HUMAN PAPILLOMAVIRUS ONCOPROTEIN E7 DYSREGULATES IMMUNE RESPONSES THROUGH EPIGENETIC MANIPULATION by LOUIS J CICCHINI B.S., University of Arizona, 2009 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Molecular Biology 2016 This thesis for the Doctor of Philosophy degree by Louis J Cicchini has been approved for the Molecular Biology Program by Rytis Prekeris, Chair Dohun Pyeon, Advisor James Hagman Thomas E. Morrison Xiao-Jing Wang Date: August 18, 2016 ii Cicchini, Louis J. (PhD, Molecular Biology) Human Papillomavirus Oncoprotein E7 Dysregulates Immune Responses through Epigenetic Manipulation Thesis directed by Associate Professor Dohun Pyeon ABSTRACT High-risk human papillomaviruses (HPVs) are causally associated with multiple human cancers. Previous studies have shown that the HPV oncoprotein E7 induces immune suppression; however, the underlying mechanisms remain unknown. We report that, while expression of many proinflammatory chemokines increases throughout HPV-positive cancer progression, CXCL14 is dramatically downregulated by promoter hypermethylation in an E7- dependent manner. Our in vivo mouse models revealed that restoration of Cxcl14 expression in HPV-positive mouse oropharyngeal carcinoma cells clears tumors in immunocompetent syngeneic mice, but not in Rag1-deficient mice. Further, restoration of Cxcl14 expression significantly increases natural killer (NK), CD4+ T, and CD8+ T cell infiltration into the tumor-draining lymph nodes in vivo. In vitro transwell migration assays show that restoration of Cxcl14 expression induces chemotaxis of NK, CD4+ T, and CD8+ T cells. These findings suggest that high-risk HPV E7 is likely to dysregulate host gene expression in order to persist by modulating host DNA methylation. To investigate the extent of gene expression dysregulated by HPV E7-induced DNA methylation, we performed parallel global gene expression and methylome analyses with HPV-positive and -negative normal immortalized keratinocyte lines, NIKS, NIKS-16, NIKS-18, and NIKS-16∆E7. We show that expression of the MHC class-I genes HLA-A, -B, -C, -E, and -G is downregulated in HPV-positive keratinocytes in an E7-dependent manner. Additional methylome analysis revealed hypermethylation at a distal CpG island (CGI) near the HLA-E gene in HPV16- positive keratinocytes (NIKS-16) cells compared with NIKS and NIKS-16∆E7 cells. The HLA- E CGI functions as a promoter exhibiting transcription factor binding sites and DNase iii hypersensitivity. Promoter activity of the HLA-E CGI is considerably decreased by DNA methylation. HLA-E protein expression also is downregulated by high-risk HPV16 and HPV18 E7 expression, but not by low-risk HPV6 and HPV11 E7 expression. Conversely, demethylation in the HLA-E CGI by treatment with 5-aza-β’-deoxycytidine restores HLA-E protein expression in HPV-positive keratinocytes. Because HLA-E plays an important role in antiviral immunity, epigenetic downregulation of HLA-E by high-risk HPV E7 may contribute to HPV-induced immune suppression during HPV persistence. Our findings provide a new mechanistic understanding of virus-induced immune evasion that contributes to cancer progression. The form and content of this abstract are approved. I recommend its publication. Approved: Dohun Pyeon iv TABLE OF CONTENTS CHAPTER I. INTRODUCTION……………………..……………..……………………………………….1 Human Papillomavirus: Disease and Global Implications .......................................... 1 The Molecular Biology of Papillomavirus .................................................................. 3 Papillomavirus Oncogenes and Mechanisms of Transformation ............................... 7 E5 and Dysregulation of Immune Signaling ................................................... 8 E6 Abrogation of p53 and Evasion of Apoptosis ............................................ 9 Transformation by E7 through Dysregulation of Host Gene Expression .......10 Papillomavirus-Induced Neoplastic Transformation ......................................15 Viral Persistence and Cancer Progression ...............................................................17 Evaluating General Risk Factors for HPV-Associated Cancer Progression ..................................................................................................17 HPV Persistence and Host Epigenetic Consequences .................................18 Virus Evasion of Immune Detection .........................................................................21 Evasion of Resident Antigen Presenting Cells ..............................................22 Dysregulation of Antigen Presentation .........................................................25 Chemokines and Inflammation.................................................................................27 Inflammation and HPV-Associated Cancer Progression ...............................27 CXCL14 is a Potential Tumor Suppressor ....................................................28 Dissertation Goals and Objectives ...........................................................................29 II. MATERIALS AND METHODS .................................................................................32 Cell and Tissue Culture ...........................................................................................32 Conventional PCR and Quantitative Reverse Transcription-PCR (RT-qPCR) ..........33 Bisulfite Modification and Assessment of Methylated DNA ......................................34 Vectors and Plasmids ..............................................................................................35 Luciferase Reporter Vectors .........................................................................35 v Lentiviral Vectors ..........................................................................................36 Enzyme-Linked Immunosorbent Assay (ELISA) ......................................................36 Cell Migration Assays ..............................................................................................37 Scratch Assay ..............................................................................................37 Transwell Migration ......................................................................................37 Mice and Treatment .................................................................................................38 Flow Cytometry ........................................................................................................39 Antibodies ....................................................................................................39 Sample Preparation and Flow Analysis ........................................................39 Array Preparation and Analysis ................................................................................40 Genome-wide Expression and DNA Methylation Arrays ...............................40 Data Processing and Statistical Analysis ......................................................40 Bioinformatics ..............................................................................................41 Immunoblotting ........................................................................................................42 Statistical Analysis ...................................................................................................42 Generation of HPV16 Reporter Virions ....................................................................43 Reagent Acknowledgements ...................................................................................43 III. SUPPRESSION OF ANTITUMOR IMMUNE RESPONSES BY HUMAN PAPILLOMAVIRUS THROUGH EPIGENETIC DOWNREGULATION OF CXCL14 ...................................................................................................................44 Importance ..............................................................................................................44 Introduction ..............................................................................................................44 Results ....................................................................................................................46 Proinflammatory Chemokines are Upregulated During CxCa Progression ..................................................................................................46 CXCL14 Expression is Downregulated in HPV-Associated Cancer Progression ..................................................................................................47 CXCL14 Downregulation in HPV-Positive Keratinocytes is Associated with Promoter Hypermethylation ..................................................................50 vi CXCL14 Expression Hinders Cell Migration in vitro ......................................53 Restoration of Cxcl14 Expression Clears HPV-Positive Tumors in Immunocompetent Mice, but not in Rag1-Deficient Mice ..............................55 Restored Expression of Cxcl14 Increases Natural Killer (NK), CD4+ T, and CD8+ T Cells in Tumor-Draining Lymph Nodes in vivo...........................57 Expression of Cxcl14 Induces Chemotaxis of NK, CD4+ T, and CD8+ T Cells in vitro .................................................................................................59 Discussion ...............................................................................................................62 IV. HIGH-RISK HUMAN PAPILLOMAVIRUS E7 ALTERS HOST DNA METHYLOME AND REPRESSES