DISCERNING THE ROLE OF FOXA1 IN MAMMARY GLAND DEVELOPMENT AND BREAST CANCER by GINA MARIE BERNARDO Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Adviser: Dr. Ruth A. Keri Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY January, 2012 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Gina M. Bernardo ______________________________________________________ Ph.D. candidate for the ________________________________degree *. Monica Montano, Ph.D. (signed)_______________________________________________ (chair of the committee) Richard Hanson, Ph.D. ________________________________________________ Mark Jackson, Ph.D. ________________________________________________ Noa Noy, Ph.D. ________________________________________________ Ruth Keri, Ph.D. ________________________________________________ ________________________________________________ July 29, 2011 (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. DEDICATION To my parents, I will forever be indebted. iii TABLE OF CONTENTS Signature Page ii Dedication iii Table of Contents iv List of Tables vii List of Figures ix Acknowledgements xi List of Abbreviations xiii Abstract 1 Chapter 1 Introduction 3 1.1 The FOXA family of transcription factors 3 1.2 The nuclear receptor superfamily 6 1.2.1 The androgen receptor 1.2.2 The estrogen receptor 1.3 FOXA1 in development 13 1.3.1 Pancreas and Kidney 1.3.2 Liver 1.3.3 Lung 1.3.4 Brain 1.3.5 Gastrointestinal Tract 1.3.6 Prostate 1.4 The mammary gland 24 1.4.1 Stages of mammary gland development iv 1.4.2 Mammary epithelial hierarchy 1.4.3 The estrogen receptor in mammary gland development 1.5 FOXA1 in cancer 32 1.5.1 FOXA1 in cancers other than breast and prostate 1.5.2 FOXA1 in prostate cancer 1.6 FOXA1 in breast cancer 42 1.6.1 The molecular subtypes of breast cancer 1.6.2 FOXA1 expression in breast cancer 1.6.3 FOXA1 and the estrogen receptor 1.6.4 Additional roles of FOXA1 in breast cancer 1.7 Statement of Purpose 58 Chapter 2 FOXA1 is an Essential Determinant of ERα Expression 67 and Mammary Ductal Morphogenesis 2.1 Abstract 67 2.2 Introduction 69 2.3 Materials and methods 72 2.4 Results 78 2.5 Discussion 87 2.6 Acknowledgements 92 Chapter 3 FOXA1 Represses Basal Breast Cancer Characteristics 115 3.1 Abstract 115 3.2 Introduction 117 3.3 Materials and methods 121 v 3.4 Results 126 3.5 Discussion 134 3.6 Acknowledgments 140 Chapter 4 Summary and Future Directions 188 4.1 Summary 188 4.2 Is FOXA1 expression necessary for maintaining the 191 mammary epithelial lineage? 4.3 Does manipulation of FOXA1 alter breast cancer subtype 195 tumor progression? 4.4 How does FOXA1 repress basal breast cancer gene expression? 200 4.5 How is FOXA1 differentially regulated in breast cancer? 204 4.6 Concluding Remarks 210 Bibliography 213 vi LIST OF TABLES Table 1.1 Overview of Foxa1 mouse models of development 59 Table 1.2 Overview of FOXA1 in human cancer 60 Table 3.1 Genes commonly decreased upon knockdown of FOXA1 in 141 MCF7, T47D, MB-453 and SKBR3 cells (p<0.001). Table 3.2 Genes commonly increased upon knockdown of FOXA1 in 147 MCF7, T47D, MB-453 and SKBR3 cells (p<0.001). Table 3.3 Basal A, basal B and luminal classifier genes (RN) whose 155 expression is changed upon knockdown of FOXA1 in MCF7, T47D, MB-453 and SKBR3 cells (p<0.05). Table 3.4 Classifier gene lists (RN) used to discriminate the luminal, 156 basal A, and basal B molecular subtypes for GSEA. Table 3.5 Lum(B)-ECJ classifier gene list used for GSEA. 157 Table 3.6 Bas-ECJ classifier gene list used for GSEA. 158 Table 3.7 Lum(M)-ECJ classifier gene list used for GSEA. 159 Table 3.8 Mes-ECJ classifier gene list used for GSEA. 160 Table 3.9 GSEA of classifier gene lists that are discriminatory 161 of luminal v. basal breast cancer molecular subtypes. Table 3.10 Potential binding sites in basal and luminal signature 162 genes regulated by FOXA1. Table 3.11 Gene order on Luminal (B)-ECJ heatmap in Figure 3.7. 163 Table 3.12 Gene order on Basal-ECJ heatmap in Figure 3.7. 165 vii Table 3.13 Primers used for PCR amplification of DNA 167 that has been subject to FOXA1 ChIP. viii LIST OF FIGURES Figure 1.1 Mammary gland terminal end bud (TEB) formation and 61 ductal invasion Figure 1.2 FOXA1/AR signaling in prostate cancer 63 Figure 1.3 FOXA1/ERα signaling in breast cancer 65 Figure 2.1 FOXA1 is expressed in the developing mammary gland in 93 conjunction with ERα. Figure 2.2 FOXA1 is not necessary for embryonic development of 95 the mammary gland. Figure 2.3 FOXA1 is required for mammary ductal outgrowth in an 97 orthotopic transplantation model. Figure 2.4 FOXA1 is required for TEB formation and ductal invasion. 99 Figure 2.5 FOXA1 is not required for luminal or basal/myoepithelial 101 lineage specification. Figure 2.6 Pubertal mice heterozygous for the Foxa1 null allele display 103 decreased mammary ductal invasion. Figure 2.7 FOXA1 is not required for alveolar differentiation during 105 pregnancy. Figure 2.8 FOXA1 is required for expression of ERα in the normal 107 mammary gland. Figure 2.9 FOXA1 regulates transcription of ESR1. 109 Figure 2.10 FOXA1 regulates transcription of ESR1 in T47D cells. 111 ix Figure 2.11 Schematic of the mammary epithelial cell hierarchy 113 Figure 3.1 FOXA1 is expressed in the absence of ERα in breast 168 tumors and luminal cell lines. Figure 3.2 FOXA1 expression correlates with the luminal subtype 170 in breast cancer cell lines. Figure 3.3 Loss of FOXA1 increases migration and invasion of 172 luminal breast cancer cells. Figure 3.4 Identification of a FOXA1-dependent luminal transcriptome. 174 Figure 3.5 Loss of FOXA1 decreases enrichment for luminal genes, 176 while increasing enrichment for basal genes. Figure 3.6 Loss of FOXA1 induces changes in RN classifier 178 gene expression. Figure 3.7 Loss of FOXA1 induces changes in ECJ classifier 180 gene expression. Figure 3.8 GSEA enrichment plots for BasB-RN, BasAB-RN, 182 Lum(M)-ECJ and Mes-ECJ classifier lists. Figure 3.9 Loss of FOXA1 induces basal mRNA and protein expression. 184 Figure 3.10 FOXA1 binds to luminal and basal genes in 186 luminal breast cancer cells. Figure 4.1 FOXA1 is required for expression of cytokeratin 5/6 211 in the normal mammary gland. x ACKNOWLEDGEMENTS My journey to the Ph.D. has been influenced by so many. I must first thank all my professors from Washington & Jefferson College, who thoroughly prepared me for what was to follow. Vinnedge Lawrence, thank you for pushing me to my limits and helping me see my potential. Roy Ickes, for always being so insightful as an advisor. Steve Malinak, for making chemistry tolerable. Candy DeBerry and Alice Lee, thank you for the constant support and providing me with the opportunity to assist in your laboratory classes. Dennis Trelka, I will forever owe you for directing me towards UCLA. I must also thank my roommate from W&J, Kisa Lape, for your friendship over the years and pulling so many all nighters with me! Second, I was incredibly fortunate to have had the opportunity to work at UCLA before coming to CWRU. Dennis Slamon, thank you for exposing me to translational breast cancer research. Cindy Wilson, there are no words to express my gratitude for your patience, guidance and friendship. You are an inspiration. Raul Ayala, thank you for safely introducing me to mouse work and East LA cuisine! Chuck Ginther and Lee Anderson, you have taught me so much about science and life. Especially the joys of food and wine! Thank you for accepting me into your family and for the countless hours of insightful conversation. I especially want to thank you for being such great collaborators over the past couple years. It was so nice working together! Lisa Pinelli, thank you for being the best roommate and friend, and for putting up with so many evenings of science babble. xi My appreciation to all the members of the Keri lab, you were always there to provide advice and technical help. Marjorie Montañez-Wiscovich and Jonathan Mosley, thank you for your friendship and guidance, especially through my early years in the lab. Ruth, you have been a great mentor! Thank you so much for allowing me to be independent in the lab, but then always being there when I needed you (which we both know was quite often). I am much obliged to my committee members and the entire Department of Pharmacology. Also, to my dearest Pharmacology friends, Payal Gandhi, Andrea Moomaw, Elizabeth Sabens and Tara Ellison, thanks for all your advice and help with my research, but most of all, for the great times shared outside of the lab! Of course, I owe all the thanks in the world to my family. Mom and dad, you have put up with so much over the years! Thank you for never giving up on me, even when I seemed impossible to deal with, and for sacrificing so much so that I could take advantage of every opportunity thrown my way. Dad, thank you for bringing me up with strength and perseverance, Mom, for teaching me that is it important to counterbalance strength with grace and for always listening when I needed to vent, Dan, for keeping the world in perspective. Thank you grandparents, aunts and uncles for your unwavering love and support.