Identification of an NNK-associated gene signature and tumor microenvironmental features in head and neck squamous cell carcinoma.

BACKGROUNDTobacco exposure is a major risk factor for head and neck squamous cell carcinoma (HNSCC), and the tobacco-specific nitrosamine NNK is an established carcinogen. However, the molecular features linking NNK-associated targets with HNSCC transcriptomic alterations and tumor microenvironmental states remain incompletely understood.

METHODSNNK-related targets and HNSCC-associated genes were collected from public databases and integrated with GEO transcriptomic datasets. Differential expression analysis, LASSO regression, SHAP interpretation, and external validation were used to identify candidate genes. Immune infiltration analysis, molecular docking, molecular dynamics simulation, comparative docking, single-cell RNA-seq analysis, Human Protein Atlas immunohistochemistry, and western blotting were performed to characterize the prioritized candidate.

RESULTS71 overlapping genes were identified between NNK-related targets and HNSCC-associated genes. Integrated analyses retained a six-gene signature comprising MAOB, MMP13, RGS4, ALDH1A1, CXCL8, and PTGS2, which showed diagnostic relevance across validation datasets. Immune deconvolution suggested heterogeneous gene-immune association patterns across the selected genes. Docking suggested that NNK could be accommodated within a predicted MAOB pocket, and 100 ns molecular dynamics simulation supported relative stability of the predicted pose. Single-cell analysis showed that MAOB expression was limited in malignant epithelial cells but relatively enriched in mast cells, glandular cells, keratinocytes, and endothelial cells. Immunohistochemistry and western blotting further supported reduced and heterogeneous MAOB protein expression in HNSCC-related contexts.

CONCLUSIONThis study identifies a six-gene NNK-associated HNSCC signature and highlights MAOB as a candidate molecular feature associated with tobacco carcinogen-related transcriptomic alterations, immune-context associations, predicted ligand accommodation, and cell-type-specific expression.