Ann Lab Med.  2024 Sep;44(5):426-436. 10.3343/alm.2023.0443.

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

• Park SH
• Lee J
• Yun HJ
• Kim SH
• Lee JH

• KMID: 2559149
• DOI: http://doi.org/10.3343/alm.2023.0443

Abstract

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.

Keyword

E3 ligase; Immunotherapy; Metformin; PD-L1; PD-1; Tumor immune evasion

Figure

• 

  Fig. 1 Metformin inhibits both PD-L1 expression on cancer cells and PD-1 expression on infiltrating immune cells in the TME. (A) Schematic diagram of the experimental procedures. B16F10 murine melanoma cells were subcutaneously injected into C57BL/6 mice. From day 3 after tumor implantation, the mice received an intraperitoneal injection of metformin or PBS daily. On day 20, after tumor implantation, the mice were sacrificed. (B) Representative images of B16F10 tumors. Scale bar, 1 cm. (C) Growth curve of subcutaneous B16F10 tumors derived from metformin-treated (red line; N=5) and PBS-treated (blue line; N=5) mice. (D) Tumor weight of subcutaneous B16F10 tumors on day 20 derived from metformin-treated (red line; N=5) and PBS-treated (blue line; N=5) mice. (E) Cell-surface analysis of PD-L1 protein expression in B16F10 single-cell suspensions derived from B16F10 tumors using flow cytometry. A representative histogram (left panel) and graph (right panel) are shown. (F) Cell-surface analysis of PD-1 protein expression in NK or CD8+ T single-cell suspensions derived from B16F10 tumors using flow cytometry. A representative histogram (left panel) and graph (right panel) are shown. *P<0.05; **P<0.01; ***P<0.001, Student’s t-test. Abbreviations: PD-L1, programmed death ligand 1; PD-1, programmed death 1; TME, tumor microenvironment; PBS, phosphate-buffered saline; NK, natural killer.

• 

  Fig. 2 Metformin inhibits transcriptional expression of PD-L1 in an AMPK-downregulated AKT/β-catenin signaling-dependent manner. (A, B) The indicated cells were treated or not with metformin for 12 hrs. Then, mRNA and protein expression levels of PD-L1 were determined using quantitative real-time PCR (A) and immunoblotting (B) with the indicated primers and antibodies, respectively. (C) A431 cells were treated or not with metformin for 12 hrs and subjected to immunoblotting analysis using the indicated antibodies. (D) A431 cells were transfected with TOP-FLASH or FOP-FLASH, followed by metformin (10 mM) treatment for 12 hrs. Luciferase activity was measured. Relative luciferase activity levels were normalized to levels in untreated cells and to Renilla luciferase activity levels. (E–G) A431 cells stably expressing control vector or CA-β-catenin were transfected with TOP-FLASH or FOP-FLASH, followed by metformin (10 mM) treatment for 12 hrs. Luciferase activity was measured. Relative luciferase activity levels were normalized to levels in untreated cells and to Renilla luciferase activity levels (E). mRNA and protein expression levels of PD-L1 were determined using quantitative real-time PCR (F) and immunoblotting (G) with the indicated primers and antibodies, respectively. (H–J) A431 cells stably expressing the control vector or HA-myr-AKT1 were transfected with TOP-FLASH or FOP-FLASH, followed by metformin (10 mM) treatment for 12 hrs. Luciferase activity was measured. Relative levels of luciferase activity were normalized to levels in untreated cells and to Renilla luciferase activity levels (H). The mRNA and protein expression levels of PD-L1 were determined using quantitative real-time PCR (I) and immunoblotting (J) with the indicated primers and antibodies, respectively. (K, L) A431 cells were pretreated or not pretreated with compound C (5 μM) for 1 hr and then treated or not pretreated with metformin (10 mM) for 12 hrs. Protein and mRNA expression levels of PD-L1 were determined using immunoblotting (K and bottom panel of L) and quantitative real-time PCR (L, top panel) with the indicated antibodies and primers, respectively. Data are presented as mean±SD of three independent experiments (A, D, E, F, H, I, L). *P<0.05; **P<0.01; ***P<0.001, Student’s t-test or one-way ANOVA followed by Tukey post-hoc tests. Abbreviations: PD-L1, programmed death ligand 1; AMPK, adenosine monophosphate-activated protein kinase; PCR, polymerase chain reaction; CA, constitutively active; HA, hemagglutinin.

• 

  Fig. 3 Cancer cells enhance PD-1 protein stability in NK and T cells. (A) NK-92 and Jurkat cells were treated or not treated with CM (50%) for the indicated periods. Protein and mRNA expression levels of PD-1 were determined using immunoblotting (upper panel) and quantitative real-time PCR (bottom panel) with the indicated antibodies and primers, respectively. Data are presented as mean±SD of three independent experiments. (B) NK-92 and Jurkat cells were pretreated with DMSO or CHX (100 μg/mL) for 1 hr and then treated with CM (50%) for 6 hrs. Immunoblotting analyses were performed using the indicated antibodies. (C) NK-92 and Jurkat cells were pretreated with CM (50%) for 12 hrs and then treated with CHX (100 μg/mL) for the indicated periods. Immunoblotting analyses were performed using the indicated antibodies (left panel). Quantification of PD-1 levels relative to tubulin levels is shown (right panel). Band intensities were quantified using the ImageJ software. Data are presented as mean±SD of three independent experiments. **P<0.01, Student’s t-test. (D) NK-92 and Jurkat cells were treated or not treated with CM (50%) for 12 hrs. Immunoprecipitation analyses were performed using an anti-PD-1 antibody, followed by immunoblotting analyses using the indicated antibodies. (E) NK-92 and Jurkat cells were treated with CM (50%) for the indicated periods. The cells were incubated with MG132 (10 μM) for 6 hrs before they were harvested using a guanidine-HCl-containing buffer. Immunoprecipitation was performed using an anti-PD-1 antibody, followed by immunoblotting analyses using the indicated antibodies. Abbreviations: PD-1, programmed death 1; NK, natural killer; CM, conditioned medium; DMSO, dimethyl sulfoxide; CHX, cycloheximide.

• 

  Fig. 4 Metformin inhibits cancer-enhanced PD-1 expression via AMPK-dependent deregulation of PD-1 protein stability. (A) NK-92 and Jurkat cells were treated or not with CM (50%) or metformin (10 mM) for 12 hrs. Immunoblotting analyses were performed using the indicated antibodies. (B) NK-92 and Jurkat cells were pretreated with DMSO or MG132 (10 μM) for 1 hr and then treated or not with CM (50%) or metformin (10 mM) for 12 hrs. Immunoblotting analyses were performed using the indicated antibodies. (C) NK-92 and Jurkat cells were pretreated or not with CM (50%) or metformin (10 mM) for 12 hrs and then treated with CHX (100 μg/mL) for the indicated periods. Immunoblotting analyses were performed using the indicated antibodies (left panel). Quantification of PD-1 levels relative to tubulin levels is shown (right panel). Band intensity was quantified using the ImageJ software. Data are presented as mean±SD of three independent experiments. *P<0.05 and **P<0.01, Student’s t-test. (D) NK-92 and Jurkat cells were treated or not with CM (50%) and metformin (10 mM) for 12 hrs. Immunoprecipitation was performed using an anti-PD-1 antibody, followed by immunoblotting analyses using the indicated antibodies. (E) NK-92 and Jurkat cells were treated or not with CM (50%) or metformin (10 mM) for 12 hrs. The cells were incubated with MG132 (10 μM) for 6 hrs before they were harvested using a guanidine-HCl-containing buffer. Immunoprecipitation was performed using an anti-PD-1 antibody, followed by immunoblotting analyses using the indicated antibodies. (F) 293T cells stably expressing V5-PD-1 and Flag-vector or Flag-CA-AMPK were treated or not with CM (50%) for 12 hrs. The cells were incubated with MG132 (10 μM) for 6 hrs before they were harvested using a guanidine-HCl-containing buffer. Immunoprecipitation was performed using an anti-V5 antibody, followed by immunoblotting analyses using the indicated antibodies. (G) AMPK WT and AMPK DKO MEFs were transfected or not with V5-PD-1. These cells were incubated with MG132 (10 μM) for 6 hrs before they were harvested using a guanidine-HCl-containing buffer. Immunoprecipitation was performed using an anti-V5 antibody, followed by immunoblotting analyses using the indicated antibodies. (H) NK-92 and Jurkat cells were pretreated with DMSO or compound C (5 μM) for 1 hr and then treated or not with CM (50%) and metformin (10 mM) for 12 hrs. Immunoblotting analyses were performed using the indicated antibodies. (I) 293T cells stably expressing V5-PD-1 were pretreated with DMSO or compound C (5 μM) for 1 hr and then treated or not with CM (50%) or metformin (10 mM) for 12 hrs. The cells were incubated with MG132 (10 μM) for 6 hrs before they were harvested using a guanidine-HCl-containing buffer. Immunoprecipitation was performed using an anti-V5 antibody, followed by immunoblotting analyses using the indicated antibodies. Abbreviations: PD-1, programmed death 1; AMPK, adenosine monophosphate-activated protein kinase; CM, conditioned medium; DMSO, dimethyl sulfoxide; CHX, cycloheximide; CA, constitutively active; WT, wild-type; DKO, double knockout.

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  Fig. 5 Schematic representation of the proposed mechanisms.

Reference

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