Chloride Intracellular Channel 1 as a privileged receptor mediating metformin antiproliferative action on glioblastoma cells

Metformin is the first-line drug for type 2 diabetes. Retrospective analyses based on diabetic patients’ clinical data show that daily use of metformin reduces the incidence of various solid tumors. Although it is widely accepted that metformin must be internalized to achieve its pharmacological effects, direct evidence regarding metformin’s membrane permeability or the presence of a specific membrane receptor in cancer cells remains lacking. Here, we demonstrate that the transmembrane form of Chloride Intracellular Channel 1 (tmCLIC1) functions as a key metformin receptor in glioblastoma stem-like cells. We found that metformin inhibits tmCLIC1 activity through a specific binding involving arginine 29. Mutation of this site, which prevents metformin from binding and blocking tmCLIC1, abolishes the biguanide’s ability to inhibit glioblastoma cell proliferation in both 2D and 3D models, as well as the effect of metformin on mitochondrial respiration. Additionally, we show direct binding between the drug and its target. Through in vivo experiments with zebrafish embryos and mice orthotopically engrafted with glioblastoma cells and treated with metformin, we prove that metformin binding to tmCLIC1 is essential for metformin’s antineoplastic action. Given tmCLIC1’s role in glioblastoma progression, this work lays the groundwork for developing strategies to enhance metformin-tmCLIC1 interactions, thereby further boosting metformin's therapeutic potential.