HPV sensitizes OPSCC cells to cisplatin-induced apoptosis by inhibiting autophagy through E7-mediated degradation of AMBRA1

Head and neck (HN) cancers are the 6th most common malignancy worldwide. Despite improvements in treatments, the survival rate for this disease is less than 50%, calling for novel therapies. A widespread head and neck cancer is the oropharyngeal squamous cell carcinoma (OPSCC). Until two decades ago, the major risk factor for OPSCC was represented by alcohol and smoking abuse. Lately, oncogenic human papillomavirus (HPV) is emerged as new risk factor for it and the infection with high risk human papillomavirus (hrHPV) represents a key factor in OPSCC development. Nervertheless, patients with HPV-related OPSCC show better prognosis and response to treatment compared to HPV-negative counterparts. The hrHPV proteins E6 and E7 are involved in cancer development, modulating the stability and activity of proto-oncogenes and tumor suppressors, including p53, pRB, c-MYC and PP2A. In addition, hrHPV infection associates with reduced expressions of autophagy markers in human cervical squamous cell carcinoma, due to the expression of early genes of hrHPV. AMBRA1 is one of the critical regulators of autophagy through its interaction with ULK1 and BECLIN1. AMBRA1 plays a key role in tumorigenesis, by regulating c-Myc activity and stability and it has an important role in the crosstalk between autophagy and apoptosis. Recent discoveries, show the involvement of AMBRA1 in the control of the stability of cyclin D1, a phenomenon which is related to cell cycle and proliferation. More broadly, autophagy was described to play different role in tumorigenic cells: it is employed by cancer cells as a highly plastic and dynamic mechanism to either repress initial steps in carcinogenesis or support the survival and growth of established tumors. Several studies have also described that autophagy inhibition allows sensitization to apoptosis during cancer therapy. The mechanism by which HPV confers a less aggressive phenotype in OPSCC patients and so a greater response to treatments remains enigmatic. The hrHPV infection has been described to reduce autophagy in human cervical SCCs, while high autophagy was associated with a poor survival of OPSCC patients. In line with these data, we investigated a possible cause effect relationship among HPV positivity, autophagy and prognosis. In this project, we mainly used four different cell lines: two are OPSCC cellular system related to HPV, and 2 not. In a preliminary step, we defined that autophagy levels are reduced in HPV-positive cell. We found out that the expression of AMBRA1 is strongly affected in these cell lines due to the HPVE7 mediated degradation of AMBRA1 by calpains. HPV E7 also directly binds AMBRA1, and competes with BECLIN1 for its interaction. In order to investigate the role of HPV and autophagy in OPSCC resistance to chemotherapeutic agents, we firstly tested the ability of our cellular systems PhD in Cellular and Molecular Biology 7 to activate cell death in response to cisplatin. As expected, cells derived from HPV-negative OPSCC are more resistant to cisplatin compared to the HPVpositive ones. Prompted by this result, we tested the ability of AMBRA1, and more in general of autophagy, to modulate the response to cisplatin in HPVnegative OPSCC. Either autophagy inhibition or the downregulation of AMBRA1 or BECN1 in HPV-negative OPSCC cells induces an increased rate of cell death when cells were exposed to cisplatin. Similarly, two different semi-quantitative immunohistochemical analysis of primary OPSCCs confirmed AMBRA1 expression is reduced in HPV-positive tumors. Altogether, these results indicated that autophagy is inhibited in hrHPVpositive OPSCC and, the degradation of AMBRA1 mediated by E7 through calpains could be relevant in this phenomenon. In addition, we highlighted that autophagy impairment correlates with a higher response to cisplatin exposition in HPV-positive OPSCC-derived cell lines. These data suggest autophagy modulation as viable therapeutic strategy to improve the clinical outcome of HPV-negative OPSCC.