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.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance mediated by T790M-independent mechanisms remains a major challenge in the treatment of non-small cell lung cancer (NSCLC). We identified a targetable mechanism of EGFR inhibitor resistance whereby stress hormones activate beta2-adrenergic receptors (β2-AR) on NSCLC cells, which cooperatively signal with mutant EGFR, resulting in the inactivation of the tumor suppressor, liver kinase B1 (LKB1), and subsequently induce IL-6 expression. We show that stress and β2-AR activation promote tumor growth and EGFR inhibitor resistance, which can be abrogated with beta blockers or IL-6 inhibition. IL-6 was associated with worse outcome in EGFR TKI-treated NSCLC patients, and beta blocker use was associated with lower IL-6 concentrations and improved benefit from EGFR inhibitors. These findings provide evidence that chronic stress hormones promote EGFR TKI resistance via β2-AR signaling by an LKB1/CREB/IL-6-dependent mechanism and suggest that combinations of beta blockers with EGFR TKIs merit further investigation as a strategy to abrogate resistance.

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Evading host immune surveillance is one of the hallmarks of cancer. Immune checkpoint therapy, which aims to eliminate cancer progression by reprogramming the antitumor immune response, currently occupies a solid position in the rapidly expanding arsenal of cancer therapy. As most immune checkpoints are membrane glycoproteins, mounting attention is drawn to asking how protein glycosylation affects immune function. The answers to this fundamental question will stimulate the rational development of future cancer diagnostics and therapeutic strategies.

Triple-negative breast cancer (TNBC) lacks a well-defined molecular target and is associated with poorer outcomes compared to other breast cancer subtypes. Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade therapy shows a 10% to 20% response rate in TNBC patients. Our previous studies show that PD-L1 proteins are heavily glycosylated in TNBC, and the glycosylation plays an important role in the PD-L1 protein's stability and immunosuppressive function.However, a strategy for PD-L1 deglycosylation in TNBC is poorly defined. Here we found that a saccharide analog, 2-deoxy-D-glucose (2-DG), inhibits glycosylation of PD-L1 and its immunosuppressive function by combining with EGFR inhibitor, gefitinib.Interestingly, 2-DG/gefitinib-induced deglycosylation of PD-L1 decreased the expression level of PD-L1 protein as well as its binding with PD-1. However, there was no significant decrease in 4-1BB expression and its binding with 4-1BBL by 2-DG/gefitinib. Furthermore, we demonstrated that the combination treatment of 2-DG/gefitinib and 4-1BB antibody enhances antitumor immunity in TNBC syngeneic murine models.Together, our results suggest a new immunotherapeutic strategy to enhance antitumor immunity by PD-L1 deglycosylation and 4-1BB stimulation in TNBC. K E Y W O R D S4-1BB, glycosylation, PD-L1, triple-negative breast cancer