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

Abstract

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.

Keywords: cancer; epigenetics; gene expression; human papillomavirus; methylation.

Figure 1. Organization of the CDKN2A and CDKN2B loci

The cyclin-dependent kinase inhibitor 2A (CDKN2A) locus encodes two functionally unrelated protein products named p16 INK4A and p14 ARF. The adjacent CDKN2B locus encodes p15 INK4B. This gene cluster also encodes a long non-coding RNA transcribed in the antisense direction named CDKN2B-AS or antisense non-coding RNA in the INK4 locus (ANRIL). The position of transcripts (black lines), exons (black vertical bars), coding regions (thick black vertical bars), and orientation (direction of arrowheads) of the major transcripts in this region of chromosome 9 are indicated.

Figure 2. HPV perturbation of expression of the CDKN2A and CDKN2B genes in human head & neck and cervical carcinomas

Normalized RNA-Seq data extracted from the TCGA database for the HNSC (A) and CESC (B) cohorts for HPV+, HPV-, and normal control tissues. Numbers in brackets refer to the number of samples included in each analysis. * p≤0.05, ** p≤0.01, *** p≤0.001, ns- not significant.

Figure 3. HPV perturbation of expression of polycomb components that may regulate CDKN2A transcription in human head & neck and cervical carcinomas

Normalized RNA-Seq data extracted from the TCGA database for the HNSC (A) and CESC (B) cohorts for HPV+, HPV-, and normal control tissues. Numbers in brackets refer to the number of samples included in each analysis. * p≤0.05, ** p≤0.01, *** p≤0.001, ns - not significant, red bracket indicates a comparison that did not achieve significance with a power value <0.8.

Figure 4. HPV perturbation of expression of the lysine demethylases KDM6A and KDM6B in human head & neck and cervical carcinomas

Normalized RNA-Seq data extracted from the TCGA database for the HNSC (A) and CESC (B) cohorts for HPV+, HPV-, and normal control tissues. Numbers in brackets refer to the number of samples included in each analysis. * p≤0.05, ** p≤0.01, *** p≤0.001, ns- not significant, red brackets indicate a comparison that did not achieve significance with a power value <0.8.

Figure 5. HPV perturbation of DNA methylation at the CDKN2A and CDKN2B loci in human head & neck and cervical carcinomas

HPV+ cancers exhibit markedly altered methylation from HPV- tumors or normal control tissues at two regions flanking the CDKN2A locus. (A) Location of the transcripts (black lines), CpG islands and methylation probes used in this study. Normalized methylation data was extracted from the TCGA database for the HNSC (B) and CESC (C) cohorts. The mean methylation levels for HPV+, HPV-, and normal control tissues for all probes located in the vicinity of the CDKN2A and CDKN2B loci are shown. ** p≤ 0.01, *** p≤ 0.001, **** p ≤ 0.0001.