USE OF A TRANSGENIC MOUSE MODEL OF OVARIAN HYPERSTIUMLUATION TO IDENTIFY THERAPEUTIC TARGETS AND MECHANISMS IN HORMONE-INDUCED MAMMARY CANCER by ERIN LEE MILLIKEN Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Advisor: Dr. Ruth A. Keri Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY August, 2005 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ______________________________________________________ candidate for the ________________________________degree *. 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DEDICATION I dedicate this dissertation to Matt, whose patience and optimism I have drawn on repeatedly over the past six years and to my grandmother, Virginia Gerrity iii TABLE OF CONTENTS Dedication iii List of Tables v List of Figures vi Acknowledgments viii Abstract x Chapter I Introduction 1 Chapter II Ovarian Hyperstimulation by Luteinizing 35 Hormone Leads to Mammary Gland Hyperplasia and Cancer Predisposition in Transgenic Mice Chapter III EB1089, a Vitamin D Receptor Agonist, Reduces 77 Proliferation and Decreases Tumor Growth Rate in a Mouse Model of Hormone-Induced Mammary Cancer Chapter IV Centrosome Amplification and p53 Signaling in a 98 Model of Hormone-Induced Mammary Cancer Chapter V Summary, Future Directions, and Conclusions 150 Reference List 177 iv LIST OF TABLES Table II-1 Serum levels of estrogen, progesterone, and 60 prolactin are increased in LH-overexpressing animals Table IV-1 Genes associated with p53, 120 centrosomes/microtubules/polarity, and genomic instability Table IV-2 Genes upregulated in mammary glands of LH- 122 overexpressing relative to wild-type mice Table IV-3 Genes downregulated in mammary glands of LH- 129 overexpressing relative to wild-type mice v LIST OF FIGURES Figure I-1 Mammary gland development 29 Figure I-2 Chemical structures of 1, 25-dihydroxyvitamin D3 31 and EB1089 Figure I-3 Generation of LH-overexpressing mice 33 Figure II-1 Mammary gland development is accelerated in 61 LH-overexpressing mice Figure II-2 The mammary glands in adult, virgin LH- 63 overexpressing mice display a mid-pregnancy morphology Figure II-3 The mammary glands of adult, virgin LH- 65 overexpressing mice display an increase in epithelial cell proliferation, but no change in apoptosis Figure II-4 The mammary glands of adult virgin LH- 67 overexpressing mice display a mid-pregnancy phenotype at the molecular level Figure II-5 Mammary gland hyperplasia in LH- 69 overexpressing mice is dependent on ovarian input Figure II-6 LH-overexpressing mice are predisposed to 71 mammary cancer Figure II-7 DMBA-induced and spontaneous mammary 73 cancers in LH-overexpressing mice Figure II-8 Spontaneous mammary tumors from LH- 75 overexpressing largely lack expression of progesterone and estrogen receptors Figure III-1 Mammary gands of LH-overexpressing mice 92 demonstrate elevated expression of vitamin D vi receptor Figure III-2 EB1089 decreases mammary epithelial cell 94 proliferation in LH-overexpressing mice Figure III-3 EB1089 displays anti-tumorigenic activity in a 96 subset of hormone-induced mammary tumors Figure IV-1 Pre-neoplastic mammary glands and mammary 139 tumors from LH-overexpressing mice display centrosome amplification Figure IV-2 A mutant form of p53 does not alter the latency of 141 hormone-induced mammary tumorigenesis Figure IV-3 mRNA levels of regulators of p53 stability in 143 mammary glands and tumors of LH- overexpressing mice Figure IV-4 p53 is activated in the mammary glands of wild- 145 type and LH-overexpressing mice by ionizing radiation Figure IV-5 Mammary epithelial cells of wild type and LH- 147 overexpressing animals demonstrate variable apoptotic response to ionizing radiation Figure V-1 Tamoxifen inhibits ductal branching and alveolar 171 bud formation in LH-overexpressing mice Figure V-2 Hormone-induced mammary gland hyperplasia 173 persists in the presence of EB1089 Figure V-3 EB1089 fails to inhibit the formation of hormone- 175 induced mammary gland hyperplasia vii ACKNOWLEDGEMENTS My graduate school experience has been influenced by a number of people. First and foremost among these is my thesis advisor and mentor, Ruth Keri. Ruth has given me endless support and encouragement, as well as much needed sympathy throughout my time in her lab. In addition to molding me into a scientific thinker, she has taught me, by example, the importance and benefit of being open to the constructive criticism of others. I would also like to acknowledge John Nilson, my scientific “grandfather”, whose confidence in me has been unwavering and who has provided me with numerous opportunities to experience the world of science outside the laboratory. I also appreciate the guidance and input of the remaining members of my thesis committee – Amy Wilson-Delfosse, Lloyd Culp, and Paul MacDonald. Interacting with other students in a variety of settings has been the most intellectually stimulating component of my graduate education. I didn’t really understand the concept of “critical analysis” until I experienced prelim I preparation with Helai Mohammad and Amelia Sutton. These sessions and the many others that followed with many different students always challenged me and taught me more than I learned in any class. The students in my own lab have had a particularly important part in my education and life over the past several years. Melissa Landis has been an example and a friend to me from my first day in the Pharmacology Department. Her thoughtfulness and careful consideration of scientific questions, as well as her patience and positive attitude have amazed and inspired me. Jonathan Mosley, questioner of all ideas and viii conventions, has, much to my benefit, afforded me many stimulating discussions in and beyond the realm of science. My protracted tenure as a graduate student has also allowed me to learn from “younger” students, like Jen Yori and Marjorie- Montanez-Wiscovich, as they ask questions I’ve never thought to ask. The work presented in this dissertation was made possible by contributions from many other people. Kristen Lozada has tirelessly monitored all of my mice and has made sure that I had all the reagents and equipment I needed, even at a moments notice. Much of the early work on the CTP mice was done by former Keri lab members Alireza Behrooz and Becky Ameduri. The VDR work was done in conjunction with Xiaoxue Zhang in the Paul MacDonald lab. The MRI experiments were made possible by Chris Flask and Jeff Duerk. Ira Whitten contributed substantially to the assessment of centrosome amplification in the CTP mammary glands. Melissa Landis performed the breeding and palpating of the p53R172H/CTP mice. Finally, although their contributions may not appear in any of the figures presented here, I would like to acknowledge other members of the Keri lab: Lenka Yunk, my faithful undergraduate, and Darcie Seachrist, who has provided not only expert technical advice, but a great deal of encouragement and support. In ending, I would like to acknowledge the incredible support I have received from my family – Matt, Mom, Dad, Daniel, Phil, Evan, and all the Gerritys, McCallions, Lees, Caseys, Fines, and Millikens. I am ever thankful that their love does not depend on whether or not my experiments work and that, even though they’re proud of me, this PhD is immaterial. ix Use of a Transgenic Mouse Model of Ovarian Hyperstimulation to Identify Therapeutic Targets and Mechanisms in Hormone-Induced Mammary Cancer Abstract By Erin L. Milliken Epidemiological studies and clinical trials have revealed the critical impact that reproductive hormones have on breast cancer. The work described in this dissertation characterizes a transgenic mouse model, the LH-overexpressing mouse, which develops mammary gland hyperplasia and tumorigenesis in response to ovarian hyperstimulation, making it a unique model of hormone- induced mammary cancer. The hyperplasia in these mice is ovary-dependent and is due to a dramatic increase in proliferation of mammary epithelial cells. Most of the spontaneous mammary tumors that form in this model are mammary intraepithelial neoplasias that lack expression of both estrogen and progesterone receptors. The LH-overexpressing mice have been utilized to investigate potential therapeutic targets and identify mechanisms that contribute to hormone-mediated mammary gland pathology. Similar to observations made in human breast cancer, the pre-neoplastic mammary glands and tumors of LH-overexpressing mice demonstrate increased expression of vitamin D receptor, supporting the notion that this protein is a potential target for therapy. Treatment with EB1089, a vitamin D receptor agonist, results in decreased proliferation in the pre-neoplastic x mammary glands of LH-overexpressing mice and reduces the growth rate of a subset of established mammary tumors, providing evidence for both chemopreventive and chemotherapeutic benefits of activating the vitamin D receptor. Analysis of pre-neoplastic mammary glands of LH-overexpressing mice has revealed the presence of centrosome