THE EFFECTS OF THE FEMALE REPRODUCTIVE HORMONES ON OVARIAN CANCER INITIATION AND PROGRESSION IN A TRANSGENIC MOUSE MODEL OF THE DISEASE Laura Allison Laviolette Thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Cellular Molecular Medicine Department of Cellular and Molecular Medicine Faculty of Medicine University of Ottawa April 2011 © Laura Allison Laviolette, Ottawa, Canada, 2011 Authorization It is with the rights granted by the Assignment of Copyright Agreement of The Endocrine Society (Endocrinology. 2010 Mar;151(3):929-38) that the manuscript that constitutes Chapter 2 is reproduced. It is with the rights granted by the Copyright Transfer Agreement of Lippincott Williams & Wilkins (Menopause, accepted for publication in May 2011; Volume 18.5) that the manuscript that comprises Chapter 4 is reproduced. Regardless of the formatting requirements of the journals in which the manuscripts contained herein have been submitted or published, the references for this entire document have been formatted into a single list and Canadian English spelling has been used throughout the thesis. ii Contributions of Co-authors All of the studies contained in this thesis were conducted under the supervision of Dr. Barbara Vanderhyden. Unless otherwise specified, all of the experiments were performed by Laura Laviolette. Laura Laviolette designed the experiments, interpreted and analysed the data and wrote the manuscripts, with supervision and revisions from Dr. Barbara Vanderhyden. Chapter 2 Title: 17β-estradiol accelerates tumour onset and decreases survival in a transgenic mouse model of ovarian cancer Journal: Endocrinology (2010 Mar;151(3):929-38) Authors: Laura A. Laviolette, Kenneth Garson, Elizabeth A. Macdonald, Mary K. Senterman, Kerri Courville, Colleen A. Crane, and Barbara C. Vanderhyden Kenneth Garson and Kerri Courville generated the tgCAG-LS-TAg transgenic mouse model. Elizabeth Macdonald performed the radioimmunoassays. Mary Senterman assessed the histology of the mouse ovarian tumours. Colleen Crane assisted with the embedding, sectioning and IHC staining of the tumours. Chapter 3 Title: 17β-estradiol accelerates ovarian tumour progression in vivo through the upregulation of Greb1 Journal: Unpublished manuscript (prepared for submission to Cancer Research) Authors: Laura A. Laviolette, Kendra M. Hodgkinson, Neha Minhas, Carolina Perez- Iratxeta, and Barbara C. Vanderhyden Kendra Hodgkinson performed the survival study in the ovariectomized immunodeficient mice and performed the Q-PCR validation of the microarray targets. Neha Minhas performed the in vitro proliferation assay. Carolina Perez- Iratxeta analysed the microarray data. iii Chapter 4 Title: Induction of a menopausal state alters the growth and histology of ovarian tumours in a mouse model of ovarian cancer Journal: Menopause (accepted for publication in Volume 18.5, May 2011) Authors: Laura A. Laviolette, Jean-François Ethier, Mary K. Senterman, Patrick J. Devine and Barbara C. Vanderhyden Jean-François Ethier performed the xenograft study in ovariectomized immunodeficient mice. Mary Senterman analyzed and diagnosed the histology of the mouse ovarian tumours. Patrick Devine assisted with the interpretation and analysis of results and provided the protocols for the mouse VCD treatments. iv Abstract Ovarian cancer is thought to be derived from the ovarian surface epithelium (OSE), but it is often diagnosed during the late stages and therefore the events that contribute to the initiation and progression of ovarian cancer are poorly defined. Epidemiological studies have indicated an association between the female reproductive hormones and ovarian cancer etiology, but the direct effects of 17β-estradiol (E2), progesterone (P4), luteinizing hormone (LH) and follicle stimulating hormone (FSH) on disease pathophysiology are not well understood. A novel transgenic mouse model of ovarian cancer was generated that utilized the Cre/loxP system to inducibly express the oncogene SV40 large and small T-Antigen in the OSE. The tgCAG-LS-TAg mice developed poorly differentiated ovarian tumours with metastasis and ascites throughout the peritoneal space. Although P4 had no effect; E2 significantly accelerated disease progression in tgCAG-LS-TAg mice. The early onset of ovarian cancer was likely mediated by E2’s ability to increase the areas of putative preneoplastic lesions in the OSE. E2 also significantly decreased survival time in ovarian cancer cell xenografts. Microarray analysis of the tumours revealed that E2 mainly affects genes involved in angiogenesis and cellular differentiation, proliferation, and migration. These results suggest that E2 acts on the tumour microenvironment in addition to its direct effects on OSE and ovarian cancer cells. In order to examine the role of the gonadotropins in ovarian cancer progression, the tgCAG-LS-TAg mice were treated with 4-vinylcyclohexene-diepoxide (VCD) to v induce menopause. Menopause slowed the progression of ovarian cancer due to a change in the histological subtype from poorly differentiated tumours to Sertoli tumours. Using a transgenic mouse model, it was shown that E2 accelerated ovarian cancer progression, while P4 had little effect on the disease. Menopause (elevated levels of LH and FSH) altered the histological subtype of the ovarian tumours in the tgCAG-LS-TAg mouse model. These results emphasize the importance of generating animal models to accurately recapitulate human disease and utilizing these models to develop novel prevention and treatment strategies for women with ovarian cancer. vi Table of Contents Authorization .............................................................................................................. ii Contributions of Co-authors ....................................................................................... iii Abstract .......................................................................................................................v Table of Contents ...................................................................................................... vii List of Figures ............................................................................................................ xi List of Tables ........................................................................................................... xiii List of Abbreviations and Chemical Formulae ...........................................................xiv Acknowledgements ...................................................................................................xix Chapter 1: Introduction ...............................................................................................1 Literature Review .....................................................................................................1 Ovarian cancer .....................................................................................................1 Incidence and mortality ....................................................................................1 Standard of treatment for ovarian cancer .........................................................1 The etiology of ovarian cancer .............................................................................2 Tissues of origin of epithelial ovarian cancers ..................................................2 Histological subtypes .......................................................................................4 Progression of ovarian cancer .........................................................................7 Theories for the origin of epithelial ovarian cancer ...............................................8 Theory of incessant ovulation ...........................................................................8 Gonadotropin theory ........................................................................................9 Risk factors for the development of ovarian cancer ..............................................9 Mutations in BRCA1 and BRCA2 .....................................................................9 Menopause ..................................................................................................... 10 Hormone replacement therapy ....................................................................... 11 Oral contraceptives ........................................................................................ 13 Pregnancy ...................................................................................................... 13 Reproductive hormones and ovarian cancer........................................................ 14 17β-estradiol .................................................................................................. 14 Progesterone .................................................................................................. 18 Luteinizing hormone ....................................................................................... 20 vii Follicle stimulating hormone .......................................................................... 21 Mouse models of menopause ............................................................................. 22 In vivo models of ovarian cancer ........................................................................ 25 Transgenic mouse models of cancer ................................................................... 26 Transgenic mouse models of ovarian cancer ......................................................