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

Abstract

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.

Keywords: Bladder cancer; CD276; Fenofibrate; Fibrate lipid-lowering drugs; Immunotherapy.

Fig. 1

Fenofibrate (FNF) Induced Mitochondrial Dysfunction and Oxidative Stress in MB49 Bladder Cancer Cells. A CCK-8 assay for MB49 cell viability after 24 h treatment with different fibrate concentrations. B Mitochondrial respiratory chain complexes I, II, IV activities after 50 µM fenofibrate treatment. C Intracellular ADP/ATP ratio analysis after 12 h treatment with fenofibrate (0, 50, 100 µM). D DCFH-DA-based ROS detection in MB49 cells after 12 h fenofibrate (0, 50, 100 µM) treatment; scale bar = 200 μm. E JC-1 staining for mitochondrial membrane potential (MMP) changes in MB49 cells after 8 h fenofibrate (0, 50, 100 µM) treatment; scale bar = 50 μm. ns, no significance; ***, p < 0.001

Fig. 2

Fenofibrate (FNF) inhibits CD276 expression by activating AMPK/mTOR signaling pathway. A-D Scatter plots depicting the correlation between CD276 and mitochondrial complex I-related genes (NDUFA1, NDUFA2, NDUFS1, NDUFS4) in bladder cancer tissues. E Confocal immunofluorescence microscopy images of CD276 in MB49 cells treated with 0, 50, or 100 µM fenofibrate (FNF) for 24 h; scale bar = 50 μm. F Western blot (WB) analysis of CD276, phosphorylated AMPK (p-AMPK), AMPK, phosphorylated mTOR (p-mTOR), and mTOR protein expression in MB49 cells treated with 0, 50, or 100 µM FNF for 24 h. G WB analysis of the aforementioned proteins in MB49 cells treated with 50 µM FNF in the presence or absence of the AMPK inhibitor (10 µM Compound C) for 24 h. H Cell viability of MB49 cells treated with 100 µM FNF in the presence or absence of the AMPK inhibitor (10 µM Compound C) for 24 h. ***, p < 0.001

Fig. 3

Fenofibrate (FNF) enhanced T cell-mediated antitumor immunity in bladder cancer. After T24 cells were pretreated with 0, 50, or 100 µM fenofibrate (FNF) for 24 h: A-B ELISA was used to detect the secretion levels of IFN-γ and TNF-α in the culture supernatants; C Pretreated T24 cells were co-cultured with activated T cells to evaluate the effect of FNF on T cell-mediated cytotoxicity against T24 cells; scale bar = 100 μm. ***, p < 0.001

Fig. 4

Knockdown of CD276 inhibited bladder cancer growth in vitro and in vivo. A RT-qPCR analysis of CD276 mRNA levels in MB49 cells transfected with CD276-shRNA or NC-shRNA B Western blot (WB) analysis of CD276 protein levels in MB49 cells transfected with CD276-shRNA or NC-shRNA. C CCK-8 assay for viability of MB49 cells after CD276 knockdown. (C-G) Xenograft model: Nude mice (n = 5) were injected subcutaneously with shCD276- or NC-transfected MB49 cells and monitored for 14 days. D Representative images of excised tumors. E Tumor growth curves. F Tumor weights on day 14. G Body weight changes. H H&E and immunohistochemistry of tumor sections; scale bar = 100 μm. ***, p < 0.001

Fig. 5

In vivo antitumor effect of fenofibrate (FNF) and its modulation of immune microenvironment in bladder cancer. A Analysis of renal/hepatic function markers (CR, BUN, ALT, AST) in mice after 48 h PEG/FNF treatment (n = 3). B Tumor growth curves of MB49 xenografts monitored over 14 days (n = 5). C Tumor weights on day 14 (n = 5). D Representative images of excised tumors (n = 5). E Body weight changes in tumor-bearing mice (n = 5). F Representative H&E and immunohistochemistry (IHC) images of tumor sections; scale bar = 100 μm. G Immunofluorescence staining for CD3+/CD4+/CD8 + T cell infiltration in tumors; scale bar = 100 μm. *, p < 0.05; ***, p < 0.001

Fig. 6

The mechanism model of fenofibrate (FNF) mediating T cell activation by inhibiting the expression of CD276