HPV16‐E7 Oncoprotein Enhances Ceramide‐Mediated Lethal Mitophagy by Regulating the Rb/E2F5/Drp1 Signaling Axis

Abstract

The five‐year survival rate of head and neck squamous cell carcinoma (HNSCC) patients is only 50% and has not increased in the past several decades. This is due, in part, to high‐resistance to treatment and a lack of advances in targeted molecular therapies. Human papillomavirus (HPV) infection is implicated in 40–60% of HNSCC cases, which express the viral oncogenes E6 and E7. Interestingly, patients with HPV‐associated HNSCC respond better to treatment compared to HPV‐negative HNSCC patients, though the molecular mechanism for this phenomenon has not been defined. It is known that dihydroceramide‐synthase‐1 (CerS1) and its product C18‐ceramide are associated with positive response of HNSCC to chemotherapeutic treatment. Recently, the mechanism for this response was determined to be a novel form of lethal mitophagy, which is induced by CerS1 or the synthetic sphingolipid C18‐pyridinium‐ceramide (C18‐pyr‐cer).ObjectiveAs no studies have examined sphingolipid signaling or mitophagy in HPV‐associated HNSCC, we asked whether ceramide‐mediated lethal mitophagy may be involved in the improved response of HPV‐associated HNSCC.MethodsCisplatin, a common chemotherapeutic for HNSCC, or C18‐pyridinium‐ceramide was used to treat cell lines developed from either HPV‐negative or HPV‐positive HNSCC tumors. Knockdown or overexpression of key mitophagy mediators, HPV oncoproteins, and their downstream targets were utilized to determine roles of these proteins in cell response. Mitophagy assays, including live cell confocal microscopy for co‐localization of mitotracker and lysotracker dyes, as well as trypan blue counts, were used to detect lethal mitophagy, and protein associations were examined by co‐immunoprecipitation and proximity ligation assays.ResultsWe found that Cisplatin treatment in HPV‐positive cells resulted in CerS1‐dependent lethal mitophagy and accumulation of CerS1 and ceramide in the mitochondria. Treatment with C18‐pyr‐cer, which localizes to the mitochondria due to its positive charge, also resulted in lethal mitophagy. HPV‐positive HNSCC cells were more sensitive to either cisplatin or C18‐pyr‐cer treatment when compared with HPV‐negative HNSCC cells. Interestingly, ceramide‐induced lethal mitophagy was attenuated by knockdown of E6/E7 oncoproteins in HPV‐positive cells and enhanced by E7 expression alone in HPV‐negative cells. The Rb/E2F5 signaling axis, which is downstream of E7 signaling, affected this cell response, particularly Drp1‐oligomerization. Unexpectedly, E2F5 was found to stabilize this oligomerization by associating with Drp1.ConclusionThis work demonstrates that Cisplatin induces CerS1‐dependent lethal mitophagy, and that the HPV E7 oncoprotein functions to sensitize HNSCC cells to ceramide via the E7/Rb/E2F5/DRP1 signaling axis. By manipulating HPV‐negative HNSCC cells to mimic HPV‐positive HNSCC cells through either expressing E7, knocking down Rb, or overexpressing E2F5, we were able to sensitize them to treatment. This is the first demonstration, to the best of our knowledge, of a link between the enhanced response of HPV‐associated HNSCC and sphingolipid signaling. We hope these data will provide groundwork for an adjuvant therapy to sensitize HPV‐negative HNSCC to treatment.Support or Funding InformationNIDCR grant 3R01DE016572‐07S1 MUSC Dental Training Grant 5T32DE017551‐08