A keratinocyte life cycle model identifies novel host genome regulation by human papillomavirus 16 relevant to HPV positive head and neck cancer

Abstract

Many aspects of the HPV life cycle have been characterized in cervical cell lines (W12, CIN612) and in HPV immortalized primary foreskin keratinocytes. There is now an epidemic of HPV positive oropharyngeal cancers (HPV16 is responsible for 80-90% of these); therefore increased understanding of the HPV16 life cycle in oral keratinocytes is a priority. To date there have been limited reports characterizing the HPV16 life cycle in oral keratinocytes. Using TERT immortalized "normal" oral keratinocytes (NOKs) we generated clonal cell lines maintaining the HPV16 genome as an episome, NOKs+HPV16. Organotypic raft cultures demonstrated appropriate expression of differentiation markers, E1^E4 and E2 expression along with amplification of the viral genome in the upper layers of the epithelium. Using this unique system RNA-seq analysis revealed extensive gene regulation of the host genome by HPV16; many of the changes have not been observed for HPV16 before. The RNA-seq data was validated on a key set of anti-viral innate immune response genes repressed by HPV16 in NOKs+HPV16. We show that the behavior of these NOKs+HPV16 lines is identical to HPV16 immortalized human tonsil keratinocytes with regards innate gene regulation. Finally, using The Cancer Genome Atlas (TCGA) data we examined gene expression patterns from HPV positive and negative head and neck cancers and demonstrate this innate immune gene signature set is also downregulated in HPV positive cancers versus negative. Our system provides a model for understanding HPV16 transcriptional regulation of oral keratinocytes that is directly relevant to HPV positive head and neck cancer.

Human papillomavirus 16 E6 and E7 synergistically repress innate immune gene transcription

“…This transcription factor complex then enters the nucleus and binds to the control elements of interferon stimulated genes (ISGs) to activate their transcription. These ISGs are also downregulated in HPV16 positive N/Tert-1 cells (30, 31). These genes are repressed by E6, E7 and E2 and it was unclear what viral protein is responsible for this repression in the context of the entire HPV16 genome.…”

“…Our previous work in N/Tert-1 cells demonstrates that there is down regulation of innate immune gene expression at various stages of the interferon signaling pathway by HPV16 (30, 31). Following treatment of cells with interferon there is an activation of ISGF3 (interferon stimulated gene factor 3) which is a complex composed of STAT1, STAT2 and IRF9 (32–34).…”

“…These were transfected into the hTERT immortalized foreskin keratinocyte cell line N/Tert-1 to establish stable cell lines containing wild type HPV16 and the assorted E6/E7 mutants. We have demonstrated that HPV16 represses IIGs in these cells (30, 31). These experiments required the use of already immortalized keratinocytes as HPV16 cannot immortalize primary keratinocytes in the absence of E6 and E7 expression.…”

“…(29). Recently we demonstrated that repression of IIGs by HPV16 persists in HPV16 positive tumors and that this repression could contribute to the immune evasion by the HPV16 tumor (30, 31). We also recently demonstrated that, as well as E6 and E7, E2 can also repress expression of IIGs (30).…”

Prevalence of human papillomavirus type 16 in persistent oral lesions arising in patients with tobacco, areca nut, and alcohol habits

“…The healthy and nonaddicted oral mucosa shows some resistance toward HPV and HPV16 infections . The cancer‐precancer lesions and microenvironment of oral cavity are immunocompromised and the immune system of host plays an important role in enabling oral keratinocytes to escape or delay the immortalization of oral keratinocytes by HPV16 …”