DECODING THE TRANSCRIPTIONAL LANDSCAPE OF TRIPLE-NEGATIVE BREAST CANCER USING NEXT GENERATION WHOLE TRANSCRIPTOME SEQUENCING Milan Radovich Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Medical and Molecular Genetics Indiana University August 2011 Accepted by the Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. __ _____________________________ Bryan P. Schneider, MD, Chair David A. Flockhart, MD, PhD Doctoral Committee Mircea Ivan, MD, PhD June 15, 2011 Brittney-Shea Herbert, PhD Brenda R. Grimes, PhD Harikrishna Nakshatri, PhD ii ACKNOWLEDGEMENTS As always, the success of our endeavors are built upon the support and love of so many people who help make life possible. Through my graduate career, I have undeservedly received an abundance of encouragement, support, and resources from so many which has allowed me to be where I am today. To my mentor Dr. Bryan Schneider, we have been together since the lab first started, and what an amazing time it has been! Through victories and failures, and time of joy and sorrow, you have always been there. I truly appreciate your friendship and mentorship through the years. Your persistence and support is what has allowed me to be the translational scientist I am today. Thank you for believing in me and for giving me the opportunity to pursue my scientific aspirations. I would not be graduating if it wasn’t for you, I will forever be indebted. To my labmates, Nawal Kassem, Brad Hancock, and Lateef Aregbe, I couldn’t have done this without you guys. Thank you for enduring the many days of my frustration when things just didn’t seem to work. Also, thank you for always being there, whether it was my talks or presentations or just needing someone to chat with, your presence was also so appreciated. Thank you for always making me laugh and always letting me know that I was loved by you all. The memories, I will never forget. Whether it was our trips to Washington, D.C., Denver, San Antonio, or Orlando, or just the many times we chatted in lab or at lunch, it is the camaraderie that made coming to lab everyday a joy. To my collaborators Drs. Susan Clare, George Sledge, and Mircea Ivan, and also to Connie Rufenbarger, my project would have not been possible with you. Thank you for taking the risk and investing in a young graduate student with a crazy idea. I can’t tell you how much it meant to me when you decided that I was worth your investment. iii Thank you for the countless conversations and always lending yet another good scientific idea. I will always be deeply indebted, and I only hope that I can return many fold what you gave to me. A special thanks goes to Dr. David A. Flockhart, I wouldn’t have been in graduate school if it wasn’t for you. I will forever be indebted to your kindness and generosity. I hope you will always know how appreciative I am of what you gave. To my committee, Drs. Bryan Schneider, David Flockhart, Mircea Ivan, Brittney- Shea Herbert, Brenda Grimes, Harikrishna Nakshatri, thank you for your time and sacrifice in ensuring that I was becoming the best scientist that I can be. Thank you for your thoughtful suggestions and encouragement, and also thank you so much for making sure that I wasn’t going too overboard in my scientific ambitions. I am indebted to the time that you gave while never expecting anything in return. To my wife Betsy and my daughter Grace, thank you for being there for me through this entire time. I know it was so busy, and there wasn’t always a ton of time, but we always knew it would be done soon. Now that I will finally have a real job, we can finally go on that family vacation. To my mother Desanka, brother Alex, and my In-laws, Jim & Micki Geise thank you for your love and unwavering support. It has always been one of my greatest joys to know that I have made you proud. I hope that I will continue to do so. I know there are probably others that I am missing, but it goes without mentioning that so many were involved in making this project happen. To the volunteers and staff of the Susan G. Komen Tissue Bank who tirelessly collected and prepared the normal breast tissues, thank you for all you work and for the many many days of LCM! To the folks at Cofactor Genomics and Applied Biosystems whose technical expertise in next- generation sequencing made so much of this work possible, thank you for being such great collaborators and for being great friends. To the Cancer Biology Training Program iv and the Indiana CTSI who provided the funding that allowed me to be a successful student. Finally, to the Indiana University Simon Cancer Center, the Department of Medical & Molecular Genetics, and the Department of Surgery, thank you for providing the resources to make this project happen… and ensuring that the work still continues. v ABSTRACT Milan Radovich DECODING THE TRANSCRIPTIONAL LANDSCAPE OF TRIPLE-NEGATIVE BREAST CANCER USING NEXT GENERATION WHOLE TRANSCRIPTOME SEQUENCING Triple-negative breast cancers (TNBCs) are negative for the expression of estrogen (ER), progesterone (PR), and HER-2 receptors. TNBC accounts for 15% of all breast cancers and results in disproportionally higher mortality compared to ER & HER2- positive tumours. Moreover, there is a paucity of therapies for this subtype of breast cancer resulting primarily from an inadequate understanding of the transcriptional differences that differentiate TNBC from normal breast. To this end, we embarked on a comprehensive examination of the transcriptomes of TNBCs and normal breast tissues using next-generation whole transcriptome sequencing (RNA-Seq). By comparing RNA- seq data from these tissues, we report the presence of differentially expressed coding and non-coding genes, novel transcribed regions, and mutations not previously reported in breast cancer. From these data we have identified two major themes. First, BRCA1 mutations are well known to be associated with development of TNBC. From these data we have identified many genes that work in concert with BRCA1 that are dysregulated suggesting a role of BRCA1 associated genes with sporadic TNBC. In addition, we observe a mutational profile in genes also associated with BRCA1 and DNA repair that lend more evidence to its role. Second, we demonstrate that using microdissected normal epithelium maybe an optimal comparator when searching for novel therapeutic targets for TNBC. Previous studies have used other controls such as reduction mammoplasties, adjacent normal tissue, or other breast cancer subtypes, which may be sub-optimal and have lead to identifying ineffective therapeutic targets. Our data vi suggests that the comparison of microdissected ductal epithelium to TNBC can identify potential therapeutic targets that may lead to be better clinical efficacy. In summation, with these data, we provide a detailed transcriptional landscape of TNBC and normal breast that we believe will lead to a better understanding of this complex disease. Bryan P. Schneider, MD, Chair vii TABLE OF CONTENTS List of Tables ................................................................................................................... x List of Figures ................................................................................................................. xi List of Abbreviations ..................................................................................................... xiii Chapter 1: Introduction 1.1 Triple-Negative Breast Cancer ....................................................................... 1 1.1.1 Clinical characteristics and treatment of triple-negative breast cancer ...............................................................................................1 1.1.2 Histological and molecular characterization of TNBC ........................ 8 1.1.3 Hereditary and somatic mutation profile of TNBC ............................ 13 1.2 The normal breast in cancer research .......................................................... 17 1.2.1 Overview of normal epithelium in the breast .................................... 17 1.2.2 The use of normal breast tissue in cancer research ........................ 22 1.3 Next-generation whole transcriptome sequencing (RNA-Seq) ...................... 24 1.3.1 Overview of next-generation sequencing and its role in cancer research .......................................................................................... 24 1.3.2 RNA-sequencing overview and chemistry of the Applied Biosystems SOLiD sequencer ......................................................... 29 1.3.3 Overview of SOLiD RNA-seq bioinformatics and data analysis ....... 33 1.4 Statement of purpose ................................................................................... 44 Chapter 2: Differential gene expression of the transcriptomes of TNBC and normal breast tissue 2.1 Introduction .................................................................................................. 45 2.2 Materials and methods ................................................................................. 47 2.3 Results ......................................................................................................... 58 viii 2.3.1 RNA sequencing output of TNBCs and normals .............................. 58 2.3.2 Profiling and differential expression of known genes from the RefSeq database ......................................................................