Work

Gαi3 acts as a metabolic checkpoint orchestrating TAMs immunosuppression and lipid metabolism in glucose-deprived hepatocellular carcinoma

Year: 2026

Type: article

Abstract: Background Tumor-associated macrophages (TAMs) are abundant in the hepatocellular carcinoma (HCC) tumor microenvironment (TME) and undergo metabolic reprogramming to support an immunosuppressive phenotype. While G protein-coupled receptors are known sensors of environmental cues, the specific role of the inhibitory G protein subunit alpha i3 (Gαi3) in linking metabolic stress to TAMs-mediated immunosuppression remains undefined.Methods Clinical associations were evaluated by multiplex immunohistochemistry, Kaplan-Meier analysis, and multivariable Cox regression. Myeloid-specific Gnai3-deficient mice were studied in DEN/CCl4 or DEN/HFD-induced and orthotopic HCC models. Metabolic reprogramming was assessed by targeted metabolomics, Seahorse analysis, and pharmacological inhibition of fatty acid oxidation. Mechanistic studies included co-immunoprecipitation, a cell-free kinase assay using recombinant AMPK and Gαi3, chromatin immunoprecipitation, promoter-reporter assays, cycloheximide-chase analysis, surface-biotinylation-based EGFR recycling assays, and GBF1 knockdown/rescue experiments.Results Gαi3 was upregulated in TAMs within glucose-deprived HCC regions and was associated with M2-like polarization, CD8 +T cell exhaustion, and shorter overall survival. High TAM Gαi3 remained associated with overall survival after adjustment for clinical covariates. Myeloid-specific Gnai3 deletion suppressed HCC progression and restored cytotoxic CD8+T cell activity. Etomoxir phenocopied Gnai3 deficiency and attenuated macrophage-mediated T-cell suppression, supporting a requirement for fatty acid oxidation. Mechanistically, glucose deprivation activated AMPK-dependent phosphorylation of Gαi3; the cell-free kinase assay, mass spectrometry, and S143A mutagenesis together supported Ser143 as the relevant regulatory site. Active Gαi3 stabilized EGFR through a GBF1-associated trafficking program, promoted EGFR-dependent PKM2 nuclear translocation, and enhanced formation of a nuclear PKM2/HIF-1α transcriptional complex that activated Pparg and sustained an immunosuppressive fatty acid oxidation program.Conclusions Our study identifies Gαi3 as a critical metabolic checkpoint that orchestrates TAMs immunosuppression in the glucose-deprived TME. The Gαi3-EGFR-PKM2-PPARγ signaling axis drives metabolic reprogramming towards lipid oxidation, thereby inhibiting antitumor immunity. Targeting Gαi3 represents a novel immunotherapeutic strategy to reverse TAMs-mediated immunosuppression and improve outcomes in HCC.

Source: Journal for ImmunoTherapy of Cancer

DOI: 10.1136/jitc-2026-015875