Metformin Suppresses Both PD-L1 Expression in Cancer Cells and Cancer-Induced PD-1 Expression in Immune Cells to Promote Antitumor Immunity

Background: Metformin, a drug prescribed for patients with type 2 diabetes, has potential efficacy in enhancing antitumor immunity; however, the detailed underlying mechanisms remain to be elucidated.Therefore, we aimed to identify the inhibitory molecular mechanisms of metformin on programmed death ligand 1 (PD-L1) expression in cancer cells and programmed death 1 (PD-1) expression in immune cells.Methods: We employed a luciferase reporter assay, quantitative real-time PCR, immunoblotting analysis, immunoprecipitation and ubiquitylation assays, and a natural killer (NK) cell-mediated tumor cell cytotoxicity assay.A mouse xenograft tumor model was used to evaluate the effect of metformin on tumor growth, followed by flow-cytometric analysis using tumor-derived single-cell suspensions.Results: Metformin decreased AKT-mediated β-catenin S552 phosphorylation and subsequent β-catenin transactivation in an adenosine monophosphate-activated protein kinase (AMPK) activation-dependent manner, resulting in reduced CD274 (encoding PD-L1) transcription in cancer cells.Tumor-derived soluble factors enhanced PD-1 protein stability in NK and T cells via dissociation of PD-1 from ubiquitin E3 ligases and reducing PD-1 polyubiquitylation.Metformin inhibited the tumor-derived soluble factor-reduced binding of PD-1 to E3 ligases and PD-1 polyubiquitylation, resulting in PD-1 protein downregulation in an AMPK activation-dependent manner.These inhibitory effects of metformin on both PD-L1 and PD-1 expression ameliorated cancer-reduced cytotoxic activity of immune cells in vitro and decreased tumor immune evasion and growth in vivo.Conclusions: Metformin blocks both PD-L1 and PD-1 within the tumor microenvironment.This study provided a mechanistic insight into the efficacy of metformin in improving immunotherapy in human cancer.