Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1

Abstract

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.

Keywords: ER accumulation; ERAD; PD-L1; cancer immunotherapy; glycosylation; immune checkpoint blockade; metformin.

Figure 1.. Metformin increases CTL activity through the AMPK/PD-L1 axis.

(A) 4T1_luc2 cells were injected into mice (n = 6 mice per group) on day 0, and metformin administered as indicated. (B) Tumor volume was measured on the indicated time points. Data represent mean ± SD. (C) Quantification (top) of bioluminescence imaging. Bottom, endpoint images shown (D) Immunostaining of cleaved caspase 3 in the 4T1 tumor mass. Hoechst: nuclear counter staining (pseudo-color: green). Scale bar, 200 μm (inset, 50 μm). (E) Immunostaining of CD8 (CTL marker) and granzyme B (activity of T cell) in the 4T1 tumor mass. Scale bar, 200 μm (inset, 50 μm). For (D) and (E), data represent mean ± SD. n = 12; 3 tissue slides per tumor, 4 mice per group. Unit = 466,038 μm² in (D) and 262,144 μm² in (E). (F) T cell-mediated cancer cell killing assay. MDA-MB-231 WT and AMPKα KO cells co-cultured with activated T cell for 48 hr with or without metformin (5 mM) were subjected to crystal violet staining. MDA-MB-231 to T-cell ratio, 1:5. Data represent mean ± SD. n = 3. (G) Correlation analysis between PD-L1 and AMPKα T172-p expression in 14 breast cancer cell lines. (H) BT-549 and MDA-MB-231 were treated with increasing concentrations of metformin (1.25 to 5 mM) for 24 hr. (I) MDA-MB-231 parental, AMPKα WT and KO cells were treated with metformin (5 mM) for 24 hr. Compound C (Com C) AMPK inhibitor was pretreated 6 hr before metformin treatment. (J) Membrane PD-L1 expression by flow cytometric analysis after metformin (5 mM) treatment for 24 hr. (K) Right, quantitation of binding of green fluorescent-labeled PD-1/Fc on MDA-MB-231 treated with or without metformin for 24 hr. Data represent mean ± SD. n = 3. Left, representative images shown. Scale bar, 100 μm. (L) MDA-MB-231 WT and PD-L1 KO cells co-cultured with activated T cells for 48 hr with or without metformin (5 mM) were subjected to crystal violet staining. MDA-MB-231 to T- cell ratio, 1:3. *P, 0.01~0.05, **P, 0.001~0.01, ^#P, < 0.001, and NS, not significant, Student’s t test.

Figure 2.. AMPK activated by metformin directly phosphorylates serine 195 of PD-L1.

(A) BT-549 cells were treated with metformin (5 mM) for the indicated time. Detection of endogenous AMPKα and PD-L1 binding (red dots) by Duolink II assay. Three different positions were randomly selected at each point, and the number of red dots were divided by the number of nuclei. Data represent mean ± SD. n = 3. *P, 0.01~0.05, **P, 0.001~0.01, and ^#P, < 0.001, Student’s t test. Scale bar, 20 μm. Right, MDA-MB-231 WT, PD-L1 KO and AMPKα KO cells were used as negative controls. Scale bar, 25 μm. (B) MDA-MB-231 cells were cultured for 6 hr with or without metformin (5 mM) and MG132 (10 μM). Endogenous PD-L1 and AMPKα were immunoprecipitated and their binding was analyzed with immunoblotting. (C) In vitro kinase activity of AMPK toward PD-L1 with ³²P-labeled ATP. (D) Kinetics of PD-L1 phosphorylation by AMPK. Acetyl-CoA carboxylase (ACC) was used as positive control. Km and Vmax were calculated using the Michaelis-Menten equation. (E) In vitro phosphorylation assay and phospho-tag gel shifting assay. W, PD-L1/WT. A, PD-L1/S195A. (F) PD-L1/S195 phosphorylation was examined using anti-PDL1/S195-p antibody at different time points after metformin (5 mM) treatment. (G) Western blot analysis of MDA-MB-231 WT and AMPKα KO cells after metformin treatment (5 mM) for 8 hr. Endogenous PD-L1 purified by IP was subjected to immunoblotting with PDL1 S195-p antibody after PNGase F reaction. (H) PD-L1 and AMPK subcellular localization of MDA-MB-231 WT and AMPKα KO cells. (I) Trypsin digestion of ER fractions with or without permeabilization. (J, K) BT-549 cells were treated with metformin (5 mM) for 3 hr. (J) BT-549 cells were subjected to Duolink II assay combined with immunofluoresence staining using markers for ER (HSP90B1), Golgi (TNG46), and nuclei (Hoechst). Scale bar, 20 μm (inset, 10 μm). (K) Duolink assay with antibodies specific for ECD (Ab205921 and 86744S) and ICD (13684S and GTX104763) of PD-L1. Scale bar, 50 μm (inset, 25 μm). Red dots: AMPK-PD-L1 binding in (J) and (K).

Figure 3.. Phosphorylation of PD-L1 S195 induces its abnormal glycosylation and blocks its ER-to-Golgi translocation.

(A) WT, S195A S195D, S195E, and 4NQ PD-L1 stable cells were treated with or without tunicamycin (5 μg/ml) for 24 hr. (B) Schematic diagram of PD-L1 showing the position of S195 and the 4 N-glycosylation sites. (C) Comparison of the glycan structure between WT and S195E PD-L1 by IP/Mass analysis. (D) BT-549 and MDA-MB-231 stable cells expressing WT, S195E, or S195A PD-L1 were treated with metformin (5 mM) for 24 hr. (E) Expression pattern of PD-L1 in MDA-MB-231 WT, S195A and S195E PD-L1 stable cells by IF staining. (F) MDA-MB-231 stable cells co-stained with antibodies against PD-L1 and Golgi markers (GM130: cis, Giantin: medial, TNG46: trans). (G) IF staining with antibodies against PD-L1 and ER marker (HSP90B1) (H) Flow cytometric analysis of membrane PD-L1 in MDA-MB-231 WT, S195A and S195E PD-L1 stable cells. Data represent mean ± SD. n = 3. (I) Binding of green fluorescent-labeled PD-1/Fc to MDA-MB-231 WT, S195A and S195E PD-L1 stable cells was quantified. Data represent mean ± SD. n = 3. (J) PD-L1 localization in MDA-MB-231 expressing WT, S195E or NXT motif mutant (glycosylation site mutant) PD-L1 by IF staining. For experiments shown in (E), (F), (G) and (J), MG132 (10 μM) was added 6 hr prior to fixation to prevent degradation of PD-L1. Hoechst: nuclear counter staining. Scale bar, 20 μm (inset, 10 μm). *P, 0.01~0.05, **P, 0.001~0.01, and ^#P, < 0.001, Student’s t test. NS, not significant.

Figure 4.. Phosphorylation of S195 induces ER-associated degradation of PD-L1

(A) IP-MS analysis showing candidates with increased binding to PD-L1 S195E compared to PD-L1 S195A. ERAD components are shown in red. (B) IPA based on the mass data from panel (A) showing unfolding protein response and ubiquitination pathways closely related to ERAD were activated (C) MDA-MB-231 stable cells were treated with 50 μM cycloheximide (CHX) for the indicated time. The band intensity was quantified by Image J analysis. Data represent mean ± SD. n = 3. **P, 0.001~0.01, and NS, not significant, Student’s t test. (D) MDA-MB-231 stable cells were treated with proteasome inhibitor MG132 (10 μM) and ERAD inhibitor eeyarestatins (Eer I, 20 μM) for the indicated time. (E, F) MDA-MB-231 cells were treated with or without metformin (5 mM) for 24 hr and MG132 (10 μM) for 6 hr. Endogenous PD-L1 (E) or Flag-tagged WT, S195A, or S195E PD-L1 (F) was pulled down by the PD-L1 antibody (E) or Flag M2 magnetic bead (F), respectively, followed by Western blotting to detect ERAD components (SEL1L, HRD1, ERLEC1, and OS9). (G) MDA-MB-231 cells expressing exogenous HA-ubiquitin were cultured with or without metformin (5 mM) for 24 hr followed by MG132 (10 μM) for 6 hr. Ubiquitination of endogenous PD-L1 was examined by HA immunoblotting after IP with antibody against PD-L1. (H) Ubiquitination of WT, S195A, and S195E PD-L1 was examined by HA immunoblotting after IP with Flag M2 magnetic bead. (I) Control or HRD1 siRNA was transfected into MDA-MB-231 cells expressing exogenous HA-ubiquitin. Ubiquitination of endogenous PD-L1 in each transfectant was examined by HA immunoblotting after IP with PDL1 antibody. (J) Control or HRD1 siRNA was transfected into MDA-MB-231 WT and S195E PD-L1 stable cells expressing exogenous HA-ubiquitin. Ubiquitination of WT and S195E PD-L1 was examined by HA immunoblotting after IP with Flag M2 magnetic bead. (K) siRNA targeting SEL1L, HRD1, OS9 or ERLEC1 was transfected into MDA-MB-231 (L) Each siRNA for ERAD components was transfected into MDA-MB-231 S195E PD-L1 stable cells followed by immunoblotting with the indicated antibodies. M, mock. W, WT PD-L1. A, S195A PD-L1. E, S195E PD-L1.

Figure 5.. The reduction of PD-L1 by metformin-activated AMPK is physiologically significant and clinically relevant

(A) 4T1 WT or S194E mPD-L1 stable cells (5 × 10⁴) were injected into BALB/c mice (n = 7 mice per group) (B) 4T1 WT or S194A mPD-L1 stable cells (5 × 10⁴) were injected into BALB/c mice (n = 7 mice per group) on day 0, and metformin (200 mg/kg) administered by i.p. injection starting at day 4 for 18 days. Tumor volume was measured on the indicated time points. Data represent mean ± SD. (C) The relative tumor volume (%) at the end point (day 22) of panel (B). (D) Left, representative images of immunostaining of CD8 and GB in the 4T1 tumor mass. Hoechst: counter staining. Right, CD8 and GB were quantified using Image J. Data represent mean ± SD. n = 12. Three tissue slides per tumor, 4 mice per group. Unit = 262,144 μm². (E) AMPKα T172-p and PD-L1 levels before and after metformintreatment in tumor tissues of breast cancer patients. −, score 0; +, score 1; ++, score 2; +++, score 3; ↑, up; ↓, down; X, no change. (F) Representative IHC images of the cases shown in panel (E). Scale bar, 50 μm. (G) Plot of IHC scores of AMPKα T172-p and PD-L1 expression levels in responders before and after metformin treatment. n = 7 (H) PBS and metformin were administered to a pair of SCID mice with same patient tumor for 7 days. Immunostaining of AMPKα T172-p and PD-L1 in the PDX tumor mass. Hoechst:counter staining. Scale bar, 200 μm. (I) Western blotting of lysates from PDX tumors in (H) with the indicated antibodies. *P, 0.01~0.05, **P, 0.001~0.01, and ^#P, < 0.001, Student’s t test.

Figure 6.. The combination of metformin and CTLA4 blockade effectively suppresses tumor growth in vivo

(A) Tumor growth of 4T1 cells in immunocompetent BALB/c mice treated with control, metformin, anti-CTLA4, or the combination. Data represent mean ± SD. n = 8 mice per group. (B) Quantification (top) of bioluminescence imaging (endpoint images shown). The maximum and minimum values were excluded for more accurate analysis. Data represent mean ± SD. n = 6 mice per group. (C) Survival of mice bearing 4T1-derived tumor following treatment with control, metformin, anti-CTLA4, or the combination. n = 8 mice per group, log-rank test. (D) Top, immunostaining of PD-L1, CD8, and GB in the 4T1 tumor mass. Hoechst: nuclear counter staining. Scale bar, 50 μm (inset, 200 μm). Bottom, quantification of PD-L1, CD8, and GB using Image J. Data represent mean ± SD. n = 12 (4 mice per group). Three tissue slides per tumor. Unit = 182,047 μm². (E) Cells were extracted from 4T1-tumors by Percoll density gradient centrifugation. PD-L1 levels on extracted cells were evaluated by FACS. Cells were gated based on the FSC (forward scattered light) vs. SSC (side-scattered light). Membrane PD-L1 is shown as median fluorescence intensity (MFI). Data represent mean ± SD. n = 5 per group. (F) FACS analysis of CD45⁺CD3⁺CD8⁺ CTL activity as indicated by the activity intracellular IFN-γ in the leukocyte fractions from Percoll density gradient separation. Detailed gating information is shown in Figure S6B. Data represent mean ± SD. n = 7 per group. Two or three tumors were collected for one sample for the combination group. (G) Tumor growth of B16F10 melanoma cells in immunocompetent C57BL/6J mice treated with control, metformin, anti-CTLA4, or the combination. Data represent mean ± SD. n = 6 mice per group. (H) Tumor growth of CT26 colorectal cancer cells in immunocompetent BALB/C mice treated with control, metformin, anti-CTLA4, or the combination. Data represent mean ± SD. n = 6 mice per group. (I) Survival rate of mice bearing CT26-derived tumor following treatment with control, metformin, anti-CTLA4, or the combination. n = 10 mice per group, log-rank test. *P, 0.01~0.05, **P, 0.001~0.01, and ^#P, < 0.001, Student’s t test.