Lnc-EPCAM AND Lnc-BHLHE41 AS RNA REGULATORS OF BREAST CANCER AND BREAST CANCER PREVENTION A Dissertation Submitted to the Temple University Graduate School Board In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY By MARIA BARTON May 2017 Examining Committee Members: Jose Russo, MD, FACP, Advisor, The Irma H. Russo MD Breast Cancer Research Laboratory, Fox Chase Cancer Center Dianne Soprano, PhD, Advisor, Department of Biochemistry, Lewis Katz School of Medicine, Temple University Ana Gamero, PhD, Department of Biochemistry, Lewis Katz School of Medicine, Temple University Nora Engel, PhD, Department of Cancer Biology, Genetics and Epigenetics, Fels Institute, Lewis Katz School of Medicine, Temple University Richard Katz, PhD, External Member, Department of Epigenetics, Fox Chase Cancer Center Raghbir Athwal, PhD, External Member, Department of Pathology and Laboratory Medicine, Fels Institute, Lewis Katz School of Medicine, Temple University © Copyright 2016 By MARIA BARTON All Rights Reserved ii ABSTRACT The objective of this study was to unveil a novel area of gene regulation in breast cancer and breast cancer prevention through the study of a recent discovered class of genetic regulators named long non-coding RNAs (lncRNAs). LncRNAs are RNA molecules longer than 200 nucleotides that are not translated into proteins, but regulate the transcription of genes involved in different cellular processes, including differentiation, cancer initiation and progression. The link between lncRNAs and cancer is well documented in the literature. More recently, their relevance in the transcription field is beginning to be explored and their roles have been found to vary from guiding proteins to the genome to scaffolding proteins complexes needed for the transcription of a specific gene. Initial transcriptome analysis of normal breast of parous and nulliparous postmenopausal women revealed that several lncRNAs are differentially expressed in the parous breast. This observation provided evidence of a potential role of lncRNAs in the regulation of transcription and their function in pregnancy’s preventive effect in reducing the lifetime risk of developing breast cancer. Specifically, RNA sequencing of healthy postmenopausal breast tissue biopsies from eight parous and eight nulliparous women using Illumina platform was performed. The sequencing results showed that there are 42 lncRNAs differentially expressed between parous and nulliparous breast tissue. These data led to the hypothesis that these novel lncRNAs may be drivers in the process of development that occurs in the mammary gland during pregnancy, providing protection against breast cancer. After analysis of these 42 lncRNAs using bioinformatics tools, review of the scientific literature, and real-time PCR analysis, two lncRNAs (lncBHLHE41 and lncEPCAM) were selected to be tested in vitro, using different molecular techniques in human epithelial breast cell lines to determine their relevance in breast cancer. This project provided novel information on lncRNAs induced by pregnancy in the breast tissue, and identified two lncRNAs as potential key regulators in breast differentiation and cancer progression. The manipulation of these lncRNAs led to evidence of their function in vitro and, using xenograft studies, we determined their relevance in vivo. Although treatment for cancer using lncRNAs as targets is in its infancy at the clinic, the advancement in knowledge and technology to study their relevance in disease could lead to the development of therapeutics for breast cancer and breast cancer prevention in the near future. iii To my little “chinis” who has changed my life forever In memory of Miguel R. Barton iv ACKNOWLEDGEMENTS I feel this is the hardest part for me to write. I am so grateful to so many people who have contributed to me getting to this point. I first want to start by acknowledging my undergraduate mentor Dr. Sonia Rodriguez who sprinkled on me her passion for research. The few years I was her mentoree, and had the pleasure of interacting with her diverse team, ignited the fire for research that now burns in me. I have been very lucky with the mentors I have had and that is why my Master’s Degree mentor Dr. Robert H. Silverman also deserves my gratitude for the achievements during my PhD. It was in his laboratory, a great experience at the Cleveland Clinic, that I learned how to start walking the path of becoming an independent researcher and melting into the challenging working environment of a research-based institution. That is why, when I met Dr. Russo at Fox Chase Cancer Center, I already felt at home. Dr. Russo was welcoming from the very beginning. That is just who he is. Although I was nervous thinking of the responsibilities ahead of me, I felt comfortable during our first encounter, which feels like yesterday. Thank you for letting me (and even encouraging me) to always speak my mind. I owe you a great deal of my achievements and I will be forever grateful for the opportunity of working in The Irma H. Russo MD Breast Cancer Research Laboratory. In addition to my mentors, I want to deeply thank my advisory committee, Dr. Dianne Soprano, Dr. Nora Engel and Dr. Ana Gamero for their continuous support and suggestions at every step during my graduate work. I took your comments into action and your input undoubtedly gave shape to this very challenging project. Thank you all for your caring guidance and advice. To my external dissertation readers, Dr. Richard A. Katz and the generous Dr. Raghbir Athwal, it has been a pleasure meeting you both. I am so grateful to all the faculty, postdoctoral fellows and graduate students at Fox Chase Cancer Center for their availability and cooperative work ethics and Dr. Katz is a true embodiment of the institution’s values and mission. As I said, Fox Chase felt like home on my first visit, and time didn’t prove me wrong. Another “home” away from home was Fels Institute. I am very grateful to my outside research proposal sponsor, Dr. Xavier Grana, not only for his invaluable contributions and guidance during my ORP project but also for taking me under the CBGN wing. I felt I had the best of two worlds being a Biochemistry student in a Cell, Molecular, Genetics and Epigenetics cluster. v To my past and present lab members, I wouldn’t have been able to do this without your suggestions, constructive criticism, and friendship. Julia, Su, Tom, Theresa, and Dr. Irma Russo helped me design and/or carry out experiments. In particular I want to thank Ricardo from the bottom of my heart for always being available (in the most broad way) to discuss results and future directions for this project. In the Russo lab, I was honored to mentor many undergraduate and graduate students and these experiences have shaped me into a more solid scientist. Special thanks to Dominic, Olivia and Tyler for reminding me how much I love teaching. To my graduate fellows in this great adventure, I want to thank Danielle Feather, Elsie Samakai, Umme Ayesa, and Jenny Shrestha for their words of support and encouragement at every step. I’m happy we will be finding the light at the end of the tunnel and wish you all success in your careers. Last but not least, I want to thank the drivers and energy fillers of my life. To the love of my life, Javier, and the little princess we added to the family, my beloved Zoe. Javier, your determination to be the best professional you possibly can have always inspired me to find the best scientist in me. I’m so proud of our “all-terrain” family. To my sisters, Victoria and Luciana, my beautiful niece Sofia and especially my mother Susana. Mom, you have been my utmost supporter, several times flying overseas so that I could get to this point. Gracias. vi TABLE OF CONTENTS Page ABSTRACT ................................................................................................................... iii DEDICATION ………………………………………………………………………………..... iv ACKNOWLEDGEMENTS ............................................................................................... v LIST OF TABLES ………………………………………………………………………….….. x LIST OF FIGURES ………………………………………………………………………….… xi LIST OF ABBREVIATIONS ......................................................................................... xiv HYPOTHESIS ……………………………………………………………………………….. xvi SPECIFIC AIMS …………………………………………………………………………….. xvii CHAPTER 1: INTRODUCTION ...................................................................................... 1 1.1. Incidence and mortality rates in recent years ..............................................................2 1.2. Breast Cancer Subtypes ................................................................................................2 1.3. Breast Cancer Prevention ..............................................................................................3 1.4. Role of lncRNA in Development and Disease ...............................................................4 1.4.1. Molecular interactions and functional roles of lncRNA ...................................................... 6 1.4.1.i- Epigenetic Transcriptional Regulation .......................................................................... 7 1.4.1.ii - Enhancer-associated lncRNA ...................................................................................... 8 1.4.1.iii - mRNA Processing ....................................................................................................... 8 1.4.1.iv - miRNA Sequestration .................................................................................................