Exploring estrogen receptor gene regulatory mechanism in breast cancer ______________________________________ A Dissertation Presented to the Faculty of the Department of Biology and Biochemistry University of Houston _______________________________________ In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy _______________________________________ By Anne Chinenye Katchy August 2013 Exploring estrogen receptor gene regulatory mechanism in breast cancer ______________________________________________ Anne Chinenye Katchy APPROVED ______________________________________________ Dr. Cecilia Williams, Chairman ______________________________________________ Dr. Robert Schwartz ______________________________________________ Dr. Paul Webb Methodist Research Institute, Houston, TX ______________________________________________ Dr. Xiaolian Gao ______________________________________________ Dean, College of Natural Science and Mathematics ii Acknowledgements This dissertation was made possible because of the guidance from my advisor, guidance from my committee members, and help and support from family and friends. I would like to express my deepest gratitude and appreciation to my advisor, Dr. Cecilia Williams, for believing in me and letting me do my dissertation in her lab, for her patience and excellent guidance and support. I would like to thank you, Cecilia, for making this dissertation possible, and for your time and effort that you have put into preparing me for this moment. I am forever grateful to you. I would also like to thank my committee members: Dr. Robert Schwartz, Dr. Paul Webb, and Dr. Xiaolian Gao. I want to thank you all for your guidance, advice, and time you have given me to make this dissertation possible. I am very grateful to you all. I would like to thank all my dear friends and colleagues Trang, Philip, Jun, Eylem, Karin, Kim, Caroline, Ka, Lucy, Fotis, Efi, Sharanya, Stella, Ejike, and colleagues at CNRCS. Thank you all for the discussions, advice, and support. I appreciate you all. I would like to thank Ana, Esra, and Bukky. I have never known such good friends. I thank you all for your love and support. I remember when we first started, and look at us now. I love you all. I would most especially like to thank my family. I would like to thank my parents for their everlasting love and care. I thank you both for giving me the opportunity to study abroad and for believing in my dreams. I thank you both for everything. I would like to say a special thank you to my older sister, Ngozi. You have been an inspiration to me. I thank you for your encouraging words, and for never letting me give up on anything. You iii are my strength and I would always love you. To my brothers, Nnamdi and Kenechukwu, I would like to say thank you for your love and support, and especially for always being protective of me. To my baby sister, Oby, I thank you for your love and for being you. The sky is the limit for you, my dear. And finally, I would like to thank my friend, Laura, for being a sister to me. I thank you for your support, love, and care. You are the true definition of what a friend should be. You are my family and I appreciate you. iv Exploring estrogen receptor gene regulatory mechanism in breast cancer ______________________________________ An Abstract of a Dissertation Presented to the Faculty of the Department of Biology and Biochemistry University of Houston _______________________________________ In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy _______________________________________ By Anne Chinenye Katchy August 2013 v ABSTRACT Estrogen has vital roles in development and maintenance of mammary gland and supports the growth of the majority of primary breast cancer. It carries out its function through the estrogen receptors (ER): ERα and ERβ. In this dissertation, we focused on identifying the functional and genome wide effects of both receptors in breast cancer cell lines (T47D and MCF7). First, we aimed at identifying microRNAs (miRNAs) that are significantly associated with normal or disrupted estrogen signaling in breast cancer cells, and are regulated by ERα and ERβ. Despite the fact that 24h estrogen treatment results in strong changes to expression of about 900 protein-coding transcripts, we found no significant changes in mature miRNA expression levels by 17β-estradiol (E2)-activated ERα in neither T47D nor MCF7 breast cancer cells. On the other hand, when studying the effects of exogenously expressed ERβ, we identified miR-135a, miR-21, miR-200c, and miR- 522, among others, as being regulated. Most of the ERβ effects were ligand-independent, but we observed a significant response to E2 in the MCF7-ERβ cells. Also, the MCF7- ERβ showed enhanced stem cell abilities in comparison to the MCF7-Control cells as illustrated by increased mammospheres formation during several generations. Secondly, we aimed at investigating environmental factors and its effect on the risk for breast cancer. We identified the gene expression response for each of these compounds: BPA, genistein (Gen), E2, and soy formula extract (SF) in MCF7 cells, and found that each regulated similar genes in the same manner. Many target genes regulated vi by BPA, Gen, and SF, were involved in important biological processes identical to those of estrogen. Investigation of non-ERα mediated regulations for these EDCs suggested that these compounds may regulate gene transcription solely through ERα, in the cells and doses used. Furthermore, we found that these compounds acted together in a sub- additive manner, and tumor clustering with the gene expression profile of these EDC compounds revealed a significantly lower disease free survival. Altogether, the data presented in this dissertation would aid in increasing the knowledge of early risk factors for breast cancer. Our work provides data for future use of the estrogen receptors in clinical applications by providing new candidates for pharmaceutical drug development, as well as, biomarkers for diagnosis and prognosis. vii Table of Contents Page Chapter 1 1 Introduction 1 1.1 Breast cancer 2 1.1.1 Origin and causes 3 1.1.2 Molecular characteristics of breast cancer 7 1.1.3 Treatment approaches 12 1.2 MicroRNA 13 1.2.1 miRNA biogenesis and mechanism of action 14 1.2.2 miRNA and breast cancer 17 1.3 Molecular functions of estrogen in breast cancer 20 1.3.1 Estrogen synthesis 20 1.3.2 Estrogen receptors 21 1.3.3 Estrogen receptor and miRNA 25 1.3.4 Influence of endocrine disruptive chemicals 26 Chapter 2 30 Materials and methods 30 2.1 Materials 31 2.2 Cell cultures 32 2.3 RNA preparation and cDNA synthesis for mRNA and miRNA analysis 33 2.4 Gene expression analysis 34 2.4.1 qPCR for mRNA and miRNA expression analysis 34 2.4.2 Microarray experiment for mRNA and miRNA analysis 35 2.5 Bioinformatics 36 Chapter 3 37 Estradiol-activated ERα does not regulate mature miRNAs in T47D breast cancer cells 37 viii Page 3.1 Abstract 38 3.2 Introduction 39 3.3 Methods 41 3.3.1 Cell culture 41 3.3.2 RNA extraction 42 3.3.3 cDNA synthesis 42 3.3.4 miRNA microarray analysis 42 3.3.5 qPCR analysis of mRNA and miRNA expression 43 3.3.6 Bioinformatics 44 3.4 Results 45 3.4.1 Strong ERα-mediated transcription of protein-coding genes occur at 24h in T47D cells 45 3.4.2 Profiling showed minimal ERα-mediated regulation of miRNAs at 24h 47 3.4.3 qPCR confirms non-significant ERα regulation of miRNAs 48 3.4.4 Previously reported estrogen-induced miRNA regulations could not be reproduced 51 3.4.5 Time-course study of miRNA reveals a slight variation in miRNA expression 55 3.4.6 miRNAs with an ERα chromatin-binding site nearby are not regulated in T47D cells 57 3.4.7 miRNAs can differentiate between non-tumor and tumor cell lines 60 3.5 Discussion 60 3.6 Conclusion 64 3.7 Supplemental figures to Chapter 3 65 Chapter 4 68 Estrogen Receptor β regulates mature miRNAs in breast cancer cells 68 4.1 Abstract 69 4.2 Introduction 70 4.3 Methods 73 ix Page 4.3.1 Cell culture 73 4.3.2 miRNA microarray analysis 73 4.3.3 qPCR analysis of mRNA and miRNA expression 74 4.3.4 siRNA analysis 74 4.3.5 Mammosphere formation assay 74 4.4 Results 76 4.4.1 Strong ERβ-mediated transcription of protein coding genes occurs at 24h in T47D and MCF7 cells 76 4.4.2 miRNA profiling reveals possible ERβ mediated regulation of miRNAs 78 4.4.3 qPCR confirms ERβ mediated regulation of miRNAs in T47D cells 80 4.4.4 Presence of ERβ may be responsible for miRNA regulation in T47D cells line 82 4.4.5 MCF7 showed a more pronounced ERβ-mediated regulation of miRNA 82 4.4.6 ERβ mediates a ligand-dependent regulation of miRNAs in MCF7 cells 85 4.4.7 ERβ affects mammosphere formation abilities in breast cancer cells 85 4.4.8 miR-135a in breast cancer cells 90 4.5 Discussion 97 4.6 Conclusion 100 Chapter 5 101 Bisphenol A and Phytoestrogens mediate identical and sub-additive effects on ERα-mediated transactivation 101 5.1 Abstract 102 5.2 Introduction 103 5.3 Methods 105 5.3.1 Cell culture 105 5.3.2 RNA preparation and cDNA synthesis 105 5.3.3 qPCR analysis for mRNA expression 106 5.3.4 Microarray experiment and analysis 106 x Page 5.3.4 Bioinformatics 107 5.3.5 Survival cluster analysis 107 5.4 Results 108 5.4.1 Comparison of dose-dependent transcriptional activation of endogenous ERα by BPA, genistein, and soy formula extract 108 5.4.2 Sub-additive effects of EDCs on ERα-mediated transcriptional activation 110 5.4.3 Transcriptional regulation by BPA, genistein, and SF are solely through an ERα-mediated pathway