University of Massachusetts Amherst ScholarWorks@UMass Amherst Doctoral Dissertations Dissertations and Theses 5-8-2020 INTER-INDIVIDUAL VARIATION IN RESPONSE TO ESTROGEN AND IMPLICATIONS FOR BREAST CANCER RISK Amye L. Black University of Massachusetts Amherst Follow this and additional works at: https://scholarworks.umass.edu/dissertations_2 Part of the Cancer Biology Commons, and the Developmental Biology Commons Recommended Citation Black, Amye L., "INTER-INDIVIDUAL VARIATION IN RESPONSE TO ESTROGEN AND IMPLICATIONS FOR BREAST CANCER RISK" (2020). Doctoral Dissertations. 1976. https://doi.org/10.7275/gxe7-4m14 https://scholarworks.umass.edu/dissertations_2/1976 This Open Access Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks@UMass Amherst. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. INTER-INDIVIDUAL VARIATION IN RESPONSE TO ESTROGEN AND IMPLICATIONS FOR BREAST CANCER RISK A Dissertation Presented by AMYE L. BLACK Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2020 Molecular and Cellular Biology © Copyright by Amye L. Black 2020 All Rights Reserved INTER-INDIVIDUAL VARIATION IN RESPONSE TO ESTROGEN AND IMPLICATIONS FOR BREAST CANCER RISK A Dissertation Presented by AMYE L. BLACK Approved as to style and content by: ____________________________________ D. Joseph Jerry, Chair ____________________________________ Sallie S. Schneider, Member ____________________________________ Kathleen Arcaro, Member ____________________________________ Thomas Zoeller, Member ____________________________________ Scott Garman, Director Molecular and Cellular Biology DEDICATION To my family, for their never-ending support. ACKNOWLEDGMENTS I would like to thank my advisor, D. Joseph Jerry, for welcoming me into the lab and giving me this great opportunity to work with primary human tissue and cell models. He has always encouraged me to push myself, and in doing so has helped me develop as a scientist. I am also grateful for the support of Karen Dunphy, whose advice has been instrumental to my growth as a scientist. In addition, I need to thank all the Jerry lab members, past and present, who have helped me along the way including Amy Roberts, Libby Daniele, Aliza Majewski, Prabin Dhangada Majhi, Aman Sharma, Ceren Goral, Mary Hagen, and Shakirah Ssebyala. Through lab meeting discussions and informal conversations, you helped me develop project ideas and push forward with this work. I am especially thankful for the support of Amy Roberts, who has helped me out in the lab more times than I can count. I am also thankful for the guidance of my committee members Sallie Schneider, Kathleen Arcaro, and Thomas Zoeller throughout this journey. Their support and helpful suggestions at our meetings were crucial to finishing this work. I especially thank Sallie Schneider and her lab members Stephanie Morin, Kelly Gregory, and Jennifer Ser- Dolansky for their collaboration over the years. I also need to thank Michele Markstein for her support during my first lab rotation and beyond. Lastly, I would like to thank my family and friends. I would not have been able to finish this work without your support. v ABSTRACT INTER-INDIVIDUAL VARIATION IN RESPONSE TO ESTROGEN AND IMPLICATIONS FOR BREAST CANCER RISK MAY 2020 AMYE L. BLACK, B.S., UNIVERSITY OF CONNECTICUT Ph.D., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: D. Joseph Jerry Breast cancer is the second leading cause of cancer related death among women and the most prevalent cancer type in women worldwide. Many risk factors for breast cancer are related to estrogen exposure, including early menarche, late menopause, nulliparity, hormone replacement therapy, and high serum estrogen levels. These factors all involve prolonged or high levels of estrogen exposure. However, estrogen exposure during early pregnancy lowers risk by up to 50% and high dose estrogen treatment is an effective antitumor treatment in postmenopausal women. The mechanisms behind estrogen’s paradoxical role in both contributing to and reducing breast cancer risk are currently unknown. We hypothesized that a subset of women may be more sensitive to estrogen exposure, and the following experiments were designed to test this hypothesis and determine if sensitivity to estrogen is due to estrogen receptor levels or downstream regulation of estrogen signaling using 3 human breast tissue or cell models. Our first model used to characterize responses to estrogen treatment was the human breast ex-vivo explant model. Donor human breast explants were treated with control or estrogen for 4 days and then transcriptional and functional responses were compared between individuals. Up to 80 or 100-fold variation in estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) mRNA levels were observed in explants. Responses to vi estrogen also varied among individuals, although estrogen receptor target gene expression (AREG, PGR, and TGFβ2), progesterone receptor protein (PR) expression, proliferation, and irradiation induced apoptotic responses were not significantly correlated with estrogen receptor levels. These results indicated that there are variable responses to estrogen exposure among individuals but that these responses do not appear connected to estrogen receptor expression levels. Our second model, conditionally immortalized primary human mammary epithelial cells (ciHMEC), also demonstrated variable responses in luciferase reporter assays with estrogen and xenoestrogen treatment, though the responses were more consistent than those observed in the human breast explant model. This was likely due to transfection of saturating levels of estrogen receptor. Lastly, we examined estrogen-induced responses in 4 inducible ERα HMEC cell lines. While all 4 lines were able to activate luciferase reporter assays and expressed ERα protein, only 3 out of 4 demonstrated a proliferation response to estrogen. Expression of estrogen receptor target genes (AREG and PGR) however, was not regulated by estrogen treatment. Conditioned growth media assays demonstrated that this proliferative response was not due to secretion of factors into the media but is instead driven by intracellular factors. As others have implicated the pioneer factors FOXA1 and GATA3 in restoring estrogen-induced responses, we examined mRNA expression of these factors in our cells and found no correlation with estrogen-induced proliferation. These results imply that while FOXA1 and GATA3 are not sufficient to induce biological responses to estrogen. Collectively, our work demonstrated individual variation in estrogen receptor expression and estrogen and xenoestrogen-induced responses. Sensitivity to estrogen treatment was not driven by estrogen receptor expression levels. Further, our results suggest intracellular vii factors, such as estrogen receptor coregulators, are important for modulation of estrogen- induced biological responses. viii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ...................................................................................................v ABSTRACT...................................................... ................................................................. vi LIST OF TABLES ............................................................................................................ xii LIST OF FIGURES ......................................................................................................... xiii CHAPTER 1. INTRODUCTION .........................................................................................................1 Estrogen Exposure and Breast Cancer Risk.............................................................1 Familial Breast Cancer Risk ....................................................................................2 Estrogen Sensitivity in Rodents ...............................................................................3 Estrogen Receptors Alpha and Beta ........................................................................5 Estrogen Receptor Signaling....................................................................................7 Pioneer Factors, Coregulators, and Estrogen Receptor Signaling ...........................9 Estrogen Receptor Levels in Normal Breast Epithelium .......................................11 Human Models for Estrogenic Responses in the Normal Breast ...........................12 Hypothesis and Objectives .....................................................................................16 2. INTER-INDIVIDUAL VARIARION IN ESTROGEN-INDUCED RESPONSES IN HUMAN BREAST EXPLANT CULTURES ...................................................................24 Introduction/Rationale ...........................................................................................24 Materials and Methods ...........................................................................................27 Sample Collection ......................................................................................27 Microarray Analysis...................................................................................27 Explant Culture ..........................................................................................28 RT-qPCR....................................................................................................29