Human papillomavirus dysregulates the cellular apparatus controlling the methylation status of H3K27 in different human cancers to consistently alter gene expression regardless of tissue of origin

High-risk human papillomaviruses (HPV) cause cancer at multiple distinct anatomical locations. Regardless of the tissue of origin, most HPV positive (HPV+) cancers show highly upregulated expression of the p16 product of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene. Paradoxically, HPV+ tumor cells require continuous expression of this tumor suppressor for survival. Thus, restoration of normal p16 regulation has potential therapeutic value against HPV induced cancers. Normally, p16 transcription is tightly controlled at the epigenetic level via polycomb repressive complex-mediated tri-methylation of histone 3 lysine 27 (H3K27me3). Although a mechanism by which HPV induces p16 has been proposed based on tissue culture models, it has not been extensively validated in human tumors. In this study, we used data from over 800 human cervical and head and neck tumors from The Cancer Genome Atlas (TCGA) to test this model. We determined the impact of HPV status on expression from the CDKN2A locus, the adjacent CDKN2B locus, and transcript levels of key epigenetic regulators of these loci. As expected, HPV+ tumors from both anatomical sites exhibited high levels of p16. Furthermore, HPV+ tumors expressed higher levels of KDM6A, which demethylates H3K27me3. CpG methylation of the CDKN2A locus was also consistently altered in HPV+ tumors. This data validates previous tissue culture studies and identifies remarkable similarities between the effects of HPV on gene expression and DNA methylation in both cervical and oral tumors in large human cohorts. Furthermore, these results support a model whereby HPV-mediated dysregulation of CDKN2A transcription requires KDM6A, a potentially druggable target.

[1]  S. Sanjosé,et al.  Human papillomavirus 16 is an aetiological factor of scrotal cancer , 2017, British Journal of Cancer.

[2]  J. Durzynska,et al.  Human papillomaviruses in epigenetic regulations. , 2017, Mutation research. Reviews in mutation research.

[3]  Giulia Basile,et al.  Intragenic DNA methylation prevents spurious transcription initiation , 2017, Nature.

[4]  Steven J. M. Jones,et al.  Integrated genomic and molecular characterization of cervical cancer , 2017, Nature.

[5]  D. Hayes,et al.  Nonpromoter methylation of the CDKN2A gene with active transcription is associated with improved locoregional control in laryngeal squamous cell carcinoma , 2017, Cancer medicine.

[6]  Dylan M. Marchione,et al.  Impaired H3K36 methylation defines a subset of head and neck squamous cell carcinomas , 2017, Nature Genetics.

[7]  P. Buckhaults,et al.  Identification and characterization of HPV-independent cervical cancers , 2017, Oncotarget.

[8]  A. Lorincz Virtues and Weaknesses of DNA Methylation as a Test for Cervical Cancer Prevention , 2016, Acta Cytologica.

[9]  M. Einstein,et al.  Novel epigenetic changes in CDKN2A are associated with progression of cervical intraepithelial neoplasia. , 2016, Gynecologic oncology.

[10]  Brian O'Sullivan,et al.  Human Papillomavirus Genotype Association With Survival in Head and Neck Squamous Cell Carcinoma. , 2016, JAMA oncology.

[11]  E. Burd Human Papillomavirus Laboratory Testing: the Changing Paradigm , 2016, Clinical Microbiology Reviews.

[12]  D. Salyakina,et al.  Non-coding RNAs profiling in head and neck cancers , 2016, npj Genomic Medicine.

[13]  A. Mäkitie,et al.  Association of BMI-1 and p16 as prognostic factors for head and neck carcinomas , 2016, Acta oto-laryngologica.

[14]  D. Salyakina,et al.  Human Papilloma Viruses and Breast Cancer , 2015, Front. Oncol..

[15]  M. Stoler,et al.  Risk Stratification By p16 Immunostaining of CIN1 Biopsies: A Retrospective Study of Patients From the Quadrivalent HPV Vaccine Trials , 2015, The American journal of surgical pathology.

[16]  Kan Zhai,et al.  HPV and lung cancer risk: a meta-analysis. , 2015, Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology.

[17]  Steven J. M. Jones,et al.  Comprehensive genomic characterization of head and neck squamous cell carcinomas , 2015, Nature.

[18]  Thomas M. Harris,et al.  Epigenetic changes in the CDKN2A locus are associated with differential expression of P16INK4A and P14ARF in HPV-positive oropharyngeal squamous cell carcinoma , 2015, Cancer medicine.

[19]  Lukas Burger,et al.  Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation , 2015, Nature.

[20]  R. Sobti,et al.  Implication of high risk Human papillomavirus HR-HPV infection in prostate cancer in Indian population- A pioneering case-control analysis , 2015, Scientific Reports.

[21]  Yuanyuan Chen,et al.  Increased expression of oncogene-induced senescence markers during cervical squamous cell cancer development. , 2014, International journal of clinical and experimental pathology.

[22]  E. Speel,et al.  P16INK4A immunostaining is a strong indicator for high‐risk‐HPV‐associated oropharyngeal carcinomas and dysplasias, but is unreliable to predict low‐risk‐HPV‐infection in head and neck papillomas and laryngeal dysplasias , 2014, International journal of cancer.

[23]  S. Willems,et al.  Differences in methylation profiles between HPV-positive and HPV-negative oropharynx squamous cell carcinoma , 2014, Epigenetics.

[24]  Margaret E McLaughlin-Drubin,et al.  Tumor suppressor p16INK4A is necessary for survival of cervical carcinoma cell lines , 2013, Proceedings of the National Academy of Sciences.

[25]  K. Ogawa,et al.  Viral load, physical status, and E6/E7 mRNA expression of human papillomavirus in head and neck squamous cell carcinoma , 2013, Head & neck.

[26]  M. Lerman,et al.  Human papillomavirus-associated oral intraepithelial neoplasia , 2013, Modern Pathology.

[27]  T. Stöver,et al.  The role of p16 expression as a predictive marker in HPV-positive oral SCCHN--a retrospective single-center study. , 2013, Anticancer research.

[28]  Wim Quint,et al.  The biology and life-cycle of human papillomaviruses. , 2012, Vaccine.

[29]  M. Plummer,et al.  Global burden of human papillomavirus and related diseases. , 2012, Vaccine.

[30]  Peter A. Jones Functions of DNA methylation: islands, start sites, gene bodies and beyond , 2012, Nature Reviews Genetics.

[31]  S. McDade,et al.  Evidence for Alteration of EZH2, BMI1, and KDM6A and Epigenetic Reprogramming in Human Papillomavirus Type 16 E6/E7-Expressing Keratinocytes , 2011, Journal of Virology.

[32]  K. Münger,et al.  Human papillomavirus E7 oncoprotein induces KDM6A and KDM6B histone demethylase expression and causes epigenetic reprogramming , 2011, Proceedings of the National Academy of Sciences.

[33]  Xiao Zhang,et al.  Comparison of Beta-value and M-value methods for quantifying methylation levels by microarray analysis , 2010, BMC Bioinformatics.

[34]  J. Gil,et al.  Epigenetic regulation of the INK4b-ARF-INK4a locus , 2010, Epigenetics.

[35]  W. Rittgen,et al.  p16INK4a overexpression predicts translational active human papillomavirus infection in tonsillar cancer , 2010, International journal of cancer.

[36]  A. Psyrri,et al.  E6 and e7 gene silencing and transformed phenotype of human papillomavirus 16-positive oropharyngeal cancer cells. , 2009, Journal of the National Cancer Institute.

[37]  F. Hoppe-Seyler,et al.  Activation of the enhancer of zeste homologue 2 gene by the human papillomavirus E7 oncoprotein. , 2008, Cancer research.

[38]  K. Münger,et al.  Human Papillomavirus Type 16 E7 Oncoprotein Associates with E2F6 , 2008, Journal of Virology.

[39]  L. Meng,et al.  RNA interference against HPV16 E7 oncogene leads to viral E6 and E7 suppression in cervical cancer cells and apoptosis via upregulation of Rb and p53 , 2008, Apoptosis.

[40]  T. Swigut,et al.  H3K27 Demethylases, at Long Last , 2007, Cell.

[41]  Robert E. Brown,et al.  Senescence and apoptosis in carcinogenesis of cervical squamous carcinoma , 2007, Modern Pathology.

[42]  Edgar Erdfelder,et al.  G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences , 2007, Behavior research methods.

[43]  S. Syrjänen Human papillomavirus (HPV) in head and neck cancer. , 2005, Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology.

[44]  T. Enomoto,et al.  Correlation between p14ARF/p16INK4A expression and HPV infection in uterine cervical cancer , 2004 .

[45]  Eileen M. Burd,et al.  Human Papillomavirus and Cervical Cancer , 1988, The Lancet.

[46]  J. Milner,et al.  Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference , 2002, Oncogene.

[47]  H. Hausen Papillomaviruses and cancer: from basic studies to clinical application , 2002, Nature Reviews Cancer.

[48]  Karl Münger,et al.  Biological activities and molecular targets of the human papillomavirus E7 oncoprotein , 2001, Oncogene.

[49]  M. von Knebel Doeberitz,et al.  Overexpression of p16INK4A as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri , 2001, International journal of cancer.

[50]  D. DiMaio,et al.  Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[51]  D. DiMaio,et al.  Rapid induction of senescence in human cervical carcinoma cells. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[52]  D. Sidransky,et al.  Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. , 2000, Journal of the National Cancer Institute.

[53]  T. Nakajima,et al.  Expression status of p16 protein is associated with human papillomavirus oncogenic potential in cervical and genital lesions. , 1998, The American journal of pathology.

[54]  Yue Xiong,et al.  ARF Promotes MDM2 Degradation and Stabilizes p53: ARF-INK4a Locus Deletion Impairs Both the Rb and p53 Tumor Suppression Pathways , 1998, Cell.

[55]  Tony Kouzarides,et al.  Retinoblastoma protein recruits histone deacetylase to repress transcription , 1998, Nature.

[56]  D. Wazer,et al.  E7 protein of human papilloma virus-16 induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway. , 1996, Cancer research.

[57]  Kathleen R. Cho,et al.  Human papillomavirus 16 E6 expression disrupts the p53-mediated cellular response to DNA damage. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[58]  K. Münger,et al.  Homologous sequences in adenovirus E1A and human papillomavirus E7 proteins mediate interaction with the same set of cellular proteins , 1992, Journal of virology.

[59]  N. Muñoz,et al.  Role of Human Papillomavirus in Penile Carcinomas Worldwide. , 2016, European urology.

[60]  A. Botezatu,et al.  Histone lysine demethylases as epigenetic modifiers in HPV-induced cervical neoplasia , 2015 .

[61]  The Cancer Genome Atlas Research Network,et al.  Comprehensive molecular characterization of urothelial bladder carcinoma , 2014, Nature.

[62]  T. Stöver,et al.  The Role of p 16 Expression as a Predictive Marker in HPV-positive Oral SCCHN – A Retrospective Single-center Study , 2013 .

[63]  Vincenzo Pirrotta,et al.  Polycomb silencing mechanisms and the management of genomic programmes , 2007, Nature Reviews Genetics.

[64]  H. zur Hausen,et al.  Papillomavirus infections--a major cause of human cancers. , 1996, Biochimica et biophysica acta.