HPV Infection Leaves a DNA Methylation Signature in Oropharyngeal Cancer Affecting Both Coding Genes and Transposable Elements

SIMPLE SUMMARY: The HPV oncoproteins E6 and E7 can modulate the expression and activity of the maintenance DNA methyltransferase 1, suggesting that HPV carcinogenic mechanisms may include aberrant DNA methylation. Some studies previously proposed both gene-associated DNA methylation signatures and a global hypermethylation profile in HPV-positive head and neck cancer, but the validation of such signatures and a more detailed analysis of the methylation profile of transposable elements (TEs) in oropharyngeal squamous cell carcinoma (OPSCC) are still missing. TEs account for approximately 50% of the human genome and their hypomethylation and reactivation have been consistently reported in cancer, usually being associated with worse prognosis. Based on this, this study aimed at validating a previously established 5-CpG methylation signature in FFPE OPSCC from a middle-income population, in which the frequency of HPV infection is only 6.1%, and dissecting the methylation profile of TEs, focusing on their impact on gene expression and overall survival. ABSTRACT: HPV oncoproteins can modulate DNMT1 expression and activity, and previous studies have reported both gene-specific and global DNA methylation alterations according to HPV status in head and neck cancer. However, validation of these findings and a more detailed analysis of the transposable elements (TEs) are still missing. Here we performed pyrosequencing to evaluate a 5-CpG methylation signature and Line1 methylation in an oropharyngeal squamous cell carcinoma (OPSCC) cohort. We further evaluated the methylation levels of the TEs, their correlation with gene expression and their impact on overall survival (OS) using the TCGA cohort. In our dataset, the 5-CpG signature distinguished HPV-positive and HPV-negative OPSCC with 66.67% sensitivity and 84.33% specificity. Line1 methylation levels were higher in HPV-positive cases. In the TCGA cohort, Line1, Alu and long terminal repeats (LTRs) showed hypermethylation in a frequency of 60.5%, 58.9% and 92.3%, respectively. ZNF541 and CCNL1 higher expression was observed in HPV-positive OPSCC, correlated with lower methylation levels of promoter-associated Alu and LTR, respectively, and independently associated with better OS. Based on our findings, we may conclude that a 5-CpG methylation signature can discriminate OPSCC according to HPV status with high accuracy and TEs are differentially methylated and may regulate gene expression in HPV-positive OPSCC.