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Human papillomavirus 8 (HPV8) is associated with the development of squamous cell carcinoma (SCC) of the skin. HPV-infected keratinocytes are able to override normal checkpoint control mechanisms and sustain cell cycle activity, allowing for synthesis of cellular proteins necessary for viral genome amplification. To study how HPV8 may disrupt cell cycle control, we analyzed the impact of HPV8 early gene expression on one of the key regulators of cell cycle and DNA damage response, checkpoint kinase-1 (CHK1). We found that expression of E1, E1̂E4, E2, E6 or E7 individually did not affect CHK1; however, keratinocytes expressing the complete early genome region (CER) of HPV8 showed a profound loss of CHK1 protein levels, that proved to be mediated by E6E7 co-expression. Neither CHK1 promoter regulation nor the ubiquitin-proteasome pathway are involved in HPV8-mediated CHK1 repression. However, CHK1 protein repression in organotypic skin cultures was paralleled by downregulation of the autophagy marker LC3B. Treatment of HPV8-CER expressing cells with the autophagy inhibitor Bafilomycin A1 rescued CHK1 expression and led to LC3B accumulation. Taken together, our data implicate that CHK1 autophagic degradation is enhanced by HPV8, which may contribute to the oncogenic potential of the virus., (© 2019 UICC.)

Human papillomavirus type 16 (HPV16) is a major risk for development of oropharyngeal squamous-cell-carcinoma (OPSCC). Although HPV + OPSCC metastasize faster than HPV - tumors, they have a better prognosis. The molecular and cellular alterations underlying this pathobiology of HPV + OPSCC remain elusive. In this study, we examined whether expression of HPV16-E6E7 targets the number of migratory and stationary cancer stem cells (CSC). Furthermore, we wanted to elucidate if aberrantly expressed miRNAs in migratory CSC may be responsible for progression of OPSCCs and whether they may serve as potential novel biomarkers for increased potential of metastasis. Our studies revealed that HPV16-E6E7 expression leads to an increase in the number of stationary (CD44 high /EpCAM high ) stem cells in primary keratinocyte cultures. Most importantly, expression of E6E7 in the cell line H357 increased the migratory (CD44 high /EpCAM low ) CSC pool. This increase in migratory CSCs could also be confirmed in HPV + OPSCC. Differentially expressed miRNAs from HPV16-E6E7 positive CD44 high /EpCAM low CSCs were validated by RT-qPCR and in situ hybridization on HPV16 + OPSCCs. These experiments led to the identification of miR-3194-5p, which is upregulated in primary HPV16 + OPSCC and matched metastasis. MiR-1281 was also found to be highly expressed in HPV + and HPV - metastasis. As inhibition of this miRNA led to a markedly reduction of CD44 high /EpCAM low cells, it may prove to be a promising drug target. Taken together, our findings highlight the capability of HPV16 to modify the phenotype of infected stem cells and that miR-1281 and miR3194-5p may represent promising targets to block metastatic spread of OPSCC., (© 2018 UICC.)