Background: Colorectal cancer (CRC) remains one of the most prevalent malignancies worldwide. Ferroptosis, a form of regulated cell death driven by lipid peroxidation, has emerged as a critical determinant of tumor progression, particularly through its interplay with dysregulated cholesterol metabolism. However, the role of the immune checkpoint molecule B7-H3 (CD276) in regulating ferroptosis in CRC remains poorly defined. This study aimed to characterize the expression profile of B7-H3 in CRC and elucidate its role and underlying mechanisms in ferroptosis regulation, with a focus on cholesterol metabolic signaling.

Methods: B7-H3 expression was assessed in normal colonic epithelial cells and CRC cell lines by Western blotting. Stable CD276 knockdown and overexpression models were established using lentiviral transduction. Ferroptosis sensitivity was evaluated using Cell Counting Kit-8 (CCK-8) assays following RSL3 treatment, along with measurements of malondialdehyde (MDA), intracellular Fe²⁺ levels, and ferroptosis-related gene expression. Cholesterol metabolism was assessed by quantifying total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels. Activation of the AKT–SREBP2 signaling axis was examined using Western blotting. Mechanistic validation was performed using the SREBP2 inhibitor betulin and the AKT inhibitor perifosine. An in vivo xenograft model under a high-cholesterol diet was further employed to evaluate the role of B7-H3 in tumor growth and regulation of ferroptosis.

Results: B7-H3 expression was significantly elevated in CRC cell lines (RKO, HCT116, SW480) compared with normal NCM460 cells (p < 0.001), with the highest expression observed in HCT116 cells. Stable CD276 knockdown and overexpression models were successfully established (p < 0.001). CD276 silencing markedly reduced cell viability, enhanced ferroptosis sensitivity, and increased levels of MDA, Fe²⁺, TC, and LDL-C (p < 0.05), accompanied by upregulation of PTGS2, FTL, and FTH mRNA (p < 0.001) and downregulation of glutathione peroxidase 4 (GPX4) (p < 0.05). In contrast, CD276 overexpression produced the opposite phenotype and significantly protected cells from RSL3-induced viability loss (p < 0.001). Mechanistically, CD276 knockdown increased nuclear SREBP2 (n-SREBP2) levels while reducing the p-AKT/AKT ratio (p < 0.001), whereas CD276 overexpression showed the opposite effect (p < 0.01). Betulin significantly reversed the elevation in TC, LDL-C, MDA, and Fe²⁺ induced by CD276 knockdown (p < 0.001), while perifosine restored n-SREBP2 expression that had been suppressed by CD276 overexpression (p < 0.01). In vivo, under a high-cholesterol diet, CD276 knockdown significantly reduced tumor volume and weight (p < 0.01), while increasing SREBP2 and COX-2 staining intensity, as well as MDA and Fe²⁺ levels in tumor tissues (p < 0.001).

Conclusions: B7-H3 is upregulated in CRC and promotes tumor progression by suppressing ferroptosis through modulation of the AKT–SREBP2 axis and cholesterol metabolism. Targeting B7-H3 may represent a promising therapeutic strategy to induce SREBP2-dependent ferroptosis in colorectal cancer.