Head and neck squamous cell carcinoma (HNSCC) is frequently managed with radiotherapy, but the emergence of radioresistant cancer cells with cancer stem cell (CSC) properties remains a major clinical obstacle. Here, we established a radioresistant FaDu cell line (F-IRR) by repeated fractionated irradiation to investigate the molecular basis of radiation resistance in HNSCC. Irradiated colony-forming assays confirmed the radioresistant phenotype of F-IRR cells. Although F-IRR cells proliferated more slowly than parental cells, they exhibited markedly enhanced CSC activity, as demonstrated by sphere formation, limiting dilution, and soft agar assays. Among multiple stem cell-associated markers, Musashi-1 (MSI1)-an ribonucleic acid (RNA)-binding protein with established roles in CSC maintenance-was the most prominently upregulated, as confirmed by western blot, reverse transcription-polymerase chain reaction (RT-PCR), and immunocytochemistry. Functional studies demonstrated that siRNA-mediated knockdown of MSI1 suppressed stemness and radioresistance in F-IRR cells, whereas stable MSI1 overexpression in parental FaDu and CAL27 cells conferred enhanced stemness and radiation resistance. RNA sequencing and pathway analysis identified hyperactivation of the mitogen-activated protein kinase (MAPK) pathway in F-IRR cells, and targeted inhibition experiments showed that the c-Jun N-terminal kinase (JNK) signaling pathway was the primary upstream regulator of MSI1 expression. JNK inhibition with SP600125 reduced MSI1 levels and attenuated stemness and radioresistance in F-IRR cells. In vivo xenograft experiments further confirmed that F-IRR cells possessed greater tumorigenic potential and radioresistance than parental cells, with high expression of MSI1 and phospho-JNK in tumor tissues. Collectively, these findings identify the JNK-MSI1 axis as a critical driver of CSC-mediated radioresistance in HNSCC and a promising therapeutic target for overcoming treatment failure.
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