fateme salehi a thesis submitted in conformity with the ......ii immunohistochemical expression of...

IMMUNOHISTOCHEMICAL EXPRESSION OF PITUITARY TUMOR TRANSFORMING

GENE IN PITUITARY AND BRAIN TUMORS

BY

Fateme Salehi

A thesis submitted in conformity with the requirements

for the degree of Master’s of Science

Graduate Department of Institute of Medical Science

University of Toronto

© Copyright by Fateme Salehi 2009

ii

Immunohistochemical Expression of Pituitary Tumor Transforming Gene In Pituitary And Brain

Tumors

Fateme Salehi

Master’s of Science, Institute of Medical Science

Faculty of Medicine, University of Toronto, 2009

Abstract

The purpose of this study was to investigate a) PTTG expression in pituitary adenoma subtypes,

b) the correlation between PTTG expression and clinico-pathological variables in patients with

Cushing’s disease with ACTH secreting pituitary adenomas, and c) PTTG expression in brain

tumour subtypes. PTTG expression was investigated in 89 pituitary and 88 brain tumours, and

54 ACTH adenomas of patients with Cushing’s disease. Our results show that PTTG is

expressed in the cytoplasm of pituitary adenoma cells, and at higher levels in GH adenomas, with

lower PTTG expression levels being exhibited by PRL adenomas. Significant differences were

noted between PTTG expression in medically treated and non-treated GH adenomas. However,

PTTG expression in ACTH adenomas did not correlate with clinico-pathological variables,

making it an unsuitable prognostic indicator in such tumours. In brain neoplasms, PTTG was

expressed at higher levels in malignant tumours, whereas in less aggressive tumours, PTTG

expression was considerably lower.

iii

Acknowledgments

I would like to thank my supervisors, Dr. Kovacs and Dr. Cusimano, for their guidance and

support. To Dr. Kovacs, I am forever indebted for your enthusiastic teaching, support,

contagious love for the pituitary gland, and for inspiring critical thinking.

To Dr. Cusimano, I am most grateful for your mentorship, and for inspiring me to always strive

to be better.

My gratitude goes to Dr. Anne Agur. Your kind support, encouragement and insights have been

instrumental to my progress.

My thanks also go to my current and past committee members Dr. Ng, Dr. Fornasier, and Dr.

Asa.

Special thanks to Dr. Scheithauer and Dr. Lloyd for their expert teaching and generous support.

I also would like to thank everyone at the Injury Prevention Research Office for providing a

stimulating and supportive environment. My special thanks to my good friends, David Cantelmi

and Safraz Mohammed. Thank you Nada Elfeki for all your great help.

Thank you also to the members of the Departments of Laboratory Medicine and Neurosurgery.

And last, but not least, I would like to thank my family for their unwavering support throughout

my endless years of study.

iv

Table of Contents

Page

Abstract ii

Acknowledgements iii

Table of Contents iv

List of Figures xiii

List of Tables xv

List of Abbreviations xvii

Chapter 1: Introduction 1

1.1 Introduction 1

Chapter 2: Literature review 3

2.1 The normal human pituitary gland 3

2.2 Adenohypophysial cytodifferentiation 4

2.3 The relevance of pituitary adenomas to normal pituitary cytodifferentiation 6

2.4 Pathogenesis of pituitary adenomas: the role of PTTG and other factors 7

v

2.5 PTTG functions and pituitary adenomas 9

2.5.1. PTTG in pituitary tumourigenesis: mouse models 9

2.5.2. PTTG and estrogen 11

2.5.3. PTTG and insulin 12

2.6 PTTG tumourigenic mechanisms as relevant to pituitary adenomas 13

2.6.1 Apoptosis and p53 13

2.6.1.1 Pituitary and p53 14

2.6.1.2 Pituitary and apoptosis 14

2.6.1.3 PTTG and p53-dependent and –independent apoptosis 16

2.6.2 Angiogenesis and pituitary adenomas 19

2.6.2.1 VEGF in pituitary adenomas 19

2.6.2.2 FGF in pituitary adenomas 21

2.6.2.3 PTTG and angiogenesis, VEGF and FGF 23

2.6.3 Genetic instability 24

2.6.3.1 Genetic instability and pituitary adenomas 24

vi

2.6.3.2 PTTG and genetic instability 25

2.6.4 Tumour invasion 25

2.6.4.1 Tumour invasion in pituitary adenomas 25

2.6.4.2 PTTG and Tumour invasiveness 27

2.7 PTTG and pituitary tumours: correlations with tumour subtype 28

2.7.1 PTTG in stem cells 30

2.8 PTTG as a clinico-pathological marker in ACTH-secreting pituitary adenomas 31

2.8.1 ACTH-secreting pituitary adenomas 31

2.8.2 PTTG, Cortisol and Cushing’s disease 32

2.8.3 PTTG as a clinico-pathological marker in neoplasms 33

2.8.4 PTTG clinico-pathological correlations in pituitary adenomas 33

2.9 PTTG cellular localization 35

2.10 The importance of PTTG as a potential targeted therapeutic 38

2.11 Highlights of PTTG association with molecular factors linked to pituitary

adenomas

39

Chapter 3: Immunohistochemical expression of PTTG in brain neoplasms 41

vii

3.1 Brain neoplasms 41

3.2 WHO classification of brain tumours 41

3.3 PTTG expression in the normal and the neoplastic brain 42

3.4 PTTG and cell proliferation in the CNS 44

3.5 Cellular localization of PTTG in the CNS 44

3.6 The role of PTTG in brain tumourigenesis 45

3.6.1 PTTG and p53-dependent and –independent apoptosis 45

3.6.2 46

3.6.3 PTTG and c-myc 46

3.6.4 PTTG and IGF-1 47

3.6.5 PTTG and genetic instability 47

3.6.6 PTTG and general tumourigenic pathways 47

3.6.7 PTTG as an immunohistochemical maker of tumour behaviour 48

Chapter 4: Objectives and Hypothesis 50

4.1 Rationale 50

4.1.1 Pituitary Tumors 50

viii

4.1.1 Brain Tumors 51

4. 2 Objectives 53

4.3 Hypothesis 53

Chapter 5: Materials and Methods 54

5.1 Materials 54

5.1.1 Pituitary adenomas 54

5.1.2 ACTH secreting pituitary adenomas of patients with Cushing’s

disease

54

5.1.3 Brain Tumours 54

5.2 Methods 55

5.2.1 Clinico-pathological data of patients with Cushing’s disease

caused by ACTH-secreting pituitary adenomas

55

5.2.2 Morphologic studies 55

5.2.2.1 Pituitary adenomas 55

5.2.2.2 ACTH secreting tumours of patients with

Cushing’s disease

56

5.2.2.3 Brain tumours 56

ix

5.2.3 Microscopy 56

5.2.3.1 Pituitary tumours 57

5.2.3.2 Brain tumours 57

5.2.4 Statistical analysis 59

Chapter 6: Results: Pituitary adenomas 61

6.1 PTTG cellular localization 61

6.2 Percentage of PTTG Immunopositive Cells 61

6.3 PTTG immunoexpression: Intensity of immunopositive cells 63

6.4 PTTG Histoscore 64

6.5 PTTG histoscore in medically treated ad untreated GH and PRL

adenomas

64

6.6 PTTG immunopositivity percentage-intensity scatter plots 64

6.7 Correlations between PTTG immunostaining and clinico-pathological

variables in patients with Cushing’s disease due to ACTH secreting

adenomas

65

6.8 Inter- and intra-observer reliability 69

Chapter 7: Results: Brain tumours 70

x

7.1 PTTG immunoexpression intensity in brain tumour subtypes 73

7.2 PTTG Cellular Localization 73

7.3 PTTG nuclear expression and tumour aggressiveness 75

7.4 Intra- and inter-observer variability 77

Chapter 8: Discussion: Pituitary tumours 78

8.1 PTTG immunostaining and clinico-pathological variables in ACTH-

secreting adenomas

78

8.2 PTTG expression in different adenomas subtype 78

8.3 Lowest and highest PTTG expression in PRL and GH adenomas

respectively

81

8.4 Medically Treated vs. Non-treated GH and PRL adenomas 83

8.5 PTTG Cellular Localization 88

8.6 Limitations 89

8.7 Summary and implications 90

Chapter 9: Discussion: Brain tumours 92

9.1 Discussion 92

xi

9.2 PTTG immunohistochemical expression is highest in medulloblastomas 92

9.3 Nuclear PTTG expression is higher in malignant tumours compared to

benign tumours

95

9.4 Lower PTTG immunoreactivity in pilocytic astrocytomas 97

9.5 Meningiomas 97

9.6 PTTG expression in schwannomas 98

9.7 PTTG expression in ependymomas 99

9.8 Lack of PTTG expression in hemangiopericytomas 88

9.9 Limitations 100

9.10 Summary and implications 100

Chapter 10: Conclusions 102

10.1 Conclusions: Pituitary adenomas 102

10.2 Conclusions: Brain tumours 102

Chapter 11: Future directions 104

References

105

xii

Appendix A: Pituitary tumour-transforming gene in endocrine and other

neoplasms: a review and update (Published manuscript)

120

Appendix B: Biomarkers of pituitary adenomas (Part II). (Under review for

publication in “Neurosurgery”)

143

xiii

List of Figures

Chapter 2 Page

Figure 2.1 Schematic overview of pituitary cytodifferentiation 5

Figure 2.2 Pituitary PTTG content correlates with gland plasticity and tumour

formation potential

10

Figure 2.3 PTTG molecular pathways in pituitary adenomas 40

Chapter 5

Figure 5.1 PTTG immunostaining intensity in pituitary adenomas 58

Figure 5.2 PTTG immunostaining intensity in brain tumours 59

Chapter 6

Figure 6.1 PTTG immunohistochemistry in pituitary adenomas 62

Figure 6.2 Percentage of PTTG immunopositivity in pituitary adenoma subtypes 63

Figure 6.3 Intensity of PTTG immunohistochemical staining in pituitary adenoma

subtypes

65

Figure 6.4 Intensity distribution of PTTG immunohistochemical staining by

intensity score.

66

Figure 6.5 Intensity score distribution by pituitary adenoma subtype 66

Figure 6.6 PTTG histoscores in pituitary adenomas 67

Figure 6.7 PTTG intensity, percentage and histoscore in GH and PRL adenomas 67

Figure 6.8 PTTG percentage-intensity scatter plots 68

Chapter 7

xiv

Figure 7.1 PTTG immunostaining in brain tumours 71

Figure 7.2 PTTG immunostaining intensity distribution in brain tumours 73

Figure 7.3 Nuclear and cytoplasmic PTTG expression in brain tumours 75

Figure 7.4 Nuclear and cytoplasmic PTTG expression in glial and non glial

tumours

76

Figure 7.5 Nuclear and cytoplasmic PTTG expression in glial and non glial

tumours

76

Figure 7.6 Increased PTTG immunoreactivity in glialversus non-glial tumours 77

Chapter 8

Figure 8.1 PTTG role in pituitary cytodifferentiation 82

Figure 8.2 PTTG association with molecular pathways in pituitary adenoma

subtypes

82

xv

List of Tables

Chapter 2 Page

Table 2.1 PTTG and p53 dependent and independent apoptosis 17

Table 2.2 VEGF expression in pituitary adenomas 20

Table 2.3 FGF and FGFR expression in pituitary adenomas 22

Table 2.4 MMP expression in pituitary adenomas 26

Table 2.5 PTTG cellular localization in cell lines 37

Table 2.6 PTTG cellular localization in normal and neoplastic pituitary tumours 38

Chapter 6

Table 6.1 Clinico-pathological correlations in patients with Cushing’s disease 69

Chapter 7

Table 7.1 Immunohistochemical expression of PTTG in brain tumours 72

Table 7.2 PTTG immunoreactivity in glial and non-glial brain tumours. 74

xvi

List of Abbreviations

3D 3 Dimensional

ACTH Adenocorticotropic hormone

Alpha-SU Alpha subunit of glycoprotein hormone

Alpha-GSU Alpha-glycoprotein subunit

Beta-SU Beta subunit of glycoprotein hormone

bFGF Basic fibroblast growth factor

BMP Bone morphogenic protein

BrdU Bromodeoxyuridine

CUTE Corticotroph upstream transcription element

Cox-2 Cyclooxygenase-2

CNS Central nervous system

c-myc c-myc protein

CDK Cyclin dependent kinase

ER-alpha Estrogen receptor alpha

EGF Epidermal growth factor

E2 Estradiol

ESCC Esophageal small cell carcinoma

EGFR Epidermal growth factor receptor

xvii

E Embryologic day

FGF Fibroblast growth factor

FSH Follicle stimulating hormone

Flk-1 Fetal liver kinase-1 (a VEGF receptor)

FGFR Fibroblast growth factor receptor

FGF-2 Fibroblast growth factor-2

FTC133 Human follicular cancer ell line

GH Growth hormone

GHF Growth hormone factor

GBM Glioblastoma multiforme

HUVEC Human umbilical vein endothelial cells

HNSCC Head and neck squamous cell carcinoma

HIF-alpha Hypoxia inducible factor-alpha

IGF-1 Insulin growth factor-1

ID3 Inibitor of DNA binding-3

IGFR-2 Insulin growth factor receptor-2

IGF-2 Insulin growth factor-2

Ki-67 Ki-67 protein (proliferation marker)

KDR Kinase insert domain receptor (VEGF receptor)

LH Luteinizing hormone

xviii

MMP-9 Matrix metaloproteinase-9




MAPK Mitogen-activated protein kinase

MAPKK Mitogen-activated protein kinase kinase

MEK1 Mitogen activated protein kinase kinase 1

NSCLC Non small cell lung carcinoma

PTTG Pituitary tumour transforming gene

PRL Prolactin

Pit-1 Pituitary-specific positive transcription factor 1

PTTG3 Pituitary tumour transforming gene-3

PNS Peripheral nervous system

PDGF Platelet derived growth factor

PTEN Phosphatase and tensin homolog

PI3K Phosphatidylinositol 3- and 4-kinase

Rb Retinoblastoma

Rb+/- Retinoblastoma heterozygous knockout

Rb +/- PTTG +/- Retinoblastoma heterozygous PTTG heterozygous knockout

Rb +/- PTTG -/- Retinoblastoma heterozygous PTTG homozygous knockout

xix

SU subunit

SF-1 Steroidogenic factor-1

siRNA Small interfering RNA

SCLC Small cell lung carcinoma

TSH Thyroid stimulating factor

TGF Transforming growth factor

TSP-1 Thrombospondin-1

TRßPV/PV Mutant thyroid hormone receptor kindred PV;

UV Ultraviolet

UCLA University of California at Los Angles

VEGF Vascular endothelial growth factor

Wnt Wnt protein

1

CHAPTER 1: INTRODUCTION

1.1 Introduction

Pituitary tumor transforming gene (PTTG) was first isolated from rat GH3 pituitary

adenoma cells by the Melmed group (1). Despite considerable studies of the role of PTTG in

pituitary tumorigenesis, its precise function in the pituitary gland remains unknown. Empirical

evidence suggests for PTTG to be involved in cell-specific functions and here, the evidence is

reviewed to provide a rationale for the present study.

PTTG has been shown to be overexpressed in most neoplasms studied to date, and its

expression has been shown to be closely correlated with several clinicopathological parameters

including patient survival, tumor recurrence, and metastasis (2-6). PTTG is the human analog of

yeast securin, and is integral to cell cycle control by regulating sister chromatid separation.

PTTG inhibits separase which cleaves sister chromatids. Since its discovery, PTTG has been

shown to be a proto-oncogene, promoting tumor formation in nude mice subcutaneously injected

with PTTG-expressing cells. PTTG also plays a role in several cellular processes including cell

cycle progression, chromosomal stability, apoptosis, DNA damage pathways, and angiogenesis

(7-11). PTTG induces transcription of several factors including c-myc, FGF as well as p53 and

is highly expressed in early tumorigenesis in estrogen-injected rats that harbor pituitary tumors

(2, 10, 12-14). The role of PTTG in promotion of pituitary tumorigenesis is well established

following several genetic mouse models of PTTG knockout and PTTG overexpression (15-19).

Whereas PTTG overexpression promotes pituitary tumorigenesis in Rb-/+ mice that are prone to

pituitary tumor formation, PTTG knockout in these Rb-/+ mice inhibits pituitary tumor

formation.

2

Several studies have examined the expression of PTTG in pituitary adenomas, but the

findings have been largely contradictory and inconsistent, with respect to PTTG nuclear versus

cytoplasmic expression, as well as PTTG expression in different pituitary adenoma cell types.

Whereas some investigators have found PTTG to be differentially expressed in adenoma cell

types, its levels being higher in GH adenomas, others have found no significant differences.

Additionally, some studies have reported solely nuclear PTTG, whereas others have

demonstrated only cytoplasmic or both nuclear and cytoplasmic PTTG expression.

3

CHAPTER TWO: LITERATURE REVIEW

2.1 The Normal Human Pituitary Gland

The pituitary gland is an endocrine organ consisting of two lobes, the adenohypophysis, also

known as the anterior pituitary, and the neurohypophysis, also known as the posterior pituitary.

Approximately 80% of the pituitary gland is comprised of the adenohypophysis, which contains

six distinct hormonal cell types that secrete growth hormone (GH), prolactin (PRL),

adenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), luteinizing hormone

(LH) and follicle stimulating hormone (FSH). The corresponding cells are as follows:

somatotroph cells (GH), lactotrophs (PRL), mammosomatotrophs (GH and PRL), corticotrophs

(ACTH), thyrotrophs (TSH), and goandotrophs (LH and FSH). It is of note that the three

glycoprotein hormones TSH, LH and FSH consist of two subunits (SU), alpha- and beta, with the

beta-SU being hormone-specific and alpha-SU being the same in all three subtypes. Hence the

alpha-SU is produced by TSH and LH/FSH cell types, whereas each subtype produces hormone-

specific beta-SU. The presence of a myriad of different cell types each producing different

hormones within a small endocrine organ is thought to be closely regulated by transcription and

other factors that act to stimulate and abrogate hormone expression in these cell types early in

development as well as later on in life (20). Thus, in any molecular study of the

adenohypophysis, it is critical to distinguish each cell type.

2.2 Adenohypophysial Cytodifferentiation

This section discusses the process of pituitary cell lineage differentiation that produces

the six cell types that contain and/or secrete GH, PRL, ACTH, TSH, and FSH./LH. As discussed

below, estrogen receptor (ER) is shown to play an important role in adenohypophysial

4

cytodifferentiation, providing grounds for suspicion of a role for PTTG in cytodifferentiation as

well, since PTTG is shown to be closely involved early on, during estrogen induced pituitary

tumorigenesis. The following paragraphs outline anterior pituitary cell differentiation with a

focus on the role of ER. Later sections show how ER and PTTG are closely associated in

pituitary tumorigenesis as well.

Studies of early pituitary development have shown that cell types producing specific

hormones arise in a highly regulated fashion (20) (Figure 2.1). Following early pituitary

development, Rathke’s pouch stem cells give rise to three main cell lineages, including the

differentiated corticotrophs and gonadotrophs, as well as the somatotroptroph stem cells that in

turn could differentiate further into somatotrophs, mammosomatotrophs, and thyrotrophs (Figure

2.1). Additionally, mamosomatrophs are thought to give rise to lactotrophs. The first cells to

differentiate are ACTH-containing cells at 6-7 weeks, followed by differentiation of GH

containing cells at 8 weeks. Although PRL-containing cells appear at 12 weeks, they also

contain GH and are bihormonal. Further differentiation is thought to give rise to PRL containing

cells at 24 weeks (20).

Growth hormone factor (GHF) (Pit-1) is selectively expressed in human pituitary gland

cells that produce GH, PRL, and beta-TSH. GH cell lineage appears earlier than that of PRL and

beta-TSH, indicating that the expression of latter requires other factors in addition to Pit-1 (21).

To date, factors that induce PRL expression in the Pit-1 expressing cells remain largely

unknown. One such factor is thought to be estrogen which is closely associated with PTTG.

Estrogen acts via its receptor, estrogen receptor-alpha (ER-alpha) on the PRL gene promoter

region. In normal

5

Figure 2.1. A schematic overview of human adenohypophysial cell differentiation. Oral ectoderm gives rise to

Rathke’s pouch stem cells which in turn give rise to corticotrophs, somatotrophs ad gonadotrophs. The

somatototroph stem cells further differentiate into 3 cell lineages under the influence of ER-alpha and other factors.

A “GH-repressor” is thought to inhibit GH expression by somatotrophs. Adapted from Asa et al (20). Copyright

2009. Karger.

pituitary cells, ER-alpha is localized by immunohistochemistry in PRL-cells producing and

gonadotropin/beta-SU-expressing cells as well as some GH-producing cells. It is thought that

GH cells immunopositive for ER-alpha are mammosomatotrophs existing in the pituitary, a

notion supported by the observation that GH-releasing cells that do not produce PRL are devoid

of ER-alpha (22, 23). Thus, evidence supports the role of estrogen in mediating PRL production

by somatotrophs expressing Pit-1. Clearly, factors that silence GH expression in PRL cell

lineage following transition from mammosomatotrophs to lactotrophs must also exist (20). It is

6

of note that pituitary tumor transforming gene (PTTG) is closely regulated by estrogen in

pituitary adenomas, providing a rationale for further investigation of the role and association of

PTTG and pituitary hormonal cell cytodifferentiation. PTTG is also shown to induce PRL

expression and suppress GH production, further supporting its involvement in PRL and GH cell

type differentiation (24).

2.3 The relevance of pituitary adenomas to normal pituitary cytodifferentiation

Pituitary adenomas comprise approximately 15-25% of all intracranial lesions (25).

Incidental pituitary adenomas are found in upto 27% of in autopsy material, indicating a high

prevalence of asymptomatic pituitary adenomas in the general population (26). Pituitary tumors,

although benign and rarely metastasizing, can be associated with high morbidity and if untreated,

mortality rates. Small microadenomas are often detected due to their excessive hormone

secretion which manifests with endocrine clinical symptoms. Non-functioning adenomas, which

do not secrete any hormones, are usually detected based on symptoms arising due to their mass

effects, including visual disturbance and headache secondary to the large tumor size.

Pituitary adenomas were thus classified based on their hormonal secretion as well as

histological and ultrastructural features by Kovacs and Horvath (27). Based on their

recommendations to the World Health Organization (WHO), pituitary adenomas are classified

into subtypes associated with cell lineage of origin (Table 2.1). Although most pituitary

adenomas are monoclonal in origin, arising from de novo somatic genetic changes in a single

pituitary cell, the clonal origin of pituitary adenomas is still debated (27). It is unclear whether

pituitary adenomas are monoclonal or polyclonal, arising from a cell population as a result of

trophic factors. Therefore, a complicating factor in any study of pituitary adenomas as a model

of hormonal cell differentiation remains the potential for inclusion of a heterogeneous cell

7

population not reflected in tumor histopathological features. As such, research on differential

expression of factors in distinct hormonal cell types will reveal insight into mechanisms affecting

cell differentiation in the pituitary gland. PTTG is one such factor whose differential expression

in different pituitary cell lines is supported by empirical evidence discussed in future sections.

2.4 Pathogenesis of pituitary adenomas

Pituitary adenomas are mostly monoclonal in origin, arising from cells with genetic

mutations (28). To date, factors initiating such genetic mutations and contributing to tumor

progression remain largely unknown. Generally, elements thought to play a role in pituitary

tumorigenesis are among growth factors and cytokines, cell cycle regulators, and elements of

cellular signaling pathways, including G proteins, FGF, TGF-alpha, p27, p21, p53 and PTTG.

Among growth factors and cytokines that play a role in pituitary tumorigenesis, a

mutation found in a small number of GH secreting adenomas, is that of the alpha subunit of the

G(s) protein (29, 30). G-proteins are intimately involved in signal transduction from cell

membrane to intracellular effectors (30). The G(s) protein mutation renders it constitutively

active (30, 31). Another rare mutation found primarily in pituitary carcinomas is seen in the

signaling protein Ras, which is also involved in tranduction of signals from cell membrane to

downstream effectors (32). Among growth factors with aberrant expression in pituitary

adenomas, epidermal growth factor (EGF) family and transforming growth factor (TGF)-alpha

are thought to play an important role in pituitary tumorigenic processes (32, 33). TGF-alpha

overexpression is implicated in formation of lactoroph adenomas (34). Moreover, fibroblast

growth factors (FGFs) and FGF receptors (FGFRs) are shown to mediate lactoroph adenoma

proliferation, and basic FGF (bFGF) is markedly elevated in patients with pituitary tumors (34,

35). An emerging factor important in the process of pituitary tumor development and

8

progression is PTTG which appears to be regulated by FGF and EGF (36, 37). PTTG has been

also closely associated with FGF in an autocrine loop, PTTG and FGF promoting their respective

levels in a loop (36).

Among regulators of cell cycle, p27, a cyclin-dependent kinase inhibitor is often found to

be downregulated in pituitary neoplasms. However, no mutations in the p27 gene have been

detected in pituitary tumors (38), pointing to post-translational modifications such as increased

degradation of p27. Expression levels of p21, another inhibitor of cyclin-dependent kinases and

cell cycle arrest factor, have been reported to be lower in non-functioning adenomas as compared

to functioning ones (39, 40). No mutations in the p21 protein have been detected thus far (38).

Although mutations of p53, an important factor in cell cycle arrest following DNA damage, have

not been detected in pituitary carcinomas (41), p53 expression has been linked to aggressive

tumor behaviour. Several studies have demonstrated a significant association between tumor

behaviour and p53 expression (42-44). Another cell cycle protein, recently implicated in

pituitary tumorigenesis is PTTG. PCR analysis of PTTG gene from 25 pituitary tumors (2 GH, 5

PRL, 2 ACTH, 2 TSH, 14 nonfunctioning) failed to show any insertions/deletions or specific

mutations (45). Thus, aberrant PTTG overexpression in pituitary adenomas may have other

causes, such as hypomethylation or may be a result of other epigenetic factors (45).

PTTG is yet the most recent factor implicated in pituitary tumorigenesis, and

interstingly, several lines of evidence support its role in pituitary cytodifefrentiation. It is well-

known that during neoplastic processes, elements of early cell differentiation and prolifereation

are overexpressed, and hence it would be reasonable for PTTG , which is overexpressed in

pituitary tumors, to be involved in early pituitary cytodifferentiation. Empirical support of this

notion is discussed in section 2.5

9

2.5 PTTG functions and pituitary adenomas

This section presents the evidence in the literature on the potential role of PTTG in

pituitary cytodifferentiation. First, the role of PTTG in pituitary tumor development and

progression is presented, as there is consensus that factors associated with neoplasia are also

implicated in early cell differentiation from progenitor cells. Further, the relationship between

PTTG and estrogen as well as insulin is discussed. Both estrogen and insulin are implicated in

pituitary adenoma tumorigenic processes in a cell-type specific fashion, and thus examination of

the evidence in the literature on the relationship between these factors and PTTG emphasizes the

importance of investigating PTTG expression in pituitary adenoma hormonal cell types.

2.5.1 PTTG in pituitary tumorigenesis: mouse models

Mouse models of pituitary tumors have contributed to our understanding of PTTG action

(Figure 2.2) (15, 17, 19, 46-50). In the Rb+/- mouse model, PTTG deficiency was protective for

pituitary tumor development, delaying and reducing their incidence through inhibition of cellular

proliferation (15)(Chesnokova et al. 2005). In a recent study, Chesnokova et al. demonstrated that

PTTG deficiency in mice is associated with pituitary cell senescence, thereby restraining

development of pituitary tumors (17). In keeping with the pro-tumorigenic role of PTTG in the

pituitary, pretumurous pituitary of Rb+/-PTTG+/+ mice had elevated PTTG levels when

compared to 2-4 month old wildtype mice (15).

10

PTTG targeted overexpression in gonadotrophs and thyrotrophs, using the alpha-GSU promoter,

was associated with focal hyperplasia and increased neoplasia (46). The hyperplastic and

neoplastic LH and TSH cells overexpressed PTTG. Surprisingly, using double immunostaining,

GH cells also expressed PTTG (46). Similarly, pituitary-targeted PTTG overexpression in the

Rb+/- also using the alpha-GSU promoter, resulted in focal pituitary hyperplasia. The incidence

of anterior pituitary tumor development was increased in alpha-GSU.PTTGxRb+/– mice when

compared to Rb+/- animals (46). These findings clearly support the possible role of PTTG in

Figure 2.2. Pituitary PTTG content correlates with gland plasticity and tumor formation potential. On the

left side of the inverted triangle are listed mouse models with descending pituitary PTTG content, with or

without the combination with tumorigenic Rb+/–. Horizontal bars represent observed effects of the different

genotypes on pituitary trophic status, which correlates with pituitary tumorigenic potential (arrow).

Copyright 2009. The Endocrine Society.

11

pituitary tumorigenesis (Figure 2.2). Factors associated with tumor initiation and progression are

classically associated with cell cytodifferentiation as well. Therefore, investigation of PTTG

expression in different types of pituitary tumors will provide more information on the association

between PTTG and adenohypophysial cytodifferentiation.

2.5.2 PTTG and Estrogen

A factor associated with early pituitary cytodifferentiation is estrogen, acting via its

receptor (ER-alpha) (23). Estrogen is thought to mediate PRL production by early cells that

initially only produce GH (somatotrophs), followed by production of both GH and PRL

(mammosomatotrophs). Interestingly, estrogen was shown to regulate PTTG expression within

the context of PRL adenoma development (51, 52). Estrogen increased PTTG levels concordant

with early lactotrophic hyperplastic response (36). In a dose- and time- dependent manner,

lactrotroph tumors induced by estrogen in Fischer rats showed PTTG expression within 24 hours

after estrogen treatment, reaching maximal levels after 48 hours (36). Estrogen induced bFGF,

which in turn promoted expression of PTTG (36). Moreover, PTTG mediated secretion of bFGF,

which augmented lactotroph cell proliferation and PRL secretion as well as angiogenesis (36). .

PTTG in turn is known to stimulate angiogenesis via VEGF and bFGF (2, 7, 24, 49, 53). Thus,

PTTG is an important regulatory element in PRL cell tumorigenesis, as it is expressed and

mediated early during PRL adenoma development (36). In a study by Yin et al, in 2 of the 4 rat

strains examined, estrogen increased pituitary PTTG mRNA levels in parallel with estrogen

induced pituitary tumor development (54). Further, estrogen induced PTTG transcription in

growth hormone (GH)/ prolactin (PRL) secreting GH3 cells and pituitary PTTG mRNA levels

both increased concomitantly with the proestrus serum estradiol surge (51). Estrogen regulation of

PTTG expression may be cell specific. For example, in U87 glioma cells, 17-beta estradiol does

12

not affect PTTG mRNA levels (55). Furthermore, PTTG is induced in oopherectomized rats

treated with estrogen in a strain-dependent manner similar to the ability of estradiol to induce

pituitary tumors in these rats, suggesting that PTTG expression may mediate estradiol-induced

pituitary tumorigenesis in a cell and organism specific manner (56). Most pituitary tumors,

especially PRL adenomas, express high levels of estrogen receptors as well as PTTG, and given

the association of PTTG with early PRL adenoma development, PTTG may play a role in early

pituitary cytodifferentiation. Whether PTTG is associated with cytodifferentiation of

mamosommatotrophs producing both PRL and GH may be clarified by studying PTTG expression

in GH and PRL adenomas.

2.5.3 PTTG and insulin

An effect of insulin and insulin growth factor (IGF-1) on PTTG expression has been

demonstrated in several cell lines (57, 58). For example, two insulin-responsive sequences have

been found in the PTTG promoter region, implicating insulin and IGF-1 regulation in PTTG

expression (59). Treatment of human glioma cell lines (LN405 and U87MG) with IGF-1 or

insulin resulted in PTTG protein expression via the PI3K and MAPK pathways (57). In addition,

in MCF-7 breast cancer cells, insulin and IGF-1 treatment each stimulated PTTG mRNA and/or

protein synthesis in a dose- and time-dependent fashion, peak PTTG mRNA levels being seen 48

hrs after insulin administration (58). IGF-1 plays an essential role in the negative feedback loop

effect of GH on somatotroph cells in the pituitary gland (60). It potently inhibits GH gene

transcription, by directly acting on the GH gene promoter (60). IGF also inhibits GH production

via abrogation of Pit-1 mRNA levels, a transcription factor that mediates pituitary specific GH

expression (60). Given the elevated levels of IGF-1 in patients with GH-producing adenomas,

and that PTTG is overexpressed in these neoplasms, it is important to investigate PTTG

13

expression in pituitary hormonal cell types to unveil any associations between PTTG and cell-

specific hormone production and/or secretion. The literature findings of associations between

PTTG and insulin, as well as between IGF-1 and GH producing cells support the notion that

PTTG must be involved in cellular mechanisms in a cell-specific fashion, and make it a suitable

candidate for involvement in adenohypophysial differentiation.

2.6 PTTG tumorigenic mechanisms as relevant to pituitary adenomas

Although several neoplastic processes have been identified to play a role in pituitary

tumor development and progression, whether such mechanisms are cell-specific have not been

explored in detail. This is an important factor to consider in any study of pituitary adenomas, as

the adenohypophysis contains six distinct cell types that are surely regulated uniquely. Thus, this

section highlights the findings of literature on tumorigenic processes implicated in pituitary

adenomas, underlining any cell-type specific findings. In parallel sections, the connection

between PTTG and each of such neoplastic processes implicated in pituitary adenomas is

explored, demonstrating that PTTG is central to all tumorigenic mechanisms identified to date,

and highlighting the need for investigation of cell-specific expression of PTTG. The latter notion

would then guide research on how different pituitary cell types may involve specific tumorigenic

processes, including apoptosis, angiogenesis, genetic instability, and tumor invasiveness.

2.6.1 Apoptosis and p53

This section first examines the role of p53 (2.6.1.1) and apoptosis (2.6.1.2) in the

neoplastic pituitary and then shows the evidence of PTTG association with p53-dependent and –

independent apoptosis (2.6.1.3).

14

2.6.1.1 Pituitary and p53

P53 expression has been linked to aggressive tumor behaviour. Thapar et al.

demonstrated a significant association between tumor behaviour and p53 expression, labeling of

0%, 15.2% and 100% being seen in non-invasive and invasive adenomas and carcinomas,

respectively (p<0.001) (42). With respect to pituitary adenoma subtypes, p53 immunoreactivity

was found in one PRL, one GH, one ACTH, and one null cell adenoma, as well as 4 PRL and 3

ACTH carcinomas, no correlations being noted between p53 expression and cell type.

Recently, Wierinckx et al. reported a significantly higher p53 and nuclear PTTG

expression in “aggressive-invasive” tumors as compared to those with less aggressive behaviour

(p<0.0001) (43). Studying 41 pituitary tumors, Ozer et al. showed that elevated p53 expression

was an independent indicator of local relapse (p=0.002), thus suggesting that p53 status is

associated with tumor progression (44). Interestingly, expression of the anti-apoptotic Bcl-2

was significantly increased in GH adenomas and decreased in PRL adenomas (44). This finding

is of significance, as PTTG induces gene expression of the pro-apoptotic factor bax, which

counteracts Bcl-2 effects (10). Whether PTTG is involved in p53-dependent apoptosis and/or its

downstream pathways may be determined by investigating the expression of PTTG in different

pituitary adenoma cell types, since evidence indicates that such pathways may be cell-type

dependent.

2.6.1.2 Pituitary and apoptosis

It is of note that the role of p53-mediated apoptosis in early pituitary differentiation has

been recently established (61), but studies of apoptosis in pituitary tumors are limited in number.

Vidal et al, examining over 8000 pituitary tumor biopsies, reported that most apoptotic activity

15

was observed in corticotroph adenomas, only occasional examples being seen in PRL or

gonadotroph adenomas (62). Several studies have investigated the relevance of apoptotic

activity as a clinicopathologic marker (63, 64). Kontogeorgos et al noted higher apoptotic

activity in aggressive, drug-resistant adenomas, indicating that apoptosis may be a useful

prognostic marker (65). Similar findings were reported by Kulig et al (64) who observed a

fourfold increase in apoptotic activity in pituitary carcinomas as compared to adenomas .

Correspondingly, lower expression of Bcl-2 anti-apoptotic factor, was seen in pituitary

carcinomas as compared to adenomas and the non-tumoral pituitary gland (64). On the other

hand, Ibrahim et al. found that apoptotic indices were not predictive of the growth rate of non-

functioning pituitary tumors (63). These findings echo those of Nakabayashi et al, who in a

series of 48 pituitary adenomas, found no significant difference between apoptotic indices in

recurring and non-recurring adenomas (66). Lastly, in a series of 51 pituitary adenomas, Losa et

al. found no significant difference between the apoptotic indices of ACTH macroadenomas and

microadenomas (67).

With respect to the functional status of pituitary tumor, Kontogoergos et al. (65) showed

that hormone secreting adenomas had higher indices than did non-functioning tumors; highest

apoptotic indices were observed in TSH adenomas, followed by GH, PRL, and mixed GH/PRL

adenomas (65). Similarly, Sambaziotis et al found that functioning adenomas exhibit higher

apoptotic indices than ones non-functioning (68). In contrast, Green et al. found a higher

apoptotic index in nonfunctioning tumors compared to GH adenomas (33% vs. 11%), a

difference not statistically significant (69).

The frequency and significance of p53 expression and apoptosis and the interdependence

of the two in different pituitary adenoma subtypes remain unclear. PTTG is known to be

16

associated in both inhibition and stimulation of p53 and its functions, including p53-dependent

apoptosis. Given the elevated expression of PTTG in pituitary adenomas, examination of PTTG

expression patterns in these tumors will provide more insight into the apoptotic process and p53

expression, which may be dependent on PTTG levels rather than expression.

2.6.1.3 PTTG and P53-dependent and –independent apoptosis

Evidence supports a link between PTTG with that of p53 (Table 2.3) (9, 10, 13, 70-72).

The above findings of variable apoptotic indices among pituitary tumor cell types is relevant to

PTTG expression in pituitary adenoma subtypes, as PTTG has been shown to both stimulate and

abrogate p53-dependent apoptosis and/or the expression of p53 and bax in several in vitro studies

(10) (Table 2.3). A clear picture of PTTG expression profile in pituitary adenoma cell types may

provide a map of potential cell-specific mechanisms involved associated with PTTG and p53-

dependent and/or independent mechanism. For example, the finding of parallel overexpression

of PTTG and apoptosis in pituitary adenoma cell types would further implicate PTTG in pituitary

cell apoptosis.

17

Table 2.1 PTTG correlations with p53

A. Studies showing PTTG has no effect on p53

Study Cell Line System PTTG P53 Apoptosis

Mu et al, 2003 (73) A549 1 PTTG OX ↑ NC -

Mu et al, 2003 (73) HeLa 2 PTTG OX ↑ NC -

B. Studies showing PTTG upregulates p53


Yu et al, 2000 (71) MG-63 (p53 -/-) 3 PTTG OX ↑ P53-/- NC

Yu et al, 2000 (71) MCF-7 (p53+/+) 4 PTTG OX ↑ ↑ ↑

Yu et al, 2006 (74) MIN6 5 PTTG OX ↑ - ↑

Hamid et al, 2004 (10, 74) HEK293 (p53+/+) 6 PTTG OX ↑ ↑ ↑

Hamid et al, 2004 (10, 74) MCF-7 (p53+/+) 4 PTTG OX ↑ ↑ ↑

Hamid et al, 2004 (10) PC-3 (p53-/-) 7 PTTG OX ↑ NC NC

C. Studies showing PTTG inhibits p53 or apoptosis


Bernal et al, 2002 (9) H1299 8 PTTG OX ↑ ↓ ↓

Bernal et al, 2002 (9) HCT166 (PTTG-/-) 9 ↑p53 dependent apoptosis** ↓ ↑ ↑

Cho-Rok et al, 2006(75) SH-J1 10 PTTG siRNA transfection ↓ ↑ ↑

Kho et al, 2004 (70) HCT166 9 si RNA PTTG ↓ - ↑

Bernal, JA, 2007 HCT166 (PTTG-/-) 9 PTTG-/- ↓ ↑ -

D. Studies showing PTTG suppression by p53


Kho et al, 2004 (70) HCT166 9 Microarray analysis after ↑p53 ** ↓ by p53 ↑ -

Zhou et al, 2003 (72) U2OS (p53+/+) 3 Doxorubicin & bleomycin *** ↓ ↑ -

Zhou et al, 2003 (72) HCT116 (p53+/+) 9 Doxorubicin & bleomycin *** ↓ ↑ -

Zhou et al, 2003 (72) Saos2 (p53-/-) 3 Doxorubicin & bleomycin *** NC p53 -/- -

Zhou et al, 2003 (72) DLD-1 (p53-/-) 9 doxorubicin & bleomycin *** NC p53 -/- -

Chiu et al, 2006 (76) RKO (p53+/+) 9 Oxaliplatin * ↓ ↑ -

Chiu et al, 2006 (76) SW480 (p53-/-) 9 Oxaliplatin * NC - -

1 lung cancer, 2 cervical cancer, 3 osteosarcoma, 4 breast cancer, 5 insulinoma, 6 embryonic kidney, 7 prostate cancer, 8 non-small cell lung carcinoma, 9 colorectal

cancer, 10 hepatoma, ↑ : overexpression, ↓ : depletion/decrease , NC: No Change, -: not reported, OX: overexpression, * clinical anti-cancer drug, ** genotoxic stress

due to 5-flurouracil, *** (DNA damaging drugs) that induce strand breaks and activate, p53, -: no change.

18

PTTG overexpression induced p53 upregulation and translocation to the nucleus, as well

as apoptosis in MCF-7 breast cancer cells that expressed wild-type p53 (71). PTTG also induced

p53-independent apoptosis in MG-63 osteosarcoma cells deficient in p53, wherein p53

expression did not affect PTTG-induced apoptosis rate (77). In insulin-secreting insulinoma

MIN6 cells PTTG tranfection induced apoptosis, which increased over time (74). Similarly,

PTTG transfection of HEK293, a human embryonic kidney cell line expressing wildtype p53,

induced elevated p53 protein expression and apoptosis, whereas transfection of PC-3 cells

expressing a mutant p53 form did not (10). Analysis of p53 promoter showed increased activity

in MCF7, PC3 and HEK293 cells, implicating transcriptional activation of p53 by PTTG (10).

Additionally, PTTG transfection induced enhanced transcription and expression of the bax gene,

a pro-apoptotic factor, in a dose-dependent manner in MCF7 and HEK293 cells. In contrast,

there was no change in bax expression in PC-3 cells that lacked a functional p53, suggesting that

PTTG modulation of the pro-apoptotic factor occurs through p53 (10).

In contrast, inhibition of apoptosis by PTTG has also been shown. PTTG knockdown

using siRNA enhanced apoptosis in UV irradiated HeLa cells, whereas PTTG overexpression

suppressed UV-induced apoptosis in these cells (78). Similarly, transfection of SH-J1 hepatoma

cells with siRNA against PTTG activated p53 and induced apoptosis (75). In HCT116 colorectal

cancer cells, PTTG depletion resulted in p53-dependent cytotoxicity (75). Bernal et al. similarly

found that PTTG tranfection of H1299 cells inhibited p53-dependent apoptosis in a dose-

dependent way (9). P53 apoptotic effects were further enhanced in PTTG-/- cells (9). These

results suggest that PTTG may exert its oncogenic effects at least in part by affecting p53-

dependent pathways (9).

19

That studies have found correlations between pituitary tumor subtype and p53 expression,

combined with the intimate association of PTTG with p53, calls for investigation into PTTG

expression in different pituitary adenoma subtypes to provide more insight into potential

correlations.

2.6.2 PTTG, angiogenesis and pituitary adenomas

Angiogenesis is defined as the formation of new blood vessels from existing ones and is

associated with tumor progression (79). It is axiomatic that tumor growth requires

neovascularization in order to supply tumor cells with necessary nutrients and oxygen.

Therefore, tumor vascularity is often associated with tumor growth, aggressive behaviour and

metastatic potential (79). However, findings of pituitary tumor vascularity are heterogenous,

with some investigators reporting increased tumor vasculogenesis, and others observing lower

microvascular density. It is well-established that PTTG promotes angiogenesis and angiogenic

factors including VEGF, FGF and FGF receptors (FGFR) (7, 14, 80, 81). This section

examines the findings of studies on the role of angiogenesis and its factors in pituitary adenomas

(2.6.2.1 and 2.6.2.2), and the inconsistencies in literature reports of differential VEGF, FGF

and/or FGFR in a cell-specific manner. The interplay between PTTG and such factors is also

presented (2.6.2.3), thus highlighting the need for investigation of PTTG in pituitary adenomas

and specifically, different hormonal cell-types.

2.6.2.1 Vascular endothelial growth factor (VEGF) in pituitary adenomas

Vascular endothelial growth factor (VEGF) is an important angiogeneic factor that

mediates endothelial cell proliferation as well as permeability and motility. Its expression is

related to tumor angiogenesis and often to aggressive behaviour (79). In pituitary adenomas,

20

several studies have found correlations between VEGF expression and clinico-pathological

parameters including tumor invasiveness and proliferation rate (Table 2.4). In a series of 148

pituitary tumors including 6 pituitary carcinomas, VEGF expression was seen to be higher in

carcinomas than adenomas, which in turn showed lower VEGF expression than pituitary tissue

(82). Similarly, another study of VEGF expression in adenomas showed it to be associated with

invasion (83). In contrast, although Iuchi et al. showed that VEGF correlated with tumor

proliferation in octreotide-treated GH adenomas, it was not associated with cavernous sinus

invasion (84). Thus, although VEGF may not directly contribute to tumoral invasion, it may

regulate pathways that do increase tumor volume or mediate invasiveness. This notion is

supported by the observation that VEGF expression is not strictly associated with endothelium

and vessels, but is expressed by adenoma cells as well (85).

Table 2.2. Clinicopathologic correlations with VEGF expression in pituitary tumors.

Study

N

Subtype

Niveiro et al. 2005 47 60 ↓ PRL

↑ in NF

Pan et al, 2005 52 45 -

Arita et al, 2004 1 - -

Fukui et al. 2003 13 48 -

McCabe et al. 2002 42 103 ↑ NF vs. normal pit

↓ TSH than normal

Iuchi et al. 2000 22 25 -

Lloyd et al. 1999 39 148 ↑ in GH, ACTH, non-oncocytic null cell

↑ in treated GH and PRL

N: number of cases; GH = growth hormone adenoma; PRL=prolactin adenoma; ACTH=

adenocorticotropin hormone adenoma; FSH=follicle stimulating hormone adenoma; LH= luteinizing

21

Expression of VEGF receptors in pituitary adenomas has also been investigated. For

example, higher expression of Flk-1 (fetal liver kinase-1), a form of VEGF receptor that

mediates mitogenesis and affects endothelial cell morphology, was associated with extrasellar

extension (81). Furthermore, Flk-1 was significantly higher in non-functioning as compared to

functioning tumors (81). Studies regarding the expression of VEGF and its receptors in the

various pituitary adenoma subtypes are limited. In one study, VEGF expression differed in the

subtypes, thus implicating different mechanisms of VEGF expression and/or action (82). The

strongest VEGF immunoreactivity was exhibited by GH, ACTH, silent subtype II, and non-

oncocytic null cell adenomas (82). Octreotide-treated GH adenomas showed lower VEGF

expression than non-treated ones (82). Comparatively weak VEGF immunopositivity was seen

in PRL, gonadotroph, TSH, and oncocytic cell adenomas (82) (Table 2.4). Given the strong

evidence on the regulatory role of PTTG in VEGF mediated-pathways, and the limited literature

on cell-type specific expression of VEGF, expression of PTTG in pituitary tumor subtypes may

provide more information on the interplay between PTTG, VEGF and cell type.

2.6.2.2 FGF in pituitary adenomas

Studies have shown associations between pituitary tumor behaviour and the expression of

bFGF (basic fibroblast growth factor), a well-characterized angiogenic growth factor, and its

receptor, bFGFR (86, 87). Levels of serum FGF2 are elevated in patients with pituitary tumors

(Table 2.5). The expression of higher bFGF and/or FGFR levels has been shown to correlate

with pituitary tumor recurrence, larger tumor size, tumor invasivenes ((86, 88-90), as well as

poor outcome (2, 86, 88-91). Tumors with higher labeling indices of Ki-67, a proliferation

22

marker, also showed increased FGF-R expression (89) (Table 2.5). One study reported elevated

FGF expression in only TSH adenomas as compared to normal TSH secreting cells (2).

Table 2.3 Clinicopathological correlations with FGF, FGFR expression in pituitary

tumors.

Study

N

Invasion

Recurrence

Subtype

Other

Correlations

Qian et al. 2004 54 137 FGFR4

↑ in invasive (ns)

-

-

↑ size (except

PRL)

↑ Ki-67

McCabe et al.

2003 43

121 FGFR1

↑ invasive

↑ TSH (vs normal)

Fukui et al. 2002 12

64

-

bFGF

↑ Recurrent

-

↑ Poor outcome

↑ size

Susui et al. 1994 61

30 - - - bFGF with

FGFR1

N: number of cases; PRL=prolactin adenoma; TSH=thyrotropin stimulating hormone; bFGF=basic fibroblast growth

factor; FGFR1=fibroblast growth factor 1; FGFR4=fibroblast growth factor 4; ↑= Higher, ↓= Lower; ns: statistically

not significant

FGF-2 stimulates PRL production as well as proliferation of lactotroph cells (36), thus

acting at the cell-specific level. Given the strong correlation between expression of PTTG and

FGF in estrogen induced PRL adenomas in mice, characterization of PTTG expression profile in

PRL adenomas will contribute to our understanding of molecules involved in pituitary

development and tumorigenesis.

23

2.6.2.3 PTTG, angiogenesis, VEGF and FGF

PTTG is involved in angiogenesis at least in part by induction of bFGF and VEGF

(vascular endothelial growth factor) angiogenic factors (49) (8) (81) (92). Transactivation of

FGF-2 by PTTG is well documented (14, 36, 93). In a mouse model of follicular thyroid cancer,

PTTG deficiency resulted in reduced FGF-2, FGFR-1 (FGF receptor) and VEGF levels (49).

Thus an autocrine mechanism involving PTTG and bFGF may exist (36). Both PTTG and bFGF

expression was increased in acute leukemia, a positive correlation being noted between their

expression levels (94). Concordant PTTG and FGF-2 expression patterns were detected in

developing fetal brain cortex (95). PTTG dependent bFGF and/or VEGF expression was

observed in several cell lines (24, 36, 81, 96). It was found that PTTG promoted human

umbilical vein endothelial cell (HUVEC) proliferation and tube formation, as well as in vitro

vessel growth (93). In pituitary adenomas, a significant positive correlation was found between

VEGF and PTTG mRNA expression, as well as between PTTG and KDR (a VEGF receptor)

mRNA (7). Also, VEGF and PTTG showed colocalization in pituitary adenomas (92). PTTG

upregulation of ID3 (inhibitor of DNA-binding-3), an important mediator of VEGF, induced

angiogenesis, and downregulation of TSP-1 (thrombospondin-1), an angiogenic inhibitor, has

been demonstrated in FTC133 human follicular cancer cells (7). Thus PTTG appears to play an

essential role in tumor angiogenesis and may provide a therapeutic target in neoplasms with high

vascular density.

Given the evidence on cell specific expression of VEGF as well as FGF (82) and the

importance of PTTG in pituitary tumor development and progression and its role in induction of

VEGF and FGF, it is essential to investigate cell type specific PTTG expression in pituitary

24

adenomas to illuminate on the interplay of cell type, VEGF, FGF and PTTG as elements of

pituitary angiogenesis and/or tumorigenesis.

2.6.3 Genetic Instability

This section provides the evidence on the incidence of genetic instability in pituitary

adenoma subtypes (2.6.3.1) and the central role of PTTG in genetic instability (2.6.3.2), thus

presenting the need for investigation of PTTG expression in pituitary adenoma hormonal

subtypes.

2.6.3.1 Genetic instability in pituitary adenomas

It is remarkable that over 50% of pituitary tumors show aneuploidy (97). It is debatable

whether aneuploidy and genetic instability are causal to tumor formation, or are rather a result of

the tumorigenic process (97). Some investigators have observed higher rates of aneuploidy in

PRL and/or GH adenomas. Anniko et al (98) found that aneuploidy was significantly higher in

PRL adenomas (71%), whereas GH adenomas showed aneuploidy in 41% of the cases.

Interestingly, 67% of mamosommatotroph tumors producing both GH and PRL exhibited

aneuploidy. Finelli et al also found a greater incidence of aneuploidy in PRL adenomas (99).

Similarly, Rock et al. found aneuploidy in 8/11 GH, 2/2 PRL, 6/7 GH/PRL and only 2/7 null cell

adenomas, suggesting a higher incidence in GH and/or PRL secreting adenomas(100). These

findings support the cell-specific incidence of aneuploidy. As PTTG is increasingly associated

with aneuploidy in neoplasms, including those of colon and thyroid, its expression in pituitary

tumor subtypes requires thorough investigation. As empirical evidence points to differences in

the incidence of aneuploidy in pituitary adenoma types, cell-type examination of PTTG is

important.

25

2.6.3.2 PTTG and genetic instability

The association between PTTG overexpression and increased aneuploidy as well as

genetic instability in tumors and cell lines is well established (101-103). In colorectal cancer

patient, such instability correlated with higher tumor grade, indicating that PTTG and genetic

instability may be linked in these tumors (101). PTTG expression also correlated with

aneuploidy in

melanomas and in follicular as well as papillary thyroid carcinomas (102). In vitro transfection

of colorectal (101) and follicular thyroid carcinoma cells (102) as well as of HeLa and A549

cells induced both genetic instability and aneuploidy. Indeed, cell transfection with a non-

degradable PTTG mutant was shown to result in incomplete cytokinesis and lack of

chromosomal segregation in 51 of 55 cultured cells (103). Interestingly, aneuploidy was also

evident in the pituitary gland cells of PTTG deficient mice (16). Thus, aberrant PTTG expression

is associated with aneuploidy which may significantly contribute to its tumorigenic potential

(103).

2.6.4 Tumor invasion

The following sections present the evidence on the role of tumor invasiveness factors,

matrix metalloproteinase’s (MMPs) in pituitary adenomas with respect to cell types (2.6.4.1),

followed by the role of PTTG in induction of invasiveness (2.6.4.2), providing further support

for the cell-type specific investigation of PTTG expression in pituitary neoplasms.

2.6.4.1 Tumor invasion in pituitary adenomas

Matrix metalloproteinases (MMP) are proteolytic enzymes that break down basement

membrane and connective tissue, thus facilitating invasive growth (104). They do so by

26

breaking down extracellular matrix and selectively remodeling it (104). In a recent microarray

analysis and gene clustering study, Hussaini et al found a robust, eightfold increase in MMP-9

expression in invasive as compared to non-invasive pituitary adenomas (105) a result in keeping

with the findings of earlier studies (Table 2.6).

Table 2.4 . Correlations between expression of MMPs and clinicopathological parameters

in pituitary tumors.

Study N Invasiveness Tumor size

Recurrence Subtype

Gong et al. 2007 14 73 ↑ MMP-9 MMP-9 (NF)

↑ MMP-9

(in secondary tissue)

-

Hussaini et al. 2007 19 6 ↑ MMP-9 - - -

Liu et al. 2005 37

54 ↑MMP-2 - - -

Pan et al, 2005 52 45 ↑ MMP-9 - - -

Knappe et al. 2003 29 84 ↓TIMP-2 - - MMP-2, -9

(↑ ACTH)

MMP-2 , -9

(↓ in NF)

TIMP-2 (↑ ACTH)

Turner et al. 2000 66

55 ↑ MMP-2

(macroPRL)

- ↑ MMP-9

(in secondary tissue)

-

Beaulieu et al, 1999 3 28 ↑ MMP-9 - - -

Kawamoto et al. 199626

22 ↑ MMP-9 - - -

N= number of cases; GH= growth hormone adenoma; PRL=prolactin adenoma; ACTH=adenocorticotropin hormone adenoma; TSH= thyrotropin stimulating horome ; FSH=follicle stimulating hormone adenoma; LH= luteinizing hormone adenoma; α-SU= α subunit of glycoprotein hormone; Mix= adenoma with 2 or more hormones ; NF=nonfunctioning adenoma; ↑=Higher, ↓= Lower ; MMP= Matrix metalloproteinase; TIMP = Tissue inhibitor of matrix metalloproteinase ;

27

Studies relating MMP-9 expression and functional status and/or tumor subtype are

limited (Table 2.6). In one study, compared to all other tumors, MMP-9 expression was higher

in ACTH adenomas (106), and this may contribute to the relatively high frequency of ACTH

secreting adenomas that are invasive despite their small size (106). Knappe et al. found

significantly lower MMP-9 expression in clinically non-functioning adenomas compared to

hormone-secreting tumors, an observation in keeping with the generally lower invasion rates of

the former (Table 2.6). In contrast, Kawamoto et al., found no difference in MMP-9 expression

between functioning and non-functioning adenomas (107).

Other members of the matrix metalloproteinase family, such as MMP1, 2 and 3 have also

been shown to be expressed differentially in pituitary adenoma subtypes. MMP-2 expression is

significantly higher in clinically functioning than non-functioning adenomas (P<0.05) (108).

Beaulieu et al. found lower MMP-1, -2 and -3 levels in GH radiological grade II tumors as

compared to lesions of grades 0, I, III and IV (109). However, MMP-1, -2, -3 levels were

unrelated to tumor invasiveness (109). The inconsistent findings of these studies highlight the

differences in actions of molecular factors in various pituitary adenoma subtypes. As PTTG is

one such factor implicated both in MMP upregulation and pituitary adenoma pathogenesis, the

cell-specific phenotype of PTTG will reveal more information on its potential role in tumor

invasiveness, and in particular, MMP-mediated invasion.

2.6.4.2 PTTG and tumor invasiveness

Several studies have shown a correlation between tumor PTTG expression and tumor

invasiveness. This is true of endocrinologically functioning pituitary adenomas as well as

colorectal and breast carcinomas (110-112). In the TRßPV/PV model of follicular thyroid

carcinoma, PTTG deficiency attenuates tumor vascular invasion and the occurrence of lung

28

metastasis (49). Recently, PTTG regulation of matrix metalloproteinase-2 (MMP-2) has been

shown which may account for decreased tumor invasiveness in the PTTG-deficient TRßPV/PV

mice (47). In vivo, PTTG overexpressing HEK293 cells injected into nude mice were seen to

induce increased MMP-2 expression and tumor formation (11). PTTG also facilitated endothelial

tubule formation and growth in 3D Matrigel matrix, effects mediated through MMP-2 expression

(11). Lastly, PTTG depletion by siRNA reduces tumor invasiveness in several glioma cell lines

(113). Thus PTTG may stimulate mediators of tumor invasiveness, contributing to enhanced

invasive potential of tumors. A complicating factor in the study of the role of PTTG in pituitary

adenoma invasiveness is the inherent difference in molecular pathways of the different hormonal

and non-hormonal subtypes, and as such, cell-type specific study of PTTG in pituitary neoplasm

will address this issue and provide a clear picture of PTTG expression patterns in such cell types.

2.7 PTTG in pituitary adenomas: correlations with tumor subtype

This section reviews the preliminary evidence on differential expression of PTTG in

different pituitary adenoma subtypes, providing further support for the present study that focuses

on PTTG expression in pituitary adenoma cell types.

Although there is consensus in literature about the overexpression of PTTG in pituitary

adenomas, whether PTTG expression is differentially expressed in various adenoma subtypes

remains unclear. Few studies have investigated a potential correlation between PTTG expression

and pituitary tumor subtype. PTTG mRNA levels in 40 pituitary tumors (12 GH, 5 PRL, 5

ACTH, 18 nonfunctioning) showed significantly elevated expression in GH-secreting adenomas

(2.7 fold) compared to nonfunctioning adenomas (24), suggesting cell type-dependent expression

of PTTG. In another study, although PTTG expression was higher in GH adenomas as compared

to PRL and ACTH adenomas, this difference was not statistically significant (114). In keeping

29

with these studies, Uccella et al. also found that compared to other pituitary adenoma subtypes,

GH and PRL adenomas exhibited elevated PTTG expression levels and aneuploidy (102, 115) . It

should be noted that a higher incidence of abnormal chromosome number has been reported in

GH and PRL adenomas in previous studies as well (97, 116), supporting the notion of PTTG cell-

specific expression/function. Additionally, Zhang et al. found a significant difference in PTTG

association with tumor stage between non-functioning and hormone-secreting pituitary neoplasms

(112). This distinction suggests differences in the molecular mechanisms underlying PTTG-

mediated tumor initiation and/or progression. These findings allude to a role for PTTG as a factor

in pituitary cell type differentiation, warranting further study of PTTG expression in pituitary

adenoma cell types.

In addition to clinical investigations, animal models also support an association between

PTTG cell-specific expression/function in the pituitary gland and adenomas arising therein.

Targeted PTTG overexpression in the pituitary gland, using the alpha-SU promoter, induced focal

expression of PTTG not only in LH cells, as expected, but also in GH-producing cells (46). GH

producing cells, arising from the somatotroph cell lineage during early development of the

pituitary gland, are not associated with the alpha-SU promoter, which is involved in gonadotroph

cell differentiation (46). That PTTG should be expressed in GH cells despite early

cytodifferentiation of gonadotroph and somatotroph cell lineages, suggests a link between PTTG

and the GH producing somatotroph cell type.

Furthermore, in vitro studies also provide evidence for a connection between PTTG and GH and

PRL cell types in the pituitary gland (52). In rat GH3 cells that secret PRL and GH,

overexpression of PTTG C-terminus, a dominant negative that competes with endogenous PTTG

and inhibits its action, suppressed PRL secretion by 10-fold, whereas GH secretion was

30

upregulated. These findings suggest that PTTG mediates PRL and GH production. In contrast to

the mounting evidence supporting PTTG involvement in cell hormonal phenotype, a study by

Minematsu et al showed a lack of correlation between PTTG expression and pituitary adenoma

cell type (92). Examining 101 pituitary adenomas (29 GH, 12 PRL, 6 ACTH, 1 FSH, 3 TSH, and

50 non-functioning), Minematsu et al. found that while elevated in most pituitary adenoma

subtypes, PTTG mRNA levels were not significantly different. However, several in vivo and

animal studies suggest a role for PTTG in cell-specific hormone expression in the pituitary gland,

meriting further investigation of a potential function.

2.7.1 PTTG in stem cells

There is cumulating evidence on the importance of PTTG expression in early progenitor

cells, and this section explores the relevant literature that implicates PTTG as an early element in

cytodifferentiation, making it a candidate in pituitary hormonal cell type differentiation.

That PTTG-/- mice develop organ-specific hypoplasia suggests a role for PTTG in

progenitor cell function and cytodifferentiation (50). Indeed, the PTTG gene is transcribed in

the one-cell stage of mouse development (117), the transcript showing a 39% increase in the

zygote as compared to the oocyte. Furthermore, gene profiling of immature and mature oocytes

showed differential expression of PTTG3 during oocyte meiosis (118). Thus PTTG may play a

role in the zygote-stage gene activation (117). In a recent study, PTTG was shown to be

essential for maintenance and proliferation of bone marrow stem cells (119). Gene profiling of

proliferating and differentiated neural stem cells has shown PTTG to be one of the genes

upregulated in proliferating neural progenitor cultures as compared to differentiated neural cells

(120). Similarly, the PTTG gene is seen to be upregulated following treatment of human

31

mesenchymal stem cells with bone morphogenic protein (BMP), the upregulation being

associated with stem cell proliferation (121). These findings cumulatively suggest that PTTG

expression in stem and progenitor cells is associated with their proliferative potential. They also

raise the possibility that PTTG plays a role early in pituitary cytodifferentiation from progenitor

cells into the mature hormonal cell lineages, though yet unknown mechanisms.

2.8 PTTG as a cinico-pathological indicator in ACTH-secreting pituitary adenomas

This section provides an outline of ACTH-secreting adenomas, and the need for

prognostic indicators in patients with Cushing’s disease with ACTH-secreting adenomas. The

role of PTTG as such an indicator is explored.

2.8.1 ACTH-secreting pituitary adenomas

Cushing’s disease is caused by endocrinologically functional adenocorticotropic hormone

(ACTH)- producing pituitary corticotroph adenomas. Most occur in women (80-90%) at a mean

age of 35 to 44 years. The majority (75-90%) are microadenomas; marcoadenomas have been

reported to occur in 9% to 22% (122-127).

Long-term management of patients with Cushing’s disease remains a challenge for the

clinician. Cushing’s disease is associated with muscle atrophy, hypertension, diabetes mellitus,

obesity, depression and osteoporosis, as well as psycho-cognitive defects. If left untreated,

mortality in a five-year period is up to 50% (128, 129). Studying 215 Cushing’s disease patient’s

with ACTH-secreting adenomas, Patil et al found recurrence in 25% of patients who had

achieved remission following transsphenoidal surgical removal of their tumor (130).

32

While increasingly immunohistochemical markers have been employed as prognostic

indicators of parameters such as recurrence in patients with several tumours, few such

immunohistochemical markers have been investigated for prognostic correlation in functional

ACTH-secreting pituitary adenomas. Given the elevated expression of PTTG in pituitary

adenomas, and its utility in several neoplasms as a marker of tumor aggressive behaviour, it is

important to examine such clinico-pathological correlations in patients with Cushing’s disease.

We had complete patient records of over 54 patients with Cushing’s disease, and investigated the

utility of PTTG as an immunohistochemical prognostic indicator with respect to patient age, sex,

tumor size, invasiveness, and recurrence. PTTG immunohistochemical expression was studied

in these ACTH secreting adenomas and its usefulness as a marker of tumor aggressive baheviour

was examined.

2.8.2 PTTG, cortisol, and Cushing’s disease

Cortisol, a glucocorticoid elevated in patients with Cushing’s disease due to increased

plasma ACTH levels, binds to its receptor on ACTH secreting pituitary cells, thereby enforcing a

negative feedback effect on ACTH secreting pituitary and tumor cells. It is of particular interest

that hydrocortisone, a cortisol derivative is found to inhibit PTTG levels in T-cells (131, 132).

Thus, it would be expected that ACTH secreting cells express suppressed PTTG levels, as

cortisol is implicated in PTTG suppression. However, PTTG inhibition in T cells treated with

hydrocortisone may be only secondary to the anti-proliferative effects of hydrocortisone in T

cells (131). Investigation of cell type specific expression of PTTG will illuminate on the effect

on PTTG expression of elevated cortisol levels in patients with Cushing’s disease caused by an

ACTH secreting pituitary adenoma.

33

2.8.3 PTTG as a clinico-pathological marker in neoplasms

PTTG is found to be expressed in almost all neoplasms studied to date (2, 57, 110, 112,

133, 134). Several authors have found PTTG to be a valuable prognostic indicator with respect

to patient survival time, tumor recurrence and invasiveness, as well as metastatic potential of

neoplasms (2, 6, 43, 49, 102, 112, 135).

PTTG is promising as a prognostic marker in a variety of neoplasms examined. In lung

and hepatocellular carcinomas as well as esophageal small cell carcinoma (ESCC), PTTG

expression levels are predictive of patient survival. Additionally, PTTG levels are directly

correlated with tumor stage in HNSCC, ESCC, and colorectal carcinomas. Further studies are

needed to better characterize and standardize the predictive role of PTTG as a marker of

aggressive tumor behaviour, especially in pituitary adenomas.

2.8.4 PTTG clinico-pathological correlations in pituitary adenomas

Several studies have investigated the potential predictive value of PTTG expression in

pituitary adenomas. A recent study of 25 PRL-secreting pituitary tumors showed that although

PTTG expression did not predict aggressive behaviour per se, nuclear PTTG staining was one of

the histological features (numerous mitoses, high Ki-67 index, nuclear labeling of PTTG and

P53) distinguishing aggressive-invasive PRL tumors from those behaving less aggressively

(125)(Wierinckx et al. 2007). A comparative RT-PCR study of 54 pituitary adenomas (13 GH,

10 PRL, and 1 ACTH, 30 nonfunctioning) showed no correlation between tumor stage and

PTTG mRNA levels in nonfunctioning adenomas, whereas in hormone-secreting tumors

significantly higher PTTG expression was seen in invasive tumors (112)(Zhang et al. 1999a).

With respect to tumor recurrence, examination of 45 pituitary tumors (14 PRL, 8 GH, 6 ACTH,

34

11 luteinizing hormone (LH) / follicle stimulating hormone (FSH), 6 nonfunctioning)

demonstrated nuclear PTTG immunoreactivity (using a monoclonal antibody) in 89% of the

tumors (27)(Filippella et al. 2006); its expression showed a strong correlation with Ki-67

immunopositivity, and was higher in recurrent tumors. The cut-off value separating recurring

and non-recurring tumors was 3.3% for PTTG immunopositivity (60% sensitivity, 76%

specificity). There was no significant correlation between PTTG immunopositivity and tumor

size or radiologic grade, patient age and sex or treatment (27)(Filippella et al. 2006). In 27 of the

45 patients for whom 1-year follow-up was available, a cut-off of 2.9% for both PTTG and Ki-67

positivity predicted recurrence vs. non-recurrence, Ki-67 being the superior predictor

(27)(Filippella et al. 2006). These findings indicate the need for thorough investigation of the

predictive value of PTTG as a marker of aggressive pituitary tumor behaviour, and underscore

the necessity of employing a uniform methodological approach to the assessment of PTTG

expression in order to maximize its utility as a prognostic indicator. Thus, in our study, we

examined the value of PTTG as a prognostic indicator in ACTH secreting adenomas of patients

with Cushing’s disease, with respect to age, sex, tumor size, invasiveness, and recurrence. It is

of note that the studies investigating such correlations thus far, have included no or very few

ACTH-secreting pituitary adenomas (27, 125, 134). ACTH-secreting pituitary adenomas

account for up to 15% of all pituitary adenomas, and are the cause of Cushing’s disease.

Although rare, Cushing’s disease is associated with sever clinical manifestations

including elevated blood pressure, truncal obesity, hirsuitism, abdominal striations, osteoporosis,

fatigue and weakness. Left untreated, Cushing’s disease is associated with a 5-year

cardiovascular mortality of up to 50% (130). ACTH-secreting adenomas tend to be slowly

growing and are rarely invasive, but a recent study of long- term follow up of 215 subjects with

Cushing’s disease demonstrated a recurrence rate of 25% in patients initially cured after

35

transsphenoidal surgery. However, to date, no single immunohistochemical marker for

prediction of recurrence in ACTH secreting adenomas exists. Most studies investigating the

prognostic value of immunohistochemical markers in pituitary adenomas include a small number

of ACTH adenomas, making it difficult to draw definitive conclusions (67). Much research has

been devoted to finding the prognostic value of markers routinely used in several neoplasms, but

has proved to be of limited use in pituitary adenomas. Such factors include the Ki-67

proliferation marker, as well as markers of angiogenesis (microvascular density, VEGF, bFGF,

EGF, Cox-2, HIF-alpha), matrix metalloproteinases (MMP2 and MMP9), and cell cycle

regulators (p21, p27, and p53). It is essential to investigate the role of PTTG as a potential

indicator of tumor behaviour in ACTH adenomas, given that, a) there currently exists no

prognostic indicator for patients with ACTH secreting pituitary adenomas, b) the high rate of

recurrence in these neoplasms, c) recent studies have shown a high level of PTTG expression in

pituitary adenomas, d) the involvement of PTTG in several tumourigenic mechanisms, and e) its

demonstrated value as a prognostic indicator in other neoplasms.

2.9 PTTG cellular localization

This section presents a literature overview of PTTG nuclear versus cytoplasmic

localization, providing a rationale for the importance of investigating cellular localization.

Cellular PTTG localization, particularly the significance of its cytoplasmic versus nuclear

expression, remains controversial. Nuclear PTTG is thought to function as a transcriptional

activator, as well as a securin, inhibiting premature sister chromatid separation, whereas the role

of cytoplasmic PTTG remains unclear (136). While differential PTTG localization may be due

to variations in cell lines and tumor types examined, cell cycle dependent expression of PTTG

may also account for the reported differences (Table 2.5 and Table 2.6). A summary of reported

36

PTTG expression patterns is found in Tables 2.5 and 2.6. Clearly, whether PTTG is a primarily

nuclear or cytoplasmic protein or both remains the topic of much controversy. In pituitary

aenomas, recently, Wierinckx et al., found only nuclear PTTG expression to be predictive of

aggressive pituitary tumor behaviour (43), with cytoplasmic PTTG expression not being a

predictive factor. Thus, PTTG subcellular localization appears to be a significant factor in

determination of its tumourigenic role.

To date, PTTG cellular localization has not been established in pituitary adenomas (Table

2.6). Whereas some studies have reported cytoplasmic PTTG expression predominantly, with

minor expression in cell nuclei (43, 112, 133, 137), others have found PTTG expression to be

only nuclear (135). It is essential therefore, to establish PTTG localization in pituitary tumor

cells, as its presence in the nucleus and/or the cytoplasm will have vastly different implications

for its functional role in physiological as well as tumourigenic processes, as well as its role as a

potential therapeutic target.

37

Table 2.5 PTTG cellular localization in cell lines

Table 2.6 PTTG cellular localization in adenomas

38

2.10 The importance of PTTG as a potential targeted therapeutic

Several in vitro and in vivo models have demonstrated the therapeutic potential of PTTG

(8, 52, 55, 75, 113, 138-141). Disruption of PTTG pathways in PRL- and GH-secreting pituitary

GH3 cells using a C-terminus truncated form of PTTG suppressed PRL promoter activity as well

as secretion (52). In addition, subcutaneous injection GH3 cells transfected with truncated PTTG

demonstrated the formation of smaller pituitary tumors in rats (52). These results suggest that

PTTG may be therapeutic target in the treatment of PRL-secreting pituitary adenomas and/or

control of their hypersecretion (52). Yet another study showed PTTG depletion by siRNA in U87

glioma cells, resulting in inhibition of cell proliferation associated with reduced BrdU

incorporation (55). Related studies showed similar results. For example: a) siRNA knockdown of

PTTG in FTC133 follicular thyroid carcinomas cells suppressed ID3 levels and increased TSP-1

levels, a result in keeping with PTTG stimulation of angiogenic factors (8) , b) PTTG anti-sense

transfection of the SK-OV-3 ovarian carcinoma cell line both reduced bFGF expression by 53%

and suppressed colony formation (142), c) PTTG knockout using siRNA in the lung cancer cell

line H1299 resulted in reduced colony formation as assayed by the soft agar colony formation

assay (138), d) nude mice injected with PTTG siRNA transfected H1299 cells inhibited tumor

development and growth without adverse effects (138), and e) expression of Ki-67, bFGF and

CD34 were significantly reduced in PTTG siRNA transfected tumors (138).

Transfection of HeLa-S3 human cervical cancer cells with anti-sense PTTG mRNA

decreased cell viability and reduced the mitotic index. Alterations in cell phenotype included

aberrations in sister chromatid separation, DNA bridges, and apoptosis (139). Due to its anti-

tumor effects, PTTG antisense has been proposed as a potential therapeutic target (139).

39

Similarly, simply depleting PTTG in T98G, ON12 and U251 glioma cell lines using the siRNA

technique resulted in decreased cell proliferation and reduced invasion as studied by cell counts

and the Matrigel invasion assay (113). Antisense injection of mice was unassociated with side

effects (113). Adenoviral vector encoding siRNA to silence the PTTG gene resulted in PTTG

depletion and induced apoptosis in the SH-J1 hepatoma cell lines. In HCT116 colorectal cancer

cells, PTTG gene silencing also showed dose-dependent cytotoxicity. In vivo, growth inhibition

of Huh-7 hepatoma cell lines and of SH-J1 tumor xenografts in nude mice was achieved by PTTG

gene silencing via siRNA. The results promise the therapeutic potential of PTTG gene silencing

in the treatment of hepatic tumors as well as other neoplasms expressing high levels of PTTG

(75). It is warranted therefore to establish PTTG expression patterns in different pituitary

adenoma subtypes, which may provide a rationale for the utility of PTTG-targeted therapy in

these neoplasms.

2.11 Highlights of PTTG association with molecular factors linked to pituitary adenomas

Figure 2.3 summarizes the molecular pathways which link PTTG and pituitary adenomas

subtypes, especially GH and PRL adenomas. In sum, of interest is the elevated expression levels

of VEGF and Bcl-2 in GH adenomas, and relatively lower levels of these two factors in PRL

adenomas. VEGF is an angiogenic factor shown to be upregulated by PTTG, and its expression

is reported to be higher in GH adenomas and lower in PRL ones. Similarly, Bcl-2, an anti-

angiogenic factor inhibited by p53, is expressed at higher levels in GH adenomas and relatively

40

Figure 2.3. A summary of pathways in which PTTG is implicated in the context of GH and PRL adenomas. PRL

adenomas show relatively lower VEGF and Bcl-2 expression levels, and elevated levels of genetic instability (G.I.)

and bFGF expression. GH adenomas on the other hand show relatively higher VEGF and Bcl-2 levels, as well as

IGF1 and genetic instability. PTTG is known to both upregulate and downregulate p53, which in turn inhibits Bcl-2

production and IGF-1 levels.

lower levels in PRL adenomas. Whether p53 mediates Bcl-2 expression in pituitary

adenomas as a downstream effect of PTTG remains unknown.

Overexpresssion of IGF1 is also associated with GH adenomas, and IGF1 in turn

upregulates PTTG expression. Additionally, genetic instability is found to be higher in G and

PRL adenomas among all pituitary tumours.

Given that a number of molecular pathways are associated with PTTG, and that these

factors, namely VEGF, Bcl-2, IGF1, bFGF, as well as genetic instability are found to be

distinctly linked to GH and PRL adenomas, the present study aims to determine PTTG

expression in such tumours.

GH PRL

41

CHAPTER 3: IMMUNOHISTOCHEMICAL EXPRESSION OF PTTG IN BRAIN

NEOPLASMS

3.1 Brain neoplasms

Tumors of the central nervous system (CNS) and peripheral nervous system (PNS) arise

from neurons, meninges, as well as glial cells that provide support and protection for neurons

and form the blood brain barrier. Glial cells include astrocytes, oligodendrocytes, and

ependymal cells in the CNS, as well as Schwann cells in the PNS. These can give rise to

astrocytomas, oligoastrocytomas, oligodendrogliomas, ependymomas and schwannomas. More

prevalent non-glial tumors of the nervous system include meningiomas and

hemangiopericytomas, arising from meninges, and medulloblastomas that are embryologic in

origin. Malignant gliomas, derived from glial cells, account for up to 70% of new cases of

malignant primary brain tumors in the United States (143). They are often associated with poor

prognosis with a median survival of only 12 to 15 months in patients with glioblastoma (WHO

grade 4), and 2 to 5 years for those with anaplastic gliomas (WHO grade III), despite surgical

and/or radiotherapeutic intervention, necessitating new modes of therapy. Therefore,

understanding the mechanisms that underlie tumourigenesis will aid development of new

biomarkers and treatments.

3.2 WHO classification of brain tumors

Brain tumors are classified and graded by the WHO largely based on histological features

including mitotic activity, cellularity, atypical nuclei, vascularity and necrosis (144) (Table 3.1).

Grading takes into consideration both clinical and histologic malignancy (145). Whereas grade I

and II neoplasms are deemed largely benign, grade III and IV tumors are most often lethal, with

42

varying survival times (145). As such, grading and subtyping influences therapeutic choices

profoundly (145). Although WHO classification of brain tumors serves as a standard,

uncertainties arise in clinical diagnosis of brain tumors by pathologists. Thus, there is a pressing

need for development of markers of brain neoplasms. Given that PTTG is increasingly proposed

as an immunohistochemical marker in various neoplasms including those of breast, lung, colon

and thyroid, and that it is overexpressed in neoplastic astrocytomas as compared to normal

astrocytes, it is important to investigate its utility as a marker to differentiate between various

brain tumors.

3.3 PTTG expression in normal and neoplastic brain

Only a few studies have investigated PTTG expression in normal brain tissue and brain

tumors, the latter focusing only on tumors of astrocytic origin. Expression of PTTG has been

demonstrated in fetal as well as adult brain (2, 146). In the ventricular zone of the developing

ferret cortex, PTTG was expressed during embroyologic day (E) 11.5-E13.5, peaked in its

expression at E15.5, and was mainly found in mitotically active compared to differentiated tissue

(147). Levels of PTTG began to decrease at E18.5 and were detectable in postnatal day 2 (P2),

but were no longer expressed in adult brains (147). In contrast, in human fetal brain, PTTG

mRNA and protein expression was detectable from 7 weeks of gestation, significantly lower

PTTG expression being seen in the first and second trimesters as compared to the adult cerebral

cortex (adult, N=12; fetal, N=61) (2). Interestingly, expression patterns of FGF-2, thought to be

involved in PTTG pathways in an autocrine loop, parallels that of PTTG. In vitro, PTTG

expression upregulates FGF-2 in fetal neuronal NT-2 cells. Human adult brain shows strong

expression of FGF-2, particularly in CNS neurons and the cerebellar purkinje cells (148) and

FGF-2 knockout in mice results in neurological defects (149, 150). Additionally, FGF-2 is often

43

overexpressed in low-grade gliomas (WHO grade I and II) (151). In cell culture, FGF-2 also

cooperates with PDGF to prevent differentiation (151). Thus, parallel expression patterns of

PTTG and FGF-2 highlight the importance of PTTG in human brain development, making it a

potential neoplastic factor in brain tumor development and progression (151). It is thus essential

to investigate PTTG expression profiles in brain tumors of different subtypes. Indeed, Tfelt-

Hansen et al. found PTTG expression to be higher in neoplastic than in normal astrocytes (55).

In their study, Tfelt-Hansen et al. reported elevated PTTG in 3 astrocytomas and 6 glioblastoma

multiforme (IV) compared to normal astrocytes (55). Limitations of this study include the low

number of cases examined, as well as an absence of WHO classification of the astrocytomas

reported. The latter has implications for patient prognosis and clinical management (144). Our

study addresses these shortcomings by including a large number of astrocytomas of all grades as

well as other CNS tumors, as classified by WHO, in order to clearly establish the pattern of

PTTG expression in brain neoplasms. In another study, Genkai et al. also reported

overexpression of PTTG in 44 gliomas (9 astrocytomas, 9 anaplastic astrocytomas, 26

glioblastomas) (113). Their results showed nuclear PTTG expression to be significantly higher

in glioblastomas than in the lower grade astrocytomas (113). In that PTTG immunoexpression

showed a significant correlation with patient survival (113), it was proposed to be a prognostic

marker in these patients (113). Our work expands on these findings by investigating brain

tumors of various subtypes, including non-glial tumors, in order to examine PTTG expression in

a wider spectrum of brain tumors, and provide insight into the potential differences between

PTTG expression in tumors of both glial and non-glial origin in CNS and PNS.

44

3.4 PTTG and cell proliferation in the CNS

It is currently unclear whether PTTG is associated with increased or decreased cellular

proliferation. One study suggests that the effect of PTTG on cell proliferation is dose-dependent

in neuronal cells (95). Whereas transfection with lower PTTG doses stimulates NT-2 cell

proliferation (150% compared to a vector transfected control), higher doses inhibit cell

proliferation (86% compared to control) (95). The important role of PTTG in brain cell

proliferation was further supported in a study of murine PTTG-/- model, where overexpression

of separase and Rad21, involved in sister chromatid separation and cohesion respectively, was

seen (146). In another study, PTTG depletion in T98G, ON12, and U251 glioma cell lines

resulted in a decrease in cell proliferation and invasion as assessed by cell counts and Matrigel

invasion assay (113). In contrast, in fetal neuronal NT-2 cells, PTTG repression by way of

siRNA method resulted in increased Rad21 and separase levels as well as stimulation of cell

proliferation (113, 146) These contradictory results can be explained by the dose-dependent

association of PTTG levels and cell proliferation. Determination of PTTG expression patterns in

brain tumors of various subtypes with different proliferation potentials will also illuminate on the

role of PTTG in cell proliferation.

3.5 Cellular localization of PTTG in the CNS

Cellular localization of PTTG is important for determination of its role in pathways

associated with tumor development and progression, as PTTG in nucleus would implicate its role

in mechanisms at the DNA and/or RNA level, whereas in the cytoplasm, PTTG would have a

direct role in regulation of other proteins. The limited number of studies investigating the

expression of PTTG in brain tissue and neoplasms present contradictory results. Whereas Genkai

et al. found PTTG to be solely expressed in tumor cell nuclei, Chamaon et al, reported nuclear as

45

well as cytoplasmic PTTG expression (57, 113). Others have not reported PTTG cellular

localization in their investigations (95, 147, 152). Whereas nuclear localization implicates PTTG

in processes including transcriptional tranactivation and mitotic activity, its presence in the

cytoplasm will have yet other consequences, including a role in a myriad of signaling pathways

that take place within the cell cytoplasm. Thus our work aims to establish PTTG cellular

localization. Given the contradictory findings reported in various cell lines and neoplasms, it is

possible that PTTG is translocated between cell nucleus and cytoplasm in a regulated fashion.

Determination of PTTG cellular localization may shed light on the balance between nuclear and

cytoplasmic PTTG expression, and provide a framework for further investigations into

subcellular functions of PTTG.

3.6 The role of PTTG in brain tumourigenesis

In a limited number of studies, PTTG expression has been shown to be upregulated in

gliomas compared with non-neoplastic astrocytes, but its expression pattern and levels in glioma

subtypes and other neoplasms of the CNS and PNS remain unclear. Increasing evidence

suggests the involvement of PTTG in cellular pathways implicated in tumourigenesis in several

tissues including breast, lung, thyroid and pituitary. Thus, it is important to describe PTTG

expression in brain tumors in order to discern its potential role in different subtypes. Not

surprisingly, PTTG is associated with several of the above-mentioned pathways involved in

brain tumor formation and progression, as summarized below.

3.6.1 PTTG and p53-dependent and –independent apoptosis

It is established that PTTG mediates p53 transcription and pathways. Several lines of

evidence support PTTG inhibition of p53 induced apoptosis (78). PTTG knockdown enhanced

46

apoptosis in several cell lines, whereas PTTG overexpression suppressed apoptosis (9, 75, 78)

(Table 2.1). There are however, studies supporting PTTG mediated increase in p53 transcription

and apoptosis (10, 71). Overall, given the intimate association between PTTG and p53, and

involvement of p53 in brain tumors, PTTG expression patterns in brain tumors should be

investigated to shed light on its role in brain tumourigensis.

3.6.2 PTTG, FGF and EGF

PTTG is also closely associated with FGF and EGF and their receptors, implicated in

neuronal differentiation and/or brain tumourigenesis (153). Interestingly, PTTG appears to

modulate FGF-2 expression during neurogenesis (2). Like many factors that are involved in both

cellular differentiation and tumourigenic process, PTTG may also promote brain tumor

formation and/or progression through its association with FGF-2. Furthermore, the close

association between PTTG and FGF-2 is demonstrated in early prolactinoma formation in

rodents, as well as in thyroid carcinomas (36, 154). Expression of PTTG and FGF-2 are highly

correlated in a number of malignancies, including endometrial and thyroid cancers, as well as

acute leukemia (94). Similarly, with regards to EGF and EGFR, EGFR ligands induce, in a

paracrine fashion, PTTG expression in pituitary folliculostellate cells, which are thought to

contain pituitary progenitor cell population (155).

3.6.3 PTTG and C-myc

PTTG transcriptional activation of c-myc oncogene is established (12). PTTG induction

results in increased cell proliferation via c-myc oncogene activation, by binding to the c-myc

promoter region directly (12), indicating that PTTG is involved in tumourigenesis at least partly

through transcriptional activation of c-myc (12). It is also shown that PTTG activation of p53

47

and modulation of its function is achieved by regulation of c-myc expression (10). Brain tumors

often present with aberrant c-myc levels, which is shown to be upregulated by PTTG in other

cell systems, further validating investigation of PTTG in brain neoplasms.

3.6.4 PTTG and IGF-1

PTTG expression was shown to increase following IGF or insulin treatment of malignant

astrocytes, whereas in non-neoplastic rat astroctyes, PTTG only increased in response to IGF-1

treatment (55). PTTG upregulation by IGF and insulin was shown to be mediated through the

PI3K and MAPK pathways, as blocking these signaling pathways abolished PTTG

overexpression (57). These results suggest an interaction pathway between PTTG and IGF-1

and/or insulin in normal and neoplastic astrocytes, warranting investigation of PTTG expression

in brain neoplasms.

3.6.5 PTTG and genetic instability

There is growing evidence on the association between PTTG overexpression and genetic

instability in several tumor types, including those of skin, thyroid, breast, lung, colon and skin (3,

102, 103) (115, 156). In its role as a securin, PTTG prevents premature sister chromatid

separation, and its aberrant expression leads to genetic instability. Malignant brain tumors show

high prevalence of aneuploidy, but it is currently unknown whether this is a cause or effect of the

tumourigenic process. Investigation of PTTG expression in these tumors may illuminate on this

question as PTTG level aberrations are closely associated with aneuploidy in other cancers, and

thus may at least in part account for the genetic instability seen in malignant brain tumors.

3.6.6 PTTG and general tumourigenic pathways

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In addition to the factors that are specific to brain tumors, PTTG is implicated in

upregulation of several other tumourigenic pathways. These include VEGF as well as MMPs,

expression of which is increased in most types of cancers (11). PTTG is established as a

stimulatory factor in induction of VEGF expression and VEGF-induced angiogenesis, a process

required for tumor growth as well as metastasis. Similarly, PTTG induces MMPs expression and

increase its functional activity in vitro, indicating that PTTG is an important factor in promoting

cancer cell metastasis (11).

3.6.7 PTTG as an immunohistochemical maker of tumor malignancy

It is of note that higher PTTG expression in many tumors is associated with poorer

patient outcome and survival rates, as well as higher tumor grades, making PTTG promising as a

prognostic marker in a variety of neoplasms.

In a cohort of 92 Taiwanese women with breast cancer, detection of these PTTG as a part

of a four-marker subset, correlated with tumor size, histologic grade, the presence of lymph node

metastasis, and tumor node metastasis (TNM) stage(157). In another cohort of 90 breast tumors,

PTTG expression was maximal in invasive ductal carcinoma, metastatic breast carcinoma, and

breast carcinoma cell cultures. PTTG mRNA also correlated with invasion in breast tumors (134)

and demonstrated a correlation with and lymph node involvement as well as recurrence over a 5-

year period of follow-up (139). Evidently, PTTG overexpression in breast cancers is associated

with tumor invasion and metastasis, but the contribution of PTTG to these aspects of neoplasia

remains unclear. In small cell lung cancer (SCLC) as well as non-small cell lung cancer

(NSCLC patients), strong PTTG expression was associated with a longer and shorter survival

time respectively (6), as well as more aggressive NSCLC tumors, lymph node, and distant

metastases (6).

49

In esophageal small cell carcinoma (ESCC), PTTG mRNA expression was significantly

higher in tumors of high pathological stage IV disease, as compared with ones of stages 0–III, as

well as in patients with lower survival times (158). In colorectal carcinomas, increased PTTG

expression was correlated with tumor stage (101).

A study of 62 hepatocellular carcinomas showed increased PTTG expression to be an

independent prognostic marker of disease-free survival (159). In yet another series of 147

hepatocellular carcinomas, PTTG expression was found in 61% but showed no correlation with

survival (4). PTTG expression was, however, correlated with higher tumor stage (4).

In lung and hepatocellular carcinomas as well as in ESCC, PTTG expression levels are

predictive of patient survival time. Additionally, PTTG levels are directly correlated with tumor

stage in head and neck small cell carcinoma (HNSCC), ESCC, and colorectal carcinomas.

Higher PTTG levels in colorectal carcinomas are also associated with metastasis. Given the

myriad of neoplastic processes in which PTTG plays a role, it is essential to determine whether

PTTG immunohistochemical staining can serve as a marker of brain tumor type and/or stage.

50

CHAPTER 4: Hypothesis and Objectives

4.1 Rationale

4.1.1 Pituitary Adenomas

PTTG overexpression in pituitary adenomas has been demonstrated, yet its cell type

specific expression in these neoplasms remains unknown. Evidence suggests an association

between PTTG and pituitary hormone subtypes, specifically PRL and GH producing cells. In

vitro study of the effects of PTTG on PRL and GH secretion showed that PTTG stimulated PRL

expression and inhibited GH production (52), implicating PTTG in the process of pituitary

cytodifferentiation, which also involves silencing of GH production in mammosomatotrophs that

can produce both GH and PRL. It is of note that estrogen is also associated with the change in

phenotype from GH producing somatotroph cells to GH and PRL producing

mammosomatotroph cells, promoting PRL production in the pituitary gland. PTTG and estrogen

also show a similar expression pattern in mouse models of PRL pituitary adenomas, and PTTG is

stimulated by estrogen. . Moreover, PTTG mediates increased bFGF secretion, which in turn

promotes lactotroph cell proliferation and PRL secretion. It can be therefore postulated that

PTTG plays a role in early pituitary cytodifferentiation.

PTTG cellular localization in pituitary adenomas remains controversial. Whereas in the

nucleus, PTTG would be involved in cell cycle regulation and regulation of transcriptional

activity of other genes, its role in the nucleus would be vastly different. It is therefore important

to determine PTTG subcellular localization in pituitary tumors to shed light on its potential

function.

51

Additionally, currently there exist no clinico-pathological biomarkers to predict the

clinical course of Cushing’s disease in patients with ACTH-secreting pituitary adenomas. PTTG

has been shown to be associated with such parameters as tumor stage and aggressive behaviour,

and thus it can be proposed that it may also be a suitable biomarker in ACTH-secreting pituitary

adenomas.

4.1.2 Brain Tumors

Preliminary evidence shows that PTTG overexpression in glioma patients is associated

with poor prognosis (113). PTTG is also shown to be involved in neurogenesis, via its

upregulation of FGF-2. In many types of cancers, including gliomas, gene expression and

molecular profiles are similar to those of undifferentiated cells. In gliomas, several of the

affected signaling pathways are also implicated in progenitor cell differentiation and

proliferation. Thus, an improved understanding of the role of such factors will contribute to our

knowledge of cellular differentiation and tumor formation. One such factor is PTTG, shown to

be highly expressed in fetal neuronal cells as well as astrocytomas.

Several physiologic and tumourigenic pathways induced by PTTG are established as essential in

brain tumor biology. These include p53-mediated apoptosis, and factors such as c-myc, IGF-1,

VEGF and MMP. PTTG knockdown in several carcinomas has decreasedtumor metastasis and

vascularization, further supporting an important role for PTTG in neoplastic transformation as

well as aggressive behaviour. Thus, it can be postulated that PTTG will emerge as an important

factor in brain tumor development and/or progression. To our knowledge, very few investigators

to date has studied immunohistochemical expression of PTTG in brain tumors of neuroepithelial

and non-neuroepithelial origin. Our work aims to fill in this gap in research, thereby providing a

better understanding of the neoplastic processes involved in brain tumor biology.

52

Additionally, classification of brain tumors based on immunohistochemical markers is

currently limited. In the WHO scheme, tumors are graded based on histological features,

presenting with major limitations including inter-observer variability as well as molecular

differences between histologically identical tumors (144). Reliable immunohistochemical and

molecular markers are needed to differentiate between tumor subtypes. The role of PTTG in

several tumourigenic pathways makes it a potential candidate for classification of these tumors.

We aimed to determine PTTG expression patterns in brain tumor subtypes as defined by WHO

to provide insight into the utility of PTTG as a potential immunohistochemical marker for tumor

diagnosis.

Immunohistochemistry remains the method of choice for pathologists, despite the

increasing use of molecular techniques in research to identify gene expression profiles associated

with several neoplasms, including those of breast and lung. Individualized patient treatment

based on such gene profiling is gaining more support as evidence emerges on the implications

and importance of genetic variability between patients with histologically similar tumors, with

regards to diagnosis and prognosis. Similarly, molecular profiles of gliomas are used

increasingly to select patients for randomized as well as phase II trials (160). However,

immunohistochemistry remains the main tool for the pathologist, due to impracticality of

molecular analysis in individual patients because of fiscal as well as technical considerations.

To address the need for immunohistochemical profiling of brain tumors, our work

investigated PTTG immunoexpression. Given that PTTG expression and function is likely cell-

type specific, an outline of differential PTTG expression in brain tumors is essential in

investigation of PTTG tumourigenic pathways. By determining a map of PTTG expression

pattern in brain tumors, our work will provide researchers with a foundation that enables probing

53

of this pattern within the context of cellular pathways involving PTTG and tumor development

and/or progression.

4.2 Objectives

1) To determine PTTG expression in different pituitary adenoma subtypes, including GH-,

PRL-, ACTH-, TSH-, FSH/LH/alpha-SU-producing adenomas and null cell adenomas, as

well as medically treated GH and PRL adenomas.

2) To determine PTTG cellular nuclear and/or cytoplasmic localization

3) To establish the role of PTTG as a potential clinico-pathological marker in ACTH

secreting pituitary adenomas of patients with Cushing’s disease

4) To identify the expression patterns of PTTG using immunohistochemistry in brain

neoplasms and correlate PTTG mmunoexpression with WHO classification of brain

tumors

4.3 Hypothesis

The pattern of PTTG immunoexpression in human brain tumors is associated with tumor

subtypes, and is higher in more malignant tumor types such as GBM and medulloblastomas, as

compared to benign brain tumors such as low grade astrocytoma and meningiomas. The pattern

of PTTG expression in human pituitary adenomas is cell type-specific, and particularly

associated with GH- and PRL-producing cell phenotype. PTTG expression in ACTH secreting

pituitary adenomas is associated with with prognostic significance.

54

CHAPTER 5: MATERIALS AND METHODS

5.1 Materials

5.1.1 Pituitary adenomas

Eighty eight pituitary adenomas were selected from the archives of St. Michael’s

Hospital, Department of Laboratory Medicine, by obtaining all blocks available as far back as

1980. The adenomas were obtained at surgery of patients with a clinical or pathologic diagnosis

of pituitary tumor. Each tumor was classified based on the criteria of World Health Organization

(WHO). Specimens included 12 GH, 9 PRL, 10 ACTH, 11 FSH/LH/alpha-SU, and 9 TSH

adenomas. Included were also 14 null cell adenomas. In addition, 13 bromocriptine-treated

PRL-adenomas and 12 octreotide-treated GH adenomas were studied. PTTG Immunostained

specimens with minute tissue samples, and those with extensive hemorrhage and necrosis were

excluded.

5.1.2 ACTH secreting pituitary adenomas of patients with Cushing’s disease

Fifty four ACTH-secreting pituitary adenoma specimens from patients with Cushing’s

disease were selected from the archives of St. Michael’s hospital, based on availability and

completion of clinical data included in our study. All patients had clinically and histologically

verified Cushing’s disease and had undergone transsphenoidal surgery.

5.1.3 Brain Tumors

Eighty eight brain tumors were selected from the surgical archive of St. Michael’s

hospital, Department of Laboratory Medicine. The tumors were obtained at surgery of patients

diagnosed with a brain neoplasm, and were classified routinely by staff neuropathologists

55

according to criteria established by the WHO. Specimens consisted of 13 glioblastomas (WHO

grade IV), 5 anaplastic oligoastrocytomas (WHO grade III), 4 anaplastic oligodendrogliomas

(WHO grade III), 8 oligoastrocytomas (WHO grade II), 4 oligodendrogliomas (WHO grade II), 9

pilocytic astrocytomas (WHO grade I), 10 myxopapillary ependymomas (WHO grade I), 4

hemangiopericytomas, 8 schwannomas and 23 meningiomas (WHO grade I).

5.2 Methods

5.2.1 Clinico-pathological data of patients with Cushing’s disease caused by ACTH-

secreting pituitary adenomas

Clinico-pathological data retrieved from files of patients with Cushing’s disease included

patient age and sex, tumor size, invasiveness (assessed based on preoperative magnetic imaging

and on surgical findings of infiltration into the dura matter and/or the cavernous sinus), and

proliferative activity (assessed by Ki-67 labeling), as well as tumor recurrence (Table 5.1).

5.2.2 Morphologic studies

5.2.2.1 Pituitary adenomas

All specimens were fixed in 10% buffered formalin, routinely processed, paraffin

embedded, cut at 4-6 μm, and stained with hematoxylin and eosin (H&E). The streptavidin-

biotin-peroxidase complex method was used and antisera were directed against growth hormone

(GH), prolactin (PRL), adrenocorticotrophic hormone (ACTH), thyroid stimulating hormone

(TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), as well as the alpha

subunit of glycoprotein hormones (alpha-SU). Immunostaining for MIB-1 (Immunotech,

Westbrook, ME), to detect Ki-67 labeling was also performed. In many cases, electron

56

microscopy was also performed for diagnostic purposes. For immunostaining of PTTG a mouse

monoclonal antibody (1:75, Abcam, Cambridge, UK) was used. Routine deparaffinization,

rehydration, and blockade of endogenous peroxidase activity were carried out. Sections were

then microwaved in 0.1 mm sodium citrate buffer (pH 6.0), incubated with goat anti-serum and

subsequently exposed to the streptavidin-biotin peroxidase complex. Diaminobenzidine served

as the chromogen. For positive control of PTTG immunoreactivity, formalin-fixed, paraffin-

embedded mouse testicular tissue was used. Replacement of the primary antibody with PBS

served as a negative control. All samples were stained simultaneously to avoid methodological

variability.

5.2.2.2 ACTH secreting tumors of patients with Cushing’s disease

ACTH secreting pituitary adenomas were immunostained for PTTG as described earlier,

with the exception of the antibody used. For immunostaining of ACTH adenomas, a polyclonal

antibody (Dilution: 1:75, Zymed, CA) was used.

5.2.2.3 Brain tumors

Routine staining of pilocytic astrocytoma I, GBM IV, anaplastic astrocytoma III, and

anaplastic oligodendroglioma III included immunostaining for GFAP and Ki-67. Ependymoma I

tumors were routinely stained for S100. Hemangiopericytoma routine immunostaining included

vimentin and EMA (epithelial membrane antigen). Immunostaining of schwannomas included

S100. Meningiomas were stained for vimentin, cytokeratin, and EMA. PTTG immunostaining

was performed as described in the previous section.

5.2.3 Microscopy

57

5.2.3.1 Pituitary tumors

PTTG immunopositivity was evaluated using a microscope at high magnification (x400).

Both the intensity and percentage of positive cells were studied blindly and independently by two

observers (FS and DC) who assessed ten random high power fields per specimen. One observer

(the author, FS) repeated the assessments after 7 days to determine intra-observer reliability. The

intensity was evaluated on a scale of 0-3 (0=none, 1= slight, 2= moderate, 3=strong) (Figure

5.1). The percentage of cells demonstrating nuclear and/or cytoplasmic positivity was assessed

as the number of immunopositive nuclei and cell cytoplasms respectively as a fraction of the

total number of cells in each field. For each specimen, average immunopositivity intensity and

percentage were recorded as a mean of the ten high-power fields. Hot spots (focal areas of

intense immunostaining) as well as areas demonstrating necrosis, fibrosis and artifact were

excluded. The histoscore (68) for each specimen was calculated by multiplication its intensity

and percentage positivity (i.e. 1 x % of positive cells, 2 x % of positive cells, 3 x % positive

cells), with 300 being the maximum attainable histoscore. The histoscore provides a composite

of the percentage of positive cells and the intensity of positivity, providing a number that takes

into account both PTTG immunopisitivity and intensity of staining. Intra-observer reliability

was determined by re-assessment of sections by one observer after 7 days, and inter-observer

reliability by assessment of the kappa value for the values obtained by the 2 observers. The

primary author (FS) trained the second observer (DC).

5.2.3.2 Brain tumors

PTTG immunopositivty was evaluated by recording the intensity of staining. Two

observers independently evaluated each section at high magnification (x400). Nuclear and

cytoplasmic PTTG immunostaining intensity was assessed using a 0-3 scale (0= none, 1= slight,

58

2= mild, 3= moderate) (Figure 5.2). Intra-observer reliability was determined by re-assessment

of sections by one observer after 7 days, and inter-observer reliability by assessment of the kappa

value for the values obtained by the 2 observers.

Figure 5.1. PTTG immunostaining intensity scores. Immunoreactivityw as rated on a scale of 0 to 3. The figures

show microphotographs of pituitary adenomas with an immunopositivity intensity of a) none (0), b) slight (1), c)

mild (2), and d) strong (3).

59

Figure 5.2 PTTG immunopositivity in brain tumors with intensity of a) none (0), b) slight (1), c) moderate (2), and

d) strong (3).

5.2.4 Statistical analysis

Student t-test (SPSS statistical program) was used to find significant differences between

pituitary adenoma subtypes. Both the intensity and percentage of positive cells were studied

blindly and independently by two observers who assessed ten high-power fields per specimen.

Both observers rated PTTG staining intensity on a scale of 0 to 3 (0=none, 1=slight, 2=mild,

3=strong). The percentage of PTTG immunopositive cells was independently determined by

each observer by counting PTTG immunopositive cells in 10 high-power fields and calculating

an average of the 10 fields. One observer repeated the assessments after 7 days to determine

60

intra-observer reliability. For assessment of inter- and intra-observer variability for percentage

(range 0-100%) of immunopositive cells in pituitary adenomas was calculated using the

correlation coefficient (R2) was calculated. The kappa statistic was used to determine inter-

observer and intra-observer reliability with regards to the immunopositivity score (range 0-3).

Kappa sample size calculation was performed using the R statistical program to determine the

minimum sample size requiring assessment by two observers. Using R program, sample size

calculation for kappa was determined to be 43. The program required the following parameters

to be estimated (161):

a) Kappa to detect = 0.90

b) Power = 0.70

c) Proportion of positive ratings: 0.50

61

CHAPTER 6: RESULTS: PITUITARY ADENOMAS

6.1 PTTG cellular localization

PTTG immunohistochemical expression was found to be cytoplasmic in all pituitary

adenomas studied, including PRL, GH, ACTH, FSH/LH, TSH, and null cell adenomas (Figure

6.1). Predominantly a peri-nuclear pattern of PTTG immunopositivity was evident in most

adenomas regardless of subtype, with PTTG appearing to accumulate in the Golgi complex in a

peri-nuclear fashion (Figure 6.1). Nuclear PTTG immunoexpression was absent (Figure 6.1).

6.2 Percentage of PTTG Immunopositive Cells

The percentage of PTTG immunopositivity across all pituitary adenoma cell types was

52% ± 16 (range 21% to 94%) (Figure 6.2). The percentage of PTTG immunopositive cells

ranged from 0 to 100% of cells and was highest in GH adenomas (69 % ± 27) followed by TSH

and null cell adenomas (65% ± 91 and 63% ± 13 respectively). The lowest percentage of PTTG

immunoreactive cells was found in PRL adenomas (21% ± 17) (Figure 6.2).

62

Figure 6.1 PTTG immunohistochemical expression is cytoplasmic in a) GH adenoma, b) PRL c) ACTH adenoma,

d) TSH adenoma, d) FSH/LH adenoma, and e) Null cell adenoma.

63

0

20

40

60

80

100

120

PRL (9)

Treated

PRL (13

)

Treated

GH (1

1)

ACTH (10)

FSH/LH/al

pha-S

U(11)

Null (1

4)

TSH(19)

GH(12)

Total (8

9)

Perc

enta

ge P

TTG

Figure 6.2. Percentage of PTTG immunoreactive cells in various pituitary adenoma subtypes. The numbers

in parentheses refer to the number of tumors of each cell type tested. Whereas the lowest PTTG

immunostaining percentage was exhibited by PRL adenomas, GH adenomas demonstrated the highest

percentage of PTTG immunopositive cells.

6.3 PTTG immunoexpression: Intensity of immunopositive cells

The average intensity for PTTG immunostaining across all tumor types was 1.62 ± 0.66

(average ± SD) (range 0.36 to 2.4) (Figure 6.3). The highest intensity of PTTG immunostaining

was observed in GH and TSH adenomas (2.4 ± 0.58 and 2.4 ± 1.03 respectively. The lowest

PTTG intensity was exhibited by treated PRL adenomas (0.36 ± 0.79) (Figure 6.3).

Fifty one percent (45/89) of pituitary adenomas studied exhibited intensity scores of 2

(moderate), with only 4% (4/89) showing PTTG immunopositivity of 0 (none), 28% (25/89)

being 1 (slight), and 28% (15/89) being 3 (strong) (Figure 6.4).

64

The percentage of each pituitary adenoma subtype with intensity scores of 0, 1, 2 or 3 are shown

in Figure 6.5.

6.4 PTTG Histoscore

The histoscore was obtained by multiplying PTTG intensity and percentage of cells that

were immunopositive for PTTG. The mean histoscore across all pituitary tumor types was 110 ±

25 (range 34 to 200) (Figure 6.6). Null cell, TSH and GH adenomas demonstrated the highest

histoscores (200 ± 2, 159 ± 80 and 154 ± 70 respectively), and PRL adenomas had the lowest

histoscore (34 ± 40).

6.5 PTTG histoscore in medically treated and untreated GH and PRL adenomas

PTTG immunoreactivity histoscore was significantly higher in GH adenoma untreated

medically, as compared to those treated (p= 0.02) (Figure 6.6). Although PTTG histoscore was

lower in treated PRL adenomas as well, this difference was not statistically significant (p=0.13).

(Figure 6.7).

6.6 PTTG immunoposotivity percentage-intensity scatter plots

As indicated in Figure 6.7, PTTG immunoreactivity patterns in terms of intensity and

positivity was clearly variable among different pituitary adenoma subtypes. Whereas the

percentage-intensity scatter plot of medically treated GH adenomas shows no cases with

intensity of less than 1.3, that of treated GH adenomas demonstrates a wider scatter with an

apparent shift of cases to the left of the scatter plot. A look at the other plots also reveals

differences in patterns of PTTG intensity and percentage distribution among various pituitary

adenoma subtypes (Figure 6.7). For instance, the gonadotroph adenoma percentage-intensity

65

scatter plot show intensity scores of mostly 1 and 2, with a wide range of percentage PTTG

imunoreactivities, whereas plotting the TSH cases reveals mostly higher intensity scores (range:

2 to 3).

6.7 Correlations between PTTG immunostaining and clinico-pathological variables in

patients with Cushing’s disease due to ACTH secreting adenomas

No significant correlations were found between immunohistochemical expression of

PTTG and clinico-pathological variables in patients with ACTH-secreting pituitary adenomas.

PTTG expression was not correlated with patient age or sex. Furthermore, no correlations were

found with tumor invasiveness, size and recurrence (Table 6.1).

0

1

2

3

4

Trea

ted PR

L (1

3)

PRL (

9)

FSH/

LH/a

lpha

-SU

(11)

ACTH

(10)

Null (

11)

Trea

ted GH

(11)

TSH (9

)

GH (1

2)

Tota

l (89

)

Inte

nsi

ty s

core

Figure 6.3. Intensity of PTTG immunoreactive cells in various pituitary adenoma subtypes. The

numbers in parentheses refer to the number of tumors of each cell type tested. Treated and

untreated PRL adenomas exhibited the lowest intensity of PTTG immunohistochemical staining,

whereas GH adenomas demonstrated the highest intensity scores.

66

0

20

40

60

80

100

0 1 2 3

PTTG Intensity Score

Perc

enta

ge o

f Tum

ors

Figure 6.5. Intensity score distribution by pituitary adenoma subtype. Variously shaded bar

portions represent different tumor subtypes that exhibited the corresponding intensity scores.

Figure 6.4. Distribution of intensity scores by the number of cases per each intensity

score of 0 to 3. Majority of tumors demonstrated an intensity score for PTTG

67

0

50

100

150

200

250

300

Treated PRL (

13)

PRL (9)

FSH/LH/alph

a-SU(11

)

ACTH (10)

Treated G

H (11)

Null (1

4)

TSH(19)

GH(12)

Total (8

9)

PTTG

His

tosc

ore

Figure 6.6. Histoscore of pituitary adenoma subtypes. For calculation of the histoscore, average intensity and percentage scores were multiplied for each subtype. The lowest histoscore is seen in PRL adenomas, and the highest score in GH adenomas.

0

20

40

60

80

100

Treated PRL Untreated PRL Treated GH Untreated GH

Perc

enta

ge P

TTG

0

1

2

3

4


PTTG

Inte

nsity

0

100

200

300


PTT

G H

isto

scor

e

Figure 6.7: Treated and untreated GH and PRL adenomas. Statistically significant differences are seen

between treated and untreated GH adenomas when comparing PTTG intensity,percentage positivity as well

68

v

GH Adenomas: Intensity and % positivity

0

50

100

150

0 1 2 3

Intensity

% P

ositi

vity

Treated GH Adenomas: Intensity and % Positivity

0

50

100

0 1 2 3

Intensity

% P

ositi

vity

PRL adenomas: Intensity and % positivity

0

50

100

0 1 2 3

Intensity

% P

sitiv

ity

Treated PRL Adenomas: Intensity and % Positivity

0

50

100

0 1 2 3

Intensity

% P

ositi

vity

Gonadotroph Adenomas: Intensity and % Positivity

0

50

100

0 1 2 3

Intensity

% P

ositi

vity

TSh Adenomas: Intensitya nd % Positivity

0

50

100

0 1 2 3

Intensity

% P

ositi

vity

ACTH adenomas: % and Positivity

0

50

100

0 1 2 3

Intensity

% P

ositi

vity

Null Cell Adenomas: Intensity and % Positivity

0

20

40

60

80

100

0 0.5 1 1.5 2 2.5 3

Intensity

% P

ositi

vity

Figure. 6.8 Scatter plots of PTTG immunopositivity intensity and percentage in various pituitary

69

6.8 Intra- and inter-observer reliability

Intra-observer reliability was high, with a kappa value of 0.93 indicating almost perfect

agreement (161). Similarly, a high degree of inter-observer variability was seen, as indicated by

the correlation coefficient of 0.87.

Table 6.1. Clinico-pathological variables in patients with Cushing’s disease showed no correlation with PTTG

immunostaining.

Clinical Parameters N=54 R2

Age 39 0.02

Range 19-57

Sex 0.04

Males 5

Females 49

Recurrence 7 0.01

Tumor size 0.17

Macro-adenomas 15

Micro-adenomas 39

Tumor Invasion 8 0.03

Total 54

70

CHAPTER 7: RESULTS: BRAIN TUMORS

7.1 PTTG immunoexpression intensity in brain tumor subtypes

Only intensity was evaluated for brain tumors, without assessment of percentage of positive

cells, as the latter proved impractical. Nuclear PTTG immunopositivity intensity, on a scale of 0

to 3 (Figure 7.1), was highest in medulloblastoma IV (2.3 ± 1.2), followed by astrocytoma III (2

± 0.7) and glioblastoma IV (1.9 ± 0.5) (Figure 6.2 , Table 7.1). Lowest PTTG

immunoexpression was found in hemangiopericytoma III (0 ± 0) (Table 7.1).

71

Figure 7.1. PTTG

immunoexpression in a)

hemangiopericytoma, b)

pilocytic astrocytoma I,

c) ependymoma I, d)

meningioma I, e)

oligodendroglioma III, f)

oligoastrocytoma III, g)

schwannoma I, h) GBM

IV, i) astrocytoma III,

and j) medulloblastoma.

72

Table 7.1 Immunohistochemical expression of PTTG in brain tumors examined, arranged from lowest

to highest nuclear expression of PTTG. It is of note that hemangiopericytoma showed no PTTG

immunoreactivity. GBM IV: Glioblastoma Multiforme IV.

Tumor Type

Number (n)

Nuclear

(mean ± SD)

Cytoplasmic

(mean ± SD)

Hemanigiopericytoma III 4 0 ± 0 0 ± 0

Pilocytic astrocytoma I 9 0.9 ± 0.3 0.9 ± 0.3

Ependymoma I 10 0.9 ± 0.3 0.9 ± 0.3

Meningioma I 23 0.9 ± 0.3 1 ± 0.3

Oligodendroglioma III 4 1 ± 0 1 ± 0

Oligoastrocytoma III 8 1.3 ± 0.7 0.6 ± 0.7

Schwanomma I 8 1.5 ± 1.2 1.4 ± 1

GBM IV 13 1.9 ± 0.5 1.2 ± 0.6

Astrocytoma III 5 2 ± 0.7 1 ± 0.7

Medulloblastoma IV 4 2.3 ± 1.2 0 ± 0

Total 88 1.3 ± 0.8 1.03 ± 0.5

73

Figure 7.2 PTTG nuclear immunostaining intensity distribution in brain tumors examined. Majority of

tumors showed an immunostaining score of 1.

7.2 PTTG Cellular Localization

PTTG nuclear and cytoplasmic expression results are found in Figure 7.3. PTTG

immunoexpression was entirely nuclear in medulloblastomas (N=4). In glioblastoma IV,

astrocytoma III and Oligoastrocytoma III tumor types nuclear expression of PTTG was more

pronounced than its cytoplasmic expression (Table 7.2, Figure 7.4). Nuclear and cytoplasmic

PTTG expressions were not significantly different in schwannomas, ependymomas, pilocytic

astrocytomas, oligodendrogliomas, and meningiomas. Hemangiopericytomas did not show any

PTTG immunoexpression (Table 7.2, Figure 7.5). It is of note that the overall nuclear and

cytoplasmic expression was higher in glial tumors as compared to non-glial tumors (Figure 7.6).

74

Table 7.2 Comparison of nuclear and cytoplasmic PTTG expression in glial and non-glial brain

tumors. * : P value < 0.05.

2. A. Significantly different nuclear and cytoplasmic PTTG expression

Tumor Type Number (n)

Nuclear

(mean ± SD)

Cytoplasmic

(mean ± SD)

Glial 26

Glioblastoma Multiforme IV * 13 1.9 ± 0.5 1.2 ± 0.4

Astrocytoma III * 5 2 ± 0.7 1 ± 0.7

Oligoastrocytoma III * 8 1.25± 0.63 0.63 ± 0.5

Non-glial 4

Medulloblastoma IV* 4 2.3 ± 1.2 0 ± 0

2. B. No significant difference between nuclear and cytoplasmic PTTG

Tumor Type Number (n)

Nuclear

(mean ± SD)

Cytoplasmic

(mean ± SD)

Glial 13

Pilocytic Astrocytoma I 9 0.9 ± 0.3 0.9 ± 0.3

Oligodendroglioma III 4 1 ± 0 1± 0

Non-Glial 22

Schwannoma I 8 1.5 ± 1.2 1.4 ± 1

Hemangiopericytoma III 4 0 ± 0 0 ± 0

Ependymoma I 10 0.9 ± 0.3 0.9 ± 0.3

75

7.3 PTTG nuclear expression and tumor aggressiveness

As nuclear staining was significantly higher in most malignant tumors compared to low

grade ones, we compared nuclear immunostaining of PTTG only, an not cytoplasmic staining.

Among glial tumors, glioblastoma IV and astrocytoma III tumors showed the highest nuclear

PTTG intensity (1.9 ± 0.5 and 2 ± 0.7 respectively), whereas oligoastrocytoma III (1.3 ± 1.7),

oligodendroglioma III (1 ± 1), and pilocytic astrocytoma I (0.9 ± 0.3) exhibited significantly

lower nuclear expression of PTTG (P = 0.0001). Among non-glial tumors, medulloblastoma IV

had the highest PTTG immunopositivity (2.3 ± 1.2), followed by schwannoma I (1.5 ± 1.2),

meningioma I (0.9 ± 0.3), ependymoma I (0.9 ± 0.3) and hemangiopericytoma III (0 ± 0).

0

1

2

3

HPC (4)

Pilocy

tic as

trocy

toma (9

)

Epend

ymoma (

10)

Meningio

ma (23)

Oligod

endroglio

ma (4)

Oligoa

stroc

ytoma III

(8)

Schwan

omma (8)

GBM (13)

Astroc

ytoma I

II (5)

Medull

oblas

toma (

4)

Total (8

8)

Figure 7.3 Intensity of PTTG immunoexpression in different brain tumor types. The numbers in

parentheses indicate number of tumors tested. HPC: hemangiopericytoma III; GBM: Glioblastoma

Multifome IV. It is of note that hemangiopericytoma showed no PTTG immunostaining, whereas

medulloblastoma exhibited only nuclear PTTG immunoreactivity.

Nuclear

Cytoplasmic

Inte

nsity

Scor

e

76

0

1

2

3

4

GBM IV

Astrocytoma II

I

Oligoastr

ocytoma III

Medulloblastoma

Inte

nsit

y s

co

re

Nuclear

Cytoplasmic

Figure 7.4 Higher nuclear than cytoplasmic PTTG immunoexpression in the glial GBM IV, astrocytoma III, and

oligoastrocytoma III and the non-glial medulloblastoma IV. GBM IV: Glioblastoma Multiforme IV.

0

1

2

3

Piloc

ytic

Astroc

yt...

Oligod

endr

oglio

ma III

Epen

dym

oma

I

Schw

anno

ma

Meningio

ma

Heman

giop

ericy

tom

a

Inte

nsit

y s

co

re

Nuclear

Cytoplasmic

Figure 7.5 No difference between nuclear and cytoplasmic PTTG. HPC: hemangiopericytoma III; Pil Astr:

pilocytic astrocytoma I; EP: ependymoma I; Men: meningioma I; OD: oligodendroglioma III; SCH:

schwannoma I.

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Figure 7.6 Increased nuclear and cytoplasmic PTTG immunoreactivity in glial vs. non-glial tumors.

Statistically significant differences were noted between both nuclear and cytoplasmic PTTG

immunostaining in glial tumors compared to non-glial ones.

7.4 Intra- and inter-observer variability

Intra-observer reliability was high, with a kappa value of 0.98 indicating almost perfect

agreement in rating brain tumor intensity (161). Inter-observer variability was also high, as

indicated by a correlation coefficient of 0.92.

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CHAPTER 8: DISCUSSION: PITUITARY TUMORS

8.1 PTTG immunostaining and clinico-pathological variables in ACTH-secreting adenomas

Our study found no significant correlations between expression of PTTG and patient age

or sex in patients with Cushing’s disease caused by ACTH-secreting pituitary adenomas.

Similarly, no correlation was exhibited between PTTG expression and tumor size, recurrence or

invasiveness. These findings may be due to the small number of invasive and recurrent ACTH-

secreting pituitary adenomas in our study. In general, ACTH adenomas tend to be small and

non-invasive, and as such, it is not surprising to find a lack of correlation between PTTG

expression and such variables. Additionally, tumor size showed no correlation with PTTG

expression, indicating that PTTG may not be a valuable indicator of tumor growth potential.

Further studies in larger series are required to generalize the findings of our study.

8.2 PTTG expression in different adenomas subtypes

Abundant PTTG expression in pituitary adenomas has been shown in several studies

using immunohistochemical and/or PCR techniques. Here, we investigated the potential

correlation between PTTG expression levels and pituitary tumor subtype using

immunohistochemistry. Our results showed that null cell adenomas had the highest PTTG

histoscore, followed by TSH and GH adenomas. The lowest levels of PTTG expression were

apparent in PRL adenomas. Comparison of adenoma subtypes showed GH adenomas to express

significantly higher PTTG histoscores compared to PRL, FSH/LH/alpha-SU, and ACTH

adenomas as well as medically-treated GH and PRL adenomas. Moreover, TSH adenomas

showed substantially greater PTTG histoscores as compared to PRL adenomas as well as treated

GH and PRL adenomas. Our finding of higher PTTG expression in GH adenomas are in keeping

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with those of Minematsu et al., examining 101 pituitary adenomas (29 GH, 12 PRL, 6 ACTH, 1

FSH, 3 TSH, and 50 non-functioning). They found that PTTG expression level was significantly

higher in GH adenomas as well as null cell adenomas as compared to other subtypes (137).

Several other studies have investigated a correlation between PTTG expression and

pituitary adenoma cell type. Our results are in keeping with previous findings of higher PTTG

expression in GH secreting pituitary adenomas compared to other adenoma subtypes (24, 137).

Hunter et al. analyzing PTTG mRNA levels in 40 pituitary tumors (12 GH, 5 PRL, 5 ACTH, 18

nonfunctioning) showed elevated expression in GH secreting adenomas (2.7 fold) compared to

nonfunctioning adenomas (24), suggesting cell type-dependent expression of PTTG. Further, in

vitro GH secretion and PTTG expression were correlated (R=0.41, P<0.01) (24, 162).

Interestingly, targeted PTTG overexpression using the mouse alpha-SU promoter induces focal

PTTG expression not only in LH cells, but surprisingly in GH-producing cells as well (46).

Alpha-SU promoter targets PTTG expression to pituitary stem cells and is not associated with

mature somatotroph cells (46). Thus, PTTG expression in GH adenomas in this mouse model

further supports a strong link between PTTG and GH producing cells in the pituitary gland.

Uccella et al. found that GH and PRL adenomas had elevated PTTG expression levels, as

well as multiple chromosome gains, when compared to other pituitary adenoma subtypes (116).

Somatotroph and lactotroph adenomas also showed abnormal nuclear shape and increased

number of centrosomes (116). Previous studies have also shown increased chromosome number

in GH and PRL cells (163), (99, 164, 165). The elevated expression of PTTG in GH and PRL

pituitary adenomas is in line with the role of PTTG in inhibition of premature sister chromatid

separation, and its importance in maintenance of euploidy (166). Sister chromatids bind during

metaphase via cohesin molecule, which is degraded by separin to allow progression of cell cycle

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into anaphase and sister chromatid separation. PTTG has been shown to inhibit chromatid

separation by binding to separin and thereby inhibiting degradation of cohesin (167). PTTG-/-

mouse embryo fibroblasts demonstrate aneuploidy (50), and a strong correlation has been shown

between overexpression of PTTG and chromosomal aberrations in colorectal and thyroid

neoplasms (101, 102). Thus, higher PTTG histoscores found in GH adenomas may also account

for the higher reported incidence of aneuploidy in these tumors. Our findings showed the lowest

PTTG expression in PRL adenomas, which also reportedly have increased incidence of abnormal

karyotypes (99, 100). Therefore, further research into a potential correlation between aberrant

PTTG expression and ploidy status of pituitary adenomas is needed.

In vitro studies also support a relationship between PTTG and GH as well as PRL cell

types. Horwitz et al. studied the effects of PTTG on PRL and GH secretion in vitro (52).

Overexpression of PTTG C-terminus, as a dominant negative, abrogated PRL secretion 10-fold,

whereas GH secretion was increased 4-fold (52). Interestingly, subcutaneous injection of cells

overexpressing PTTG C-terminus showed elevated GH and lower PRL serum levels (52). These

results suggest that PTTG stimulates PRL expression and inhibits GH production, further

supporting the notion that PTTG is intimately associated with GH and PRL cell types in the

pituitary gland. Our study found that PTTG is expressed in highest levels among pituitary

hormonal subtypes in GH secreting adenomas and in lowest levels in PRL secreting adenomas.

Our results can be interpreted to support the in vitro findings of PTTG dependent PRL

upregulation; it may be that PTTG is upregulated in GH adenomas in an attempt to suppress the

inherent GH secretion by tumor cells, and conversely, in PRL-secreting adenomas, PTTG is

downregulated in a feedback loop in order to suppress the PRL-inducing effects of PTTG.

Although the precise mechanisms associated with PTTG upregulation of PRL and suppression of

GH are unknown, there is a relationship, which requires further investigation.

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8.3 Lowest and highest PTTG expression in PRL and GH adenomas respectively

It is of note that the highest PTTG levels were detected in GH adenomas, with PRL

adenomas showing the lowest levels of PTTG immunoexpression. Factors underlying this stark

difference in PTTG levels may exist in the process of cytodifferentiation that gives rise to PRL-

secreting cells from a common ancestor of GH secreting pituitary cells (Figure 3.2). GH and

PRL secreting pituitary cells arise from a common cell lineage expressing the transcription factor

Pit-1, which is gene is selectively expressed in pituitary adenomas expressing GH, PRL and TSH

(20). Evidence supports the role of ER-alpha in development of PRL expression in somatotrophs

expressing Pit-1 cells (20). Interestingly, estrogen is shown to regulate PTTG expression within

the context of PRL adenoma development (36, 54). Estrogen increases PTTG levels concordant

with early lactotrophic hyperplastic response. Estrogen induces bFGF, which in turn induces

expression of PTTG. Moreover, PTTG mediates secretion of bFGF, which augments lactotroph

cell proliferation and PRL secretion as well as angiogenesis. PTTG in turn is known to stimulate

angiogenesis via VEGF and bFGF. Thus, PTTG is an important regulatory element in PRL cell

tumourigenesis, as it is expressed and mediated early during PRL adenoma development (36).

Given the association of PTTG with early PRL adenoma development, it is possible that

PTTG plays a role in cytodifferntiation of mammosomatotrophs into lactotrophs (Figure 8.1).

The clear difference in PTTG levels between GH and PRL levels may stem from this yet

unknown role, and may indicate that the levels of PTTG expression are either in part responsible

for this cytodifferentiation, or are a by-product of cellular events mediating it. The differential

expression of PTTG in a cell-specific manner warrants further investigation into its role in

cellular differentiation. The notably higher expression of PTTG in GH adenomas as compared to

most other adenoma subtypes may suggest a role for PTTG in GH adenoma tumor development

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and/or progression.

Figure 8.1 A diagram of somatoroph stem cell differentiation into mammosomatotrophs and subsequently

lactotroph and somatotroph cells. Estrogen, acting through ER-alpha, promotes somatotroph stem cell

differentiation into mammosomatotrophs. A “GH-repressor” is thought to promote the differentiation of

mammosomatotrophs into lactotrophs in turn. PTTG may potentially be one such repressor, as it is closely involved

in estrogen-induced PRL adenoma formation and progression.

A summary of evidence pointing to the role of PTTG in molecular pathways implicated

in tumourigenic pathways in GH and PRL adenomas is shown in a schematic in figure 8.2.

Evidence shows upregulation to a greater extent of VEGF in GH adenomas and to a

lesser extent in PRL adenomas. This is in keeping with our findings of higher PTTG in GH

adenomas and lower levels in PRL ones, as PTTG is shown to stimulate VEGF production (81).

Similarly, the anti-apoptotic factor, Bcl-2, is found to be higher in GH adenomas and

lower in PRL adenomas. Bcl-2 expression may be mediated by PTTG indirectly, as PTTG is

known to both upregulate and downregulate p53 expression, which in turn inhibits Bcl-2

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expression. Thus, it may be that in GH adenomas, PTTG in fact inhibits p53, leading to reduced

inhibition of Bcl-2 by p53, and increased Bcl-2 expression in GH adenomas. In contrast, in RL

adenomas, PTTG may upregulate p53 expression, leading to enhanced inhibition of Bcl-2

expression by p53 and lower Bcl-2 expression in these tumours.

Among other factors known to be associated with PTTG is bFGF, which is expressed at

high levels in PRL adenomas. In turn, bFGF is shown to upregulate PTTG in an autocrine loop.

This autocrine loop has been shown to augment lactotroph cell proliferation in estrogen-induced

PRL adenoma development in mouse models.

Our findings of relatively higher PTTG expression levels in GH adenoma sand lower

levels in PRL adenomas are in keeping with the molecular pathways associated with PTTG as

well as findings of expression levels of such molecular factors in PRL and GH adenomas.

8.4 Medically Treated vs. Non-treated GH and PRL adenomas

Our results demonstrated significantly higher PTTG histoscore in GH adenomas as

compared to octreotide-treated GH adenomas (P= 0.002), indicating the effect of treatment on

PTTG expression in somatotroph adenomas. Bromocriptine-treated PRL adenomas showed a

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Figure 8.2. A schematic overview of potential PTTG pathways in GH and PRL cells. PTTG is known to

stimulate VEGF secretion, which is shown to be relatively lower in PRL cells and higher in GH cells, an effect that

may be attributed to the higher PTTG levels in GH cells and lower levels in PRL cells. Yellow arrows show the p53-

mediated pathways. P53 is shown to be both upregulated and downregulated by PTTG. In this model, p53, which

is shown to inhibit the anti-apoptotic Bcl-2, is shown to be inhibited by PTTG in GH cells and stimulated in PRL

cells, in keeping with the Bcl-2 levels, which are lower in the latter and higher in GH cells. P53 is also shown to

inhibit IGF-1 levels, and in this schematic, PTTG inhibition of p53 leads to increased IGF1, which in turn binds to

IGF1-R. Both IGF and IGF1-R are known to inhibit GH production in somatotroph cells. Another factor

upregulated in PTTG in PRL adenomas is bFGF, which in turn upregulates PTTG. Overexpression of bFGF is

associated with PRL adenomas. This model also shows increased aneuploidy in both GH and PRL cells as mediated

by aberrant PTTG expression.

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higher PTTG expression, but this difference did not reach statistical significance (P =

0.13). It is currently unknown whether the decreased PTTG expression seen in treated GH and

PRL adenomas is secondary to cellular changes seen in treated tumors. Nevertheless, the finding

of abrogated PTTG expression in treated GH adenomas supports a role for PTTG in pituitary

tumourigenic mechanisms. It appears that, following treatment, PTTG is downregulated as

evident by lower percentage of PTTG immunopositive cells as well as lower mean intensity of

PTTG expression.

Morphological and ultrastructural examination of GH adenomas treated with

somatostatin analogs (octreotide, lanreotide) demonstrates slight to mild morphological changes

including an increase in the number and size of secretory granules, indicating inhibition of

protein secretion, but no nuclear changes are seen, unlike PRL adenomas treated with dopamine

agonists (168). Several explanations may be offered with regards to decreased PTTG expression

in somatostatin analog-treated GH adenomas: A) Decreased PTTG expression in treated GH

adenomas may be a primary effect of medical treatment. It is known that somatostatin analogues

inhibit cell proliferation by inducing changes in several mechanisms, including inhibition of

DNA synthesis and cell cycle progression, thus affecting the expression level of many cell cycle

regulatory factors (169, 170). One such factor is p27, whose levels are upregulated in vitro, in

keeping with its stimulatory role in cell cycle arrest (170). It would be valuable to investigate the

correlation between the expression of the proliferation marker Ki-67 and PTTG, in treated GH

adenomas. B) Alternatively, PTTG reduction may be only secondary to cell cycle arrest and

decreased cellular proliferation in treated GH adenomas (169, 171). Reduced cellular

proliferation and cell cycle activity results in a decrease in several cell cycle proteins as detected

by immunohistochemical techniques, and one such protein may be PTTG, whose decrease may

only be a by-product of decreased cellular proliferation. C) Interestingly, somatostatin analogs

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inhibit the MAPK pathway in GH3 cells (169, 170). MAPK pathway mediates induction of

PTTG through EGFR ligands in pituitary folliculostetllate cells (37). Therefore, PTTG reduction

in somatostatin analog-treated GH adenomas may be mediated through abrogation of MAPK

pathway.

PTTG expression was found to be higher in dopamine agonist-treated PRL adenomas as

compared to non-treated ones, but the difference was not statistically significant. This finding

may appear counterintuitive, as dopamine agonist treated PRL adenomas exhibit lower mitotic

and MIB-1 indices, as well as p27 expression, suggesting inhibition of cell proliferation (172).

Dopamine agonist treatment of PRL adenomas induces notable changes in cellular morphology

of tumors, including marked reduction in rough ER (RER) and Golgi membranes, as well as

nuclear changes (168). The nuclear changes indicate that the effect of drugs is at the

transcription level (168), suggesting that the increased PTTG level in treated PRL adenomas may

be transcriptionally mediated. Although PTTG is associated with cellular proliferation as a cell

cycle protein, its anti-proliferative effects have also been demonstrated (1, 73, 74). Several

studies have showed a lack of correlation between PTTG and cellular proliferation (Kim et al.

2006b, Saez et al. 2002, Boelaert et al. 2003). In a series of 101 pituitary adenomas, no

correlation was found between PTTG expression and cell proliferation (92). PTTG may in fact

abrogate cell proliferation through induction of apoptosis and delay of mitosis, as seen in treated-

PRL adenomas in the present study(136, 168). It has been proposed that PTTG may exert its

proliferative effects dependent on its dose rather than its expression per se (95). Thus, a tendency

to see higher PTTG levels in treated PRL adenomas may point to the role of PTTG in mediating

anti-proliferative effects in this cell type.

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It should be noted that some PRL adenomas respond only mildly to medical treatment,

and the corresponding morphological changes are also modest. The tumors fail to demonstrate

alterations in their RER, Golgi apparatus, or nucleus, but do exhibit an increase in secretory

granule size and number, as well as lysosomal activity and crinophagy (168). These changes

suggest a modification at the post-translational level, corresponding to abrogated protein

secretion and increased lysosomal degradation (168). A decrease in protein secretion, as

observed in PRL adenomas mildly-responsive to dopamine agonist therapy, may have lead to

accumulation of PTTG in some of the treated PRL adenomas examined in our series.

It is remarkable that PTTG is expressed highly in GH and in much lower levels in PRL

adenomas, especially given the cytodifferentiation of GH and PRL secreting cells from a

common mammosomatotroph ancestor. Both GH and PRL are secreted by Pit-1 expressing cells

and are thought to share the same progenitor cell lineage, which later differentiates into GH-

secreting somatotroph cells, followed by a change that leads to appearance of

mammosomatotroph cells producing both GH and PRL. These cells are then thought to give rise

to lactotroph cells that only produce PRL. The factors involved in these pathways are not

understood. Estrogen, acting via its receptor (ER-alpha), regulates PRL gene expression (22,

23). ER-alpha is expressed in PRL as well as gonadotrophin beta-SU producing cells (22).

Although some studies show ER-alpha expression in GH producing cells, these are thought to be

mammosomatotrophs that produce PRL as well (173). This is supported by the observation that

GH pituitary adenomas that do not produce PRL adenomas show no ER-alpha expression (174).

Additionally, ER-alpha expression in human fetal pituitary gland appears approximately at 12

weeks, concordant with development of PRL secretion (174), further supporting the role of

estrogen in PRL cell differentiation. Mammosomatotroph transformation into lactotroph cells

requires silencing of GH production as well, and one may propose that the same factor that

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promotes PRL production, suppresses GH production. Interestingly, PTTG expression is closely

associated with estrogen in estrogen-induced PRL secreting adenomas in a murine model of

pituitary tumourigenesis (36). PTTG is induced early on by estrogen in the hyperplastic PRL

cells that transform into tumor cells, highlighting a critical role for PTTG in PRL tumor

formation. Our results may be interpreted within this context; PTTG may be relatively

downregulated in PRL adenomas to suppress its PRL promoting effects. This explanation is

supported by our findings of lowest PTTG expression in PRL adenomas among all pituitary

adenoma subtypes examined. However, our findings may simply be due to the small sample size

of our study and cannot be generalized without further investigation.

8.5 PTTG Cellular Localization

Cellular localization of PTTG is unclear. In the nucleus, PTTG acts as a transcriptional activator

as well as an inhibitor of premature sister chromatid separation (167). PTTG protein has been

identified as a global transcription factor, exerting its activity via its DNA binding domain and/or

by binding to proteins that directly interact with DNA, including PBF, p53 and Sp1 (175). As

such, PTTG nuclear localization is expected to occur with PTTG in its role as a transcription

factor. Additionally, PTTG contain a nuclear localization signal (NLS), facilitating its transport

to the nucleus (176).

However, to date, the role of cytoplasmic PTTG remains unclear. One recent study

demonstrated that pituitary PTTG is a secretory protein in pituitary tumor cells (137). This may

imply the existence of a PTTG cell surface receptor mediating its effects.

In our series of 89 pituitary adenomas, tumors only exhibited cytoplasmic PTTG

expression, with a pronounced peri-nuclear expression pattern, and no nuclear PTTG

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immunoreactivity. These results are in keeping with most other studies showing cytoplasmic

PTTG expression in pituitary tumors (88, 92, 112, 133, 137). Recently, Wierinckx et al,

studying 25 PRL secreting adenomas, reported that nuclear expression of PTTG is among a set

of prognostic indicators for tumor aggressiveness, other elements of the set being numerous

mitoses, high Ki-67 index, and p53 expression (43). It is possible that nuclear PTTG expression

is exclusive to aggressive PRL adenomas, and that our series lacked such tumors.

The peri-nuclear pattern of PTTG expression is in keeping with its expression in the

Golgi apparatus and ER, further supporting the recent findings that PTTG is a secretory

protein(137). PTTG peri-nuclear localization also may facilitate its nuclear trafficking as

needed, a phenomenon observed with other proteins (177). Alternatively, this peri-nuclear

localization may facilitate PTTG association with other factors, as seen with peri-nuclear

localization of cytosolic phospholipase A2α for coupling with cyclooxygenases (178). Research

is needed to reveal such elements that potentially interact with PTTG.

8.6 Limitations

The greatest limitation of the present study is the small number of cases included,

which may have contributed to type II errors, thus failing to detect a difference in PTTG

immunostaining when in fact, there would have been such a difference. As such, the small

sample size may have contributed to the lack of a finding of statistically significant difference

between medically treated GH and PRL adenomas, where it was expected.

Similarly, the small number of recurrent ACTH adenomas and macroadenomas, may

have resulted in Type II error, in keeping with our results that there was no statistically

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significant difference between recurrent and non-recurrent adenomas as well as macro-and

microadenomas. PTTG overexpression has been correlated with recurrence and more aggressive

tumor behaviour in many neoplasms , but in our series of ACTH adenomas of patients with

Cushing’s disease, no such correlation was noted, perhaps due to the small number of recurrent

tumors and `. However, our series contained a relatively large number of ACTH adenomas, and

ACTH adenomas do tend to be non-recurring and also smaller than 1 cm in size

(microadenomas).

8.7 Summary and Implications

The present study showed that PTTG immunoexpression in pituitary adenomas is

primarily cytoplasmic, in keeping with the findings of most other investigators . As such, the

nuclear role of PTTG as a securin, inhibiting sister chromatid separation, does not apply to

pituitary tumors. Similarly, the role of PTTG as a nuclear activator of transcriptional activity

would be unlikely in pituitary neoplasms, given its expression in the cytoplasm. However, the

latter cannot be ruled out, as PTTG may actually regulate downstream effectors that in trun cold

play a role in nuclear transcriptional activity.

The present study also exhibited higher PTTG expression in Gh adenomas a compared to

PRL adenomas, a finding relevant to pituitary cell lineage differentiation. PRL and GH cells

have a common progenitor ancestor, the mammosomatotroph cells. The regulatory factors that

mediate mammosomatotroph differentiation into somatotroph (GH) and lactotroph (PRL) cells

are largely unknown. Estrogen is one such factor that is though to promote PRL gene

transcription and lactotroph differentiation. The presence of a “GH repressor” is also generally

agreed on, although such a fractor remains unidentified. Our findings of reciprocal PTTG

expression levels in GH and PRL adenomas make PTTG a potential candidate in

91

cytodifferentiation of mammosomatotroph cells into lactotrophs and somatotrophs, and further

studies to dissect the molecular pathways in which PTTG is involved are needed.

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CHAPTER 9: DISCUSSION: BRAIN TUMORS

9.1 Discussion

This study described the immunohistochemical expression of PTTG in brain tumors and

demonstrated that PTTG expression is different in different tumor subtypes. Additionally, PTTG

expression in the nucleus and cytoplasm was different in several brain tumor subtypes.

9.2 PTTG immunohistochemical expression is highest in medulloblastomas

All of the medulloblastomas studied showed high-intensity, nuclear PTTG

immunoexpression only, in contrast to all other tumor types examined that had both nuclear and

cytoplasmic expression, or no PTTG expression at all (hemangiopericytoma, n=4).

Medulloblastomas are tumors of the cerebellum and are classified by WHO as one of the five

tumors of embryonal origin. Medulloblastomas are grade IV (WHO) due to their aggressive

behaviour and are the best characterized brain malignancy in terms of molecular mechanisms of

pathogenesis (151). Although the low number of medulloblastomas in our study (n=4) presents a

limitation of our findings, there are several pathways via which PTTG may be involved in

promotion of medulloblastoma formation and/or progression. Pathways implicated in

medulloblastoma formation and progression include the sonic hedgehog (SHH) signaling

pathway, Wnt/beta-catenin pathway, c-myc and n-myc upregulation, defective DNA repair

pathways and aneuploidy (179). The association between PTTG and all these pathways is

documented.

First, the sonic hedgehog (SHH) signaling pathways is the best-characterized molecular

pathways in medulloblastoma formation and progression (179). SHH signaling is associated

with stem cell differentiation and embryonic patterning, and modulates the downstream effects

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of bone morphogenic protein (BMP) signaling to induce cell proliferation without differentiation

in progenitor blood cells (180). Interestingly, BMP-2 treatment of human mesenchymal stem

cells resulted in increased PTTG expression, attributed to increased cell proliferation of these

cells (121). However, BMP-2 treatment of the U87 astrocytoma cell line failed to induce any

change in PTTG mRNA levels (55). These contradictory findings may be due to the different

cell lines examined; the pathways involved in mesenchymal stem cells are expected to be more

similar to those associated with medulloblastoma formation as this is an embryonal neoplasm,

whereas U87 astrocytoma cells are postulated to harbor pathways similar to astrocytic

neoplasms. Thus, given that SHH is strongly associated with medulloblastoma formation and

that BMPs are downstream of SHH, and that BMP-2 upregulates PTTG in stem cells, it can be

proposed that PTTG plays an essential role in medulloblastoma formation. This is supported by

our observations of intense nuclear PTTG expression in all medulloblastomas studied.

Second, PTTG may also be involved in medulloblastoma formation and/or progression

via upregulation of c-myc. It is of note that c-myc is functionally interchangeable with n-myc in

murine development (181, 182), and that upregulation of c-myc downstream of SHH results in

enhanced cell proliferation in medulloblastomas (183, 184). Thus, one may propose that PTTG

is associated with medulloblastoma neoplastic processes via induction of c-myc transcription that

synergistically results in enhanced cellular proliferation (179). Similarly, it has been shown that

IGF pathways result in elevated n-myc levels and enhance the effects of SHH signaling-induced

n-myc in formation of medulloblastomas (185, 186). Moreover, upregulation of c-myc is

implicated in approximately 15% of medulloblastomas (187-190), which supports the role of

PTTG induced c-myc in medulloblastoma development and/or progression. C-myc upregulation

in medulloblastoma is downstream of the WNT/beta-catenin pathway, which is shown to

promote PTTG promoter activity (179). PTTG expression also colocalizes with beta-catenin in

94

esophageal small cell carcinoma (N=69) (191). Thus, there is strong support for PTTG as a

target of the beta-catenin pathway, which is implicated in initiation of many neoplasms.

A third pathway that provides a potential link between PTTG and formation of

medulloblastomas is defective DNA repair mechanisms. PTTG is pivotal in inhibition of DNA

repair mechanisms, which have recently been shown to play a role in medulloblastoma formation

(192-194). Cerebellum has been shown to be highly susceptible to genotoxic stress, and mice

that lack Ligase IV, a DNA repair enzyme, as well as p53 (Lig4(-/-)p53(-/-)) develop

medulloblastomas as early as 21 days post-natally (192-194). Other mouse models of DNA

damage deficiency also show elevated medulloblastoma formation when crossed with p53-/-

mice (192-194). Thus, a deficiency in DNA repair pathways appears to be critical in

medulloblastoma formation. As stated previously, PTTG is essential in suppression of DNA

repair mechanisms. PTTG binds to Ku70, the regulatory subunit of DNA-PK, which is involved

in repair of double-stranded DNA breaks (195). The PTTG binding of Ku70 is inhibited by

dsDNA breaks, with resultant freeing and activation of Ku70 and DNA repair mechanisms (195).

Etoposide-induced dsDNA damage was elevated in PTTG-transfected HCT116 cells and human

fibroblasts and associated with lower Ku70 function, thus suggesting that PTTG suppresses

Ku70 activity (101). Similarly, PTTG-deficient bone marrow stem cells (119), as well as

pituitary gland cells (17) showed upregulation of genes involved in DNA damage and repair

pathways, supporting a role for PTTG in suppression of these pathways. PTTG inhibition and/or

proteosomal degradation as a result of DNA damage has also been demonstrated (72, 101, 195).

Additionally, a recent study by Bernal et al., demonstrated that loss of PTTG in cell culture lead

to hindered cell proliferation after genotoxic stress (196), providing a potential rationale for

PTTG overexpression in many malignancies. The wealth of evidence on the importance of

PTTG in suppression of DNA damage pathways implicate it as a strong candidate in induction of

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medulloblastomas. Further research on the role of PTTG in this process is warranted.

Yet another mechanism that links PTTG to medulloblastoma development and/or

progression is aneuploidy. PTTG overexpression strongly correlates with aneuploiy in a number

of neoplasms. In medulloblastomas, aneuploidy is suggested to be linked to prognosis in several

studies. A recent study showed that increased aneuploidy was associated with decreased survival

rate in 60 medulloblastomas that were stratified into 2 groups based on chromosomal instability

(197).

Our study demonstrated the highest levels of PTTG among brain tumors in

medulloblastomas, providing evidence for the importance of PTTG in mechanisms involved in

medulloblastoma development. Further studies to dissect the PTTG-associated pathways in

medulloblastomas are clearly warranted.

9.3 Nuclear PTTG expression is higher in malignant tumors compared to benign tumors

In most types of malignant gliomas studied including GBM IV, astrocytoma III and

oligoastrocytoma III, except in oligodendroglioma III (n=4), PTTG expression was more

pronounced in the nucleus as compared to the cytoplasm. There may be several reasons for an

increased nuclear/cytoplasmic balance of PTTG expression. First, PTTG in the nucleus of these

tumors may specifically be involved in promotion of transcriptional activity of other factors

involved in oncogenesis such as FGF family and c-myc. In gliomas, FGF-2 and its receptors are

commonly elevated (198, 199). Interestingly, elevated PTTG levels are parallel to those of FGF-

2 levels in the developing brain cortex, and in NT-2 neuronal cells, where PTTG stimulates FGF-

2 expression (95). Increased bFGF expression in primary astrocytic tumors is associated with

shorter survival times in patients (200). PTTG association with bFGF expression and regulation

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is supported by several lines of evidence. PTTG and bFGF levels are correlated in acute

leukemia (94). A significant correlation between PTTG and bFGF expression in pituitary

adenomas is also reported (36). In cultured leiomyoma cells, bFGF and PTTG are involved in a

positive autocrine feedback loop that is thought to promote tumourigenesis (201). Thus, nuclear

PTTG expression in brain tumors, and specifically in astrocytic tumors, may elicit elevated

bFGF and FGF2 expression in these tumors and contribute to the tumourigenic process.

PTTG in the nucleus may also play a role in increasing transcriptional activation of c-

myc, another proto-oncogene that is implicated in astrocytoma tumourigenesis (202). A study of

140 primary astrocytomas showed c-myc upregulation in 65% of these tumors that increased

with tumor grade and malignancy (202). Other factors whose regulation may be mediated by

nuclear PTTG may include angiogenic factors including VEGF, ID3, and TSP-1. A microarray

study of thyroid carcinomas identified ID-3, a pro-angiogenic factor, and TSP-1, an anti-

angiogenic gene, as being up- and downregulated by PTTG respectively (7). Provided that

angiogenesis is an important factor in progression of gliomas and malignancy grading, elevated

PTTG expression in the nuclei of such aggressive gliomas is expected. One should note that

since PTTG is also involved in cell cycle regulation by inhibition of premature sister chromatid

separation, its elevation in malignant gliomas may simply be a reflection of the greater

proliferative index of these highly mitotic tumors. Additionally, elevated nuclear PTTG

expression in malignant gliomas may be mediated by yet unidentified mechanisms.

Although this study failed to show increased nuclear PTTG expression in

oligodendroglioma III, which is a malignant glioma, this may be due to the low number of

oligodendrogliomas included (n=4), compared to other types of brain tumors.

Oligodendrogliomas III show high expression levels of bFGF and EGFR, and are highly vascular

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tumors. PTTG association with increased vascularity has been found in most vascular

neoplasms studied including thyroid, lung, hepatocellular and colorectal carcinomas (49, 111,

159). It is possible that due to the low number of oligodendrogliomas included in this study,

increased PTTG expression was not observed when compared to benign brain neoplasms.

9.4 Lower PTTG immunoreactivity in pilocytic astrocytomas

Among astrocytic tumors, pilocytic astrocytomas exhibited the lowest PTTG

immunoexpression, with similar nuclear and cytoplasmic expression levels. This observation

may be due to the low proliferation rate of these tumors, as PTTG is implicated in tumor

proliferation and aggressive behaviour. This lower PTTG expression may also highlight the

differences in molecular pathways involved in their tumourigenesis and tumor progression when

compared to those tumors that demonstrated higher nuclear PTTG expression. For example,

pilocytic astrocytomas (WHO grade I) and diffuse astrocytomas (WHO grade II) differ from

GBM IV by expression of 5 genes (fibronectin, osteopontin, YKL-40, keratoepithilim and

fibromodulin) involved in invasion and angiogenesis, as shown by gene expression analysis in

one study (203). PTTG appears to be yet another gene differentially expressed in these tumors.

It is of note that low grade gliomas usually progress to resemble high-grade gliomas with respect

to histological and clinical characteristics (151), and it can be proposed that elevated PTTG in

higher-grade gliomas as compared to lower-grade ones may be acquired over time.

9.5 Meningiomas

Albeit low when compared to most malignant tumors, PTTG immunoexpression was

exhibited by meningiomas. Meningiomas are common CNS tumors and mostly benign, with

only 1-2% being graded as WHO grade III (204). Meningioma incidence is higher in females,

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and is increased during pregnancy. Additionally, over 30% of meningiomas express estrogen

receptors. Estrogen is known to mediate PTTG upregulation in PRL-secreting pituitary

adenomas, and as such, it may be postulated that PTTG in meningiomas may interact with

estrogen pathways and contribute to tumourigenic mechanisms of meningiomas. Another

oncogenic mechanism, increased genetic instability, is highly correlated with tumor grade in

meningiomas. It is proposed that aberrant chromosomal segregation may underlie such genetic

instability. PTTG expression in most other neoplasms, including those of thyroid, breast and

colon exhibits strong correlation with aneuploidy and genetic instability (101, 102, 197).

Therefore, the role of PTTG expression in aneuploidy seen in meningiomas needs further

investigation. Furthermore, PTTG quantification and its correlation with aneuploidy in

meningiomas may provide a new method of meningioma classification.

9.6 PTTG expression in schwannomas

Immunoexpression of PTTG in schwannoma was significantly higher than in pilocytic

astrocytoma, oligodendroglioma II, meningioma, ependymoma, pilocytic astrocytoma and

hemangiopericytoma. The average intensity of PTTG immunostaining in the schwannomas in

our series was 1.5 (nuclear) and 1.4 (cytoplasmic) with no significant difference between the

nuclear and cytoplasmic PTTG expression. This elevated PTTG expression in schwannomas

compared to several benign brain tumor types is notable. Schwannomas are also generally

benign, and tend to grow slowly, with very little being known about their molecular pathways.

Schwannomas are tumors of the peripheral nervous system, and are caused by the loss of NF2

gene that encodes the Schwann cell protein, Merlin. Our findings of relatively high PTTG

expression in schwannomas contributes to the body of knowledge on schwannomas, and creates

a context for further investigation of PTTG associated pathways that may contribute to

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schwannoma development and progression.

9.7 PTTG expression in ependymomas

PTTG expression in ependymoma was lower than most other types of brain tumors, and

was similar in the nuclear and cytoplasmic components. Ependymomas are generally slowly

growing tumors of neuroepithelial origin, and like schwannomas, little is known about the

molecular pathways associated with their neoplastic initiation and progression. Our work

demonstrated that PTTG is present in ependymomas, providing a rationale for further study of

the role of PTTG in the development and progression of these neoplasms.

9.8 Lack of PTTG expression in hemangiopericytomas

We did not find any PTTG expression in the 4 hemangiopericytoma tumors examined.

Hemangiopericytomas are rare vascular tumors associated with high rates of metastasis and

recurrence. To date, PTTG expression has been shown to be increased in all aggressive

behaving tumors examined. However, our immunohistochemical study detected no PTTG

expression in hemangiopericytomas. There may be several reasons for this, including the low

sensitivity of immunohistochemistry to very low levels of proteins in tissue. It is possible that

PTTG is expressed in very low levels undetectable by immunohistochemistry in

hemangiopericytomas, and thus could not be visualized by immunohistochemistry. However,

absent or low PTTG levels in hemangiopericytoma may highlight a yet unknown fundamentally

different tumourigenic mechanism between these tumors and all the other aggressive neoplasms

studied for PTTG expression. That PTTG is highly expressed in all other malignant tumors

investigated, and not in hemangiopericytomas may also be a cell specific effect, indicating that

PTTG is not associated with cells of hemangiopericytoma origin which currently remain

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unknown. Given the stark absence of PTTG in these vascular, often metastatic tumors,

microarray studies that compare hemangiopericytoma gene expression profiles to those of other

such vascular tumors that do express PTTG may provide insight into the differences in the

associated neoplastic processes.

9.9 Limitations

A limitation of this study was the small number of some brain tumors types included in the

study, which may have resulted in failure to detect a difference in PTTG immunohistochemical

staining despite an actual difference (Type II error). Our study included 4 of each

medulloblastoma, hemangiopericytoma, and oligodendrogliomas, due to availability of

specimens. This may have partly explained the absence of PTTG expression in

hemangiopericytomas which are malignant entities. Similarly, in the 4 oligodendroglioma

(WHO grade III) studied, PTTG expression levels appeared to be relatively low (intensity of 1

(slight)), whereas these tumors tend to behave aggressively, and PTTG expression is often

associated with aggressive tumor behaviour. However, despite the small sample size of some of

the brain tumor types, we still found differences and a trend towards higher PTTG expression in

more malignant tumors, a finding in keeping with many studies of other neoplasms.

9.10 Summary and Implications

The present study reported findings of higher PTTG expression in most malignant brain

tumors as compared to benign ones, in keeping with findings of elevated PTTH

immunoreactivity in several other neoplasms studied to date . PTTG expression was highest in

medulloblastomas, which are aggressive tumors of the CNS. In contrast, pilocytic astrocytomas,

which are benign, exhibited low PTTG immunoexpression, confirming the findings of other

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studies that show limited PTTG expression in non-aggressive neoplasms. Meningiomas (WHO

grade I) also showed low PTTG expression, in keeping with their slowly growing and benign

nature.

In sum, among astrocytic tumors, the WHO grade III and IV tumors (GBM IV and

astrocytoma III) exhibited the highest PTTG immunoexpression levels, whereas benign pilocytic

astrocytoma WHO garde I, showed the lowest PTTG immunoreactivity. Our findings may have

implications for PTTG as a biomarker of brain tumor aggressiveness, and further research is

required to examine the potential correlations between PTTG expression levels and clinico-

pathological variables such as patient survival and recurrence.

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CHAPTER 10: CONCLUSIONS

10.1 Conclusion: Pituitary adenomas

The present study demonstrated that PTTG is consistently expressed in the cytoplasm of

pituitary adenomas but is expressed at different levels in various adenoma cell types. The

highest PTTG immunoexpression was found in GH adenomas and the lowest in PRL adenomas.

Medically treated GH adenomas showed a decreased in PTTG expression. Our results highlight

that the role of PTTG in regulation of tumourigenic mechanisms is different in various pituitary

cell types even among similar lineages of pituitary adenomas, and that any further studies of

PTTG in pituitary adenomas must distinguish between different pituitary adenoma cell types.

10.2 Conclusions: Brain Tumours

This study finds evidence of a correlation between immunohistochemical expression of

PTTG and human brain tumor grade, specifically documenting elevated PTTG expression in

malignant astrocytomas of grade III and IV as compared to lower grade astrocytomas (I and II).

PTTG has been associated with several tumourigenic mechanisms including cellular

proliferation, angiogenesis, invasion, apoptosis, and DNA repair mechanisms. Determination of

the precise role of PTTG in brain tumors will require dissection of these pathways in various

brain tumor subtypes. Mouse models of PTTG deficiency have tremendously advanced our

understanding of pituitary tumor development and progression, and similar models need to be

developed to reveal the role of PTTG in brain tumor formation and progression. In gliomas,

knockdown of PTTG using the siRNA technique results in reduced cell proliferation in cell

culture. Thus PTTG appears to be a promising target in treatment of tumors that demonstrate its

103

elevated expression. Evidently, more research is required to demonstrate the role of PTTG in

oncogenic pathways involved in brain tumourigenesis.

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CHAPTER 11: FUTURE DIRECTIONS

The findings of the present study provide the faremwork for further research into the specific

role of PTTG in pituitary cell differentiation as well as correlations between PTTG

immunoexpression and clinical variables such as patients survival. The following are potential

future studies stemming from the present work.

1. Inclusion of more cases of both pituitary and other brain tumor types should be

considered, in order to decrease the probability of Type II errors, and to uncover

additional potential differences in PTTG expression in various tumor subtypes.

2. Furthermore, clinical correlations between patients outcome measures such as disease-

free survival, and expression of PTTG should be investigated to determine te utility of

PTTG as a prognostic indicator of pituitary and other brain tumours.

3. In addition to immunohistochemistry, PCR technique should be employed to quantify

levels of PTTG in tumor subtypes, as PCR is a more sensitive factors. This is true

expecially in the case of hemangiopericytomas, where the present study failed to detect

any PTTG immunoexpression.

4. Since PTTG localization has implications for its potential actions, electron microscopy

should be utilized to specify PTTG subcellular localization in cells.

5. Importantly, molecular studies should be undertaken to clarify the role (if any) of PTTG

association with cell lineage, and its potential action in mammosomatotroph

differentiation into somatotroph and lactotroph cells.

105

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