YBX1 Promotes Malignant Progression of HNSCC via Stabilizing NSUN2‐m5C Modified ATF4 mRNA

Head and neck squamous cell carcinoma (HNSCC) remains a major therapeutic challenge because of its aggressive metastatic behavior and the paucity of effective targeted therapies. The present study identifies YBX1 as a critical 5‐methylcytosine (m5C) reader that cooperates with the m5C methyltransferase NSUN2 to promote malignant progression through mRNA stabilization. Integrated analysis of The Cancer Genome Atlas datasets demonstrated significant upregulation of YBX1 in HNSCC. Clinical validation in a cohort of 77 patients further showed that elevated YBX1 expression was a significant risk factor for poor overall survival, with a hazard ratio of 1.184. Functional analyses in vitro and in vivo revealed that YBX1 depletion markedly suppressed proliferation, migration, and invasion, whereas YBX1 overexpression enhanced these oncogenic properties in multiple HNSCC cell lines and xenograft models. Mechanistic investigations using m5C methylated RNA immunoprecipitation sequencing (m5C‐MeRIP‐seq) combined with YBX1‐RIP‐seq identified ATF4 mRNA as a direct YBX1 target containing m5C modifications within its coding region. NSUN2‐dependent m5C deposition significantly increased YBX1 binding affinity, thereby stabilizing ATF4 transcripts ( p < 0.001). Luciferase reporter assays confirmed the specificity of YBX1 recognition of m5C‐modified ATF4 mRNA. Collectively, these findings define an epitranscriptomic regulatory pathway in which YBX1 decodes NSUN2‐installed m5C marks to enhance oncogenic mRNA stability and support tumor progression. Dual inhibition of YBX1 and NSUN2 is therefore proposed as a potential therapeutic strategy for HNSCC.