Characterization of 17β- Survival Signaling in Medulloblastoma: Relation to Tumor Growth and IGF1 Signaling

A dissertation submitted to the

Graduate School

of the University of Cincinnati

in partial fulfillment of the

requirements for the degree of

Doctor of Philosophy (Ph.D.)

in the Department of Pharmacology and Cell Biophysics

of the College of Medicine

2015

By

Clifford James Cookman

B.S., University of Cincinnati, Cincinnati, OH 2009

Doctoral Thesis Advisor

Scott M. Belcher, Ph.D.

Doctoral Thesis Committee:

Robert Rapoport, Ph.D. (Chair)

Nira Ben-Jonathan, Ph.D.

Terence L. Kirley, Ph.D.

Jo El Schultz, Ph.D. Abstract

Medulloblastoma has been identified as an -responsive tumor that expresses β and whose growth is regulated by 17β-estradiol both in vitro and in vivo. However, several clear gaps of knowledge exist with regard to the role of 17β-estradiol in medulloblastoma. These include the precise mechanism responsible for the growth promoting effect of 17β-estradiol in medulloblastoma as well as the potential for estrogen receptor inhibition in medulloblastoma treatment. To address these gaps of knowledge and gain a better understanding of the role of 17β-estradiol in medulloblastoma, several model systems were utilized.

Extensive pharmacological studies that utilized the human medulloblastoma cell line, D283Med, were performed to characterize the effect of 17β-estradiol on cell death and the mechanism responsible for any observed effect. Estrogen protected D283Med cells from cell death and this cytoprotection was observed to be due to the activation of estrogen receptor β and downstream up-regulation of the insulin-like growth factor 1 pathway. To characterize the efficacy of estrogen receptor inhibition and clarify the role of estrogen receptor β in medulloblastoma, mouse models of medulloblastoma were utilized. It was observed that inhibition of estrogen receptors by or loss of estrogen receptor β expression decreased tumor growth. This decreased tumor growth is likely due to decreased activation of the insulin-like growth factor pathway and downstream mediators that increase survival. Lastly, to further support the notion that estrogen receptor inhibition may be an effective medulloblastoma treatment, the effect of estrogen on the chemosensitivity of D283Med cells was evaluated. These experiments were conducted with cisplatin, vincristine and lomustine, which are chemotherapeutics that are currently utilized in medulloblastoma treatment. Estrogen decreased the

i chemosensitivity of D283Med cells while inhibitors of estrogen receptor β blocked this effect.

Taken together, these studies support the notion that medulloblastoma is an estrogen-responsive tumor and suggest that therapies that inhibit estrogen receptor β may be effective in inhibiting medulloblastoma tumor growth in humans. Furthermore, estrogen receptor β inhibition may improve the response rate to current treatments due to its sensitizing effect on medulloblastoma with regard to the cytotoxic effects of chemotherapeutic compounds.

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Acknowledgements

I would like to thank my mentor, Dr. Scott M. Belcher, for providing the resources required to complete my graduate studies. I would also like to thank the members of my dissertation committee: Drs Nira Ben-Jonathan, Terry Kirley, Jo El Schultz, and Robert

Rapoport for their dedication, un-ending support and helpful guidance. Current and past members of the laboratory including Dr. Eric Kendig, Charles Lo, Cam Harris, Jessica

Kendziorski, Robin Gear, Susie Christie, and Dana Buesing are also acknowledged for their friendship, encouragement and contributions to the work presented here. I would also like to acknowledge the Department of Pharmacology and Cell Biophysics, the

National Institutes of Health and Dr. Peter Stambrook for their support. Lastly, I would also like to thank my family, including my parents, Clifford Ray Cookman and Helen Marie

Fink Cookman, my sister, Jennilee Cookman, and most importantly my wife and son,

Stacey Lynette Naylor-Cookman and William Walter Cookman, for their unconditional love, patience and understanding during the difficult process of graduate school.

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TABLE OF CONTENTS

Abstract i

Acknowledgments iv

Table of Contents v

List of Abbreviations xi

List of Tables xviii

List of Figures xviii

CHAPTER 1: INTRODUCTION 1

1 Overview 2

1.1 Estrogen 5

1.1.1 Serum levels of estrogen 6

1.1.2 Estrogen biosynthesis in normal and malignant tissue 7

1.2 Estrogen’s effects in normal tissues 11

1.2.1 Reproductive tissues 11

1.2.2 Central nervous system 13

1.3 Estrogen Receptors 15

1.3.1 Gene and Protein Structure 17

1.3.2 The N-terminal transactivation and DNA binding domains 19

1.3.3 The ligand binding domain 19

1.4 Mechanisms of estrogen actions 21

1.4.1 Ligand-activated transcription factor 22

1.4.2 Transcription factor co-regulator 24

1.4.3 Ligand independent transcriptional regulation 25

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1.4.4 Rapid activation of intracellular signaling pathways 25

1.5 The insulin-like growth factor pathway 27

1.6 The cerebellum as an estrogen-responsive tissue 29

1.6.1 Development and structure of the cerebellum 30

1.6.2 Actions of estrogen in the cerebellum 31

1.7 Pro-survival signaling by estrogen and insulin-like growth factor 32

1.7.1 Cytoprotective effect of estrogen in cancer 32

1.7.2 Neuroprotective effect of estrogen and insulin-like growth factor 34

1.7.2.1 Neuroprotective actions of estrogen and insulin-like growth factor

in the cerebellum 36

1.8 Estrogen Receptor signaling in cancer 38

1.8.1 The role of ERβ in cancer 40

1.8.1.1 Colon Cancer 41

1.8.1.2 Ovarian and Endometrial Cancer 42

1.8.1.3 Prostate Cancer 44

1.8.1.4 Lung Cancer 46

1.8.1.5 Breast Cancer 49

1.9 Estrogenic endocrine disrupting chemicals 51

1.9.1 52

1.9.2 53

1.10 Medulloblastoma 54

1.10.1 Medulloblastoma classification 57

1.10.1.1 Histological classification 58

1.10.1.2 Molecular classification 60

1.10.2 Medulloblastoma etiology 64

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1.10.2.1 Sonic Hedgehog pathway in medulloblastoma 64

1.10.2.2 The wingless (WNT) pathway in medulloblastoma 67

1.10.2.3 The insulin-like growth factor pathway and additional regulators of

medulloblastoma carcinogenesis and growth 68

1.10.3 Current medulloblastoma treatment 69

1.10.3.1 Emerging medulloblastoma treatments 71

1.10.4 Expression of ERs in medulloblastoma 73

1.10.5 Effect of 17β-E2 in medulloblastoma 75

1.11 Rationale, Hypothesis and Goals 77

1.11.1 Overall Rationale 77

1.11.2 Hypothesis 78

1.11.3 Specific Goals 78

CHAPTER 2: MATERIALS AND METHODS 80

2.1 and pharmacological agents 81

2.2 Cell culture conditions 81

2.3 Treatment of tumor cell lines 82

2.3.1 Analysis of viability and proliferation 82

2.3.2 Analysis of caspase-3 activity 83

2.4 Proliferation and viability studies 84

2.4.1 Viable cell counts 84

2.4.2 Caspase-3 activity assay 85

2.4.3 Bromodeoxyuridine incorporation assay 86

2.4.4 MTT assay 87

2.4.5 Clonogenic assay 88

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2.5 mRNA expression studies 92

2.5.1 mRNA isolation and purification 92

2.5.2 Quanti