277 HPV8 E6 induced STAT3 activation leads to hair follicle junctional zone keratinocyte stem cell proliferation and expansion in actinic keratoses

Actinic keratoses (AKs) represent the basis for 80% of squamous cell carcinoma (SCC) and 30% of basal cell carcinoma, yet the cellular basis for AKs remains to be determined. In a cohort of 79 AK samples, HPV8 was detected in all cases where there were koliocytes (n = 42) but not wherein koilocytes were absent, using a highly sensitive and validated PM polymerase chain reaction reverse hybridization assay can identify 25 separate β-HPV genotypes. We have previously shown that selective Lrig1+ hair follicle junctional zone keratinocyte stem cell (HFJZKSC) proliferation and expansion, using the K14-HPV8 complete early region (HPV8tg), phenocopies the pathology of AKs. Lineage tracing with HPV8tg:Lrig1-CreERT2:R26R-LoxP-flanked-Brainbow2.1 (Confetti reporter) and HPV8tg: K15-CreERT2:R26R-LoxP-flanked-Brainbow2.1, showed the presence of perifollicular and infundibulum clones emanating only from the Lrig1 HFJZKSC (n=7). Paired RNA sequencing of HPV8tg flow-sorted skin for Lrig1 and CD34 KSC with bioinformatic analysis using IPA identified transcriptional regulators cMYC in wild type but STAT3 in HPV8tg mice (n=6, p<0.01). STAT3 binding of the p63 promoter was dependent on nuclear YAP. As expected STAT3+/-:HPV8tg do not exhibit HFJZKSC proliferation and does not develop papilloma or SCC. To study the upstream pathway, we identified Lrig1 HFJZKSC proliferation and expansion in the HPV8 mouse model with the single early region gene E6, but not E2, E4, and E7. E6 bound p300 acetylated STAT3, resulting in its subsequent phosphorylation. STAT3 and vector siRNA treatment of E6 transduced HaCaT cell lines showed that ΔNP63 was dependent upon STAT3. Collectively, our findings show the mechanism of HPV8-associated AKs.