Musashi‐1 Drives Radioresistance and Stemness in Head and Neck Squamous Cell Carcinoma via a Radioresistant FaDu Model

Vollständiger Abstract

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ABSTRACT 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.