UNIVERSITY OF CINCINNATI Date:_9/4/07______________ I, _Yuxin Feng________________________________________________, hereby submit this work as part of the requirements for the degree of: Doctor of Philosophy in: Cell and Cancer Biology It is entitled: Mechanism of estrogen receptor alpha action and the consequence of its conditional deletion on Mammary gland development and function This work and its defense approved by: Chair: Sohaib Khan, Ph.D.____________ Karen E.Knudsen, Ph.D.________ Elwood V. Jensen, Ph.D._______ Susan Waltz, Ph.D.____________ Nelson Horseman, Ph.D.________ Robert Brackenbury, Ph.D._____ Mechanism of estrogen receptor-alpha action and the consequence of its conditional deletion on mammary gland development and function A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (Ph.D.) in the Department of Cell and Cancer Biology of the College of Medicine 2007 by Yuxin Feng M. S., Nankai University, 1997 B. S., Ocean University of Qingdao, 1990 Committee Chair: Sohaib Khan, Ph.D. ABSTRACT ERα is a critical regulator in breast cancer and mammary gland development. Deregulation of ER signaling correlates with abnormal mammary gland development and breast cancer. However, the role of epithelial ER remains to be clarified in vivo and the mechanism of ER signaling regulation is far from comprehensive. We hypothesize that 1) mammary epithelial ER plays critical roles in mammary gland development during pregnancy and lactation and that 2) novel, as yet identified factors in ER transcriptional regulation are involved in breast cancer development. The loxP-Cre system was used to generate epithelial ERKO mice. The well characterized MMTV-Cre and WAP-Cre transgenic mice were used to delete ER in mammary epithelial cells at different developmental stages. Early expression of MMTV-Cre arrested mammary gland development at the neonatal stage. Successive pregnancy and lactation activated epithelial ER ablation, which compromised side-branching, alveolar development, and epithelial proliferation. Further analysis revealed a massive loss of luminal epithelial cells presumably caused by apoptosis. The abnormal mammary gland development decreased milk production, thereby, caused growth retardation in the offspring. Similar phenotypes were also observed in MMTV-ERKO females in lactation. Thus, we concluded that epithelial ER is essential for mammary gland development during pregnancy and lactation stages. To further pursue the molecular mechanism of ER signaling regulation, a human mammary gland cDNA library was screened to identify novel factors that interact with ER. One novel ERα binding protein identified in the screen contains two conserved LXXLL motifs (NR-box) and a coiled-coil domain. The protein product, which we named NRCC, consists of 3 isoforms that vary in their N-terminal region. NRCC is 2 conserved in vertebrates and its mRNA was detected in human breast cancer cells and mouse breast tumors. We found that NRCC-A interacts with ERα and enhances ERα transcriptional activity in human cancer cells. Moreover, NRCC-A co-localized with ERα in the cell nucleus and was recruited to ER target gene promoters. SiRNA analysis indicated that NRCC proteins are important for endogenous ERα-mediated transcriptional activity and estrogen dependent cell proliferation. Taken together, these data indicate that NRCC-A is a novel coactivator for ERα. 3 4 DEDICATION To my grandparents and my father 5 ACKNOWLEDGMENTS So many brilliant and giving people guided and supported me for my research. I would like to specifically thank my advisor Professor Sohaib Khan for laying the foundation for this study. I am grateful for his guidance and patience in these years. He has been a great advisor and mentor. His commitment, determination and dedication shape me to become a scientist. I am sincerely grateful to the following persons and institutions: To my wife, my mother, and my daughters (Selena and Hannah) for their support and patience; To my committee for their brilliant advice and enlightened discussion: Drs. Elwood Jensen, Nelson Horseman, Robert Brackenbury, Susan Waltz and Karen Knudsen; To the current and previous members of Khan lab for their help and friendship: Drs. David Manka, David Singleton, Gina Bell, Elizabeth Shaughnessy, Robin Tharakan, Jun Yang, Peihong Jin and Pierig Lepont; To the Dept. of Cancer and Cell Biology and Cell and Molecular Biology graduate program: Management team: Susan Seiler, Sasha Simms, Jaynee Tolle, Barbara Carter, Amy Itescu; Zhang lab; Ip lab; Stambrook lab; Ben-Jonathan lab; Eric and Karen Knudsen lab; Joe Closson: without whom our network would not run; UC Center for Biological Microscopy: Birgit Ehmer and Nancy Kleene; To all the Graduate students in Cancer and Cell Biology; To the UC Gene Targeting Core: Phil Sanford and Dr. Tom Doetschman To the UC Comparative Pathology: Rita Angel and Dr Greg. Boiwin To the Children’s Pathology Core: Lisa and Dr. Stringer; To the National Institutes of Health Grant T32 HD07463; To my family; To all my friends. 6 TABLE OF CONTENTS Abstract…………………………………………………………………………………....2 Acknowledgments…………………………………………………………………………6 Table of contents…………………………………………………………………………..7 List of figures……………………………………………………………………………...9 Abbreviations…………………………………………………………………………….11 Chapter I: Introduction…………………………………………………………………...12 A. Breast cancer and estrogen receptor.………………………………………...13 B. Estrogen signaling……………………………………………………………15 C. Estrogen receptors……………………………………………………………16 D. Hormone receptor co-factors………………………………………………...17 1. Corepressors………………………………………………………...........17 2. Coactivators……………………………………………………………...18 i. P160 family…………………………………………………..19 ii. Co-integrators………………………………………………..20 iii. TRAP/mediators……………………………………………..20 iv. Chromatin remodeling factors……………………………….21 v. Secondary coactivators………………………………………21 vi. Other coactivators……………………………………………22 E. Morphogenesis of mammary gland development……………………………23 F. ERα in mammary gland development……………………………………….25 G. ER-related signaling pathways in mammary gland development…………....26 H. Introductory conclusions and hypothesis…………………………………….29 I. References……………………………………………………………………32 Chapter II: NRCC-A enhances estrogen receptor alpha (ERα) transcriptional activity……………..43 A. Abstract………………………………………………………………………44 B. Introduction…………………………………………………………………..45 C. Results………………………………………………………………………..47 D. Discussion……………………………………………………………………53 E. Materials and methods……………………………………………………….57 F. References……………………………………………………………………60 G. Figures and legends…………………………………………………………..63 Chapter III: Estrogen receptor-α expression in the mammary epithelium is required for ductal and alveolar morphogenesis in pubertal, pregnant and lactating mice……………………….73 A. Abstract………………………………………………………………………74 B. Introduction…………………………………………………………………..76 C. Results………………………………………………………………………..79 D. Discussion……………………………………………………………………85 7 E. Materials and methods……………………………………………………….89 F. References……………………………………………………………………92 G. Figures and legends…………………………………………………………..95 Chapter IV: Mechanism of ERα-dependent developmental changes in mammary gland…………...109 A. Abstract……………………………………………………………………..110 B. Introduction…………………………………………………………………111 C. Results………………………………………………………………………115 D. Discussion…………………………………………………………………..119 E. Materials and methods……………………………………………………...124 F. References…………………………………………………………………..126 G. Figures and legends…………………………………………………………130 Chapter V: Summary and conclusions………………………………………………….139 A. Summary……………………………………………………………………139 B. Conclusion…………………………………………………………………..139 C. References…………………………………………………………………..150 Chapter VI: Future directions…………………………………………………………..153 A. The role of ERα in AIB1-induced tumors………………………………….153 B. The anti-inflammation effect of ERα in AIB1-induced mammary tumorigenesis……………………………………………………………….154 C. The role of ERα in mammary stem cells and epithelial progenitor cells…...156 D. References…………………………………………………………………..158 8 List of figures: Chapter I: Introduction Figure 1 Estrogen induced cascade of cellular events Figure 2 Functional domains of ER Figure 3 Transcription cycle of estrogen-dependent activation of pS2 gene Figure 4 Stages of postnatal mouse mammary gland development Figure 5 ERα signaling in mammary gland development Chapter II: NRCC is a novel ERα coactivator Figure 1 NRCC protein structure and mRNA expression in different tissues and cells Figure 2 NRCC-A interacts with ERα in mammalian cells Figure 3 NRCC-A enhances ERα and AR transcriptional activity Figure 4 NRCC-A and ERα co-localized in cell nucleus Figure 5 NRCC-A is a nuclear receptor coactivator Figure 6 NRCC-A promotes cancer cell proliferation Chapter III: Estrogen receptor-α expression in the mammary epithelium is required for ductal and alveolar morphogenesis in pubertal, pregnant and lactating mice Figure 1 Targeted and conditional disruption of the mouse ERα gene using the Cre-loxP recombination system Figure 2 ERα is required for mammary gland development Figure 3 Dilation and inadequate branching of ducts in WAP-ERKO mice during 2nd lactation Figure 4 Growth retardation in WAP-ERKO pups from the 2nd litter is attributable to mother’s genotype Figure 5 Loss of ERα and PR immunohistochemical staining in WAP-ERKO (WAP fl/fl), but not ERαfl/fl (fl/fl) mammary glands of the 2nd lactation, day 1 Figure 6 Introduction of loxP sites into the ERα