Determining the mechanism for YAP1 activation by HPV E7 in oropharyngeal carcinoma

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PROJECT SUMMARY Human papillomavirus (HPV) is a driver of carcinogenesis in oropharyngeal squamous cell carcinoma (OPSCC). HPV infects epithelial keratinocytes and alters host cell signaling to suit viral replication. HPV simultaneously promotes basal cell growth and represses cell differentiation programming causing increased basal cell retention within stratified squamous epithelia and tumorigenesis from high-risk HPVs. The HPV protein E7 is a major driving oncogenic factor responsible for promoting these effects. While it has been documented that high-risk E7 causes degradation of host retinoblastoma tumor suppressor RB1, leading to increased cell growth and DNA replication, RB1 degradation does not solely cause E7 tumorigenic activity. E7 activity is more tumorigenic than RB1 loss alone. In addition to growth stimulation, HPV requires repression of cell differentiation. Our lab has documented E7 facilitates degradation of an additional tumor suppressor, PTPN14. PTPN14 degradation by E7 represses cell differentiation and induces translocation of YAP1, an oncogenic transcription coactivator. The mechanism connecting PTPN14 to YAP1 regulation is unknown. My preliminary data indicates PTPN14 degradation by E7 suppresses YAP1 inhibition. Phosphorylation of YAP1 at S127 coincides with YAP1 exclusion from the nucleus. YAP1 inhibition is induced by the Hippo kinase cascade. I have shown PTPN14 loss is transformative and E7-mediated degradation promotes dephosphorylation of YAP1 at S127. I have also shown PTPN14 induces YAP1 phosphorylation at S127. My results show these effects in immortalized human foreskin keratinocytes and my goal is to apply my findings to oral keratinocyte models. The specific aims of this proposal are to 1) determine if E7-mediated PTPN14 degradation promotes YAP1 dephosphorylation, stability and nuclear translocation in HPV positive OPSCC and 2) determine the mechanism of YAP1 phosphorylation by PTPN14. In Aim 1 I will establish effects of PTPN14 degradation in an oral keratinocyte cell model, quantifying phosphorylation levels of YAP1 and YAP1 stability by cycloheximide-chase experiments. I will test rescue of YAP1 inhibition on E7 knockdown and also perform rescue experiments with an E7-binding mutant of PTPN14 to reestablish normal YAP1 regulation in HPV positive OPSCC cells. YAP1 localization in three-dimensional organoid cultures of OPSCC cells will also be analyzed by immunofluorescence microscopy. In Aim 2 I will determine how PTPN14 promotes YAP1 S127 phosphorylation and subsequent nuclear exclusion in oral keratinocytes. PTPN14 mutants containing single deletions of function domains will be expressed with YAP1 phosphorylation quantified by immunoblot. I will determine interactors of PTPN14 important for its ability to induce YAP1 phosphorylation by performing BioID biotin labelling of proteins under conditions of phosphorylated YAP1 and dephosphorylated YAP1.