Cell-specific multi-target mechanisms of cotinine in cervical cancer oncogenesis and progression

Cervical cancer is the fourth most common malignant tumor in women globally, with tobacco smoke being a major environmental risk factor. The tobacco-specific metabolite cotinine may promote cancer progression through mechanisms such as chronic inflammation and genomic instability, but its precise molecular targets and interactions remain unclear.

We integrated network toxicology and multi-omics for target prediction, validated by molecular biology experiments and dynamic simulations. The workflow included database screening of cotinine-related targets, identification of key modules via single-cell analysis and hdWGCNA, integration of differential gene analysis to obtain potential targets, determination of core targets using protein-protein interaction networks, pathway enrichment and machine learning, transcriptomic profiling of cotinine-induced cell type-specific pathway regulation in H8 and HeLa cells, and further validation through molecular docking, molecular dynamics simulations, and in silico knockout.

Initial screening identified 151 cotinine-related targets, refined to 17 key candidates via integrated multi-omics analysis and machine learning. Molecular studies revealed multi-level carcinogenic mechanisms: in H8 cells, cotinine promoted tumorigenesis via chronic inflammation, differentiation defects, ECM remodeling, and signaling dysregulation; in HeLa cells, it accelerated progression/metastasis through dysregulated proliferation, matrix degradation, impaired metal ion transport, and cytokine storms. Validated targets—KAT2B (in H8 cells) and CA9, CCNB1, LAGE3 (in HeLa cells)—were significantly downregulated in cervical tumors. Molecular docking and dynamics simulations confirmed stable cotinine-target binding.

This study systematically demonstrates that cotinine exerts its carcinogenic effects by targeting KAT2B, CA9, CCNB1, and LAGE3, thereby dysregulating multiple signaling pathways to induce tumorigenesis in epithelial cells and accelerate progression/metastasis in malignant cells. Elucidation of this multi-target toxicity mechanism provides a critical theoretical foundation for developing targeted detoxification strategies against tobacco-associated cervical cancer.