Hypoxia, metastatic origin and HPV16 E6/E7 expression differentially shape the radiation response in head and neck...

Hypoxia, a common feature of head and neck squamous cell carcinoma (HNSCC), significantly compromises radiotherapy efficacy by modulating DNA repair, cell‑cycle progression, and inflammatory signaling pathways. In addition, intrinsic tumor heterogeneity-including tumor origin and human papillomavirus (HPV) oncogene expression-further shapes therapeutic response. Despite these recognized factors, the integrated impact of oxygen tension, tumor origin, and viral oncogene expression on radiation response remains poorly defined. We examined the effects of hypoxia (1% O₂) and gamma-irradiation (6 Gy) on proliferation, cell-cycle progression, apoptosis, transcriptomic profiles, and cytokine secretion in three HNSCC cell lines: FaDu (primary origin), Detroit-562 (metastatic origin), and 2A3, an isogenic FaDu derivative stably expressing HPV-16 E6/E7 oncogenes. Hypoxia prolonged cell doubling time across all lines, independent of origin or E6/E7 expression, while irradiation induced features of an intermediate epithelial-mesenchymal transition phenotype. Compared with FaDu, 2A3 cells exhibited distinct transcriptional responses, reduced cytokine secretion (IL-8, Serpin E1), and altered cell-cycle regulation following irradiation. Detroit-562 cells secreted markedly higher cytokine levels and showed no increase in cleaved caspase-3 under hypoxia after irradiation. Our findings demonstrate that oxygen tension and biological factors-including metastatic origin and E6/E7 expression-modulate radiation response in HNSCC cell lines. These results highlight the need for biomarker-driven strategies that consider both hypoxic tumor microenvironment and key biological factors such as metastatic origin and HPV‑related E6/E7 oncogene expression.