Integrated transcriptomic and in silico structural analysis identifies NFS1 and HSPA9 as potential regulators...

Colorectal cancer (CRC) exhibits extensive metabolic reprogramming and resistance to regulated cell death, contributing to tumor progression and therapeutic failure. Ferroptosis, an iron-dependent and mitochondria-associated form of cell death, has emerged as a potential therapeutic vulnerability; however, its integrated molecular regulation in CRC remains poorly understood. In this study, an integrative transcriptomic and in silico structural analysis was performed using two GEO datasets (GSE290002 and GSE65632). Differential expression analysis combined with curated mitochondrial and ferroptosis-related gene sets identified 69 mitochondrial ferroptosis-associated genes dysregulated in CRC. Functional enrichment analyses revealed significant involvement in oxidative phosphorylation, TCA cycle activity, iron-sulfur cluster assembly, and redox homeostasis. Protein-protein interaction and co-expression analyses identified HSPA9 and NFS1 as central hub genes associated with mitochondrial stress adaptation and ferroptosis resistance. Survival and stage-specific analyses further supported their prognostic significance in CRC progression. Structural and pathogenicity analyses of prioritized nsSNPs demonstrated that NFS1 variants may disrupt catalytic stability and ligand interactions, whereas HSPA9 variants predominantly affected conformational flexibility and protein-protein interaction interfaces. Collectively, these findings highlight mitochondrial ferroptosis dysregulation as a key mechanistic feature of colorectal cancer and identify HSPA9 and NFS1 as potential biomarkers and therapeutic targets. This study provides a comprehensive systems-level framework for understanding mitochondrial ferroptosis regulation and its translational relevance in CRC.