast cancer risk (88–96).

Recent meta-analyses and case control studies suggest a moderate

strongest for use lasting greater than 8 years compared to non-users (95). The risk reduction seems to be similar for acetyl-salicylic acid (ASA) containing NSAIDs (such as aspirin or sodium salicylate), and non-ASA NSAIDs (ibuprofen, diclofenac, or indomethacin).

Potential weaknesses of all these forms of study include the fact that patients taking NSAIDs may generally be more health conscious, and therefore enter a lower risk group for breast cancer based on lifestyle, and social group, and numerous sources of bias, including selection, information, and recall bias.

270

reduction in risk of breast cancer with use of NSAIDs of up to 24% (95, 96). This appears to qualify for use of NSAIDs for any duration, and

TO TREAT AND PREVENT BREAST CANCER

Prospective studies give conflicting results – the Women’s Health Study reported no effect of aspirin on breast cancer incidence over the study term (97). Aspirin in this study was given at low dose (100mg) on alternate days only, to a large group of healthy, female health care pro-fessionals. Pharmacological studies of COX-2 indicate that suppression of COX-2-dependent physiological processes requires much larger doses of aspirin and more frequent dosing interval, due to decreased sensitivity of COX-2 to aspirin, and rapid re-synthesis of the enzyme by nucleated cells (98). It is possible, therefore, that in addition to incorrect targeting of high breast cancer-risk patients by these prospective studies, there is also simply insufficient COX-2 inhibition to unmask any preventative effect.

COX-2 SUPPRESSION AS A STRATEGY

breast cancer cell line Hs578T, with cDNA for COX-1 or COX-2, leads

271

few trials examining the issue. Preliminary reports from the celecoxib anti-aromatase neoadjuvant (CAAN) trial indicate that the combination of a COX-2 and aromatase inhibitor in postmenopausal females with hormone-sensitive breast cancer may be more effective in inducing a complete clinical response compared to an aromatase inhibitor alone (102), findings which clearly need substantiation.

It appears attractive to minimise the loss of protective effects of the COX-1 isoenzyme by using selective COX-2 inhibitors in malignancy. Experimental studies have shown however, that dysregulated COX-1 activity is also present in breast tumours. COX-1 activity is upregulated

in cell lines leads to overproduction of MMP-2 (85). This leads one to consider whether inhibition of COX-2 alone in the established malignant state would be effective, or indeed, whether this strategy would encourage consequent overactivity of the COX-1 enzyme system. Cell line experi-ments with COX-1 or COX-2 null cells have shown that PGE2 production remains high in the cells, as a consequence of increased transcription of the remaining functional gene (104)

Finally, any analysis of the potential benefits of COX-2 suppression as a therapeutic strategy, needs to consider possible toxic effects of the medications involved.

Re-analysis of the celecoxib long term arthritis safety study (CLASS), uncovered flaws in the original design of the study, and therefore in conclusions made regarding the superiority of COX-2 inhibitors compared to traditional NSAIDs. Revised data indicate that COX-2 inhibitors may have a similar incidence of ulcer complications to traditional NSAIDs (105). Furthermore, recent prospective clinical trials of COX-2 inhibitors have demonstrated serious cardiovascular effects (106, 107), which have led to the withdrawal of a large number of these agents from clinical use.

REFERENCES

1. Sasco AJ. Breast cancer and the environment. Horm Res. 2003; 60 Suppl 3: 50. 2. Picot D, Loll PJ, Garavito RM. The X-Ray crystal structure of the membrane

protein prostaglandin H2 synthase-1. Nature 1994; 367 (6460): 243–249. 3. Hla T, Bishop-Bailey D, Liu CH, Schaefers HJ, Trifan OC. Cyclooxygenase-1

and -2 isoenzymes. Int J Biochem Cell Biol 1999; 31 (5): 551–557.


in tumour tissue (26, 103), and transfection of both COX-1 and COX-2

effect (99–101). Translating these results to humans is more complex, with

With regard to selective COX-2 suppression using COX-2 inhibitors, data from animal studies supports both treatment and chemo-preventative

Clearly, although we are aware that COX-2 expression is a funda-mental step in breast cancer pathogenesis, further work is required to esta-blish how this enzyme system can be best manipulated for therapeuticbenefit.


6.

(12): 7991–7994. 7. Herschman HR. Prostaglandin synthase-2. Biochim Biophys Acta 1996; 1299

(1): 125–140. 8. Appleby SB, Ristimaki A, Neilson K, Narko K, Hla T. Structure of the human

cyclooxygenase-2 gene. Biochem J 1994; 302 (3): 723–727. 9. Yamagata K, Andreasson KI, Kaufmann WE, Barnes CA, Worley PF. Expression

of a mitogen-inducible cyclooxygenase in brain neurones: regulation by synaptic activity and glucocorticoids. Neuron 1993; 11 (4): 371–386.

10. Simmons DL, Xie W, Chipman JG, Evett GE. Prostaglandins, leukotrienes,

1991. 11. Walenga RW, Kester M, Coroneos E, Butcher S, Dwivedi R, Statt C.

Prostaglandins 1996; 52 (5): 341–359. 12. Harris RC, McKanna JA, Akai Y, Jacobson HR, Dubois RN, Breyer MD.

Cyclooxygenase-2 is associated with the macula densa of rat kidney and increases with salt restriction. J Clin Invest 1994; 94 (6): 2504–2510.

13. Spencer AG, Woods JW, Arakawa T, Singer II, Smith WL. Subcellular locali-sation of prostaglandin endoperoxidase H synthases-1 and -2 by immunoelectron

14. Fitzpatrick FA, Soberman R. Regulated formation of eicosanoids. J Clin Invest 2001; 107 (11): 1347–1351.

15. Murakami M, Naraba H, Tanioka T, Semmyo N, Nkatari Y, Kojima F, Ikeda T, Fueki M, Ueno A, Oh S, Kudo I. Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostalglandin E2 synthase that acts in concert with cyclooxygenase-2. J Biol Chem 2000; 275 (42): 32783–32792.

16. cation of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis. J Biol Chem 2000; 275 (42): 32775–32782.

17. Dinchuk JE, Car BD, Focht RJ, Johnston JJ, Jaffee BD, Covington MB, Contel NR, Eng VM, Collins RJ, Czemiak PM. Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase-2. Nature 1995; 378 (6555): 406–409.

18. Lim H, Paria BC, Das SK, Dinchuk JE, Langenbach R, Trzaskos JM, Dey SK. Multiple female reproductive failures in cyclooxygenase-2 deficient mice. Cell 1997; 91 (2): 197–208.

19. Davis BJ, Lennard DE, Lee CA, Tiano HF, Morham SG, Wetsel WC,

2685–2695. 20. Morham SG, Langenbach R, Loftin CD, Tiano HF, Vouloumanos N, Jennette

JC, Mahler JF, Kluckman KD, Ledford A, Lee CA. Prostaglandin synthase-2 gene disruption causes severe renal pathology in the mouse. Cell 1995; 83 (3): 473–482.

21. Loftin CD Trivedi DR, Tiano HF, Clark JA, Lee CA, Epstein JA, Morham SG, Breyer MD, Nguyen M, Hawkins BM. Failure of ductus arteriosus closure and remodelling in neonatal mice deficient in cyclooxygenase-1 and -2. Proc Natl Acad Sci USA 2001; 98 (3): 1059–1064.

272

lipoxins and PAF (Bailey JM, editor). pgs. 67–8. Plenum Press, New York,

microscopy. J Biol Chem 1998; 273 (16): 9886–9893.

by prostaglandin E2 and interleukin-1 beta. Endocrinology 1999; 140 (6): Langenbach R. Anovulation in cyclooxygenase-2 deficient mice is restored

Kujubu DA, Herschman HR. Dexamethasone inhibits mitogen induction of theTS10 prostaglandin synthase/cyclooxygenase gene. J Biol Chem 1992; 267

Tanioka T, Nakatani Y, Semmyo N, Murakami M, Kudo I. Molecular identifi-

4. Smith WL, Garavito M, Dewitt DL. Prostaglandin endoperoxide-H synthases-1

5. Appleby SB, Ristimaki A, Neilson K, Narko K, Hla T. Structure of the human cyclooxygenase-2 gene. Biochem J 1994; 302 (3): 723–727.

and -2. J Biol Chem 1996; 271 (52): 33157–33160.

273

24. Munroe DG, Wang EY, MacIntyre JP, Tam SS, Lee DH, Taylor GR, Zhou L, Plante RK, Kazami SM, Bauerle PA. Novel intracellular signalling function of prostaglandin H synthase-1 in NF-kappa β activation. J Inflamm 1995; 45 (4): 260–268.

25. Josephy PD, Eling TE, Mason RP. Co-oxidation of benzidine by prostaglandin synthase and comparison with the action of horseradish peroxidase. J Biol Chem 1983; 258 (9): 5561–5569.

26. Hwang D, Scollard D, Byrne J, Levine E. Expression of cyclooxygenase-1 and

455–460. 27. Soslow RA, Dannenberg AJ, Rush D, Woerner BM, Khan KN, Masferrer J,

Koki AT. Cyclooxygenase-2 is expressed in human pulmonary, colonic and

28. Ristimaki A, Sivula A, Lundin J, Lundin M, Salminen T, Haglund C, Joensuu H, Isola J. Prognostic significance of elevated cyclooxygenase-2 expression in breast cancer. Cancer Res 2002; 62 (3): 632–635.

29.

30. Boland GP. Butt IP, Prasad R, Knox WF, Bundred NJ. COX-2 expression is associated with an aggressive phenotype in ductal carcinoma in situ. Br J Cancer 2004; 90: 423–429.

31. Kelly LM, Hill ADK, Kennedy S, Connolly EM, Ramanath R, Teh S, Dijkstra B, Purcell R, McDermott EW, O’Higgins N. Lack of prognostic effect of COX-2 expression in primary breast cancer on short-term follow-up. Eur J Surg Oncol 2003; 29: 707–710.

32. Singh Ranger G, Thomas V, Jewell A, Mokbel K. Elevated cyclooxygenase-2 expression correlates with distant metastases in breast cancer. Anticancer Res.

33. Kirkpatrick K, Ogunkolade W, Bustin S, Jenkins P, Ghilchik M, Mokbel K. The mRNA expression of cyclooxygenase-2 and vascular endothelial growth

34. Watanabe O, Shimizu T, Imamura H, Kinoshita J, Utada Y, Okabe T, Kimura K, Hirano A, Yoshimatsu K, Aiba M, Ogawa K. Expression of cyclooxygenase-2 in malignant and benign breast tumours. Anticancer Res 2003; 23(4): 3215–3221.

35. Yoshimura N, Sano H, Okamoto M, Akioka K, Ushogome H, Kadotani Y, Yoshimura R, Nobori S, Higuchi A, Ohmori Y, Nakamura K. expression of cyclooxygenase-1 and -2 in human breast cancer. Surg Today 2003; 33: 805–811.

36. Zhao XQ, Pang D, Xue YW. Expression of the cyclooxygenase-2 gene in human breast carcinoma. Zhongua Wai Ke Za Zhi 2003; 41 (6): 427–429.

37.

38. Singh Ranger G, Kirkpatrick KL, Clark GM, Mokbel K. Cyclooxygenase-2 (COX-2) mRNA expression correlates with progesterone receptor positivity in human breast cancer. Curr Med Res Opin 2003; 19 (2): 131–134.


2004; 24 (4): 2349–2351.

factor in human breast cancer. Breast Cancer Res Treatment 2001; 69 (3): 373.

mammary tumours. Cancer 2000; 89 (12): 2637–2645.

cyclooxygenase-2 in human breast cancer. J Natl Cancer Institute 1998; 90 (6):

22. Rocca B, Spain LM, Pure E, langenbach R, Patrono C, Fitzgerald GA. Distinct roles of prostaglandin H synthases-1 and -2 in T-cell development. J Clin Invest 1999; 103 (10): 1469–1477.

23. Ohki S, Ogino N, Yamamoto S, Hayaishi O. Prostaglandin hydrperoxidase, an integral part of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J Biol Chem 1979; 254 (3): 829–836.

Half E, Tang XM, Gwyn K, Sahin A, Wathen K, Sinicrope FA. Cyclo-oxygenase-2 expression in human breast cancers and adjacent ductal carcinomain situ. Cancer Research 2002; 62: 1676–1681.

Guo GL, Yao ZX, Wu J. The clinical significance of expression of cyclo-oxygenase-2 gene in breast cancer. Zhongua Yi Xue Za Zhi 2003; 83 (19):1661–1664.


43. Srivasta SK, Tetsuka T, Daphna-Iken D, Morrison AR. IL 1 beta stabilises COX II mRNA in renal mesangial cells; role of 3’ untranslated region. Am J Physiol 1994; 36: F504–F508.

44. Evett GE, Xie W, Chipman JG, Robertson DL, Simmons DL. Prostaglandin GH synthase isoenyme 2 expression in fibroblasts: regulation by dexamethasone, mitogens and oncogenes Arch Biochem Biphy 1993; 306: 169–177.

45.

46. Sorlie T, Perou CM, Tibshirani R. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. PNAS USA 2001; 98: 10869–10874.

47. Brueggemeier RW, Richards JA, Petrel TA. Aromatase and cyclooxygenases: enzymes in breast cancer. J Steroid Biochem Mol Biol 2003; 86: 501–507.

48. Read DL, Greene GL, Katzenellenbogen BS. Regulation of estrogen receptor messenger ribonulcelic acid and protein levels in human breast cancer cell lines by sex steroid hormones, their antagonists, and growth factors. Mol Endocrinol 1989; 3 (2): 295–304.

49. Katzenellenbogen BS, Norman MJ. Multihormonal regulation of the progesterone receptor in MCF-7 human breast cancer cells: interrelationships among insulin/insulin-like growth factor-I serum, and estrogen. Endocrinology 1990; 126 (2): 891–898.

50. Cho H, Aronica SM, Katzenellenbogen BS. Regulation of progesterone receptor gene expression in MCF-7 breast cancer cells: A comparison of the effects of cyclic adenosine 3’, 5’-Monophosphate, Estradiol, Insulin-like growth factor-I serum, and serum factors. Endocrinology 1994; 134 (2): 658–664.

51. Vienonen A, Syvala H, Miettinen S, Tuohimaa P, Ylikomi T. Expression of progesterone receptor isoforms A and B is differentially regulated by estrogen in different breast cancer cell lines. J Steroid Biochem Mol Biol 2002; 80: 307–313.

52. Robertson FM, Parrett ML, Joarder FS, Ross M, Abou-Issa HM, Alshafie G, Harris RE. Ibuprofen-induced inhibition of cyclooxygenase isoform gene expression and regression of rat mammary carcinomas. Cancer Letters 1998; 122 (1-2): 165–175.

53. Nakatsugi S, Ohta T, Kawamori T, Mutoh M, Tanigawa T, Watanabe K, Sugie S, Sugimura T, Wakabayashi K. Chemoprevention by nimesulide, a selective cyclooxygenase-2 inhibitor, of 2-amino-1-methyl-6-phenylimidazo [4,5,6] pyridine (PhIP)-induced mammary gland carcinogenesis in rats. Japanese J Cancer Res 2000; 91 (9): 886–892.

54. Howe LR, Crawford HC, Subbaramaiah K, Haassell JA, Dannenberg AJ, Brown AMC. PEA-3 is upregulated in response to Wnt1 and activates the expression of cyclooxygenase-2. J Biol Chem 2001; 276 (23): 20108–20115.

55. Liu CH, Chang SH, narko K, Trifan OC, Wu MT, Smith E, Haudenschild C, Lane TF, Hla T. Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice. J Biol Chem 2001; 276 (21) 18563–18569.

274

39. Appleby SB, Ristimaki A, Neilson K, Narko K, Hla T. Structure of the human cyclooxygenase-2 gene. Biochem J 1994; 302 (3): 723–727.

40. Kosaka T, Miyata A, Ihara H, Hara S, Sugimoto T, Takedo O, Takahashi E, Tanabe T. Characterisation of the human gene (PTGS2) encoding prostaglandin-endoperoxide synthase 2. Eur J Biochem 1994; 221: 889–897.

41. Chen C-Y, Shyu A-B. AU-rich elements; characterisation and importance in mRNA degradation. Trends Biochem Sci 1995; 20: 465–470.

42. Ristimaki A, Garfinkel S, Wessendorf J, Maciag T, Hla T. Induction of cyclooxygenase-2 by interleukin 1-alpha. Evidence for post-transcriptional regulation. J Biol Chem 1994; 269: 11769–11775.

genase-2 transcript isoforms by dexamethasone: evidence for post-transcriptionalRistimaki A, Narko K, Hla T. Downregulation of cytokine-induced cyclooxy-

regulation. Biochem J 1996; 318 (1): 325–331.

275

59. Perrone G, Santini D, Vincenzi B, Zagami M, La Cesa A, Bianchi A, Altomare V, Primavera A, Battista C, Vetrani A, Tonini G, Rabitti C. COX-2 expression in DCIS: correlation with VEGF, HER2 / neu, prognostic molecular markers and clinicopathological features. Histopathology 2005; 46: 561–568.

60. Howe LR, Subbaramaiah K, Brown AM, Dannenberg AJ. Cyclooxygenase-2: a target for the prevention and treatment of breast cancer. Endocr Relat Cancer.

61. Subbaramaiah K, Telang N, Ramonetti JT, Araki R, DeVito B, Weksler BB, Dannenberg AJ. Transcription of cyclooxygenase-2 is enhanced in transformed mammary epithelial cells. Cancer Res 1996; 56 (19): 4424–4429.

62. Sheng H, Williams CS, Shao J, Liang P, Dubois RN, Beauchamp RD. Induction of cyclooxygenase-2 by activated Ha-ras oncogene in Rat-1 fibroblasts and the role of the mitogen-activated protein kinase pathway. J Biol

63. Vadlamudi R, Mandal M, Adam L, Steinbach G, Mendelsohn J, Kumar R. Regulation of the cyclooxygenase-2 pathway by the HER-2 receptor. Oncogene 1999; 18 (2): 305–314.

64. Mestre JR, Subbaramaiah K, Sacks PG, Schantz SP, Tanabe T, Inoue H, Dannenberg AJ. Retinoids suppress epidermal growth factor-induced transcription of cyclooxygenase-2 in human oral squamous carcinoma cells. Cancer Res 1997; 57 (14): 2890–2895.

65. Xie W, Herschmann HR. v-src induces prostaglandin synthase-2 gene expression by activation of the c-Jun N-terminal kinase and the c-Jun transcription factor.

66. Xie W, Herschmann HR. Transcriptional regulation of prostaglandin synthase-2 gene expression by platelet derived growth factor and serum. J Biol Chem

67. Subbaramaiah K, Altorki N, Chung WJ, Mestre JR, Sampat A, Dannenberg AJ. Inhibition of cyclooxygenase-2 gene expression by p53. J Biol Chem 1999;


Chem 1998; 273 (34): 22120–22127.

J Biol Chem 1995; 270 (46): 27622–27628.

1996; 271 (49): 31742–31748.

2001; 8 (2): 97–114.

274 (16): 10911–10915.

56. Oshima M, Dinchuk JE, Kargman SL, Oshima H, Hancock B, Kwong E, Trzaskos JM, Evans JF, Taketo MM. Suppression of intestinal polyposis in APC delta 716 knockout mice by inhibition of cyclooxygenase-2 (COX-2). Cell 1996; 87 (5): 803–809.

57. Shim V, Gauthier ML, Sudilovsky D, Mantei K, Chew KL, Moore DH, Cha I, Tlsty TD, Esserman L. Cyclooxygenase-2 expression is related to nuclear grade in ductal carcinoma in situ and is increased in its normal adjacent epithelium. Cancer Res 2003; 63: 2347–2350.

58. Tan KB, Yong WP, Putti TC. Cyclooxygenase-2 expression: a potential prognostic and predictive marker for high-grade ductal carcinoma in situ of the breast. Histopathology 2004; 44(1): 24–28.

68. Nolan RD, Danilowicz RM, Eling TE. Role of arachidonic acid metabolism in the mitogenic response of BALB/c 3T3 fibroblasts to epidermal growth factor. Molecular Pharmacology 1988; 33 (6): 650–656.

69. Goin M, Pignataro O, Jimenez de Asua L. Early cell cycle diacyglycerol (DAG) content and protein kinase C (PKC) activity enhancement potentiates prostaglandin F2 alpha (PGF2 alpha) induced mitogenesis in Swiss 3T3 cells. FEBS Letters 1993; 316 (1): 68–72.

70. Bandyopadhyay GK, Imagawa W, Wallace D, Nandi S. Linoleate metabolites enhance the in vitro proliferative response of mouse mammary epithelial cells to epidermal growth factor. J Biol Chem 1987; 262 (6): 2750–2756.

71. Huang M, Sharma S, Mao JT, Dubinett SM. Non-small cell lung cancer-derived soluble mediators and prostaglandin E2 enhance peripheral blood lymphocyte IL-10 transcription and protein synthesis. J Immunol 1996; 157 (12): 5512–5520.


76. Zhao Y, Agarwal VR, Mendelsohn CR, Simpson ER. Transcriptional regulation of CYP19 (aromatase) gene expression in adipose stromal cells in primary culture. J Steroid Biochem Mol Biol 1997; 61 (3-6) 203–210.

77.

induction of cyclooxygenase-2 in MC3T3-E1 cells. J Biol Chem1995; 270 (52): 31315–31320.

78. Prescott SM, Fitzpatrick FA. Cyclooxygenase-2 and carcinogenesis. Biochim

79. Wiese FW, Thompson PA, Kadlubar FF. Carcinogen substrate specificity of human cyclooxygenase-1 and cyclooxygenase-2. Carcinogenesis 2001; 22 (1): 5–10.

80. Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, DuBois RN. Cyclooxygenase

705–716. 81. Gately S. The contributions of cyclooxygenase-2 to tumour angiogenesis.

Cancer Metastasis Rev 2001; 19 (1-2): 19–27. 82. Kirkpatrick K, Ogunkolade W, Bustin S, Jenkins P, Ghilchik M, Mokbel K.

The mRNA expression of cyclooxygenase-2 and vascualr endothelial growth

83. Williams CS, Tsujii M, Reese J, Dey SK, DuBois RN. Host cyclooxygenase-2 modulates carcinoma growth. J Clin Invest 2000; 105 (11): 1589–1594.

84. Marnett LJ. Aspirin and the potential role of prostaglandins in colon cancer.

85. Takahashi Y, Kawahara F, Noguchi M, Miwa K, Sato H, Seiki M, Inoue H, Tanabe T, Yoshimoto T. Activation of matrix metalloproteinase-2 in human breast cancer cells overexpressing cyclooxygenase-1 or -2. FEBS Letters 1999; 460 (1): 145–148.

86. Hong BK, Kwon HM, Lee BK, Kim D, Kim IJ, Kang SM, Jang Y, Cho SH, Kim HK. Co-expression of cyclooxygenase-2 and matrix metalloproteinases in human aortic atherosclerotic lesions. Yonsei Med J 2000; 41 (1): 82–88.

87. Iawata H, Kobayashi S, Iwase H, Masaoka A, Fujimoto N, Okada Y. Production of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human breast carcinomas. Jpn J Cancer Res 1996; 87 (6): 602–611.

88. Friedman GD, Ury HK. Initial screening for carcinogenicity of commonly used drugs. J Natl Cancer Inst 1980; 65 (4): 723–733.

89. Paganini-Hill A, Cho A, Ross RK, Henderson BE. Aspirin use and chronic diseases: a cohort study of the elderly. Br Med J 1989; 299 (6710): 1247–1250.

90.

276

regulates angiogenesis-induced by colon cancer cells. Cell 1998; 93 (5):

factor in human breast cancer. Breast Cancer Res Treatment 2001; 69 (3): 373.

Biophys Acta. 2000 Mar 27; 1470 (2): M69–78.

Cancer Res. 1992; 15: 52 (20): 5575–5589.

72. Stolina M, Sharma S, Zhu L, Dubinett SM. Lung cancer cyclooxygenase-2 dependent inhibition of dendritic cell maturation and function. Proc Am Ass

73. Rolland PH, Martin PM, Jacquerier J, Roland AM, Toga M. Prostaglandin production in human breast cancer: evidence suggesting that an elevated prostaglandin production is a marker of high metastatic potential for neoplastic cells. J Natl Cancer Institute 1980; 64 (5): 1061–1070.

74. James VHT, Reed MJ. Steroid hormones and human cancer. Progress in Cancer Research and Therapy 1980; 14: 471–487.

75. Zhao Y, Agarwal VR, Mendelsohn CR, Simpson ER. Oestrogen biosynthesis proximal to a breast tumour is stimulated by PGE2 via cyclic AMP, leading to activation of promoter II of the CYP19 (aromatase) gene. Endocrinology 1996; 137 (12): 5739–5742.

Cancer Res 2000; 41: 619.

of nuclear factor-interleukin-6 in the tumour necrosis factor α-dependent Yamamoto K, Arakawa T, Veda N, Yamamoto S. Transcriptional roles

Rosenberg L, Palmer JR, Zauber AG, Warshauer ME, Stolley PD, Shapiro S. A hypothesis: nonsteroidal anti-inflammatory drugs reduce the incidence of large bowel cancer. J Natl Cancer Inst 1991; 83 (5): 355–358.

277

98. Patrono C, Coller B, Dalen JE, FitzGerald GA, Fuster V, Gent M, Hirsh J, Roth

99. Harris RE, Alshafie GA, Abou-Issa H, Seibert K. Chemoprevention of breast

(8): 2101–2103. 100. Suzui N, Sugie S, Rahman KM, Ohnishi M, Yoshimi N, Wakabayashi K, Mori

H. Inhibitory effects of diallyl disulfide or aspirin on 2-amino-1-methyl-6-phenyl-imidazole [4,5-b] pyridine-induced mammary carcinogenesis in rats.

101. Alshafie GA, Abou-Issa HM, Seibert K, Harris RE. Chemotherapeutic evaluation of celecoxib, a cyclooxygenase-2 inhibitor in a rat mammary tumour model.

102. Chow LW, Wong JL, Toi M. Celecoxib anti-aromatase neoadjuvant (CAAN) trial for locally advanced breast cancer: preliminary report. J Steroid Biochem

103. Parret ML, Harris RL, Joarder FS, Ross MS, Clausen KP, Robertson FM. Cyclooxygenase-2 gene expression in human breast cancer. Int J Oncol 1997; 10: 503–508.

104. Kirtikara K, Morham SG, Raghow R, Laulederkind SJF, Kanekura T, Goorha S, Ballou LR. Compensatory prostaglandin E2 biosynthesis in cyclooxygenase-

105. Juni P, Rutjes AWS, Dieppe PA. Are selective COX-2 inhibitors superior to traditional non steroidal anti-inflammatory drugs? BMJ 2002; 324: 1287–1288.

106.

107. Anderson WF, Zauber A, Hawk E, Bertagnolli M; Adenoma Prevention with Celecoxib (APC) Study Investigators. Cardiovascular risk associated with celecoxib in a clinical trial for colorectal adenoma prevention. N Engl J Med 2005; 352 (11): 1071–1080.

cancer: the Women’s Health Study: a randomized controlled trial. JAMA. 2005; 6: 294(1): 47–55.

effects. Chest. 2001; 119(1 Suppl): 39S–63S. G. Platelet-active drugs: the relationships among dose, effectiveness, and side

Oncol Rep 2000; 7 (6): 1377–1381.

1 or cyclooxygenase-2 null cells. J Exp Med 1998; 187 (4): 517–523.

Solomon SD, McMurray JJ, Pfeffer MA, Wittes J, Fowler R, Finn P,

Levesque LE, Brophy JM, Zhang B. the risk of myocardial infarction with cyclooxygenase-2 inhibitors: a population study of elderly adults. Ann Intern Med 2005; 142 (7): 481–489.

Jpn J Cancer Res 1997; 88 (8): 705–711.

Mol Biol. 2003; 86 (3-5): 443–447.

91. Thun MJ, Namboodiri MM, Calle EE, Flanders WD, Heath CW. Aspirin use and risk of fatal cancer. Cancer Res 1993; 53 (6): 1322–1327.

92. Harris RE, Kasbari S, Farrar WB. Prospective study of nonsteroidal anti-inflammatory drugs and breast cancer. Oncol Rep 1999; 6 (1): 71–73.

93. Schreinmachers DM, Everson RB. Aspirin use and lung, colon and breast cancer incidence in a prospective study. Epidemiology 1994; 5 (2): 138–146.

94. Sharpe CR, Collett JP, McNutt M, Belzille E, Bowin JF, Hanley JA. Nested case-control study of the effects of nonsteroidal anti-inflammatory drugs on breast cancer risk and stage. Br J Cancer 2000; 83 (1): 112–120.

95. Cotterchio M, Krieger N, Sloan M, Steinegart A. Non-steroidal anti-inflammatory drug use and breast cancer risk. Cancer Epidemiol Biomarkers Prev 2001; 10 (11): 1213–1217.

96. Khuder SA, Mutgi AB. Breast cancer and NSAID use: a metaanalysis. Brit J

97. Cook NR, Lee IM, Gaziano JM, Gordon D, Ridker PM, Manson JE, Hennekens CH, Buring JE. Low-dose aspirin in the primary prevention of

Cancer 2001; 84 (9): 1188–1192.


cancer in rats by celecoxib, a cyclooxygenase-2 inhibitor. Cancer Res 2000; 60

Chapter 14

IN HUMAN BREAST CANCER

Soe Maunglay, Douglas C Marchion, and Pamela N Münster Experimental Therapeutics and Breast Medical Oncology, Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center, 12902 Magnolia Dr, Tampa, FL 33612, USA

Abstract: More than 210,000 Americans will be diagnosed with breast cancer each year and more than 41,000 will die from this disease. Over the last two decades many novel therapies have been introduced. However, due to the limited availability and accuracy of prognostic and predictive markers, many patients will have to be treated for the benefit of a few. The lack of precise predictive markers not only pertains to the use of chemotherapy, but also to hormonal or targeted therapy. Still more than half of the patients treated will not derive a benefit. Currently, the prognosis and treatment plan for early stage breast cancer is based on the number of involved lymph nodes, the size of the tumor, the histological grade and type, as well as lymphatic and vascular invasion which will determine the potential benefits from adjuvant chemo- and radiation therapy. Further therapeutic intervention, targeting estrogen receptor and HER2 signaling pathways, are based on the qualitative and quantitative assessment of the estrogen receptor (ER) and progesterone receptor (PR) status and HER2 expression by immunohistochemistry or the HER2 amplification by

prognostic and predictive values of gene expression patterns and the better definition of therapeutic targets will lead to significant change in the assessment and treatment of breast cancer in the near future.

1. INTRODUCTION

The 10-year risk of recurrence of stage I and II breast cancer even in the absence of lymph node involvement or angiolymphatic invasion remains as high as 25% with a corresponding breast-cancer-specific mortality of 10%.

© 2007 Springer.


PROGNOSTIC AND PREDICTIVE FACTORS

fluorescence in situ hybridization. The rapid emergence of data on the

Key words: breast cancer, prognostic factors, predictive factors, estrogen receptor, HER2expression, progesterone receptor, gene expression patterns, gene profiling

280 In contrast, a subgroup of patients will not recur despite the presence of many adverse factors (1). The better understanding of prognostic and predictive factors would therefore vastly benefit patients with breast cancer regardless of their age. Furthermore, while advances in the last decade have improved the disease-free and overall survival for a collective group of patients, the increase in toxicities associated with more extensive therapies may place many individual patients at increased risk without directly adding a personal benefit. Over 40% of the patients with breast cancer are over the age of 65 (2). While long-term sequelae may pose a significant problem with more aggressive therapy in younger women, the presence of co-morbidities from other health issues may render older women more vulnerable to the toxicities of aggressive therapy. The ultimate goal of therapy should not only be the disease-free survival, but also overall survival and quality of life. Hence, to select patients who will most likely benefit from adjuvant systemic therapy while avoiding toxicities in those less likely to benefit is now a central focus of many studies. The advances and limitations in the currently available prognostic and predictive factors will be discussed.

2. PROGNOSTIC FACTORS

Prognostic factors for breast cancer are in part derived from the tumor and in part from the specific environment provide by the host (Table 1). The approach to breast cancer is based on the assessment of several factors, the most important of which are the age of the patients, the

histology may further weight in on decision making if there are uncertainties. Other factors that will be discussed are less significant in the context of prognostic factors or remain investigational.

Table 1. Factors with prognostic value and factors with both prognostic and predictive value in breast cancer (*investigational)

Maunglay, Marchion, and Münster

of the tumor. Other factors such as angiolymphatic invasion or tumor menopausal status, the number of involved lymph nodes and the size

14. Prognostic and predictive factors 281 2.1. Nodal involvement status

Macroscopic involvement

The involvement of axillary node by surgical-pathological assessment remains one of the most significant prognostic factors. Mammograms for axillary node assessment may only identify grossly involved lymph nodes. The sensitivity of computed tomography (CT) for axillary metastases is low. Positron emission tomography (PET) with or without CT has a high

specificity, but the low sensitivity does not allow accurate assessment of small nodal metastases (3). The 5-year survival for patients with node-negative disease is 83% compared with 73% for those with 1–3 involved lymph nodes suggesting a role for more extended adjuvant therapy in patients with lymph node involvement (4). The 5-year survival further declines to 46% for 4–12 positive nodes and 28% for patients with 13 or more positive nodes according to the National Surgical Adjuvant Breast and Bowel Project (NSABP) data (4). More recent studies categorize risk groups in node-negative versus node-positive. Patients with node-positive tumors are further subgrouped into 1–3 involved, 4–9 involved, or 10 and more involved lymph nodes, which is commonly used by clinicians as a risk assessment tool (www.adjuvantonline.com). Not only the number of lymph nodes, but also the percentage of tumor involvement in each individual lymph node appears to correlate with adverse outcome. An involvement of more than 25% of the lymph node by tumors was associated with a higher distant recurrence rate (53% versus 30%) and a lower overall survival rate (43% versus 63%) (5).

However, as axillary lymph node dissection (ALND) has been associated with an increase in morbidity, the use of limited sampling employing sentinel lymph node biopsies (SLNB) has become the standard for axillary staging for operable breast cancer patients (6–11). Overall and disease-free survival in node-negative breast cancer patients who received lymph node resection by SLNB were comparable to those treated with ALND (9). Several studies validated the efficacy and safety of sentinel-node biopsy (9, 12). While the overall accuracy of the sentinel-node assessment was found to be 97%, the sensitivity was 91%, and the specificity 100% in one randomized study compared to routine ALND (12). Furthermore, a recent single-center study showed increased lymph node positivity after SLNB compared to complete ALND in patients with T1a/T1b tumors (10% versus 3%), supporting earlier reports that SLNB may not only be associated with less morbidity but also higher sensitivity (13). When analyzed by an experi-enced pathologist with serial sectioning and immunohistochemical evalu-ation, SLNB was found to be the most accurate detection tool used in

2.1.1. Axillary nodal involvement

staging of breast cancer (14). To validate these data, a large multi-center

282

debate, whether or not there are therapeutic implications of axillary lymph node removal, most centers will consider a complete ALND in patients with positive lymph nodes identified by SLNB.

Micrometastases Involvement Using immunohistochemistry (IHC) for cytokeratin staining, tumor

deposits less than 0.2 mm or isolated tumor cells are defined as submicro-metastatic, and tumor foci of 0.2 mm –2 mm are considered micrometa-static (16). In early studies involving complete ALND, the presence of micrometastatic disease was associated with outcomes similar to node-negative disease (17). However, the common use of SLNB has confounded

micrometastatic foci found with SLNB had further macrometastatic nodal involvement (8, 16). Despite the low incidence of positive ALND with single micrometastatic foci found by SLNB, most patients will proceed to a complete ANLD.

Mammographic or ultrasonic evidence of intramammary lymph node involvement warrants histopathological assessment. In a recent single center retrospective study, isolated intramammary lymph node involve-ment was documented in 6 out of 106 patients (5%). The presence of intramammary lymph nodes was an independent adverse prognostic indicator in multivariate analysis with poorer 5-year rates for disease-free survival (54% versus 89%) and overall survival (64% versus 88%), respectively (18).

The number of lymph nodes involved with tumor may be confounded by the number of examined lymph nodes. Data from a retrospective analysis of 83,686 patients undergoing axillary node dissection demon-strated that nodal involvement of 10% of the sampled lymph nodes was

findings were reported by other investigators and is currently being further evaluated (3, 20, 21).

2.2. Tumor Size

Tumor diameter and lymph node status may act as independent but interlinked prognostic indicators. Several studies suggested tumor size as

the relevance of micrometastatic disease, as 9%–12% of patients with

90%–100% of the sampled nodes suggested a 45% mortality (19). Similarassociated with 5% breast cancer mortality, whereas involvement of

2.1.2. Intramammary lymph node involvement

2.1.3. Ratio of involved to sampled lymph nodes


phase III, randomized trial comparing axillary resection with a stan-dardized method of SLNB is underway (15). Due to the ongoing

≤

14. Prognostic and predictive factors 283 an independent prognostic factor in node-negative patients. Patients with tumors measuring 1.0 cm or less had a significantly better 20-year recurrence-free survival (88%) than those with tumors 1.1–2.0 cm (72%) (22). The 20-year risk of recurrence for tumors measuring 2.1–3.0 cm was 33%, and 44% for patients tumors between 3.1 and 5.0 cm (23). A correlation between tumor size and outcome was further suggested in a single institution study involving over 3000 patients (24). While tumor size should be taken into consideration for treatment planning, a review of Surveillance, Epidemiology, and End Results (SEER) data suggested that elderly patients with node-negative tumors measuring less than 2 cm may have a similar overall survival to women without breast cancer (25).

2.3. Tumor Histological Grade and Type

The most commonly used grading system is the Scarff-Bloom-Richardson(SBR) scheme which was based on morphologic features such as degree of tumor tubule formation, tumor mitotic activity, and nuclear pleomorphism (nuclear grade) of tumor cells with a score of 1 to 3 for each. Combined scores converts to the SBR differentiation/grade. Scores of 3 to 5 equals to well-differentiated (SBR low grade), 6 to 7 moderately differentiated (SBR intermediate grade) and 8 to 9 equals to poorly differentiated (SBR high grade). A multivariate analysis in 1,262 patients with operable, invasive ductal breast carcinoma suggested that nodal metastases and SBR scores were the two most important factors for metastasis-free survival (26).

Patients with mucinous, tubular, or papillary cancers had a better prognosis than those with tumors that were not otherwise specified (NOS) or atypical medullary tumors. Survival for those with typical medullary, NOS combinations, or lobular invasive cancers was intermediate (27).

2.4. Lymphatic and Vascular Invasion (LVI)

A study involving 1,704 women with early stage breast cancer revealed vascular invasion (lymphatic and/or blood vessel) on routine hematoxylin and eosin sections in the tumors of 23% of the examined patients; and its

2.3.1. Tumor Grade

2.3.2. Tumor Histology

The NSABP B-06 study in node-negative invasive breast cancer pati-ents demonstrated three prognostic categories for histologic tumor type.

284 presence was associated with both decreased survival and increased local recurrence (28). Similarly, the recurrence rate in 644 women with stage I breast cancer was found to be significantly higher (38% compared to 22%) in those with vascular invasion than those without (29). A recent small study suggested that immunohistochemical detection of blood

vascular invasion with anti-D2-40 (lymphatic endothelium) antibodies found in peritumoral tissue were independent determinants of lymph node metastases (30).

Tumor histology and grade as well as lymphovascular invasion may be used for therapeutic consideration if the tumor size and the lymph node involvement leave the treatment decision ambiguous.

2.5. Age at diagnosis

A retrospective study analyzed data on 1,398 early-stage breast cancer patients treated with breast-conserving therapy. This study suggested that patients younger than 35 had a worse prognosis, with a higher overall recurrence rate as well as a greater risk for developing distant metastases, when compared with older patients (31). The analysis of 8,738 patient from the San Antonio database further suggested that younger women more often had a higher number of involved lymph nodes, larger tumors, and negative hormone receptors (32). Furthermore, node-negative premeno-pausal patients with endocrine responsive tumors tend to have larger tumors of higher grade and carry a worse prognosis, when under the age of 35 (33). In contrast, a diagnosis of small (less than 2 cm) node-negative breast cancer did not impact the overall survival of women over the age of 70 (25).

2.6. Race and ethnicity

While the breast cancer mortality continues to decline in women of all

African American women remains higher than in Caucasian women (34). Whether this is due to a difference in the nature of the tumors or due to a difference in access to care or both is currently being intensely studied.

2.7. Proliferation Markers

Higher SPF is generally adversely related with disease-free and overall survival. Higher SPF was correlated with worse tumor grade and larger tumor size, nodal involvement, and negative hormone-receptor (35–37).

vessel invasion (BVI) with anti-CD34 (pan-endothelium) and lympho-

races and ethnicity in the USA, the breast cancer-specific death rates in

2.7.1. S-phase fraction (SPF)


14. Prognostic and predictive factors 285

The relevance of TLI was addressed in a retrospective evaluation of 523 women receiving adjuvant therapy for early stage node-positive breast cancer. Patients with a lower TLI had significantly better 5-year relapse-free survival (66% versus 50%), and overall survival (85% versus 73%) (38). In addition, high TLI also inversely affected prognosis in patients with node-negative tumors (39). Larger prospective studies will be needed to reach definite conclusions. A multi-center prospective study with median follow-up of 118 months in 516 patients with lymph node-negative breast cancer showed that a MAI above 10 was associated with a higher recurrence rate and mortality (40).

An overview involving 40 studies evaluating more than 11,000 patients suggested that Ki-67 may have independent prognostic significance, however, in relation to other prognostic factors it was less important (41). Ki-67 is currently not routinely used in the pathological assessment of primary breast cancer. High PCNA labeling index was associated with shorter relapse-free and overall survival in at least two studies (42, 43).

2.8. Cathepsin D

levels greater than 70 pmol/mg protein were associated with a 4.5-fold increase in relapse rate in patients with node-negative tumors, particularly high cytosolic cathepsin-D values were associated with poor prognosis (44, 45). Furthermore, higher Cathepsin D levels were seen in the cytosol of tumors that were larger and involved lymph nodes. However, Cathepsin D levels were also higher in hormone receptor-positive tumors (46). While Cathepsin D may be an adverse prognostic factor, its role as a predictive factor of anti-hormonal therapy has not been established. However, due to technical challenges in reproducibility of the test, the assessment of Cathepsin D currently remains investigational.

2.9.

The tumor-induced blood vessel formation, angiogenesis, has become an exciting target for cancer therapy and many novel compounds have entered

2.7.2. Thymidine labeling index (TLI) and Mitotic Activity Index (MAI)

2.7.3.antigen (PCNA) Ki-67 nuclear antigen and Proliferating cell nuclear

Angiogenesis markers — Microvascular density (MVD), basic fibroblast growth factor (bFGF, FGF-2), and vascular endothelial growth factor (VEGF)

an independent prognostic factor in several studies. Cathepsin D protein Cathepsin D, a lysosomal proteolytic enzyme has been identified as

286 the clinical arena. In particular, tumors with high MVD and VEGF were thought to be primary targets for therapy (47). For IHC staining to measure MVD, various angiogenic markers are used such as cluster designations 31 (CD31), 34 (CD34), and 105 (CD105), von Willebrand factor (VWF), factor VIII, type IV collagen, and VEGF. In a prospective, blinded study of 165 consecutive patients with invasive breast carcinoma, MVD was detected via IHC staining of factor VIII-related antigen. In this study, MVD was found to be an independent and highly significant prognostic indicator for overall and relapse-free survival in patients with early-stage breast cancer (48). An early study had linked the expression of cytosolic levels of VEGF to poor prognosis in node-negative patients (49). MVD expression measuring factor VIII, type IV collagen, and VEGF 3 by IHC suggested that the MVD involvement was positively related to the presence of axillary lymph node metastases (50).

However, when intratumoral MVD was assessed using factor VIII-related antigen, only MVD index in the lymph node metastases, not the MVD index in the primary breast tumor was found to be adversely related to outcome (51). Furthermore, no predictive value for either VEGF, or MVD was found in node-negative high-risk or node-positive patients treated with two different adjuvant chemotherapy regimens (52).

These varying results suggest that further studies are required to consider the use of angiogenesis markers in clinical decision making.

2.10. Bone marrow micrometastases

A study involving the role of bone marrow micrometastases reported the presence of tumor cells in the bone marrow of 203 (55%) of 367 lymph node-positive patients and in 112 (31%) of 360 lymph node-negative patients (53). The presence of tumor cells was determined by monoclonal antibody 2E11, directed against the polymorphic epithelial mucin, TAG12. After a median follow-up of 36 months, tumor cell detection in bone marrow was found to be an independent prognostic indicator for both distant disease-free survival and overall survival and also has superior

It was found to be an important predictor of outcome among the patients with tumors less than 2 cm in diameter. Of note, most of these patients received adjuvant therapy.

The relevance of bone marrow micrometastases was further evaluated in a pooled analysis, deriving data from nine studies involving 4,703 patients with early-stage breast cancer (54). The presence of bone marrow micrometastasis was a significant adverse prognostic factor with poorer overall and breast-cancer-specific survival in all groups. Most notably in subgroup analysis, patients with stage I breast cancer who were not receiving adjuvant chemotherapy had significantly shortened survival.

predictive value to tumor stage, tumor grade, and axillary lymph node status.



While these are important findings, further data is required to assess the diagnostic value of bone marrow sampling and bone marrow sampling should not be used routinely if it will not lead to a change in the therapeutic management of the patient.

3. PROGNOSTIC AND PREDICTIVE FACTORS

3.1. Estrogen receptor (ER) and progesterone receptor (PR) status

Until the definition of the role of HER2, the expression of estrogen (ER) and progesterone receptor (PR) has been the only validated predictive marker for therapy. The majority of patient will present with an estrogen or progesterone positive breast cancer, and more so in the postmenopausal patient population (Figure 1) (55). While the ER/PR expression are predominantly predictive markers of response, their roles as a favorable prognostic factors were determined in studies before hormonal intervention became an integral part of therapy. The National Surgical Adjuvant Breast and Bowel Project Protocol (NSABP) B-06 study evaluated the outcome of 1,157 node-negative breast cancer patients who were treated with adequate local therapy, however without systemic adjuvant therapy. This study suggested that patients with ER-positive tumors had a longer disease-free survival and overall survival at 5 years

beyond 5 years (56).

Figure 1. Percentage distribution of estrogen receptor and progesterone receptor status.

In addition to a potential role as a prognostic factor, the expression of

ER and PR status is by far the most important predictive factor until recently. Hormone receptor positivity is strongly correlated with response to endocrine therapies and manipulations. The assessment of ER and PR should be considered standard of care and be assessed in all patients with

(74% versus 66% and 92% versus 88%). The differences became minimal

Reported in the Surveillance, Epidemiology, and End Results Program, 1990–2000 (55).

288 breast cancer. A reassessment of ER and PR status upon a reoccurrence should be strongly considered due to a possibility in the change in the hormone and HER2 receptor status between the primary tumor and the metastatic sites.

In patients with ER-positive breast cancer, exposure to 5 years of adjuvant tamoxifen, a selective estrogen receptor modulator (SERM), reduced the annual risk of recurrence of breast cancer by 50% and the breast cancer death rate by 31%. This relative risk reduction was observed independent of age, tumor characteristics, or progesterone receptor status and whether these patients received chemotherapy (57). The cumulative reduction in mortality continued beyond the 5 years of tamoxifen treatment with a doubling of the relative benefits by 15 years (57).

More recent data suggests that the benefits of chemotherapy, in particular those regimens containing a taxane, may be less pronounced in hormone receptor-positive patients. A retrospective analysis, of Cancer and Leukemia Group B (CALGB) and US Breast Cancer Intergroup trial data, compared the outcomes of 6,644 node-positive breast cancer patients who received adjuvant treatment. The overall mortality rate reduction associated with chemotherapy improvements was 55% and 23% among ER-negative and ER-positive patients, respectively (58). A higher 10-year recurrence risk in premenopausal women with ER-positive compared to ER-negative patients was also seen in an adjuvant study comparing CMF (cyclophosphamide, methotrexate, 5-fluorouracil) to CEF (cyclo-phosphamide, epirubicin, 5-fluorouracil) (59), suggesting that postmeno-pausal patients with ER-positive tumors may have less benefits from chemotherapy. These data are currently being explored and suggest a need for the further development of hormonal interventions. The predictive value of hormone receptor expression was further demonstrated by the MD Anderson group reporting a 8% versus 24% likelihood to achieve a complete pathological response rate to anthracycline- containing neoadju-vant therapy for hormone receptor positive compared to hormone receptor negative patients (60).

While the benefits of systemic chemotherapy in hormone-sensitive breast cancer will be further evaluated, a collective body of data indicates that adjuvant hormonal therapy is not beneficial in patients with hormone receptor-negative tumors (57). Furthermore, the loss of either ER or PR in recurrent breast cancers were both associated with poor response to endocrine therapy (61).

3.2. The c-erbB-2 (HER2/neu) proto-oncogene overexpression

The HER2 receptor is a member of the epidermal growth factor receptor family of receptors. Overexpression of HER2 has been found in 18%–25% of all breast cancer and has been associated with a higher risk of nodal involvement and poorer survival (62–65). Overexpression of HER2 has


14. Prognostic and predictive factors 289 been further associated with hormone receptor negativity (64). More relevantly, HER2 is a predictive factor for trastuzumab response in breast cancer.

HER2 overexpression has been evaluated using either the detection of its protein by immunohistochemistry (IHC) or its mRNA amplification in relation to chromosome 17 centromere using fluorescence in situ hybridization (FISH). Due to the higher variability in the IHC assays, FISH testing is a more reliable test. Several studies have evaluated the concordance between FISH testing and IHC testing. A survival benefit has only been seen in patients whose tumor was FISH amplified or 3+ overexpressed by IHC. About 12%–25% of patients with IHC 2+ will be FISH-amplified and about 3% of patients with negative IHC (0 or 1) are believed to be FISH positive (Figure 2) (66, 67).

There are some conflicting data over influence of HER2 expression on

endocrine therapy (68). Preclinical studies suggest overexpression of HER2 promotes tamoxifen resistance in ER-positive human breast cancer cells by phosphorylation of the ER, ligand-independent activation, or regulation of ER expression (69–71). A recent meta-analysis of current data suggested that HER2 positive metastatic breast cancer may be less responsive to any type of endocrine treatment including patients with positive or unknown steroid receptors (72). Simultaneous interruption of both the ER and HER2 pathways have an enhanced inhibitory effect on cell proliferation in pre-clinical studies, and is currently being studied in the clinical setting (73).

While amplification of HER2 in breast-cancer cells is associated with a poorer prognosis regardless of the type of chemotherapy, as demons-trated in the MA.5 trial performed by the National Cancer Institute of Canada involving 710 patients with early stage breast cancer, the use of an anthracycline-containing regimen appears preferable in patients with HER2 amplification (74). Furthermore, the overexpression of HER2 may be

Figure 2. Concordance in the methods of HER2 testing (66, 67).

290 associated with anthracycline-sensitivity due to an interaction between HER2 and topoisomerase IIα (topo IIα) (75).

The overexpression of HER2 is clearly associated with response to trastuzumab, a recombinant humanized monoclonal antibody directed against HER2, either alone or when used in combination with chemo-therapy. Several studies have shown increased efficacy of trastuzumab and chemotherapy versus chemotherapy alone, not only in the metastatic, but also in the adjuvant and neoadjuvant setting (76–79).

The NCCN (National Comprehensive Cancer Network) guidelines define the most recent recommendation on testing and interpretation of the HER2 status (80). The role of HER2 overexpression as a predictive marker for endocrine therapy is not yet clearly defined, however HER2 status

HER2 targeting therapy.

3.3. and plasminogen activator inhibitor type 1 (PAI-1)

In a prospective study of 247 breast cancer patients, uPA and PAI-1 were found to be independent prognostic factors and lower levels of both antigens were linked to a low risk of relapse (93% disease-free survival at 3 years) in contrast to patients with high levels (55% disease-free survival at three years) (81). The roles of these markers were further evaluated in a pooled analysis of more than 8,000 patients with breast cancer and several multi-center prospective randomized trials in node-negative breast cancer patients. Low antigen levels of uPA and PAI-1 were found to be independently associated with a low risk of recurrence, whereas patients with elevated uPA/PAI-1 antigen levels carried an increased risk of disease recurrence and a benefit from adjuvant chemotherapy (82–84). High tumor levels of uPA and PAI-1 were also associated with resistance to tamoxifen therapy when used as first line therapy for metastatic hormone-positive breast cancer (85). A study in 898 breast cancer patients with HER2-positive tumors further suggested that uPA mRNA expression may also be an adverse prognostic indicator in HER2-positive tumors (86).

high levels may also indicate poor response to hormonal therapy and may be an indication to use adjuvant chemotherapy. Currently the enzyme immunoassays using monoclonal antibodies to human uPA as the capture reagents are available commercially in both USA and Europe. To date however, only the assessment of ER, PR, and HER2 are recommended as initial work up (http://www.nccn.org/professionals).

Invasion factors: Urokinase-type plasminogen activator (uPA)

These data suggest low uPA/PAI-1 antigen levels may be used to avoid aggressive therapy in low grade node-negative breast cancer whereas

should be determined in every patient who might be a candidate for


14. Prognostic and predictive factors 291 3.4. Gene expression profiling

predictive markers determined by immunohistochemistry has suggested a need for alternative approaches to define patients who will benefit from therapy. Furthermore, the definition of a specific receptor or pathway may be too narrow. Hence, much emphasis has been placed on defining gene patterns that correlate with outcomes. Gene array assessment may include DNA microarrays. DNA array are best performed on fresh frozen tissue, however newer techniques have now allowed the use paraffin-embedded tissues. Furthermore, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) methods may be used to confirm expression of select genes or assessing a preselect limited number of genes.

The assessment of a multigene array involving 21 predefined genes to predict recurrence and response to chemotherapy and hormonal therapy (Oncotype DX™) has recently been added as a new clinical tool. The like-lihood of recurrence is expressed in the recurrence score ranging from low (0–18), intermediate (28–31), to high (>31) risk. The risk assessment tool was developed based on the expression of a predefined gene array of 16 cancer-related genes and 5 reference genes on banked tumor samples from two original NSABP studies (B14 and B20) (87) (Figure 3). The B14 study involved women with node-negative, ER-positive tumors who were treated with either tamoxifen or placebo. The second study involved a similar patient population receiving tamoxifen and adjuvant chemotherapy. Based on the RS score, patients treated with tamoxifen had a 10-year disease recurrence rate of 7%, 14%, or 31% (87) (Figure 3). These recur-ence rates were independent of age and tumor size. These studies further suggested that patients with higher RS had a 28% absolute benefit from adjuvant chemotherapy (CMF or MF), whereas patients with low-RS tumors derived minimal benefit from chemotherapy (88). While the large confi-dence intervals for the intermediate group renders the benefits statistically not significant the numerical benefit in this risk group may have to be re-evaluated in larger, prospective studies. Based on these data, the Onco [type] DX assay may be used to distinguish which ER-positive, node-negative patients may benefit from chemotherapy or where chemotherapy could be avoided. Fifty-four percent of the patients were in the low risk group hence chemotherapy may be avoided in a large group of patients (87). However, it has to be kept in mind that the Onco[type] DX test has not involved patients receiving anthracycline-containing benefits or an

3.4.1. Pre-select gene arrays

The unpredictability in outcome in patients with very similar pro-gnostic and predictive markers and the variability in the expression of

292 aromatase inhibitor and this assay has not been validated in node-positive patients. Therefore, the benefits of adjuvant therapy of node-positive patients should not involve Onco[type] DX testing. To further validate this test, several prospective studies involving Oncotype DX as a risk assessment and therapy-response tool are ongoing (TAILORx) (http://www.clinicaltrials.gov/ct/show/ NCT00310180).

Figure 3. 21-gene array. Gene expression panel, patient distribution, and 10-year distant

A further classification of risk groups was proposed using DNA micro-

(mostly ER-negative). These subtypes were associated with distinct dif-ferences in prognosis and response to therapy, with the luminal B, basal-like and HER2-positive having significantly worse outcomes (89–91). By using previously established 70-gene prognosis profiles, van de Vijver categorized patients with primary breast carcinomas into good and poor prognosis gene-expression signatures. The study suggested 10-year survival rates for the poor and the good prognosis groups of 55% and 95%, res-pectively, and distant disease-free survival of 51% and 85%, respectively

disease-free survival (87, 88)


3.4.2. Gene pattern array

positive), luminal B, basal-like (mostly ER-negative), and HER2 positive

arrays. Based on gene expression patterns breast cancer was subclassifiedinto different groups, including luminal A, normal-like (mainly ER-

14. Prognostic and predictive factors 293 (92, 93). Wang et al. analyzed samples from lymph-node-negative patients who had not received adjuvant systemic treatment and developed a 76-gene signature consisting of 60 genes for patients positive for estrogen ER and 16 genes for ER-negative patients (94). This study identified patients developing distant metastases at 5 years with a sensitivity and specificity of 93% and 48%, respectively. These findings were validated in a subsequent study suggesting a 5- and 10-year distant metastases-free survival of 96% and 94% for the good prognosis group compared to 74% and 65% for the poor profile group (95).

Collectively these studies suggest that the assessment of prognosis and response to therapy may be significantly enhanced by the addition of gene expression profiles. While further standardization of the DNA microarrays and correlation with response in prospective clinical studies may be requir-ed to select the optimal clinical test, these methods should rapidly transition into routine medical care. 3.5. Circulating tumor cells

In recent years, several studies have evaluated the role of circulating tumors cells as a prognostic and predictive marker of response. Initial studies have shown that the presence of more than five circulating tumor cells per 7.5 ml of blood collected from patients with metastatic breast cancer were associated with a poorer progression-free and overall survival (96–99). Furthermore, a decrease in the number of circulating cells during therapy for metastatic breast cancer predicted better progression-free survival and overall survival. The commercially available CellSearch™ system may be used to quantify circulating tumor cells. The system allows the enumeration of CD45-, EpCAM+, and cytokeratins 8, 18+, and/or 19+ containing cells in peripheral blood. However, the testing kit is only approved in patients with metastatic breast cancer. To date the studies on the predictive and prognostic value of circulating tumor cells for early stage breast cancer have not been conclusive and further studies are ongoing (http://www.clinicaltrials.gov/ct/ show/NCT00353483).

3.6. Mutation in p53

Expression of mutant p53 protein studied with nuclear immunostaining was associated with high tumor proliferation rate, early disease recurrence, and early death in node-negative breast cancer (100). Tumors with p53 mutation were associated with poor response to adjuvant systemic therapy and especially to tamoxifen (101), and a significantly increased local failure rate (102). The role of p53 as a predictive marker of response was evaluated in patients treated with dose-dense sequential chemotherapy (103). Overexpression of p53 was detected in 27% of the patients and was

294 linked to negative hormonal status, worse histologic grade, and a higher risk of disease recurrence and death (103).

Although a p53 mutation may have both prognostic and predictive signi-ficance, it has not found a place as a routine marker in the clinical assess-ment due to multiple factors, including technical difficulties of testing.

3.7. Circulating Angiogenic Factors (CAF)

Reports on the predictive value of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in the serum have been contradictory to date, while it may be useful to monitor response for chemotherapy (104), it has not been shown to predict response to letrozole (105). These studies may require further prospective confirma-tory testing and standardizing of techniques for their measurement.

3.8. Topoisomerase II-alpha (topo IIα) expression

The topoisomerase II alpha gene (topo IIα) is located adjacent to the HER2 oncogene at chromosome 17q12-q21. Topo IIα is thought to be the primary molecular target of anthracyclines, as the exposure of cells to topo II inhibitors results in stabilization of covalently bound cleavable complexes and subsequent DNA double-strand breaks (106) and sensitivity to topo II inhibitors including anthracyclines was associated with topo IIα expression (75, 107–109). Furthermore, amplification of the topo IIα gene has been found in breast cancers with HER2 amplification.

In small studies, coamplification of erbB-2 and topo IIα was found to be significantly associated with favorable local response to anthracycline based therapy in locally advanced breast cancer (110, 111). In a larger retrospective study from the Danish Breast Cancer Cooperative Group, topo IIα amplification was found to have increased recurrence-free and overall survival, respectively, if treated with epirubicin-based regimes

Group trial (9401) in women with high-risk breast cancer found topo IIα coamplification in 37% of HER2–amplified tumors (113). Topo IIα ampli-fication was associated with better relapse-free survival in patients treated with tailored FEC (fluorouracil, epirubicin, and cyclophosphamide) (113).

A more recent study has further suggested an important role for topo IIα as a predictive marker. As the anthracyclines and trastuzumab have been associated with cardiac toxicity, BCIRG 06 addressed the question of whether an anthracycline could be omitted in patients with HER2 over-expressing breast cancer. A combination of four cycles of doxorubicin and cyclophosphamide followed by four cycles of docetaxel and trastuzumab were compared to six cycles of docetaxel, carboplatin, and trastuzumab

compared to non-epirubicin-based regimens (112). A Scandinavian Breast


14. Prognostic and predictive factors 295 in patients with HER2 positive early-stage breast cancer (114). While the two trastuzumab-containing arms did not differ statistically, there was a numerical benefit towards the anthracycline-containing arm. Preliminary analyses suggested that in particular those patients with topo IIα and HER2 co-amplification appeared to benefit from the anthracycline-containing regimen (114, 115). These findings are currently being evalua-ted. A standardized test of topo IIα expression is not yet currently available.

Breast cancer (BRCA) gene mutation

of all patients with a newly diagnosed breast cancer. They are associated with a 50%–85% lifetime risk of developing breast cancer (116, 117).

Breast cancer patients with germ-line mutations in BRCA1 or BRCA2 have a high risk of developing ipsilateral and contralateral second primary tumors (118).

Prophylactic mastectomy was associated with a reduction in the inci-dence of breast cancer of at least 90% in a retrospective study of women with a family history of breast cancer (119). In a prospective cohort of women with germ-line mutations in BRCA1 or BRCA2 and no previous cancer diagnosis, bilateral prophylactic salpingo-oophorectomy improved overall survival and cancer-specific survival (120). While the emotional and social implication of carrying a gene mutation have to be considered, the potential to prevent further cancers in the patient and possibly also in the patient’s relatives by prophylactic measures should warrant a discussion about genetic testing in patients with suspected BRCA mutations.

4. SUMMARY

Most clinical practice guidelines warrant adjuvant systemic chemothe-rapy for the majority of node-positive patients. For patients with tumors of 1 cm or larger, 5–10 years of hormonal therapy should be considered in patients with hormone-receptors positive tumors and trastuzumab for one year in those with HER2 overexpression. Less aggressive therapy may be often considered in older patients with hormone-sensitive tumors.

The patients under discussion are those with node-negative tumor size 0.6–1.0 cm, moderate or poorly differentiated or unfavorable features such as angiolymphatic invasion, high nuclear grade, and high histological grade. While determination of tumor grade, type, ER, PR, and HER2 status at the time of initial work up are recommended, there are no specific recommendation for gene profiling using approved and commercially available assays such as Onco[type] DXTM or other gene array assays as

3.9.

BRCA1 or BRCA2 gene germ-line mutations can be identified in 5–10%

296 of yet. However, the possibility of avoiding chemotherapy in many node-negative, ER-positive breast cancer with a low risk of recurrence has led to a more frequent use of Onco[type] DXTM testing in these women. Other tumor risk assessment by gene expression profiling is underway. The measurement of topo IIα overexpression to assist in the selection of the optimal chemotherapy regimen has not yet become standard practice. The assessment of topo IIα may be particularly relevant in patients with HER2 overexpression to discern whether an anthracycline should be used, despite increased cardiac toxicity. There are no standard guidelines for routine BRCA1 or BRCA2 gene testing in younger women with breast cancer or those with a significant family history. However as the proper management of mutation carriers has been associated not only with prevention of future cancers but also with improved survival, genetic testing should be considered in carefully selected individuals. The assessment of circulating tumor cells, serum HER2 antigen, anti-angiogenic, or proliferation markers in the tumor or the serum, and the presence of micrometastases in the bone marrow will require further standardization and management guidelines. As the majority of patients will be offered adjuvant therapy, great emphasis should be placed on standardizing current and exploring novel predictive factors.

REFERENCES

1. Chia SK, Speers CH, Bryce CJ, Hayes MM, Olivotto IA. Ten-year outcomes in a population-based cohort of node-negative, lymphatic, and vascular invasion-negative early breast cancers without adjuvant systemic therapies. J Clin Oncol. 2004; 22(9): 1630–1637

2. Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, Thun MJ. Cancer statistics. 2006, CA Cancer J Clin. 2006; 56(2): 106–130

3. Wahl RL, Siegel BA, Coleman RE, Gatsonis CG. Prospective multicenter study of axillary nodal staging by positron emission tomography in breast cancer: a report of the staging breast cancer with PET Study Group. J Clin Oncol. 2004; 22(2): 277–285

4. Fisher B, Bauer M, Wickerham DL, Redmond CK, Fisher ER, Cruz AB, Foster R, Gardner B, Lerner H, Margolese R, et al., Relation of number of positive axillary nodes to the prognosis of patients with primary breast cancer. An NSABP update. Cancer. 1983; 52(9): 1551–1557

5. Truong PT, Berthelet E, Lee J, Kader HA, Olivotto IA. The prognostic significance of the percentage of positive/dissected axillary lymph nodes in breast cancer recurrence and survival in patients with one to three positive axillary lymph nodes. Cancer. 2005; 103(10): 2006–2014

6. Nieweg OE, Jansen L, Valdes Olmos RA, Rutgers EJ, Peterse JL, K. Hoefnagel A, Kroon BB. Lymphatic mapping and sentinel lymph node biopsy in breast cancer. Eur J Nucl Med. 1999; 26(4 Suppl): S11–16

7. Cox CE, Pendas S, Cox JM, Joseph E, Shons AR, Yeatman T, Ku NN, Lyman GH, Berman C, Haddad F, Reintgen DS. Guidelines for sentinel node biopsy



and lymphatic mapping of patients with breast cancer. Ann Surg. 1998; 227(5): 645–651; discussion 651–643

8. de Widt-Levert L, Tjan-Heijnen V, Bult P, Ruers T, Wobbes T. Stage migration in breast cancer: surgical decisions concerning isolated tumour cells and micro-

9. Cox C, White L, Allred N, Meyers M, Dickson D, Dupont E, Cantor A, Ly Q, Dessureault S, King J, Nicosia S, Vrcel V, Di.z N, Survival outcomes in node-negative breast cancer patients evaluated with complete axillary node dissection versus sentinel lymph node biopsy. Ann Surg Oncol. 2006; 13(5): 708–711

10. Varghese P, Mostafa A, Abdel-Rahman AT, Akberali S, Gattuso J, Canizales A, Wells CA, Carpenter R. Methylene blue dye versus combined dye-radioactive tracer technique for sentinel lymph node localisation in early breast cancer. Eur J Surg Oncol. (2006), in press

11. PB, McMasters KM. Sentinel lymph node biopsy for breast cancer: impact of the number of sentinel nodes removed on the false-negative rate. J Am Coll Surg. 2001; 192(6), 684–689; discussion 689–691

12. Veronesi U, Paganelli G, Viale G, Luini A, Zurrida S, Galimberti V, Intra M, Veronesi P, Robertson C, Maisonneuve P, Renne G, De Cicco C, De Lucia F, Gennari R. A randomized comparison of sentinel-node biopsy with routine axillary dissection in breast cancer. N Engl J Med. 2003; 349(6), 546–553

13. Vanderveen KA, Schneider PD, Khatri V P, Goodnight JE, Bold R J. Upstaging and improved survival of early breast cancer patients after implementation of sentinel node biopsy for axillary staging. Ann Surg Oncol. 2006; 13(11), 1450–1456

14. Amersi F and Hansen NM. The benefits and limitations of sentinel lymph node biopsy. Curr Treat Options Oncol. 2006; 7(2), 141–151

15. Krag DN, Julian TB, Harlow SP, Weaver DL, Ashikaga T, Bryant J, Single RM, Wolmark N. NSABP-32: Phase III, randomized trial comparing axillary resection with sentinal lymph node dissection: a description of the trial. Ann Surg Oncol. 2004; 11(3 Suppl), 208S–210S

16. Dabbs DJ, Fung M, Landsittel D, McManus K, Johnson R. Sentinel lymph node micrometastasis as a predictor of axillary tumor burden. Breast J. 2004; 10(2), 101–105.

17. Rosen PP, Saigo PE, Braun DW, Weathers E, Fracchia AA, Kinne DW. Axillary micro- and macrometastases in breast cancer: prognostic significance of tumor size. Ann Surg. 1981; 194(5): 585–591

18. Shen J, Hunt KK, Mirza NQ, Krishnamurthy S, Singletary SE, Kuerer HM, Meric-Bernstam F, Feig B, Ross MI, Ames FC, Babiera GV. Intramammary lymph node metastases are an independent predictor of poor outcome in patients with breast carcinoma, Cancer. 2004; 101(6): 1330–1337

19. Vinh-Hung V, Verschraegen C, Promish DI, Cserni G, Van de Steene J, Tai P, Vlastos G, Voordeckers M, Storme G, Royce M. Ratios of involved nodes in early breast cancer. Breast Cancer Res. 2004; 6(6): R680–688

20. Voordeckers M, Vinh-Hung V, Van de Steene J, Lamote J, Storme G. The lymph node ratio as prognostic factor in node-positive breast cancer. Radiother

21. Woodward WA, Vinh-Hung V, Ueno NT, Cheng YC, Royce M, Tai P, Vlastos G, Wallace AM, Hortobagyi GN, Nieto Y. Prognostic value of nodal ratios in node-positive breast cancer. J Clin Oncol. 2006; 24(18), 2910–2916

22. Rosen PP, Groshen S, Kinne DW, Norton L. Factors influencing prognosis in node-negative breast carcinoma: analysis of 767 T1N0M0/T2N0M0 patients with long-term follow-up. J Clin Oncol. 1993; 11(11): 2090–2100

metastases in the sentinel lymph node. Eur J Surg Oncol. 2003; 29(3): 216–220

Wong SL, Edwards MJ, Chao C, Tuttle TM, Noyes RD, Carlson DJ, Cerrito

Oncol. 2004; 70(3): 225–230

298 Maunglay, Marchion and Münster 23. Rosen PP, Groshen S, Kinne DW. Prognosis in T2N0M0 stage I breast

carcinoma: a 20-year follow-up study. J Clin Oncol. 1991; 9(9): 1650–1661 24. Tubiana M and Koscielny S. The natural history of breast cancer: implications

for a screening strategy. Int J Radiat Oncol Biol Phys. 1990; 19(5): 1117–1120 25. Diab SG, Elledge RM, Clark GM. RESPONSE: Re: Tumor Characteristics and

Clinical Outcome of Elderly Women With Breast Cancer. J Natl Cancer Inst. 2001; 93(1): 65–66

26. Le Doussal V, Tubiana-Hulin M, Friedman S, Hacene K, Spyratos F, Brunet M. Prognostic value of histologic grade nuclear components of Scarff-Bloom-Richardson (SBR). An improved score modification based on a multivariate analysis of 1262 invasive ductal breast carcinomas. Cancer. 1989; 64(9): 1914–1921

27. Fisher ER, Redmond C, Fisher B, Bass G. Pathologic findings from the National Surgical Adjuvant Breast and Bowel Projects (NSABP). Prognostic discriminants for 8-year survival for node-negative invasive breast cancer patients. Cancer. 1990; 65(9 Suppl): 2121–2128

28. Turner RR, Chu KU, Qi K, Botnick LE, Hansen NM, Glass EC, Giuliano AE. Pathologic features associated with nonsentinel lymph node metastases in patients with metastatic breast carcinoma in a sentinel lymph node. Cancer. 2000; 89(3): 574–581

29. factors in stage I (T1N0M0) and stage II (T1N1M0) breast carcinoma: a study of 644 patients with median follow-up of 18 years. J Clin Oncol. 1989; 7(9): 1239–1251

30.

lymph vessel invasion in breast cancer: a prospective immunohistochemical study. Br J Cancer. 2006; 94(11): 1643–1649

31. Nixon AJ, Neuberg D, Hayes DF, Gelman R, Connolly JL, Schnitt S, Abner A, Recht A, Vicini F, Harris JR. Relationship of patient age to pathologic features of the tumor and prognosis for patients with stage I or II breast cancer. J Clin Oncol. 1994; 12(5): 888–894

32. Albain KS, Allred DC, Clark GM. Breast cancer outcome and predictors of

33. Colleoni M, Rotmensz N, Peruzzotti G, Maisonneuve P, Orlando L, Ghisini R, Viale G, Pruneri G, Veronesi P, Luini A, Intra M, Cardillo A, Torrisi R, Rocca A, Goldhirsch A. Role of endocrine responsiveness and adjuvant therapy in very young women (below 35 years) with operable breast cancer and node negative disease. Ann Oncol. 2006; 17(10): 1497–1503

34. Trends in breast cancer by race and ethnicity: update 2006. CA Cancer J Clin. 2006; 56(3): 168–183

35. Baldetorp B, Stal O, Ahrens O, Cornelisse C, Corver W, Falkmer U, Ferno M. Different calculation methods for flow cytometric S-phase fraction: prognostic implications in breast cancer? The Swedish Society of Cancer Study Group, Cytometry. 1998; 33(4): 385–393

36. Chassevent A, Jourdan ML, Romain S, Descotes F, Colonna M, Martin PM, Bolla M, Spyratos F. S-phase fraction and DNA ploidy in 633 T1T2 breast cancers: a standardized flow cytometric study. Clin Cancer Res. 2001; 7(4): 909–917

37. Asselain B. S-phase fraction as an independent prognostic factor of long-term overall survival in patients with early-stage or locally advanced invasive breast carcinoma. Cancer. 2005; 105(6): 476–482

Rosen PP, Groshen S, Saigo PE, Kinne DW, Hellman S. Pathological prognostic

Van den Eynden GG, Van der Auwera I, Van Laere SJ, Colpaert CG, van Dam P, Dirix LY, Vermeulen PB, Van Marck EA. Distinguishing blood and

outcome: are there age differentials? J Natl Cancer Inst Monogr. 1994; 16, 35–42

Smigal C, Jemal A. Ward E, Cokkinides V, Smith R, Howe HL, Thun M.

Vielh P, Carton M, Padoy E, de Rycke Y, Klijanienko J, El-Naggar AK,

14. Prognostic and predictive factors 299 38. Silvestrini R, Daidone MG, Valagussa P, Di Fronzo G, Mezzanotte G, Mariani L,

Bonadonna G. 3H-thymidine-labeling index as a prognostic indicator in node-

39. Bilir A, Ozmen V, Kecer M, Eralp Y, Cabioglu N, Ahishali B, Camlica H, Aydiner A. Thymidine labeling index: prognostic role in breast cancer. Am J Clin Oncol. 2004; 27(4): 400–406

40. Baak JP, van Diest PJ, Voorhorst FJ, van der Wall E, Beex LV, Vermorken JB, Janssen EA. Prospective multicenter validation of the independent prognostic value of the mitotic activity index in lymph node-negative breast cancer patients younger than 55 years. J Clin Oncol. 2005; 23(25): 5993–6001

41. Urruticoechea A, Smith IE, Dowsett M. Proliferation marker Ki-67 in early breast cancer. J Clin Oncol. 2005; 23(28): 7212–7220

42. carcinomas. An immunohistochemical study with correlation to histopathological features and prognostic factors. Virchows Arch. 1994; 424(1): 39–46

43. Horiguchi J, Iino Y, Takei H, Maemura M, Takeyoshi I, Yokoe T, Ohwada S, Oyama T, Nakajima T, Morishita Y. Long-term prognostic value of PCNA labeling index in primary operable breast cancer. Oncol Rep. 1998; 5(3): 641–644

44.

2810 patients. Br J Cancer. 1999; 79(2): 300–307 45. Foekens JA, van Putten WL, Portengen H, de Koning HY, Thirion B,

Alexieva-Figusch J, Klijn JG. Prognostic value of PS2 and cathepsin D in 710 human primary breast tumors: multivariate analysis. J Clin Oncol. 1993; 11(5): 899–908

46. Rodriguez J, Vazquez J., Corte MD, Lamelas M, Bongera M, Corte MG, Alvarez A, Allende M, Gonzalez L, Sanchez M, Vijande M, Garcia Muniz J, Vizoso F. Clinical significance of cathepsin D concentration in tumor cytosol of primary breast cancer. Int J Biol Markers. 2005; 20(2): 103–111

47. Lorincz T, Toth J, Szendroi M, Timar J. Microvascular density of breast cancer in bone metastasis: influence of therapy. Anticancer Res. 2005; 25(4): 3075–3081

48. Weidner N, Folkman J, Pozza F, Bevilacqua P, Allred EN, Moore DH, Meli S, Gasparini G. Tumor angiogenesis: a new significant and independent prognostic indicator in early-stage breast carcinoma. J Natl Cancer Inst. 1992; 84(24): 1875–1887

49. Manders P, Beex LV, Tjan-Heijnen VC, Geurts-Moespot J, Van Tienoven TH, Foekens JA, Sweep CG. The prognostic value of vascular endothelial growth factor in 574 node-negative breast cancer patients who did not receive adjuvant systemic therapy. Br J Cancer. 2002; 87(7): 772–778

50. Nathanson SD, Zarbo RJ, Wachna DL, Spence CA, Andrzejewski TA, Abrams J. Microvessels that predict axillary lymph node metastases in patients with breast cancer, Arch Surg. 2000; 135(5): 586–593; discussion 593–584

51. Guidi AJ, Berry DA, Broadwater G, Perloff M, Norton L, Barcos MP, Hayes DF. Association of angiogenesis in lymph node metastases with outcome of breast cancer. J Natl Cancer Inst. 2000; 92(6): 486–492

52. Ludovini V, Sidoni A, Pistola L, Bellezza G, De Angelis V, Gori S, Mosconi AM, Bisagni G, Cherubini R, Bian AR, Rodino C, Sabbatini R, Mazzocchi B, Bucciarelli E, Tonato M, Colozza M. Evaluation of the prognostic role of vascular endothelial growth factor and microvessel density in stages I and II breast cancer patients. Breast Cancer Res Treat. 2003; 81(2): 159–168

53. Diel IJ, Kaufmann M, Costa SD, Holle R, von Minckwitz G, Solomayer EF, Kaul S, Bastert G. Micrometastatic breast cancer cells in bone marrow at primary surgery: prognostic value in comparison with nodal status. J Natl Cancer Inst. 1996; 88(22): 1652–1658

positive breast cancer. J Clin Oncol. 1990; 8(8): 1321–1326

Haerslev T and Jacobsen GK. Proliferating cell nuclear antigen in breast

Klijn JG. Cathepsin-D in primary breast cancer: prognostic evaluation involving Foekens JA, Look MP, Bolt-de Vries J, Meijer-van Gelder ME, van Putten WL,

300 Maunglay, Marchion and Münster 54. Braun S, Vogl FD, Naume B, Janni W, Osborne MP, Coombes RC, Schlimok G,

Diel IJ, Gerber B, Gebauer G, Pierga JY, Marth C, Oruzio D, Wiedswang G, Solomayer EF, Kundt G, Strobl B, Fehm T, Wong GY, Bliss J, Vincent-Salomon A, Pantel K. A pooled analysis of bone marrow micrometastasis in breast cancer. N Engl J Med. 2005; 353(8): 793–802

55. Grann VR, Troxel AB, Zojwalla NJ, Jacobson JS, Hershman D, Neugut AI. Hormone receptor status and survival in a population-based cohort of patients

56. Fisher B, Redmond C, Fisher ER, Caplan R. Relative worth of estrogen or progesterone receptor and pathologic characteristics of differentiation as indicators of prognosis in node negative breast cancer patients: findings from National Surgical Adjuvant Breast and Bowel Project Protocol B-06. J Clin Oncol. 1988; 6(7): 1076–1087

57. Early Breast Cancer Trialists’ Collaborative Group (EBCTCG). Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Lancet. 2005; 365(9472): 1687–1717

58. Berry DA, Cirrincione C, Henderson IC, Citron ML, Budman DR, Goldstein LJ, Martino S, Perez EA, Muss HB, Norton L, Hudis C, Winer EP. Estrogen-receptor status and outcomes of modern chemotherapy for patients with node-positive breast cancer. JAMA. 2006; 295(14): 1658–1667

59. Levine MN, Pritchard KI, Bramwell VH, Shepherd LE, Tu D, Paul N, Randomized trial comparing cyclophosphamide, epirubicin, and fluorouracil with cyclophosphamide, methotrexate, and fluorouracil in premenopausal women with node-positive breast cancer: update of National Cancer Institute of Canada Clinical Trials Group Trial MA5. J Clin Oncol. 2005; 23(22): 5166–5170

60. Guarneri V, Broglio K, Kau SW, Cristofanilli M, Buzdar AU, Valero V, Buchholz T, Meric F, Middleton L, Hortobagyi GN, Gonzalez-Angulo AM. Prognostic value of pathologic complete response after primary chemotherapy in relation to hormone receptor status and other factors. J Clin Oncol. 2006; 24(7): 1037–1044

61. Kuukasjarvi T, Kononen J, Helin H, Holli K, Isola J. Loss of estrogen receptor in recurrent breast cancer is associated with poor response to endocrine therapy. J Clin Oncol. 1996; 14(9): 2584–2589

62. Slamon DJ and Clark GM. Amplification of c-erbB-2 and aggressive human breast tumors? Science. 1988; 240(4860): 1795–1798

63. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene, Science. 1987; 235(4785): 177–182

64. Witton CJ, Reeves JR, Going JJ, Cooke TG, Bartlett JM. Expression of the HER1-4 family of receptor tyrosine kinases in breast cancer, J Pathol. 2003; 200(3): 290–297

65. Andrulis IL, Bull SB, Blackstein ME, Sutherland D, Mak C, Sidlofsky S, Pritzker KP, Hartwick RW, Hanna W, Lickley L, Wilkinson R, Qizilbash A, Ambus U, Lipa M, Weizel H, Katz A, Baida M, Mariz S, Stoik G, Dacamara P, Strongitharm D, Geddie W, McCready D. neu/erbB-2 amplification identifies a poor-prognosis group of women with node-negative breast cancer. Toronto Breast Cancer Study Group, J Clin Oncol. 1998; 16(4):1340–1349

66. Yaziji H, Goldstein LC, Barry TS, Werling R, Hwang H, Ellis GK, Gralow JR, Livingston RB, Gown A M. HER-2 testing in breast cancer using parallel tissue-based methods. JAMA. 2004; 291(16): 1972–1977

67. Mass RD, Press MF, Anderson S, Cobleigh MA, Vogel CL, Dybdal N,

with breast carcinoma. Cancer. 2005; 103(11): 2241–2251

Leiberman G, Slamon DJ. Evaluation of clinical outcomes according to HER2


detection by fluorescence in situ hybridization in women with metastatic breast cancer treated with trastuzumab. Clin Breast Cancer. 2005; 6(3): 240–246

68. Love RR, Duc NB, Havighurst TC, Mohsin SK, Zhang Q, DeMets DL, Allred DC. Her-2/neu overexpression and response to oophorectomy plus tamoxifen adjuvant therapy in estrogen receptor-positive premenopausal women with operable breast cancer. J Clin Oncol. 2003; 21(3): 453–457

69. Benz CC, Scott GK, Sarup JC, Johnson RM, Tripathy D, Coronado E, Shepard HM, Osborne CK. Estrogen-dependent, tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected with HER2/neu. Breast Cancer Res Treat. 1992; 24(2): 85–95

70. Liu Y, el-Ashry D, Chen D, Ding IY, Kern FG. MCF-7 breast cancer cells overexpressing transfected c-erbB-2 have an in vitro growth advantage in estrogen-depleted conditions and reduced estrogen-dependence and tamoxifen-sensitivity in vivo. Breast Cancer Res Treat. 1995; 34(2): 97–117

71. Pietras RJ, Arboleda J, Reese DM, Wongvipat N, Pegram MD, Ramos L, Gorman C M, Parker MG, Sliwkowski MX, Slamon D J. HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone-independent growth in human breast cancer cells. Oncogene. 1995; 10(12): 2435–2446

72. De Laurentiis M, Arpino G, Massarelli E, Ruggiero A, Carlomagno C, Ciardiello F, Tortora G, D’Agostino D, Caputo F, Cancello G, Montagna E,

the interaction between HER-2 expression and response to endocrine treatment in advanced breast cancer. Clin Cancer Res. 2005; 11(13): 4741–4748

73. Witters LM, Kumar R, Chinchilli VM, Lipton A. Enhanced anti-proliferative activity of the combination of tamoxifen plus HER-2-neu antibody. Breast Cancer Res Treat. 1997; 42(1): 1–5

74. Pritchard KI, Shepherd LE, O’Malley FP, Andrulis IL, Tu D, Bramwell VH, Levine MN. HER2 and responsiveness of breast cancer to adjuvant chemotherapy. N Engl J Med. 2006; 354(20): 2103–2111

75. Jarvinen TA, Tanner M, Rantanen V, Barlund M, Borg A, Grenman S, Isola J, Amplification and deletion of topoisomerase IIalpha associate with ErbB-2 amplification and affect sensitivity to topoisomerase II inhibitor doxorubicin in breast cancer. Am J Pathol. 2000; 156(3): 839–847

76. Buzdar AU, Ibrahim NK, Francis D, Booser DJ, Thomas ES, Theriault RL, Pusztai L, Green MC, Arun BK, Giordano SH, Cristofanilli M, Frye DK, Smith TL, Hunt KK, Singletary SE, Sahin AA, Ewer MS, Buchholz TA, Berry D, Hortobagyi GN. Significantly higher pathologic complete remission rate after neoadjuvant therapy with trastuzumab, paclitaxel, and epirubicin chemotherapy: results of a randomized trial in human epidermal growth factor receptor 2-positive operable breast cancer. J Clin Oncol. 2005; 23(16): 3676–3685

77. Romond EH, Perez EA, Bryant J, Suman VJ, Geyer CE, Jr., Davidson NE, Tan-Chiu E, Martino S, Paik S, Kaufman PA, Swain SM, Pisansky L.

Brown AM, Dakhil SR, Mamounas EP, Lingle WL, Klein PM, Ingle JN, Wolmark N. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med. 2005; 353(16): 1673–1684

78. Piccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, Smith I, Gianni L, Baselga J, Bell R, Jackisch C, Cameron D, Dowsett M, Barrios CH, Steger G, Huang CS, Andersson M, Inbar M, Lichinitser M, Lang I, Nitz U, Iwata H, Thomssen C, Lohrisch C, Suter TM, Ruschoff J, Suto T, Greatorex V, Ward C, Straehle C, McFadden E, Dolci MS, Gelber RD.

Fehrenbacher TM, Kutteh LA, Vogel VG, Visscher DW, Yothers G, Jenkins RB,

N Engl J Med. 2005; 353(16): 1659–1672 Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer.

Malorni L, Zinno L, Lauria R, Bianco AR, De Placido S. A meta-analysis on

302 Maunglay, Marchion and Münster 79. Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A,

Fleming T, Eiermann W, Wolter J, Pegram M, Baselga J, Norton L. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med. 2001; 344(11): 783–792

80. Carlson RW, Moench SJ, Hammond ME, Perez EA, Burstein HJ, Allred DC, Vogel CL, Goldstein LJ, Somlo G, Gradishar WJ, Hudis CA, Jahanzeb M, Stark A, Wolff AC, Press MF, Winer EP, Paik S, Ljung BM. HER2 testing in breast cancer: NCCN Task Force report and recommendations. J Natl Compr

81. Janicke F, Schmitt M, Pache L, Ulm K, Harbeck N, Hofler H, Graeff H. Urokinase (uPA) and its inhibitor PAI-1 are strong and independent prognostic factors in node-negative breast cancer. Breast Cancer Res Treat. 1993; 24(3): 195–208

82. Harbeck N, Kates RE, Schmitt M, Gauger K, Kiechle M, Janicke F, Thomassen C, Look MP, Foekens JA. Urokinase-type plasminogen activator and its inhibitor type 1 predict disease outcome and therapy response in primary breast cancer. Clin Breast Cancer. 2004; 5(5): 348–352

83. Janicke F, Prechtl A, Thomssen C, Harbeck N, Meisner C, Untch M, Sweep CG, Selbmann HK, Graeff H, Schmitt M. Randomized adjuvant chemotherapy trial in high-risk, lymph node-negative breast cancer patients identified by urokinase-type plasminogen activator and plasminogen activator inhibitor type 1. J Natl Cancer Inst. 2001; 93(12): 913–920

84. Zemzoum I, Kates RE, Ross JS, Dettmar P, Dutta M, Henrichs C, Yurdseven S, Hofler H, Kiechle M, Schmitt M, Harbeck N. Invasion factors uPA/PAI-1 and HER2 status provide independent and complementary information on patient outcome in node-negative breast cancer. J Clin Oncol. 2003; 21(6): 1022–1028

85. Meijer-van Gelder ME, Look MP, Peters HA, Schmitt M, Brunner N, Harbeck N, Klijn JG, Foekens JA. Urokinase-type plasminogen activator system in breast cancer: association with tamoxifen therapy in recurrent disease. Cancer Res. 2003; 64(13): 4563–4568

86. Urban P, Vuaroqueaux V, Labuhn M, Delorenzi M, Wirapati P, Wight E, Senn HJ,

urokinase-type plasminogen activator mRNA determines adverse prognosis in ErbB2-positive primary breast cancer. J Clin Oncol. 2006; 24(26): 4245–4253

87. Paik S, Shak S, Tang G, Kim C, Baker J, Cronin M, Baehner FL, Walker MG, Watson D, Park T, Hiller W, Fisher ER, Wickerham DL, Bryant J, Wolmark NA multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med. 2004; 351(27): 2817–2826

88. Paik S, Tang G, Shak S, Kim C, Baker J, Kim W, Cronin M, Baehner FL, Watson D, Bryant J, Costantino JP, Geyer CE, Jr, Wickerham DL, Wolmark N. Gene expression and benefit of chemotherapy in women with node-negative, estrogen receptor-positive breast cancer. J Clin Oncol. 2006; 24(23): 3726–3734

89. Fan C, Oh DS, Wessels L, Weigelt B, Nuyten DS, Nobel AB, van't Veer LJ, Perou CM. Concordance among gene-expression-based predictors for breast cancer. N Engl J Med. 2006; 355(6): 560–569

90. Brenton JD, Carey LA, Ahmed AA, Caldas C. Molecular classification and molecular forecasting of breast cancer: ready for clinical application? J Clin Oncol. 2005; 23(29): 7350–7360

91. Sorlie T. Molecular portraits of breast cancer: tumour subtypes as distinct disease entities. Eur J Cancer. 2004; 40(18): 2667–2675

92. van de Vijver MJ, He YD, van’t Veer LJ, Dai H, Hart AA, Voskuil DW, Schreiber GJ, Peterse JL, Roberts C, Marton MJ, Parrish M, Atsma D, Witteveen A, Glas A, Delahaye L, van der Velde T, Bartelink H, Rodenhuis S,

Canc Netw. 2006; 4 Suppl 3(S1-22; quiz S23–24)

Benz C, Eppenberger U, Eppenberger-Castori S. Increased expression of


Rutgers ET, Friend SH, Bernards RA gene-expression signature as a predictor of survival in breast cancer. N Engl J Med. 2002; 347(25): 1999–2009

93. van’t Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, Mao M, Peterse HL, van der Kooy K, Marton MJ, Witteveen AT, Schreiber GJ, Kerkhoven RM, Roberts C, Linsley PS, Bernards R, Friend SH. Gene expression profiling predicts clinical outcome of breast cancer. Nature. 2002; 415(6871): 530–536

94. Wang Y, Klijn JG, Zhang Y, Sieuwerts AM, Look MP, Yang F, Talantov D, Timmermans M, Meijer-van Gelder ME, Yu J, Jatkoe T, Berns EM, Atkins D, Foekens JA. Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet. 2005; 365(9460): 671–679

95. Foekens JA, Atkins D, Zhang Y, Sweep FC, Harbeck N, Paradiso A, Cufer T, Sieuwerts AM, Talantov D, Span PN, Tjan-Heijnen VC, Zito AF, Specht K, Hoefler H, Golouh R, Schittulli F, Schmitt M, Beex LV, Klijn JG, Wang Y. Multicenter validation of a gene expression-based prognostic signature in lymph node-negative primary breast cancer, J Clin Oncol. 2006; 24(11): 1665–1671

96. Budd GT, Cristofanilli M, Ellis MJ, Stopeck A, Borden E, Miller MC, Matera J, Repollet M, Doyle GV, Terstappen LW, Hayes DF. Circulating Tumor Cells versus Imaging--Predicting Overall Survival in Metastatic Breast Cancer. Clin Cancer Res. 2006; 12(21): 6403–6409

97. Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller MC, Reuben JM, Doyle GV, Allard WJ, Terstappen LW, Hayes DF. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 2004; 351(8): 781–791

98. Cristofanilli M, Hayes DF, Budd GT, Ellis MJ, Stopeck A, Reuben JM, Doyle GV, Matera J, Allard WJ, Miller MC, Fritsche HA, Hortobagyi GN, Terstappen LW. Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer. J Clin Oncol. 2005; 23(7): 1420–1430

99. Hayes DF, Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Miller MC, Matera J, Allard WJ, Doyle GV, Terstappen LW. Circulating tumor cells at each follow-up time point during therapy of metastatic breast cancer patients predict progression-free and overall survival. Clin Cancer Res. 2006; 12(14 Pt 1): 4218–4224

100. Allred DC, Clark GM, Elledge R, Fuqua SA, Brown RW, Chamness GC, Osborne C K, McGuire W L. Association of p53 protein expression with tumor cell proliferation rate and clinical outcome in node-negative breast cancer. J Natl Cancer Inst. 1993; 85(3): 200–206

101. Bergh J, Norberg T, Sjogren S, Lindgren A, Holmberg L. Complete sequencing of the p53 gene provides prognostic information in breast cancer patients, particularly in relation to adjuvant systemic therapy and radiotherapy. Nat Med. 1995; 1(10): 1029–1034

102. Zellars RC, Hilsenbeck SG, Clark GM, Allred DC, Herman TS, Chamness GC, Elledge RM. Prognostic value of p53 for local failure in mastectomy-treated breast cancer patients. J Clin Oncol. 2000; 18(9): 1906–1913

103. Malamou-Mitsi V, Gogas H, Dafni U, Bourli A, Fillipidis T, Sotiropoulou M, Vlachodimitropoulos D, Papadopoulos S, Tzaida O, Kafiri G, Kyriakou V, Markaki S, Papaspyrou I, Karagianni E, Pavlakis K, Toliou T, Scopa C, Papakostas P, Bafaloukos D, Christodoulou C, Fountzilas G. Evaluation of the prognostic and predictive value of p53 and Bcl-2 in breast cancer patients participating in a randomized study with dose-dense sequential adjuvant chemotherapy. Ann Oncol. 2006; 17(10): 1504–1511

104. Granato AM, Frassineti GL, Giovannini N, Ballardini M, Nanni O, Maltoni R, Amadori D, Volpi A. Do serum angiogenic growth factors provide additional information to that of conventional markers in monitoring the course of metastatic breast cancer? Tumour Biol. 2006; 27(6): 302–308

304 Maunglay, Marchion and Münster 105. Alba E, Llombart A, Ribelles N, Ramos M, Fernandez R, Mayordomo JI,

Tusquets I, Gil M, Barnadas A, Carabante F, Ruiz M, Vera R, Palomero I, Soriano V, Gonzalez J, Colomer R. Serum endostatin and bFGF as predictive factors in advanced breast cancer patients treated with letrozole. Clin Transl Oncol. 2006; 8(3): 193–199

106. Cummings J, Smyth JF. DNA topoisomerase I and II as targets for rational design of new anticancer drugs. Ann Oncol. 1993; 4(7): 533–543

107. Jarvinen TA, Tanner M, Barlund M, Borg A, Isola J. Characterization of topoisomerase II alpha gene amplification and deletion in breast cancer. Genes Chromosomes Cancer. 1999; 26(2): 142–150

108. Di Leo A, Larsimont D, Gancberg D, Jarvinen T, Beauduin M, Vindevoghel A, Michel J, Focan CH, Ries F, Gobert PH, Closon-Dejardin MT, Dolci S, Rouas G, Paesmans M, Lobelle JP, Isola J, Piccart MJ. HER-2 and topo-isomerase IIalpha as predictive markers in a population of node-positive breast cancer patients randomly treated with adjuvant CMF or epirubicin plus cyclophosphamide. Ann Oncol. 2001; 12(8): 1081–1089

109. Di Leo A, Gancberg D, Larsimont D, Tanner M, Jarvinen T, Rouas G, Dolci S, Leroy JY, Paesmans M, Isola J, Piccart MJ. HER-2 amplification and topoisomerase IIalpha gene aberrations as predictive markers in node-positive breast cancer patients randomly treated either with an anthracycline-based therapy or with cyclophosphamide, methotrexate, and 5-fluorouracil. Clin Cancer Res. 2002; 8(5): 1107–1116

110. Coon JS, Marcus E, Gupta-Burt S, Seelig S, Jacobson K, Chen S, Renta V, Fronda G, Preisler HD. Amplification and overexpression of topoisomerase IIalpha predict response to anthracycline-based therapy in locally advanced breast cancer. Clin Cancer Res. 2002; 8(4): 1061–1067

111. Park K, Kim J, Lim S, Han S. Topoisomerase II-alpha (topoII) and HER2 amplification in breast cancers and response to preoperative doxorubicin chemotherapy. Eur J Cancer. 2003; 39(5): 631–634

112. Knoop AS, Knudsen H, Balslev E, Rasmussen BB, Overgaard J, Nielsen KV, Schonau A, Gunnarsdottir K, Olsen KE, Mouridsen H, Ejlertsen B. retrospective analysis of topoisomerase IIa amplifications and deletions as predictive markers in primary breast cancer patients randomly assigned to cyclophosphamide, metho-trexate, and fluorouracil or cyclophosphamide, epirubicin, and fluorouracil: Danish

113. Tanner M. Isola J, Wiklund T, Erikstein B, Kellokumpu-Lehtinen P, Malmstrom P, Wilking N, Nilsson J, Bergh J. Topoisomerase IIalpha gene amplification predicts favorable treatment response to tailored and dose-escalated anthracycline-based adjuvant chemotherapy in HER-2/neu-amplified breast cancer: Scandinavian Breast Group Trial 9401. J Clin Oncol. 2006; 24(16): 2428–2436

114. Slamon DJ, Eiermann W, Robert N, Pienkowski T, Martin M, Press MF, Bernstein L, Sauter G, Zhou JY, Pawlicki M, Chan M, Smylie M, Liu M, Falkson C, Pinter T, Fornnander T, Shiftan T, Valero V, machey J, Tabah-Fisch I, Buyse M, Lindsay MA, Riva A, Bee V, Pegram M, Press MF, Crown J. Phase III randomized trial comparing doxorubicin and cyclophosphamide followed by docetaxel (AC T) with doxorubicin and cyclophosphamide followed by docetaxel and trastuzumab (AC TH) with docetaxel, carboplatin and trastuzumab (TCH) in HER2 positive early breast cancer patients: BCIRG 006 study. San Antonio Breast Cancer Symposium. 2005; A1 (abstract)

115. Press MF, Bernstein L, Sauter G, Zhou JY, Eiermann W, Pienkowski T, Crown J, Robert N, Bee V, Taupin H, Villalobos I, Seelig S, Pegram M, Slamon DJ. Topoisomerase II-alpha gene amplification as a predictor of responsiveness to anthracycline-containing chemotherapy in the Cancer International Research

Breast Cancer Cooperative Group. J Clin Oncol. 2005; 23(30): 7483–7490


Group 006 clinical trial of trastuzumab (herceptin) in the adjuvant setting. San Antonio Breast Cancer Symposium. 2005; A 1045 (abstract)

116. Claus EB, Schildkraut JM, Thompson WD, Risch NJ. The genetic attributable risk of breast and ovarian cancer. Cancer. 1996; 77(11): 2318–2324

117. Lee JS, Wacholder S, Struewing JP, McAdams M, Pee D, Brody LC, Tucker MA, Hartge P. Survival after breast cancer in Ashkenazi Jewish BRCA1 and BRCA2 mutation carriers. J Natl Cancer Inst. 1999; 91(3): 259–263

118. Haffty BG, Harrold E, Khan AJ, Pathare P, Smith TE, Turner BC, Glazer PM, Ward B, Carter D, Matloff E, Bale AE, Alvarez-Franco M. Outcome of conser-vatively managed early-onset breast cancer by BRCA1/2 status. Lancet. 2002; 359(9316): 1471–1477

119. Hartmann LC, Schaid DJ, Woods JE, Crotty TP, Myers JL, Arnold PG, Petty PM, Sellers TA, Johnson JL, McDonnell SK, Frost MH, Jenkins RB. Efficacy of bilateral prophylactic mastectomy in women with a family history of breast cancer. N Engl J Med. 1999; 340(2): 77–84

120. Domchek SM, Friebel TM, Neuhausen SL, Wagner T, Evans G, Isaacs C, Garber JE, Daly MB, Eeles R, Matloff E, Tomlinson GE, Van’t Veer L, Lynch HT, Olopade OI, Weber BL, Rebbeck TR. Mortality after bilateral salpingo-oophorectomy in BRCA1 and BRCA2 mutation carriers: a prospective cohort study. Lancet Oncol. 2006; 7(3): 223–229

Chapter 15

MOLECULAR IMAGING IN METASTATIC BREAST CANCER

C.P. Schröder1, G.A.P. Hospers1, P.H.B. Willemse1, P.J. Perik1, E.F.J. de Vries2, P.L. Jager2, W.T.A. van der Graaf 1, M.N. Lub-de Hooge2, and E.G.E. de Vries1 1Department of Medical Oncology, 2Department of Hospital Pharmacy, Nuclear

The Netherlands

Abstract: Breast cancer is the most common cause of cancer death among women worldwide. Therapeutic decisions in breast cancer are based on stage and specific tumour characteristics. In addition to conventional imaging and histopathological evaluation, potentially non-invasive molecular imaging of tumour metabolism (by means of the [18F] fluorodeoxyglucose (FDG)-

disease evaluation in the future. Molecular imaging provides a functional, dynamic aspect that might be useful for diagnostic purposes, treatment selection, and for monitoring treatment response at a molecular level. This is of particular interest in view of the dynamics of tumour metabolism and biomarker expression during progression and treatment of breast cancer. For staging of recurrent and metastatic breast cancer, FDG-PET imaging of tumour metabolism can be of value in selected cases, with its high

required to confirm this. Molecular imaging of tumour HER2 and the oestrogen receptor was shown to be feasible in metastatic breast cancer. Imaging of these biomarkers may allow a more tumour specific detection than with FDG-PET or conventional imaging, but its use in breast cancer staging- or treatment requires further evaluation. Future options for molecular imaging in breast cancer include monitoring of other significant biomarkers (such as the progesterone receptor), or direct treatment evaluation by radiolabelling targeted therapeutic drugs (such as the anti-vascular endothelial growth factor antibody bevacizumab). To establish molecular imaging in practical (breast) cancer care, more extensive research is needed, but clearly the possibilities are extensive.

© 2007 Springer.


Medicine and Molecular Imaging, University Medical Centre Groningen, Groningen,

markers (oestrogen- and progesterone receptor, HER2) can be used for positron emission tomography, FDG-PET) and known predictive bio-

sensitivity, but varying specificity. For response monitoring and pro-gnostic evaluation, FDG-PET may be useful, but future studies are

Schröder et al.

(SPECT), [18F] fluorodeoxyglucose (FDG), oestrogen receptor (ER), human epidermal growth factor receptor (HER2)

1. INTRODUCTION

Breast cancer is the most common cause of cancer death among women worldwide. One third of all new cancer cases in women are breast cancer. Despite significant advances in primary and adjuvant treatment for local breast cancer, many patients suffer a systemic relapse. In addition, metastatic disease is diagnosed at presentation in 1–5% of women. According to present guidelines (1–3), staging of advanced disease, comprises a radionuclide bone scan, chest X-ray, and imaging of the liver (by means of ultrasound or computed tomography), in addition to routine physical examination and blood tests. Molecular imaging techniques, including positron emission tomography (PET), are not routinely recommended as yet. However, all routinely used staging procedures are known to have a non-perfect diagnostic yield (4). Also, specific tumour characteristics increasingly determine the optimal systemic therapeutic strategy next to disease stage. Molecular imaging of biomarkers and metabolism can possibly be of additional value in this respect (5).

At diagnosis, the tumour is characterised by histology and immuno-histochemistry for the presence of receptors for oestrogen (ER), pro-gesterone (PR), and immunohistochemistry, FISH or CISH for the human epidermal growth factor receptor (HER2). In addition, different biologic phenotypes in breast cancer can be identified by means of a number of gene-expression profiling methods (6). The predictive value of these biologic phenotypes is increasingly recognised. Most likely, gene-expression profiling will be used in the near future for further treatment differentiation (7). However, tumour characteristics of breast cancer can vary between different tumour localisations in the body, and these characteristics can change over time as well. Discordance of biomarker expression between primary tumours and corresponding metastases has already been described (8). In addition to intrapatient heterogeneity, tumour metabolism, biomarker expression, and drug resistance profiles can change during the course of chemo- or hormonal therapy (9–11). In view of these dynamics, the present guidelines advise repetitive biopsies to guide treatment differentiation during the course of metastastic breast cancer (2, 12). However, this is not always practically feasible. Non-invasive technologies such as PET, allow biomarker- and metabolism imaging of the whole tumour mass (in contrast to biopsy material), in the

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Keywords: breast cancer, metastastasis, molecular imaging, biomarkers, positron emission tomography (PET), single photon emission computed tomography

15. Molecular imaging in metastatic breast cancer 309

treatment. Patients can be spared needless treatment, and (costly) therapy can be allocated to those patients likely to respond. It is clear, that monitoring of changes in relevant tumour characteristics during the course of the disease can have an effect on treatment management.

This chapter explores current and future methods for molecular imaging of metastatic breast cancer. The emphasis will be on imaging of tumour metabolism with the well-known [18F] fluorodeoxyglucose (FDG)-PET tracer as well as imaging of the known predictive biomarkers:

factors in breast cancer).

2.

Radioisotopes in PET imaging emit positrons during radioactive decay. After combining with an electron, the positron and electron are annihilated and their combined masses are converted into two gamma rays that travel in opposite directions (E = mc2). The gamma rays thus produced are detected by a PET camera, when opposite detectors register a gamma ray in coincidence (i.e., within a few nanoseconds) (13). The registered gamma rays are subsequently converted into 3D tomographic images. FDG-PET visualises the increased glycolytic metabolism in

phosphorylated by hexokinases. In contrast to glucose-6-phosphate, FDG-6-phosphate is not further metabolised and thus ‘trapped’ in the cell. The entrapment of FDG-6-phosphate can be detected with a PET camera. Under physiological conditions, FDG predominantly accumulates in tissues with high glucose metabolism, such as the brain. A lower grade uptake is seen in muscle, myocardium, liver, and kidneys.

In a pre-operative setting, high FDG tumour uptake was observed particularly in ductal carcinomas (14) of all stages. The quantity of FDG uptake in tumours was positively correlated with the pathologic grade, and the proliferation index (Ki-67) (14, 15). However, FDG uptake itself is not tumour-specific, and the distinction between malignant and benign breast cells can be difficult- particularly in situations of breast hyper-metabolism (breast feeding, mastitis) (16, 17). Also, false positive results can be caused by the accumulation of FDG in activated inflammatory cells such as granulocytes and macrophages (4).

In early stage breast cancer, the value of FDG-PET detection of micrometastatic disease and small lymph nodes is limited by the spatial resolution of PET imaging systems (about 5 mm). For initial staging of

ER, PR, and HER2 (see also chapter 13 on prognostic and predictive

FDG-PET IN METASTATIC BREAST CANCER

whole patient, in a more dynamic way than hitherto possible. This may have therapeutic impact and improve the efficacy of breast cancer

cell membrane by glucose transporter proteins and is enzymatically cancer cells compared to normal cells. FDG is transported across the

Schröder et al.

value of FDG-PET for detecting and staging metastatic breast cancer (Figure 1), a number of reports exist. Moon et al. (19) assessed the accuracy of FDG-PET detection in 57 patients. Sensitivity was 85%, and specificity was 79%, when compared with routine imaging follow-up and histology as standard reference. False negative results in bone were parti-cularly due to osteoblastic bone lesions. When FDG-PET was compared with magnetic resonance imaging (MRI; with histology as standard reference) in 32 patients, sensitivity was 94% (versus 79% for MRI), and specificity was 72% (versus 94% for MRI). In recent, larger studies,

while sensitivity of FDG-PET may vary with tumour biological character-istics (such as tumour type, proliferation index, as mentioned above), overall acceptable and generally superior sensitivity is reported compared to conventional imaging for metastatic breast cancer. Specificity is (highly) variable in different reports, and therefore histologic or cytologic confirmation of PET positive lesions is advised in breast cancer, similar as in other tumour types (21). For the detection of osseous metastases, particularly osteolytic or mixed type, FDG-PET may have a specificity advantage over the conventional bone scan (22). Limited anatomical information by FDG-PET alone is increasingly improved by integration of PET with CT imaging (23).

Assessment of therapeutic response can also be studied by means of FDG-PET. A relative decrease of the standardised (FDG) uptake value of 20% compared to baseline, is considered to indicate a response (5).

Metabolic response determined with FDG-PET was more predictive of histopathological response than clinical examination or ultrasound

patients with primary and metastatic breast cancer (25). However, in a recent report, histopathological tumour response could not be predicted in patients with low initial FDG uptake (in a large part of the patients: 57 out of 96) (26). Thus far, no studies have been performed in which treatment differentiation in breast cancer was based on tumour response assessment by means of FDG-PET. Therefore, the role of FDG-PET in this setting remains to be evaluated. Increased metabolic activity, or “metabolic flare”, detected by FDG-PET in response to hormonal treatment was shown to be predictive for tumour response (11). However, differentia-tion of early tumour progression from metabolic flare in this setting may

310

cancer) (18). In contrast, sensitivity was up to 100% (20). Therefore,

specificity varied from 95% (n = 80 patients, 12 with metastatic breast cancer) (20) to as low as 38% (n = 200 patients, 33 with metastatic breast

line, in responding breast cancer patients (n = 51 patients) (24).

imaging. Similar results were previously shown by Smith et al., in 30

breast cancer, FDG-PET has limited additional value compared to con-ventional imaging and especially sentinel node analysis which allows relatively easy detection of micrometastatic disease (18). With regard to the

In one study in a neo-adjuvant setting, uptake of FDG was signifi-cantly decreased after one course of chemotherapy compared to base-


or metastatic breast cancer is a reimbursable oncological application for PET scanning in for instance the USA.

and abdominal metastases. One study has recently been published with regard to the prognostic

role of FDG-PET in 47 metastatic breast cancer patients, treated with high-dose chemotherapy. Cachin et al. showed a significantly superior prognostic value of complete metabolic response measured with FDG-PET before- and one month after completion of chemotherapy, as compared with conventional imaging techniques. Mean survival was 10

10 months in non-responders. Although these differences appear small,

Figure 1. FDG-PET showing extensive skeletal, pulmonary, mediastinal, supraclavicular,

months without metabolic response (n = 13), versus 24 months with response (n = 34 patients). In patients with response measured by conventional imaging (n = 31), median survival was 21 months, versus

be difficult. Also in the setting of hormonal therapy, the usefulness of FDG-PET for therapeutic decision making has not yet been shown. Nonetheless, monitoring of response, as well as staging of recurrent

in a multivariate analysis only metabolic response was an independent predictor of response (p < 0.0001) (27).

Schröder et al.

staging and imaging techniques. Sensitivity of this detection technique is generally reported to be superior to other techniques, but with varying specificity, histological confirmation appears necessary. In summary, FDG-PET is potentially useful for response monitoring and prognostic evaluation, however, future studies are required to confirm this.

MOLECULAR IMAGING OF BIOMARKERS

HER2, the human epidermal growth factor receptor, is encoded for by the HER2 proto-oncogene (HER2/neu or c-erbB-2), and has growth stimulating activity. Overexpression of HER2 as a result of HER2 amplification has been shown in 25–30% of breast cancer patients, and is associated with a worse prognosis and more aggressive clinical behaviour. The anti-HER2 monoclonal antibody trastuzumab (Herceptin®) binds specifically to HER2. The addition of trastuzumab to chemotherapy is effective in the treatment of patients with HER2-overexpressing breast cancer, both in the metastatic and in the adjuvant setting (28).

However, HER2 expression in breast cancer is a dynamic entity that can vary within one patient. Discordance of HER2 expression between primary breast cancers and corresponding metastases was found in 14% of the patients (8). In patients with HER2 negative primary tumours, HER2 positive cells have been detected in bone marrow (9) and in the circulation (29). Also, patients with HER2 positive primary tumours can have metastases with different HER2 expression levels, or they can convert during therapy to a HER2 negative tumour or a tumour with less HER2 expression in about 25% (30). Variation in time of HER2 expression was also shown by Rasbridge et al. (31). Neo-adjuvant anthracycline containing chemotherapy as well as hormonal therapy may induce HER2 expression (9). During cancer progression, nearly 40% of breast cancer patients whose primary tumour was HER2 negative acquired HER2 gene amplification (32). Heterogeneity of tumour tissue can also lead to over or underestimation of HER2 expression, as a result of sampling error (33).

In vivo testing of HER2 expression in a non-invasive fashion may circumvent these problems. Various approaches have been described to image HER2, including intact monoclonal antibodies and more recently: antibody fragments. The anti-HER2 antibody trastuzumab has been radio-labelled with various isotopes, both with diagnostic and therapeutic objec-

124I- and 99m

312

3.1 HER2/neu imaging

3.

At present, FDG-PET imaging can be of value in selected cases for staging of recurrent and metastatic disease, in addition to conventional

Tc-ICR12 (a rat tives. Immunolocalisation studies with


data are limited. In a recent study, immuno-SPECT (single photon emi-ssion computed tomography) has been performed using radiolabelled trastuzumab (111Indium-trastuzumab) in 17 patients with HER2 positive metatastatic breast cancer (36). With this technique, 45% of single tumour lesions, detected with conventional imaging, could be shown (Figure 2). However, new tumour lesions were discovered in 13 of 15 patients. Although the number of patients in this study was small, these results may indicate a role for tumour-specific detection by means of radio-labelled trastuzumab. Further studies, including histological samples of the detected lesions, are needed to confirm this. Currently, researchers are optimising the HER2 imaging technique by developing PET tracers based on trastuzumab or Fab fragments – either by labelling of intact antibodies or antibody fragments with PET isotopes (e.g., 68Ga, 18F, 89Zr) (37). Smaller size fragments may improve sensitivity of HER2 imaging. Although preclinical data are promising, this requires further study in humans.

Figure 2. HER2 SPECT performed 5 days after radiolabelled trastuzumab (111Indium), showing bone, supraclavicular and possibly intrapulmonary lesions (arrows).

breast carcinoma xenografts showed specific tracer uptake (34), which was strongly correlated with HER2 expression level (35). In humans however,

anti-HER2 antibody) in athymic mice bearing HER2 overexpressing human

Schröder et al.

bearing tumours. Measurement of hormone receptor expression (ER and PR) at the time of primary diagnosis is the standard of clinical care. Selection of appropriate therapy is based on receptor expression, which is predictive of response to anti-hormonal treatment in up to 70% of patients with a new diagnosis of breast cancer. In recurrent breast cancer, the response rate to anti-hormonal treatment is only 7–21% (38). The present guidelines indicate that new histology is needed at the time of relapse (2, 12), as ER expression can vary between primary tumour and recurrence in 30% of cases (39). As already indicated, it is not always practically feasible to perform a repetitive biopsy. Also, sampling error may be a potential problem, as ER expression can differ between primary tumour and synchronous metastases. In one study, the discordance between the ER status of the primary tumour and the distant metastases was 41% in cases of bone marrow metastases, and 44% in liver metastases (40). In addition, anti-hormonal treatment induces loss of ER in a number of patients with acquired resistance to this treatment (41). In view of these issues, molecular imaging of ER expression in the whole patient may be of value. The PET tracer 16-a-[18F] fluoro-17beta -estradiol (FES) was developed for this purpose. Few studies with FES-PET imaging have been performed in the human setting so far. FES uptake was previously shown to correlate well with ER density (42). When FES uptake was compared with FDG uptake in the human setting, 85% of FDG positive lesions were also found FES positive (11). No direct comparison between FES-PET and conventional imaging techniques has been performed. With regard to monitoring of response to anti-hormonal therapy, few data are available. In a recent study, FES-PET was evaluated in 47 immuno-histochemically ER positive metastatic breast cancer patients (43). Imaging was performed before and after 6 months of individualised anti-hormonal treatment (mainly aromatase inhibition in 77% of patients, but also tamoxi-fen in 11%). In patients with low initial uptake (n = 15), no response was shown. There were responders and non-responders in patients with a high initial uptake. Particularly those patients with HER2 co-expression did not show treatment response, in spite of high initial uptake. Apart from receptor status, downstream effects will also determine tumour res-ponse. This might be in line with oestrogen-independent growth, possibly through the epidermal growth factor receptor pathway (44). This study shows the feasibility of FES-PET in humans and suggests the possibility to select a group of non-responders. However, the number of patients in this study is too small to draw definite conclusions, particularly in view of the heterogeneous anti-hormonal treatment. It is clear that a much larger study is required, which should take in account other factors such as HER2 as

Nearly two-thirds of breast cancer patients have hormone receptor-

3.2 ER imaging

314

15. Molecular imaging in metastatic breast cancer 315 well, to define the precise role of ER imaging in breast cancer treatment. Given the increasing options for individualising hormonal treatment, this is of major interest. With regard to the prognostic value of FES-PET, no data are available yet. In conclusion, thus far FES-PET cannot be regarded as a routine imaging technique for metastasised breast cancer. However, it is certainly a technique that deserves to be explored more extensively.

4. FUTURE PERSPECTIVES

contrast agents can be specifically attached to cellular biomarkers by anti-bodies, peptides, and drugs acting as ligands, allowing ligand-directed accumulation (45). The biomarkers used ideally provide a good reflection of a certain (patho)biological process. Functional imaging of these pro-cesses displays the biochemical and physiological abnormalities underlying the cancer, in contrast to anatomic imaging, which can only show the structural consequences of these abnormalities (46). In this light, molecular imaging clearly has the potential to add a functional, informative com-ponent to conventional detection techniques for cancer. Increasingly,

by means of CT-PET. Sequential imaging of relevant molecular markers, such as hormone receptors and HER2, may provide insight in the dynamics of marker expression in individual patients, which may have therapeutic consequences. Finally, molecular imaging may clearly improve the insight in molecular effects of cancer therapy, with regard to commonly used cancer therapeutics as well as new targeted drugs.

For instance, imaging of anti-angiogenesis directed therapy may be of significance. Tumour neo-vascularisation is of major importance for tumour growth. An important factor contributing to this process is the vascular endothelial growth factor (VEGF) group, particularly VEGF-A, produced by the tumour. As such, VEGF is used as a target for site-specific therapy by means of bevacizumab, the antibody against VEGF-A. VEGF is fre-quently overexpressed in breast cancer, and bevacizumab has been shown to improve response when added to chemotherapy in metastatic breast cancer (28, 47). Radiolabelled VEGF has been examined in animal models, showing an excellent tumour- to organ ratio (48). Further studies are presently initiated with radiolabelled VEGF in the human setting.

Another example is the imaging of the hormone receptor PR. PR is con-sidered to be the result of the expression of an active ER and appears to predict hormonal sensitivity better than the presence of ER itself. Hormonal therapy can therefore better be initiated based on the presence of PR. In this light, functional imaging of the PR may be of value. In the 1990s,

these features of molecular and anatomic imaging are literally combined

The emergence of molecular imaging has coincided with the deve-lopment of molecular targeted therapy in cancer. Therapeutic and/or

Schröder et al. 21-[18F]fluoro-16α-ethyl-19norprogesterone was evaluated as a PET tracer for the PR. Despite promising pre-clinical results, this tracer proved unsuitable for imaging due to extensive metabolism in humans (49). In a recent study, the synthesis of novel radiolabelled PR ligands was described (50). However, no data are available in the diagnostic setting. This has to do with the major challenge for (radio)chemists/ pharmacists to transform these ligands to clinically applicable compounds.

In conclusion, molecular imaging is a rapidly evolving field of cancer imaging, stimulated in particular by the development of new targeted drugs. It adds a functional component to conventional imaging, and can be potentially useful for diagnostic purposes, treatment selection, and for monitoring treatment response at a molecular level. More extensive research is needed to establish this form of imaging in practical (breast) cancer care, but it is clear that its future possibilities are extensive.

REFERENCES

1. National Comprehensive Cancer Network. Clinical Practive Guidelines in Oncology V.2.2006, section Invasive Breast Cancer. www.nccn.org.

2. PDQ: Cancer Information Summaries: Adult Treatment, National Cancer Institute. http://www.cancer.gov/cancertopics/pdq/adulttreatment.

3. DeVita VT, Hellman S, Rosenberg SA (editors). Cancer, principles and practice of oncology (7th ed.). Ch 33.2, 2005, Lippincott Williams and Wilkins. 1454–1455.

4. Barentz J, Takahaxhi S, Oyen W, Mus R, de Mulder P, Oudkerk M, Mali W. Commonly used imaging techniques for diagnosis and staging. J Clin Oncol 2006; 24: 3234–3244.

5. Weber WA. Positron Emission Tomography as an imaging biomarker. J Clin Oncol 2006; 24: 3282–3292.

6. Perou CM. Concordance among gene-expression-based predictors for breast cancer. N Engl J Med 2006; 355: 560–569.

7. O’Shaughnessy JA. Molecular signatures predict outcomes of breast cancer. N Engl J Med 2006; 355: 615–617.

8. Zidan J, Dashkovsky I, Stayerman C, Basher W, Cozacov C, Hadary A. Comparison of HER-2 overexpression in primary breast cancer and metastatic sites and its effect on biological targeting therapy of metastatic disease. Br J Cancer. 2005; 93: 552–556.

9. Solomayer EF, Becker S, Pergola-Becker G, Bachmann R, Kramer B, Vogel U, Neubauer H, Wallwiener D, Huober J, Fehm TN. Comparison of HER2 status between primary tumor and disseminated tumor cells in primary breast cancer patients. Breast Cancer Res Treat 2006; 9: 179–184.

10. Smith IC, Welch AE, Hutcheon AW, Miller ID, Payne S, Chilcott F, Waikar S, Whitaker T, Ah-See AK, Eremin O, Heys SD, Gilbert FJ, Sharp PF. Positron emission tomography using [(18)F]-fluorodeoxy-D-glucose to predict the pathologic response of breast cancer to primary chemotherapy. J Clin Oncol

11. Dehdashti F, Flanagan FL, Mortimer JE, Katzenellenbogen JA, Welch MJ, Siegel BA. Positron emission tomographic assessment of “metabolic flare” to predict response of metastatic breast cancer to antiestrogen therapy. Eur J Nucl Med. 1999; 26: 51–56.

316

Fan C, Oh DS, Wessels L, Weigelt B, Nuyten D, Nobel AB, van’t Veer L,

2000; 18: 1676–1688.

15. Molecular imaging in metastatic breast cancer 317 12. UpToDate®, Inc. founded by Burton D. Rose, MD Joseph M. Rush, MD. General

13. Brix G, Henze M, Knopp MV, Lucht R, Doll J, Junkermann H, Hawighorst H, Haberkorn U. Comparison of pharmacokinetic MRI and [18F] fluorodeoxyglucose PET in the diagnosis of breast cancer: initial experience. Eur Radiol. 2001; 11:

14. Buck A, Schirrmeister H, Kuhn T, Shen C, Kalker T, Kotzerke J, Dankerl A, Glatting G, Reske S, Mattfeldt T. FDG uptake in breast cancer: correlation with biological and clinical prognostic parameters. Eur J Nucl Med Mol Imaging.

15. Crippa F, Seregni E, Agresti R, Chiesa C, Pascali C, Bogni A, Decise De Sanctis V, Greco M, Daidone MG, Bombardieri E. Association between [18F]fluorodeoxyglucose uptake and postoperative histopathology, hormone receptor status, thymidine labelling index and p53 in primary breast cancer: a

16. Hicks RJ, Binns D, Stabin MG. Pattern of uptake and excretion of (18)F-FDG

17. Bakheet SM, Powe J, Kandil A, Ezzat A, Rostom A, Amartey J. F-18 FDG

18. Carr CE, Conant EF, Rosen MA, Schnall MD, Davidson R. Impact of FDG PET in the staging of breast cancer. J Clin Oncol 2006 ASCO Annual meeting proceedings I; 24 (18S): 530.

19. Moon DH, Maddahi J, Silverman DH, Glaspy JA, Phelps ME, Hoh CK. Accuracy of whole-body fluorine-18-FDG-PET for the detection of recurrent or metastatic breat carcinoma. J Nucl med 1998; 39: 431–435.

20. Port ER, Yeung H, Gonen M, Liberman L, Caravelli J, Borgen P, Larson S. 18F-2-fluoro-2-deoxy-D-glucose positron emission tomography scanning affects surgical management in selected patients with high-risk, operable breast carcinoma. Ann Surg Oncol. 2006;13: 677–684.

21. Van der Hoeven JJ, Krak NC, Hoekstra OS, Comans EF, Boom RP, van Geldere D, Meijer S, van der Wall E, Buter J, Pinedo HM, Teule GJ, Lammertsma AA. 18F-2-fluoro-2-deoxy-d-glucose positron emission tomography

22. Minn H, Soini I. [18F]fluorodeoxyglucose scintigraphy in diagnosis and follow

23. Endo K, Oriuchi N, Higuchi T, Iida Y, Hanaoka H, Miyakubo M, Ishikita T, Koyama K. PET and PET/CT using 18F-FDG in the diagnosis and management of cancer patients. Int J Clin Oncol 2006; 11: 286–296.

24. Rousseau C, Bodet-Milin C, Bennouna J, Ferrer L, Campion L, Bridji B, Sagan C, Ricaud M, Resche I, Kraeber-Bodere F, Campone M. Evaluation of FDG-PET in early axillary lymph node response to neo-adjuvant chemotherapy in locally advanced breast cancer patients. J Clin Oncol 2006 ASCO Annual meeting proceedings I; 24 (18S): 10500.

25. Smith IC, Welch AE, Hutcheon AW, Miller ID, Payne S, Chilcott F, Waikar S, Whitaker T, Ah-See AK, Eremin O, Heys SD, Gilbert FJ, Sharp PF. Positron emission tomography using [(18)F]-fluorodeoxy-D-glucose to predict the pathologic response of breast cancer to primary chemotherapy. J Clin Oncol.

26. McDermott GM, Welch A, Staff RT, Gilbert FJ, Schweiger L, Semple SI, Smith A, Hutcheon AW, Miller ID, Smith IC, Heys SD. Monitoring primary breast cancer throughout chemotherapy using FDG-PET. Breast Cancer Res

2002; 29: 1317–1323.

in staging of locally advanced breast cancer. J Clin Oncol. 2004; 22: 1253–1259.

up of treatment in advanced breast cancer. Eur J Nucl Med. 1989; 15: 61–66.

2000; 18: 1676–1688.

principles of management of metastatic breast cancer. www.uptodate.com.

2058–2070.

preliminary observation. Eur J Nucl Med. 1998; 25: 1429–1434.

in the lactating breast. J Nucl Med. 2001; 42: 1238–1242.

Treat. 2006 9; [Epub ahead of print].

uptake in breast infection and inflammation. Clin Nucl Med. 2000; 25:100–103.

Schröder et al. 27. Cachin F, Prince HM, Hogg A, Ware RE, Hicks RJ. Powerful prognostic

stratification by [18F]fluorodeoxyglucose positron emission tomography in

28. Bernard-Marty C, Lebrun F, Awada A, Piccart MJ. Monoclonal antibody-based targeted therapy in breast cancer: current status and future directions. Drugs. 2006; 66: 1577–1591.

29. Wulfing P, Borchard J, Buerger H, Heidl S, Zanker KS, Kiesel L, Brandt B. HER2-positive circulating tumor cells indicate poor clinical outcome in stage I to III breast cancer patients. Clin Cancer Res. 2006; 15: 1715–1720.

30. Burstein HJ, Harris LN, Gelman R, Lester SC, Nunes RA, Kaelin CM, Parker LM, Ellisen LW, Kuter I, Gadd MA, Christian RL, Kennedy PR, Borges VF, Bunnell CA, Younger J, Smith BL, Winer EL. Preoperative therapy with trastuzumab and paclitaxel followed by sequential adjuvant doxorubicin/ cyclophosphamide for HER2 overexpressing stage II or III breast cancer: a pilot study. J Clin Oncol. 2003; 21: 46–53.

31. Rasbridge SA, Gillett CE, Seymour AM, Patel K, Richards MA, Rubens RD, Millis RR. The effects of chemotherapy on morphology, cellular proliferation, apoptosis and oncoprotein expression in primary breast carcinoma. Br J Cancer

32. Meng S, Tripathy D, Shete S, Ashfaq R, Haley B, Perkins S, Beitsch P, et al. HER-2 gene amplification can be acquired as breast cancer progresses. Proc

33. Sekido Y, Umemura S, Takekoshi S, Suzuki Y, Tokuda Y, Tajima T, Osamura RY. Heterogeneous gene alterations in primary breast cancer contribute to discordance between primary and asynchronous metastatic/recurrent sites: HER2 gene amplifi-cation and p53 mutation. Int J Oncol. 2003; 22: 1225–1232.

34. Bakir MA, Eccles S, Babich JW, Aftab N, Styles J, Dean CJ, Lambrecht RM, Ott RJ. C-erbB2 protein overexpression in breast cancer as a target for PET using iodine-124-labeled monoclonal antibodies. J Nucl Med 1992; 33: 2154–2160.

35. Allan SM, Dean C, Fernando I, Accles S, Styles J, McCready VR, Baum M, Sacks N. Radioimmunolocalisation in breast cancer using the gene product of c-erbB2 as the target antigen. Br J Cancer 1993; 67: 706–712.

36. Perik PJ, Lub-de Hooge MN, Gietema JA, van der Graaf WTA, de Korte MA,

positive metastatic breast cancer. J Clin Oncol 2006; 24: 2276–2282. 37. Tang Y, Wang J, Scollard DA, Mondal H, Holloway C, Kahn HJ, Reilly RM.

Imaging of HER2/neu-positive BT-474 human breast cancer xenografts in athymic mice using (111)In-trastuzumab (Herceptin) Fab fragments. Nucl Med Biol 2005; 32: 51–58.

38. Nabholtz JM, Buzdar A, Pollak M, Harwin W, Burton G, Mangalik A, Steinberg M, Webster A, von Euler M. Anastrozole is superior to tamoxifen as first-line therapy for advanced breast cancer in post-menopausal women: results of a North American multicenter randomized trial. Arimidex Study Group. J Clin Oncol 19: 2001; 3357–3366.

39. Spataro V, Price K, Goldhirsch A, Cavalli F, Simoncini E, Castiglione M, Rudenstam CM, Collins J, Lindtner J, Gelber RD. Sequential estrogen receptor determinations from primary breast cancer and at relapse: prognostic and therapeutic relevance. The International Breast Cancer Study Group (formerly

318

Ludwig Group). Ann Oncol. 1992; 9: 733–740.

J Clin Oncol. 2006; 24: 3026–3031. patients with metastatic breast cancer treated with high-dose chemotherapy.

1994; 70: 335–341.

Natl Acad Sci USA. 2004; 101: 9393–9398.

de Vries EGE. 111In-labeled trastuzumab scintigraphy in patients with HER2-Jonkman S, Kosterink JGW, van Veldhuisen DJ, Sleijfer DT, Jager PL,

15. Molecular imaging in metastatic breast cancer 319 40. Brunn Rasmussen B, Kamby C. Immunohistochemical detection of estrogen

receptors in paraffin sections from primary and metastatic breast cancer. Pathol

41. Normanno N, Di Maio M, De Maio E, De Luca A, de Matteis A, Giordano A, Perrone F; NCI-Naple Breast Cancer Group. Mechanisms of endocrine resistance and novel therapeutic strategies in breast cancer. Endocr Relat Cancer. 2005;

42. Dehdashti F, Mortimer JE, Siegel BA, Griffeth LK, Bonasera TJ, Fusselman MJ, Detert DD, Cutler PD, Katzenellenbogen JA, Welch MJ. Positron tomographic assessment of estrogen receptors in breast cancer: comparison

43. Linden HM, Stekhova SA, Link JM, Gralow JR, Livingston RB, Ellis GK, Petra PH, Peterson LM, Schubert EK, Dunnwald LK, Krohn KA, Mankoff DA. Quantitative fluoroestradiol positron emission tomography imaging predicts res-

44. Osborne CK, Shou J, Massarweh S, Schiff R Crosstalk between estrogen receptor and growth factor receptor pathways as a cause for endocrine therapy

45. Atri M. New technologies and directed agents for applications of cancer imaging. J Clin Oncol 2006; 24: 3299–3308.

46. Sullivan DC. Molecular imaging in oncology. Ann Oncol 2006; 17: 287–292. 47. Miller KD, Chap LI, Holmes FA, Cobleigh MA, Marcom PK, Fehrenbacher L,

Dickler M, Overmoyer BA, Reimann JD, Sing AP, Langmuir V, Rugo HS. Randomized phase III trial of capecitabine compared with bevacizumab plus capecitabine in patients with previously treated metastatic breast cancer. J Clin Oncol. 2005; 23: 792–799.

48. Collingridge DR, Carroll VA, Glaser M, Aboagye EO, Osman S, Hutchinson OC, Barthel H, Luthra SK, Brady F, Bicknell R, Price P, Harris AL. The develop-ment of [(124)I]iodinated-VG76e: a novel tracer for imaging vascular endothelial growth factor in vivo using positron emission tomography. Cancer Res. 2002;

49. Verhagen A, Studeny M, Luurtsema G, Visser GM, De Goeij CC, Sluyser M, Nieweg OE, Van der Ploeg E, Go KG, Vaalburg W. Metabolism of a [18F]fluorine labeled progestin (21-[18F]fluoro-16 alpha-ethyl-19-norprogesterone) in humans:

50. Zhou D, Carlson KE, Katzenellenbogen JA, Welch MJ.Bromine- and iodine-substituted 16alpha,17alpha-dioxolane progestins for breast tumor imaging and radiotherapy: synthesis and receptor binding affinity. J Med Chem. 2006; 49: 4737–4744.

with FDG-PET and in vitro receptor assays. J Nucl Med. 1995; 10: 1766–1774.

ponse to endocrine treatment in breast cancer. J Clin Oncol. 2006; 18: 2793–2799.

resistance in breast cancer. Clin Cancer Res. 2005; 11: 865s–870s.

a clue for future investigations. Nucl Med Biol. 1994; 21: 941–952.

Res Pract. 1989; 185: 856–859.

4: 721–747.

62: 5912–5919.

Chapter 16

DETECTION OF DISSEMINATED TUMOR CELLS IN THE BONE MARROW AND BLOOD OF BREAST CANCER PATIENTS

Volkmar Müller1 and Klaus Pantel2 1Department of Gynecology, 2Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany

Abstract: Early tumor cell dissemination can be detected in patients with breast cancer using immunocytochemical and molecular assays based on the use of monoclonal antibodies or PCR. Studies involving more than 4,000 breast cancer patients have demonstrated now that the presence of disseminated tumor cells (DTC) in bone marrow (BM) identified with immuncyto-chemical assays at primary diagnosis is a strong prognostic factor. The published studies for the detection of disseminated tumor cells in bone marrow fulfill the highest level of evidence as prognostic markers in primary breast cancer. In addition, various assays for the detection of circulating tumor cells in the peripheral blood have been recently developed and some studies suggest a potential clinical relevance of this parameter as prognostic and predictive factor. Advanced methods for molecular characterization of single tumor cells have been developed lately and this approach allows new insights into the metastatic cascade and characteri-zation of targets for therapeutic approaches. These findings provide the basis for the implementation of DTC in BM or blood as markers for stratification and assessment of therapies in prospective clinical trials. The valuable information derived from these trials should help to improve future treatment of breast cancer patients.

disseminated tumor cells, bone marrow, cytokeratin, immunocytochemistry,

1. BACKGROUND

The first step in metastatic spread of breast cancer is tumor cell dissemination via the regional lymph nodes or/and by tumor circulation in

© 2007 Springer.


Keywords: circulating tumor cells

322 Müller and Pantel the blood followed by homing in on secondary distant organs. Among these distant organs, bone marrow (BM) is a common homing organ for disseminated tumor cells (DTC) and is therefore a potential indicator organ for the presence of disseminated tumor cells throughout the body.

The most recent TNM classification for breast cancer (1) does not qualify the presence of single cancer cells in peripheral blood or BM as metastasis (stage M0), but it optionally reports the presence of such cells together with their detection method, e.g., M0(i+) for the immunocyto-chemical detection or M0(mol+) for the detection by molecular methods.

In this chapter, the methods and implications of DTC detection in BM and of circulating tumor cells in the blood (CTC) for staging and therapy of breast cancer patients will be discussed.

2.

Many different assays have been applied to detect DTC in breast

cancer and other solid tumors. One major approach to identify DTC from BM includes density gradient centrifugation with subsequent immunocyto-chemical staining using monoclonal antibodies against epithelial or tumor-associated antigens (Figure 1). Different monoclonal and polyclonal antibodies or antibody cocktails were used for immunocytochemical identification of DTC in BM. Groups have used antibodies against EMA, directed against an epithelial cell-surface antigen (2), TAG12, a tumor-associated glycoprotein (3), and cytokeratins (CK), the structural proteins of the epithelial cytoskeleton (4, 5). To date, cytokeratins have become the most widely accepted protein marker in such immunocyto-chemical assays. A combination of several antibodies to various CK antigens or an antibody against a common epitope present on various CK proteins (e.g., A45B/B3 directed among others against CK8, 18, 19) seems to be superior to monospecific antibodies directed against a single

considerable antigenic heterogeneity of solid tumor cells. With this app-roach, one single DTC can be detected in the background of millions of hematopoetic cells. However, different staining techniques can result in specificity variations. Hematopoietic cells can be directly reactive to alka-line phosphatase (8) or produce endogenous peroxidase (9), conesquently resulting in false-positive staining in alkaline phosphatase-based or peroxi-dase-based methods, if these enzymes were not fully blocked. Several international organizations have recognized the need for standardization of

2.1. Immunocytochemical staining

cytokeratin protein (e.g., CK2, against CK18) (5–7), because of the

OF DISSEMINATED TUMOR CELLS METHODS FOR THE DETECTION

16. Detection of disseminated tumor cells in the BM and blood 323 the immunocytochemical assay and for its evaluation in prospective studies (10, 11).

The use of new automated devices for the microscopic screening of immunostained slides may help to read slides more rapidly and to increase reproducibility of the read-out (12–17). Another way to improve current detection assays for single tumor cells is to develop better tumor cell enrichment procedures using improved density gradients (18) and antibody-coupled magnetic particles (12, 19, 20). At present, it is unclear whether these new enrichment techniques provide more clinically relevant information than the standard density gradient procedure used to isolate the mononuclear cell fraction.

process begins with a Ficoll density gradient centrifugation to isolate mononuclear cells and uses cytokeratins as markers of DTC. The detection of the stained DTC can be performed automatically.

Figure 1. Immunocytochemical detection of DTC in patients with breast cancer. The

324 Müller and Pantel 2.2. PCR approach

An alternative to immunocytochemical assays for the detection of DTC are molecular detection procedures. The nucleic acid from a sample can be amplified by PCR, so that tumor cells can be detected with high sensitivity in a heterogeneous population of cells. However, the tumor cells must have changes in its DNA or mRNA expression pattern that distinguish them from the surrounding hematopoetic cells. At the DNA level, breast carcinomas are genetically heterogeneous, with no universally applicable DNA marker available. Therefore, research has focused on RNA markers. A multimarker approach with a panel of tumor-specific mRNA markers may improve the sensitivity for the detection of DTC over single marker assays (21, 22). Many transcripts have been evaluated as “tumor-specific” markers like CK18, CK19, CK20, Mucin-1, and carcinoembryonic antigen (23–25). However, many of these transcripts can also be identified by RT-PCR in normal BM, blood, and lymph node tissue (26–28). Preanalytical depletion of the disturbing normal cell fraction (e.g., granulocytes that express CK20) or quantitative RT-PCR determinations could solve this problem.

2.3. Molecular characterization of DTC

Multiple characterization approaches of DTC in BM show a considerable phenotypic heterogeneity. A detailed molecular description of DTC in BM of breast cancer patients without clinical signs of overt metastases demonstrated that these cells are genetically heterogeneous (29) and lacked genomic aberrations observed in the primary tumors (30). In particular the HER-2/neu proto-oncogene appears to define a very aggressive subset of DTC with an increased invasive capability (31) and has gained substantial importance as biological target for systemic therapy in breast cancer (32, 33). Furthermore, there is also evidence for a prognostic effect of HER-2/neu-positive DTC in BM and CTCs in stage I to stage III breast cancer (34, 35). Furthermore, most DTC and CTC do not express the proliferation antigen Ki-67 and may therefore be resistant to chemotherapy (36, 37).

By applying gene expression analysis on primary breast tumors in relation to the presence or absence of DTC in BM, a specific gene signature in primary tumors of patients with DTC in BM was observed (38). These findings challenge the traditional concept that tumor cells acquire their metastatic genotype and phenotype late during tumor development, and support the alternative concept that tumor cells acquire the genetic changes relevant to their metastatic capacity early in tumorigenesis (39), so that the metastatic potential of human tumors is encoded in the bulk of a primary tumor (39). This concept could also explain the presence of DTC in BM at early stages of breast cancer.

16. Detection of disseminated tumor cells in the BM and blood 325 3. CLINICAL RELEVANCE OF DTC

DETECTION IN BM

Despite the progress made in therapy of breast cancer, the prognosis of breast cancer patients even with small primary tumors is still limited by metastatic relapse, which indicates an early tumor cell spread. It was shown that the presence of DTC in BM was detectable in 20–40% of breast cancer patients without signs of distant metastases (5). Interestingly, a similar prevalence was found in several other carcinoma types studied, and until now, no report has demonstrated a solid tumor type without immunocytochemically detectable epithelial cells in BM. In fact, DTC have also been found in the BM of patients with colon cancer that rarely metastasize to the bone (40). It seems that BM is an important reservoir that allows DTC to adapt and disseminate into other organs.

A negative BM finding may therefore represent an additional clinical marker to identify those node-negative patients who are cured by surgery alone and need no additional adjuvant chemotherapy (5, 43). In the context of adjuvant therapy, it is also of interest that several studies found DTC in BM even several years after surgery and adjuvant therapy and it seems that the presence of DTC after adjuvant treatment might be useful to identify patients with an increased risk for recurrence (44, 45). These results

Many studies have demonstrated a correlation between the presence of DTC in BM and an impaired prognosis (Table 1). Nevertheless, there are also a few studies that could not confirm BM as an independent prognostic indicator (2, 41). A previous meta-analysis including 20 older studies of nearly 2,500 patients suggested that the detection of DTC offer no additional prognostic information over the established prognostic factors (42). However, these studies are associated with inherent problems, in that the detection methods, antibodies used, and the number of cells analyzed was not according to procedures currently regarded as standards. Furthermore, the patient cohorts were small and the follow-up data, relatively short. A recent pooled analysis of more than 4,700 breast cancer patients with stage I, II, or III disease without manifest metastases from nine independent studies demonstrated that the presence of DTC in BM was associated with larger tumors, with a higher histologic grade, with lymph node metastases and with hormone-receptor-negative tumors (5). Subgroup analysis showed that DTC in BM were associated with worse outcomes at all risk levels, even among those with small tumors and without lymph node involvement indicating prognostic relevance in all subgroups. In these analyzed large trials most investigators used anti-bodies against cytokeratins to detect DTC in BM.

326 Müller and Pantel demonstrate that DTC can reside in a latent state of dormancy for many years before they grow out into overt skeleton metastases.

Table 1. Examples for reports demonstrating a prognostic relevance of DTC in BM of breast cancer patients without overt metastases (TNM-stage M0)

Reference Number of patients

Detection rate (%)

Technique Prognostic value

Landys et al. (3) 128 19 IHC DFS*, OS*

Diel et al. (46) 727 43 ICC DFS*, OS*

Mansi et al. (47) 350 25 ICC DFS, OS

Gebauer et al. (2) 393 42 ICC DFS*, OS

Cote et al. (48) 49 37 ICC DFS

Braun et al. (7) 552 36 ICC DDFS*, OS*

Harbeck et al. (49) 100 38 ICC DFS*, OS*

Gerber et al. (4) 484 31 ICC DFS*, OS*

Wiedswang et al. (19) 817 13 ICC DDFS*, OS*

Pooled Analysis (5) 4703 31 ICC DDFS, OS

Abreviations: CK, cytokeratin; DFS, disease-free survival; DDFS, distant disease-free survival; ICC, immunocytochemistry; IHC, immunohistochemistry; OS, overall survival *Prognostic value supported by multivariate analysis

4. CLINICAL RELEVANCE OF CIRCULATING TUMOR CELLS IN THE BLOOD

Peripheral blood would be an ideal source for the detection of minimal residual disease since it is easier to obtain than BM. This is of relevance especially in the context of repeated examinations in order to monitor treatment. However, the relevance of CTC so far is much less clear than for DTC in BM. Blood is only a transient compartment for tumor cells, and it is possible that only a small fraction of CTC survives and is subsequently capable to form detectable metastases.

The clinical relevance of CTC in patients with primary, nonmetalsta-tic breast cancer is currently under investigation. Preliminary comparative studies examining DTC in BM and CTC in blood in these patients found a correlation between the presence of tumor cells in both compartments

16. Detection of disseminated tumor cells in the BM and blood 327 (37, 50), but the current findings do not support the replacement of BM analysis by blood testing (50, 51). However, it seems possible that blood analysis could deliver additional information and the monitoring of adjuvant therapy with repeated examinations deserves further attention.

In metastatic breast cancer, it was demonstrated that the detection of CTC correlate with tumor progression and could therefore provide clinically relevant information (37, 52). By using an automated enrichment and analysis system, prognostic information was obtained (52, 53). Clinical studies must now show that the prognostic information derived from CTC detection can improve outcome of patients, e.g., by earlier change of treatment.

5. FUTURE CLINICAL POTENTIAL

Consensus is now obtained regarding quality control issues and criteria for acceptable technical assay performance in order to permit multicenter clinical studies (11). With these developments, the final step toward implementation into the clinical setting will be taken. In addition to standardization of technical issues, more detailed marker implement-tation into current risk classification systems, such as the Tumor-Node-Metastasis (TNM) Classification System, is needed.

BM and blood can be obtained repeatedly in the postoperative course of treatment. Therapeutic efficacy of adjuvant systemic therapy can be assessed currently only retrospectively in large-scale clinical trials following a long observation period. The potential of a surrogate marker assay that permits immediate assessment of therapy-induced effects is therefore evident. For example, it could be possible to identify patients who need additional adjuvant therapy, e.g., bisphosphonate treatment which might be able to eliminate tumor cells in bone marrow persisting after adjuvant treatment. Prospective clinical studies must evaluate whether eradication of DTC in BM and blood after systemic therapy translates into a longer disease-free period and overall survival. An additional important goal is the possibility of identifying tumor specific targets to improve therapy regimens. Studies have shown that it is possible to identify therapeutic targets on DTC and some evidence suggests that single DTC show different properties than cells of the primary tumor (35, 36, 54). This is of importance, e.g., in the context of new therapeutic approaches, like antibody treatment directed against HER-2/neu which has been demonstrated to reduce relapse rates in the adjuvant setting.

In addition, the research in the field of tumor cell dissemination should lead to an increased understanding of the metastatic cascade. This could allow the development of new therapeutic approaches suppressing the

328 Müller and Pantel development of metastatic disease when applied in the early stage of micrometastases before the development of manifest metastatic disease.

REFERENCES

1. Singletary SE, Allred C, Ashley P, et al. Revision of the American Joint

3628–3636. 2. Gebauer G, Fehm T, Merkle E, et al. Epithelial cells in bone marrow of breast

cancer patients at time of primary surgery: clinical outcome during long-term follow-up. J Clin Oncol 2001; 19:3669–3674.

3. Landys K, Persson S, Kovarik J, Hultborn R, Holmberg E. Prognostic value of bone marrow biopsy in operable breast cancer patients at the time of initial diagnosis: Results of a 20-year median follow-up. Breast Cancer Res Treat 1998; 49:27–33.

4. Gerber B, Krause A, Muller H, et al. Simultaneous immunohistochemical detection of tumor cells in lymph nodes and bone marrow aspirates in breast cancer and its correlation with other prognostic factors. J Clin Oncol 2001; 19:960–971.

5. Braun S, Vogl FD, Naume B, et al. International Pooled Analysis of Prognostic Significance of Bone Marrow Micrometastasis in Patients with Stage I, II, or III Breast Cancer. N Engl J Med 2005; 353:793–802.

6. cytochemical screening for disseminated epithelial tumor cells in bone marrow. J Hematother 1994; 3:165–173.

7. Braun S, Pantel K, Muller P, et al. Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer. N Engl J Med 2000;

8. Borgen E, Beiske K, Trachsel S, et al. Immunocytochemical detection of isolated epithelial cells in bone marrow: non-specific staining and contribution by plasma cells directly reactive to alkaline phosphatase. J Pathol 1998; 185:427–434.

9. Braun S, Pantel K. Micrometastatic bone marrow involvement: detection and

10. Borgen E, Naume B, Nesland JM, et al. Standardization of the immuno-cytochemical detection of cancer cells in BM and blood: I. establishment of objecive criteria for the evaluation of immunostained cells. Cytometry 1999;

11. disseminated tumor cells in bone marrow of patients with primary breast cancer and its clinical implementation. In press 2006.

12. Witzig TE, Bossy B, Kimlinger T, et al. Detection of circulating cytokeratin-positive cells in the blood of breast cancer patients using immunomagnetic enrichment and digital microscopy. Clin Cancer Res 2002; 8:1085–1091.

13. Kraeft SK, Ladanyi A, Galiger K, et al. Reliable and sensitive identification of occult tumor cells using the improved rare event imaging system. Clin Cancer Res 2004; 10:3020–3028.

14. Borgen E, Naume B, Nesland JM, et al. Use of automated microscopy for the detection of disseminated tumor cells in bone marrow samples. Cytometry 2001; 46:215–221.

15. Kraeft SK, Sutherland R, Gravelin L, et al. Detection and analysis of cancer cells in blood and bone marrow using a rare event imaging system. Clin Cancer Res 2000; 6:434–442.

Pantel K, Schlimok G, Angstwurm M, et al. Methodological analysis of immuno-

342:525–533.

Fehm T, Braun S, Müller V, et al. A concept for the standardized detection of 1:377–388.

Committee on Cancer staging system for breast cancer. J Clin Oncol 2002; 20:

prognostic significance. Med Oncol 1999; 16:154–165.

16. Detection of disseminated tumor cells in the BM and blood 329 16. Bauer KD, de la Torre-Bueno J, Diel IJ, et al. Reliable and sensitive analysis of

occult bone marrow metastases using automated cellular imaging. Clin Cancer Res 2000; 6:3552–3559.

17. Mehes G, Luegmayr A, Ambros IM, Ladenstein R, Ambros PF. Combined auto-matic immunological and molecular cytogenetic analysis allows exact identifi-cation and quantification of tumor cells in the bone marrow. Clin Cancer Res 2001; 7:1969–1975.

18. Rosenberg R, Gertler R, Friederichs J, et al. Comparison of two density gradient centrifugation systems for the enrichment of disseminated tumor cells in blood. Cytometry 2002; 49:150–158.

19. Wiedswang G, Borgen E, Karesen R, et al. Detection of isolated tumor cells in bone marrow is an independent prognostic factor in breast cancer. J Clin Oncol 2003; 21:3469–3478.

20. Woelfle U, Breit E, Zafrakas K, et al. Bi-specific immunomagnetic enrichment of micrometastatic tumour cell clusters from bone marrow of cancer patients. J Immunol Methods 2005; 300:136–145.

21. Symmans WF, Liu J, Knowles DM, Inghirami G. Breast cancer heterogeneity: evaluation of clonality in primary and metastatic lesions. Hum.Pathol. 1995; 26:210–216.

22. Braun S, Hepp F, Sommer HL, Pantel K. Tumor-antigen heterogeneity of disse-minated breast cancer cells: implications for immunotherapy of minimal residual disease. Int J Cancer 1999; 84:1–5.

23. Datta YH, Adams PT, Drobyski WR, et al. Sensitive detection of occult breast cancer by the reverse-transcriptase polymerase chain reaction. J Clin Oncol 1994; 12:475–482.

24. Stathopoulou A, Ntoulia M, Perraki M, et al. A highly specific real-time RT-PCR method for the quantitative determination of CK-19 mRNA positive cells in peripheral blood of patients with operable breast cancer. Int J Cancer 2006.

25. Xenidis N, Perraki M, Kafousi M, et al. Predictive and prognostic value of peripheral blood cytokeratin-19 mRNA-positive cells detected by real-time polymerase chain reaction in node-negative breast cancer patients. J Clin Oncol 2006; 24:3756–3762.

26. Zippelius A, Kufer P, Honold G, et al. Limitations of reverse-transcriptase polymerase chain reaction analyses for detection of micrometastatic epithelial cancer cells in bone marrow. J Clin Oncol 1997; 15:2701–2708.

27. Bostick PJ, Chatterjee S, Chi DD, et al. Limitations of specific reverse-transcriptase polymerase chain reaction markers in the detection of metastases in the lymph nodes and blood of breast cancer patients. J Clin Oncol 1998; 16:2632–2640.

28. Jung R, Kruger W, Hosch S, et al. Specificity of reverse transcriptase polymerase chain reaction assays designed for the detection of circulating cancer cells is influenced by cytokines in vivo and in vitro. Br J Cancer 1998; 78:1194–1198.

29. Klein CA, Blankenstein TJ, Schmidt-Kittler O, et al. Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer. Lancet 2002; 360:683–689.

30. Gangnus R, Langer S, Breit E, Pantel K, Speicher MR. Genomic profiling of viable and proliferative micrometastatic cells from early-stage breast cancer patients. Clin Cancer Res 2004; 10:3457–3464.

31. Brandt B, Roetger A, Heidl S, et al. Isolation of blood-borne epithelium-derived c-erbB-2 oncoprotein-positive clustered cells from the peripheral blood of breast cancer patients. Int J Cancer 1998; 76:824–828.

32. Piccart-Gebhart MJ, Procter M, Leyland-Jones B, et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 2005; 353:1659–1672.

33. Romond EH, Perez EA, Bryant J, et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 2005; 353:1673–1684.

330 Müller and Pantel 34. Wülfing P, Borchard J, Buerger H, et al. HER2-positive circulating tumor cells

indicate poor clinical outcome in stage I to III breast cancer patients. Clin Cancer Res 2006; 12:1715–1720.

35. Braun S, Schlimok G, Heumos I, et al. ErbB2 overexpression on occult metastatic cells in bone marrow predicts poor clinical outcome of stage I-III breast cancer patients. Cancer Res. 2001; 61:1890–1895.

36. Pantel K, Schlimok G, Braun S, et al. Differential expression of proliferation-associated molecules in individual micrometastatic carcinoma cells. J Natl Cancer Inst 1993; 85:1419–1424.

37.

Systemic Therapy and Low Proliferative Activity. Clin Cancer Res 2005; 11:

38. Woelfle U, Cloos J, Sauter G, et al. Molecular signature associated with bone marrow micrometastasis in human breast cancer. Cancer Res 2003; 63:5679–5684.

39. Bernards R, Weinberg RA. A progression puzzle. Nature 2002; 418:823. 40. Calaluce R, Miedema BW, Yesus YW. Micrometastasis in colorectal carcinoma: a

review. J Surg Oncol 1998; 67:194–202. 41. Molino A, Pelosi G, Turazza M, et al. Bone marrow micrometastases in 109

breast cancer patients: correlations with clinical and pathological features and prognosis. Breast Cancer Res Treat 1997; 42:23–30.

42. Funke I, Schraut W. Meta-analyses of studies on bone marrow micrometastases: an independent prognostic impact remains to be substantiated. J Clin Oncol 1998; 16:557–566.

43. Lugo TG, Braun S, Cote RJ, Pantel K, Rusch V. Detection and measurement of occult disease for the prognosis of solid tumors. J Clin Oncol 2003; 21:2609–2615.

44. Braun S, Hepp F, Kentenich CR, et al. Monoclonal antibody therapy with edrecolomab in breast cancer patients: monitoring of elimination of disseminated cytokeratin-positive tumor cells in bone marrow. Clin Cancer Res 1999; 5:3999–4004.

45. Wiedswang G, Borgen E, Karesen R, et al. Isolated tumor cells in bone marrow three years after diagnosis in disease-free breast cancer patients predict unfavorable clinical outcome. Clin Cancer Res 2004; 10:5342–5348.

46. Diel IJ, Kaufmann M, Costa SD, et al. Micrometastatic breast cancer cells in bone marrow at primary surgery: prognostic value in comparison with nodal status. J Natl Cancer Inst 1996; 88:1652–1658.

47. Mansi JL, Gogas H, Bliss JM, et al. Outcome of primary-breast-cancer patients with micrometastases: a long-term follow-up study. Lancet 1999; 354:197–202.

48. Cote RJ, Rosen PP, Lesser ML, Old LJ, Osborne MP. Prediction of early relapse in patients with operable breast cancer by detection of occult bone marrow micrometastases. J Clin Oncol 1991; 9:1749–1756.

49. Harbeck N, Untch M, Pache L, Eiermann W. Tumour cell detection in the bone marrow of breast cancer patients at primary therapy: results of a 3-year median follow-up. Br J Cancer 1994; 69:566–571.

50. Pierga JY, Bonneton C, Vincent-Salomon A, et al. Clinical significance of immunocytochemical detection of tumor cells using digital microscopy in peripheral blood and bone marrow of breast cancer patients. Clin Cancer Res 2004; 10:1392–1400.

51. Wiedswang G, Borgen E, Schirmer C, et al. Comparison of the clinical signi-ficance of occult tumor cells in blood and bone marrow in breast cancer. Int J Cancer 2006; 118:2013–2019.

52. Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 2004; 351:781–791.

Cancer: Correlation to Bone Marrow Micrometastases, Heterogeneous Response to Müller V, Stahmann N, Riethdorf S, et al. Circulating Tumor Cells in Breast

3678–85.

16. Detection of disseminated tumor cells in the BM and blood 331 53. Cristofanilli M, Hayes DF, Budd GT, et al. Circulating tumor cells: a novel

prognostic factor for newly diagnosed metastatic breast cancer. J Clin Oncol 2005; 23:1420–1430.

54. Meng S, Tripathy D, Shete S, et al. HER-2 gene amplification can be acquired as breast cancer progresses. Proc Natl Acad Sci USA 2004; 101:9393–9398.

Chapter 17

Amit Goyal and Robert E. Mansel

Sentinel lymph node biopsy (SLNB) is the current standard of care for nodal staging in early-stage breast cancer patients who are clinically node-negative. Data from three randomised controlled trials conclusively demon-strates that SLNB is associated with less arm morbidity and better quality of life than axillary lymph node dissection (ALND). Large observational studies have shown that SLNB is associated with low local recurrence rate and similar survival to ALND. Appropriately identified patients with negative results of SLNB need not undergo completion ALND. Micrometastasis and isolated tumour cells detected by pathologic examination of the SLN with use of immunohistochemical staining or RT-PCR are currently of unknown clinical significance and they are not a required part of SLN

evaluation for breast cancer at this time.

breast cancer, blue dye, isotope, lymphatic mapping, sentinel lymph node

1.

Accurate assessment of the status of axillary lymph nodes forms an integral part of the management of breast cancer. The status of the axillary lymph nodes is the single most important predictor of survival, and the presence of lymph node metastasis dictates the need for adjuvant chemo-therapy. For patients with invasive breast cancer, the standard approach to the axilla has been axillary lymph node dissection, which consumes considerable resources and causes both acute and late morbidities for the patient. Complications of axillary lymph node dissection include lympho-

© 2007 Springer.


CF14 4XN, UK Department of Surgery, School of Medicine, Cardiff University, Heath Park, Cardiff,

Abstract:

Keywords: biopsy

INTRODUCTION

edema, pain, numbness, and restricted shoulder movement (1–5). Most

SENTINEL LYMPH NODE BIOPSY IN EARLY-STAGE BREAST CANCER

334 Goyal and Mansel

The best ideas in clinical medicine are often simple, and the sentinel node concept is of no exception. Sentinel lymph node biopsy is a mini-mally invasive alternative to axillary lymph node dissection as a way of staging breast cancer in clinically node-negative patients. A sentinel lymph node is defined as any lymph node that receives direct lymphatic drainage from a primary tumour site (Figure 1). Therefore, if the sentinel lymph node (SLN) is not involved with metastatic disease, the remainder of the lymph nodes should also be negative. Likewise, if the SLN is positive, there is a risk that higher order nodes may be involved with metastatic disease.

Figure 1. Sentinel lymph node (SLN) receives direct lymph drainage from the primary tumour.

Cabanas (6) introduced the concept of “sentinel node” in 1977 when he used lymphangiograms performed via dorsal lymphatics of the penis to demonstrate the existence of a specific node or group of nodes associ-ated with the superficial epigastric vein that predicted the nodal status of penile carcinoma. In 1992, Morton et al. (7) described lymphatic mapping utilising an intradermal isosulfan blue dye injection technique for mali-gnant melanoma and were the first to employ this concept to localise SLNs in patients with malignant melanoma. The authors demonstrated a high

women with early-stage breast cancer are node-negative, and axillary dissection in these women exposes them to the complications of this procedure without any benefit.

17. 335

tive lymphatic mapping using blue dye and applied it to breast cancer. Giuliano injected isosulfan blue dye into the breast tumour and the surroun-ding parenchyma in 174 patients. An incision was made in the axilla and all blue lymphatic channels were identified and traced to a blue node (Figure 2). A sensitivity of 88% and a false-negative rate of 6.5% were found. Subsequently, large studies have shown that using both blue dye and radioisotope together improves the SLN detection rate (percentage of patients in whom a sentinel lymph node is found) and reduces the false-negative rate (number of patients with a negative sentinel lymph node who actually have undetected axillary nodal metastases).

Figure 2. Blue-stained lymphatic leading to a blue sentinel lymph node.

Krag et al. (9) then applied radiolocalisation to the staging of breast cancer. Giuliano et al. (10) in 1994 modified Morton’s technique of intraopera-

Sentinel lymph node biopsy in early-stage breast cancer

success rate in both identifying a SLN (82%) and in achieving low false-negative rate (1%). In 1993, Alex and Krag (8) introduced the use of a radioactive tracer 99mTechnetium sulphur colloid injected intradermally around a primary melanoma site, followed by imaging and subsequent intraoperative use of a gamma probe to localise and extirpate the SLN.


Sentinel lymph node biopsy (SLNB) appears to reliably identify node-negative patients who can be spared the morbidity resulting from axillary lymph node dissection. Non-randomised studies of sentinel node biopsy followed by axillary lymph node dissection have demonstrated that one or more SLNs can be identified in more than 90% of patients with invasive

2.1 Learning curve

The data show that there is a definite learning curve for sentinel node biopsy that cannot be ignored (13–15). The ALMANAC study group has

tive training can decrease the learning curve. The surgeon’s first procedure is at higher risk of failure (especially a false negative, than subsequent ones (16). The number of procedures of the learning curve cannot be fixed for all surgeons. It has been suggested that surgeons should attend a formal course on SLNB technique and perform a minimum of 20–30 SLNB procedures in combination with axillary lymph node dissection (ALND) or with mentoring to reduce the risk of false-negative results. In the UK, a national sentinel lymph node biopsy (SLNB) training progra-mme, NEW START, was launched in October 2004. This unique surgical educational programme provides structured, multiprofessional, regional (and local) training in SLNB, supported by high quality educational DVD. The programme consists of:

• A theory day: lectures, discussions, and hands-on training models •

procedure •

procedure •

(Administration of Radioactive Substances Advisory Committee) standards: o <10% false negative in 30 consecutive cases

Minimum of 10 cases must be node positive o >90% localisation rate in 30 consecutive cases

The aim is to meet audit criteria in 30 consecutive cases. Once success-ful completion of training has been confirmed the surgeon can proceed to

2. FACTORS AFFECTING SUCCESS AND ACCURACY OF SENTINEL LYMPH NODE BIOPSY

breast cancer, with a false-negative rate of 10% or less (9, 11, 12).

Audit (validation series): 30 cases benchmarked against ARSAC

shown that standardised training programme of in-house proctored opera-

Validation phase: further 25 cases – SLNB with standard axillary

In-house proctoring: first 5 cases – SLNB with standard axillary

17. 337

2.2 Blue dye, radioisotope, or both

Many studies have sought to determine the optimal technique for SLNB. Using a combination of isotope and blue dye for sentinel node locali-sation drastically reduces the rates of failed and false-negative proce-dures. In the ALMANAC study, the success rate of harvesting the SLN by blue dye alone was 86%, by radioactive mapping alone was 86%, and

mately 4% of patients the positive SLN was found by dye alone and in 3% by isotope alone; these would have been missed by relying on a single technique of localisation. This is in line with other studies which show that the combination of radiolabeled colloid, lymphoscintigraphy, and blue dye offers the highest success rate with the fewest false negatives

dye alone was compared with a combination of blue dye and radiolabeledcolloid showed that the combined technique significantly improved the intraoperative SLN identification rate (100% vs. 86%; P = 0.002) (19).

Figure 3. Intradermal injection of radioisotope.


by a combination method was 96% (11). More importantly, in approxi-

(12, 17, 18). A small prospective randomised study in which the use of blue

offer stand-alone SLNB. Ongoing performance auditing (minimum 25 cases per annum) is encouraged though there is insufficient data to recom-mend specific volume levels to maintain proficiency.

338 Goyal and Mansel 2.3 Injection technique

The various injection techniques include intraparenchymal, dermal or subdermal, and subareolar. All three techniques have been shown to be reliable in experienced hands. The dermal technique has been shown by McMasters et al. (20) to identify the SLN in the axilla with increased frequency as compared to the peritumoral injection technique (98% vs. 90%). The dermal technique (Figure 3) results in significantly higher counts in the SLN and compares favourably with the peritumoral injection for concordance and false negatives. The subareolar technique offers many of the advantages of the dermal injection: it is easy, avoids the need for image guided injection, and increases the distance of the tumour from the axillary SLN thus eliminating shine through from upper outer quadrant lesions. The transit time is also quicker than the peritumoral technique (21). In spite of the many advantages of the dermal or subareolar technique, some institutions continue to utilise an intraparenchymal injection, because this is the only technique that will identify internal mammary lymph nodes.

2.4 Lymphoscintigraphy

Arguments have been made in favour of preoperative lymphoscinti-graphy as a ‘road map’ for surgeons (Figure 4). SLN visualisation on pre-operative lymphoscintigraphy significantly improves the intraoperative

image with a camera, it should be easily detected with the intraoperative probe.

The question is whether lymphoscintigraphy should be done at all since

and most surgeons are concerned with mapping only to the axilla. In add-ition, the demonstration of extraaxillary lymphatic drainage only becomes important when a treatment decision is to be made based on the finding. Given the time and cost required to perform preoperative lymphoscintigra-phy its routine use does not appear to be justified. It may be valuable for surgeons in the learning phase to decrease the learning curve and in pati-ents who have an increased risk of intraoperative failed localisation (obese or old patients). A negative preoperative lymphoscintiscan predicts inability to localise with the hand held gamma probe. Therefore, if SLN is not visualised on lymphoscintigraphy then the addition of intraoperative blue dye is recommended to increase the likelihood of SLN identification (23).

2.5 Lymphatic tumour burden

It has been suggested that the accurate identification of the SLN by lymphatic mapping could be compromised if there is extensive tumour

SLN identification rate (22, 23). If a SLN takes up enough radiocolloid to

SLNs are still identified in the majority of image negative patients (22–25)

17. 339 infiltration of afferent lymphatic channels or draining lymph nodes. Distal obstruction of the lymphatics by tumour and extensive tumour infil-tration of the draining lymph nodes may prevent the migration of blue dye and radioisotope to the SLN, adversely affecting SLN identification. Lymph fluid re-routing may cause an alternative non-sentinel node to become “sentinel”, increasing the risk of false-negative biopsy (26). We found that in the individual SLN, the percentage replacement by tumour and extranodal invasion of tumour are markers of lymphatic obstruction and are significantly associated with reduced radioisotope uptake (27). More than 50% replacement of the node by tumour will compromise the lymphatic flow and may lead to failed localisation of the node if the radioisotope is used alone (Figure 5). However, SLN identification using blue dye is not compromised by increased SLN tumour burden. The afferent lymphatic leading to the blocked node may be patent. The surgeon can identify the tumour-replaced node by following the blue lymphatic leading to the node. This result further supports combination of blue de and radioisotope being used to optimise the localisation rate.

2.6 Multiple sentinel lymph nodes

The issue regarding number of sentinel nodes requiring removal conti-nues to be hotly debated. One might hypothesise that a regional lymphatic basin first drains to a single SLN, but in actual experience surgeons mostly

We found that most patients have multiple SLNs (29). This may reflect migration of dye or isotope from the “true” SLN into secondary echelon nodes, or simply normal anatomic variation in which the lymphatics of a given site drain simultaneously to more than one SLN.

In the ALMANAC study, the false-negative rate was significantly less in patients who had multiple SLNs (three or more) removed than in

results suggest that removal of several SLNs decreases the false-negative rate. That is not to say that more than one SLN must be removed in every patient. Rather, these data suggest that more than one SLN is present in the majority of patients, and that identification of multiple SLNs is impor-tant for reducing the false-negative rate.

Although removal of multiple SLNs is important to accurately stage the axilla, indiscriminate removal of inordinate number of axillary nodes is not justified as they may be secondary echelon nodes and may potentially worsen the morbidity of SLNB. Therefore, the surgeon will naturally wonder if it is safe to stop after removing two, three, or four SLNs, assume-ing that any metastasis would be contained in these nodes, or whether one should continue until all blue or hot SLNs have been removed.

those with one or two removed (1.1% vs. 10.1% and 8.5%) (29). These


identify more than one SLN regardless of the technique used (9, 11, 28).


A B

Figure 4. Lymphoscintiscan (Anterior View) showing multiple “hot” sentinel lymph nodes. A: “hot” sentinel lymph nodes; B: Radioisotope injection site.

17. 341

Figure 5. Modelling of nodal invasion.



Only 50 (6.2%) patients in the above study had more than 4 SLNs removed (29). Removal of three SLNs in this series identified 98.5% of node-positive patients and four SLNs identified 99.6% of the node-positive patients. Only one patient had the first positive SLN beyond this, at node number six. Removal of more than four SLNs did not appreciably increase

McCarter et al. (30). In their series, removal of three SLNs identified 98% of node-positive patients and four SLNs identified 99%. These data suggest that for identification of “true” SLNs the surgeon can stop after sampling four nodes.

2.7 Tumour location, size, and grade

Tumour location and size influences the successful identification of the SLN but not the false-negative rate. Tumours located in the upper outer quadrant appear to have a higher SLN identification rate compared to other tumour locations (ALMANAC trial: upper outer 98.2% vs. 89.1% lower inner, p = 0.008; NSABP-32 trial: 98.7% outer central vs 93.9%

shorter transit distance for the blue dye or radioisotope from the injection site to the axilla. Tumour size did not affect successful identification of SLN in the ALMANAC study but was found to adversely affect SLN identi-

Tumour grade does not influence successful identification of the SLN but may adversely affect the false-negative rate (false-negative rate grade I 0% vs. grade II 4.7% vs. grade III 9.6%, p = 0.022) (11). grade III tumours have a higher incidence of nodal metastases, thereby have an increased risk of lymphatic obstruction and re-routing of tracer leading to a false-negative result. High false-negative rates may have a direct adverse impact on patient care including accurate staging, treatment decision making and long-term outcomes including survival. Therefore, caution is required when applying the SLNB procedure in patients at considerably increased risk of lymph node positive disease.

3.1 Morbidity

of complications associated with SLNB when compared with ALND (32–34). SLNB reduces but does not completely eliminate the risk of

lower inner, p < 0.0001) (11,31). The simplest explanation relates to the

fication in the NSABP-32 trial (T3 95.9% vs. T2 98.4%, p = 0.03) (11, 31).

3. PROS AND CONS OF SENTINEL LYMPH NODE BIOPSY

Data from 3 RCTs conclusively demonstrates a marked diminution

the accuracy of finding a positive node. Similar findings were reported by

17. 343

morbidity associated with SLNB was compared with that associated with

showed that less lymphoedema, shoulder discomfort, sensory deficits, and infections were associated with SLNB than with ALND. Quality of life was found to be superior for patients who had SLNB. Moderate or severe lymphoedema was reported more frequently by patients in the standard axillary treatment group than by patients in the SLNB at 1, 3, 6, and 12 months after surgery (all P<.001). The relative risk of any lymphoe-dema for the SLNB group compared with the standard axillary treatment group at 12 months was 0.37 (95% CI = 0.23 to 0.60). Sensory loss at 1 month after surgery was reported by 18% of patients in the SLNB, compared with 62% of patients in the standard axillary treatment group. At 12 months after surgery, the percentage of patients reporting sensory loss declined to 11% in the sentinel lymph node biopsy group and to 31% in the standard axillary treatment group; at all time points, statistically significantly more patients in the standard treatment group than in the SLNB groups reported sensory deficit (P<.001 for all). The relative risk of sensory deficit at 12 months was 0.37 (95% CI = 0.27 to 0.50) in favour of the SLNB group. Compared with patients in the sentinel node biopsy group, those in the standard axillary treatment group displayed statistically significant impairment of shoulder flexion and abduction on the ipsilateral side at 1 month after surgery (P = 0.004 and 0.001, respectively). However, shoulder flexion and abduction improved rapidly at the subsequent timepoints in both groups, and differences between the groups were no longer statistically significant.

3.2 Local recurrence and survival

It is premature to make a definitive comment on local recurrence rate or survival following SLNB. These questions will be answered by the ongoing National Surgical Adjuvant Bowel and Breast Project (NSABP-32) and the American College of Surgeons Oncology Group (ACOSOG) trials (Table 1).

However, 12-month follow-up data from the ALMANAC trial suggests that sentinel lymph node biopsy is associated with a low local recurrence rate and survival is similar to that after ALND. A large non-randomised study from Memorial Sloan–Kettering, New York, reported only 0.1% local recurrence in sentinel lymph node-negative patients at a median follow-up of 31 months (35), and an Italian study of 953 patients had a similar recurrence rate of 0.3% at a median follow-up of 38 months (36).

Lymphatic Mapping Against Nodal Clearance) trial (34) in which the

conventional ALND were recently published (34). Analysis at 12 months


lymphoedema. Early results from the large ALMANAC (Axillary


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3.3 Allergic reactions

Allergic reactions to the dye occur in no more than 1–2% of patients who have SLNB; most of these reactions are hives, which are often strik-ingly blue and respond to antihistamines. True anaphylactic reactions are rare, occurring in approximately 0.25–0.5% of patients (37).

3.4 Radiation hazard

Radiation exposure to the patient, family and friends, surgeon, staff, and pathologist appears to be small. For mapping with a radiolabeled colloid,

bone scan (38). No isolation, precautions, or special radiation monitoring are required.

4.

4.1 Multicentric cancer

In the past, multicentric or multifocal carcinoma was considered a contraindication to SLNB on the assumption that tumours located in dif-ferent breast quadrants drain to different lymph node sites and not to the same sentinel node. Therefore SLNB would result in inaccurate axillary

of the sentinel node for the subareolar injection site and the peritumoral injection site. The SLN was successfully identified in 89.9% using the blue dye and 94.2% using the technetium radiocolloid. The concordance rate between the two techniques was 89.9%. Borgstein et al. (40) demonstrated a 100% concordance in localising the SLN utilizing an intradermal injection of blue dye and an intraparenchymal injection of radioisotope; the concordance rate in the study by Linehan et al. (41) was 95%. As the different SLNB techniques accurately lead to identification of the same SLN, these studies support the hypothesis that all quadrants of the breast drain through common afferent channels to a common axillary sentinel node. In the ALMANAC trial validation phase (42), 75 of the 842 patients had multifocal or multicentric lesions on histopathologic examination. SLN was successfully identified in 95% (71/75) patients with multifocal tumours. SLNB with the peritumoral injection technique accurately pre-dicted lymph node status in 95.8% (68/71) patients with multifocal or multicentric breast cancer. The false-negative rate (8.8%) and the negative predictive value (92.5%) were similar to that in the unifocal group.

17.

345

IN SPECIFIC CLINICAL SCENARIOS ROLE OF SENTINEL LYMPH NODE BIOPSY

(3.7–37 MBq) is approximately 4% of that administered for a conventional an injected dose of technetium (99mTc) in the range of 0.1–1.0 mCi

lymph node staging. Klimberg et al. (39) reported equal identification rates


346 Goyal and Mansel Therefore, lymphatic mapping in patients with multifocal or multicentric cancers may also be possible, allowing these patients to be spared the morbidity of ALND. Several other non-randomised series have demons-

4.2

Patients with DCIS have a low (1%) incidence of lymph node metastasis using conventional hematoxylin-eosin (H&E) staining and have an excellent long-term prognosis (98% survival). Given this background, SLNB in all patients with DCIS cannot be justified. However, 10–38% of patients with DCIS will be found at definitive surgery to have an invasive cancer. SLNB following lumpectomy is associated with increased failed locali-sation and false-negative rates (31) and is impossible after a mastectomy. Therefore, if SLNB is not performed at the time of the definitive operative procedure, a significant number of patients found to have an invasive cancer will require a second operative procedure and, in all likelihood, axillary lymph node dissection. The combination of low morbidity and greater risk for invasive carcinoma at the time of definitive resection make SLNB an important consideration in high-risk patients with DCIS. Historically, risk factors reportedly associated with invasive disease have included large tumours, high-grade tumours, tumours with comedo-type necrosis, and presence of palpable mass or mass that is appreciated by imaging studies (45–49).

It is difficult to compare different series as biopsy techniques, grading system for DCIS, and patient populations have varied. Furthermore, most series numbers are not large enough to provide the power to detect signi-ficance. At the present time there is no consensus on the predictive factors and this issue remains controversial. In a large study reported recently, we found two independent predictors of invasive cancer in patients with an initial diagnosis of DCIS: clinically palpable mass and mammographic mass (50). Presence of a clinically palpable mass increased the risk of invasive carcinoma 5-fold (odds ratio 5.09, 95% CI 3.06–8.48); while a mammographic mass increased the risk of invasive disease 7-fold (odds ratio 7.37, 95% CI 3.27–16.64). SLNB should be performed at the time of the initial procedure in this subgroup of patients to avoid a second opera-tive procedure for axillary nodal staging. In addition, SLNB should be performed in patients undergoing breast reconstruction or mastectomy because both preclude SLNB if invasive disease is subsequently discovered.

4.3 Male breast cancer

Carcinoma of the male breast is a relatively rare disease that accounts for less than 1% of all cases of cancer in men. Optimal management of breast cancer in men is unknown because the rarity of the disease precludes

Ductal carcinoma in situ (DCIS)

trated that SLNB can be applied in women with multifocal disease (43, 44).

17. 347 large randomised trials. Most information has been obtained from small, single-institution, retrospective studies or by extrapolation from breast cancer trials in women. There are increasing reports in the literature confir-ming the feasibility and accuracy of SLNB in male breast cancer patients with negative axillae, suggesting that SLNB may be offered as a surgical management option to male breast cancer patients by surgeons who are

underwent SLNB in the ALMANAC trial. SLN was identified in all of them and there was no false-negative result (53).

4.4 Neoadjuvant chemotherapy

Neoadjuvant chemotherapy has been considered a relative contraindi-cation to sentinel lymph node mapping as there are some concerns that this may adversely affect SLN localisation and false-negative rates. How-ever, an increasing body of data suggests that SLNB accurately predicts the status of the axillary nodes in patients who have received preoperative chemotherapy. Overall, the studies of preoperative chemotherapy suggest that the failed localisation rates (85–96%) and the false-negative rates (0–33%) may be slightly higher in this setting (54–59). However, the SLNB is clearly not the major factor determining the use of adjuvant therapy in these patients, so the slightly increased false-negative rate is unlikely to cause harm. Neoadjuvant therapy may eradicate foci of disease in axillary lymph nodes, the long-term clinical significance of negative findings on SLNB after preoperative treatment is less clear. This potential loss of prognostic information may complicate clinical decision making for local treatment, such as, whether completion ALND is indicated, whether radiation is indicated after mastectomy, or which regions should be irradiated after lumpectomy. Therefore, SLNB should be considered before primary systemic therapy, with ALND performed after chemo-therapy if disease is present in the SLN (60).

4.5 Internal mammary node

Interest in evaluating internal mammary nodes (IMNs) has recently been rejuvenated with the advent and widespread acceptance of lymphatic mapping and SLNB in breast cancer. The lymphoscintiscan demonstrates mapping to IMNs in 0–35% patients (61–63). In contrast to traditional thin-king, internal mammary drainage can occur with tumours in any quadrant (61). Most patients with drainage to internal mammary nodes also have axillary drainage and surgeons are reluctant to perform internal mammary lymph node biopsies even if drainage to this site is demonstrated because this procedure is not performed currently. Determination of internal mam-mary nodal involvement may alter adjuvant therapy. However, this repre-sents <1% of patients as few patients have an internal mammary SLN


experienced with the technique (51, 52). Nine men with breast cancer


Moreover, many patients currently receive adjuvant systemic therapy basedupon tumour characteristics (size and grade), even if node negative. The fact that IMN dissection does not improve survival (64) poses a problemfor indication of adjuvant radiotherapy to this basin. The effect on survivalof radiation therapy on the internal mammary chain is the subject of the ongoing EORTC 22922 trial. Therefore, internal mammary sentinel lymphnode biopsy is not recommended in patients with drainage to this basin.

4.6 Prior breast or axillary surgery

Lymphatic drainage of the breast may be altered because of previous surgery. NSABP-32 data (31) suggests that prior excisional biopsy signific-antly increases the false-negative rate (FNA/Core 8.0% vs. Excisional biopsy 15.2%, p = 0.02) while not affecting the failed localisation rate. On the basis of these data, SLNB should not be performed after excisional biopsy.

SLNB after axillary surgery has not been widely studied, and therefore cannot be recommended in this setting.

4.7 Pregnancy

There is minimal data on safety and test performance of SLNB during pregnancy. Blue dyes are contraindicated in pregnant patients but radio-isotopes are probably safe. Keleher et al. (65) showed that the potentially absorbed radiation dose to the fetus is less than 50mGy threshold absorbed dose for adverse effects to the fetus. Recent reports suggest that SLNB is feasible in pregnant patients without adversely affecting the

SLNB cannot be recommended in pregnant women with breast cancer.

4.8 Older age and obesity

High BMI adversely influences successful mapping of SLNs. Patient age did not alter SLN localisation in the ALMANAC trial (11), though it has been reported in several other studies which have shown that accurate identification of the SLN decreases with increasing age as well as weight

are unclear. Sentinel node identification may be difficult in obese women because of the higher content of subcutaneous and axillary adipose tissue. Furthermore, the increased fatty tissue may impede the flow of the tracer through the lymphatics in the breasts of these patients. Additionally, the lymph nodes in obese patients may have undergone fatty degeneration reducing their capacity to concentrate the tracer.

containing metastatic cancer when the axillary SLN is negative (61, 63).

fetus (66, 67). However, due to small numbers and insufficient follow-up,

(31, 68, 69). The specific causes for mapping failure in overweight patients

17. 349

Older women (>50 years) are more likely to have failure of SLN visuali-sation on preoperative lymphoscintigraphy (23). There is a decrease in tissue turgor in older women, with a resultant decrease in the hydrostatic intralymphatic pressure that drives the mapping agent into the node. Even if the agent is delivered successfully to the node, it may not be concen-trated because of the limited sinusoidal space that remains in a fat-replaced node, another feature found more commonly in older patients.

These findings, however, do not contraindicate SLNB in these indivi-duals as the rate of successful localisation remains high and unsuccessful mapping does not adversely affect their prognosis or treatment.

4.9 Pathological considerations

The role of immunohistochemical (IHC) staining of the SLNs and signi-ficance of micrometastases remains an area of significant controversy. SLNB and a focused pathologic evaluation have resulted in upstaging of approximately 10–20% of breast cancer patients. The question is whether this detectable disease is clinically significant. All the present literature is retrospective in nature and the results are very inconclusive. Prospective studies are ongoing to evaluate the prognostic significance of micrometa-stases (IBCSG 23–01 trial, ACOSOG Z0010 trial) (Table 1). Therefore, until we have good data, clinical decisions should not be made based on inadequate studies.

4.10 Completion ALND in patients with a positive SLN

The need for completion ALND in patients with a positive SLN who will receive systemic cytotoxic therapy is also controversial. Our data show that in 52% of patients the sentinel node is the only positive

removed and the size of metastasis in the SLN were significant positive predictors for additional positive nodes in the axilla, while the number of negative sentinel nodes removed was a significant negative predictor. Unfortunately, neither of these characteristics, alone or in combination,

ment to identify a subset of patients who can safely forgo completion ALND (70). As a result, the role of completion ALND in these patients is controversial. This important clinical question is being addressed by the ACOSOG Z0011 trial (Table 1). In this trial, patients with a positive SLN are randomised to completion ALND or no additional axillary therapy.

was a strong enough predictor of non-sentinel lymph node tumour involve-

node (70). On multivariate analysis, the number of positive sentinel nodes


350 Goyal and Mansel 5. CONCLUSION

Sentinel lymph node biopsy accurately stages the axilla and is associated with reduced arm morbidity and better quality of life compared with axillary lymph node dissection. It should be the treatment of choice for patients who have early-stage breast cancer with clinically negative nodes. Breast cancer patients with no metastatic disease in the SLNs need not undergo completion ALND.

REFERENCES

1. Warmuth MA et al. Complications of axillary lymph node dissection for carcinoma of the breast: a report based on a patient survey. Cancer 1998; 83:

2. Velanovich V, Szymanski W. Quality of life of breast cancer patients with

3. Roses DF, Brooks AD, Harris MN, Shapiro RL, Mitnick J. Complications of level I and II axillary dissection in the treatment of carcinoma of the breast. Ann Surg 1999; 230: 194–201.

4. Hack TF, Cohen L, Katz J, Robson LS, Goss P. Physical and psychological morbidity after axillary lymph node dissection for breast cancer. J Clin Oncol 1999; 17: 143–149.

5. Maunsell E, Brisson J, Deschenes L. Arm problems and psychological distress after surgery for breast cancer. Can J Surg 1993; 36: 315–320.

6. Cabanas RM. An approach for the treatment of penile carcinoma. Cancer 1977; 39: 456–466.

7. Morton DL et al. Technical details of intraoperative lymphatic mapping for early stage melanoma. Arch Surg 1992; 127: 392–399.

8. Alex JC, Krag DN. Gamma-probe guided localization of lymph nodes. Surg Oncol 1993; 2: 137–143.

9. N Engl J Med 1998; 339: 941–946.

10. Giuliano AE, Kirgan DM, Guenther JM, Morton DL. Lymphatic mapping and sentinel lymphadenectomy for breast cancer. Ann Surg 1994; 220: 391–398.

11. Goyal A, Newcombe RG, Chhabra A, Mansel RE. Factors affecting failed localisation and false-negative rates of sentinel node biopsy in breast cancer - results of the ALMANAC validation phase. Breast Cancer Res Treat 2006; 99: 203–208.

12. McMasters KM et al. Defining the optimal surgeon experience for breast cancer sentinel lymph node biopsy: a model for implementation of new surgical techniques. Ann Surg 2001; 234: 292–299.

13. Morrow M et al. Learning sentinel node biopsy: results of a prospective randomized trial of two techniques. Surgery 1999; 126: 714–720.

14. Simmons RM. Review of sentinel lymph node credentialing: how many cases are enough? J Am Coll Surg 2001; 193: 206–209.

15. Tafra L, McMasters KM, Whitworth P, Edwards MJ. Credentialing issues with sentinel lymph node staging for breast cancer. Am J Surg 2000; 180: 268–273.

16. Clarke D, Newcombe RG, Mansel RE. The learning curve in sentinel node

1362–1368.

lymphedema. Am J Surg 1999; 177: 184–187.

biopsy: the ALMANAC experience. Ann Surg Oncol 2004; 11: 211S–215S.

Krag D et al. The sentinel node in breast cancer–a multicenter validation study.

17. 351

18. Derossis AM et al. A trend analysis of the relative value of blue dye and isotope localization in 2,000 consecutive cases of sentinel node biopsy for breast cancer. J Am Coll Surg 2001; 193: 473–478.

19. Hung WK et al. Randomized clinical trial comparing blue dye with combined dye and isotope for sentinel lymph node biopsy in breast cancer. Br J Surg 2005; 92: 1494–1497.

20. McMasters KM et al. Dermal injection of radioactive colloid is superior to peritumoral injection for breast cancer sentinel lymph node biopsy: results of a multiinstitutional study. Ann Surg 2001; 233: 676–687.

21. Kern KA. Lymphoscintigraphic anatomy of sentinel lymphatic channels after subareolar injection of Technetium 99m sulfur colloid. J Am Coll Surg 2001; 193: 601–608.

22. Birdwell RL et al. Breast cancer: variables affecting sentinel lymph node visuali-zation at preoperative lymphoscintigraphy. Radiology 2001; 220: 47–53.

23. Goyal A et al. Role of routine preoperative lymphoscintigraphy in sentinel node biopsy for breast cancer. Eur J Cancer 2005; 41: 238–243.

24. Burak WE, Jr. et al. Routine preoperative lymphoscintigraphy is not necessary prior to sentinel node biopsy for breast cancer. Am J Surg 1999; 177: 445–449.

25. McMasters KM et al. Preoperative lymphoscintigraphy for breast cancer does not improve the ability to identify axillary sentinel lymph nodes. Ann Surg 2000; 231: 724–731.

26. Borgstein PJ et al. Sentinel lymph node biopsy in breast cancer: guidelines and pitfalls of lymphoscintigraphy and gamma probe detection. J Am Coll Surg 1998; 186: 275–283.

27. Goyal A, Douglas-Jones AG, Newcombe RG, Mansel RE. Effect of lymphatic tumor burden on sentinel lymph node biopsy in breast cancer. Breast J 2005;

28. Giuliano AE, Jones RC, Brennan M, Statman R. Sentinel lymphadenectomy in breast cancer. J Clin Oncol 1997; 15: 2345–2350.

29. Goyal A, Newcombe RG, Mansel RE. Clinical relevance of multiple sentinel nodes in patients with breast cancer. Br J Surg 2005; 92: 438–442.

30. McCarter MD, Yeung H, Fey J, Borgen PI, Cody HS, III. The breast cancer patient with multiple sentinel nodes: when to stop? J Am Coll Surg 2001; 192: 692–697.

31. Julian TB et al. Preliminary technical results of NSARP B-32, a randomized phase III clinical trial to compare sentinel node resection to conventional axillary dissection in clinically node-negative breast cancer patients. Breast Cancer Res Treat 2004; 88: S11–S12.

32. Veronesi U et al. A randomized comparison of sentinel-node biopsy with

33. Purushotham AD et al. Morbidity after sentinel lymph node biopsy in primary breast cancer: results from a randomized controlled trial. J Clin Oncol 2005; 23: 4312–4321.

34. Mansel RE et al. Randomized multicenter trial of sentinel node biopsy versus

35. Naik AM et al. The risk of axillary relapse after sentinel lymph node biopsy for breast cancer is comparable with that of axillary lymph node dissection: a follow-up study of 4008 procedures. Ann Surg 2004; 240: 462–468.

36. Veronesi U et al. Sentinel node biopsy in breast cancer: early results in 953 patients with negative sentinel node biopsy and no axillary dissection. Eur J Cancer 2005; 41: 231–237.

11: 188–194.

routine axillary dissection in breast cancer. N Engl J Med 2003; 349: 546–553.


17. Hill AD et al. Lessons learned from 500 cases of lymphatic mapping for breast cancer. Ann Surg 1999; 229: 528–535.

J Natl Cancer Inst 2006; 98: 599–609. standard axillary treatment in operable breast cancer: the ALMANAC Trial.


39. Klimberg VS et al. Subareolar versus peritumoral injection for location of the sentinel lymph node. Ann Surg 1999; 229: 860–864.

40. Borgstein PJ, Meijer S, Pijpers R. Intradermal blue dye to identify sentinel lymph-node in breast cancer. Lancet 1997; 349: 1668–1669.

41. Linehan DC et al. Intradermal radiocolloid and intraparenchymal blue dye injection optimize sentinel node identification in breast cancer patients. Ann Surg Oncol 1999; 6: 450–454.

42. Goyal A et al. Sentinel lymph node biopsy in patients with multifocal breast cancer. Eur J Surg Oncol 2004; 30: 475–479.

43. Kumar R et al. Retrospective analysis of sentinel node localization in multifocal, multicentric, palpable, or nonpalpable breast cancer. J Nucl Med 2003; 44: 7–10.

44. Schrenk P, Wayand W. Sentinel-node biopsy in axillary lymph-node staging for patients with multicentric breast cancer. Lancet 2001; 357: 122.

45. Hoorntje LE et al. The finding of invasive cancer after a preoperative diagnosis of ductal carcinoma-in-situ: causes of ductal carcinoma-in-situ underestimates with stereotactic 14-gauge needle biopsy. Ann Surg Oncol 2003; 10: 748–753.

46. Mittendorf EA, Arciero CA, Gutchell V, Hooke J, Shriver CD. Core biopsy diagnosis of ductal carcinoma in situ: an indication for sentinel lymph node biopsy. Curr Surg 2005; 62: 253–257.

47. Renshaw AA. Predicting invasion in the excision specimen from breast core needle biopsy specimens with only ductal carcinoma in situ. Arch Pathol Lab Med 2002; 126: 39–41.

48. Wilkie C, White L, Dupont E, Cantor A, Cox CE. An update of sentinel lymph node mapping in patients with ductal carcinoma in situ. Am J Surg 2005; 190: 563–566.

49. Yen TW et al. Predictors of invasive breast cancer in patients with an initial diagnosis of ductal carcinoma in situ: a guide to selective use of sentinel lymph node biopsy in management of ductal carcinoma in situ. J Am Coll Surg 2005; 200: 516–526.

50. Goyal A et al. Is there a role of sentinel lymph node biopsy in ductal carcinoma in situ?: analysis of 587 cases. Breast Cancer Res Treat 2006; 98: 311–314.

51. Albo D et al. Evaluation of lymph node status in male breast cancer patients: a role for sentinel lymph node biopsy. Breast Cancer Res Treat 2003; 77: 9–14.

52. Hill AD, Borgen PI, Cody HS, III. Sentinel node biopsy in male breast cancer. Eur J Surg Oncol 1999; 25: 442–443.

53. Goyal A et al. Sentinel lymph node biopsy in male breast cancer patients. Eur J Surg Oncol 2004; 30: 480–483.

54. Fernandez A et al. Gamma probe sentinel node localization and biopsy in breast cancer patients treated with a neoadjuvant chemotherapy scheme. Nucl Med Commun 2001; 22: 361–366.

55. Balch GC, Mithani SK, Richards KR, Beauchamp RD, Kelley MC. Lymphatic mapping and sentinel lymphadenectomy after preoperative therapy for stage II and III breast cancer. Ann Surg Oncol 2003; 10: 616–621.

56. Haid A et al. Is sentinel lymph node biopsy reliable and indicated after preopera-tive chemotherapy in patients with breast carcinoma? Cancer 2001; 92: 1080–1084.

57. Tafra L, Verbanac KM, Lannin DR. Preoperative chemotherapy and sentinel lymphadenectomy for breast cancer. Am J Surg 2001; 182: 312–315.

58. Nason KS et al. Increased false negative sentinel node biopsy rates after preoperative chemotherapy for invasive breast carcinoma. Cancer 2000; 89: 2187–2194.

37. Montgomery LL et al. Isosulfan blue dye reactions during sentinel lymph node mapping for breast cancer. Anesth Analg 2002; 95: 385–388.

38. Waddington WA et al. Radiation safety of the sentinel lymph node technique in breast cancer. Eur J Nucl Med 2000; 27: 377–391.

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61. Goyal A, Newcombe RG, Mansel RE. Clinical relevance of internal mammary node drainage in sentinel node biopsy for breast cancer. Journal of Clinical Oncology 2005; 23: 8S.

62. Klauber-DeMore N, Bevilacqua JL, Van Zee KJ, Borgen P, Cody HS, III. Compre-hensive review of the management of internal mammary lymph node metastases in breast cancer. J Am Coll Surg 2001; 193: 547–555.

63. Mansel RE, Goyal A, Newcombe RG. Internal mammary node drainage and its role in sentinel lymph node biopsy: the initial ALMANAC experience. Clin Breast Cancer 2004; 5: 279–284.

64. Veronesi U, Marubini E, Mariani L, Valagussa P, Zucali R. The dissection of internal mammary nodes does not improve the survival of breast cancer patients. 30-year results of a randomised trial. Eur J Cancer 1999; 35: 1320–1325.

65. Keleher A, Wendt R, III, Delpassand E, Stachowiak AM, Kuerer HM. The safety of lymphatic mapping in pregnant breast cancer patients using Tc-99m sulfur colloid. Breast J 2004; 10: 492–495.

66. Gentilini O et al. Safety of sentinel node biopsy in pregnant patients with breast cancer. Ann Oncol 2004; 15: 1348–1351.

67. Mondi MM, Cuenca RE, Ollila DW, Iv JH, Levine EA. Sentinel Lymph Node Biopsy During Pregnancy: Initial Clinical Experience. Ann Surg Oncol 2006.

68. Cox CE et al. Age and body mass index may increase the chance of failure in sentinel lymph node biopsy for women with breast cancer. Breast J 2002; 8: 88–91.

69. Derossis AM, Fey JV, Cody HS, III, Borgen PI. Obesity influences outcome of sentinel lymph node biopsy in early-stage breast cancer. J Am Coll Surg 2003; 197: 896–901.

70. Goyal A, Douglas-Jones A, Newcombe RG, Mansel RE. Predictors of non-sentinel lymph node metastasis in breast cancer patients. Eur J Cancer 2004; 40: 1731–1737.


59. Miller AR et al. Analysis of sentinel lymph node mapping with immediate pathologic review in patients receiving preoperative chemotherapy for breast carcinoma. Ann Surg Oncol 2002; 9: 243–247.

60. Sabel MS et al. Sentinel node biopsy prior to neoadjuvant chemotherapy. Am J Surg 2003; 186: 102–105.

Chapter 18

Adam I. Riker1, SuHu Liu1, Mona Hagmaier1, Matthew J. D’Alessio1,and Charles E. Cox2 1University of South Alabama-Mitchell Cancer Institute, 301 North University Blvd.,

2

Center, 13902 Magnolia Drive, Tampa, FL 33612, USA Mitchell Cancer Institute,University of South Alabama, Mobile, AL, USA

Abstract: Metastatic breast cancer can be a difficult problem to manage surgically as the sole mode of treatment. In certain cases, surgical management is the initial treatment of choice, such as sentinel lymph node mapping and nodal dissections for locoregional control. Other situations, such as metastatic breast cancer to solid organs, such as the brain, liver, and lung, are dictated by individual patient characteristics and the overall clinical situation. Clearly, surgery has become a part of the broader management schema in treating patients with metastatic breast cancer, with developing technologies, such as stereotactic radiosurgery, greatly enhancing our ability to treat such patients. Surgery is a single, but important, tool that should be combined with other modes of therapy, such as hormonal therapy and radiation therapy, to optimize treatment strategies and patient outcomes. There remains a fair amount of controversy as to the clinical significance of micrometastatic disease within the lymph nodes and bone marrow, a relevant topic that is actively being examined by clinicians and researchers from around the world. Regardless, the role of surgery for patients with metastatic breast cancer has become a central part of the multidisciplinary care of such patients, with this review providing an overview of the various aspects of surgical management of metastatic disease.

Keywords: metastastic breast cancer, micrometastatic, sentinel lymph node, surgical management, locoregional recurrence, bone metastasis, lung metastasis, liver metastasis

© 2007 Springer.


SURGICAL MANAGEMENT OF PATIENTS WITH METASTATIC BREAST CANCER

MSB 2015, Mobile, AL 36688, USA; University of South Florida, Moffitt Cancer

356 1. EFFECT OF PRIMARY BREAST CANCER

EXTIRPATION IN STAGE IV PATIENTS

Metastatic breast cancer is considered by many to be an incurable disease and therefore the treatment of such patients, whether with systemic therapy (chemotherapy, hormonal therapy), or with surgery is considered palliative (1). However, it has been suggested by others that there may be role for curative surgery in the treatment of selected patients with metastatic breast cancer (2, 3). Khan et al. were one of the first to describe an aggressive local surgical approach to metastatic breast cancer as a possible way of improving overall survival. They retrospect-tively examined over 16,000 patients with stage IV breast cancer derived from the National Cancer Data Base between 1990–1993, finding that 43% received either no operation or a variety of other diagnostic or palliative procedures and 57% underwent either a partial or total mastectomy (3). This study revealed that those patients who had free surgical margins had an improved overall 3-year survival compared to those not surgically treated. Multivariate analysis also showed that the overall number of metastatic sites, the type of metastatic burden, and the extent of resection of the primary tumor as significant independent prognostic factors.

Others began to focus on the true impact of local surgery on overall survival of women who initially presented with metastatic breast cancer and an intact primary tumor. Babiera et al. retrospectively analyzed 224 patients with metastatic breast cancer, of which 82 (37%) underwent surgical removal of the primary and the remainder (63%) were treated without surgery (4). They found that the patients who underwent removal of the primary tumor had a significant improvement in metastatic progression-free survival compared to the group that did not undergo surgery. Additionally, this study contradicts the notion that surgical removal of the primary is associated with an enhancement of distant metastatic tumor growth, a concept supported by several previous studies (5–7).

Rapiti et al. compared 300 patients with metastatic breast cancer who presented with an intact primary tumor of which 58% had no surgical intervention of the primary and 42% had a complete surgical removal of the primary (8). They found that the 5-year survival for those undergoing surgery with negative surgical margins was 27%, 16% with positive surgical margins, 12% with an unknown margin status, and 12% for women who did not undergo any form of surgery for their primary tumor. Thus, surgery of the primary tumor (with negative surgical margins) in patients with metastatic breast cancer was significantly linked to a >50% reduction in breast cancer mortality compared to women who did not have surgical treatment.

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18. Surgical management of metastatic breast cancer 357

It has long been suggested that tumor cells from various histologies are capable of producing immunosuppressive cytokines and able to escape recognition by the host immune system through a variety of cellular mechanisms (9–11). Indeed, several lines of evidence suggest that early and complete surgical resection of distant metastatic disease may provide a survival benefit for patients with melanoma. Danna et al. elegantly demonstrate that the surgical removal of the primary tumor in mice (BALB/c-derived transplantable tumor 4T1 mammary carcinoma) with metastatic disease resulted in rebounding of antibody and cell-mediated responses and restoration of immunocompetence (12).

This approach, best described as “complete cytoreductive immuno-therapeutic surgery (CCIS),” which removes the bulk of tumor burden in most cases, is hypothesized to allow for an improved overall function of

CCIS of metastatic disease may allow the host immune system to over-come tumor-induced immunosuppression, recently describing his results from the premature closure of the onamelatucel-L (Canvaxin) trial designed to assess the efficacy of this vaccine in both stage III and stage IV melanoma patients. Although both trials were closed to further accrual early due to an interim analysis that revealed no probable efficacy over placebo, some very interesting results were nonetheless found in stage IV patients. The design of the Canvaxin trial for stage IV patients required that all patients receive definitive surgical removal of all metastatic disease prior to entry into the trial. Although there was no advantage to receiving the Canvaxin vaccine over placebo, they found

but instead to complete surgical resection of metastatic disease (14). This provides a clear proof-of-principle that CCIS of metastatic disease may be a critical part of the overall strategy for improvement of long-term survival in selected patients with advanced cancer.

2. PATTERNS OF BREAST CANCER METASTASIS

For early stage breast cancer, the most common pattern of metastatic spread is to regional, ipsilateral draining lymph nodes. Identification and treatment of nodal disease with sentinel node mapping has resulted in a true paradigm shift in the staging and surgical management of early-stage breast cancer (15). For more advanced disease, breast cancer is readily capable of metastasizing to other organs of the body, with a particular prevalence for the bone, lung, liver, and brain. Autopsy studies of women dying of breast

the host antitumor immune response (13). Morton et al. hypothesize that

that a remarkably high 40% of all patients (in both arms) were alive at5 years, suggesting that prolonged survival may be due not to the vaccine,

358

liver (61%), and brain (30%) as the four most common metastatic sites

2.1 Bone metastases

The bone is the most common site of breast cancer metastasis, with a typical clinical presentation of the new development and onset of bone pain. Through poorly understood mechanisms, breast cancer cells are able to induce osteoclastic activity that results in the destruction of the bony cortex and marrow with subsequent increased propensity for pathological fractures as the bony matrix is weakened (19, 20). Although pathological fractures can develop anywhere within the bony structures, breast cancer has a propensity to metastasize to the spine, long bones, and joints (21). It is likely that breast cancer cells metastasize regularly to the bone marrow, with several investigators able to isolate and identify such cells in 13–43% of early and 40–60% of late stage breast cancer patients (22–24). Additionally, others have shown that tumor cells can be readily identified in the peripheral blood, as well as, within the bone marrow, paralleling significant differences in clinical outcomes when found (24–26).

Skeletal breast cancer metastases may be fairly indolent in many instances, with many patients exhibiting minimal symptoms throughout their clinical course. Isolated lesions tend to respond well to hormonal therapy, chemotherapy, and radiation therapy with overall longer survival noted in these patients (27). For example, bisphosphonate

frequency of skeletal-related events and symptomatic and palliative relief of metastatic bone pain (28, 29). A comparison was performed between solitary and multiple skeletal metastatic lesions in 703 metastatic breast cancer patients, revealing that 41% had solitary skeletal metastasis and 59% with multiple metastases (27). They show that the sternum is the most common site of solitary metastasis and the thoracic spine and ribs as the most common site for multiple metastases. Radiotherapy also may play an important role in palliation of painful bone metastases (30).

Surgical intervention for metastatic breast cancer to the bone is usually reserved for either the fixation of pathologic fractures, stabilization of weight-bearing bones with impending fractures, or for acute spinal cord compressions which may result in life-threatening or significant functional neurologic compromise that can lead to bowel or urinary incontinence (31, 32). It is estimated that 5% of all cancer patients will develop meta-static spinal cord compression during the course of their disease, with urgent

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stases below. (16–18). We will describe these patterns of locoregional and distant meta-

the treatment regimen, providing a durable reduction in the overalltherapy for metastatic bone disease has become an effective part of

cancer reveal widespread disease involving the bones (70%), lungs (66%),


histologies, with long course radiotherapy as the preferred modality of treatment (34). Surgical decompression of the affected segment has only a limited role in acute management, with the preferred immediate treat-ment being radiotherapy in most cases (34–36).

The sternum is the most common site of isolated bony metastasis (27), with several reported cases of large primary breast cancers capable of extending into the sternum or local recurrences extensively involving the sternum (37–40). When sternal resection of metastatic disease is under-taken with potentially curative intent, consideration should be given to the complexity of the reconstructive options available, such as the com-bined use of autologous tissue transplants and synthetic mesh (Marlex with or without methylmethacrylate) (2, 41–43). These complex and poten-tially morbid procedures can occasionally result in long-term survivors, with several studies showing a durable median survival of ~30 months with many patients surviving >6 years (27, 37–43).

Locoregional recurrence of breast cancer involving the chest wall can be found in about 5–40% of patients and is generally thought to have a poor overall prognosis and outcome (44). However, a subgroup of patients may exist that will benefit from an aggressive surgical approach to therapy, with survival greatly influenced by numerous factors, such as the disease-free interval between mastectomy and chest-wall recurrence, primary tumor size, axillary nodal status, and number of recurrences (45). A study by Chagpar et al. examined 130 patients with isolated chest-wall recurrences following mastectomy, showing 5- and 10-year survival rates of 47% and 29%, respectively (44). The significant factors associated with a worse overall survival were positive initial node status, lack of radiotherapy for the treatment of chest-wall recurrence, and use of systemic therapy for treatment of the primary tumor.

Full-thickness chest-wall resection for locally recurrent breast cancer can provide long-term palliation and occasional cure for select patients. Pameijer et al. performed such resections with reconstruction in 22 women with isolated chest-wall recurrences from breast cancer, reporting a 5-year disease-free survival of 67% and an overall survival of 71% (46).

resection of chest-wall disease to negative margins when possible, follo-wed by reconstruction with autologous tissue or synthetic mesh and/or methylmethacrylate to fill the defect. Advanced techniques involve the use of extended and V-Y latissimus dorsi myocutaneous flap reconstruc-tion, rectus abdominus myocutaneous flap reconstruction, and cutaneous

2

(47, 48). Complex chest wall reconstruction will usually involve radical

Others have performed similar chest-wall resections with complex plasticreconstruction report similar survival rates, ranging from 47% to 62%

(49–51). thoraco-abdominal flaps to cover very large defects up to 600 cm

treatment required in most cases (33–35). Breast cancer patients however seem to have an improved median survival compared to other tumor

360 2.2 Lung metastases

Isolated lung metastases from breast carcinoma occurs in about 10–20% of all women, with 60–74% of patients who die of breast carcinoma found to have pulmonary metastases (52, 53). An early study of 96 patients with isolated pulmonary metastases from breast cancer stratified them based upon their surgical therapy into complete resection (N=28),

the mean survival time for those who underwent a complete resection

patients with metastatic breast cancer only to the lung who underwent surgical resection with curative intent (55). Of the 125 patients, 96 underwent a complete resection with no evidence of a significant improve-ment in survival when compared to the remainder of the patients who had an incomplete resection or were deemed unresectable. Additionally, they report an overall median survival for the entire group of 4.2 years, with a 5-year survival of 45% (55). Significant prognostic factors of survival included the size of the largest metastasis and the disease-free interval.

The International Registry of Lung Metastases have reported on a large series of breast cancer patients who have undergone lung metastasec-tomy (isolated, multiple, bilateral lung, or combined with other organs), with 84% (390/467) of patients receiving a complete resection (56). They report a 5-, 10-, and 15-year survival of 38%, 22%, and 20% (median survival of 37 months), respectively, for patients undergoing a complete surgical resection of isolated lung metastases (56). Additionally, they established several prognostic groupings based upon risk factors, showing that the group with the best overall survival had a complete surgical resection and a disease-free interval of ≥36 months (5-year survival of 50%, median survival of 59 months). Similar studies examin-ing the role of pulmonary metastasectomy for breast cancer have shown similar survival outcomes, ranging from 5-year survival rates of 36–54% to median survival times of 42–70 months, with higher rates achievable with postoperative systemic therapy (57–61).

2.3 Liver metastases

Of all patients with metastatic breast cancer, only about 5% will develop isolated hepatic metastases as the only site of metastatic disease (62–64). An early study of 17 patients with isolated hepatic metastases secondary to breast cancer who underwent curative surgical intent revealed a 5-year overall survival of 22% (65). Although similar to the outcomes of other non-surgical treatment options, 75% of the patients experienced a recur-ence of their disease (median time to recurrence of 8 months) with the

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incomplete resection (N=34), and no resection (N=34). They found that

was 79 months with a 5- and 10-year survival of 80%, and 60%, res-pectively (54). A more recent report by Planchard et al. examined 125


hepatectomy for metastatic breast cancer summarizes the clinical outcome of a total of 240 patients (individual series ranging from 6 to 65 patients) from 9 series, reporting a 5-year survival range of 27–51% and an average 5-year survival of 35% (66). Interestingly, there did not seem to be any correlation with achieving negative surgical resection margins and an adverse impact on survival. Possible explanations include the hypo-thetically significant role of cytoreductive surgery and tumor debulking in improving the efficacy of adjuvant chemo/hormonal therapy, an idea strongly supported by others (67–69).

Ercolani et al. surgically treated 21 metastatic breast cancer patients to the liver with curative intent (major, partial, or segmental hepatectomy), yielding a median survival of 40 months and a 5-year survival of 20% (70). Vlastos et al. support an aggressive surgical approach to therapy of similar patients as part of a multimodal regimen, often including chemo/ hormonal (neo-)/adjuvant systemic therapy (64). In their series of 31 patients who underwent hepatic resection of metastatic breast cancer, 87% received either preoperative or postoperative systemic therapy as part of their overall regimen of treatment. They report a median survival of 63 months with a 2-year and 5-year survival rate of 86% and 61%, respectively, and a disease-free 2- and 5-year survival rate of 39% and 31%, respectively (64). However, it should be noted that over half of the patients (52%) eventually developed recurrent disease, usually outside of the liver, with a mean time to recurrence of 21 months. Thus, the role of hepatic metastasectomy for breast cancer seems appropriate for a highly select subgroup of patients.

2.4 Brain metastases

The true incidence of brain metastases from stage IV breast cancer patients is about 10–15%, with autopsy findings revealing a higher incidence of about 18–30% (17, 18, 71). It is estimated that brain meta-stases (either isolated or as the first site of disease) occur relatively infrequently with an incidence ranging from 3% to 12% (72–74). The International Breast Cancer Study Group recently attempted to identify prognostic factors in patients with early stage breast cancer that were possibly associated with a higher risk of developing central nervous system (CNS) metastases (75). They found a 10-year incidence of CNS recurrence of 5.2% and identified several risk factors for the develop-ment of CNS metastases as the first site of spread:

1. Node-positive disease 2. estrogen receptor (ER)-negative tumor 3. tumor size >2cm 4. tumor grade 3, 4. <35 years old, HER2-positive tumor 5. ER(-) + Node (+)

liver accounting for 67% of these recurrences. A recent database review of

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It is interesting to note the increased risk of CNS metastases due to HER2 overexpression by tumor cells. This could be due to the possible increased aggressive nature of HER2+ breast cancer, or a biological predisposition for metastasizing to the CNS (75, 76). Furthermore, patients with advanced breast cancer treated with trastuzumab have been shown to develop higher rate of isolated CNS metastases (76–78). This may be reflective of an improvement in peripheral tumor control with trastuzumab-based therapy, or the tendency of HER2-positive tumors to home in on the CNS due to the inability of trastuzumab in penetrating the blood-brain barrier. A third possibility may be due to the prolonged survival of trastuzumab-treated patients, allowing for the delayed development of brain metastases. Yau et al. examined the incidence, pattern, and timing of brain metastases among 87 patients with advanced breast cancer treated with trastuzumab (77). They found that 30% of all patients developed brain metastases within a year of therapy, with brain metastases as the first site of recurrence in 21% of the patients who derived a clinical benefit from trastuzumab therapy. While others have shown a similarly high rate of brain metastases in trastuzumab-treated patients, ranging from 25% to 34%, there is current information that refutes such data, not showing an increased risk of CNS metastases in trastuzumab-treated patients (78–81). Thus, there is still controversy as to the possible associ-ation of the increased risk of developing brain metastases secondary to trastuzumab therapy.

The majority of patients with metastatic breast cancer will develop brain metastases later in their clinical course, often having involvement of other solid organs such as the bone, lungs, and liver antecedent to their CNS involvement. Thus, isolated brain metastases are relatively uncommon, often precluding a major role for surgical intervention. However, for the small group of patients with isolated brain metastases, strong consideration should be given to the surgical removal of all metastatic parenchymal brain disease. An important trial has addressed the role of surgery for isolated metastases to the brain (10% with breast cancer), randomly assigning 48 patients with single brain metastases to surgery followed by whole brain irradiation (WBRT) versus no surgical intervention with WBRT alone (82). They revealed a significant increase in functional independence and improved survival for the combined group, a finding that is supported by several other studies (83–85). Although more controversial, the role of surgery for multiple brain metastases has been examined, showing only limited benefit in most cases (86, 87).

The development of newer technologies, such as stereotactic radio-surgery (SRS) has greatly enhanced our ability to treat patients with either single or multiple brain metastases. Several studies have shown the utility of SRS in treating such patients in addition to providing a role as

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single brain metastasis appear to have equal efficacy, with the actual choice of treatment based primarily upon the size of the lesion, location,

2.5 Other sites of metastases

Primary breast cancer has been reported to metastasize to rare sites, such as the pancreas, with seven reported cases in the literature (92). Patients will clinically present with obstructive jaundice secondary to metastasis of the pancreatic head, with palliative surgical resection, or biliary bypass the mainstay of treatment for patients with a good per-formance status. Other rare locations for breast cancer metastases include the gall bladder and primary biliary tract, thyroid gland, adrenal gland, colon, small bowel, stomach, and endobronchus, most treated with surgical resection in symptomatic patients (93). Occasionally, patients will present with recurrent breast cancer to the axilla that is unable to be resected due to the rapidity of growth and aggressive nature of the tumor. Such advanced cases can be debilitating to the patient who may experience intractable pain, chronic debilitating lymphedema, and concomitant vascular and neurological dysfunction that leads to a nonfunctioning extremity. In the absence of metastatic disease at other sites, these patients can benefit from a forequarter amputation of the extremity as a means to obtain locoregional disease control and efficient palliation of chronic unremitting pain (94).

Sentinel lymph node (SLN) mapping for breast cancer has become the primary means for accurately assessing for nodal metastasis. This technique has been extensively evaluated throughout the world, with several studies confirming the overall accuracy of this procedure (95). The NSABP B-32 Trial and the ACOSOG Z0010 in the USA, the Milan SLNB trial for

Australian Trial of SLN mapping have all been recently reported on at the 5th Biennial International Sentinel Node Society Meeting in Rome, Italy(1–4 Nov. 2006). The accumulated studies represent >20,000 cases of early-stage breast cancer with an overall accuracy of 93–97% and a false-negative rate ranging from 3% to 9% with definitive singular publications forth-coming. The systematic review performed by a panel of designated experts from ASCO included 69 eligible trials examining SLNB in early-stage breast cancer, representing a total of 8,059 patients, finding an overall accu-racy of 96% in successfully identifying the SLN and a false-negative rate of 7.3% (96). Current guidelines would suggest that if a patient has evidence (by routine H&E analysis) of metastatic breast cancer within a SLN(s), they

2.6 Metastasis to the draining lymph node basin

breast cancer in Italy, the ALMANC trial in the UK, and the SNAC I and II

salvage therapy following neurosurgery with or without WBRT (88–91). Overall, the studies evaluating SRS versus surgery for the treatment of a

and functional status of the patient (88, 89).

364 should undergo a definitive complete axillary lymph node dissection (CALND).

However, a more thorough evaluation of the SLN will often yield very small areas of metastatic disease, considered by many to represent “micrometastatic” disease. The precise definition of micrometastatic disease is the finding of breast cancer cells within a SLN that measure between 0.2–2.0 mm in diameter, with submicroscopic tumor cells (isolated tumor cells) measuring <0.2 mm. The advent of SLNB combined with immuno-histochemical (IHC) analysis and other diagnostic tools, such as step-sectioning, have allowed the pathologist to examine a select few SLNs (as opposed to all of the nodes within a CALND), more thoroughly. This has resulted in the detection of additional microscopic and submicro-scopic disease, down to areas of even a few isolated tumor cells (ITC) and/or clusters of cells (CTC) within a single SLN.

Most studies have concluded that the pathological results obtained are a true representation of the remaining lymph node basin, with the full realization that there is an intrinsic rate of false-negativity in the SLNB procedure itself as well as within the staining method utilized. Thus, if a SLN is found to be negative for metastatic disease by routine histological and CK-IHC analysis, the chances of finding a positive non-SLN (NSLN) is negligible, with longer follow-up of such patients having an overall incidence of local axillary recurrence with a CK-IHC negative SLN of <0.1–0.2% (98). These findings clearly support that a CK-IHC- negative SLN accurately reflects the status (whether positive or negative) of the remaining nodal basin. However, question remains as to the detection of

is a high likelihood (75%) of missing an occult lymph node metastasis measuring <0.02 mm utilizing standard light microscopy, with a 61% chance of missing a metastasis measuring ≤0.10mm (99). Thus, it is important to realize the limitations of our current technology for analyzing lymph nodes, noting that pathologists may frequently miss metastatic disease that measures ≤0.10mm in greatest diameter. The clinical signi-ficance of this finding is unknown at this time, but recently clarified by Jakub et al. who show that the net effect of identifying submicroscopic disease eliminates an inherent 2% cause for false-negativity by the SLN procedure (102).

Currently, the need for further treatment of the axilla for patients found to have submicroscopic disease within the SLN is unclear. Houvenaeghel et al. have analyzed 700 patients to determine the rate of non-SLN involve-ment in patients with SLN micrometastases and to identify prognostic factors that might be predictive of positive non-SLN involvement (100). Several such factors were identified that were predictive of non-SLN involvement, such as the size of the primary tumor, lymphovascular invas-ion, and histologic subtype in addition to the method of detection of

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part of the NSABP B-32 Quality Assurance Study, has shown that thereoccult sentinel lymph node micrometastases by IHC. Weaver et al., as

18. Surgical management of metastatic breast cancer 365 micrometastases (by H&E or IHC). They conclude that the omission of a CALND is possible for patients at minimal risk of additional disease, specifically those with a pT1a, or pT1b tumor, and pT1a-b-c tumors with a tubular, colloidal, or medullary subtype. Furthermore, they found no correlation between the size of micrometastases and chance of finding a non-SLN, inclusive of those patients with submicrometastatic disease.

Other studies have also addressed the role of performing a CALND for patients with CK-IHC (+) disease only in the SLN, with conflicting data and no true consensus on the optimal surgical management (101–104). Recent evidence by van Rijk retrospectively examined the SLNs in 2,150 patients with breast cancer, finding a total of 649 patients (30%) with tumor-positive SLN of which 23% had micrometastatic disease and 16% had submicrometastatic disease (101). A total of 106 patients from the group with micrometastatic disease underwent a CALND, of which 20 (19%) were found to have additional disease involving the non-SLNs. The latter group of patients with submicrometastatic disease who underwent a CALND comprised a total of 4% of patients. Regardless of the finding of submicrometastatic disease and subsequent upstaging in these patients, changes in the clinical management were made in 0% (0/2150) and 0.4% (9/2150) of the total study population and 7% overall. They conclude that patients with micrometastatic disease should undergo

should not. In contrast, Jakub et al. examined 971 patients with H&E- negative SLNs, finding 78 (8%) with CK-IHC disease only and 62/78 undergoing a complete lymph node dissection (102). A total of 9 patients (14.5%) had further metastases identified in the CALND by H&E, con-cluding that CK-IHC should be used to evaluate SLNs and that CALND should be considered when the SLNs are positive by CK-IHC only. Cox et al. have recently analyzed the clinical significance of CK-IHC positive micrometastatic disease in breast cancer patients who have under-gone SLNB (105). Of the 6,781 patients reviewed, 3,047 patients were classified as having no evidence of nodal disease [N0(i-)], evidence of nodal disease by CK-IHC only [N0(i+)], or positive for nodal disease by standard histological criteria (H&E +) [N1mi]. Of the 2,627 patients who were treated after 1996, 90% were N0(i-), 16% were N0(i+), and 4% were N1mi. The overall and disease-free survival of patients with N1mi SLN differed significantly from patients with N0(i-) SLN ( p = 0.037 and p = 0.09, respectively). Of the 3,047 patients who were considered to be SLN-negative by routine histological analysis with H&E, the addition of CK-IHC detected micrometastatic disease (pN1mi) in 132, ITCs in 153, and no evidence of disease in 2,762. The results indicate that pN0(i+) disease in the SLN has no significant effect upon the survival of breast cancer pati-ents, but that the results can be utilized to define a subset of T1-T3 patients who should receive a CALND. Indeed, they found that 10.6% of T1c and

a CALND and those with submicrometastatic disease within their SLN

366 20.4% of T2 lesions will have additional H&E positive non-SLNs in the remainder of the nodal basin upon CALND. Other studies have also addressed the issue as to the biological significance of occult micrometa-stases, with some showing that micrometastatic disease is indeed clinically significant with others finding no such correlation (106–108). Prospective trials are currently being evaluated as to the overall clinical significance of micrometastatic disease, specifically the American College of Surgeons Oncology Group Z0010 trial and the NSABP B-32 trial, both having completed accrual. These studies were formulated to specifi-cally address the role of CK-IHC staining of SLNs and as to the overall clinical significance of such findings.

The critical roles of the surgeon to accurately stage the nodal basin and provide local control of the disease are accomplished without com-promise in a disease which in 15 to 20 years may ultimately bear the

decision-making process; a classic example of this being the management of the internal mammary lymph nodes in breast cancer. The logical extension of the philosophy to accurately stage and achieve maximal local control resulted previously in surgeons performing very extensive and radical procedures, such as the classic Halsted radical mastectomy. Thirty years of data and careful longitudinal follow-up has lead to an improved understanding as to the clinical significance of internal mammary lymph node metastases, with the utility of the SLN procedure being critical in identifying such nodes. The conclusion of such outcomes is that we must exhibit caution in terms of what is truly in the patients best interest, as often only longer periods of time are necessary in order to give us the correct answers. We must always err on the side of the principle of noncompromise for the patient, discussing the latest test and procedures available, and utilizing all possible tools available to us in order to provide the most accuracy in staging and achieving maximal locoregional control. This principle may be the patient’s best defense against a disease such as breast cancer, with the final recourse and outcome known 30 years hence.

REFERENCES

1. Wood WC, Muss HB, Solin LJ, et al. Cancer of the Breast. In DeVita VT, Hellman S, Rosenberg SA (eds) Cancer Principles & Practice of Oncology. Lippincott Williams and Wilkins, Philadelphia, 2005, 1453–1462.

2. Singletary SE, Walsh G, Vauthey JN, Curley S, Sawaya R, Weber KL, Meric F, Hortobagyi GN. A role for curative surgery in the treatment of selected patients with metastatic breast cancer. The Oncologist 2003, 8(3):241–251.

3. Khan SA, Stewart AK, Morrow M. Does aggressive local therapy improve survival in metastatic breast cancer? Surgery 2002, 132(4):620–626.

Riker et al.

reality of the situation. We cannot allow length-time bias to infiltrate the

18. Surgical management of metastatic breast cancer 367 4. Babiera GV, Rao R, Feng L, Meric-Bernstam F, Kuerer HM, Singletary SE,

Hunt KK, Ross MI, Gwyn KM, Feig BW, et al. Effect of primary tumor extirpation in breast cancer patients who present with stage IV disease and an intact primary tumor. Ann Surg Oncol 2006, 13(6):776–782.

5. Demicheli R, Valagussa P, Bonadonna G. Does surgery modify growth kinetics of breast cancer micrometastases? Br J Cancer 2001, 85(4):490–492.

6. Retsky M, Bonadonna G, Demicheli R, Folkman J, Hrushesky W, Valagussa P. Hypothesis. induced angiogenesis after surgery in premenopausal node-positive breast cancer patients is a major underlying reason why adjuvant chemotherapy works particularly well for those patients. Breast Cancer Res 2004, 6(4):372–374.

7. O’Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, Lane WS, Cao Y, Sage EH, Folkman J. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma Cell 1994,

8. Rapiti E, Verkooijen HM, Vlastos G, Fioretta G, Neyroud-Caspar I, Sappino AP, Chappuis PO, Bouchardy C. Complete excision of the primary breast tumor improves survival of patients with metastatic breast cancer at diagnosis. J Clin Oncol 2006, 24(18):2743–2749.

9. Marincola FM, Jaffee EM, Hicklin DJ, Ferrone S. Escape of human solid tumors from T-cell recognition: molecular mechanisms and functional significance. Adv Immunol 2000, 74:181–273.

10. Ochsenbein AF, Sierro S, Odermatt B, Pericin M, Karrer U, Hermans J, Hemmi S, Hengartner H, Zinkernagel RM. Roles of tumor localization, second signals and cross priming in cytotoxic T-cell induction. Nature 2001, 411(6841):1058–1064.

11. Pardoll D. Does the immune system see tumors as foreign or self ? Annu Rev Immunol 2003, 21:807–839.

12. Danna EA, Sinha MG, Gilbert M, Clements VK, Pulaski BA, Ostrand-Rosenberg S. Surgical removal of primary tumor reverses tumor-induced immunosuppression despite the presence of metastatic disease. Cancer Res 2004, 64(6):2205–2211.

13. and adjuvant immunotherapy. A new management paradigm for metastatic melanoma. CA Cancer J Clin 1999, 49(2):101–116.

14. Morton DL. Surgery prolongs survival in stage IV melanoma. Symposium at the Society of Surgical Oncology, San Diego 2006.

15. Leong SPL. Paradigm shift of staging and treatment for early breast cancer in the sentinel node era. Breast J 2006, 12(2):S128–S133.

16. Leong SP, Cady B, Jablons DM, Garcia-Aguilar J, Reintgen D, Jakub J, Pendas S, Duhaime L, Cassell R, Gardner M, et al. Clinical Patterns of Metastasis. Cancer Metastasis Rev 2006, 25(2):221–232.

17. Lee Y-T. Breast carcinoma: Patterns of metastasis at autopsy. J Surg Oncol 1983, 23:175–180.

18. Cho SY, Choi HY. Causes of death and metastatic patterns in patients with mammary cancer. Am J Clin Pathol 1980, 73(2):232–234.

19. Theriault RL, Hortobagyi GN. Bone metastasis in breast cancer. Anticancer Drugs 1992, 3(5):455–462.

20. Theriault RL, Biermann JS, Brown E, Brufsky A, Demers L, Grewal RK, Guise T, Jackson R, McEnery K, Podoloff D, et al. NCCN task force report: Bone health and cancer care. J Natl Compr Canc Netw 2006, 4(2):S1–20.

21. Durr HR, Muller PE, Lenz T, Baur A, Jansson V, Refior HJ. Surgical treatment of bone metastases in patients with breast cancer. Clin Orthop Relat 2002, 396:191–196.

79(2):315–328.

Morton DL, Ollila DW, Hsueh EC, Essner R, Gupta RK. Cytoreductive surgery

368 22. Yu JJ, Brennan M, Christos P, Osborne MP, Hoda S, Simmons RM. Bone marrow

micrometastases and adjuvant treatment of breast cancer. Breast J 2004, 10(3): 181–185.

23. Wiedswang G, Borgen E, Karesen R, Kvalheim G, Nesland JM, Qvist H, Schlichting E, Sauer T, Janbu J, Harbitz T, et al. Wiedswang G, Borgen E, Karesen R, et al. Detection of isolated tumor cells in bone marrow is an independent prognostic factor in breast cancer. J Clin Oncol 2003, 21(18): 3469–3478.

24. patients. J Am Coll Surg 2006, 203 (2):240–249.

25. Cristofanilli M, Hayes DF, Budd GT, Ellis MJ, Stopeck A, Reuben JM, Doyle GV, Matera J, Allard WJ, Miller MC, et al. Circulating tumor cells: A novel prognostic factor for newly diagnosed metastatic breast cancer. J Clin Oncol 2005, 23(7): 1420–1430.

26. Cristofanilli M. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. Semin Oncol 2006, 33(3 Suppl 9):S9–14.

27. Koizumi M, YoshimotoM, Kasumi F, Ogata E. Comparison between solitary and multiple skeletal metastatic lesions of breast cancer patients. Ann Oncol 2003, 14(8):1234–1240.

28. Body JJ. Breast cancer: bisphosphonate therapy for metastatic bone disease. Clin Cancer Res 2006, 12 (20 Suppl):6258s–6263s.

29. Selvaggi G, Scagliotti G. Management of bone metastases in cancer: a review. Critical Reviews in Oncology/Hematology 2005, 56(3):365–378.

30. Hoskin PJ, Yarnold JR, Roos DR, Bentzen S. Second Workshop on Palliative Radiotherapy and Symptom Control: Radiotherapy for bone metastases. Clin Oncol 2001, 13(2):88–90.

31. Bohm P, Huber J. The surgical treatment of bony metastases of the spine and limbs. J Bone Joint Surg Br 2002, 84(4):521–529.

32. Wedin R, Bauer HCF, Rutqvist LE. Surgical treatment of skeletal breast cancer metastases: A population-based study of 641 patients. Cancer 2001, 92(2): 57–262.

33. Hill ME, Richards MA, Gregory WM, Smith P, Rubens RD. Spinal cord compression in breast cancer: A review of 70 cases. Br J Cancer 1993, 68(5): 969–973.

34. Rades D, Fehlauer F, Schulte R, Veninga T, Stalpers LJ, Basic H, Bajrovic A, Hoskin PJ, Tribius S, Wildfang I, et al. Prognostic factors for local control and survival after radiotherapy of metastatic spinal cord compression. J Clin Oncol 2006, 24(21):3388–3393.

35. Kovner F, Spigel S, Rider I, Otremsky I, Ron I, Shohat E, Rabey JM, Avram J, Merimsky O, Wigler N, et al. Radiation therapy of metastatic spinal cord compression: Multidisciplinary team diagnosis and treatment. J Neurooncol 1999, 42(1):85–92.

36. Rades D, Veninga T, Stalpers LJ, Schulte R, Hoskin PJ, Poortmans P, Schild SE, Rudat V. Prognostic factors predicting functional outcomes, recurrence-free survival, and overall survival after radiotherapy for metastatic spinal cord com-pression in breast cancer patients. Int J Radiat Oncol Biol 2006, 64(1):182–188.

37. Tsuda M, Satou S, Ichiki K, Doki Y, Misaki T, Matsui K, Tei S. [Sternal meta-stasis of breast cancer: report of a case]. Kyobu Geka 2005, 58(4): 341–343.

38. Tenpaku H, Maze Y, Sato T. [Recurrent breast cancer to the sternum 15 years after radical mastectomy and primary lung cancer; report of a case]. Kyobu Geka 2004, 57(12):1165–1167.

39. Carbognani P, Vagliasindi A, Costa P, Pascarella L, Pazzini L, Bobbio A, Rusca M. Surgical treatment of primary and metastatic sternal tumors. J Cardiovasc Surg 2001, 42(3):411–414.

Riker et al.

Singletary SE. Isolated tumor cells in bone marrow of early stage breast cancer

18. Surgical management of metastatic breast cancer 369 40. Yatsuyanagi E, Hirata S, Yamazaki K, Sugimoto H, Koshiko S, Sasajima T,

Kubo Y. Full thickness chest wall resection for solitary sternal metastasis in breast carcinoma. Eur J Surg 2000, 166(6):501–503.

41. Takanami I, Ohnishi H. [Study of surgical resection of sternal metastasis from carcinoma of the breast]. Gan No Rinsho 1989, 35(15):1735–1738.

42. Incarbone M, Nava M, Lequaglie C, Ravasi G, Pastorino U. Sternal resection for primary or secondary tumors. J Thorac Cardiovasc Surg 1997, 114(1):93–99.

43. Noguchi S, Miyauchi K, Nishizawa Y, Imaoka S, Koyama H, Iwanaga T. Results of surgical treatment for sternal metastasis of breast cancer. Cancer 1988, 62(7):1397–1401.

44. Chagpar A, Meric-Bernstam F, Hunt KK, Ross MI, Cristofanilli M, Singletary SE, Buchholz TA, Ames FC, Marcy S, Babiera GV, et al. Chest wall recurrence after mastectomy does not always portend a dismal prognosis. Ann Surg Oncol 2003, 10(6): 628–634.

45. Magno L, Bignardi M, Micheletti E, Bardelli D, Plebani F. Analysis of prognostic factors in patients with isolated chest wall recurrence of breast cancer. Cancer 1987, 60(2):240–244.

46. Pameijer CR, Smith D, McCahill LE, Bimston DN, Wagman LD, Ellenhorn JD. Full-thickness chest wall resection for recurrent breast carcinoma: an institutional review and meta-analysis. Am Surg 2005, 71(9):711–715.

47. Toi M, Tanaka S, Bando M, Hayashi K, Tominaga T. Outcome of surgical resection for chest wall recurrence in breast cancer patients. J Surg Oncol 1997, 64(1):23–26.

48. Kolodziejski LS, Wysocki WM, Komorowski AL. Full-thickness chest wall resection for recurrence of breast malignancy. Breast J 2005, 11(4):273–277.

49. Woo E, Tan B-K, Koong HN, Yeo A, Chan MYP, Song C. Use of the extended V-Y latissimus dorsi myocutaneous flap for chest wall reconstruction in locally advanced breast cancer. Ann Thorac Surg 2006, 82(2):752–755.

50. Sakai S, Ando K, Natori M, Sakai S. Cosmetic reconstruction after resection of breast cancer: Use of the ELD-MC flap and EVRAM flap. Int J Clin Oncol 2005, 10(5):298–303.

51. Persichetti P, Cagli B, Tenna S, Fortunato L, Vitelli CE. Role of cutaneous thoraco-abdominal flap in the surgical treatment of advanced stage breast tumors. Suppl Tumori, 2005 4(3):S177.

52. Carter CL, Allen C, Henson DE. Relation of tumor size, lymph node status, and survival in 24,740 breast cancer cases. Cancer 1989, 63(1):181–187.

53. of indications and results of surgical treatment for patients with pulmonary metastasis]. Pneomonol Alergol Pol 1999, 67(5–6):228–236.

54. Murabito M, Salat A, Mueller MR. Complete resection of isolated lung meta-stases from breast carcinoma results in a strong increase in survival. Minerva Chir 2000, 55(3):121–127.

55. Planchard D, Soria JC, Michiels S, Grunewald D, Validire P, Caliandro R, Girard P, Le Chevalier T. Uncertain benefit from surgery in patients with lung metastases from breast carcinoma. Cancer 2004, 100(1):28–35.

56. Friedel G, Pastorino U, Ginsberg RJ, Goldstraw P, Johnston M, Pass H, Putnam JB, Toomes H. Results of lung metastasectomy from breast cancer: prognostic criteria on the basis of 467 cases of the International Registry of Lung Metastasis. Eur J Cardiothorac Surg 2002, 22(3):335–344.

57. Lanza LA, Natarajan G, Roth JA, Putnam JB Jr. Long-term survival after resection of pulmonary metastases from carcinoma of the breast. Ann Thoracic Surg 1992, 54(2):244–248.

Kolodziejski L, Goralczyk J, Dyczek S, Duda K, Dymek H, Nabialek T. [Analysis

370 58. Livartowski A, Chapelier A, Beuzeboc P, Dierick A, Asselain B, Dartevelle P,

Pouillart P. [Surgical excision of pulmonary metastasis of cancer of the breast: apropos of 40 patients]. Bull Cancer 1998, 85(9):799–802.

59. McDonald ML, Deschamps C, Ilstrup DM, Allen MS, Trastek VF, Pairolero PC. Pulmonary resection for metastatic breast cancer. Ann Thoracic Surg 1994, 58(6):1599–1602.

60. Simpson R, Kennedy C, Carmalt H, McCaughan B, Gillett D. Pulmonary resection for metastatic breast cancer. Aust NZJ Surg 1997, 67(10):717–719.

61. Staren ED, Salerno C, Rongione A, Witt TR, Faber LP. Pulmonary resection for metastatic breast cancer. Arch Surg 1992, 127(11):1282–1284.

62. Hoe AL, Royle GT, Taylor I. Breast liver metastases-incidence, diagnosis and outcome. J R Soc Med 1991, 84(12):714–716.

63. O’Reilly SM, Richards MA, Rubens RD. Liver metastases from breast cancer: the relationship between clinical, biochemical and pathological features and survival. Eur J Cancer 1990, 26(5):574–577.

64. Vlastos G, Smith DL, Singletary SE, Mirza NQ, Tuttle TM, Popat RJ, Curley SA, Ellis LM, Roh MS, Vauthey JN. Long-term survival after an aggressive surgical approach in patients with breast cancer hepatic metastases. Ann Surg Oncol 2004, 11(9):869–874.

65. Elias D, Maisonnette F, Druet-Cabanac M, Ouellet JF, Guinebretiere JM, Spielmann M, Delaloge S. An attempt to clarify indications for hepatectomy for liver metastases from breast cancer. Am J Surg 2003, 185(2):158–164.

66. Selzner M, Morse MA, Vredenburgh JJ, Meyers WC, Clavien P-A. Liver metastases from breast cancer: Long-term survival after curative resection. Surgery 2000, 127(4):383–389.

67. Metcalfe M, Mullin EJ, Maddern GJ. Hepatectomy for metastatic noncolorectal gastrointestinal, breast and testicular tumors. ANZ J Surg 2006, 76(4): 246–250.

68. Pocard M, Pouillart P, Asselain B, Falcou MC, Salmon RJ. Hepatic resection

126(5):413–420. 69. Pocard M, Salmon RJ. [Hepatic resection fro breast cancer metastasis. The

concept of adjuvant surgery]. Bull Cancer 1997, 84(1): 47–50. 70. Ercolani G, Grazi GL, Matteo Ravaioli M, Ramacciato G, Cescon M, Varotti

G, Del Gaudio M, Vetrone G, Pinna AD. The role of liver resections for noncolorectal nonneuroendocrine metastases: Experience with 142 observed cases. Ann Surg Oncol 2005, 12(6):1–8.

71. Lin NU, Bellon JR, Winer EP. CNS metastases in breast cancer. J Clin Oncol 2004, 22(17):3608–3617.

72. Kamby C. The patterns of metastases in human breast cancer: methodological aspects and influence of prognostic factors. Cancer Treat Rev 1990, 17(1):37–61.

73. Sastre-Garau X, Jouve M, Asselain B, Vincent-Salomon A, Beuzeboc P, Dorval T, Durand JC, Fourquet A, Pouillart P. Infiltrating lobular carcinoma of the breast: clinicopathologic analysis of 975 cases with reference to data on consecutive therapy and metastatic patterns. Cancer 1996, 77(1):113–120.

74. Carty NJ, Foggitt A, Hamilton CR, Royle GT, Taylor I. Patterns of clinical metastasis in breast cancer: an analysis of 100 patients. Eur J Surg Oncol 1995, 21(6):607–608.

75. Pestalozzi BC, Zahrieh D, Price KN, Holmberg SB, Lindtner J, Collins J, Crivellari D, Fey MF, Murray E, Pagani O, et al. Identifying breast cancer patients at risk for central nervous system metastases in trials of the International Breast Cancer Study Group. Ann Oncol 2006, 17(6):935–944.

76. Burstein HJ, Lieberman G, Slamon DJ, Winer EP, Klein P. Isolated central nervous system metastases in patients with HER2-overexpressing advanced breast cancer treated with first-line trastuzumab-based therapy. Ann Oncol

Riker et al.

for breast cancer metastases: results and prognosis (65 cases). Ann Chir 2001,

2005, 16(11):1772–1777.

18. Surgical management of metastatic breast cancer 371 77. Yau T, Swanton C, Chua S, Sue A, Walsh G, Rostom A, Johnston SR,

O’Brien MER, Smith IE. Incidence, pattern and timing of brain metastases among patients with advanced breast cancer treated with trastuzumab. Acta Oncologica 2006, 45(2):196–201.

78. Bendell JC, Domchek SM, Burstein HJ, Harris L, Younger J, Kuter I, Bunnell C, Rue M, Gelman R, Winer E. Central nervous system metastases in women who receive trastuzumab-based therapy for metastatic breast carcinoma. Cancer 2003, 97(12):2972–2977.

79. Clayton AJ, Danson S, Jolly S, Ryder WD, Burt PA, Stewart AL, Wilkinson PM, Welch RS, Magee B, Wilson G, et al. Incidence of cerebral metastases in patients treated with trastuzumab for metastatic breast cancer. Br J Cancer 2004, 91(4):639–643.

80. Shmueli E, Wigler N, Inbar M. Central nervous system progression among patients with metastatic breast cancer responding to trastuzumab treatment. Eur J Cancer 2004, 40(3):379–382.

81. Tham Y-L, Sexton K, Kramer R, Hilsenbeck S, Elledge R. Primary breast cancer phenotypes associated with propensity for central nervous system meta-stases. Cancer 2006, 107(4):696–704.

82. Patchell RA, Tibbs PA, Walsh JW, Dempsey RJ, Maruyama Y, Kryscio RJ, Markesbery WR, Macdonald JS, Young B. A randomized trial of surgery in the treatment of single metastases to the brain. N Engl J Med 1990, 322(8):494–500.

83. Noordijk EM, Vecht CJ, Haaxma-Reiche H, Padberg GW, Voormolen JH, Hoekstra FH, Tans JT, Lambooij N, Metsaars JA, Wattendorff AR, et al. The choice of treatment of single brain metastases should be based on extracranial tumor activity and age. Int J Radiat Oncol Biol Phys 1994, 29(4):711–717.

84. Pieper DR, Hess KR, Sawaya RE. Role of surgery in the treatment of brain metastases in patients with breast cancer. Ann Surg Oncol 1997, 4(6):481–490.

85. Wronski M, Arbit E, McCormick B. Surgical treatment of 70 patients with brain metastases from breast carcinoma. Cancer 1997, 80(9):1746–1754.

86. Bindal RK, Sawaya R, Leavens ME, Lee JJ. Surgical treatment of multiple brain metastases. J Neurosurg 1993, 79(2):210–216.

87. Hazuka MB, Burleson WD, Stroud DN, Leonard CE, Lillehei KO, Kinzie JJ. Multiple brain metastases are associated with poor survival in patients treated with surgery and radiotherapy. J Clin Oncol 1993, 11(2):369–373.

88. Lin NU, Bellon JR, Winer EP. CNS Metastases in Breast Cancer. J Clin Oncol 2004, 22(17):3608–3617.

89. Chang EL, Lo S. Diagnosis and management of central nervous system meta-stases from breast cancer. The Oncologist 2003, 8(5):398–410.

90. Lederman G, Wronski M, Fine M. Fractionated radiosurgery for brain metastases in 43 patients with breast carcinoma. Breast Cancer Res Treat 2001, 65(2): 145–154.

91. Firlik KS, Kondziolka D, Flickinger JC, Lunsford LD. Stereotactic radiosurgery for brain metastases from breast cancer. Ann Surg Onc 2000, 7(5):333–338.

92. Tohnosu N, Narushima K, Sunouchi K, Saito T, Shimizu T, Tanaka H, Maruyama T, Watanabe Y, Kato T, Shimizu S, et al. A case of breast cancer metastatic to the tail of the pancreas. Breast Cancer 2006, 13(2):225–229.

93. Uygun K, Kocak Z, Altaner S, Cicin I, Tokatli F, Uzai C. Colonic metastasis from carcinoma of the breast that mimicks a primary intestinal cancer. Yonsei Med J 2006, 47(4):578–582.

94. Goodman MD, McIntyre B, Shaughnessy EA, Lowy AM, Ahmad SA. Forequarter amputation for recurrent breast cancer: A case report and review of the literature. J Surg Oncol 2005, 92(2):134–141.

372 95. Sabel MS. Locoregional therapy of breast cancer: maximizing control, minimizing

morbidity. Expert Rev Anticancer Ther 2006, 6(9):1261–1279. 96. Kim T, Giuliano AE, Lyman GH. Lymphatic mapping and sentinel lymph node

biopsy in early-stage breast carcinoma. A Meta-analysis. Cancer 2006, 106(1): 4–16.

97. Turner RR, Ollila DW, Krasne DL, Giuliano AE. Histopathologic validation of the sentinel lymph node hypothesis for breast carcinoma. Ann Surg 1997, 226(3):271–276.

98. Naik AM, Fey J, Gemignani M, Heerdt A, Montgomery L, Petrek J, Port E, Sacchini V, Sclafani L, VanZee K, et al. The risk of axillary relapse after sentinel lymph node biopsy for breast cancer is comparable with that of axillary lymph node dissection: a follow-up study of 4008 procedures. Ann Surg 2004, 240(3):462–468.

99. Weaver DL, Krag DN, Manna EA, Ashikaga T, Waters BL, Harlow SP, Bauer KD, Julian TB. Detection of occult sentinel lymph node micrometastases by immunocytochemistry in breast cancer: An NSABP B-32 quality assurance study. Cancer 2006, 107:661–667.

100. Houvenaeghel G, Nos C, Mignotte H, Classe JM, Giard S, Rouanet P, Lorca FP, Jacquemier J, Bardou VJ. Micrometastases in sentinel lymph node in a multicentric study: Predictive factors of nonsentinel lymph node involvement-Groupe des chirurgiens de la federation des centres de lutte contre le cancer. J Clin Oncol 2006, 24:1814–1822.

101. vanRijk MC, Peterse JL, Nieweg OE, Oldenburg HS, Rutgers EJ, Kroon BB. Additional axillary metastases and stage migration in breast cancer patients with micrometastases or submicrometastases in sentinel lymph nodes. Cancer 2006, 107:467–471.

102. Jakub JW, Diaz NM, Ebert MD, Cantor A, Reintgen DS, Dupont EL, Shons AR, Cox CE. Completion axillary lymph node dissection minimizes the likelihood of false negatives for patients with invasive breast carcinoma and cytokeratin positive only sentinel lymph nodes. Am J Surg 2002, 184(4):302–306

103. Teng S, Dupont E, McCann C, Wang J, Bolano M, Durand K, Peltz E, Bass SS, Cantor A, Ku NN, Cox CE. Do cytokeratin-positive-only sentinel lymph nodes warrant complete axillary lymph node dissection in patients with invasive breast cancer? Am Surg 2000, 66(6):574–578.

104. Imoto S, Ochiai A, Okumura C, Wada N, Hasebe T. Impact of isolated tumor cells in sentinel lymph nodes detected by immunohistochemical staining. EJSO (2006), doi:10.1016/j.ejso.2006.08.006.

105. Cox CE. Fine tuning treatment of the axilla in an era of sentinel node biopsy. San Antonio Breast Cancer Symposium. Issue 3, December 10th, 2005.

106. Galea MH, Ellis Io, Elston Cw, Blamey RW. Occult axillary lymph node micrometastases in breast cancer. Lancet 1990; 336:435–441.

107. Gershenwald JE, Colome MI, Lee JE, Mansfield PF, Tseng C, Lee JJ, Balch CM, Ross MI. Patterns of recurrence following a negative sentinel lymph node biopsy in 243 patients with stage I or II melanoma. J Clin Oncol 1998, 16(6): 2253–2260.

108. Kahn HJ, Hanna WM, Chapman JW, Trudeau M, Lickley HL, Mobbs BG, Murray D, Pritchard KI, Sawka CA, McCready DR, Marks A. Biological significance of occult micrometastases in histologically negative axillary lymph nodes in breast cancer patients using the recent American Joint Committee on Cancer Breast Cancer Staging System. The Breast J 2006, 12(4):294–301.

Riker et al.

Chapter 19

BREAST CANCER: CHEMOTHERAPY

Robert C.F. Leonard and Thinn P. Pwint

Abstract: The main treatment aims in managing metastatic breast cancer (MBC) are to relieve symptoms by controlling disease and prolonging survival while maintaining quality of life. Endocrine therapy is considered as a primary option for oestrogen-receptor positive MBC except in those with rapidly progressive visceral disease. Chemotherapy is considered for women with hormone-receptor-negative disease, and in those whose cancer is refractory to endocrine therapy or those with symptomatic or rapidly progressive visceral metastases. The major chemotherapy programmes routinely include anthracycline or taxane but capecitabine, gemcitabine, vinorelbine, and carboplatin are also increasingly used. For HER2- overexpressing breast

see comments about safe combinations later. Compared with single agent chemotherapy, combination regimens show a greater tumour response rate and improved time to progression in women with metastatic breast cancer,

increasingly being investigated for clinical use and potentially leading to the evolution of more effective treatment.

Keywords: relieve symptoms; prolong survival; quality of life; chemotherapy; endocrine therapy; anthracycline; taxane; capecitabine; gemcitabine; vinorelbine; carboplatin; HER2; trastuzumab; single agent chemotherapy; combination; oestrogen-receptor

Each year nearly 800,000 women worldwide are diagnosed with breast cancer, and 314,000 die from the disease. Breast cancer is one of the most frequently diagnosed types of cancer and a second leading cause of cancer death in women after lung cancer (1). Although some improvement of long- term survival has been noted over the past 40 years with the development © 2007 Springer.


THERAPEUTIC ASPECTS OF METASTATIC

cancer, trastuzumab-containing chemotherapy is used as a standard but

a modest improvement in overall survival but significantly worse toxi-cities. The newly developed targeted therapies, e.g., Lapatinib are

MBC

General principles of management, treatment of specific pro-blems related to metastatic disease and systemic treatment of

South West Wales Cancer Institute, Singleton Hospital, Swansea, Wales, UK

374 Leonard and Pwint of newer therapies and improved management of the disease, MBC is still an incurable disease.

Approximately 10% of newly diagnosed breast cancer patients have locally advanced and/or metastatic disease and up to 30% of patients who are diagnosed with early breast cancer will later develop metastatic disease. The likelihood of developing metastatic disease depends on the initial stage, tumour biology, and treatment strategy used (2).

Breast cancer is characterised by a very wide natural history even after systemic relapse. The prognoses of patients who develop recurrent disease vary partly depending on the site of relapse. Patients with local/regional

or bone-only recurrences have more favourable prognoses than patients who relapse with visceral or central nervous system (CNS) metastases (3) (Table1). Studies have also shown that duration of survival is inversely

proportional to the number of anatomical sites of metastasis (4, 5). Other important clinical indicators of prognosis include endocrine

receptor status, HER2 protein expression status, and duration of survival from diagnosis to relapse; less than 2 years being relatively unfavourable.

The median survival for all patients after the discovery of metastases is generally around 2 years (6, 7), although for a select group of patients (1%–3%), long-term survival is possible (8, 9). Younger age, good perfor-mance status, and lower tumour burden predicts a higher probability of response to chemotherapy although this response is rarely for more than a few months.

The main goals of treatment for patients with MBC are to prolong

disease progression, and to palliate symptoms.

bone metastasis 19 soft tissue disease 15

10 liver metastasis 8 brain metastasis 3

1. GENERAL PRINCIPLES OF MANAGEMENT

The optimal management of MBC remains a significant therapeutic

challenge. There is no single standard of care for patients with MBC; treat-ment plans require an individualised approach based on multiple factors.

These include patient-related factors, such as age and co-morbidities, and characteristics of the tumour itself (e.g., hormone receptor status, HER2 status), sites of metastasis, degree of tumour-related symptoms, tumour

survival while trying to maintain or improve quality of life (QoL), to delay

Table 1. Median survival associated with sites of metastasis in MBC

Site of relapse Survival (months)

lung metastasis

19. Therapies of metastatic breast cancer: chemotherapy 375 burden, risk for toxicity, history of prior therapy and response, and patient preferences.

1.1 Treatment of specific problems related to metastatic disease

Local radiotherapy should be considered for patients with troublesome local recurrence, symptomatic localised bone disease, CNS disease, and spinal cord compression. In the case of painful bone metastasis with radiological evidence of large lytic cortical deposits in weight-bearing long bones, it is often necessary to precede radiotherapy by orthopaedic fixation to minimise the risk of fracture. As a rule of thumb, alarm bells sound when 50% or more of cortical bone thickness is eroded at the site of the metastasis. Impending fracture is often signalled by a rapid worsen-ing of pain. A symptomatic recurrent pleural effusion can be managed with thoracocentesis followed by talc pleurodesis to relieve symptoms.

Brain metastases occur in about 15% of patients with MBC during the

cancer. Radiotherapy is beneficial in approximately two thirds of patients. Patients with isolated brain metastases could be considered for suitability of surgical resection followed by brain irradiation or stereotactic radiotherapy.

Approximately 70% of patients with advanced breast cancer develop bone metastases at some time during the course of the illness (10). Skeletal- related events (SREs) include bone pain, pathological fractures, spinal cord compression, and hypercalcaemia of malignancy, and are associated with significant reduction in a patient’s QoL. Bisphosphonates such as clodronate, ibandronate, pamidronate, and zoledronic acid have been

patients with bone metastases from advanced breast cancer (11). In a randomised study, zoledronic acid has been shown to be superior to pamidronate for reducing skeletal complications and can be infused in a much shorter period of time (12). Based on the current data, oral ibandronte (50 mg od) appeared to be equally effective as zolindronic acid in sup-pressing bone resorption and is associated with favourable renal safety profile. But the results of ongoing, randomised, phase III comparisons

relative clinical efficacy of the two drugs in reducing SREs in patients with breast cancer and bone metastasis.

The most serious side effect of some amino-bisphophonates (pamidro-nate, zoledronate) is renal impairment, which is uncommon and only seen with intravenous amino-bisphonates after protracted (more than 2 years) therapy. Other toxicities are bone and joint pain, gastrointestinal problems (oral agents only), and injection site problems such as swelling, redness, and, rarely but with increasing awareness, osteonecrosis of the jaw has been reported such that it is now advised that patients needing dental

shown to delay or reduce the risk for developing of SREs by 20–40% in

course of treatment and more often in women with HER2-positive breast

(e.g., ZICE trial and SWOG trial S0308) are required to determine the

376 Leonard and Pwint extractions should be advised to suspend their bisphosphonate therapy prior to that going ahead.

In addition to giving systemic treatment, appropriate attention and treatment must be given to control pain, dyspnoea, cachexia, depression, and other symptoms that frequently are associated with cancer spread. A team approach including participation of the nurse, the psychologist, and ultimately, the palliative team provides optimal care for patients with metastases and supports their families.

1.2 Systemic Treatment of MBC

In principle, palliative therapy of advanced disease is always determined by a judgement as to the balance of effectiveness against toxicity. Usually, therefore, we always consider the possibility of using hormone therapy as first choice as it is both, effective and usually well-tolerated. However many patients may present with life threatening complications that

therapy may be more effective as this approach typically produces a more

chemotherapy is routinely preferred is the patient who presents with hormone receptor-negative disease or where endocrine therapy has been used, but is now failing.

2. CHEMOTHERAPY

For women with hormone-receptor-negative disease, whose cancer is refractory to endocrine therapy or those with symptomatic or rapidly pro-gressive visceral metastases chemotherapy is considered to be the first treatment option (13). With the advent of trastuzumab, biological therapy alone or in conjunction with chemotherapy is an option for patients whose breast cancers overexpress the HER2 protein, a consequence of

Over the past decade, increase in number and diversity of cytotoxic agents has opened the door to more effective management of MBC. Effec-tive first- and second-line, chemotherapeutic agents include the taxanes (paclitaxel and docetaxel), anthracyclines, vinorelbine, capecitabine, gem-citabine, and carboplatin. Examples of commonly used chemotherapy regimens for metastatic breast cancer are shown in Table 2.

With chemotherapy, a balance must be made between achieving a high rate of response and limiting the side effects. An important consideration is whether to use them in combination or in single-agent. Examples of randomised trials of different treatments in metastatic disease showing a survival benefit are shown in Table 3.

require intervention with a rapidly acting anti-cancer drug. Here, chemo-

rapid anti-cancer effect than endocrine manipulation. Other settings where

over-amplification of the HER2 gene.

Tabl

e 2.

Com

mon

che

mot

hera

py re

gim

ens f

or m

etas

tatic

bre

ast c

ance

r (M

BC

)

Reg

imen

s R

espo

nse

Tox

icity

C

omm

ents

1s

t lin

e:

C

MF

40

–50%

al

opec

ia+;

n+;m

+;m

u+

less

toxi

c FE

C/F

AC

/AC

/EC

40

–70%

al

opec

ia++

;n++

;m+;

mu+

+;ca

rd+

sche

dule

s var

y 1s

t or 2

nd li

ne:

D

ocet

axel

3

5–60

%

al

opec

ia+;

n+;m

++;m

u+;n

eu+

caut

ion

if liv

er im

pairm

ent;

grea

ter r

espo

nse

but i

ncre

ased

D

ocet

axel

+C

apec

itabi

ne

50

–60%

alop

ecia

+; n

++; m

++; m

u++;

ne

u+;

HFS

+ to

xici

ty if

com

bine

d w

ith c

apec

itabi

ne

D

ocet

axel

+ T

ratz

50

–80%

al

opec

ia+;

n+;m

++;m

u+;n

eu+;

card

+ fo

r HER

2+ve

MB

C

Pacl

itaxe

l 20

–60%

al

opec

ia+;

n+;m

+;m

u+;n

eu++

va

ries a

ccor

ding

to sc

hedu

le

Pacl

itaxe

l +G

emci

tabi

ne

40–5

0%

alop

ecia

+;n+

;m+;

mu+

;neu

+ in

crea

sing

evi

denc

e fr

om w

eekl

y sc

hedu

le

Pacl

itaxe

l + T

ratz

50

–80%

al

opec

ia+;

n+;m

+;m

u+;n

eu++

;car

d+

for H

ER2+

ve M

BC

U

sual

ly a

s 2nd

or 3

rd li

ne:

Cap

ecita

bine

20

–35%

n+

;v+;

m+;

mu+

+;di

ar+,

HFS

+ G

emci

tabi

ne

20–3

0%

n+;v

+;m

+;m

u+

Vin

orel

bine

30

–50%

n+

;m+;

neu+

C

arbo

plat

in

20–3

5%

n+;m

+;ne

phro

+

Abb

revi

atio

ns:

card

=car

diot

oxic

ity

CM

F=cy

clop

hosp

ham

ide/

met

hotre

xate

/5-f

luor

oura

cil

FEC

/FA

C/A

C/E

C=d

oxor

ubic

ine

or e

piru

bici

n w

ith C

F H

FS=h

and-

foot

synd

rom

e m

=mye

loto

xici

ty

n=na

usea

m

u=m

ucoc

itis

neph

ro=n

ephr

otox

icity

ne

u=ne

urot

oxic

ity

Trat

z=Tr

astu

zum

ab

v=vo

miti

ng

19. Therapies of metastatic breast cancer: chemotherapy

377

Tabl

e 3.

Che

mot

hera

py tr

ials

show

ing

surv

ival

ben

efit

in m

etas

tatic

bre

ast c

ance

r

Firs

t A

utho

r

Tri

al

OS

(mon

ths)

P-

Val

ue

O’S

haug

hnes

sy J

Cap

/Doc

> D

oc

14.5

vs 1

1.5

0.01

3 N

abho

ltz JM

39

2 (2

nd-li

ne)

Doc

> M

ito/V

inb

11.4

vs 8

.7

0.00

97

Bis

hop

JF

209

(1st

-line

)*

Pac

> C

MFP

17

.3 v

s 13.

9 0.

025

Jass

em J

267

(1st

-line

) A

Pac

> FA

C

23.3

vs 1

8.3

0.01

3 A

lbai

n K

52

9 (1

st-li

ne)*

G

em/P

ac>P

ac

18.5

vs 1

5.8

0.01

8 Jo

nes S

44

9 (1

st-li

ne)*

D

oc>

Pac

15

.4 v

s 12.

7 0.

03

Stew

art D

J 24

9 (1

st-li

ne)

CA

F >

CM

xF

15.2

vs 1

0.9

0.00

3 Sl

amon

D

469

(1st

-line

)**

Trat

z/Pa

c>Pa

c

25.1

vs 2

0.3

0.04

6 M

arty

M

186

(1st

-line

) Tr

atz\

Doc

>Doc

31

.2 v

s 22.

7 0.

0325

A

bbre

viat

ions

: C

AF=

cycl

opho

spha

mid

e/do

xoru

bici

n;

Cap

=Cap

ecita

bine

;

Doc

= D

ocet

axel

; D

oxo=

doxo

rubi

cin;

Ep

i=Ep

irubi

cin;

FA

C=

fluor

oura

cil/d

oxor

ubic

in/c

yclo

phos

pham

ide;

G

em=G

emci

tabi

ne;

Mito

/Vb=

mito

myc

in/v

inbl

astin

e;

Pac=

Pacl

itaxe

l.

*= p

rior

adju

vant

or n

eoad

juva

nt c

hem

othe

rapy

incl

udin

g an

thra

cycl

ine

**=

who

rec

eive

d ad

juva

nt a

nthr

acyc

line

rece

ived

pac

litax

el a

nd w

ho n

ot, r

ecei

ved

Dox

o or

Epi

plu

s C

Leonard and Pwint378

511

(2nd

-line

)*

CM

FP=c

yclo

phos

pham

ide/

met

hotre

xate

/5-f

l uor

oura

cil/p

redn

ison

e;

CM

xF=

cycl

opho

spha

mid

e/m

itoxa

ntro

ne/5

-flu

orou

raci

l;

No.

of P

atie

nts

19. Therapies of metastatic breast cancer: chemotherapy 379

2.1

loss, nausea, and vomiting (15, 16). The taxane/antimetabolite combinations have been shown to offer

clinically meaningful survival advantages with a manageable safety profile.

O’Shaughnessy et al. reported that a combination of capecitabine and docetaxel (1,250 mg/m2 bd and 75 mg/m2, respectively) improves RR (42% vs 30%, P = 0.006), median TTP (6.1 vs 4.2 months, P = 0.0001) and prolongs median survival (14.5 vs 11.5 months P = 0.013) compared with docetaxel (100 mg/m2) alone in anthracycline pretreated patients. The combination therapy was more toxic with respect to gastrointestinal adverse events and hand-foot syndrome, but the QoL scores were similar in the two treatment arms (26).

On the basis of this trial, this docetaxel/capecitabine regimen was approved in the UK by the National Institute for Clinical Excellence (NICE).

2.1.2 Gemcitabine/paclitaxel combination vs paclitaxel

A trial by Albain et al. comparing first-line gemcitabine (1,250 mg/m2) plus paclitaxel (175 mg/m2) with paclitaxel alone in patients with

improvement was also seen with the combination regimen. These data also correlated with an improvement in overall survival in an interim analysis (18.5 vs 15.8 months P = 0.018). Although there was more haematological toxicity with the combination, overall toxicities in both arms were manageable (17).

2.1.3 Anthracycline/Taxane combination vs anthracycline or taxane

In women with no prior anthracycline chemotherapy, clinical trials have shown that anthracycline-taxane combinations are more effective than anthracyclines or taxanes alone but often more toxic. A clinical trial by Jassem et al. reported higher RR (68% vs 55%), TTP (8.3 vs 6.2) and improvement in OS (23.3 vs 18.3 moths) with doxorubicin and paclitaxel

Combination sequential mono-therapy

2.1.1 Docetaxel/capecitabine combination vs docetaxel

P <0.0003) and ORR (39% vs 23%; P = 0.007). Global QoL and pain MBC showed a significant improvement in TTP (5.2 vs 2.9 months;

This remains a controversial issue and the decision must be indivi-dualised for each patient (14).

Clinical trials have shown that, compared with single-agent chemo-therapy, combinations have a better response rate and greater time toprogression and modest improvement in overall survival but at the expense of some increased treatment-related toxicities of leucopenia, hair

380 Leonard and Pwint

Sledge et al. reported no significant survival benefit (OS: 18.9 vs 22.2 vs 22.0 months) with combination regimen of doxorubicin, 50 mg/m , plus paclitaxel, 150 mg/m2/day when compared with either doxorubicin

2 2

Although improved response with combination therapy often translates into improved symptom control, quality of life, and functional status, there is insufficient evidence to comment on the impact of the regimens on the net clinical benefit from the patients’ perspectives.

Based on the available evidence, it seems reasonable to recommend that combination therapy may be particularly useful in patients needing a prompt reduction in tumour burden (extensive and rapidly progressive disease) as they are less likely to be eligible for a second line following failure to first line, whereas sequential mono-therapy might be applied in slowly progressing disease.

Currently, docetaxel plus capecitabine combination is the NICE recommended first-line therapy in the UK for anthracycline treated MBC patients. However in practice, most practice in the UK is single-agent docetaxel. On disease progression, it is usually followed by capecitabine or less often vinorelbine but whose neurotoxicity and myelotoxicity may be problematic after taxotere unlike capecitabine.

Gemcitabine is only occasionally used alone but this agent together with paclitaxel may be slightly better-tolerated combination alternative to capecitabine/docetaxel although it is not NICE approved in the UK at present.

2.2 Chemotherapeutic agents commonly used in MBC

2.2.1 Anthracyclines

Anthracycline antibiotics (doxorubicin and epirubicin) are among the most active drugs in MBC and cause anti-tumour effects by a variety of mechanisms including generation of oxygen free radicals and impairing DNA replication.

Recent studies have conferred that overexpression of topoisomerase II alpha (TOP2A) gene may provide as a rational predictive marker for

when compared to fluorouracil, doxorubicin, and cyclophosphamide (FAC) as first-line therapy for women with MBC (32).

However, there is no proof from randomised clinical trials that com-bination chemotherapy is superior in terms of overall survival comparedwith the same agents used sequentially on disease progression.

60mg/m and (paclitaxel 175mg/m at progression), or paclitaxel (doxoru-bicin at progression). But the combination regimen resulted in a superiorresponse rate (47% vs 36% vs 34%; P = 0.007) and time to treatmentfailure (8.0 vs 5.8 vs 6.0 months; P = 0.009) and no difference betweenthe treatment arms in terms of safety or QoL (18).

2


They have been shown to retain significant activity in women who develop metastases more than 12 months after receiving adjuvant anthracyclines, but their efficacy in women who have an anthracycline-free interval of less than 12 months is uncertain (19–22).

Fossati et al. in 1998 demonstrated that first-line treatment with anthracycline-containing combination regimens had superior efficacy compared with mono-chemotherapy (15).

Systematic Reviews 2006 showed a statistically significant advantage of anthracycline containing regimens over non-anthracycline regimens for tumour response and TTP but no improvement in OS and was also associated with greater toxicity (24).

Their use is limited by cumulative dose-dependent cardio toxicity, above 450 mg/m2 of doxorubicin or 1,000 mg/m2 of epirubicin. They are also associated with significant acute toxicity (nausea and vomiting, myelotoxicity, alopecia).

Myocet (liposome- encapsulated doxorubicin) (75mg/m2 every 3 2

mg/m2 every 3 weeks) have shown within randomised trials to be equally

2.2.2 Taxanes

Paclitaxel and docetaxel, are microtubule inhibitors, and also inhibit tumour angiogenesis.

In patients whose disease has progressed during or following anthracycline therapy or who are ineligible for further anthracycline

anthracycline-resistant breast carcinoma. Taxanes are also increasingly being used, either as single agents or together with other agents as first-line treatment for MBC.

A systematic review of 21 randomised MBC trials by Ghersi et al. in

response to anthracycline therapy. It is often co-amplified in 35% of

HER2 negative cancer is not well defined. Approximately, 30–40% of chemotherapy-naive patients with meta-

static breast cancer respond to anthracycline therapy with a median overall survival of 22 months.

rates in 40–70% of the patients (23).

weeks) and Caelyx (PLD 50 mg/m every 4 weeks doxorubicin 60

effective and significantly less cardiotoxic than conventional doxocubicinin metastatic breast cancer.

and docetaxel have shown a 30–50% response rate in patients with therapy, taxanes have been considered the agent of choice. Paclitaxel

2005, comparing taxane with non-taxane regimens showed evidence in favour of taxane-based regimens in terms of OS and RR. But if

The most commonly used combinations are AC/EC (doxorubicin/

5-fluorouracil plus doxorubicin or epirubicin), which have yielded responseepirubicin plus cyclophosphamide) or CAF/CEF (cyclophosphamide/

tumours with HER-2 amplification. But its level of overexpression in


The efficacy of docetaxel and paclitaxel can be increased while decreasing drug-related toxicity, by adapting dose-dense schedules of drug administration.

2.2.2.1 Docetaxel

Docetaxel is now generally considered as the standard treatment for antracycline pretreated MBC. As a single agent, it produces objective responses in up to 60% of chemotherapy naive patients with MBC.

capecitabine combination showed a better RR and OS compared to docetaxel alone.

In a mono-therapy trial in anthracycline-naive MBC patients, docetaxel (100 mg/m2 every 3 weeks) produced a superior response rate (48% vs 33%; p = 0.008) but similar survival when compared with

2

term use is often limited by haematological toxicities, peripheral neuropathy, fatigue, nail toxicity, and fluid retention. There is randomised evidence that the weekly docetaxel regimen is at least as effective as the every-3-weeks regimen and causes less toxicity, but requires weekly outpatient visits.

2.2.2.2 Paclitaxel

Paclitaxel has been shown to produce equivalent RR, TTP, and survival to CMFP (cyclophosphamide, methotrexate, fluorouracil, and prednisolone) chemotherapy as front-line treatment for patients with MBC (30).

But, with the 3-weekly schedule, at the dose of 175 mg/m2, paclitaxel has been unable to demonstrate superiority over either doxorubicin or docetaxel in the first-line metastatic setting.

Weekly paclitaxel regimen at doses ranging between 80 and 90 mg/m2/week has demonstrated higher activity and less toxicity for MBC

also effective and well tolerated in heavily pretreated refractory disease

chemotherapy for metastatic breast cancer the difference is no longer statistically significant (25).

Combinations of taxanes and targeted biologic agents, such as trastuzumab, present clinical synergism with significant improvements in overall survival in patients with MBC.

the analysis is limited to the trials in women receiving first-line

In a trial including first- and second-line therapy, docetaxel and

docetaxel to combinations such as mitomycin/vinblastine or methotrexate/fluorouracil have favoured the taxane in treating MBC after anthracycline

Docetaxel although generally well tolerated as a single agent, long-resistance (26–29).

doxorubicin (75 mg/m 3 weekly) (20). Comparisons of single-agent

in both nonrandomised and in randomised clinical trials (55,56). It is


A newer albumin-bound formulation of paclitaxel (ABI-007) showed the safety of a high dose of 300 mg/m2 infused over 30 minutes without pre-medication. A significantly higher RR, TTP, and lower incidence of grade 4 neutropenia and no hypersensitivity reactions were seen when compared to paclitaxel (31).

2.2.3 Capecitabine

The oral capecitabine is designed to generate 5FU preferentially in tumour tissue and to mimic continuous infusion 5FU through activation pathway involving thymidine phosphorylase.

Capecitabine monotherapy at 1,250 mg/m2 twice daily for 14 days every 21 days has been well tolerated. When used following failure of anthracycline and taxane therapy, it has achieved response rates of 15% – 26% and a median survival in excess of 1 year (33–36). The common adverse events are hand-foot syndrome, diarrhoea, and nausea but Myelo-suppression and alopecia are rare.

Capecitabine a dose of 1,000 mg/m2 twice daily has been shown to be safe and effective in the 70 years and older patients who are candidates for cytotoxic therapy and do not have severely impaired renal function (37).

2.2.4 Gemcitabine

Gemcitabine, a nucleotide analogue that inhibits DNA synthesis has produced an ORR of 20–30% in those with previous exposure to chemo-therapy, and 40% as first-line treatment in MBC (40–42). It is well tolerated in general including in the elderly patients. It is associated with a low incidence of nausea, vomiting, and alopecia. The most common dose-limiting toxicities are usually neutropenia and thrombocytopenia. The combination of gemcitabine plus paclitaxel has shown a 68% overall

and in elderly patients or those with poor performance status. There is evidence for better outcomes when used in combination with gemcitabine as a first-line treatment in MBC.

The side effects of paclitaxel include hypersensitivity reactions (such as shortness of breath or skin rash), myelosupression, peripheral neuropathy, cardiac rhythm disturbances, joint or muscle pain, diarrhoea, nausea and vomiting, or hair loss. Patients often receive premedication before receiving paclitaxel to prevent possible allergic reactions.

Capecitabine plus docetaxel significantly increased survival comparedwith docetaxel alone (14.5 vs 11.5 months) in patients with metastaticdisease recurring after anthracycline therapy.

Capecitabine/trastuzumab combination therapy has produced pro-

patients with HER2 overexpressing metastatic breast cancer (38, 39). mising efficacy and safety results for first- or second-line therapy in


14) plus same dose docetaxel, but with significantly less drug-related hand-foot syndrome, mucositis, and diarrhoea (43).

2.2.5 Vinorelbine

The efficacy of vinorelbine, a third generation vinca alkaloid, alone or in combination with other agents has been extensively investigated in

peripheral neuropathy are the primary toxicities (44). Addition of Vinorelbine to epiribicin has been shown to confer higher

RR and PFS but no OS benefit. An oral formulation of vinorelbine, an

2.2.6 Carboplatin

Platinum compounds or alkylating agents have shown significant

Recent phase III data suggest that adding carboplatin to a paclitaxel/ trastuzumab regimen produces superior efficacy than paclitaxel/ trastuzumab alone for patients with HER2-positive metastatic disease. Myelosuppression is more common with this three-drug regimen.

However, preliminary results from the NCCTG 98-32-52 trial suggest that a weekly schedule of carboplatin/paclitaxel plus trastuzumab produces at least as effective results as the 3-weekly schedule but with much less toxicity.

It has been suggested that “triple negative” (TN) breast cancers, (ER-ve, PR-ve, HER2 –ve) may be particularly sensitive target for platinum

possibility of TN breast cancers sharing BRCA1-associated DNA repair defects, which may be targeted with DNA cross-linking agents like platinum compounds. Currently there is an ongoing clinical trial by CRUK using carboplatin for patients with metastatic genetic breast cancer with BRCA1 or 2 mutations.

response rate as first-line chemotherapy and a 48% as second-line

Gemcitabine has also been shown to be active in combination with docetaxel (ORR: 36–79%) and a phase III trial has shown that gemcitabine plus docetaxel (1,000 mg/m2 on days 1 and 8 plus docetaxel 75 mg/m2) is as effective as capecitabine (1,250 mg/m2 bd day 1 through

therapy (17).

effective and well-tolerated agent, was evaluated as first-line chemotherapyfor MBC offering an alternative to the intravenous route.

a first-line single agent. Myelosuppression, gastrointestinal toxicities, and MBC. Response rates, varying from 35–50%, have been demonstrated as

activity in previously untreated patients with MBC. Single-agent carbo-platin produces ORR of 20–35% (45–47). Retrospective review of clinicaltrials by Perez revealed that carboplatin combined with paclitaxelor docetaxel was more effective than carboplatin or taxanes alone,with ORR of 53–62% in the first-line treatment of MBC.

compound. This is based on preclinical evidence which suggests the


Slamon et al. demonstrated the addition of trastuzumab to paclitaxel significantly increased TTP ( p < 0.001; median 6.9 months versus 3.0 months) and RR ( p < 0.001; 49% versus 17%), and resulted in improved OS (median 25 months vs 18 months) in anthracycline-pretreated patients with HER2 positive MBC (49). Marty et al. observed similar results, in a study comparing trastuzumab plus with docetaxel alone as first-line therapy for HER2 positive MBC (50).

Combinations of trastuzumab and other cytotoxic agents, including, gemcitabine, carboplatin, and vinorelbine, are also highly active first- or second-line therapies in patients with HER2 overexpressing MBC.

Despite impressive tumour response rates, most of the metastatic patients develop disease progression during treatment. Although a few retrospective analyses supporting the practice of continuing trastuzumab alone or combined with other drugs beyond disease progression, in the absence of data from randomised trials is at least debatable.

2.2.8 Lapatinib

Lapatinib, an oral dual inhibitor for EGFR1 and HER2 has shown efficacy when used either alone or in combination with conventional chemotherapy, in patients with HER2-positive breast cancer who had progressed during trastuzumab treatment (51). A phase II trial of lapatinib as first-line treatment of HER2-positive MBC showed a 35% response rate and is undergoing further evaluation in Phase III clinical trials (52).

There is preliminary evidence of clinical effect to suggest that lapatinib is active in HER2-positive patients with new or progressive brain metastases (53). Further investigation of lapatinib in HER2-positive CNS disease is ongoing.

The newly developed targeted therapies including inhibitors of angiogenesis, e.g., Bevacizumab and several other agents (alone and in combination) are also being investigated, and are expected to lead to the evolution of more effective therapies in MBC.

REFERENCES:

1. National Cancer Institute: SEER Cancer Statistics Review (1975–2000). Available at: http://seer.cancer.gov/csr.

2. Honig SF. Treatment of metastatic disease. In: Harris JR, Lippman ME, Morrow M et al. and Eds. Diseases of the Breast. Philadelphia: Lippincott-

2.2.7 Trastuzumab

Trastuzumab is a humanised monoclonal antibody directed against HER2 glycoprotein, overexpressed in 20–25% of human breast cancers. As a single agent it has a response rate around 35% in selected MBC patients (48). The addition of trastuzumab to chemotherapy results in significant improvements in clinical outcomes, including overall survival.

Raven Publishers, 1996; 669–734.


6. Diaz-Canton EA, Valero V, Rahman Z, et al. Clinical course of breast cancer patients with metastases confined to the lungs treated with chemotherapy. The

7. Valagussa P, Bonadonna G, Veronesi U. Patterns of relapse and survival following radical mastectomy. Analysis of 716 consecutive patients. Cancer 1978; 41:1170–1178.

8. Yeatman TJ. The natural history of locally advanced primary breast carcinoma and metastatic disease. Surg Oncol Clin N Am 1995; 4:569–589.

9. Kamby C, Ejlertsen B, Andersen J, et al. The pattern of metastases in human breast cancer. Influence of systemic adjuvant therapy and impact on survival.

10. Coleman RE. Metastatic bone disease: clinical features, pathophysiology and

11. Pavlakis N, Schmidt R, Stockler M. Bisphosphonates for breast cancer. Cochrane Database Syst Rev 2005; (3).

12. Rosen LS, Gordon D, Kaminski M, et al. Long-term efficacy and safety of zoledronic acid compared with pamidronate disodium in the treatment of skeletal complications in patients with advanced multiple myeloma or breast carcinoma: a randomised, double-blind, multicentre, comparative trial. Cancer 2003; 98:1735–1744.

13. GN. Chemotherapy of Metastatic Breast Cancer: What to Expect in 2001 and

14. Miles D, von Minckwitz G, Seidman AD. Combination versus sequential

6):13–16. 15. Fossati R, Confalonieri C, Torri V, et al. Cytotoxic and hormonal treatment for

metastatic breast cancer: A systematic review of published randomised trials involving 31,510 women. J Clin Oncol 1998; 16:3439–3460.

16. Carrick S, Parker S, Wilcken N, Ghersi D, Marzo M, Simes J. Single agent versus combination chemotherapy for metastatic breast cancer. Cochrane Database of Systematic Reviews 2005.

17. gemcitabine plus paclitaxel (T) as frontline therapy for metastatic breast cancer (MBC): first report of overall survival. J Clin Oncol 2004;22:5s.

18. and the combination of doxorubicin and paclitaxel as front-line chemotherapy for metastatic breast cancer (MBC): an Intergroup trial (E1193). J Clin Oncol

19. compared with paclitaxel in metastatic breast cancer. J Clin Oncol 2005; 23:5542–5551.

20. versus doxorubicin in patients with metastatic breast cancer. J Clin Oncol 1999; 17:2341–2354.

21.

3. Cardoso F, Di LA, Lohrisch C, et al. Second and subsequent lines of chemotherapy for metastatic breast cancer: what did we learn in the last two decades? Ann Oncol 2002; 13:197–207.

4. premenopausal women with metastatic breast cancer; an Eastern Cooperative.

5. Hortobágyi GN. Can we cure limited metastatic breast cancer? J Clin Oncol 2002; 20:620–623.

Falkson G, Holcroft C, Gelman RS, et al. Ten-year follow-up study of

Oncology Group study. J Clin Oncol 1995; 13:1453–1458.

the literature. Ann Oncol 1998; 9:413–418. University of Texas M.D. Anderson Cancer Centre experience and review of

Acta Oncol 1988; 27:715–719.

treatment strategies. Cancer Treat Rev 2001; 27:165–176.

single-agent therapy in metastatic breast cancer. The Oncologist 2002; 7(suppl

Albain KS, Nag S, Calderillo-Ruiz G, et al. Global phase III study of

Sledge GW, Neuberg D, Ingle J, et al. Phase III trial of doxorubicin, paclitaxel

2003; 21:588–592. Jones S, Erban J, Overmoyer B, et al. Randomised phase III study of docetaxel

Chan S, Friedrichs K, Noel D, et al. Prospective randomised trial of docetaxel

Paridaens R, Biganzoli L, Bruning P, et al. Paclitaxel versus doxorubicin as first

Esteva FJ, Valero V, Pusztai L, Boehnke-Michaud L, Buzdar AU, Hortobagyi

first line single-agent chemotherapy for metastatic breast cancer: A European

Beyond. The Oncologist Apr 2001; 6:133–146.


25.

26. O’Shaughnessy J, Miles D, Vukelja S, et al. Superior survival with capecitabine

27. Nabholtz JM, Senn HJ, Bezwoda WR, et al. Prospective randomised trial of docetaxel versus mitomycin plus vinblastine in patients with metastatic breast cancer progressing despite previous anthracycline-containing chemotherapy. 304 Study Group. J Clin Oncol 1999; 17:1413–1424.

28. Sjöström J, Blomqvist C, Mouridsen H, et al. Docetaxel compared with sequential methotrexate and 5-fluorouracil in patients with advanced breast cancer after anthracycline failure: a randomised phase III study with crossover on progression by the Scandinavian Breast Group. Eur J Cancer 1999; 35:1194–1201.

29. Bonneterre J, Roche H, Monnier A, et al. Docetaxel vs 5-fluorouracil plus vinorelbine in metastatic breast cancer after anthracycline therapy failure. Br J Cancer 2002; 87:1210–1215.

30. Bishop JF, Dewar J, Toner GC, et al. Initial paclitaxel improves outcome compared with CMFP combination chemotherapy as front-line therapy in untreated metastatic breast cancer. J Clin Oncol 1999; 17:2355–2364.

31. Gradishar WJ, Tjulandin S, Davidson N, et al. Superior efficacy of albumin-bound paclitaxel, ABI-007, compared with polyethylated castor oil-based paclitaxel in women with metastatic breast cancer: results of a phase III trial. J Clin Oncol 2005; 23.

32. Jassem J, Pienkowski T, Pluzanska A, et al. Doxorubicin and paclitaxel versus fluorouracil, doxorubicin, and cyclophosphamide as first line therapy for women with metastatic breast cancer: Final results of a randomised phase III multicentre trial. J Clin Oncol 2001; 19:1707–1715.

33. Blum JL, Jones SE, Buzdar A. Capecitabine (Xeloda) in 162 patients with paclitaxel-pretreated MBC: updated results and analysis of dose modification. Eur J Cancer 2001; 37:S190a.

34. Blum JL, Dieras V, Lo Russo PM, et al. Multicentre, phase II study of capecitabine in taxane-pretreated metastatic breast carcinoma patients. Cancer 2001; 92:1759–1768.

35. Reichardt P, Von Minckwitz G, and Luck HJ, et al. Capecitabine: the new standard in metastatic breast cancer failing anthracycline and taxane containing chemotherapy? Mature results of a large multicentre phase II trail. Eur J Cancer 2001; 37(suppl 6): S191a.

36. Leonard RCF, Twelves C, Breddy J, et al. Capecitabine named-patient programme for patients with advanced breast cancer: The UK experience. Eur J Cancer 2002; 38:2020–2024.

37. Bajetta E, Procopio G, Celio L, et al. Safety and efficacy of two different doses

Organization for Research and Treatment of Cancer randomised study with cross-over. J Clin Oncol 2000; 18:724–733.

22. Tabernero J, Climent MA, Lluch A, et al. A multicentre, randomised phase II studies of weekly or 3-weekly docetaxel in patients with metastatic breast

23. Chemotherapy of Metastatic Breast Cancer: What to Expect in 2001 and Beyond: The Oncologist April 2001; 6(2): 133–146.

24. Lord S, Ghersi D, Gattellari M, Wortley S, Wilcken N, Simes J. Anti–tumour antibiotic containing regimens for metastatic breast cancer. Cochrane Database of Systematic Reviews 2004, Issue 4.

cancer. Ann Oncol Sep 2004; 15(9): 1358–1365.

Ghersi D, Wilcken N, Simes J, Donoghue E. Taxane containing regimens for meta-static breast cancer. Cochrane Database of Systematic Reviews 2005, Issue 2.

with advanced breast cancer: phase III trial results. J Clin Oncol 2002; plus docetaxel combination therapy in anthracycline-pretreated patients

20:2812–2823.

J Clin Oncol 2005; 23:2155–2161. of capecitabine in the treatment of advanced breast cancer in older women.


42. Spielmann M, Llombart-Cussac A, Kalla S. Single-agent gemcitabine is active in previously treated metastatic breast cancer. Oncology 2001; 60:303–307.

43. capecitabine plus docetaxel (CD) for anthracycline-pretreated metastatic breast cancer (MBC) patients (pts): Results of a European phase III study. J Clin Oncol 2005; 23:581.

44. advanced breast cancer in women 60 years of age or older. Ann Oncol 1999; 10:397–402.

45. Kolaric K, Vukas D. Carboplatin activity in untreated metastatic breast cancer patients—results of a phase II study. Cancer Chemotherapy Pharmacology 1991; 27:409–412.

46. carcinoma of the breast, Ann Oncol 1990; 1:P3: 33.

47. Martin M, Diaz-Rubio E, and Casado A, et al. Carboplatin: an active drug in metastatic breast cancer, J Clin Oncol 1992; 10:433–437.

48. Vogel CL, Cobleigh MA, Tripathy D, et al. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol 2002; 20:719–726.

49. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001; 344:783–792.

50. Marty M, Cognetti F, Maraninchi D, et al. Randomised phase II trial of the efficacy and safety of trastuzumab combined with docetaxel in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer administered as first-line treatment: the M77001 study group. J Clin Oncol 2005; 23:4265–4274.

51.

52. Gomez H, Chavez M, Doval D. A phase II, randomised trial using the small molecule tyrosine kinase inhibitor lapatinib as a first-line treatment in patients with FISH positive advanced or metastatic breast cancer. Am Soc Clin Oncol 2005; 23:3046.

53. Phase II trial of lapatinib for brain metastases in patients with HER2+ breast cancer. ASCO Clinical Science Symposium – Saturday, June 3, 2006: The ASCO Abstracts2View™.

54. ABC of Breast Diseases. 55.

paclitaxel in patients with locally advanced or metastatic breast cancer by Verril et al. NCRI Birmingham, UK 2006 (abstract).

56. Dose-dense therapy with weekly 1-hour paclitaxel infusions in the treatment of metastatic breast cancer. J Clin Oncol. Oct 1998; 16(10): 3353–3361. Review.

38. Yamamoto T, Iwase S, Kitamura K, et al. Multicentre phase II study of trastuzumab (H) and capecitabine (X) as first- or second-line treatment in HER2 over-expressing metastatic breast cancer (Japan Breast Cancer Study Group: JBCSG-003). J Clin Oncol, 2005; 23:78s.

39. Schaller G, Bangemann N, Weber J, et al. Efficacy and safety of trastuzumab plus capecitabine in a German multicentre phase II study of pre-treated metastatic breast cancer. J Clin Oncol 2005; 23:57s

40. Phase II study of gemcitabine as first-line chemotherapy in patients with advanced or metastatic breast cancer. Anticancer Drugs 1999; 10:155–162.

41. Blackstein M, Vogel CL, and Ambinder R. Gemcitabine as first-line therapy in patients with metastatic breast cancer: A phase II trial. Oncology 2002; 62:2–8.

Chan S, Romieu G, Huober J. Gemcitabine plus docetaxel (GD) versus

Vogel C, O’Rourke M, Winer E, et al. Vinorelbine as first-line chemotherapy for

Carmo-Pereira J, Dittrich C, Keizer J, et al. Phase II trial of carboplatin in

Burris HA III. Dual kinase inhibition in the treatment of breast cancer: Initial experience with the EGFR/ErbB-2 inhibitor lapatinib. Oncologist 2004; 9:10–15.

Anglo-Celtic IV: an NCRN randomised phase 3 trial of weekly versus 3 weekly

Erratum in: J Clin Oncol May 10 2006; 24(14):2220.

ASCO 2006 Annual Meeting: N.U. Lin, L.A. Carey, M.C. Liu, J. Younger, S.E.

Chapter 20

THE DIAGNOSIS AND TREATMENT OF BONE METASTASES IN BREAST CANCER

Allan Lipton, MD Pennsylvania State University, College of Medicine, Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, USA

Abstract: Bone metastases from breast cancer can cause clinically relevant skeletal morbidity. These skeletal-related events (SREs) can undermine patient quality of life and functional independence. Early diagnosis of bone metastases could provide important benefits to patients. Although there is no clear consensus, some guidelines suggest bone scans for patients with advanced breast cancer. Skeletal scintigraphy should be the first choice for screening, and confirmation of diagnosis should be made with plain radiography, computed tomography, or magnetic resonance imaging. All patients with advanced metastatic disease should receive systemic anticancer therapy. Furthermore, supplementation of systemic anticancer therapy with bisphosphonates or other therapeutic approaches is suggested for prevention of SREs. Bisphosphonates have demonstrated significant reductions in the risk of SREs in patients with bone metastases from breast cancer. Zoledronic acid has demonstrated benefits beyond those of pamidronate in patients receiving cytotoxic or hormonal therapy for advanced breast cancer. Other palliative therapies and other therapies that reduce skeletal morbidity associated with malignant bone lesions are also discussed. Considering the increasing survival of patients with breast cancer achieved with emerging therapies, monitoring for, and treatment of, bone metastases to maintain the quality of patient survival are critical.

Keywords:

© 2007 Springer.


events, zoledronic acid bisphosphonates, bone metastases, breast cancer, screening, skeletal-related

390 Lipton

1. INTRODUCTION

The bone microenvironment can provide a fertile “soil” in which metastasizing cancer cells may grow (1). The majority (~70%) of patients with advanced breast cancer develop bone metastases (2), which can have devastating consequences for their quality of life (QoL) and functional independence (3). Metastatic bone disease from breast cancer typically involves the ribs, spine, pelvis, skull, and proximal limbs, and can compromise skeletal integrity (4). Patients with bone metastases have an increased risk of skeletal-related events (SREs), which include pathologic fractures, the requirement for palliative radiotherapy or surgery to bone,

metastases from breast cancer, SREs occurred in 68% of patients

Similarly, approximately 50% of patients with bone metastases from breast cancer (N = 113) who received standard anticancer therapy without bisphosphonates experienced an SRE (7). With a median survival in this population of approximately 2 years, patients typically survive long enough to experience multiple SREs from their bone lesions (2,8). For example, in two published reports, patients treated only with standard anticancer therapy experienced 1.1 (7) and 3.7 (6) SREs/year.

spinal cord compression, and hypercalcemia of malignancy (5). In a

(N = 384) who did not receive bone-specific therapy (Figure 1) (6).

2-year, large-scale, randomized clinical trial in patients with bone

Figure 1. Skeletal-related events (SREs) are a serious threat to patients with bone metastases from breast cancer. HCM = hypercalcemia of malignancy. Data from Lipton A,et al (6).

391

As metastatic disease progresses, the incidence of skeletal complications increases (9), and each type of SRE has been associated with decreases in multiple domains of health-related QoL (10). For example, in a retrospec-tive analysis of patients with advanced breast cancer receiving chemo-therapy or hormonal therapy in a recent study (N = 1,130), experiencing a fracture was associated with a significant increase in the risk of death

addition, patients who experience an SRE are at higher risk of develop-ing subsequent events (12,13). In a retrospective analysis of patients with breast cancer treated with 4 mg zoledronic acid or 90 mg pamidronate (N = 1,122), patients with an SRE prior to study entry (N = 760; 68%) had a two fold increased risk of developing an SRE on study versus patients without a prior SRE (hazard ratio = 2.08) (12). Therefore, screening and diagnosis of bone metastases allow early initiation of therapy and are essential for optimal disease management. Although several treatments are available for palliating symptoms of bone metastases (e.g., analgesics, radiotherapy), bisphosphonates are the standard of care for treatment of bone metastases because they treat the underlying pathology and protect patients from skeletal morbidity. Maintaining skeletal health in patients with advanced breast cancer may preserve QoL and functional independence.

2. SCREENING OF BONE METASTASES

metastases, the associated risk factors have not been clearly identified. Therefore, there is no clear consensus regarding when to screen for bone metastases from breast cancer. Current American Society of Clinical Oncology (ASCO) guidelines do not recommend routine bone scan surveillance (14), whereas the National Comprehensive Cancer Network (NCCN) guidelines state that bone scans are recommended for patients with stage III or IV breast cancer but are optional for patients with stage I or II breast cancer (15). The NCCN guidelines also state that for posttherapy surveillance and follow-up, routine bone scans provide no advantages in survival or pain relief for asymptomatic patients and are not recommended (15).

There is also no consensus on the optimal imaging modality for screening of bone metastases in patients with breast cancer. An expert panel per-formed a literature-based review evaluating current imaging modalities in terms of statistical strength of study design and scientific strength of assessed endpoints (16). Based on the resulting algorithm, skeletal scintigraphy should be the first choice for screening, and confirmation of diagnosis should be made with plain radiography, computed tomography, or magnetic resonance

20. Diagnosis and treatment of bone metastases

compared with patients without a fracture (32%; P = 0 .003) (11). In

Although most patients with advanced breast cancer develop bone

392 Lipton

No studies of biochemical markers have been able to diagnose or predict the development of bone metastasis, although several markers are being evaluated as possible predictors of bone metastasis. A recent study indicated a significant linear association between urinary levels of alpha C-telopeptide (CTX) and number of bone metastases (P < 0.001) in patients with breast cancer (N = 90) (17). At this time, no marker has clearly demonstrated a predictive correlation. Therefore, the use of bone markers in screening for bone metastases and patient response to therapy is not recommended outside the context of a clinical trial.

Figure 2. Algorithm for detection of bone metastases. *Bone biopsy may be required for confirmation. †Indications for CT versus MRI are not well defined. In general, CT is indicated for lesions in weight-bearing or chest-wall bones, and MRI is indicated for spinal lesions. XR = Plain radiography; CT = Computed tomography; MRI = Magnetic resonance imaging; SS = Skeletal scintigraphy. Adapted with permission from Hamaoka T, et al. Bone imaging in metastatic breast cancer. J Clin Oncol 2004, 22(14):2942–2953. Reprinted with permission from the American Society of Clinical Oncology (16).

Although many physicians might not consider screening for bone

metastases in patients with no distant metastases from breast cancer and no symptoms of bone metastases, earlier diagnosis of skeletal lesions could provide important benefits. For example, treatment with bisphosphonates has been shown to significantly delay the onset of potentially debilitating SREs, including pathologic fractures (18). Pathologic fractures typically cannot heal without intervention, and may require extended convalescence.

imaging (Figure 2) (16). Use of routine positron emission tomography scanning outside the clinical trial setting was not supported by the literature (16).

393 Moreover, pathologic fractures were associated with significant reductions in survival in patients with advanced breast cancer in a recent exploratory analysis of a phase III clinical trial database (11). Delaying the onset of SREs may preserve functional independence and may possibly provide survival benefits. Therefore, defining tools to aid in the early diagnosis of bone metastasis is critical.

3. TREATMENT OF BONE METASTASES

All patients with advanced metastatic disease should receive systemic anticancer therapy, which can palliate bone pain and may delay disease progression, and prevent or delay the development of bone metastases (19). However, cytotoxic chemotherapy or hormonal therapy is rarely sufficient for prevention of SREs in patients with bone metastases. For treatment of bone metastases from breast cancer, supplementation of systemic anticancer therapy with bisphosphonates, or other therapeutic approaches is suggested. Several treatments are available for symptomatic management of SREs. Surgical stabilization should be used in patients with large osteolytic lesions on weight-bearing areas, and surgery can stabilize bones after fractures have occurred (19). Analgesics and radiopharmaceuticals can provide temporary pain relief, and external beam radiotherapy can both reduce pain and stabilize bone lesions (19). Radiotherapy may also reduce neurologic complications from spinal cord compression and bone pain. Bisphosphonates reduce bone pain and the need for radiotherapy (7). Furthermore, bisphosphonates directly treat the underlying disease and reduce the incidence of additional SREs.

3.1. Bisphosphonates

Bisphosphonates are a class of pharmaceutical agents that bind avidly to the bone at sites of active bone remodeling, potently inhibit osteoclast-mediated osteolysis, and have demonstrated clinical utility in patients with benign and malignant bone diseases including Paget’s disease, osteoporosis, and tumor-induced osteolysis (20). Bisphosphonates are the only treatment identified to both provide symptomatic benefit and treat malignant osteolysis, the underlying cause of SREs and bone pain, thereby being the treatment of choice for breast cancer patients with bone metastases. Early bisphos-phonates (e.g., etidronate, clodronate) lacked nitrogen atoms, and are the least potent inhibitors of osteoclast-mediated bone resorption (20). Over the course of more than 30 years, successive generations of bisphospho-nates, each with increasing clinical utility, have been introduced (21).

Among the bisphosphonates tested in the oncology setting, intravenous


394 Lipton

Both IV and oral bisphosphonates have been shown to produce significant reductions in the rate or risk of SREs in patients with bone metastases from breast cancer in randomized placebo-controlled trials (Table 1) (6,7,18,22–25). In addition to the prevention of skeletal morbidity, bisphosphonates have also demonstrated significant palliative effects on bone pain in patients with advanced breast cancer. In a randomized placebo-controlled trial in such patients (N = 228), zoledronic acid 4 mg significantly reduced Brief Pain Inventory (BPI) composite pain scores from baseline and significantly reduced BPI scores compared with placebo (P < 0.05) (7). Moreover, zoledronic acid reduced the incidence of palliative radiation to bone compared with placebo (8.8% versus 17.7%) (7). Therefore, the ASCO consensus treatment guidelines speci-fically recommend that for patients with breast cancer and plain radio-

pamidronate or 4 mg zoledronic acid should be administered (26).

3.1.1. Clodronate

Oral clodronate has demonstrated a beneficial effect on skeletal morbid-dity associated with breast cancer (22). In a randomized, placebo-controlled trial of oral clodronate 1,600 mg/day compared with placebo in patients with bone metastases from breast cancer (N = 173), clodronate significantly reduced the combined rate of all morbid skeletal events (218.6 versus 304.8 per 100 patient-years; P < 0.001) (22). In a 10-year follow-up of women with breast cancer receiving oral clodronate 1,600 mg/day or placebo (N = 299) in addition to adjuvant chemotherapy or endocrine therapy, disease-free survival was significantly lower for patients treated with clodronate compared with patients receiving placebo (45% versus 58%; P = 0.01) (27). However, the addition of clodronate did not reduce the incidence of bone metastases (32% versus 29% for placebo-treated

(IV) nitrogen-containing bisphosphonates have demonstrated the most consistent efficacy and patient compliance with therapy compared with oral agents, and only IV pamidronate and zoledronic acid (Aredia® and ZOMETA®, respectively; Novartis, Basel, Switzerland, and East Hanover, NJ) have received regulatory approval in the USA and the European Union for treatment of bone metastases from breast cancer. Intravenous and oral ibandronate (Bondronat®; Roche, Basel, Switzerland) and oral clodronate (Bonefos®, Bayer Schering Pharma, Berlin, Germany) are approved in the European Union for the treatment of bone metastases from breast cancer, but are not approved in the USA (19).

graphic evidence of bone destruction, IV treatment with 90 mg

patients; P = 0.35) (27), and there were no significant survival differences in either trial (22,27).

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395

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3.1.2. Ibandronate

In a phase II trial, patients with breast cancer metastatic to bone (N = 466) received either IV ibandronate (6 mg via 1- to 2-hour infusion or 2 mg via bolus injection) or placebo every 3–4 weeks (25). The primary endpoint was the skeletal morbidity period rate (SMPR; the number of 12-week periods with a skeletal complication divided by time on study). Ibandronate 6 mg significantly reduced the mean SMPR (1.19 versus 1.48 for placebo; P = 0.004) and significantly prolonged the median time to a first new bone event (50.6 weeks versus 33.1 weeks for

cantly reduce the proportion of patients with a skeletal complication (50.6% versus 62.0% for placebo; P = 0.052) (25). In 2 pooled phase III trials, patients with bone metastases from breast cancer were randomized to receive oral ibandronate 50 mg (N = 287) or placebo (N = 277) daily for 96 weeks (23). Ibandronate significantly reduced the mean SMPR (0.95 versus 1.18; P = 0.004) and risk of SREs (hazard ratio = 0.62; P = 0.0001) compared with placebo, but did not significantly reduce the proportion of patients with an SRE (45.3% versus 52.2% compared with placebo; P = 0.122) (23). Because ibandronate has not yet been compared with other bisphosphonates, the clinical benefit of ibandronate compared with either pamidronate or zoledronic acid is not known.

3.1.3. Pamidronate

In a randomized, placebo-controlled trial in women with stage IV breast cancer and osteolytic metastases (N = 751), women receiving pamidro-nate (90 mg via 2-hour infusion) every 3 to 4 weeks had a significant reduction in SREs compared with placebo (53% versus 68%, P < 0.001), and pamidronate extended the median time to first SRE by 5.7 months compared with placebo (P < 0.001) (6). Pamidronate also provided significant long-term palliation of bone pain compared with significantly increased mean pain scores in the placebo group (P = 0.015), and analgesic use in the pamidronate group was signifycantly lower than in the placebo group (P < 0.001).

3.1.4. Zoledronic Acid

In a randomized study in women with bone metastases from breast

15-minute infusions every 4 weeks for 1 year (7). For these analyses, hypercalcemia of malignancy was included as an SRE. Zoledronic acid significantly reduced the skeletal morbidity rate (SMR; 0.63 SRE/year for zoledronic acid versus 1.10 SREs/year for placebo; P = 0.016). Both

396 Lipton

cancer (N = 228), patients received 4 mg zoledronic acid or placebo via

placebo; P = 0.018) (25). However, ibandronate 6 mg did not signifi-

versus 52.2% for placebo; P = 0.001) and the proportion of patientsthe percentage of patients with an SRE (30.7% for zoledronic acid

397

Zoledronic acid also significantly reduced the risk of SREs by 44% compared with placebo (P = 0.009). Moreover, zoledronic acid was well tolerated in this population, with a safety profile similar to that of placebo. Zoledronic acid also significantly reduced BPI composite pain scores and reduced the incidence of palliative radiation to bone compared with placebo.

Figure 3. Proportion of patients with bone metastases from breast cancer experiencing

malignancy. Adapted with permission from Kohno N, et al. Zoledronic acid significantly reduces skeletal complications compared with placebo in Japanese women with bone metastases from breast cancer: a randomized, placebo-controlled trial. J Clin Oncol 2005, 23(15):3314–3321. Reprinted with permission from the American Society of Clinical Oncology (7).

With Bone Metastases From Breast Cancer 3.1.5. Direct Comparison of Bisphosphonates in Patients

Zoledronic acid is the only bisphosphonate to be directly compared with pamidronate for efficacy and safety in patients with advanced breast cancer in a large-scale, randomized, phase III noninferiority trial (28,29). Patients with bone metastases from breast cancer or bone lesions from multiple myeloma (N = 1,648) were randomized to receive either zoledronic

4 mg zoledronic acid (N = 114) or placebo (N = 113). HCM = Hypercalcemia ofeach type of skeletal-related event (SRE). Patients were randomized to receive either


experiencing each type of SRE were reduced (Figure 3) (7). The mediantime to SRE was not reached in patients treated with zoledronic acid versus 360 days in patients treated with placebo (P = 0.004).

398 Lipton

In a subset analysis of patients with bone metastases from breast cancer (N = 766), 4 mg zoledronic was at least as effective as 90 mg pamidronate in reducing the proportion of patients with an SRE (43% versus 45%, respectively) (30). Zoledronic acid consistently reduced the proportion of patients experiencing each type of SRE compared with pamidronate and significantly reduced the percentage of patients requiring radiation to bone below that in the pamidronate group (19% versus 27%, respectively; P = 0.011) (31). Overall, zoledronic acid produced a 40% reduction beyond that produced by pamidronate in the SMR (0.90 SRE/year for zoledronic acid versus 1.49 SREs/year for pamidronate, P = 0.125) (31). Moreover, zoledronic acid significantly reduced the risk of developing an SRE (including hypercalcemia of malignancy) by 20% compared with pamidronate in patients with breast cancer (hazard ratio = 0.799; P = 0.025) (31). Notably, zoledronic acid reduced the requirement for radiation to bone significantly more than pamidronate, suggesting that patients treated with zoledronic acid may have had better pain relief versus patients treated with pamidronate.

In a post hoc analysis of this trial in patients with bone metastases from

zoledronic acid reduced the proportion of patients with an SRE compared with pamidronate (48% versus 58%; P = 0.058) (30). Moreover, zoledronic acid significantly prolonged the time to first SRE by approximately 4.5 months compared with pamidronate (median, 310 days for zoledronic acid versus 174 days for pamidronate; P = 0.013) and significantly reduced the mean SMR (1.2 SREs/year for zoledronic acid versus 2.4 SREs/year for pamidronate; P = 0.008) (30). In addition, treatment with zoledronic acid produced a significant 30% reduction in the risk of SREs compared with pamidronate (hazard ratio = 0.704; P = 0.010) (30).

3.1.6. Independent Meta-Analysis of Bisphosphonates in Patients With Bone Metastases From Breast Cancer

breast cancer and at least 1 primarily osteolytic bone lesion (N = 528),

acid (4 mg via 15-minute infusion) or pamidronate (90 mg via 2-hour infusion) every 3–4 weeks for up to 2 years (29). Zoledronic acid was at least as effective as pamidronate in reducing the proportion of patients with an SRE and in reducing the SMR, and multiple event analysis showed that zoledronic acid reduced the risk of developing a skeletal complication by an additional 16% compared with pamidronate (risk ratio = 0.841; P = 0.030) (29).

An independent meta-analysis (18) of placebo-controlled trials of bisphosphonates in patients with bone metastases from breast cancer by the Cochrane Library concluded that bisphosphonates as a class reduce the risk of developing an SRE by 17% (risk ratio = 0.83, P < 0.00001). Although it was not based on a head-to-head analysis, zoledronic acid

399

In a british economic analysis of the associated health-care costs and quality-adjusted survival associated with commonly used bisphosphonates

(~$12,000) (32). Among the bisphosphonates, zoledronic acid was determined to be the most cost-effective, with a net monetary benefit greater than £750 compared with oral ibandronate, and greater than £2,500 compared with IV pamidronate or ibandronate.

Figure 4. Independent analysis indicates that zoledronic acid provides the greatest risk reduction in patients with breast cancer. SRE = Skeletal-related event. Adapted from Pavlakis N, et al. Bisphosphonates for breast cancer (review). Cochrane Database Syst Rev, Issue 4, 2005, Copyright Cochrane Collaboration, reproduced with permission (18).

produced the greatest reduction in the risk of SREs versus placebo (41%) of all bisphosphonates assessed (pamidronate, IV or oral ibandronate, and oral clodronate; Figure 4) (18). Overall, bisphosphonates significantly delayed the median time to an SRE and significantly improved bone pain compared with placebo. The authors concluded that bisphosphonates may improve global QoL.

or cost-effective, with a cost per quality-adjusted life year of £6,126versus no therapy, bisphosphonates were determined to be cost saving


400 Lipton 3.2. RANKL Blockade

Other signal transduction intermediates within the bone metabolism signaling pathways are being targeted by investigational therapies. One of these molecular targets is receptor activator of nuclear factor-kappaB ligand (RANKL), which drives osteoclast differentiation and activation (20). Denosumab (AMG 162) is a fully human anti-RANKL monoclonal antibody (33). This agent has primarily been investigated for the management of postmenopausal osteoporosis, in which it has been shown to increase bone mineral density and reduce bone turnover in women with low bone mineral density (34). In a 24-week study evaluating the efficacy and safety of denosumab in women with bone metastases from breast cancer (N = 255), the efficacy of denosumab in reducing the risk of SREs was not significantly different from that of IV bisphosphonates (35). Further studies are warranted to assess the activity of denosumab alone or in combination with bisphosphonates for the treatment of bone metastases from breast cancer.

3.3. Radiotherapy

Radiotherapy can palliate bone pain secondary to bone metastases. Localized radiotherapy is effective in patients whose pain is limited to a single site (19,36); however, repeated dosing to a single site for recurrent pain would not be suggested because of cumulative damage to normal tissues (19). For patients with diffuse metastatic bone pain, broad hemibody or magnafield irradiation can alleviate pain in most patients but is associated with significant myelosuppression. Moreover, the requirement for multiple visits to the radiology clinic may be considered inconvenient and uncomfortable for some patients. Therefore, patients with multiple or diffuse bone metastases may be treated with IV radiopharmaceuticals, which accumulate at sites of bone resorption. For example, strontium chloride 89 has been shown to improve pain and QoL in 89% of patients with bone pain from advanced breast cancer (19,36); however, pain flares occur in up to 20% of patients and the onset of pain relief may be delayed for several weeks (36). Therefore, radiopharmaceuticals may not be appropriate for patients with a short life expectancy.

4. CONCLUSIONS

Bone metastases from breast cancer can cause clinically relevant skeletal morbidity that can undermine patient QoL and functional independence. Early diagnosis of bone metastases could provide important benefits to patients. Although no clear consensus exists regarding screening modality,

401

be confirmed with plain radiography, computed tomography, or magnetic resonance imaging.

Although different treatments are available to palliate the symptoms of bone metastases (e.g., analgesics, radiopharmaceuticals), bone-specific therapies treat the underlying cause of these symptoms, malignant osteo-lysis. Bisphosphonates have demonstrated significant clinical benefits in patients with malignant bone disease. Zoledronic acid has demonstrated benefits beyond those of pamidronate in patients receiving cytotoxic or hormonal therapy for advanced breast cancer, and results from an independent Cochrane Database meta-analysis suggest that zoledronic acid may be the most effective bisphosphonate in patients with bone metastases from breast cancer. An increased understanding of the cycle of tumor and bone interactions has also allowed the development of additional treatments to reduce skeletal morbidity associated with mali-gnant bone lesions. Further studies are necessary to determine the optimal therapy or combination of therapies to preserve QoL and functional independence in patients with bone metastases from breast cancer.

ACKNOWLEDGMENTS

Funding support for medical editorial assistance was provided by Novartis Pharmaceuticals. I would like to thank Todd Parker, PhD, ProEd Communications, Inc., for his medical editorial assistance with this manuscript.

REFERENCES

1. Mundy GR. Mechanisms of bone metastasis. Cancer 1997, 80(suppl):1546–1556. 2. Coleman RE. Metastatic bone disease: clinical features, pathophysiology and

treatment strategies. Cancer Treat Rev 2001, 27:165–176. 3. Cheville AL. Cancer rehabilitation. Semin Oncol 2005, 32:219–224. 4. Wilson MA, Calhoun FW. The distribution of skeletal metastases in breast and

pulmonary cancer: concise communication. J Nucl Med 1981, 22:594–597. 5. Coleman RE. Management of bone metastases. Oncologist 2000, 5:463–470. 6. Lipton A, Theriault RL, Hortobagyi GN, et al. Pamidronate prevents skeletal

complications and is effective palliative treatment in women with breast carcinoma and osteolytic bone metastases: long term follow-up of two randomized, placebo-controlled trials. Cancer 2000, 88:1082–1090.

7. Kohno N, Aogi K, Minami H, et al. Zoledronic acid significantly reduces skeletal complications compared with placebo in Japanese women with bone metastases from breast cancer: a randomized, placebo-controlled trial. J Clin Oncol 2005, 23:3314–3321.


bone scans are suggested for patients with advanced breast cancer. Skeletal scintigraphy should be the first choice for screening, and diagnosis should

402 Lipton

9. Major PP, Cook R. Efficacy of bisphosphonates in the management of skeletal complications of bone metastases and selection of clinical endpoints. Am J Clin Oncol 2002, 25(suppl 1):S10–S18.

10. Weinfurt KP, Li Y, Castel LD, et al. The significance of skeletal-related events for the health-related quality of life of patients with metastatic prostate cancer. Ann Oncol 2005, 16:579–584.

11. Hei YJ, Saad F, Coleman RE, Chen YM. Fractures negatively affect survival in patients with bone metastases from breast cancer [poster]. Presented at: 28th Annual San Antonio Breast Cancer Symposium (SABCS); December 8–11, 2005; San Antonio, TX. Abstract 6036.

12. Conte P, Rosen LS, Gordon D, Zheng M, Hei Y-J. Zoledronic acid is superior to pamidronate in patients with breast cancer and multiple myeloma: analysis of patients at high risk for skeletal complications [abstract]. Ann Oncol 2004, 15:iii24. Abstract 463PD.

13. Zheng M, Rosen L, Gordon D, et al. Continuing benefit of zoledronic acid for the prevention of skeletal complications in breast cancer patients with bone metastases [poster]. Presented at: Primary Therapy of Early Breast Cancer 9th International Conference; January 26–29, 2005; St. Gallen, Switzerland. Abstract 104.

14. Khatcheressian JL, Wolff AC, Smith TJ, et al. American Society of Clinical Oncology 2006 update of the breast cancer follow-up and management guidelines in the adjuvant setting. J Clin Oncol 2006, 24:5091–5097.

15. National Comprehensive Cancer Network. NCCN clinical practice guidelines in oncology. Breast cancer. V.1.2007. Available at: http://www.nccn.org. 2006.

16. Hamaoka T, Madewell JE, Podoloff DA, Hortobagyi GN, Ueno NT: Bone imaging in metastatic breast cancer. J Clin Oncol 2004, 22:2942–2953.

17. Leeming DJ, Delling G, Koizumi M, et al. Alpha CTX as a biomarker of skeletal invasion of breast cancer: immunolocalization and the load dependency of urinary excretion. Cancer Epidemiol Biomarkers Prev 2006, 15:1392–1395.

18. Pavlakis N, Schmidt RL, Stockler M. Bisphosphonates for breast cancer (review). Cochrane Database Syst Rev 2005, CD003474.

19. Lipton A. Management of bone metastases in breast cancer. Curr Treat Options Oncol 2005, 6:161–171.

20. Green JR. Preclinical profile and anticancer potential of zoledronic acid, in: Trends in Bone Cancer Research, Birch EV, ed. Nova Science Publishers, Inc., New York, 2006, 2217–2245.

21. Fleisch H. Development of bisphosphonates. Breast Cancer Res 2002, 4:30–34. 22. Paterson AH, Powles TJ, Kanis JA, et al. Double-blind controlled trial of oral

clodronate in patients with bone metastases from breast cancer. J Clin Oncol 1993, 11:59–65.

23. Body JJ, Diel IJ, Lichinitzer M, et al. Oral ibandronate reduces the risk of skeletal complications in breast cancer patients with metastatic bone disease: results from two randomised, placebo-controlled phase III studies. Br J Cancer 2004, 90:1133–1137.

24. Tripathy D, Lichinitzer M, Lazarev A, et al. Oral ibandronate for the treatment of metastatic bone disease in breast cancer: efficacy and safety results from a randomized, double-blind, placebo-controlled trial. Ann Oncol 2004, 15:743–750.

25. Body JJ, Diel IJ, Lichinitser MR, et al. Intravenous ibandronate reduces the incidence of skeletal complications in patients with breast cancer and bone metastases. Ann Oncol 2003, 14:1399–1405.

8. Domchek SM, Younger J, Finkelstein DM, Seiden MV. Predictors of skeletal complications in patients with metastatic breast carcinoma. Cancer 2000, 89:363–368.

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27. Saarto T, Vehmanen L, Virkkunen P, Blomqvist C. Ten-year follow-up of a rando-mized controlled trial of adjuvant clodronate treatment in node-positive breast cancer patients. Acta Oncol 2004, 43:650–656.

28. Rosen LS, Gordon D, Kaminski M, et al. Zoledronic acid versus pamidronate in the treatment of skeletal metastases in patients with breast cancer or osteolytic lesions of multiple myeloma: a phase III, double-blind, comparative trial. Cancer J 2001, 7:377–387.

29. Rosen LS, Gordon D, Kaminski M, et al. Long-term efficacy and safety of zoledronic acid compared with pamidronate disodium in the treatment of skeletal complications in patients with advanced multiple myeloma or breast carcinoma: a randomized, double-blind, multicenter, comparative trial. Cancer 2003, 98:1735–1744.

30. Rosen LS, Gordon DH, Dugan W Jr, et al. Zoledronic acid is superior to pamidronate for the treatment of bone metastases in breast carcinoma patients with at least one osteolytic lesion. Cancer 2004, 100:36–43.

31. Coleman RE, Rosen LS, Gordon D, et al. Zoledronic acid significantly reduces the risk of developing a skeletal-related event compared with pamidronate in breast cancer patients with bone metastases [poster]. Presented at: 25th Annual San Antonio Breast Cancer Symposium (SABCS); December 11–14, 2002; San Antonio, TX. Abstract 355.

32. Botteman M, Barghout V, Stephens J, et al. Cost effectiveness of bisphosphonates in the management of breast cancer patients with bone metastases. Ann Oncol 2006, 17:1072–1082.

33. Body JJ, Facon T, Coleman RE, et al. A study of the biological receptor activator of nuclear factor-kappaB ligand inhibitor, denosumab, in patients with multiple myeloma or bone metastases from breast cancer. Clin Cancer Res 2006, 12:1221–1228.

34. Lewiecki EM. RANK ligand inhibition with denosumab for the management of osteoporosis. Expert Opin Biol Ther 2006, 6:1041–1050.

35. Lipton A, de Boer R, Figueroa J, et al. Extended safety and efficacy analysis of denosumab in breast cancer patients with bone metastases without prior bisphosphonate therapy [poster]. Presented at: 27th Annual San Antonio Breast Cancer Symposium (SABCS); December 8–11, 2004; San Antonio, TX. Abstract 1069.

36. Serafini AN. Therapy of metastatic bone pain. J Nucl Med 2001, 42:895–906.


26. Hillner BE, Ingle JN, Chlebowski RT, et al. American Society of Clinical Oncology 2003 update on the role of bisphosphonates and bone health issues in women with breast cancer. J Clin Oncol 2003, 21:4042–4057.

Chapter 21

HORMONAL THERAPIES OF METASTATIC

Jürgen Geisler and Per Eystein Lønning

Abstract: Endocrine therapy of metastatic breast cancer has been established for more than a century. Following the discovery of the beneficial effects of oophorectomy by Beatson in 1896 and the identification of the first estrogen receptor (ER-α) by E. Jensen 70 years later (1967), antihormonal treatment of breast cancer represented not only the first systemic treatment strategy in oncology but also the first for which a clear scientific rationale has been established. While early treatment options (either surgical or additive drug treatment) were associated with significant side effects, the partial estrogen receptor antagonist tamoxifen established modern endocrine drug therapy of metastatic breast cancer. The compound became the most widely used drug in breast cancer therapy until now. Recently, the role of tamoxifen has been challenged following development of other novel, highly potent, and selective drugs. The most important improvement has certainly been made with the implementation of the third generation of aromatase inhibitors (anastrozole, letrozole, and exemestane). These compounds are now in general used as first-line therapy in metastatic breast cancer in postmenopausal women not exposed to the compounds in

aromatase inhibitor. In addition, SERDs (selective estrogen receptor down-regulators) like fulvestrant have shown clinical efficacy and have been introduced in standard care as well. The present publication provides an overview about endocrine treatment options of metastatic breast cancer and discusses possible treatment algorithms for both pre- and postmenopausal breast cancer patients.

Keywords: endocrine therapy, aromatase inhibitors, selective estrogen receptor modifier (SERM)

© 2007 Springer.


the adjuvant setting for those relapsing after adjuvant therapy with an

Section of Oncology, Department of Medicine, Haukeland University Hospital, N–5021

THE PRESENT BREAST CANCER; THE PAST AND

Bergen, Norway

Geisler and Lønning

1. INTRODUCTION

The last decade has brought substantial improvements to all types of systemic therapy for breast cancer. Thus, hormonal therapy has been improved by development of the novel, third-generation aromatase inhibitors, initially in the metastatic, subsequently followed by successful implementation in the adjuvant setting. For chemotherapy several novel compounds, including vinorelbine and navelbine have been introduced, with the most substantial improvements achieved through implemen-tation of the taxanes. Finally, targeted therapy took a major step forward through the exiting preliminary results obtained with trastuzumab in the adjuvant setting.

Despite improvement in therapy, metastatic breast cancer remains a non-curable condition. Taking the group in total, median survival remains in the range of 2–2.5 years (1, 2). Notably, however, life expectations vary significantly between different subgroups. While life expectancy from time of diagnosis of distant metastases depends on issues like tumor burden, it is even more related to tumor-“intrinsic” biology. Thus,

in general has a slower growth rate compared to the estrogen receptor

general relapse later (more than 2 years after primary diagnosis) in comparison to the ER negative ones (3), but even more to the fact that average life expectancy from time of distant metastases appearing is longer (4, 5). ER positive disease in general is frequently associated with locoregional relapse but, in particular, distant metastases affecting the skeletal system, contrasting the predilection of ER negative disease for visceral organs (6, 7). Thus, many patients expressing skeletal metastases may live with their disease for several years. However, it should be underlined that for patients with distant metastases the long-term progno-sis is as serious for ER positive as it is for ER negative disease (4).

The fact that metastatic breast cancer remains noncurable and therapy palliative, underlines the importance of limiting toxicity of therapy. Endocrine therapy in general provides limited toxicity in comparison to chemotherapy; thus, hormonal manipulation remains no only first-line therapy but should be extended as far as possible for patients with endocrine-sensitive advanced disease. For this reason, we will provide a brief description of the different treatment options and their mechanisms

general use, as they may be of value in late sequential treatment for the subgroup of patients whose tumors may respond to multiple endocrine treatment strategies. Importantly, although most patients with initially hormone-sensitive tumors will develop acquired resistance following two or perhaps three different regimens, some patients may respond to

it has been known for decades that estrogen receptor (ER) positive tumors

negative ones. This is manifested by the fact that ER positive tumors in

of action (Fig. 1). We will include treatment options no longer in

406

21. Hormonal therapies of metastatic breast cancer 407

patients may achieve disease control for years on different endocrine regimens with no need for chemotherapy. While previous endocrine treatment options, like additive treatment with estrogens or progestins administered in pharmacological doses have been abandoned in favor of contemporary treatment options for safety reasons, notably, the discom-fort associated with such therapy is moderate compared to what most patients experience on chemotherapy.

Figure 1. Endocrine treatment options for metastatic breast cancer.

2. TYPES OF ENDOCRINE THERAPY

2.1 Ovarian suppression

Initially achieved by surgical oophorectomy (9), this represents not only the first endocrine treatment option developed empirically at a time its mechanism of action was poorly understood, but also the first systemic treatment strategy for any cancer form. Subsequently replaced by ovarian irradiation (10), today ovarian suppression in general may be achieved through “medical oophorectomy” with so-called LH-RH analog-ues; see detailed description of compounds in (11). Interestingly, while

multiple different treatments options (like additive therapies with estrogens at high doses) administered sequentially (8). Accordingly, such


should be recognized that this evidence is based on a limited number of studies involving a small number of patients.

Several issues remain unaddressed; while surgical and radiological ablation are irreversible, it is not clear for what duration LH-RH analog-ues should be administered either in the adjuvant setting or to long-term responders with metastatic disease. As continuous ovarian suppression is a requisite for many subsequent treatment options (including aromatase inhibitors), patients with metastatic disease responding to treatment with an LH-RH analogue may be candidates for subsequent surgical or radio-logical ablation, as subsequent second-line therapy with aromatase inhibitors requires suppression of ovarian function.

2.2 Adrenalectomy and hypophysectomy

Learning that the adrenal gland is a source of steroid production in postmenopausal women, surgical adrenalectomy as well as hypophy-sectomy (depriving ACTH) were considered treatment options for post-menopausal women (14, 15). At this time, the general belief was that postmenopausal estrogens together with androgens were synthesized and directly secreted by the adrenals; later, it was discovered that circulating androgens, mainly of adrenal but with a potential small contribution of testosterone from the postmenopausal ovaries (16–18), were converted into estrogens in different tissue compartments by the aromatase enzyme (Fig. 1), Both treatment options revealed significant antitumor effects but at considerable morbidity and even mortality. This led to the subsequent invention of “medical adrenalectomy” in an attempt to achieve similar antitumor effects at the cost of lower toxicity.

prednisolone 5–10 mg daily) was associated with a low response rate, with similar results from trials exploring ketoconazole-derivatives specifi-

A major breakthrough was achieved with the introduction of amino-glutethimide as treatment for metastatic disease. Due to toxic effects on the adrenocortical gland, the unsuccessful phenobarbitone antiepileptic aminoglutethimide was implemented aiming at achieving an effective “medical adrenalectomy” (20). While this drug was found to be a successful antitumor agent, the reason for its antitumour efficacy turned out to be potent inhibition of the aromatase enzyme (see below).

cally targeting 17-alpha-hydroxylase (see references and details in (19)).

However, treatment with glucocorticoids in moderate doses (prednisone/

408

specific LH-RH receptors have been identified in breast cancer (12), there is no direct evidence for a mechanistic role of LH-RH receptors in tumors during therapy with LH-RH analogues. While studies in meta-static disease have not revealed any difference in response rate between medical oophorectomy and surgical oophorectomy/irradiation (13), it


enzymes involved in adrenal steroid synthesis, circulating androgen levels were found preserved despite profound suppression of circulating estrogens (22). The reason why plasma androstenedione and testosterone levels were not affected (although there were changes in the ratio of several adrenal-derived hormones) was probably due to an increase in ACTH secretion (see (23) for a detailed description of the effects of aminoglutethimide on adrenal enzyme activities). The finding that amino-glutethimide caused estrogen suppression and, thus, antitumor effects through inhibiting the aromatase enzyme only, was a key milestone triggering the process subsequently leading to successful development of these new compounds in breast cancer therapy.

A second major event was the laboratory work of Harry and Angela Brodie, identifying androstenedione derivatives as potent aromatase inhibitors (24). These steroidal compounds act by irreversibly binding to the substrate-binding pocket on the aromatase enzyme (25), and have no effect on other enzymes involved in steroid disposition when given in the doses established in the clinic.

The main reason for subsequent development of second- and third- generation aromatase inhibitors were the side effects (aminoglutethimide) and inconvenience of parenteral administration (4-hydroxyandrostene-dione) of the two first compounds. For aminoglutethimide, interactions with adrenal steroid-synthesizing enzymes lead to glucocorticoid- and, sometimes, mineralocorticoid- deficiencies, required glucocorticoid substitution with or without mineralocorticoids (26). Even more important, the compound caused neurological as well as skin side effects (23). The most serious complication of aminoglutethimide was the rare finding of blood dyscracias (27). Thus, much effort was invested into developing less toxic compounds. For 4-hydroxyandrostenedione, the compound required 2-weekly intramuscular injections for optimal estrogen sup-pression (28), as oral administration was ineffective due to poor bioavaila-bility of the compound (29, 30).

Due to these limitations for both compounds mentioned above, a number of more selective, less toxic compounds were developed by different phar-maceutical companies. In collaboration with Professor Dowsett’s Group at the Royal Marsden Hospital, London, we developed a highly sensitive method for measurement of in vivo aromatization using a double-tracer technique (31–33). Using this system, we were able to classify different

2.3 Aromatase inhibitors

As noticed above, the first-generation aromatase inhibitor, aminoglute-thimide, was an unsuccessful, adrenotoxic antiepileptic compound imple-mented for breast cancer therapy in an attempt to achieve a “medical adrenalectomy.” While the drug was found to be an effective antitumor agent, meticulous work by Santen et al. identified aminoglutethimide as an aromatase inhibitor in vivo (21). Thus, despite interacting with several


in vivo inhibition (Table 1). These compounds were subsequently shown to be superior regarding clinical efficacy to conventional antihormonal therapy in metastatic breast cancer (40–44).

2.4 SERMS and SERDS

No other single compound has had an impact on breast cancer therapy like the first selective estrogen receptor modifier (SERM), tamoxifen. Due to its antitumor efficacy combined with a low-toxicity profile, the drug remained first-line endocrine therapy for metastatic breast cancer among pre- as well as postmenopausal women for decades (see detailed description of early and basic findings in (45), subsequently to become standard endocrine therapy in the adjuvant setting. A detailed description of the biochemical actions of tamoxifen is beyond the scope of this paper, and the readers are referred to comprehensive reviews on the subject (45–47).

The fact that tamoxifen was found of similar efficacy among pre- and postmenopausal women remains somewhat surprising. Not only do the two groups express different estrogen levels; in addition, tamoxifen was shown to elevate plasma levels of estradiol 2 to 3-fold in premenopausal

explanation is that the regular dose of tamoxifen, 20 mg daily, represents an “overdose”; thus, Descenci et al. have shown tamoxifen down to doses of 5 mg daily to exert effects on surrogate parameters resembling what is observed with the 20 mg daily dose (49). The relevance of such comparison is indirectly supported by the findings from a large phase III study comparing the second-generation SERM droloxifene (3-hydroxy-tamoxifene) to tamoxifen in pre- and postmenopausal women with meta-satic breast cancer (50). Here, droloxifene was found of similar antitumor efficacy to tamoxifen among postmenopausal patients but inferior for premenopausals. Notably, at the dose administered (40 mg daily), droloxi-fene was shown to have less effect on surrogate parameters like SHBG and the IGF-binding proteins compared to droloxifene 100 mg or tamoxi-

women due to interaction with follicular maturation (48). A possible

410

compounds based on their biochemical efficacy (Table 1); (34). Most importantly, the difference recorded was corroborated by clinical findings. While the first compounds, like fadrozole, similar to aminoglutethimide and 4-hydroxyandrostenedione caused 80–90% aromatase inhibition, these compounds were associated with reduced toxicity but did not improve antitumor efficacy (35–39). In contrast, the three so-called third-generation compounds (Fig. 2), anastrozole, letrozole, and exemestane, caused >98%

tissue. In contrast, droloxifene (40 mg daily) was able to block estrogen

the hypothesis that tamoxifen 20 mg daily allowed the drug to block fen 20–30 mg daily (51–53). These findings may be consistent with

the effect even of high premenopausal estrogen concentrations in the

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A novel class of estrogen receptor modifiers are the so-called SERDS (selective estrogen receptor downregulators), represented with fulvestrant (Fig. 3). Being a steroid derivative with a long aliphatic chain at its 7-position, fulvestrant has a chemical structure distinct from the SERMS. Importantly, fulvestrant is devoid of any intrinsic estrogen agonistic activity, and it seems to act by at least two distinct mechanisms: receptor blocking, but also receptor downregulation (57, 58).

Considering clinical trials, fulvestrant has been compared to anastrozole second-line (in patients failing tamoxifen) as well as to tamoxifen as first-line therapy for metastatic disease (59–61). Overall, results from these studies have suggested fulvestrant to be of similar clinical efficacy to anastrozole as well as tamoxifen. A major disadvantage with this compound is its need for parenteral administration.

Geisler and Lønning412

Considering other SERMS currently available, toremifene has been found of similar efficacy and with a similar side-effect profile compared to tamoxifen (54–56); thus, the two treatment options are considered similar with respect to antitumour efficacy.

competing with the high hormone levels in premenopausals. stimulation in postmenopausal patients, but was only partly effective in

Figure 2. Differences in structure of antiaromatase agents.


2.5 Additive endocrine therapy

This category includes different treatment options like progestins, andorgens, and estrogens administered at high doses. Considering pro-gestins, both medroxyprogesterone acetate (1,000 mg daily) as well as megestrol acetate (160 mg daily) represents active antitumour therapy with efficacy not different from what was achieved with aminoglutethi-mide as well as tamoxifen (62). Side effects however were significant, including weight gain in particular. This is most likely due to a signi-ficant glucocorticoid agonistic effect that may well be part of its mechanism of action; thus, megestrol acetate administered at 160 mg daily significantly suppressed adrenal steroid secretion as well as circulating estrogens (63). Whether suppression of estrogens may play a major role to its antitumour efficacy remains uncertain. The finding of a lack of complete cross-resistance (albeit probably a reduced responsive-ness) to aromatase inhibitors (64, 65) suggests estrogen suppression may play a role but with additional effects acting in concert.

Figure 3. Structures of SERMs (tamoxifen and raloxifene) and the SERD fulvestrant in comparison to estradiol.

The majority of breast cancers express androgen receptors at a level >10 fmol/mg (66), and androgens were used for breast cancer therapy before implementation of contemporary treatment options (67). The response rate however was low, and androgens cause disturbing side effects, like hirsuitism.


Estrogens administered at high doses (in general DES 15 mg daily) was a frequently used treatment option for pre- as well as postmeno-pausal breast cancer before tamoxifen came into clinical use. Thus, in a randomized trial, Ingle et al. revealed similar efficacy for estrogens and tamoxifen administered for metastatic breast cancer (68). It should be emphasized that the estrogen doses used for breast cancer therapy were at least a magnitude higher compared to doses used for hormone replacement, and the effects achieved with this strategy should never be taken as an argument in favor of safety regarding hormone replacement therapy for breast cancer patients. While the mechanism by which estrogens achieve these antitumor effects are not understood, some experimental data are of interest. It is well known that estrogen-induced growth stimulation of MCF-7 cells in vitro is expressed as a “bell-shaped” curve (69, 70). Thus, escalating the estrogen concentration above the optimal concentration for cell growth actually inhibits cell growth. In an elegant experiment, Masamura et al. (70) created an estrogen “hypersensitive” MCF-7 cell line by growing cells exposed to low hormone concentrations over time. These “LTED” cells (long-time estrogen deprived cells) achieved optimal growth stimulation by estradiol at a concentration about 0.01–0.1% the concentration needed for regular MCF-7 cells. Most importantly, in the LTED cells the whole “bell-shaped” growth stimulation curve moved to the left, meaning that estradiol at a concentration stimulating growth of regular MCF-7 cells inhibited growth of the LTED cells. Based on these findings, we designed a pilot study treating patients becoming resistant to multiple endocrine regimens, including potent third-generation aro-matase inhibitors, with DES 15 mg daily (71). Our finding that 10 out of 32 patients obtained a partial remission, confirmed many of these tumors still to be hormone sensitive, and was consistent with the hypothesis that resistance to treatment with aromatase inhibitors at least in some cases, may develop through “hypersensitivity” of the cells to estradiol.

3. ENDOCRINE THERAPY OF POSTMENOPAUSAL WOMEN WITH ADVANCED BREAST CANCER

Implementations of aromatase inhibitors either as monotherapy or in sequence with tamoxifen for adjuvant therapy has challenged our algorithm

414

Previously, for patients being treated with tamoxifen (or not having adjuvant endocrine therapy) first-line treatment in metastatic disease should mean treatment with one of the new third-generation aromatase inhibitors, anastrozole, letrozole, or exemestane. Alternatively, patients

for endocrine treatment of postmenopausal women in the metastatic setting.


relapsing more than 1 year following completion of tamoxifen therapy were rechallenged with the same compound. While currently most physicians would agree that patients not being exposed to any of the novel third-generation aromatase inhibitors should receive one of these compounds as first-line therapy in metastatic disease, we need to develop algorithms with respect to how to handle patients being exposed either to

Taking into account the high efficacy and low toxicity of third-gene-ration aromatase inhibitors as well as tamoxifen, we believe the following approaches to be reasonable:

Considering those patients having an aromatase inhibitor as mono-therapy in the adjuvant setting, first-line therapy in metastatic breast cancer could be tamoxifen-independent of the time frame between terminating treatment and time of relapse. For those being exposed to both treatment options in the adjuvant setting (e.g., tamoxifen followed by

treatment with an aromatase inhibitor, one of these compounds could beexplored as first-line treatment. For those relapsing within a time frame of 1 year or less after terminating adjuvant therapy or during therapy, while evidence is currently lacking, it may be reasonable to expect that some of these patients terminating tamoxifen several years earlier could still be sensitive to that treatment approach.

An interesting question is whether patients relapsing after treatment with an aromatase in the adjuvant setting should be treated with a different compound, preferentially a compound belonging to the “alternative class” (steroidal versus nonsteroidal compounds) in the metastatic setting. While direct evidence is lacking, based on the findings of lack of complete cross-resistance between these compounds in the metastatic setting (72–74), this seems to be a natural approach.

While the final role of fulvestrant in metastatic disease remains to be settled, this drug certainly offers an interesting treatment approach. Although fulvestrant was found to be not significantly different from anastrozole regarding efficacy in the advanced setting (75), it has not been compared to any of the other aromatase inhibitors. So far, the only study comparing two aromatase inhibitors (letrozole and anastrozole) on

letrozole but no difference regarding the primary endpoint time to pro-gression (76). Importantly, this study was conducted in the second-line

an aromatase inhibitor as monotherapy, or tamoxifen followed sequen-tially by an aromatase inhibitor, in the adjuvant setting.

a head-to-head basis found an increase with respect to response rate for

an aromatase inhibitor), provided they relapse >1 year after terminating

setting, in which it may be difficult to document a difference between two compounds. Looking at studies conducted with anastrozole as well as letrozole versus tamoxifen in metastatic disease or for primary (neoadjuvant) therapy in advanced breast cancer (40, 77–79), evidence favoring letrozole over tamoxifen is consistent. In contrast, evidence


favoring anastrozole compared to tamoxifen is weaker, with several

other hand, there is little evidence so far from the adjuvant studies indi-cating a major difference between the two aromatase inhibitor (80, 81), thus, the “jury is still out.”

While the aim of developing fulvestrant was to develop a compound causing “total estrogen blockade” with no additional agonistic effects, interestingly recent evidence suggests patients failing on fulvestrant therapy may not be totally refractory to alternative forms of endocrine therapy (82, 83). Based on in vitro data (84), the potential of combining fulvestrant with an aromatase inhibitor is currently an issue for clinical studies.

In summary, fulvestrant definitely has a role in treatment of advanced breast cancer, but there is currently limited data favoring its use over tamoxifen or third-generation aromatase inhibitors. At this stage, fulvestrant more seems a natural treatment option for patients whose tumors have become resistant to the other two treatment options (tamoxifen and AIs).

We still lack evidence regarding efficacy of progestins in high-doses for patients becoming resistant to the third-generation aromatase inhibitors. In contrast, in a pilot study we found significant clinical effects using diethylstilbestrol (DES) 15 mg daily in patients failing aromatase inhibitors (8). Side effects in general were acceptable, and estrogens in high doses are currently evaluated in larger studies. We consider additive treatment with estrogens in high-doses a feasonable

4. ENDOCRINE THERAPY OF PREMENOPAUSAL WOMEN WITH METASTATIC BREAST CANCER

For premenopausal patients, we are left with three potential options for first-line therapy; tamoxifen monotherapy, treatment with an LH-RH analogue, or the two treatment options administered in concert.

studies reporting equal efficacy for anastrozole and tamoxifen. On the

treatment alternative for advanced breast cancer. Compared to fulve-strant, with the data currently available we may consider these treatment options equal; their use based on individual patient preferences and side effects.

416

elsewhere (85). In the metastatic setting, first-line therapy with either tamoxifen or ovarian ablation is associated with similar response rates (86, 87).

The potential benefits with respect to each individual treatment approach, the combination as well as interactions with chemotherapy-induced amenorrhea in the adjuvant setting has recently been discussed in detail


Another interesting approach is to use the two treatment options in concert. Thus, Klijn et al. (88) have shown moderate superiority for combined treatment (TAM + LHRH-analogue) versus monotherapy with each individual approach. On the other hand, it is not fully clear whether a similar benefit may not be achieved through sequential application of the same treatment strategies.

Considering further treatment for premenopausal women, a natural choice should be treatment with a third-generation aromatase inhibitor. While we are left with an open question in adjuvant therapy regarding duration of treatment with an LH-RH analogue (85), based on the fact that metastatic breast cancer remains noncurable and, thus, patients responding to ovarian ablation may be candidates for subsequent aromatase inhibition, it seems reasonable to advocate permanent ovarian ablation (radiological or surgical) for those patients obtaining a clear response to treatment with an LH-RH analogue. Subsequently, such patients may be treated according to the principles outlined for postmenopausal women above.

Notably, there is currently much interest in exploring the concept of “total estrogen suppression,” meaning to combine an LH-RH analogue with an aromatase inhibitor upfront, While this is a most interesting treatment approach, awaiting evidence from phase III studies this approach should be considered experimental, and not advocated outside clinical trials at this stage.

5. CONCLUSIONS

More than a century after the seminal discovery of Beatson with respect to oophorectomy, endocrine therapy is as important as ever for treatment of breast cancer. Although chemotherapy of breast cancer is improved and targeted therapies like HER-2 antagonists have been introduced for the treatment of breast cancer, the role of endocrine therapy within the adjuvant and metastatic setting has not been weakened. Recently, the introduction of aromatase inhibitors in the adjuvant situation has challenged our treatment algorithms. For patients relapsing during or following contemporary adjuvant treatment, it will be increasingly necessary to make individual decisions based on the previous exposure to antihormonal drugs in the adjuvant setting. Still, many patients may respond to several endocrine treatment strategies given sequentially. For selected patients, additive therapies with progestins or estrogens given in high doses, remain feasible treatment options.


REFERENCES

1. Koenders PG, Beex LVAM, Kloppenborg PWC, Smals AGH, Benraad TJ, Group BCS. Human breast cancer: survival from first metastasis. Breast Cancer Res Treat 1992; 21:173–180.

2. Bruzzi P, Del Mastro L, Sormani MP, Bastholt L, Danova M, Focan C, et al. Objective response to chemotherapy as a potential surrogate end point of survival in metastatic breast cancer patients. Journal of Clinical Oncology 2005; 23(22):5117–5125.

3. May E, Mouriesse H, May-Levin F, Qian JF, May P, Delarue JC. Human breast cancer: identification of populations with a high risk of early relapse in relation to both oestrogen receptor status and c-erB-2 overexpression. Br J Cancer 1990; 62:430–435.

4. Paterson AHG, Zuck VP, Szafran O, Lees AW, Hanson J. Influence and significance of certain prognostic factors on survival in breast cancer. Eur J Cancer Clin. Oncol. 1982; 18(10):937–943.

5. Howell A, Harland RNL, Bramwell VHC, Swindell R, Barnes DM, Redford J, et al. Steroid-hormone receptors and survival after first relapse in breast cancer. Lancet 1984; 1:588–591.

6. Kamby C, Rasmussen BR, Kristensen B. Oestrogen receptor status of primary breast carcinomas and their metastases. Relation to pattern of spread and survival after recurrence. Br J Cancer 1989; 60:252–257.

7. Stewart JF, King RJB, Sexton SA, Millis RR, Rubens RD, Hayward JL. Oestrogen receptors, sites of metastatic disease and survival in recurrent breast cancer. Eur J Cancer 1981; 17(4):449–453.

8. Lønning PE, Taylor PD, Anker G, Iddon J, Wie L, Jorgensen LM, et al. High-dose estrogen treatment in postmenopausal breast cancer patients heavily exposed to endocrine therapy. Breast Cancer Res Treat 2001;67(2):111–116.

9. Beatson GT. On the treatment of inoperable cases of carcinoma of the mamma. Suggestions for a new method of treatment with illustrative cases. Lancet 1896; 2:104–107.

10. NissenMeyer R. Ovarian irradiation and its supplement by additive hormonal treatment. Hormones and Breast Cancer 1976; 58:131–158.

11. Lønning PE, Lien EA, Lundgren S, Kvinnsland S. Clinical pharmacokinetics of endocrine agents used in advanced breast cancer. Clin. Pharmacokinet. 1992; 22: 327–358.

12. Eidne KA, Flanagan CA, Millar RP. Gonadotropin-releasing hormone binding sites in human breast carcinoma. Science 1985; 229:989–992.

13. Taylor CW, Green S, Dalton WS, Martino S, Rector D, Ingle JN, et al. A multicenter randomized clinical trial of goserelin versus surgical ovariectomy in premenopausal patients with receptor-positive metastatic breast cancer: An intergroup study. J Clin Oncol 1998; 16(3):994–999.

14. Dao TL, Huggins C. Bilateral adrenalectomy in the treatment of cancer of the breast. Arch Surg 1955; 71:645–657.

15. Luft R, Olivecrona H, Sjögren B. Hypophysektomy in man. Nord Med 1952; 14:351–354.

16. Couzinet B, Meduri G, Lecce M, Young J, Brailly S, Loosfelt H, et al. The post-menopausal ovary is not a major androgen-producing gland. J Clin Endocrinol Metab 2001; 86:5060–5066.

17. Sluijmer AV, Heineman MA, Jong FHd, Evers JLH. Endocrine activity of the postmenopausal ovary: The effects of pituitary down-regulation and oophorectomy. J Clin Endocrinol Metab 1995; 80:2163–2167.

418


18. Dowsett M, Cantwell B, Lal A, Jeffcoate SL, Harris AL. Suppression of postmeno-

pausal ovarian steroidogenesis with the luteinizing hormone-releasing hormone agonist goserelin. J Clin Endocrinol Metab 1988; 66:672–677.

19. Lønning PE. New endocrine drugs for treatment of advanced breast cancer. Acta Oncol 1990; 29:379–386.

20. Cash R, Brough AJ, Cohen MNP, Satoh PS. Aminoglutethimide (Elipten-Ciba) is an inhibitor of adrenal steroidogenesis: mechanism of action and therapeutic trial. J Clin Endocrinol Metab 1967; 27:1239–1248.

21. Santen RJ, Santner S, Davis B, Veldhuis J, Samojlik E, Ruby E. Aminoglutethi-mide inhibits extraglandular estrogen production in postmenopausal women with breast carcinoma. J Clin Endocrinol Metab 1978; 47:1257–1265.

22. Samojlik E, Veldhuis JD, Wells SA, Santen RJ. Preservation of androgen secretion during estrogen suppression with aminoglutethimide in the treatment of metastatic breast carcinoma. J Clin Invest 1980; 65:602–612.

23. Lønning PE, Kvinnsland S. Mechanisms of action of aminoglutethimide as endocrine therapy of breast cancer. Drugs 1988; 35:685–710.

24. aromatase inhbitor, 4-hydroxy-androstene-3,17-dione, on estrogen-dependent pro-cesses in reproduction and breast cancer. Endocrinology 1977; 100:1684–1695.

25. Miller WR, Dixon JM. Antiaromatase agents: preclinical data and neoadjuvant therapy. Clin Breast Cancer 2000;1 (Suppl. 1):s9–s14.

26. Santen RJ, Lipton A, Kendall J. Successful medical adrenalectomy with amino-glutethimide. JAMA 1974; 230(12):1661–1665.

27. Young JA, Newcomer LN, Keller AM. Aminoglutethimide-induced bone marrow injury. Cancer 1984; 54:1731–1734.

28. Dowsett M, Cunningham DC, Stein RC, Evans S, Dehennin L, Hedley A, et al. Dose-related endocrine effects and pharmacokinetics of oral and intramuscular 4-hydroxyandrostenedione in postmenopausal breast cancer patients. Cancer Res 1989; 49:1306–1312.

29. Dowsett M, Mehta A, King N, Smith IE, Powles TJ, Stein RC, et al. An endocrine and pharmacokinetic study of four oral doses of formestane in postmenopausal breast cancer patients. Eur J Cancer 1992; 28:415–420.

30. Dowsett M, Lloyd P. Comparison of the pharmacokinetics and pharmaco-dynamics of unformulated and formulated 4-hydroxyandrostenedione taken orally by healthy men. Cancer Chemother. Pharmacol. 1990; 27:67–71.

31. Lønning PE, Skulstad P, Sunde A, Thorsen T. Separation of urinary metabolites of radiolabelled estrogens in man by HPLC. J. Steroid Biochem. 1989; 32:91–97.

32. Jacobs S, Lønning PE, Haynes B, Griggs L, Dowsett M. Measurement of aromati-sation by a urine technique suitable for the evaluation of aromatase inhibitors in vivo. J Enzyme Inhibition 1991; 4:315–325.

33. Lønning PE, Jacobs S, Jones A, Haynes B, Powles T, Dowsett M. The influence of CGS 16949A on peripheral aromatisation in breast cancer patients. Br J Cancer 1991; 63:789–793.

34. Geisler J, Lønning PE. Aromatase inhibition - translation into a successful therapeutic approach. Clin Cancer Res 2005; 11:2809–2821.

35. Thürlimann B, Beretta K, Bacchi M, Castiglionegertsch M, Goldhirsch A, Jungi WF, et al. First-line fadrozole HCI (CGS 16949A) versus tamoxifen in postmenopausal women with advanced breast cancer - Prospective randomised trial of the Swiss Group for Clinical Cancer Research SAKK 20/88. Ann Oncol 1996; 7(5):471–479.

36. Falkson CI, Falkson HC. A randomised study of CGS 16949A (fadrozole) versus tamoxifen in previously untreated postmenopausal patients with metastatic breast cancer. Ann Oncol 1996; 7(5):465–469.

Brodie AMH, Schwarzel WC, Shaikh AA, Brodie HJ. The effect of an


37. Thürlimann B, Castiglione M, HsuSchmitz SF, Cavalli F, Bonnefoi H, Fey MF, et al. Formestane versus megestrol acetate in postmenopausal breast cancer patients after failure of tamoxifen: A phase III prospective randomised cross over trial of second-line hormonal treatment (SAKK 20/90). Eur J Cancer 1997; 33(7):1017–1024.

38. Buzdar AU, Smith R, Vogel C, Bonomi P, Keller AM, Favis G, et al. Fadrozole HCL (CGS-16949A) versus megestrol acetate treatment of postmenopausal patients with metastatic breast carcinoma. Results of two randomized double blind controlled multiinstitutional trials. Cancer 1996; 77(12):2503–2513.

39. Pérez-Carrión R, Candel VA, Calabresi F, Michel RT, Santos R, Delozier T, et al. Comparison of the selective aromatase inhibitor formestane with tamoxifen as first-line hormonal therapy in postmenopausal women with advanced breast cancer. Ann Oncol 1994; 5:S19–S24.

40. Bonneterre J, Buzdar A, Nabholtz J-M, Robertson J, Thürlimann B, von Euler M, et al. Anastrozole is superior to Tamoxifen as first-line therapy in hormone receptor positive advanced breast carcinoma. Cancer 2001; 92(9):2247–2258.

41. Dombernowsky P, Smith I, Falkson G, Leonard R, Panasci L, Bellmunt J, et al. Letrozole, a new oral aromatase inhibitor for advanced breast cancer: Double-blind randomized trial showing a dose effect and improved efficacy and tolerability compared with megestrol acetate. J Clin Oncol 1998; 16(2):453–461.

42. Gershanovich M, Chaudri HA, Campos D, Lurie H, Bonaventura A, Jeffrey M, et al. Letrozole, a new oral aromatase inhibitor: Randomised trial comparing 2.5 mg daily, 0.5 mg daily and aminoglutethimide in postmenopausal women with advanced breast cancer. Ann Oncol 1998; 9(6):639–645.

43. Kaufmann M, Bajetta E, Dirix LY, Fein LE, Jones SE, Zilembo N, et al. Exemestane is superior to megestrol acetate after tamoxifen failure in post-menopausal women with advanced breast cancer: results of a phase III randomized double-blind trial. J Clin Oncol 2000; 18:1399–1411.

44. Paridaens R, Therasse P, Dirix L, Beex L, Piccart M, Cameron D, et al. First line hormonal treatment (HT) for metastatic breast cancer (MBC) with exemestane (E) or tamoxifen (T) in postmenopausal patients (pts) - a randomized phase III trial of the EORTC Breast Group. In: Am Soc Clin Oncol

45. Furr BJA, Jordan VC. The pharmacology and clinical uses of tamoxifen. Pharmac. Ther. 1984; 25:127–205.

46. Jordan VC. Tamoxifen and tumorigenicity: A predictable concern. J Natl Cancer Inst 1995; 87:623–626.

47. Osborne CK. Tamoxifen in the treatment of breast cancer. N Engl J Med 1998; 339(22):1609–1618.

48. Groom GV, Griffiths K. Effect of the anti-oestrogen tamoxifen on plasma levels of luteinizing hormone, follicle-stimulating hormone, prolactin, oestradiol and pro-gesterone in normal pre-menopausal women. J. Endocrinol 1976; 70:421–428.

49. Decensi A, Robertson C, Viale G, Pigatto F, Johansson H, Kisanga ER, et al. A randomized trial of low-dose tamoxifen on breast cancer proliferation and blood estrogenic biomarkers. J Natl Cancer Inst 2003; 95(11):779–790.

50. Buzdar A, Hayes D, El-Khoudary A, Yan S, Lonning P, Lichinitser M, et al. Phase III randomized trial of droloxifene and tamoxifen as first-line endocrine treatment of ER/PgR-positive advanced breast cancer. Breast Cancer Research and Treatment 2002; 73(2):161–175.

51. Helle SI, Anker G, Tally M, Hall K, Lønning PE. Influence of droloxifene on plasma levels of insulin-like growth factor (IGF)-I, pro-IGF-IIE, insulin-like

2004, 2004; p. 6 (Abstr 515).

J Steroid Biochem Mol Biol 1996; 57:167–171. growth factor binding protein (IGFBP)-1 and IGFBP-3 in breast cancer patients.

420


on plasma sex hormone levels in postmenopausal patients with breast cancer. J Endocrinol 1995; 146:359–363.

53. Lønning PE, Hall K, Aakvaag A, Lien EA. Influence of tamoxifen on the plasma levels of insulin-like growth factor I and Insulin-like growth factor binding protein I in breast cancer patients. Cancer Res 1992; 52:4719–4723.

54. Stenbygaard LE, Herrstedt J, Thomsen JF, Svendsen KR, Engelholm SA, Dombernowsky P. Toremifene and tamoxifen in advanced breast cancer - a double-blind cross-over trial. Breast Cancer Res Treat 1993; 25:57–63.

55. Hayes DF, Zyl JAv, Hacking A, Goedhals L, Bezwoda WR, Mailliard JA, et al. Randomized comparison of tamoxifen and two separate doses of toremifene in postmenopausal patients with metastatic breast cancer. J Clin Oncol 1995; 13:2556–2566.

56. Pyrhonen S, Valavaara R, Modig H, Pawlicki M, Pienkowski T, Gundersen S, et al. Comparison of toremifene and tamoxifen in post-menopausal patients with

Br J Cancer 1997; 76(2):270–277. 57. Curran M, Wiseman L. Fulvestrant. Drugs 2001; 61(6):807–813. 58. Liu H, Wing L, De Los Reyes A, Jordan V, Lurie R. Long-term fulvestrant

treatment results in irreversible loss of estrogen receptor alpha expression in mcf-7 human breast cancer cells. Proc Am Ass Cancer Res 2004; 45:1293.

59. Howell A, Robertson JF, Quaresma Albano J, Aschermannova A, Mauriac L, Kleeberg UR, et al. Fulvestrant, formerly ICI 182,780, is as effective as anastrozole in postmenopausal women with advanced breast cancer progressing after prior endocrine treatment. J Clin Oncol 2002; 20(16):3396–3403.

60. Osborne CK, Pippen J, Jones SE, Parker LM, Ellis M, Come S, et al. Double-blind, randomized trial comparing the efficacy and tolerability of fulvestrant versus anastrozole in postmenopausal women with advanced breast cancer progressing on prior endocrine therapy: results of a North American trial. J Clin Oncol 2002; 20(16):3386–3395.

61. Howell A, Robertson JE, Abram P, Lichinitser MR, Elledge R, Bajetta E, et al. Comparison of Fulvestrant versus tamoxifen for the treatment of advanced breast cancer in postmenopausal women previously untreated with endocrine therapy: A multinational, double-blind, randomized trial. Journal of Clinical Oncology 2004; 22(9):1605–1613.

62. Lønning PE, Lien E. Mechanisms of action of endocrine treatment in breast cancer. Crit Rev Oncol / Haematol 1995; 21:158–193.

63. Lundgren S, Helle SI, Lønning PE. Profound suppression of plasma estrogens by megestrol acetate in postmenopausal breast cancer patients. Clin Cancer Res 1996; 2:1515–1521.

64. Jones S, Vogel C, Arkhipov A, Fehrenbacher L, Eisenberg P, Cooper B, et al. Multicenter, phase II trial of exemestane as third-line hormonal therapy of postmenopausal women with metastatic breast cancer. J Clin Oncol 1999; 17(11):3418–3425.

65. Alberto P, Mermillod B, Kaplan E, Goldhirsch A, Obrecht J-P, Jungi F, et al. A clinical trial of aminoglutethimide in advanced postmenopausal breast carcinoma: Low response in patients previously treated with medroxyprogesterone. Eur J Cancer Clin Oncol 1985; 21:423–428.

66. Lea OA, Kvinnsland S, Thorsen T. Improved measurement of androgen receptors in human breast cancer. Cancer Res 1989; 49:7162–7167.

67. Nosaquo ND. Androgens and estrogens in the treatment of disseminatedmammary carcinoma. JAMA 1960; 172:135–147.

52. Geisler J, Haarstad H, Gundersen S, Raabe N, Kvinnsland S, Lønning PE. Influence of treatment with the anti-oestrogen 3-hydroxytamoxifen (droloxifene)

advanced breast cancer: a randomized double-blind, the ‘nordic’ phase III study.


postmenopausal women with advanced breast cancer. N Engl J Med 1981; 304:16–21.

69. Lippman M, Bolan G, Huff K. The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long-term tissue culture. Cancer Res 1976; 36:4595–4601.

70. Masamura S, Santner SJ, Heitjan DF, Santen RJ. Estrogen deprivation causes estradiol hypersensitivity in human breast cancer cells. J Clin Endocrinol Metab 1995; 80:2918–2925.

71. Lønning PE, Taylor PD, Anker G, Iddon J, Wie L, Jørgensen LM, et al. High-dose estrogen treatment in postmenopausal breast cancer patients heavily exposed to endocrine treatment. Breast Cancer Res Treat 2001; 67:111–116.

72. Lønning PE, Bajetta E, Murray R, TubianaHulin M, Eisenberg PD, Mickiewicz E, et al. Activity of exemestane in metastatic breast cancer after failure of nonsteroidal aromatase inhibitors: A phase II trial. J Clin Oncol 2000; 18(11):2234–2244.

73. Carlini P, Michelotti A, Giannarelli D, Conte PF, Salvadori B, Landucci E, et al. Exemestane (EXE) is an effective 3rd line hormonal therapy for post-menopausal metastatic breast cancer (MBC) patients (pts) pretreated with 3rd

5):Abstra 171p, s48. 74. Bertelli G, Garrone O, Merlano M, Occelli M, Bertolotti L, Castiglione F, et al.

Sequential treatment with exemestane and non-steroidal aromatase inhibitors in advanced breast cancer. Oncology 2005; 69(6):471–477.

75. Robertson JFR, Osborne CK, Howell A, Jones SE, Mauriac L, Ellis M, et al. Fulvestrant versus anastrozole for the treatment of advanced breast carcinoma in postmenopausal women - A prospective combined analysis of two multicenter trials. Cancer 2003; 98(2):229–238.

76. Rose C, Vtoraya O, Pluzanska A, Davidson N, Gershanovich M, Thomas R, et al. An open randomised trial of second-line endocrine therapy in advanced breast cancer: comparison of the aromatase inhibitors letrozole and anastrozole. Eur J Cancer 2003; 39(16):2318–2327.

77. Mouridsen H, Gershanovich M, Sun Y, Pérez-Carrión R, Boni C, Monnier A, et al. Superior efficacy of letrozole versus tamoxifen as first-line therapy for postmenopausal women with advanced breast cancer: Results of a phase III study of the International Letrozole Breast Cancer Group. J Clin Oncol 2001; 19:2596–2606.

78. Eiermann W, Paepke S, Appfelstaedt J, LlombartCussac A, Eremin J, Vinholes J, et al. Preoperative treatment of postmenopausal breast cancer patients with letrozole: A randomized double-blind multicenter study. Ann Oncol 2001; 12(11):1527–1532.

79. Smith IE, Dowsett M, Ebbs SR, Dixon JM, Skene A, Blohmer JU, et al. Neoadjuvant treatment of postmenopausal breast cancer with anastrozole, tamoxifen, or both in combination: The Immediate Preoperative Anastrozole, Tamoxifen, or Combined with Tamoxifen (IMPACT) multicenter double-blind randomized trial. J Clin Oncol 2005; 23(22):5108–5116.

80. Howell A, Cuzick J, Baum M, Buzdar A, Dowsett M, Forbes JF, et al. Results of the ATAC (Arimidex, Tamoxifen, Alone or in Combination) trial after

365(9453):60–62. 81. Thurlimann B, Keshaviah A, Coates AS, Mouridsen H, Mauriac L, Forbes JF,

et al. A comparison of letrozole and tamoxifen in postmenopausal women with early breast cancer. N Engl J Med 2005; 353(26):2747–2757.

completion of 5 years’ adjuvant treatment for breast cancer. Lancet 2005;

422

68.

Ingle JN, Ahmann DL, Green SJ, Edmonson JH, Bisel HF, Kvols LK, et al. Randomized clinical trial of diethylstilstilbestrol versus tamoxifen in

generation non-steroidal aromatase inhibitors (nSAI). Ann Oncol 2002; 13 (Suppl


83. Cheung KL, Owers R, Robertson JFR. Endocrine response after prior treatment with fulvestrant in postmenopausal women with advanced breast cancer: experience from a single centre. Endocrine-Related Cancer 2006; 13(1):251–255.

84. Jelovac D, Macedo L, Goloubeva OG, Handratta V, Brodie AMH. Additive antitumor effect of aromatase inhibitor letrozole and antiestrogen fulvestrant in a postmenopausal breast cancer model. Cancer Research 2005; 65(12):5439–5444.

85. Lønning PE. Adjuvant treatment of early breast cancer. Clinics of North America 2006;In press.

86. Sawka CA, Pritchard KI, Shelley W, DeBoer G, Paterson AHG, Meakin JW, et al. A randomized crossover trial of tamoxifen versus ovarian ablation for metastatic breast cancer in premenopausal women: A report of the National Cancer Institute of Canada clinical trials group (NCIC CTG) trial MA.1. Breast Cancer Res Treat 1997; 44(3):211–215.

87. Crump M, Sawka CA, DeBoer G, Buchanan RB, Ingle JN, Forbes J, et al. An individual patient-based meta-analysis of tamoxifen versus ovarian ablation as first line endocrine therapy for premenopausal women with metastatic breast cancer. Breast Cancer Res Treat 1997; 44(3):201–210.

88. Klijn JGM, Blamey RW, Boccardo F, Tominaga T, Duchateau L, Sylvester R. Combined tamoxifen and luteinizing hormone-releasing hormone (LHRH) agonist versus LHRH agonist alone in premenopausal advanced breast cancer: A meta-analysis of four randomized trials. J Clin Oncol 2001; 19(2):343–353.

82. Vergote I, Robertson JFR, Kleeberg U, Burton G, Osborne CK, Mauriac L.

sensitive to further endocrine therapy. Breast Cancer Research and Treatment 2003; 79(2):207–211.

‘Postmenopausal women who progress on fulvestrant ( Faslodex’) remain

INDEX [18F] fluorodeoxyglucose (FDG)-

PET tracer ........307, 308, 309 17 beta-hydroxysteroid dehydro-

genase ..............151, 153, 158 17-1A antigen, see also EpCAM

.........................................124 17-beta-estradiol ............99, 101 17bHSD1, see also 17 beta-

hydroxysteroid dehydrogenase .................153

3-hydroxytamoxifene...........410 4-hydroxyandrostenedione….409,

410 5-fluorouracil ......288, 304, 377,

378, 381

A32 antigen..........................120 ADAM .................................183 Adiol ....................................161 Adrenal gland, breast cancer

metastasis to.....................363 Adrenalectomy.............408, 409 AF-6, in tight junctions..........80 Agrin domain, in

matripatase .......................179 ALCAM, activated leukocyte cell

adhesion molecule, CD166......................117, 118

ALND, axillary lymph node biopsy .....281, 282, 333, 336, 342-344, 346, 347

AM, Adrenomedullin...244, 246 AMF, Autocrine Motility

Factor ...................11, 63, 124

Aminoglutethimide..... 408-411, 413

Amphiregulin...............139, 140 Anandamide.........................260 Anastrozole......... 405, 410-412,

414-416 Androgens.....36, 152, 153, 159,

408, 409, 413 Androsteonedione.......153, 409, 410 ANG1 ...............................33, 40 Angiogenesis ..2, 11, 12, 14, 34,

40, 57, 64, 82, 116, 125, 175, 188, 224, 225, 227, 230, 231, 247, 269, 285, 286, 315, 381, 385

Angiolymphatic invasion....279, 280, 295

Angiopoietin ..........................40 Anthracycline......288, 289, 291,

294-296, 312, 373, 376, 378-383, 385

Anti-HER2 antibody, for imaging ............................313

Anti-oestrogens....................206 Arachidonic acid..260, 270, 276 Aromatase...........3, 36, 37, 151,

153, 155-163, 259, 263, 265, 269, 271, 292, 314, 405, 406, 408-417

Aromatase inhibitor letrozole ....37, 294, 405, 410,

411, 414, 415 Aromatase inhibitors.....35, 158,

160-163, 271, 292, 405, 406, 408, 409, 411, 413-417

ATX, see autotaxin

Autologous tissue, in breast reconstruction ..................359

Autotaxin .........................10, 11 Axillary lymph node ......13, 39,

158, 208, 246, 281, 282, 286, 296-299, 333, 334, 336, 344-347, 350, 364

AZD0530, Src/Abl inhibitor 142 BARD1 ............................34, 36 BCF, Breast cyst fluids ........156 Bevacizumab........307, 315, 385 bFGF, basic fibroblast growth

factor ........161, 269, 285, 294 BGP, see also CEACAM1...119 Bisphosphonates .....52, 64, 241, 242, 250, 251, 327, 358, 375,

376, 389-401 Clodronate....... 375, 393-395,

399 Ibandronate ..... 375, 394-396,

399 Pamidronate ....375, 389, 391,

394-399, 401 Blood-brain barrier ........82, 362 Blue dye, in sentinel node

biopsy...............................337 BMP.....................179, 244, 248 Bone marrow ......4, 20, 48, 222,

286, 287, 296, 312, 314, 321-328, 344, 355, 358

Bone Sialoprotein ..................63 BPI, Brief Pain Inventory ...394,

397 Brain metastasis .......17, 18, 39,

361-363, 374, 375, 385 BRCA1 gene.. 2, 31-41, 62, 209,

211, 295, 296, 384 BRCA1 target genes

GADD45............................33 MAD2................................33 OPN .............................14, 33

p21CIP .................................33 pS2/TFF1 ...........................33

BRMS1 ............................16, 17 BVI, blood vessel invasion ..284 CA 15-3 ...............................125 CAAN, celecoxib anti-aromatase

neoadjuvant......................271 Cadherin........10, 47, 49, 53, 54,

60, 88, 111-116, 124 E-cadherin.......15, 16, 19, 20,

54-58, 60, 61, 88, 97, 100, 112-115, 125, 143

N-cadherin .......56-58, 60, 61,

P-cadherin 54, 57, 60-62, 58, 115, 116

VE-cadherin.....................116 CAF, Circulating Angiogenic

Factors .............294, 378, 381 Calcitonin...............................52

node dissection ........ 364-366 cAMP.......................... 267, 269 Canvaxin, trial in breast cancer

.........................................357 Capecitabine ....... 373, 376-380,

382-384 CapG......................................50 CAR, Coxsackie Adenovirus

Receptor...80, 86, 87, 88, 101 carboplatin ..........294, 373, 376,

377, 384, 385 Cardiotoxicity ......................377 CASK, in tight junctions........80 Catenin, alpha ......79-81, 88, 97,

114 Cathepsin D .............10, 52, 285 C-CAM-1, see also

CEACAM1 ......................119 CCIS, complete cytoreductive

426 Index

CALND, complete axillary lymph

100, 112, 114, 115, 118, 125

immunotherapeutic surgery...357

CCN proteins ...............244, 247 CD31, see also PECAM......120,

121, 286 CD34............................284, 286 CD44........11, 47, 121, 143, 222 CD45....................................293 CD56, see also NCAM ........119 CD62P, see also P-selectin ..121 CD66a, see also

CEACAM1 ..................... 119 CD82, see also KAI-1.....18, 59,

63 CD146..................................120 CD166, see also ALCAM....117 Cdc42.....................................51 CEACAM1, biliary

glycoprotein .....................119 Celecoxib .............................271 Cell-CAM, see also

CEACAM1 ......................119 Chemotactic factors ........13, 48,

49, 123 Chemotherapy.....5, 10, 51, 138,

208, 245, 279, 286, 288-291, 293-296, 310-312, 315, 324, 333, 347, 356, 358, 391, 393, 394, 406, 407, 416, 417

Chemotherapy 5-fluorouracil ..........288, 377,

378, 381 Capecitabine ... 373, 376-380,

382, 384 Carboplatin .....294, 373, 384,

376, 377, 385 Cyclophosphamide..........294,

373, 376, 377, 384, 385 Docetaxel ......... 294, 376-385 Doxorubicin ..... 294, 377-382 Epirubicin ................380, 381 Fluorouracil....294, 304, 378,

380, 382 Gemcitabine .... 373, 376-380,

383-385 Lapatinib ..................373, 385 Methotrexate ...........288, 377,

378, 382

Mitomycin ...............378, 382 Paclitaxel ................. 376-385 Taxanes...........288, 373, 379,

381-384, 406 Vinblastine...............378, 382 Vinorelbine .............373, 376,

377, 380, 384, 385, 406 Chemotherapy, side effects

Cardiotoxicity ..................377 myelotoxicity ...377, 380, 381 Nephrotoxicity .................377 Neurotoxicity ...........377, 380 neutropenia ......................383

Cingulin ...........................79, 80 Circulating tumor cells in the

blood (CTC)............321, 322, 324, 326, 327, 364

CK18............................322, 324 CK19....................................324 CK20....................................324 Claudin..... 12, 78, 80-82, 84-88,

91-93, 97-102 Clodronate ....375, 393-395, 399 Clostridium perfringens

enterotoxin, in the regulation of TJs......................................97

c-MET ....14, 63, 113, 137, 145, 171-173, 186, 188, 190, 191

CNS, metastasis in ......361, 362, 370, 374

Collagen........17, 47, 48, 53, 54, 122, 123, 286

Colon, breast cancer metastasis to ............122, 325

COM1, candidate of metastasis-1 ...........10, 15, 16

COX-1, cyclooxygenase-1…260, 261, 262, 270, 271

COX-2, cyclooxygenase-2......2, 3, 52, 64, 157, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271

CRSP3 ...................................16 CTC, see also Circulating tumor

cells in the blood.....322, 324, 326, 327, 364

Index 427

C-telopeptide, see CTX.........79, 80,86, 392

CTGF .....................14, 247, 251 CT-PET................................315 CTX family............................79 CTX, C-telopeptide.........79, 80,

86, 392 C-X-C motif...........................59 CXCL12.........13, 47, 59, 63, 65 CXCR1 ..........................13, 245 CXCR4 .........10, 12, 13, 47, 59,

63-65 Cyclooxygenase-1, see COX-1 Cyclooxygenase-2, see COX-2 Cyclophosphamide .....294, 300,

373, 373, 376, 377, 385 CYP19....36, 151, 155, 157, 269 Cyr61 ...................................247 Cytokeratins ..........60, 112, 293,

322-323, 325, 326 Cytokinesis ............................51 Cytoreductive surgery, for

metastatic breast cancer ...361 Cytoskeleton .......13, 47, 48, 49,

112, 114, 122, 143, 322 DARC, Duffy antigen

receptor ..............................63 DCIS, ductal carcinoma

in situ ........18, 60, 61, 91, 92, 96, 115, 121, 152, 259, 260, 266, 267, 270, 346

Dendritic cells ................87, 268

Desmocollin .........................116 Desmoplakin ........................116 Desmosomes ................112, 116 Detection of disseminated tumor

cells CK18........................322, 324 CK19................................324 CK20................................324

cytokeratins.......60, 112, 293, 321- 323, 325, 326

EMA ................................322 gradient centrifugation....322,

323 TAG12.....................286, 322

DHEA ..........................161, 162 Diclofenac............................270

Disintegrin .................53, 183 Disseminated tumor cells

(DTC)....................... 321-327 DKK-1 .................................250 DNA microarrays ...... 291-294 Docetaxel ............. 294, 376-385 Doloxifene, see also 3-

hydroxytamoxifene ..........410 Doxorubicin ......... 294, 377-382 Draining lymph node basin…363

E-cadherin...........15, 16, 19, 20,

54-58, 60, 61, 88, 97, 100, 112-115, 125, 143

EGCG ..................................250 EGF........14, 56, 60, 63, 73, 244 EGFR14, .........59, 61, 113, 123,

124, 127, 137, 139-142, 144, 145 EGFR signalling ..139, 140, 141 EGP40, see also EpCAM.....124 ELAM-1, see also E-selectin…121 EMA ....................................322 EMT, epithelial-to-mesenchymal

transition .......15, 51, 57, 112, 114, 115, 125, 139, 141, 143, 144, 150

Endobronchus, breast cancer metastasis to.....................363

Endocam, see also PECAM ...........................120

Endocrine-resistance... 137-139, 145

Endothelin............246, 248, 269 EpCAM, epithelial cell adhesion

molecule...........124, 125, 293

50, 51, 55, 59, 80, 83, 84, 97,

Denosumab (AMG 162)…249, 400

428 Index

Epirubicin ....288, 294 377, 378, 380, 381

Episialin, see also MUC1....124, 125

Epithelial membrane antigen…125 Epithelial-to-mesenchymal

transition (EMT) .........15, 51, 57, 112, 114, 115, 125, 139, 141, 143, 144, 150

Epithin..................................178 ErbB2...................113, 123, 124 ESA, see also EpCAM.........124 E-selectin .............................121 EST, estrogen

sulfotransferase ................154 Estradiol ............15, 37, 99, 101,

153, 159, 314, 410, 413, 414 Estradiol/estrone ratio ..........153 Estrogen sulfotransferase,

EST ..................................154 ET-1, endothelin-1 ......244, 246,

247, 248 ETAR...................................248 Etidronate.............................393 Exemestane ..........405, 410, 414 Factor VIII ...........................286 Fadrozole .....................410, 411 FAK, focal adhesion

kinase ..................59,142, 223 Faslodex.......................144, 145 Faslodex-resistant ........143, 144 FDG-6-phosphate ................309 FDG-PET........ 4, 307, 309-312,

317, 318 Fenretinide ...........................210 FES-PET......................314, 315 Fibronectin..............54, 60, 122,

123, 179 Fluorouracil.........294, 378, 380,

382 Formestane...........................411

Fulvestrant .......... 405, 411-413, 415, 416

GA733-2, see also EpCAM…124 Gab 1....................................174 GADD45................................33 gefitinib........................140, 141 Gelatinase B..........................52 Gemcitabine......... 373, 376-380 Gene expression profiling…..291,

296, 308 Gene pattern array ...........4, 292 Glucocorticoids.....97, 100, 157,

408, 409, 413 Gravin/AKAP12 ....................16 Grb2.....................140, 174, 223 Grb2/Ras/MAPK pathway...140 Green tea polyphenols ...........64 GSK3β .................................143 HAI-1................... 171, 176-191 HAI-2................... 171, 176-191 HAV domain..........................54 HepaCAM............................120 Hepatectomy for metastatic breast

cancer...............................361 Hepatic metastasectomy ......361 Hepatocyte growth factor .... see

HGF HER2 ...117, 139-141, 145, 146,

230, 263, 266, 267, 287-290, 292, 294, 295, 296, 300-304, 307-309, 312-316, 361, 362, 373-377, 381, 383-385

Herceptin® ...........140, 230, 312 HGF, hepatocyte growth

factor......2, 10-12, 14, 63, 91, 96, 98, 99, 101, 145, 171-191, 227

Index 429

HGFA ..... 2, 171, 176-178, 180, 181, 182, 183, 184, 185, 186, 187, 188, 190, 191

HSD, Hydroxysteroidyhydro genase .....151, 154, 158, 159, 160, 163, 203

Human milk fat globule membrane antigen............125

Hyaluronate................47, 54, 63 Hypercalcemia ....241, 242, 244,

390, 394, 396, 398 Ibandronate .......... 375, 394-396 Ibuprofen..............................270 ICAM-1, intercellular adhesion

molecule-1 ........63, 118, 119, 121, 122

IGF-1R.........................139, 206 IGF-I, Insulin-like growth

factors ......2, 56, 63, 97, 141, 161, 242

IL-1 ........................63, 244, 264 IL-4 ......................................244 IL-6, interleukin-6.........37, 156,

157, 159, 161, 244, 245, 267 IL-7 ...................... 225-227, 229 IL-8 .................. 3, 244, 245-247 IL-11 .........3, 14, 244, 245, 247,

249, 251 IL-12 ....................................244 IL-18 ................................3, 249 ILK, integrin-linked kinase....54 Immune evasion, in

metastasis .......................9, 10 Immunoglobulin-cell adhesion

molecules, ........................112 Immuno-SPECT, single photon

emission computed tomography ......................313

Indomethacin .......................270 Integrins ........ 14, 25, 47, 52-54,

63, 64, 86, 111, 122-125, 144

Intracrine system................153 Intravasation ..................49, 111 ipsilateral draining

lymph nodes.....................357 Junctional adhesion molecules ..

12, 79, 80, 82, 83, 86-88, 93, 98 KAI-1, also CD82............ 16-18 KAI1/CD82 ...........................59

KISS-1.....................16, 17, 54 Kisspeptin ..............................54 KSA, see also EpCAM ........124 Kunitz domain, in HAI .......112,

176, 179, 181, 182-184, 188, 189 Lamellipodia............ 50-52, 139 Lamellipodia, in cell

motility.................50, 51, 139 Lapatinib......................373, 385 LASP1 ...................................50 Letrozole.......37, 294, 405, 410,

411, 414, 415 Leu19, see also NCAM .......119 Leukocyte elastases, in

metastasis...........................10 LH-RH analogues .......407, 408,

416, 417 LH-RH receptors .................408 Liposome- encapsulated

doxorubicin......................381 Liver metastasis ....10, 314, 355,

360, 374

347, 353, 366 Internal mammary node......338,

Ki-67...........210, 285, 3 09, 324

430 Index

LMVD..................................230 L-selectin .....................121, 122 Lung metastasis ..........230, 245,

355, 360, 374 Lung metastasectomy ..........360 Lymphangiogenesis ............219,

224-229, 231 Lymphangiogenic factors HGF .......... 2, 10-12, 14, 63, 91,

96, 98, 99, 101, 145, 171-191, 227 IL-7 .................. 225-227, 229 VEGF-C 219, 223, 224, 225,

229, 230, 231 VEGF-D...........219, 224, 225

227, 229-231 Lymphatic drainage ....220, 334,

338, 348 Lymphatic markers

LYVE-1 ..........219, 222, 223, 226, 229, 230

podoplanin .......219, 221, 226 prox-1.......219, 221, 222, 226 VEGFR-3........ 221, 223-226,

230, 231, 245 Lymphatic microvessel density

(LMVD)...........................230 Lymphatics .........111, 219, 220,

222, 225, 227-231, 268, 334, 339, 348

Lymphedema .......................363 Lymphoscintigraphy ...........337,

338, 349 LYVE-1 ......219, 222, 223, 226,

229, 230

MAD2....................................33 MAGI-1 ...79, 80, 82, 83, 87, 88 MAGUK protein family, tight

junctions.....................81, 101 Mammography.....210, 282, 346 Management of bone metastasis

Bisphosphonates .........52, 64, 241, 242, 250, 251, 327, 358, 375, 376, 389, 401

pleurodesis .......................375 MAPK pathway ......13, 21, 139,

140, 206, 223, 225, 226 Matrilysin-1 ...........................52 Matriptase ..........2, 71, 176-188,

190, 191 MCAM.................................120 M-CSF .........................243, 245 Medroxyprogesterone ..161, 413 Mel-CAM ............................120 Met, HGF receptor..........14, 63,

113, 137, 145, 171-177, 186, 188, 190, 191

Metalloprotease, MMP ... 10-12, 20, 21, 52, 53, 57, 58, 64, 84, 113, 115, 117, 144, 179, 182, 183, 185, 269, 270, 271

Metastasectomy, hepatic......361 Metastasectomy, lung ..........360 Metastasis promoting genes…7-9 Metastasis-associated genes,

MTAs.......................8, 15, 19 Methotrexate.......288, 377, 378,

382 Micrometastasis ......4, 228, 282,

286, 296, 328, 333, 344, 349, 364-366

Microvascular density, MVD................230, 285, 286

Mitomycin ...................378, 382 MKK4.............................. 16-18 MMP, metalloproteinase….10-12,

20, 21, 52, 53, 57, 58, 64, 84, 113, 115, 117, 144, 179, 182, 183, 185, 269, 270, 271

MMP-3.................................179 MMP-7, also matrilysin ...52, 56 MMP-9, also gelati-

nase B ............20, 52, 53, 115 MMTV.............................38, 62 Molecular Imaging .................4,

307-309, 311, 312, 314-316

Index 431

432

MPGs, Metastasis promoting genes ................................ 7-9

MRI, magnetic resonance imaging ....310, 389, 392, 401

MT5-MMP.......................57, 58 MTAs, Metastasis-associated

genes ........................8, 15, 19 MT-SP1................................178 Muc-1.............................63, 125 MUC18 ................................120 MUPP-1 ...........................82, 86 MVD, Microvascular

density..................... 285, 286 Myelotoxicity.......377, 380, 381 Myocet .................................381 Myosins............................50, 51 Navelbine.............................406 N-cadherin, neural cadherin….56,

57, 58, 60, 61, 100, 112, 114, 115, 118, 125

NCAM, neural cell adhesion molecule...................119, 120

NCCN, National Comprehensive Cancer Network .......290, 391

Nectin.............80, 88, 89, 93, 96 Neoadjuvant chemotherapy…288,

347, 378 Nephrotoxicity .....................377 Neurotoxicity ...............377, 380 NFAT1...........................53, 124 NGF, nerve growth factor ......56 NKH1, see also NCAM .......119 NM23............................... 16-18 Noggin .........................244, 248 NSAIDs...............259, 261, 262,

270, 271 Nuclear factor-kappaB ligand ,

see also RANKL 17, 267, 400

Obstructive jaundice ............363 Occludin........12, 77, 79, 81, 83,

84, 85, 86, 87, 88, 91, 93, 94, 98, 99, 101

Oestrone ......................153, 159 Oncotype DX...............291, 292 Oophorectomy ......38, 295, 405,

407, 408, 417 OPG .....................244, 248, 249 OPN, see Osteopontin ORR, overall response

rates..................379, 383, 384 Osteoclast......242-251, 393, 400 Osteonectin, also

SPARC................... 10-13, 63 Osteopontin

OPN ................10, 14, 33, 52, 245, 247

Ovarian suppression 5, 407, 408 Oxygen-derived free

radicals.............................244 p120 ........................ 55, 58, 114 p450 .............................155, 156 p53 ..........18, 32, 34, 35, 40, 57,

62, 116, 208, 267, 293, 294 Paclitaxel ..................... 376-385 PAI-1, plasminogen activator

inhibitor type .............53, 290 Pamidronate ........375, 389, 391,

394-399, 401 PAR2, protease-activated receptor

2 .................................63, 179 PAR-3 ........................80, 86, 87 PAR-6 ..............................80, 86 Paracellin, see claudins....85, 93 P-cadherin, placental

cadherin .....54, 57, 58, 60-62, 115, 116

Index

PDGF ...................244, 247, 269 PDZ motifs, see ZOs..............79 PECAM, platelet endothelial cell

adhesion molecule....120, 121 PET, positron emission

tomography .................4, 281, 307-316, 392

PGE synthase .......................261 PGE2, prostaglandin E2........37,

157, 261, 265, 269, 271 PGG2, prostaglandin G2......260 PGH2, prostaglandin H2......269 Phagocytosis ..........................51 Phospholipase C-γ................174 Phospholipase C, gamma.....140 Phospholipase D ..................140 PI3-K pathway .............225, 226 Plakoglobin ....................55, 114 Plasminogen...................10, 172 plasminogen activator inhibitor

type 1, PAI-1..............53, 290 plasminogen activators ...10, 52,

177, 179, 182, 185, 290 Podoplanin ...........219, 221, 226 Ponsin ..............................80, 93 Positive surgical margins, after

breast surgery...................356 POX, peroxidase ..........262, 322

288, 290, 295, 307, 309, 314-316, 384

Predictive factors ....3, 279, 280, 285, 287, 289, 296, 321, 346

Progestagen..........................161 Prognostic factors ...10, 20, 175,

208, 230, 244, 248, 279-281, 285-287, 290, 321, 325, 356, 360, 361, 364

Promegestone.......................161

Prostaglandin synthetase.....260, 261

Prostaglandin E2, PGE2 .......37, 157, 261, 265, 269, 271

Prostaglandins...3, 37, 157, 244, 259-261, 265, 268, 269, 270

Prostanoids...........................261 Proteinases ..........10, 47, 49, 52,

53, 57 Proteolysis, in metastasis…10, 248 Prox-1 ..........219, 221, 222, 226 pS2/TFF1...............................33 PSA..............................244, 248 P-selectin ...............63, 121, 122 PTH..............................244, 248 PTHrP ............3, 244, 245, 247,

248, 249, 251 PVR, poliovirus receptor .......88 Quality of life (QOL)..........280,

333, 343, 373-375, 380, 389-391, 394, 400, 401

RANK..................243, 248, 250 RANKL, nuclear factor-kappaB

ligand ..........3, 243, 244, 245, 248, 249, 250, 400

Reconstruction, following mastectomy..............346, 359

Rho GTPase, in tight junctions.......................51, 78

Rho GTPases .............51, 52, 80 RKIP ..........................10, 16, 21 Rnd3/RhoE, in the regulation of

tight junctions ..................100 Rogletimide .........................411

PD158780, EGFR inhibitor 141

208, 209, 263-265, 279, 287, receptor..........60, 61, 92, 115,

PR, progesterone

Index 433

S100A4 ................10, 13, 24, 51 Scatter factor, see also HGF….11,

14, 63, 145, 172, 227 Selectins................10, 111, 112,

121, 124 SEMP-1......................78, 85, 91 S-Endo-1 ..............................120 Sentinel node ..............3, 4, 281,

310, 334, 336, 337, 339, 343-345, 348, 349, 357

SERM, selective estrogen receptor modifier.......56, 162, 288, 405, 410, 412, 413

Sfrp1, Soluble frizzled related protein 1 ...................249, 250

SIP1..........................55, 56, 113 siRNA ........21, 33, 36, 125, 145 Skeletal-related events ........358,

389-391, 395-399 SLN, see also sentinel

node.......... 333-335, 337-339, 342, 344, 345, 347, 348, 349, 363, 364, 365, 366

SLNB, sentinel lymph node biopsy......281, 282, 333, 337, 339, 342, 343, 345-349, 363-365

Slug ..................55, 78, 100, 113 Small bowel, breast cancer

metastasis to.....................363 Snail .....19, 55, 57, 78, 100, 113 SPARC............................. 10-13 SPF, S-phase fraction...........284 Sphingosine kinase 1 .............63 Src kinase............. 137, 141-143 SRE, skeletal-related

events .............. 358, 389-391, 395-399

SRS, stereotactic radiosurgery ..............355, 362 363

ST14.....................................178 steroid sulfatase, see

also STS ..........151, 153, 154, 160-163

Stomach, breast cancer metastasis to ..............................181, 363

Stromelysin, see also MMP-3.............................179

Stromelysin-3.........................11 STS, steroid sulfatase .........151,

153, 154, 160-163 Surgical management, metastatic

breast cancer ...........228, 347, 355, 357, 365

Symplekin..............................80

TADG-15.............................178 TAG12.........................286, 322 TAILORx.............................292 Tamoxifen.......5, 10, 15, 38, 56,

139, 141, 158, 206, 208, 210, 288, 290, 291, 293, 314, 405, 410, 412-416

Tamoxifen-resistance...........289 Tamoxifen-resistant ......15, 139,

140, 141, 142 Tangeretin..............................56 Taxanes...............288, 373, 376.

379, 381-384, 406 docetaxel.......... 294, 376-385 paclitaxel.......... 257, 376-385

TCF/LEF-1 transcription factor................................143

Tenascin.................................53 TGFα, transforming growth

factor alpha ........97, 139, 140 TGFβ, transforming growth factor β .......100, 101, 113, 241-244, 248, 251

TGF-1 ..................................269 Thrombin .............................178 Tiam-1 ...................................52 Tight Junctions .... 2, 12, 77-102 TIMP............16, 20, 21, 53, 144 TIMP-2 ................12, 20, 21, 53 TLI, Thymidine labeling

index ................................285 TNF........88, 101, 157, 159, 269 Topoisomerase.....290, 294, 380

434 Index

Toremifene...........................412 tPA .......................................177 Transforming growth factor alpha

(tgfα) ..................97, 139, 140 Trastuzumab .......289, 290, 294,

295, 312, 313, 362, 373, 376, 377, 382-385, 406

Tumor debulking, for metastatic breast cancer ....................361

Twist ..............................55, 113 TXNIP..............................16, 19 Ubiquitin ........................34, 143 uPA .....4, 10, 64, 176, 177, 244,

290

Vanillin ..................................52 Vasculogenesis ....................116 VCAM-1, vascular cell adhesion

molecule -1 ..............117, 118 VDUP1 ............................16, 19 VE-cadherin, vascular endothelial

cadherin............................116 VEGF............40, 101, 124, 219,

223, 224, 244, 245, 249, 259, 263, 267, 285, 286, 294, 315

VEGF-C......219, 223, 224, 225, 229-231

VEGF-D......219, 224, 225, 227, 229-231

VEGFR-1.....................223, 224 VEGFR-2.............224, 225, 245 VEGFR-3.....221, 223-226, 230,

231, 236, 245 Vimentin ........................57, 112 Vinblastine...................378, 382 Vinorelbine .........373, 376, 377,

380, 384, 385, 406

WBRT, whole brain irradiation.................362, 363

Wnt signaling pathway .......114, 244, 249, 250

Zoledronic acid .....64, 375, 389, 391, 394-399, 401

Zonulin...................................82 ZOs, in tight junctions ... 79-101

Index 435

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