A major challenge in cancer therapy is overcoming the profound metabolic alterations that occur in malignant cells. These cells exhibit heightened energy and biosynthetic precursor requirements relative to their non-transformed counterparts. Tumor cells achieve metabolic flexibility by shifting between glycolysis and oxidative phosphorylation (OXPHOS), processes orchestrated by transmembrane proteins that detect changes in the external cellular environment. This investigation highlights the transmembrane variant of Chloride Intracellular Channel 1 (tmCLIC1) as an indicator of malignant phenotype and a viable target for intervention. Levels of tmCLIC1 are substantially upregulated across various solid malignancies, thereby promoting tumor growth and progression, whereas its expression remains negligible in normal cells. Our work establishes that tmCLIC1 serves as the direct molecular target of metformin, an agent known to suppress OXPHOS in neoplastic cells. Single-channel patch-clamp electrophysiology, combined with NMR spectroscopy, confirmed that tmCLIC1 is the main target of metformin action in glioblastoma stem-like cells. Experiments were conducted on multiple patient-derived glioblastoma lines representing heterogeneous genetic profiles. Genetic disruption of CLIC1 via CRISPR-Cas9 or introduction of an arginine 29 point mutation eliminated the drug’s inhibitory influence on tumor cells. Subsequent analyses examined cellular proliferation, mitochondrial function, and tumorigenic potential through both in vitro assays and in vivo models, including zebrafish and mouse xenograft systems. Metformin exerts its inhibitory effect on tmCLIC1 by means of a precise physical interaction at arginine 29 within the channel’s amino acid sequence. In addition, during periods of low glucose availability, metformin triggers programmed cell death in glioblastoma cells by suppressing the Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) and concurrently stimulating the PP2A B56δ regulatory subunit. The resulting pathway drives dephosphorylation of Glycogen Synthase Kinase 3 Beta (GSK3β), which in turn destabilizes the pro-survival factor MCL-1, ultimately promoting apoptosis. Blockade of tmCLIC1 function proves indispensable for metformin’s anticancer properties, chiefly by governing the PP2A-GSK3β-MCL-1 signaling axis under nutrient-deprived conditions. Prolonged metformin exposure within tumor tissue effectively curbs in vivo expansion even at nanomolar levels. Although the potential benefit of metformin for brain tumor management remains controversial, the present results emphasize the critical need for adequate drug delivery to the tumor site. Notably, measurable suppression of tumor progression occurs in vivo at concentrations below 10 nanomolar. These observations indicate that achieving sustained therapeutic levels of metformin in the central nervous system could strengthen tmCLIC1 blockade, thereby laying the groundwork for refined therapeutic approaches that leverage metformin or structurally related molecules to augment treatment efficacy.
