NSUN2-Mediated M5c Dysregulation Promotes OSCC Ferroptosis

This study aimed to investigate the role of NSUN2-mediated 5-methylcytosine (m5C) methylation in regulating ferroptosis resistance and tumor progression in oral squamous cell carcinoma (OSCC), identifying potential epitranscriptomic therapeutic targets. Tissue microarrays and cell lines were used to analyze NSUN2 expression via immunohistochemistry, qRT-PCR, and Western blot. Functional assays (CCK8, Transwell, colony formation) and molecular techniques (RNA immunoprecipitation, m5C dot blot) were performed to assess cell proliferation, migration, and m5C methylation levels. Ferroptosis sensitivity was evaluated using erastin treatment, lipid peroxidation (C11-BODIPY) and ROS (DHE) assays, and mitochondrial morphology analysis via TEM. A xenograft mouse model was employed to validate in vivo tumorigenicity, with statistical significance determined by Student’s t-test and Kaplan-Meier survival analysis. NSUN2 was overexpressed in OSCC tissues/cells, correlating with poor prognosis, lymph node metastasis, and reduced overall survival (P < 0.01). Knockdown of NSUN2 in ferroptosis-resistant cells (SCC9, HSC6) decreased m5C methylation, enhanced ferroptosis sensitivity (increased lipid peroxidation, ROS, and mitochondrial damage), and inhibited proliferation/migration. Mechanistically, NSUN2 regulated autophagy-dependent ferroptosis by mediating m5C methylation of sequestosome 1 (SQSTM1/P62) mRNA, recruiting Y-box binding protein 1 (YBX1) to stabilize its expression; methyl donor supplementation rescued tumor growth in xenografts. The NSUN2-YBX1 axis drives ferroptosis resistance in OSCC by modulating autophagy through m5C-dependent stabilization of SQSTM1/P62. Targeting this pathway represents a novel epitranscriptomic strategy to enhance ferroptosis induction, offering potential for improving OSCC treatment and prognosis.