O04 HPV8 E6 leads to Lrig1+ keratinocyte stem cell expansion

Actinic keratoses (AKs) represent the precursor skin lesions in the pathogenesis of up to 82% of cutaneous squamous cell carcinoma (cSCCs). However, current treatment of AK lesions leads, at best, to only a 75% reduction in cSCCs for 1 year, and relies on therapies using topical chemotherapy, immune stimulants or surgical destruction. We have previously shown that 44% of AK tissue sample histologies contain koilocytes, and genotyping universally identified human papillomavirus 8 (HPV8). To determine the cell signalling basis for HPV8 Lrig1 hair follicle junctional zone keratinocyte stem cell proliferation and expansion, we sought to identify the integral early region gene(s). Skin histology of HPVtg mice expressing individual early-region genes (E2, E4, E6 and E7) identified both E2 and E6 mouse skin as having broadened hair follicle infundibulum. Whole-mount immunofluorescent labelling demonstrated Lrig1 proliferation and expansion in E6 mice only, which we confirmed by flow cytometric analysis. Compared to wild-type (WT) mice, E6 mice Lrig1 flow-sorted cells had lower expression of the differentiation marker keratin 10 but comparable levels of Sox9 and cMyc, consistent with expansion of keratinocyte stem cells, as confirmed by colony-forming efficiency assay (WT 0.05 ± 0.043 vs. E6 0.24 ± 0.11). E6-transduced, but not E7-transduced, HaCaT keratinocytes demonstrated reduced differentiation and increased p63 expression with induced differentiation using calcium shift. Compared to vector control and E7-transduced, E6-transduced HaCaT keratinocytes demonstrated increased migration and colony-forming efficiency but similar proliferation, consistent with greater expression of pSTAT3 and ΔNP63. Ingenuity pathway analysis of previously determined E6 binding partners identified p300 as a regulator of STAT3 activation. Small interfering RNA targeting P300 in E6-transduced HaCaT cells led to a reduction in pSTAT3 and ΔNP63. Pull-down experiments show that E6 bound acetylated p300, which, in turn, led to increased expression of STAT3 regulated genes. The E6K137N mutation in transgenic mice, wherein E6 does not bind p300, do not develop papilloma or cSCC. Our findings show that E6, not E7, as often observed in α-serotype HPV, was responsible for HPV8-induced Lrig1 hair follicle junctional zone keratinocyte stem cell expansion.