Mitochondrial reactive oxygen species trigger metformin-dependent antitumor immunity via activation of Nrf2/mTORC1/p62 axis in tumor-infiltrating CD8T lymphocytes

Abstract

BackgroundMetformin (Met) is the first-line treatment for type 2 diabetes mellitus and plays an effective role in treating various diseases, such as cardiovascular disease, neurodegenerative disease, cancer, and aging. However, the underlying mechanism of Met-dependent antitumor immunity remains to be elucidated.MethodsMitoTEMPO, a scavenger of mitochondrial superoxide, abolished the antitumor effect of Met, but not antiprogrammed cell death (PD-1) antibody (Ab) treatment. Consequently, we studied the mechanism of the Met-induced antitumor effect. Expressions of glucose transporter (Glut)-1, mitochondrial reactive oxygen species (mtROS), interferon (IFN)-γ, Ki67, autophagy markers, activation markers for NF-E2-related factor 2 (Nrf2), and mammalian target of rapamaycin complex 1 (mTORC1) in CD8+ tumor-infiltrating T lymphocytes (CD8TILs) were examined by flow cytometry analysis. In addition, conditional knockout mice for Nrf2 and p62 were used to detect these markers, together with the monitoring of in vivo tumor growth. RNA sequencing was performed for CD8TILs and tumor cells. Melanoma cells containing an IFN-γ receptor (IFNγR) cytoplasmic domain deletion mutant was overexpressed and used for characterization of the metabolic profile of those tumor cells using a Seahorse Flux Analyzer.ResultsMet administration elevates mtROS and cell surface Glut-1, resulting in the production of IFN-γ in CD8TILs. mtROS activates Nrf2 in a glycolysis-dependent manner, inducing activation of autophagy, glutaminolysis, mTORC1, and p62/SQSTM1. mTORC1-dependent phosphorylation of p62 at serine 351 (p-p62(S351)) is also involved in activation of Nrf2. Conditional deletion of Nrf2 in CD8TILs abrogates mTORC1 activation and antitumor immunity by Met. In synergy with the effect of anti-PD-1 Ab, Met boosts CD8TIL proliferation and IFN-γ secretion, resulting in decreased glycolysis and oxidative phosphorylation in tumor cells. Consequently, Glut-1 is elevated in CD8TILs, together with the expansion of activated dendritic cells. Moreover, tumor cells lacking in IFNγR signaling abolish IFN-γ production and proliferation of CD8TILs.ConclusionsWe found that Met stimulates production of mtROS, which triggers Glut-1 elevation and Nrf2 activation in CD8TILs. Nrf2 activates mTORC1, whereas mTORC1 activates Nrf2 in a p-p62(S351)-dependent manner, thus creating a feedback loop that ensures CD8TILs’ proliferation. In combination with anti-PD-1 Ab, Met stimulates robust proliferation of CD8TILs and IFN-γ secretion, resulting in an IFN-γ-dependent reprogramming of the tumor microenvironment.

Two distinct phenotypes of immunologically hot gastric cancer subtypes

“…Thus, active metabolism in Hot 1 tumors might be one of the reasons for T cell dysfunction via increased competition with effector T cells [ 13 ]. Therefore, targeting and controlling activated glycolysis, lipid metabolism, oxidative phosphorylation and mitochondrial function in Hot 1 tumors would be expected to improve the antitumor T cell response, and hence prognosis [ [14] , [15] , [16] ].…”

Improving Cancer Immunotherapy: Exploring and Targeting Metabolism in Hypoxia Microenvironment

“…However, metformin may inhibit tumor cell proliferation through immune-mediated mechanisms because metformin directly improves effector T-cell function in vivo . Combining metformin with an anti-PD-1 treatment directly activates CD8 + T cells and boosts IFN-γ secretion, leading to the decreased glycolysis and OXPHOS of tumor cells compared with anti-PD-1 treatment alone ( 91 ). It is found that metformin-treated animals reduce the PD-L1 stability on cancer cells, then increase the activity of cytotoxic T lymphocytes ( 92 ).…”

Metabolic Reprogramming in the Tumor Microenvironment With Immunocytes and Immune Checkpoints

“…Although glycolysis predominates in the metabolic reprogramming of cancer cells, OXPHOS is also enhanced in tumor cells, and some compounds targeting OXPHOS to suppress tumors have been studied ( 178 , 179 ). An example is metformin, which could decrease cancer cell proliferation by decreasing the NAD+/NADH ratio and inhibiting aspartate production ( 180 ), and stimulates the proliferation of CD8+ TILs with the production of mitochondrial ROS in an NF-E2-related factor 2 (Nrf2)/mTORC1/p62 pathway ( 181 ). It was also reported that metformin could further improve intratumoral T-cell function and tumor clearance when combined with ICIs ( 62 , 181 , 182 ).…”

“…An example is metformin, which could decrease cancer cell proliferation by decreasing the NAD+/NADH ratio and inhibiting aspartate production ( 180 ), and stimulates the proliferation of CD8+ TILs with the production of mitochondrial ROS in an NF-E2-related factor 2 (Nrf2)/mTORC1/p62 pathway ( 181 ). It was also reported that metformin could further improve intratumoral T-cell function and tumor clearance when combined with ICIs ( 62 , 181 , 182 ). Phase II clinical trial showed metformin therapy was associated with better-than-expected OS on non-diabetic patients with advanced-stage ovarian cancer ( 183 ).…”