HPV-mediated regulation of SMAD4 modulates the DNA damage response in head and neck cancer

Head and Neck cancer (HNC) is a fatal malignancy with poor prognosis. Human Papillomavirus (HPV) infection is becoming the prominent cause of HNC in the western world, and studying the molecular mechanisms underlying its action in cancers is key towards targeted therapy. To replicate, HPV regulates the host DNA damage repair (DDR) pathway. SMAD4 is also involved in the regulation of the DDR machinery and likely plays important role in maintaining cell viability upon genotoxic stress. In this study, we investigated the role of HPV in the upregulation of SMAD4 to control the DDR response and facilitate its lifecycle.SMAD4, Rad51 and CHK1 expression was assessed in HPV-positive and HPV-negative HNC using TCGA data, a panel of 14 HNC cell lines and 8 fresh tumour tissue samples from HNC patients. HPV16 expression was modulated by E6/E7 siRNA knock-down or transduction in HPV-positive HNC cell lines and Human Primary keratinocytes respectively. SMAD4 half-life was assessed by cycloheximide treatment in HNC cell lines, together with βTRCP1-dependent SMAD4 ubiquitination. SMAD4 siRNA knock-down was used to determine its role in HPV-mediated regulation of DDR machinery and to assess cisplatin sensitivity in HPV-positive HNC cell lines.We found that HPV increases SMAD4 expression is both HPV-positive HNC tumours and cell lines, impairing its degradation which is mediated by the E3 ubiquitin ligase βTRCP1. SMAD4 expression highly correlates with the expression of two main players of the DDR pathway, CHK1 and Rad51, which expression is also upregulated by the presence of HPV. In particular, we demonstrate that HPV stabilizes SMAD4 to increase CHK1 and Rad51 expression. In addition, SMAD4-deficient HPV-positive cells have increased sensitivity to cisplatin treatment.Our results give a clear molecular mechanism at the basis of HPV regulation of the DDR pathway. In particular, we show how HPV stabilizes SMAD4 to promote DDR protein expression, which may be used to facilitate viral replication and HNC onset. Moreover, we found that SMAD4 silencing in HPV-positive HNC cell lines increases sensitivity to cisplatin treatment, suggesting that HPV-positive HNC with low SMAD4 expression may be preferentially susceptible to similar treatments.