Abstract / Summary
Abstract Background Tobacco exposure is a major risk factor for HPV-unrelated head and neck squamous cell carcinoma (HNSCC) and is associated with poor clinical outcomes; however, the molecular regulators through which tobacco exposure drives tumor aggressiveness and immunosuppression remain poorly understood. Methods Multi-cohort transcriptomic and single-cell analyses identified tobacco-associated targets and cell type-specific alterations, followed by molecular, metabolic, and immune cell functional assays. GigaTIME -based virtual multiplex immunofluorescence characterized the tumor immune microenvironment, while 4NQO-treated HNSCC cells and orthotopic tongue tumors modeled tobacco carcinogen exposure. Results Through integrated bioinformatics analyses, AI-assisted spatial immune profiling, and functional studies, we identified MUC5B as a smoking-associated immunomodulatory mucin that contributes to tumor progression in HPV-unrelated HNSCC. Mechanistically, we found that GATA4 directly binds the MUC5B promoter and represses its transcription, whereas exposure to 4NQO disrupts this regulatory axis by reducing GATA4 expression and promoter occupancy, thereby inducing MUC5B expression. Elevated MUC5B was associated with metabolic reprogramming toward enhanced fatty acid utilization and establishment of an immunosuppressive tumor microenvironment characterized by M2-like macrophage polarization and diminished CD8⁺ T cell infiltration. Importantly, MUC5B silencing reversed these metabolic and immune alterations in vitro and in immunocompetent mouse models, resulting in reduced tumor growth under 4NQO exposure. Conclusions These findings identify a 4NQO/GATA4-MUC5B regulatory axis that links tobacco carcinogen exposure to metabolic reprogramming and immune suppression, highlighting MUC5B as a potential therapeutic target in smoking-associated HNSCC.