Mechanistic study of HIF-1α-mediated regulation of the Notch1 pathway in promoting radioresistance in head and neck squamous cell carcinoma: a comprehensive analysis based on bioinformatics and functional experiments.

BACKGROUND: Radiotherapy remains a cornerstone in the treatment of head and neck squamous cell carcinoma (HNSCC), yet resistance to irradiation significantly impairs therapeutic outcomes. Hypoxia within the tumor microenvironment is a known contributor to radioresistance, largely mediated by hypoxia-inducible factor 1-alpha (HIF-1α). However, the downstream molecular mechanisms remain incompletely defined. This study aimed to investigate whether HIF-1α promotes radioresistance in HNSCC through activation of the Notch1 signaling pathway. METHODS: Transcriptomic data from TCGA-HNSC were analyzed to identify differentially expressed genes between radiosensitive (n = 251) and radioresistant (n = 35) groups. Gene set enrichment analysis (GSEA) and Pearson correlation assessed pathway involvement. In vitro experiments using CAL27 and FaDu cell lines were conducted under normoxic and hypoxic conditions. HIF1A was silenced via shRNA, and Notch1 was overexpressed for rescue experiments. Clonogenic survival, γ-H2AX immunofluorescence, EdU incorporation, and apoptosis (Annexin V/PI) were measured post-irradiation (2 Gy). RESULTS: HIF1A and Notch1 pathway genes were upregulated in radioresistant tumors (p < 0.05). Hypoxia significantly increased HIF1A, NOTCH1, DLL4, and HES1 expression in vitro (p < 0.001). HIF1A knockdown reduced clonogenic survival (P < 0.001), increased γ-H2AX foci (p < 0.001), suppressed proliferation (p < 0.001), and elevated apoptosis (p < 0.001). Notch1 overexpression partially reversed these effects (all p < 0.01). CONCLUSION: Our findings suggest that HIF-1α promotes radiotherapy resistance in HNSCC by activating the Notch1 signaling axis. Targeting this pathway may enhance radiosensitivity and provide a new strategy for overcoming hypoxia-driven resistance.