Targeting the DEK oncogene in head and neck squamous cell carcinoma: functional and transcriptional consequences A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements to the degree of Doctor of Philosophy (Ph.D.) in the Department of Cancer and Cell Biology of the College of Medicine March 2015 by Allie Kate Adams B.S. The Ohio State University, 2009 Dissertation Committee: Susanne I. Wells, Ph.D. (Chair) Keith A. Casper, M.D. Peter J. Stambrook, Ph.D. Ronald R. Waclaw, Ph.D. Susan E. Waltz, Ph.D. Kathryn A. Wikenheiser-Brokamp, M.D., Ph.D. Abstract Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies worldwide with over 50,000 new cases in the United States each year. For many years tobacco and alcohol use were the main etiological factors; however, it is now widely accepted that human papillomavirus (HPV) infection accounts for at least one-quarter of all HNSCCs. HPV+ and HPV- HNSCCs are studied as separate diseases as their prognosis, treatment, and molecular signatures are distinct. Five-year survival rates of HNSCC hover around 40-50%, and novel therapeutic targets and biomarkers are necessary to improve patient outcomes. Here, we investigate the DEK oncogene and its function in regulating HNSCC development and signaling. DEK is overexpressed in many cancer types, with roles in molecular processes such as transcription, DNA repair, and replication, as well as phenotypes such as apoptosis, senescence, and proliferation. DEK had never been previously studied in this tumor type; therefore, our studies began with clinical specimens to examine DEK expression patterns in primary HNSCC tissue. We find that DEK is highly overexpressed in all subsets of HNSCC, independent of HPV status, stage, or patient demographics. Furthermore, an established transgenic mouse model, crossed with Dek-/- mice, examined the in vivo importance of DEK in HNSCC development in a 4-NQO model system. Transgenic Dek-/- have improved survival, fewer tumors, and reduced epidermal proliferation compared to Dek+/+ counterparts. Similarly, in vitro systems of HPV+ and HPV- cell lines demonstrated DEK loss reduced cell number and proliferation. Finally, ΔNp63 was identified as a partial mediated of this DEK-dependent cell growth. We next aimed to identify additional candidates as mediators of DEK-dependent functions. DEK is known to regulate transcription, as either a co-activator or a co-repressor, but the global impact of DEK loss on gene transcription is unknown. Therefore, RNA-Sequencing i was performed on HPV+ and HPV- HNSCC lines, either proficient or deficient for DEK, to assess the transcriptional consequences of DEK loss. From this data IRAK1 was identified as a candidate down-regulated in the absence of DEK. IRAK1 is a driver of inflammatory signaling, downstream of toll-like and interleukin-1 receptors, and activates many well-known signaling cascades including NF-κB and MAPK. These RNA-Seq results were validated by qRT-PCR and western blot analysis. IRAK1 expression was assessed by mining The Cancer Genome Atlas, which revealed IRAK1 is overexpressed in HNSCC. As IRAK1 has never previously been characterized in HNSCC, experiments were designed to identify the function of IRAK1 in this system. IRAK1 loss reduced downstream TRAF6-ubiquitination and signaling and increased apoptosis, with either shRNA or chemical inhibition. Lastly, DEK and IRAK1 cooperated to regulate ERK1/2 signaling, but independently contributed to HNSCC survival. In summary, these findings implicate two novel oncogenes as mediators in HNSCC growth and survival and that individual or combined targeting of DEK and IRAK1 may be therapeutically beneficial. ii iii Preface The following work in this dissertation is either in preparation for, or previously published, in the following peer-reviewed journals: 1. Adams AK, Wise-Draper TM, Wells, SI. Human Papillomavirus induced transformation in cervical and head and neck cancers. Review for Cancers. 2014 Sep 15;6(3):1793-820. doi: 10.3390/cancers6031793. PMID: 25226287. 2. Adams AK, Hallenbeck GE, Casper KA, Patil YJ, Wilson KM, Kimple RJ, Lambert PF, Witte DP, Xiao W, Gillison ML, Wikenheiser-Brokamp KA, Wise-Draper TM, Wells SI. DEK promotes HPV-positive and –negative head and neck cancer cell proliferation. Oncogene. 2014 Mar 10. doi: 10.1038/onc.2014.15 [Epub ahead of print]. PMID: 24608431. 3. Adams AK, Bolanos LC, Dexheimer PJ, Karns RA, Aronow BJ, Komurov K, Jegga A, Casper KA, Patil YJ, Wilson KM, Starczynowski DT, Wells SI. IRAK1 promotes HNSCC survival through a DEK-regulated network. Manuscript in preparation. iv Acknowledgements During the course of my dissertation studies, I have been privileged to collaborate with numerous research scientists and clinicians without whom my work would not have been possible. Firstly, I would like to thank my mentor Dr. Susanne (Susa) Wells at Cincinnati Children’s Hospital Medical Center. Susa has provided invaluable advice and guidance over the years, both at a scientific and personal level. She has always allowed me to be highly independent in my work and has encouraged me to pursue the experiments I was the most passionate about. I will always be grateful for all of her support and am thankful she asked me to join the Wells lab. I would also like to thank my thesis committee, Dr. Keith Casper, Dr. Peter Stambrook, Dr. Ronald Waclaw, Dr. Susan Waltz, and Dr. Kathryn Wikenheiser-Brokamp for their suggestions and feedback on my projects. They have challenged me as a scientist and were an integral part of this work inside and outside of my thesis meetings. Additionally, I thank Dr. Keith Casper and his colleagues at the University of Cincinnati Department of Otolaryngology for collaborating with our lab on the receipt of primary specimens for our studies. I believe without these tissues our work would not have been nearly as impactful--few graduates have this opportunity and I am eternally grateful for the chance to work with you. I also want to acknowledge Dr. Daniel Starczynowski and his lab for helping me with the IRAK1 story. Without their advice (and reagents) we could not have pursued my final project. To the Wells lab (past and present): thank you for your advice during the highs and lows of research. I have formed lasting friendships with many of you and my time in this lab has been full of entertainment and laughter. Many of you have been secondary mentors (Elizabeth Hoskins and Lisa Privette Vinnedge, specifically) and helped me navigate the world that is science. I dedicate this work to my parents, Randall and Susan Varner, who have provided unconditional love and support these past twenty-eight years. In times of struggle they have v always been there to listen and have encouraged me to pursue my dreams. My achievements thus far have been possible because of their help, sacrifices, and passion to see their daughter succeed. Thank you for everything! Finally, to my husband Joshua Adams—words will never be enough to express how thankful I am for all of your emotional support over 5 years of graduate school. You have been my biggest cheerleader and helped me keep the end goal in mind when I struggled with the difficulties of grad school. We met on our first day at Ohio State: two science nerds that have successfully navigated the completion of their degrees, moving to Cincinnati, surviving my qualifying exam, getting married, adopting our pup, Blue, and buying our first home. It has undoubtedly been the greatest journey of my life and I am so glad to have spent it with you. vi Table of Contents Abstract ................................ ................................................................................................................................ i Preface ................................ ............................................................................................................................... iv Acknowledgements ................................ ......................................................................................................... v List of Figures Tables and ................................ ........................................................................................... ix Chapter 1: Human Papillomavirus Induced Transformation in Cervical and Head Neck Cancers ................................ .......................................................................................................... 1 Abstract ................................ .............................................................................................................................. 2 Introduction ................................ ..................................................................................................................... 3 HPV biology ................................ ....................................................................................................................... 5 Clinical characteristics of HPV+ cancers ................................ .................................................................. 9 Prognostic comparison and staging ................................ ..................................................................................... 13 Mutational Treatment analyses variations ................................ ................................ ......................................................................................................................................................................................