Integrative transcriptomic and structural modeling reveal CASP1, TLR3, PYCARD, and CD274 as immune-modulatory drivers in breast cancer.

Breast cancer (BRCA) remains one of the most prevalent malignancies worldwide, characterized by marked molecular heterogeneity and diverse clinical outcomes. Necroptosis and ferroptosis, two regulated forms of programmed cell death (PCD), play pivotal roles in tumor progression and immune regulation; however, their combined influence on BRCA pathogenesis remains inadequately understood. To elucidate these interactions, transcriptomic datasets (GSE254218, GSE220608) were integrated to identify differentially expressed genes (DEGs) using edgeR (|log2FC|> 1, FDR < 0.05). From 805 DEGs, intersection with 614 necroptosis-related genes (NRGs) and 259 ferroptosis-related genes (FRGs) yielded 37 candidate cell death-related genes (CDRGs). CytoHubba analysis prioritized 25 hub genes, and UALCAN expression profiling identified six significantly stage-associated genes CASP1, TLR3, PYCARD, KRT5, MAP1B, and MMP13 (p < 1E - 12). Functional enrichment (GO/KEGG) and protein-protein interaction (PPI) analyses linked these genes to immune activation, inflammatory signaling, and PCD-associated pathways. Integrated survival and immune infiltration analyses (CIBERSORT, TIMER 2.0) highlighted CASP1, TLR3, PYCARD, and CD274 as key prognostic biomarkers modulating the BRCA tumor microenvironment. To evaluate genetic and structural perturbations affecting these targets, deleterious nonsynonymous SNPs (nsSNPs) were filtered using SIFT, PolyPhen-2, CADD, REVEL, MetaLR, and Mutation Assessor. Structural characterization identified nine high-impact nsSNPs (ΔΔG <  - 1.7 kcal/mol), particularly in TLR3 (L104N, L381P, L77P) and CD274 (W57S, C155S, G159D, Y112N), indicating potential disruption of receptor stability, immune checkpoint interactions, and inflammasome regulation. This integrative multi-omics and structural pharmacogenomics framework reveals a robust four-gene signature (CASP1, TLR3, PYCARD, CD274) that links necroptosis, ferroptosis, and immune modulation in BRCA, providing promising avenues for precision oncology and therapeutic target development.