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.

, Colin Purdie, Susan Bray, et al.. Evidence for biological effects of metformin in operable breast cancer: a pre-operative, window-of-opportunity, randomized trial. Breast Cancer Research and Treatment, Springer Verlag, 2011, 128 (3) AbstractMetformin may reduce the incidence of breast cancer and enhance response to neoadjuvant chemotherapy in diabetic women. This trial examined the effects of metformin on Ki67 and gene expression in primary breast cancer.Non-diabetic women with operable invasive breast cancer received pre-operative metformin. A pilot cohort of 8 patients had core biopsy of the cancer at presentation, a week later (without treatment; internal control), then following metformin 500mg o.d. for one week increased to 1g b.d. for a further week continued to surgery. A further 47 patients had core biopsy at diagnosis, were randomized to metformin (the same dose regimen) or no drug, and 2 weeks later had core biopsy at surgery. Ki67 immunohistochemistry, transcriptome analysis on formalin fixed paraffin embedded cores and serum insulin determination were performed blinded to treatment.7 patients (7/32, 21.9%) receiving metformin withdrew due to gastrointestinal upset. The mean percentage of cells staining for Ki67 fell significantly following metformin treatment in both the pilot cohort (p=0.041, paired t-test) and in the metformin arm (p=0.027, Wilcoxon rank test) but was unchanged in the internal control or metformin control arms. Messenger RNA expression was significantly down-regulated by metformin for PDE3B (phosphodiesterase 3B, cGMP-inhibited; a critical regulator of cAMP levels which affect activation of AMP-activated protein kinase, AMPK), confirmed by immunohistochemistry, SSR3, TP53 and CCDC14. By Ingenuity Pathway Analysis, the Tumour Necrosis Factor Receptor 1 (TNFR1) signaling pathway was most affected by metformin: TGFB, MEKK were up-regulated and cdc42 down-regulated; mTOR and AMPK pathways were also affected. Gene Set Analysis additionally revealed that p53, BRCA1 and cell cycle pathways also had reduced expressed following metformin. Mean serum insulin remained stable in patients receiving metformin but rose in control patients.This trial presents biomarker evidence for anti-proliferative effects of metformin in women with breast cancer and provides support for therapeutic trials of metformin.

BackgroundAMP-activated protein kinase (AMPK) acts as a cellular fuel gauge that responds to energy stress by suppressing cell growth and biosynthetic processes, thus ensuring that energy-consuming processes proceed only if there are sufficient metabolic resources. Malfunction of the AMPK pathway may allow cancer cells to undergo uncontrolled proliferation irrespective of their molecular energy levels. The aim of this study was to examine the state of AMPK phosphorylation histologically in primary breast cancer in relation to clinical and pathological parameters.MethodsImmunohistochemistry was performed using antibodies to phospho-AMPK (pAMPK), phospho-Acetyl Co-A Carboxylase (pACC) an established target for AMPK, HER2, ERα, and Ki67 on Tissue Micro-Array (TMA) slides of two cohorts of 117 and 237 primary breast cancers. The quick score method was used for scoring and patterns of protein expression were compared with clinical and pathological data, including a minimum 5 years follow up.ResultsReduced signal, compared with the strong expression in normal breast epithelium, using a pAMPK antibody was demonstrated in 101/113 (89.4%) and 217/236 (91.9%) of two cohorts of patients. pACC was significantly associated with pAMPK expression (p = 0.007 & p = 0.014 respectively). For both cohorts, reduced pAMPK signal was significantly associated with higher histological grade (p = 0.010 & p = 0.021 respectively) and axillary node metastasis (p = 0.061 & p = 0.039 respectively). No significant association was found between pAMPK and any of HER2, ERα, or Ki67 expression, disease-free survival or overall survival.ConclusionThis study extends in vitro evidence through immunohistochemistry to confirm that AMPK is dysfunctional in primary breast cancer. Reduced signalling via the AMPK pathway, and the inverse relationship with histological grade and axillary node metastasis, suggests that AMPK re-activation could have therapeutic potential in breast cancer.

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