Transcriptomic and multi-layer variant analysis identifies STAT3 and HIF1A as central regulators of regulated cell death pathways in lung squamous cell carcinoma.

Lung squamous cell carcinoma (LUSC) remains one of the most aggressive subtypes of non-small cell lung cancer (NSCLC), characterized by high mortality, therapeutic resistance, and molecular heterogeneity. Emerging evidence highlights the importance of regulated cell death (RCD) pathways including apoptosis, necroptosis, ferroptosis, and pyroptosis in determining tumor fate, immune evasion, and treatment response. However, the transcriptional regulators and genetic variants governing these cell death mechanisms in LUSC remain largely undefined. An integrative transcriptomic analysis of five GEO datasets (GSE146868, GSE204753, GSE268049, GSE236654, and GSE250509) was conducted to identify differentially expressed genes associated with apoptosis, necroptosis, ferroptosis, and pyroptosis, using curated gene sets from FerrDb v2.0, KEGG, Reactome, and GeneCards. Functional enrichment via GO and KEGG revealed strong activation of inflammatory, TNF, and necroptosis pathways. Protein-protein interaction networks (STRING) and hub analysis (Cytoscape-CytoHubba) identified core DE-RCDGs, while upstream transcriptional regulators were predicted with iRegulon and validated through UALCAN expression, hallmark enrichment, and survival datasets (TCGA, HPA). Finally, high-impact non-synonymous SNPs (nsSNPs) in key TFs were prioritized through a multi-layer bioinformatics pipeline (SIFT, PolyPhen 2, CADD, REVEL, MetaLR, Mutation Assessor) and structurally modeled to assess their functional impact. Network-based and regulatory enrichment analyses identified PPARG, STAT3, HIF1A, SMAD1, and ZBTB16 as central transcriptional regulators bridging apoptosis and necroptosis signaling in LUSC. Hallmark pathway enrichment highlighted their involvement in hypoxia response, TNF/NF-κB signaling, and metabolic reprogramming, underscoring their multi-faceted regulatory impact. Expression validation using TCGA/UALCAN confirmed significant upregulation of all five TFs in tumor tissues, with STAT3 and HIF1A exhibiting the most prominent oncogenic expression patterns. Survival analysis indicated that elevated STAT3 and HIF1A expression correlated with poorer overall survival, though independent validation via the HPA database revealed no significant survival separation, suggesting a context-dependent prognostic role. Variant analysis further revealed high-impact deleterious nsSNPs in both STAT3 (C712R; rs193922722 and Q633L; rs2144684101) and HIF1A (C255G; rs1242097924 and D55G; rs2044414353), each predicted to disrupt protein structural stability, metal-binding geometry, and interaction interfaces. Structural modeling confirmed conformational destabilization and altered residue orientation, consistent with potential impairment in transcriptional regulation and signaling fidelity. Current multi-omics analysis delineates STAT3 and HIF1A as central regulators coordinating apoptotic and necroptotic cross talk in LUSC. Deleterious nsSNPs within these TFs highlight structural vulnerabilities potentially driving tumor progression and therapeutic resistance, offering promising targets for precision-based intervention.