Mechanisms for fibrate lipid-lowering drugs in enhancing bladder cancer immunotherapy by inhibiting CD276 expression.

OBJECTIVE: This study aimed to investigate the growth-inhibitory effects of fibrate lipid-lowering drugs on bladder cancer cells and their underlying mechanisms, with a focus on exploring how fenofibrate (FNF) exerts antitumor effects by regulating mitochondrial function, the AMPK/mTOR signaling pathway, and the immune regulatory molecule CD276. METHODS: The CCK-8 assay was used to determine the growth inhibition rates of FNF, bezafibrate (BZF), and clofibric acid (CLF) on MB49 cells and calculate their half-maximal inhibitory concentration (IC50). Mitochondrial respiratory chain complex activity assays, ADP/ATP ratio analysis, DCFH-DA fluorescent probe staining, and JC-1 staining were employed to evaluate the effects of FNF on mitochondrial function and oxidative stress. Western blot analysis, immunofluorescence (IF) staining, and treatment with the AMPK inhibitor Compound C were used to investigate the regulation of the AMPK/mTOR signaling pathway and CD276 expression by FNF. T cell cytotoxicity assays and cytokine detection were performed to validate the impact of FNF on T cell antitumor activity. CD276-knockdown stable MB49 cell lines and nude mouse xenograft models were constructed to assess the inhibitory effect of CD276 depletion. Hepatorenal biochemical indices (creatinine, blood urea nitrogen, alanine transaminase, aspartate transaminase) were measured to evaluate the safety of FNF in mice. Histological characteristics, CD276 expression, and T cell infiltration in tumor tissues were analyzed via H&E staining, immunohistochemistry (IHC), and IF staining. RESULTS: All three fibrate drugs inhibited MB49 cell growth in a concentration-dependent manner, with FNF exhibiting the strongest inhibitory activity (IC50 = 129.23 ± 9.38 µM). FNF suppressed mitochondrial complex I activity, leading to impaired ATP synthesis, reactive oxygen species (ROS) accumulation, and mitochondrial membrane damage. It activated the AMPK/mTOR pathway and downregulated CD276 expression in a concentration-dependent manner, an effect reversible by the AMPK inhibitor. TIMER database analysis revealed a positive correlation between CD276 expression and genes encoding mitochondrial complex I subunits. FNF treatment enhanced the secretion of IFN-γ and TNF-α by T cells and significantly improved T cell-mediated killing of bladder cancer cells. Knockdown of CD276 suppressed bladder cancer cell proliferation in vitro and tumor growth in vivo without affecting mouse body weight. FNF showed no significant hepatorenal toxicity and exhibited a higher tumor inhibition rate (64.1%) than anti-CD276 monoclonal antibody (44.7%) in vivo, accompanied by increased infiltration of CD3+, CD4+, and CD8 + T cells in tumor tissues. CONCLUSION: Fibrate drug FNF exerts antitumor effects by targeting the mitochondrial complex I-AMPK/mTOR-CD276 axis, inducing mitochondrial dysfunction, and downregulating the immunosuppressive molecule CD276 to activate T cell-mediated antitumor immunity. This study provides a potential strategy for drug repurposing and identifies a novel target for immunotherapeutic combination strategies in bladder cancer.