Role of Environmental Estrogens and Acquired Endocrine Resistance in Breast Cancer and Implications for Treatment with Novel Antiestrogens by Thomas Lorenzo Gonzalez A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Toxicology) in the University of Michigan 2018 Doctoral Committee: Assistant Professor Justin A. Colacino, Co-Chair Associate Professor James M. Rae, Co-Chair Professor Rita Loch-Caruso Professor Bhramar Mukherjee Professor Rudy J. Richardson Thomas L. Gonzalez [email protected] ORCID iD: 0000-0003-4497-3106 © Thomas L. Gonzalez 2018 Dedication To my grandparents ii Acknowledgements First, I would like to thank my mentor, Dr. James Rae, for his guidance and unwavering support throughout the duration of my graduate work. From day one, Dr. Rae provided a research environment which allowed me to mature as a scientist, while encouraging me to explore and investigate my own scientific ideas. His commitment to supporting my academic growth has led me to seek out rewarding collaborations with other researchers and develop skills beyond what I expected of myself. I am grateful for his personal investment into my research career and I look forward to continuing our relationship both as colleagues and as friends. Moreover, I am very appreciative of the valuable guidance and time that I have spent working closely with Dr. Justin Colacino who played a critical role in helping me pursue my proposed research ideas during my training. Thank you to each of my committee members Dr. Bhramar Mukherjee, Dr. Rita Loch-Caruso, and Dr. Rudy J. Richardson for the invaluable support and guidance with my dissertation research. Thank you to Dr. Mukherjee and her graduate student, Jonathan Boss, for their expertise with statistical analysis and data interpretation. Thank you to Dr. Loch-Caruso for her valuable academic and professional guidance in the field of toxicology all these years. Thank you to Dr. Richardson for taking the time to help me understand and utilize computational toxicology techniques in my own research. I would also like to thank the members of the Rae lab, especially Dr. Jose Larios and Christina Gersch. Both Jose and Christina have both made substantial time investments iii throughout my training as a bench scientist and were individuals whom I know could reach out to for advice or help in the lab. Thank you to Siqi Sun and Molly Hancock for their time and commitment to the PROTAC project and being great students to work with. Thank you to Dr. Daniel Hayes and Dr. Erin Cobain for sharing with me their clinical expertise with me in the field of breast cancer research. A special thanks to my family, especially my parents Larry and Maria, who have provided unconditional love and support throughout my entire academic career. Lastly, I want to thank Breanna Johnson for being my foundation of support and encouragement all these years. This work is supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under Award Number T32ES007062, R01ES028802, and P30ES017885, including the Breast Cancer Research Foundation, the Michigan Lifestage Environmental Exposures and Disease (M-LEEaD) Center Pilot Project Grant, the Rackham Graduate Student Research Grant, and the Rackham Merit Fellowship Program. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Finally, I would also like to thank NSF International (Ann Arbor, MI) for their technical support and assistance for analyzing samples used in this dissertation. iv Table of Contents Dedication ...................................................................................................................................... ii Acknowledgements ...................................................................................................................... iii List of Figures .............................................................................................................................. vii List of Tables ................................................................................................................................ ix List of Abbreviations .................................................................................................................... x Abstract ......................................................................................................................................... xi Chapter I. Introduction ................................................................................................................ 1 Human Physiology of Steroid Hormones ................................................................................... 2 Adrenal Control Systems ........................................................................................................ 2 Adrenal Steroidogenesis ......................................................................................................... 3 Gonadal Control Systems ....................................................................................................... 3 Gonadal Steroidogenesis ......................................................................................................... 4 Transport of E2 in the Blood .................................................................................................. 6 Nuclear receptors .................................................................................................................... 7 The Estrogen Receptor ................................................................................................................ 8 ERß ......................................................................................................................................... 8 ERα66 ..................................................................................................................................... 9 Other ERα Isoforms and GPR30 .......................................................................................... 10 Environmental Estrogens and Anti-androgenic Endocrine Disrupting Compounds ................ 12 Phthalates .............................................................................................................................. 14 DDT and its Analogs ............................................................................................................ 15 Methoxychlor ........................................................................................................................ 17 UV-filters .............................................................................................................................. 17 Bisphenols ............................................................................................................................. 18 Parabens ................................................................................................................................ 20 The Role of Estrogen and the ER Pathway in Breast Cancer ................................................... 22 Tumor Cell Subtypes in Breast Cancer ................................................................................. 23 Discovering the Relationship Between Breast Cancer and Estrogen ................................... 24 Types of Antiestrogens ......................................................................................................... 26 Endocrine Therapy Resistance and Recurrence in Breast Cancer ........................................ 26 Rationale for Thesis .................................................................................................................. 29 References ................................................................................................................................. 32 Chapter II. Metabolites of n-Butylparaben and iso-Butylparaben Exhibit Estrogenic Properties in MCF-7 and T47D Human Breast Cancer Cell Lines ....................................... 54 Introduction ............................................................................................................................... 54 Materials and Methods .............................................................................................................. 56 v Results ....................................................................................................................................... 60 Discussion ................................................................................................................................. 65 References ................................................................................................................................. 83 Chapter III. Homology Models of Mouse and Rat Estrogen Receptor-α Ligand-Binding Domain Created by In Silico Mutagenesis of a Human Template: Molecular Docking with 17ß-Estradiol, Diethylstilbestrol, and Paraben Analogs ......................................................... 89 Introduction ............................................................................................................................... 89 Materials and Methods .............................................................................................................. 92 Results ....................................................................................................................................... 99 Discussion ..............................................................................................................................