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Chemotherapeutic resistance remains a major contributor to tumor recurrence and unfavorable clinical outcomes in colorectal cancer (CRC). Although cholesterol metabolic reprogramming has been implicated in tumorigenesis, metastasis, and drug resistance across multiple malignancies, its specific role in CRC chemoresistance requires systematic investigation. We analyzed RNA-seq data from GEO dataset GSE196900 to identify differentially expressed genes (|Log2FC| ≥ 1.5, adjusted p < 0.05). Functional enrichment analysis (GO/KEGG), protein-protein interaction (PPI) network construction, and gene set enrichment analysis (GSEA) were performed. Experimental validation using 5-fluorouracil (5-FU)-resistant CRC cell lines (HCT8/HCT15) included cholesterol/25-hydroxycholesterol (25-HC) treatments, assessed through CCK-8 proliferation assays, wound healing migration tests, quantitative real-time PCR (qRT-PCR), Western blotting, and cholesterol metabolite quantification. Integrative bioinformatics and experimental evidence revealed that 5-FU-resistant CRC cells demonstrate significant upregulation of cholesterol metabolism regulators, including lectin-type oxidized LDL Receptor 1 (LOX1), cholesterol 25-hydroxylase (CH25H), and Cytochrome P450 Family 7 Subfamily B Member 1 (CYP7B1). These cells exhibited impaired cholesterol efflux capacity and consequent intracellular cholesterol accumulation. Exogenous supplementation with cholesterol or 25-HC promoted proliferation, migration, and chemoresistance in both parental and resistant cells. Conversely, CH25H knockdown in resistant cells significantly attenuated malignant phenotypes and restored drug sensitivity. Our findings establish cholesterol metabolic dysregulation as a novel mechanistic contributor to 5-FU resistance in CRC, mediated through the LOX1-CH25H-CYP7B1 regulatory axis. These results propose that therapeutic targeting of cholesterol homeostasis may overcome chemoresistance and improve clinical management of refractory CRC patients.