apoptosis in carcinogenesis and chemotherapy filegeorge g. chen · paul b.s. lai editors apoptosis...

Apoptosis in Carcinogenesis and Chemotherapy

George G. Chen · Paul B.S. LaiEditors

Apoptosis in Carcinogenesisand Chemotherapy

Apoptosis in Cancer

123

Editors

Dr. George G. ChenChinese University of Hong KongPrince of Wales HospitalDepartment of SurgeryShatin, New TerritoriesHong Kong/PR China

Dr. Paul B.S. LaiChinese University of Hong KongPrince of Wales HospitalDepartment of SurgeryShatin, New TerritoriesHong Kong/PR China

ISBN 978-1-4020-9596-2 e-ISBN 978-1-4020-9597-9

DOI 10.1007/978-1-4020-9597-9

Library of Congress Control Number: 2008942032

c© Springer Science+Business Media B.V. 2009No part of this work may be reproduced, stored in a retrieval system, or transmittedin any form or by any means, electronic, mechanical, photocopying, microfilming, recordingor otherwise, without written permission from the Publisher, with the exceptionof any material supplied specifically for the purpose of being enteredand executed on a computer system, for exclusive use by the purchaser of the work.

Printed on acid-free paper

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Foreword

Although research on carcinogenesis has focused more on cellular proliferation thanon cell death, yet understanding the mechanism of apoptosis may have importantimplications for cancer therapy. This book brings together experts from around theworld who will discuss the common cancers encountered in clinical practice inthe laboratory setting. During the induction of these common cancers, the role ofapoptosis in cellular and molecular changes is emphasized, critically highlightingpossible anti-cancer strategies.

For those who are interested in carcinogenesis and for those who are seeking newapproaches to anti-cancer therapy, this book is an important reference. It serves notonly as a reference of the current understanding of apoptosis in common cancers butalso an important bridge between the laboratory and clinical practice.

The editors and contributors are to be congratulated in bringing together animportant pool of up-to-date knowledge to light and further our interest in thisexciting and expanding field.

Arthur K. C. LiEmeritus Professor of Surgery

The Chinese University of Hong Kong

v

Preface

The role of apoptosis in cancer development and emerging treatment strategies hasrapidly expanded over the past few years. The novel discovery in the apoptotic path-ways and their relevant molecules provides us not only the knowledge how tumorsdevelop but also the opportunity to design new therapeutic tools to prevent or inhibitthe growth of tumors with minimal side-effects. Undoubtedly, understanding theevents involved at a molecular level can permit the manipulation of apoptosis fortherapeutic purposes.

In healthy subjects, apoptosis is a normal and continuous process with complexphysiological controls. However, due to various environmental and endogenous fac-tors this process becomes out of control or develops in a manipulated directionin cancers. The imbalance between the pro-apoptosis and anti-apoptosis is oftena two-side coin. With a shift in favour of the latter, cells may growth uncontrol-lably. In contrast, with a shift in favour of the former, cells may die or sensitiveto cell death stimuli. There may be some common points of the apoptotic processin tumors of different tissue/cell types. However, different cancers often possesstheir own specific and dedicated molecules that regulate apoptosis. These specificand dedicated molecules or pathways are truly reflected in the volume of this bookwhich critically describes and summarizes the up-to-date research on an emergingtopic of “Apoptosis in Carcinogenesis and Chemotherapy” in 15 chapters, each ofthem focusing on a particular tumor, including breast, bladder, cervical, colorectal,cutaneous, esophageal, gastric, hematologic, laryngeal, liver, lung, nasopharyngeal,pancreatic, prostate and thyroid cancers.

The book, a collection of cutting-edge reviews by established leaders in the field,will be of great interest to not only clinicians interested in molecular approaches ofapoptosis in chemotherapy but also basic scientists working in the field of cancerresearch and apoptosis.

Hong Kong, PRC George G. ChenHong Kong, PRC Paul B.S. Lai

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Contents

1 Apoptotic Signaling Pathway and Resistance to Apoptosis in BreastCancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Prasanthi Karna and Lily Yang

2 Anti-Cancer Strategy of Transitional Cell Carcinoma of BladderBased on Induction of Different Types of Programmed Cell Deaths . . 25Jose A. Karam and Jer-Tsong Hsieh

3 Apoptosis in Carcinogenesis and Chemoherapy of the Uterine Cervix 51Sakari Hietanen

4 Apoptosis in Colorectal Tumorigenesis and Chemotherapy . . . . . . . . . 75Shi Yu Yang, Kevin M. Sales and Marc C. Winslet

5 Apoptosis in Cutaneous Melanoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111Michael B. Nicholl and Dave S.B. Hoon

6 Apoptosis in Carcinogenesis and Chemotherapy – EsophagealCancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127Yan Li and Robert C.G. Martin

7 Molecular Targets in Gastric Cancer and Apoptosis . . . . . . . . . . . . . . . . 157Elizabeth K. Balcer-Kubiczek and Michael C. Garofalo

8 Apoptosis and the Tumor Microenvironment in HematologicMalignancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193Danielle N. Yarde and Jianguo Tao

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x Contents

9 Bcl-2 Family Members in HepatocellularCarcinoma (HCC) – Mechanismsand Therapeutic Potentials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219Shihong Ma, George G. Chen and Paul B.S. Lai

10 Apoptosis in the Development and Treatment of Laryngeal Cancer:Role of p53, Bcl-2 and Clusterin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237Maximino Redondo, Rafael Funez and Francisco Esteban

11 Cyclooxygenase 2 and its Metabolites: Implications for LungCancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251Kin Chung Leung and George G. Chen

12 Roles of Negative and Positive Growth Regulators inNasopharyngeal Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273Mong-Hong Lee, Huiling Yang, Ruiying Zhao and Sai-Ching J. Yeung

13 Cellular Signaling Mechanisms in Pancreatic Apoptosis . . . . . . . . . . . . . 295Nawab Ali, Stewart MacLeod, R. Jean Hine and Parimal Chowdhury

14 Strategies to Circumvent Resistance to Apoptosis in ProstateCancer Cells by Targeted Necrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327Richard D. Dinnen, Daniel P. Petrylak and Robert L. Fine

15 Carcinogenesis and Therapeutic Strategies for Thyroid Cancer . . . . . 347Zhi-Min Liu and George G. Chen

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375

Contributors

Nawab Ali Graduate Institute of Technology, University of Arkansas at LittleRock, Little Rock, AR 72205, USA, [email protected]

Elizabeth K. Balcer-Kubiczek Department of Radiation Oncology, RadiationOncology Research Laboratory, University of Maryland School of Medicine, 655W. Baltimore Street, BRB, Baltimore MD 21201, USA, [email protected]

George G. Chen Department of Surgery, Prince of Wales Hospital, The ChineseUniversity of Hong Kong, Shatin, NT, Hong Kong, [email protected]

Parimal Chowdhury Department of Physiology and Biophysics,University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA,[email protected]

Richard D. Dinnen Experimental Therapeutics, Division of Medical Oncology,College of Physicians and Surgeons, Columbia University, 650 West 168th Street,BB 20-05, New York, NY 10032, USA, [email protected]

Francisco Esteban Department of Otolaryngology, Hospital Virgen delRocio, Avda. Manuel Siurot s/n CP 41013, University of Sevilla, Sevilla, Spain,[email protected]

Robert L. Fine Experimental Therapeutics, Division of Medical Oncology,College of Physicians and Surgeons, Columbia University, 650 West 168th Street,BB 20-05, New York, NY 10032, USA, [email protected]

Rafael Funez Department of Pathology, CIBER Epidemiologia y Salud Publica(CIBERESP), Hospital Costa del Sol, Carretera de Cadiz Km 187, 29600 Marbella,University of Malaga, Malaga, Spain, [email protected]

Michael C. Garofalo The Marlene and Stewart Greenebaum Cancer Center,Department of Radiation Oncology, University of Maryland School of Medicine,22 S. Greene St., Baltimore MD 21201, USA, [email protected]

Sakari Hietanen Department of Obstetrics and Gynecology, TurkuUniversity Central Hospital, Kiinamyllynkatu 4-8, 20520 Turku, Finland,[email protected]

xi

xii Contributors

Jean Hine Department of Nutrition, Health Policy and Management, College ofPublic Health Winthrop P. Rockefeller Cancer Institute, University of Arkansas forMedical Sciences, Little Rock, AR 72205, USA, [email protected]

Dave S.B. Hoon Department of Molecular Oncology, John Wayne CancerInstitute, 2200 Santa Monica Boulevard, Santa Monica, CA 90404, USA,[email protected]

Jer-Tsong Hsieh Department of Urology, University of Texas SouthwesternMedical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9110, USA,[email protected]

Jose A. Karam Department of Urology, University of Texas SouthwesternMedical Center, Dallas, TX 75390, USA, [email protected]

Prasanthi Karna Department of Surgery, Winship Cancer Institute, EmoryUniversity School of Medicine, Clinic C, Room C-4038, 1365 C Clifton Road NE,Atlanta, GA 30322, USA, [email protected]

Paul B.S. Lai Department of Surgery, Prince of Wales Hospital, The ChineseUniversity of Hong Kong, Shatin, NT, Hong Kong, [email protected]

Mong-Hong Lee Department of Molecular and Cellular Oncology, AndersonCancer Center, The University of Texas M.D., 1515 Holcombe Blvd, Houston, TX77030, USA, [email protected]

Kin Chung Leung Department of Surgery, Prince of Wales Hos-pital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong,[email protected]

Yan Li Division of Surgical Oncology, Department of Surgery, University ofLouisville School of Medicine, 511 S Floyd ST, MDR Bld, Rm 326A, Louisville,KY 40202, USA, [email protected]

Zhi-Min Liu Department of Biochemistry and Molecular Biology, The Schoolof Basic Medical Sciences, Chongqing Medical University, Chongqing, China,[email protected]

Shihong Ma Department of Surgery, Prince of Wales Hospital,The Chinese University of Hong Kong, Shatin, NT, Hong Kong,[email protected]

Stewart MacLeod Department of Obstetrics and Gynecology, ArkansasChildren’s Hospital, Little Rock, AR 72205, USA, [email protected]

Robert C.G. Martin II Department of Surgery, Division of Surgical Oncology,University of Louisville School of Medicine, 511 S Floyd ST, MDR Bld, Rm 326A,Louisville, KY 40202, USA, [email protected]

Michael B. Nicholl Department of Molecular Oncology, John Wayne CancerInstitute, 2200 Santa Monica Boulevard, Santa Monica, CA 90404, USA,[email protected]

Contributors xiii

Daniel P. Petrylak Experimental Therapeutics, Division of Medical Oncology,College of Physicians and Surgeons, Columbia University, 650 West 168th Street,BB 20-05, New York, NY 10032, USA, [email protected]

Maximino Redondo Department of Biochemistry, CIBER Epidemiologia ySalud Publica (CIBERESP), Hospital Costa del Sol, Carretera de Cadiz Km 187,29600 Marbella, University of Malaga, Malaga, Spain, [email protected]

Kevin M Sales University Department of Surgery, Royal Free and UniversityCollege Medical School, University College London, Rowland Hill Street, LondonNW3 2PF, UK, [email protected]

Jianguo Tao Hematopathology and Laboratory Medicine, H. Lee Moffitt CancerCenter and Research Institute, University of South Florida College of Medicine,12901 Magnolia Drive, MCC 2071F, Tampa, FL 33612, USA, [email protected]

Marc C. Winslet University Department of Surgery, Royal Free and UniversityCollege Medical School, University College London, Rowland Hill Street, LondonNW3 2PF, UK; Royal Free Hampstead NHS Trust Hospital, London, UK;University College Hospital, London, UK, [email protected]

Lily Yang Departments of Surgery and Radiology, Winship Cancer Institute,Emory University School of Medicine, Clinic C, Room C-4088, 1365 C CliftonRoad NE, Atlanta, GA 30322, USA, [email protected]

Shi Yu Yang University Department of Surgery, Royal Free and UniversityCollege Medical School, University College London, Rowland Hill Street, LondonNW3 2PF, UK, [email protected]

Huiling Yang Department of Pathophysiology, Sun Yat-Sen University MedicalSchool, Guangzhou, China, [email protected]

Danielle N. Yarde Hematopathology and Laboratory Medicine, H. Lee MoffittCancer Center and Research Institute, University of South Florida Collegeof Medicine, 12901 Magnolia Drive, MCC 2071F, Tampa, FL 33612, USA,[email protected]

Sai-Ching J. Yeung Endocrine Neoplasia and Hormonal Disorders, GeneralInternal Medicine, Ambulatory Treatment and Emergency Care, University ofTexas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030,USA, Syeung @mdanderson.org

Ruiying Zhao Departments of Molecular and Cellular Oncology, AmbulatoryTreatment and Emergency Care, University of Texas M. D. Anderson CancerCenter, 1515 Holcombe Blvd, Houston, TX 77030, USA, [email protected]

Abbreviations

1-BI 1-Benzylimidazole5-FU 5-Fluorouracil5-LOX 5-lipoxygenaseAA Arachidonic acidAC Adenyl cyclaseAdPC Adenomatous polyposis coliAEF Alternative-reading frame proteinAI Allelic imbalanceAIF Apoptotic inducing factorsAkt/PKB A serine/threonine protein kinase/Pritein kinase BALL Acute lymphoblastic leukemiaAML Acute myelocytic leukemiaAMPK AMP-activated protein kinaseAnt Antennapedia homeobox domainApaf-1 Apoptotic protease activating factor-1APC Anaphase-promoting complexApoptosis Programmed cell deathAPRIL A proliferation-inducing ligandASO Antisense oligonucleotidesATC Anaplastic thyroid carcinomaATG Autophagy-related geneATP Adenosine tri-phosphateBAD Bcl-2-associated death promoterBAFF B cell-activating factor of the tumor necrosis factor familyBAG Bcl-2-associated athanogeneBAK1 BAK; BCL-2 antagonist/killerBAX Bcl-2-associated X proteinBBC3 PUMA; Bcl-2 binding component 3BC BiochemotherapyBCG Bacillus Calmette-GuerinBcl-2 B-cell lymphoma 2Bcl-XL Basal cell lymphoma-extra large

xv

xvi Abbreviations

BE Barrett’s EsophagusBER Base excision repairbFGF Basic fibroblast growth factorBH domains Bcl-2 homology domainsBik Bcl-2 interacting killerBim Bcl-2 interacting mediator of cell deathBIR Baculoviral IAP repeatBIRC4 XIAP; X-linked inhibitor of apoptosisBIRC5 Survivin; baculoviral IAP repeat-containing 5Bmf Bcl-2-modifying factorBMI1 Polycomb group ring fingerBMSC Bone marrow stromal cellBN BombesinBnip Bcl-2/adenovirus E1B 19 kDa interacting proteinBok/Mtd Bcl-2-related ovarian killerB-RAF v-raf murine sarcoma viral oncogene homolog B1BV Bee venomCAM-DR Cell adhesion mediated drug resistancecAMP Cyclic adenosine monophosphateCARD Caspase recruitment domainCASP Caspase; apoptosis-related cysteine peptidaseCaspase Cysteine aspasesCCCG Colorectal Cancer Collaborative GroupCCND2 G1/S –specific cyclin D2CCNE1 Cyclin ECDC Cell cycle divisionCDH1 E-cadherinCDK Cyclin-dependent kinaseCDKN1A p21; WAF1/CIP1; cyclin-dependent kinase inhibitor 1ACDKN1B p27; kip1; cyclin-dependent kinase inhibitor 1BCDKN2A p16; INK4A; cyclin-dependent kinase inhibitor 2ACDKN2B p15; INK4B; cyclin-dependent kinase inhibitor 2bCDKs Cyclin-dependent KinasesCDNA Complementary DNACEA Carcinoembryonic antigenc-FLIP Cellular FLICE-like inhibitory proteinCIN Cervical intraepithelial neoplasiaCisplatin cis-diamminedichloroplatinumCLL Chronic lymphocytic leukemiaCLU ClusterinCML Chronic myelocytic leukemiaCOX CyclooxygenaseCOX-2 Cyclooxygenase 2CP Ceruloplasmin

Abbreviations xvii

CRC Colorectal cancerCRT ChemoradiationCSC Cigarette smoking condensateCTNNB1 β-catenin; cadherin-associated proteinCyto c Cytochrome cDAP4 Dipeptidyl-aminopeptidaseDAPK Calmodulin (CaM)-regulated Ser/Thr kinaseDCC Deleted in colorectal cancerDCF Docetaxel, cisplatin, 5-fluorouracilDCIS Ductal carcinoma in situDcR3 Fas decoy receptorsDD Death domainsDED Death effecter domainsDEDs Death effector domainsDIABLO Direct IAP binding protein with low pIDISC Death-inducing signaling complexDITC DacarbazineDNA Deoxyribonucleic acidDNMT1 DNA methyl trasferaseDOX DoxorubicinDR Death receptorDR4/DR5 Death receptor 4/Death receptor 5E HPVs encode six earlyE2F1 E2F transcription factor 1; retinoblastoma-associated

proteinEAC Esophageal adenocarcinomaEBV Epstein-Barr virusECF Epirubicin, cisplatin, 5-fluorouracilECM Extracellular matrixEDAR Ectodysplasin-A receptorEGCG Epigallocatechin-3-gallateEGF Epidermal growth factorEGFR Epidermal growth factor receptorEGFR-KI Epidermal growth factor receptor kinase inhibitorEM-DR Environment mediated drug resistanceEMT Epithelial-mesenchymal transitionEP1, EP2, EP3, EP4 PEG2 receptorsER Estrogen receptorERBB2 HER-2, erbB-2; v-erb-b2 erythroblastic leukemia

viral oncogene homolog 2ERK Extracellular signal-regulated kinaseESCC Esophageal squamous cell carcinomaFA Follicular adenomaFADD Fas-associated death domainFAP Familial adenomatous polyposis

xviii Abbreviations

FAP-1 Fas-associated phosphatase-1FasL Fas ligandFAS-R Fas receptorFC Follicular carcinomaFGF Fibroblast growth factorFHIT Fragile histidine triadFLIP Fas-associated death domain-like interleukin-

1β-converting enzyme inhibitory proteinFLIPs FADD-like ICE inhibitory proteinsFN FibronectinFP PGF2α receptorsFVPTC Follicular variant of PTCGAP GTPase-activating proteinGAST GAS; gastrinGERD Gastroesophageal reflux diseaseGI GastrointestinalGPR30 G protein-coupled receptor 30GPx Glutathione peroxidasesGRP Gastrin-releasing peptideGSI Gamma-secretase tripeptide inhibitorGSK3 Glycogen synthase kinase 3Gsp G protein subunit alphaGSTP1 Glutathione S-transferase π

HA Hurthle cell adenomaHAT Histone acetyltransferaseHC Hurthle cell carcinomaHCC Hepatocellular carcinomaHDAC Histone deacetylaseHDI Histone deacetylase inhibitorHGF/SF Hepatocyte growth factor/scatter factorHIT-T15 Pancreatic B-cell lineHMGB1 High-mobility group B1HMGI High mobility group IHNPCC Hereditary non-polyposis colonic cancerHPV Human papillomavirusHrk/DP5 HarakiriHSC Hematopoietic stem cellHSP Heat shock proteinhTERT Human telomerase reverse transcriptaseIAP Inhibitor of apoptosis proteinsId1 Inhibitor of differentiationIGFBP Insulin-like growth factor binding proteinIGF-I R Insulin-like growth factor receptorIGF-I Insulin-like growth factorIHC Immunohistochemistry

Abbreviations xix

IKB Inhibitor of NFkBIKK IκB kinaseIL-1 InterleukinIL1A Interleukin 1, α

IL1F6 Iinterleukin 1 family member 6, ε

IL1F8 Interleukin 1 family member 8, ξ

IL-6 Interleukin-6IL-8 Interleukin-8Ink4a Inhibitor of CDK4iNOP Interfering nanoparticlesIRF-3 Interferon regulatory factor-3ITF Intestinal trefoil factorKIT c-kit; v-kit Hardy-Zuckerman 4 feline sarcoma viral

oncogene homologKRAS Kirsten rat sarcoma viral oncogene homologK-sam KATO III cell-derived stomach cancer amplifiedL 2 lateLCM Laser capture microdissectionLEEP Loop electrosurgical excision procedureLF LactoferrinLIN Laryngeal intraepithelial neoplasiaLMP1 Latent membrane protein 1LNA Locked nucleic acidLOH Loss of heterozygosityLV LeucovorinMAD2 Mitotic arrest deficient 2MADDH Mothers against decapentaplegic (MAD) Drosophila

homologMALT Mucosa-associated lymphoid tissue.MAP MYH- associated polyposisMAPK Mitogen-activated protein kinaseMCC Mutated in colon carcinomaMcl-1 Myeloid cell leukemia-1MDM2 Mouse double minute 2 homologMEN Multiple endocrine neoplasiaMET c-met; hepatocyte growth factor receptorMGMT O-6-methylguanine-DNA methyltransferaseMINPP Multiple inositol polyphosphate phosphatasesMINT2 Munc18-1-interacting protein 2miR MicroRNAMITF Microphthalmia-associated transcription factorMLH1 MutL human homolog DNA mismatch repairMM Multiple myelomaMMP Mitochondrial membrane permeabilizationmPGES Microsomal prostaglandin E synthase

xx Abbreviations

MRD Minimal residual diseasemRNA Messenger RNAMSH Mismatch repair ATPase MutS familyMTC Medullary thyroid cancermTOR Mammalian target of rapamycinMUC Mucin; oligometric mucus/gel-forming)MYC c-myc; v-myc avian myelocytomatosis viral

oncogene homologMYH MutY human homolog base excision repairNAC a nucleotide binding domain and CARDN-CAM Neural cell adhesion moleculeNF-KB Nuclear factor-kappaBNFKB1 NF-κB; Nuclear factor kappa light polypeptide gene

enhancer in B-cellsNF-κB Nuclear factor kappa BNGFR Nerve growth factor receptorNHL Non-Hodgkin’s lymphomaNIS Sodium iodide symporterNix Nip3-like protein XNNK 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanoneNO Nitric oxideNOTCH Neurogenic locus notchNoxa NADPH oxidase activatorNPC Nasopharyngeal carcinomaNSAID Non-steriodal anti-inflammatory drugsNSCLC Non-small cell lung cancerOMM Outer mitochondrial membraneORF Open reading frameP53 Tumor protein p53P73 Tumor protein p73PAC PaclitaxelPAC-1 Procaspase-activating compound-1PALA N-(phosphonacetyl)-L-aspartic acidPANC-1 Human pancreatic cancer cell line-1PARP Poly-ADP ribose polymerasePCD Programmed cell deathPCNA Proliferation cell nuclear antigenPDGF Platelet-derived growth factorPGE2 Prostaglandin E2

PGES Prostaglandin synthasePGH2 Prostaglandin H2

PGI2 Prostacyclin I2

PGIS Prostacyclin synthasePI3K Phosphatidylinositol 3-kinasePI3K Phosphoinositide 3-kinase

Abbreviations xxi

PKB Protein kinasePLC Phospholipase CPML Promyelocytic leukemia tumor suppressorPPAR Peroxisome proliferator activated receptorPPARdelta Peroxisome proliferator-activated receptor deltaPPARγ Peroxisome proliferator-activated receptor-gammaPR ProgesteronePTC Papillary thyroid carcinomaPTEN Phosphatase and tensin homologPTGS2 Cyclooxygenase-2; COX2; prostaglandin endoperoxide synthase)PTHrP Parathyroid hormone related peptidePUMA p53 upregulated modulator of apoptosisRassf Ras-association domain family of proteinRASSF1A Ras association RalGDS/AF-6 domain family 1Rb RetinoblastomaRIPK1 Receptor-interacting protein kinase 1RNA Ribonucleic acidRNAi RNA interferenceROS Reactive oxygen speciesRT RadiotherapyRTK Receptor tyrosine kinaseRUNX3 Runt-related transcription factor 3)SAHA Suberoylanilide hydroxamic acidSCCL Squamous cell carcinoma of larynxSe SeleniumSHH Sonic hedgehogshRNA Short hairpin RNAsiRNA Short interfering RNASMAC Second mitochondria-derived activatior of caspaseSMAD Small mothers against decapentaplegicSP Side-populationSpike Small protein with inherent killing effectSSAT Spermidine/ spermine N1-acetyltransferaseSTAT Signal transducers and activators of transcriptionT3 TriiodothyronineT4 ThyroxinetBid Truncated BidTC Thyroid cancerTCC Transitional cell carcinomaTCF T-cell factorTERT Telomerase reverse transcriptaseTFF1 pS2; gastrointestinal trefoil protein 1TG ThyroglobulinTGFB1 TGFβ; transforming growth factor beta 1TGFBR Transforming Growth Factor-β receptors

xxii Abbreviations

TGF-β Transforming growth factor-betaTHXA2 Thromboxane A2

THXB2 Thromboxane B2

THXS Thromboxane synthaseTIL Infiltration by lymphocytesTNF Tumor necrosis factorTNFR Tumor necrosis factor receptorTNFR1 Tumor necrosis factor receptor 1TNFα Tumor necrosis factor α

TPO Thyroid peroxidaseTRADD TNFR-associated death domainTRAF1/2 TNF receptor associated factors 1 and 2TRAF2 TNF receptor–associated factor 2TRAIL TNF-related apoptosis inducing ligandTRID Decoy receptors 1 (DcR1 or TRAIL-R3)TRUNDD Decoy receptors 2 (DcR2 or TRAIL-R4)TRx Thioredoxin reductasesTSH Thyroid stimulating hormoneTSLC Tumor suppressor in lung cancerTTF-1 Thyroid transcription factor 1TUNEL Terminal deoxynucleotidyl transferase-dUTP nick end labelingTVPTC Tall cell variant of PTCTXR Thromboxane receptorVEGF Vascular endothelial growth factorwt wild typeXIAP X-linked inhibitor of apoptosis protein

Chapter 1Apoptotic Signaling Pathway and Resistanceto Apoptosis in Breast Cancer Stem Cells

Prasanthi Karna and Lily Yang

Abstract A major challenge in the treatment of human breast cancer is the develop-ment of resistant mechaims to apoptosis in cancer cells that leads to a low senstivityto therapeutic agents. Recent advances in investigation of the cellular origin of breastcancer showed that breast cancers can be derived from a few tumor initiating cells orcancer stem cells. Increasing evidence supports the notion that cancer stem cells arehighly aggressive and resistant to conventional therapies, leading to the progressionof breast cancer. Therefore, understanding the molecular mechanisms of differentialregulation of the apoptitic signaling pathway in normal mammary epithelial cells,breast cancer stem cells, and breast cancer cells representing different stages of thedisease should allow for the development of novel therapeutic approaches target-ing dysfunctional apoptotic signaling pathways in breast cancer cells and/or cancerstem cells.

Keywords Apoptosis resistance · Breast cancer stem cells · Molecular targetedtherapy

Introduction

Apoptosis is a highly regulated, energy-dependent programmed cell death in whichthe cell activates a signaling cascade that leads to cell death without triggering aninflammatory response. Apoptosis plays a critical and natural physiological role intissue homeostasis as well as in elimination of abnormal cells that are superfluous,diseased or otherwise had served their useful purpose. Apoptosis can be initiatedby a variety of stimuli, including developmental signals, cellular stress and dis-ruption of cell cycle. In contrast, execution of apoptosis is a relatively unformedsignal process, involving characteristic morphological and biochemical changes.The morphologic hallmarks include membrane blebbing, cell shrinkage, chromatin

P. Karna (B)Departments of Surgery and Radiology, Winship Cancer Institute, Emory University School ofMedicine, Atlanta, GA 30322, USAe-mail: [email protected]

G.G. Chen, P.B.S. Lai (eds.), Apoptosis in Carcinogenesis and Chemotherapy,DOI 10.1007/978-1-4020-9597-9 1, C© Springer Science+Business Media B.V. 2009

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2 P. Karna and L. Yang

condensation and DNA fragmentation (Kerr et al., 1972). A number of the keyfactors involved in the regulation, coordination and execution of these events havebeen identified (Deveraux and Reed, 1999; Igney and Krammer, 2002; Okada andMak, 2004; Peter and Krammer, 2003; Stroh and Schulze-Osthoff, 1998).

Apoptosis plays a major role in various stages of normal breast development,including the formation of the intraductal lumen during morphogenesis of breastducts, at the end of the menstrual cycle, and in the involution of mammary glandsafter cessation of lactation (Anderson, 1999; Debnath et al., 2002; Hahm andDavidson, 1998). For example, mammary epithelial cells proliferate to developadditional ductal branching and lobuloalveolar growth in mammary ductal glandsduring pregnancy. After lactation, breast ducts undergo an involution stage withmassive apoptosis and reconstruction of the ducts leading to the return of the pri-mary breast duct structure (Hahm and Davidson, 1998). Apoptosis also occurs in thelobular unit of terminal duct with proliferation of the gland epithelial cells severaldays before the menstrual cycle and a peak in apoptosis close to the end of thecycle (Anderson, 1999). During these processes, cell proliferation and apoptosisis highly regulated to maintain structural and functional characteristics of normalbreast ductal glands.

Based on the established interactions among the known mediators of apoptosis,two classic pathways of apoptotic signaling in mammalian cells have emerged. Thefirst one is extrinsic pathway, which involves signaling by interaction of apoptoticinducing ligands with their cell surface death receptors, which then activates thecaspase cascade through adapter molecules. The second intrinsic pathway is medi-ated by mitochondria, which is initiated by the withdrawal of growth factors andtreatment with some chemotherapy drugs. The activation of this pathway is initiatedby the release of cytochrome c from mitochondria, activation of Apaf-1 and trig-gering of the activation of caspases. Increasing evidence shows that the apoptosisis a dynamic process that is tightly regulated by several signal pathways. It is clearthat activation of caspases, dynamic changes in the location and levels of the Bcl-2family of proteins, levels of the inhibitor of apoptosis family (IAP) of proteins playkey roles in execution and regulation of the apoptotic cell death induced by bothpathways (Reed, 1998; Reed, 2000; Tamm et al., 1998).

Development of drugs that target and directly switch on the cell death machin-ery in tumors constitutes a novel way of cancer therapy. Induction of apoptosis bychemotherapy drugs, radiation and blocking growth factor signaling pathways hasbeen used for the treatment of breast cancer. However, it is known that some breastcancers are highly resistant to conventional therapy. The evasion of apoptosis is acritical component of oncogenic transformation and resistance to chemotherapy. Todevelop the novel therapeutic approaches for the apoptosis resistant breast cancercells, it is crucial to understand the regulation of the apoptotic pathways and themolecular mechanisms of resistance to apoptosis in these breast cancer cells.

Many of the chemotherapeutic drugs target cancer cells at multiple molecularand cellular levels. Cancer cells may escape from apoptosis in response to chemo-and radiotherapy, by downregulation of the death receptor pathway, over-expressinganti-apoptotic Bcl-2 protein family, and upregulation of the anti-apoptotic factors(Cory and Adams, 2002; Deveraux and Reed, 1999; Lowe and Lin, 2000; Peter

1 Apoptotic Signaling Pathway and Resistance to Apoptosis 3

and Krammer, 2003). Moreover, the development of breast cancer is a multistageprocess involving various genetic alternations and cellular abnormalities that pro-vide advantages for the growth and progression of tumors. Defects in the apoptoticsignaling pathway not only promote the progression of breast cancer from ductalcarcinoma in situ (DCIS) to invasive and then to metastatic stage, but also reducesensitivity of the cancer cells to commonly used chemotherapy drugs, hormonaltherapy and growth factor receptor inhibitors or blocking antibodies. At present,several mechanisms are found to be responsible for chemoresistance in cancer cells,such as modification of drug-target interactions, decreased uptake or increased elim-ination of active molecule, defects in the apoptosis, and dysfunction in other celldeath pathways including necrosis and autophagy. Presently, resistance to apoptosisis recognized as one of the major problems for cancer therapy. The effects of ther-apy on genetically unstable, rapidly dividing groups of tumor cells usually leads toonly a temporary relief of the tumor burden, because this is usually followed by theoutgrowth of a subpopulation of cells that carry advantageous genetic mutations oralternations that make them non-responsive to therapy. Loss of ability to undergocell death might be one of the key factors in the selection leading to treatmentresistance. This review outlines the basic pathways of apoptosis, and discussesmechanisms of apoptosis resistance, the concept of cancer stem cells and theirrole in resistance to treatment. Finally, potential approaches for targeting apoptosisresistant cancer stem cells are described.

Apoptotic Signal Pathway

During the last decade, many cellular factors involved in apoptosis have been iden-tified and their roles in apoptotic signaling have been elucidated. Apoptosis isinitiated when the cells receive negative signaling, and proceeds through an extrinsic(death receptor pathway) or an intrinsic (mitochondria-mediated pathway) pathway(Reed, 2000). The extrinsic pathway is triggered by ligation of cell surface deathreceptors with their specific ligands, whereas the intrinsic pathway is set off whenthe cells are under severe stress and is characterized by leakage of cytochrome cfrom mitochondria. There is also some evidence implying a crosstalk between celldeath receptors and mitochondrial pathways under certain conditions. These distinctpathways converge with the activation of the caspase cascade, but their relative con-tribution is not fully understood. Recent studies reveal a close cooperation betweenboth pathways in maintaining homeostasis, preventing autoimmunity and terminat-ing an immune response by using mice deficient for both Fas and Bim (Hugheset al., 2008; Hutcheson et al., 2008).

The Extrinsic Pathway

The extrinsic pathway is a receptor-mediated and regulated by the members of tumornecrosis factor (TNF) receptor superfamily namely, Fas (CD 95) and TNF-relatedapoptosis inducing ligand (TRAIL) receptors. The binding of the correspondingligands results in receptor trimerization and clustering of the receptor death domains.


The cytosolic domains of the death receptors form a death-inducing signaling com-plex (DISC) that links up with the adaptor molecule, Fas-associated death domain(FADD) or TNFR-associated death domain (TRADD). The DISC–FADD/TRADDcomplex, binds to the initiator caspases 8 and 10 through its death effector domains(DED, D4/D5), thereby causing the autocatalytic cleavage of procaspase-8 and acti-vation of downstream executioner caspases (Hengartner, 2000; Krammer, 2000;Thorburn, 2004).

The ligands activating the death receptor pathways include TNFα, Fas ligand,and TRAIL. TNFα is a cytokine produced by macrophages/monocytes during acuteinflammation. It regulates inflammation, survival, proliferation and apoptosis ofcells. Tumor necrosis factor receptor 1 (TNFR-1) induces both death and survivalsignals. TNFα binds to TNFR-1 or TNFR-2 and trimerizes the receptors, resultingin formation of a complex between FADD and procaspases 8/10 to activate apop-totic signals (Basile et al., 2001; Chinnaiyan et al., 1996; Sheikh and Huang, 2003).However, depending on the type of stimulus, TRADD may stimulate NF-κB activity,leading to the recruitment of TNF receptor associated factors 1 and 2 (TRAF1/2),ribosome interacting protein and c-IAP1, which interact with anti-apoptotic proteinsto prevent apoptosis. Since the level of TRAF2 is elevated in numerous tumors, thismay cause the formation of TNFR, TRADD and TRAF2 complex and activate thecell survival pathway leading to apoptosis resistant tumors.

Fas (APO-1 or CD95), a member of the TNF superfamily, is a widely expressedtransmembrane protein in cell membranes of normal and malignant cells. Fas/CD95receptor/ligand system is an important signaling pathway in the regulation of apop-tosis. Commonly used chemotherapeutic drugs may induce apoptosis by increasingFas expression. Four distinct TNF-related apoptosis inducing ligands (TRAIL-R1-4)have been identified on the surface of cells. TRAILs induce apoptosis in a variety oftransformed or tumor cells by binding to DR4 and DR5, leading to the recruitmentof adaptor proteins and activation of caspases 8, 9, 7 and 3 (Kim et al., 2000). Ithas been shown that TRAIL selectively induces apoptosis in a variety of tumorcells and transformed cells, but not in most normal cells, and therefore has garneredimmense interest as a promising agent for cancer therapy (Kim et al., 2000; Walczaket al., 1999). This selectivity could be due to a higher level of TRAIL receptorsin cancer cells compared to normal cells. Furthermore, TRAIL also interacts with‘decoy’ receptors, DcR1 and DcR2, which lack functional death domains and donot induce apoptosis, which may contribute a low sensitivity of normal cells toTRAIL-induced apoptosis (Kim et al., 2000).

The Intrinsic Pathway

In this pathway, the mitochondria play a central role in the integration and execu-tion of a wide variety of apoptotic signals (e.g., loss of growth factors, hypoxia,oxidative stress or DNA damage) and provide the energy required for execution ofthe apoptotic program and release of pro-apoptotic proteins such as cytochrome c,endonuclease G and apoptosis-inducing factor. The Bcl-2 family of proteins plays


critical roles in regulating the intrinsic pathway. Activated pro-apoptotic Bcl-2 pro-teins attach to the mitochondrial outer membrane to form conducting channels,allowing cytochrome c to translocate from the intermembrane matrix into the cyto-plasm. In the cytosol, cytochrome c combines with ATP, Apaf-1 and procaspase 9 toform an apoptosome, which activates caspase 9 and subsequently caspase 3 leadingto cell death. The mitochondrial permeability transition pore (PTP) and Bax playimportant roles in this process (Crompton, 1999; Reed and Kroemer, 2000).

Caspase Activation

The caspases are cysteine–aspartic acid-proteases that cleave proteins with specificamino acid sequences. Caspases are synthesized as inactive precursors (procas-pases), which upon proteolytic cleavage to become activated caspases to cleavevarious protein substrates. Of the 14 caspases identified in humans, two-thirds playan important role in apoptosis. Caspases are grouped into initiators or effectors ofapoptosis, depending on their point of entry into the apoptotic pathway. The initiatorcaspases 2, 8, 9 and 10 are activated by proximity-induced dimerization, and they inturn proteolytically cleave the inactive pro-forms of the effector caspases 3 and 7.The effector caspases in turn sequentially cleave protein substrates within the cell,which results in apoptosis (Boatright and Salvesen, 2003; Hengartner, 2000; Motadiet al., 2007).

The p53 Pathway

In addition to extracellular signals, cells can undergo apoptosis in response tointernal signals including genetic abnormality. Defective or inappropriate cell cycleprogression caused by a variety of genotoxic injuries can result in cell cycle arrest,and subsequent induction of apoptosis. The tumor suppressor gene p53 has beenclearly linked to these pathways leading to its designation as the ‘guardian of thegenome’. DNA strand breaks induce rapid p53 upregulation. The upregulation ofp53 is mostly post-transcriptional, involving both, an increase in translation andprolonged half-life (Sherr and McCormick, 2002). Increase in Bax transcription maybe in part responsible for p53-induced apoptosis following DNA damage (Chipuket al., 2004; Miyashita and Reed, 1995). The fact that Bcl-2 acts downstream of p53signal points out that there are additional, parallel and p53-independent pathwaysregulating DNA damage induced apoptosis in mammalian cells.

Mechanisms of Resistance to Apoptosis

Investigations into the mechanisms of emergence of the chemoresistant phenotypeshave involved traditional studies using unicellular models exposed to incrementaldoses of chemotherapeutic agent, with consequent selection for resistant clones andcell populations. Such studies have led to the identification and characterization of


many resistance mechanisms, such as decreased drug uptake, increased drug extru-sion, alterations in the drug target, alterations in drug metabolism, repair of DNAdamage, alteration of cell cycle checkpoint control, and changes in downstreammediators of apoptosis. Recent advances in molecular analysis of the apoptoticsignal pathways have revealed some dysfunctional apoptotic signals that confer alow sensitivity to apoptosis and therapeutic agents. The following are a list of somerepresentative changes in the apoptotic signal pathways.

Death Receptors

Because of the physiological role of death receptors in normal cells, downregula-tion or loss of death receptors contributes to a malignant phenotype. Death receptoractivated apoptosis is negatively regulated by cellular FLICE-like inhibitory pro-tein (c-FLIP). c-FLIP is structurally related to caspase 8 and can bind to FADD,but lacks enzymatic activity. It thus prevents apoptosis by blocking association ofcaspase 8 with the DISC. Downregulation of c-FLIP renders cells sensitive to allknown death receptors-mediated cell death including Fas, TNF-R and TRAIL-Rs.PS-341, a proteasome inhibitor, can induce a substantial reduction in c-FLIP, and hasbeen successfully combined with the death ligand TRAIL to promote tumor cells’apoptosis synergistically (Sayers et al., 2003). Recombinant TRAIL alone or incombination with chemotherapy resulted in sensitization of p53 wild type, mutant,and null cell lines to TRAIL-mediated apoptosis by downregulating expression ofc-FLIP (Galligan et al., 2005).

Further investigations indicated that human cancers have developed mechanismsto avoid Fas-mediated apoptosis since many tumor cells are shown to be resistant toFasL or Fas antibody induced apoptosis (Barnhart et al., 2004). Somatic deletionsand mutations of Fas receptor were identified in several types of human cancers(Beltinger et al., 1998; Boldrini et al., 2002; Landowski et al., 2001). In addition,some tumor cells produce a high level of soluble Fas to block interactions betweencell surface Fas receptor and FasL (Lee et al., 1999; Liu et al., 2002). A reduced levelof expression of cell surface Fas receptor is common in many tumor types, includ-ing breast cancer, due to downregulation of Fas gene expression or decreased cellsurface transportation (Bullani et al., 2002; Mullauer et al., 2000; Viard-Leveugleet al., 2003). However, increasing evidence shows that downregulation of Fas maybe a cause for resistance to apoptosis only in a small percentage of human tumorssince many tumor cells that are resistant to Fas-mediated apoptosis do not carry Fasmutations and also exhibit an adequate level of Fas expression (Elnemr et al., 2001;Muschen et al., 2001). In human normal and breast cancer cell lines and tissues, Fasand FasL are co-expressed in normal mammary epithelial and breast cancer cells.FasL is weakly expressed in normal breast ductal cells but is strongly expressedin most breast cancer cell lines and tissues. However, Fas receptor is found to beabundant in normal breast epithelial cells but is low in breast cancer cells with het-erogeneous expression levels ranging from weak to strong (Mullauer et al., 2000). Alow sensitivity to death receptor mediated apoptosis in the presence of a high level


of FasL and a moderate level of Fas receptor suggests that breast cancer cells mayhave developed anti-apoptotic mechanisms downstream of the death receptor acti-vation, which block apoptotic signaling pathway. Co-expression of Fas and FasL intumor cells that are resistant to Fas-mediated apoptosis supports the notion that thepresence of downstream inhibitory factors that block the apoptotic signal pathway(Abrams, 2005; Mullauer et al., 2000; Yang et al., 2003a).

TRAIL-induced apoptosis has a significant clinical potential. However, differ-ent human tumors have a wide range of sensitivities to TRAIL-mediated apoptosisand some human tumor cells display a low level of TRAIL expression or activity(Ibrahim et al., 2001). It has been shown that most human breast cancer cells arehighly resistant to TRAIL treatment (Bockbrader et al., 2005; Singh et al., 2003).Some tumor cells have completely lost the expression of TRAIL receptor. Further-more, some TRAIL resistant cells express high levels of both TRAIL receptor andligand (Jin et al., 2004; Singh et al., 2003). It has been shown that over-expressionof Bcl-2 blocked activation of TRAIL-mediated apoptosis by inhibiting the releaseof mitochondrial cytochrome c and the cleavage of caspase-7 (Sun et al., 2001).However, treating TRAIL resistant tumor cells with subtoxic concentrations ofchemotherapeutic drugs sensitizes them to apoptosis (Bockbrader et al., 2005;Odoux et al., 2002; Shankar and Srivastava, 2004; Singh et al., 2003).

Caspases

Resistance of cancer cells to apoptosis may also be due to failure of initiator cas-pases to activate the caspase cascade. Deficiency in the levels of expression ofprocaspase genes was detected in some tumors. In one instance, deletion or silenc-ing of the caspase 8 gene was discovered in neuroblastoma and non-small cell lungcancer (Hopkins-Donaldson et al., 2003; Iolascon et al., 2003; Teitz et al., 2002).Downregulation of the initiator caspase 8 may be responsible for resistance toapoptotic signaling. Silencing of caspase 8 expression by DNA methylation incancer cells correlated with resistance to rhTRAIL (Eramo et al., 2005; van Noe-sel et al., 2003). Suppression of caspase 8 expression was shown to occur duringthe development of neuroblastoma metastases in vivo, and reconstitution of cas-pase 8 expression in deficient neuroblastoma cells suppressed metastases formation(Iolascon et al., 2003).

Deficiency in caspase 3 was also found in human breast cancer as well as inseveral other tumor types (Fujikawa et al., 2000; Iolascon et al., 2003; Kolenkoet al., 1999). It was also demonstrated that despite the absence of caspase 3 inbreast cancer cells, the apoptotic pathway was able to proceed via sequential acti-vation of caspase 9 followed by that of caspases 7 and 6, and cells exhibited allthe morphological changes associated with apoptosis (Liang et al., 2001). It is nowgenerally agreed that the presence of processed or active caspase 3 does not alwayscoincide with the presence of cleaved substrates and apoptosis since downstreamcaspase inhibitors in the apoptotic pathway that are upregulated in human cancercells can block the apoptotic process (Yang et al., 2003a). Examination of levels


of pro-apoptotic and/or active caspases in breast carcinoma tissues from 440 breastcancer patients at different stages of the disease showed high levels of procaspasesand/or active caspases in most human breast cancer tissues. A high level of pro-caspase 3 expression is found in 58% of DCIS and 90% of invasive breast cancertissues (Bodis et al., 1996). A strong expression of procaspase-3, 6 and 8 is sig-nificantly associated with the extent of apoptosis and high grade DCIS lesions.A strong positive correlation was found between active caspase 3, 6 and XIAP,suggesting the presence of a negative feed back in breast cancer tissues. Resultsof paired or non-paired normal and breast cancer tissues by Western blot analy-sis also demonstrated the presence of active caspase 3 fragments in most breastcancer but not in normal breast tissues. Coincidentally, the breast cancer tissueswith active caspase 3 fragments also express a high level of another IAP protein,survivin (Yang et al., 2003a). At present, the significance of co-existing active cas-pase and IAP proteins in breast cancer tissues is still under investigation. Detectionof active caspases 3 and 6 indicates that the effectors upstream of the apoptoticsignaling pathway are functional and the apoptotic pathway is activated in breastcancer tissues. It is possible that abnormalities generated by genetic changes inbreast cancer cells activate the apoptotic signaling pathway and induce apoptosis inthe majority of the cancer cells. The cells that have developed apoptotic resistance,such as those expressing high levels of IAP proteins XIAP and survivin, are able toblock the caspase activity and grow into tumor masses that are highly resistant toapoptosis.

IAP Family of Proteins

IAPs are a family of proteins containing one or more conserved, cysteine andhistidine-rich baculoviral IAP repeat (BIR) N-terminal domains and a C-terminalRING domain. Members of the IAP family of proteins, including NAIP, XIAP,c-IAP1, c-IAP2, survivin, Livin and Ts-IAP, have been identified and their roles ininhibiting caspase activity have been elucidated (Deveraux and Reed, 1999; Royet al., 1997). The BIR domains of the IAPs form the zinc-figure-like structuresthat bind to active caspases to block caspase activity. The RING domain acts asan ubiquitin ligase to facilitate proteasomal degradation of caspases as well as reg-ulation of the IAP themselves (Morizane et al., 2005; Suzuki et al., 2001; Vauxand Silke, 2005). Specific interactions of BIR domains with different caspases havebeen determined by studying the structures of caspases and IAPs. For example, theproximal link region of BIR2 of XIAP protein binds and blocks the active site ofcaspase-3 and -7 while the BIR3 domain binds and inhibits active caspase-9 (Huanget al., 2001).

XIAP is the most potent endogenous caspase inhibitor (Shin et al., 2003; Tammet al., 2003) and its upregulation is found in many breast cancer cell lines and tis-sues. The pro-apoptotic protein XAF1 binds to XIAP and releases active caspasesfrom XIAP inhibition and promotes degradation of another IAP family of protein,survivin (Arora et al., 2007; Liston et al., 2001). Several studies demonstrated the


association of the a high level of XIAP with resistance to chemotherapy in humancaners (Amantana et al., 2004; Cheng et al., 2002; Notarbartolo et al., 2004; Yanget al., 2003b).

Unlike other IAPs, survivin is expressed broadly in embryonic and fetal tis-sues but is undetectable in normal adult differentiated tissues (Adida et al., 1998;Altieri, 2003). Survivin is a structurally unique member of the IAP family that actsas a suppressor of apoptosis and plays a central role in cell division. Althougha structural basis for a direct interaction between survivin and caspase-3 has notbeen defined, inhibition of caspase-3 and -7 activities has been demonstrated insurvivin protein (Shin et al., 2001). Increasing evidence suggests that survivin isclosely associated with mitochondria-dependent apoptosis. Downregulation of sur-vivin expression or function results in the activation of caspase-9 (Mesri et al., 2001;Yang et al., 2003b). It has also been shown that survivin associates with XIAPthrough the BIR domain to form a survivin-XIAP complex that promotes XIAPstability and synergistic inhibition of apoptosis (Dohi et al., 2004).

Survivin is overexpressed in most human tumor types including 70% of humanbreast cancer tissues but not in normal breast tissues and its high expression is asso-ciated with poor survival (Ambrosini et al., 1997; Li, 2003; Tanaka et al., 2000).Survivin counteracts apoptotic stimuli induced by Fas, Bax, caspases, and anticancerdrugs (Tamm et al., 1998). Therefore, Survivin may contribute significantly to thedevelopment and progression of cancer, and could become an effective therapeutictarget.

The Bcl-2 Family

The Bcl-2 family of genes encodes proteins that inhibit or promote apoptosisby regulating the mitochondrial pathway. This family of proteins shares up tofour conserved regions known as Bcl-2 homology (BH) domains. Pro-apoptoticproteins include Bax, Bak, Bad, and Bcl-xs, whereas Bcl-2 and Bcl-XL are anti-apoptotic (Cory and Adams, 2002; Reed, 1998). Overexpression of Bcl-2 protectscells from a variety of apoptotic stimuli, such as growth-factor withdrawal, expo-sure to chemotherapy agents or toxins, viral infection and inappropriate oncogeneexpression. A number of Bcl-2 related proteins have been subsequently identified(Krajewski et al., 1999; Reed, 1998) and characterized. The relative levels of pro-and anti-apoptotic Bcl-2 family of proteins have been suggested to function as a‘rheostat’ regulating the apoptotic threshold of the cell (Yang and Korsmeyer, 1996).The antagonistic function of some of the members of the family is at least in partexplained by their ability to form heterodimers. The anti-apoptotic proteins Bcl-XL

and Bcl-2 form heterodimers with pro-apoptotic Bax (Oltvai and Korsmeyer, 1994).An excess of Bax promotes cell death, but co-expression of Bcl-XL or Bcl-2 can neu-tralizes this effect. Interestingly, under certain circumstances, Bcl-2 and Bcl-XL aretargets of caspases, and cleavage of these proteins converts them from pro-survivalto pro-apoptotic molecules that are able to induce cytochrome c release from themitochondria (Cheng et al., 1997; Clem et al., 1998).


The pro-apoptotic members can be separated into two structurally distinct sub-families: (1) The ‘multi-domain’ proteins (BAX and BAK) share three BH regionsand lack the BH4 domain; (2) ‘BH3-only’ proteins (Bnip3, Nix/Bnip3L, Bid, Noxa,Puma and Bad) share only the BH3 domain (Kelekar and Thompson, 1998). TheBH3-only proteins initiate apoptosis through the activation of Bax and Bak. Studiesusing knockout mice have demonstrated that Bax and Bak are essential for apoptosisinitiation via the intrinsic pathway. Cells lacking both Bax and Bak were completelyresistant to apoptosis stimuli, while the cells lacking only Bax or Bak were notresistant to these stimuli. This suggests a functional redundancy between Bax andBak (Tobiume, 2005). Under normal conditions, Bax is present in the cytosol. How-ever, in response to apoptotic stimuli, it undergoes a conformational change thattriggers its translocation to and insertion into the outer mitochondrial membraneleading to permeabilization of the outer mitochondrial membrane and release ofpro-apoptotic proteins. On the contrary, Bak is always localized to the mitochon-dria as an integral membrane protein and has been reported to be maintained in aninactive conformation by anti-apoptotic Bcl-2 family proteins (Cheng et al., 2003;Willis et al., 2005).

Interaction of pro- and anti-apoptotic Bcl-2 family of proteins in regulation ofthe apoptotic signaling pathway has been an intensive area of research. Althoughthe roles of the Bcl-2 family of proteins in this dynamic process have yet to beelucidated, it is known that anti-apoptotic Bcl-2 proteins interact with pro-apoptoticproteins such as Bax and Bak to regulate their activity. Once the apoptotic signal isreceived, BH3-only proteins bind to and neutralize the anti-apoptotic Bcl-2 proteins,thereby releasing BAX and BAK (Kelekar and Thompson, 1998). It has been shownthat overexpression of Bcl-2 or Bcl-XL sequester BH3-only proteins and preventsBax translocation and activation (Cheng et al., 2001; Van Laethem et al., 2004).

There is an ever increasing body of literature showing that a high level ofBcl-2 not only promotes resistance to apoptosis but also increases the recurrencerate and enhances chemo- and radio-resistance in many types of human cancers(Miyake et al., 1998, 1999). However, although Bcl-2 protein is detected in over80% of breast cancers, the level of Bcl-2 expression is correlated well with thepresence of estrogen- and progesterone (PR)-receptor positive breast cancer cells(Gee et al., 1994; Krajewski et al., 1995; Villar et al., 2001). The level of its expres-sion is surprisingly associated with a good prognosis of the breast cancer patients(Krajewski et al., 1999; Schorr et al., 1999). On the other hand, the presence ofhigh levels of apoptotic cells in the breast cancer tissues for the same group ofpatients is an indication of poor prognosis factors including larger tumor sizes, moreaggressive tumor types, high proliferation rates and lymph node metastases (Joensuuet al., 1994). Therefore, the role of Bcl-2 in regulating apoptosis in breast cancercells is still controversial. Results of recent studies show that Bcl-2 is expressedmostly in the luminal and low grade breast cancer type that are positive for estrogen-and PR- receptors, which are known to have a better prognosis compared to othersubtypes of breast cancers (Alsabeh et al., 1996; Megha et al., 2002; Meijnenet al., 2008). It is possible that differential regulation of the key apoptotic regulatorsin breast cancer cells that are initiated from cellular origins along different stages of


breast stem/progenitor cells contributes the apoptotic response in breast cancer cells.However, the mechanism of the differential role of Bcl-2 in regulating apoptosis indifferent subtypes of breast cancer cells, such as basal and luminal types, has yet tobe determined.

Cancer Stem Cells

Identification of Cancer Stem Cells

Cancers originally develop from normal cells by accumulating oncogenic mutationsand gaining the ability to proliferate aberrantly, and eventually turning to malignant.The concept of initiation of many tumor types from transformed adult stem cellshas been around for a long time (Reya et al., 2001). Stem cells may be preferentialtargets of initial oncogenic mutations because in most cancer originating tissues,they are the only long-lived populations and are therefore exposed to more genotoxicstresses than their shorter-lived, differentiated progeny (Pardal et al., 2003). Likenormal adult stem cells, cancer stem cells can divide indefinitely, giving rise to bothmore cancer stem cells and progeny that ultimately differentiate into different celltypes in a tumor.

The presence of breast cancer stem cells were firstly demonstrated in a cellpopulation isolated from breast cancer patients that strongly expresses CD44 butis negative or has a low level of CD24 (CD44+/CD24−). It has been shown thattumor xenografts can grow in mice that receive as little as 100 of CD44+/CD24−

cancer cells, while CD44−/CD24+ cells do not exhibit tumor growth, even at avery high cell numbers, leading to the assumption that CD44+/CD24− cells containbreast cancer stem cells (Al-Hajj et al., 2003). Examination of gene expression pro-files of CD44+/CD24− cells isolated from human normal breast ducts and breastcancer tissues shows that those cells are estrogen negative but express high levels ofgenes that are involved in cell motility, invasion, apoptosis, and extracellular matrixremodeling. Breast cancer patients with a high percentage of CD44+ cells have apoorer clinical outcome compared to the patients whose tumors mainly composedof CD24+ cancer cells (Shipitsin et al., 2007). However, extensive research on theCD44+/CD24− cell population in human breast cancer cells and tissues suggeststhat CD44+/CD24− cell population contains the stem-like/progenitor cells for breastcancer but it is not a precise biomarker for breast cancer stem cells. A recent reportfrom studying normal and cancer human mammary epithelial cells shows that breastcancer cells that have both CD44+/CD24− and aldehyde dehydrogenase activity(ALDH) biomarkers are highly tumorigenic and produce tumor xenografts in micewhen the number of injected cells is as little as 20 CD44+/ALDH+ cells (Ginestieret al., 2007). The level of expression of ALDH1 detected in 577 breast carcino-mas by immunostaining correlated with poor prognosis of the breast cancer patients(Ginestier et al., 2007).

Recently, several groups also reported identification of cancer stem cells frombrain, prostate, head and neck, liver, and pancreatic cancers. Results of these


studies show that cancer stem cells of each tumor type display distinct surfacebiomarkers, such as human leukemia (CD34+/CD38−) (Bonnet and Dick, 1997),breast (CD44+/CD24−/ESA−) (Al-Hajj et al., 2003), head and neck (CD44+),prostate (CD44+/alpha2beta1hi/CD133+) (Collins et al., 2005; Prince et al., 2007),brain and colon (CD133+) (O’Brien et al., 2007; Singh et al., 2004), pancreatic(CD24+/CD44+/ESA+) (Li et al., 2007) liver (CD90+) (Yang et al., 2008) and lungcancers (Sca1+/CD45−/Pecam−/CD34+) (Kim et al., 2005).

Apoptosis Resistance in Cancer Stem Cells

Increasing evidence shows that cancer stem cells are highly resistant to traditionalchemo- and radio-therapy. It has been shown that subpopulation of the cancer cellswith stem cell-like properties can be selected from drug-treated cancer cells (Eramoet al., 2006; Kang and Kang, 2007; Wei et al., 2006). Although high levels ofATP-binding cassette (ABC) drug transporters found in stem cells contribute, inpart, to drug resistance, several other factors also play roles in resistance to ther-apy such as higher levels of DNA repair and a lowered ability to enter apoptosis(Keshet et al., 2008; Lou and Dean, 2007). For example, cancer stem cells isolatedfrom primary glioblastoma Multiforme (GBM) are resistant to treatment of manydrugs. It seems that drug resistance in these cells is not the result of extrusion of thedrug by (ABC) transporters since a high concentration of fluorescent chemotherapydrug, doxorubicin, is found inside the nucleus of the cells. It is likely that intrin-sic anti-apoptotic function of the stem cells play a role in drug resistance (Eramoet al., 2006). Studies on the colorectal tumors indicate that apoptosis is lower inCD44+ tumor cells as compared to CD44− cells and this suppression of apoptosisin CD44+ cells is due to a highly activated Wnt pathway (Schulenburg et al., 2007).

At present, it is still unclear whether the apoptotic signal pathway in cancerstem/progenitor cells significantly differs from other cells and what are mecha-nisms conferring their insensitivity to apoptosis and becoming self-renewal andlong-lived cells. Therefore, understanding the mechanisms of apoptosis resistancein cancer stem cells should allow for the identification of molecular targets for thedevelopment of new therapeutic approaches targeting cancer stem cells for overcom-ing resistance to apoptosis. Result of a previous study suggests that breast cancerstem/progenitor cells have different control mechanisms of cell proliferation andapoptosis and mutation in p53 may further reduce sensitivity of the cancer stemcells to apoptosis. Immunohistochemical examination of human breast cancer tis-sues from pre-invasive DCIS to invasive ductal carcinoma cases showed that breastcancer cells with a stem cell phenotype were estrogen/PR negative, p53 positiveand Bcl-2 negative. In contrast, breast cancers with more differentiated luminalcell phenotype were estrogen/PR positive, p53 negative and Bcl-2 positive (Meghaet al., 2002).

Using dye-effluxing feature of stem cells, called side-population (SP), and CD55as markers, mammary tumor stem cells are isolated from two mammary carcinomacell lines and those cells are resistant to serum depletion- or ceramide-inducing

apoptosis in carcinogenesis and chemotherapy filegeorge g. chen · paul b.s. lai editors apoptosis...

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