Head and neck squamous cell carcinoma (HNSCC) associated with human papillomavirus (HPV) infection display altered energy metabolic pathways due to numerous oncogenic events activated by viral oncoproteins (E6 and E7) and their splice variants. A subgroup of HNSCC presents with high expression levels of HPV16-E6*I, the major splice variants of HPV16-E6, and correlates with oxidative and metabolic stress pathways signatures and unfavorable prognosis. Here, we aimed to understand the effects of HPV16-E6*I overexpression on oxidative stress (OS) defense and metabolic pathways. Stable clones of HEK-293 cells overexpressing HPV16-E6*I-GFP, -E6-GFP, and control vector constructs were established and subjected to in vitro characterization by mimicking the atmospheric O₂ conditions of the tumor tissue (5% O₂ normoxia and 2% O₂ hypoxia). The effects of E6 and E6*I on OS defense pathway components and metabolic reprogramming were studied by monitoring the expression and subcellular localization of fluorescence-tagged viral proteins, immunofluorescence, live-cell imaging, ddPCR and protein expression analysis of key energy metabolic enzymes as well as cell metabolic assays. Particularly, under hypoxic conditions, overexpression of E6*I was associated with increased proliferation, increased expression of OS defense components, and altered glycolysis and OXPHOS activity. In summary, in vitro analysis of HPV16-E6*I overexpression revealed signatures of OS defense and metabolic reprogramming, which were also observed in a subset of patients with E6*I overexpression, viral host genome integration, and unfavorable prognosis. Testing for E6*I expression in patient samples could be of interest in the future to determine prognostically and therapeutically relevant subgroups.

The authors declare that there is no conflict of interest