One of the major challenges in cancer treatment is tackling the metabolic reprogramming of tumor cells, which requires higher energy and biomolecules than healthy cells. Glioblastoma (GB) cells, as well as the other cancer types, adapt their metabolism by switching between glycolysis and oxidative phosphorylation (OXPHOS). These processes are dependent on transmembrane proteins’ response to the extracellular environment. Our research identified the transmembrane form of Chloride Intracellular Channel 1 (tmCLIC1) as a marker of malignancy and a potential therapeutic target. Thus, tmCLIC1 is upregulated in several solid tumors promoting cancer growth and progression, while is mainly absent in healthy cells. Here, we found that the antidiabetic drug metformin, an OXPHOS inhibitor in cancer cells, reduces tmCLIC1 activity.

All the experiment were performed in GB cells with several CLIC1 genetic background obtained with CRISPR-Cas9-Technology. We performed NMR and patch clamp recording to demonstrate tmCLIC1-metformin interaction. Moreover, we analyzed metabolism modulation given by metformin through OCR and EACR measurment. In addition, PP2A-GSK3β-MCL-1 axis was analyzed through Western Blot. Our in vitro results were further confirmed in vivo with Zebrafish embryo and in murine.

Metformin impairs the tmCLIC1 function by a direct and specific binding coordinated by arginine 29 in the tmCLIC1 sequence. Mutation of this residue nullifies metformin inhibitory effects on cell proliferation, mitochondrial respiration, and tumor progression in in vitro and in vivo models. Additionally, in conditions of hypoglycemia, metformin induces cancer cell apoptosis by inhibiting the Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) and activating PP2A subunit B56δ. This, dephosphorylates Glycogen Synthase Kinase 3 Beta (GSK3β), leading to degradation of the pro-survival 53 protein MCL-1 and subsequent cell death. Based on previous data, it is possible to sustain a reasonable metformin mechanism of action in cancer cells. We suppose that tmCLIC1’s role in reactive oxygen species (ROS) balance is crucial for GB cells metabolic reprogramming, and its impairment sensitizes resistant tumor cells. Metformin is an extremely low-priced drug that can be repurposed as an adjuvant in cancer treatment with minor side adverse effects.

We demonstrate that tmCLIC1 is essential for this metformin-mediated antineoplastic effect, particularly through regulating the PP2A-GSK3β-MCL-1 axis under hypoglycemic conditions. Given tmCLIC1’s role in GB progression, our findings pave the way to optimize the molecular interactions between tmCLIC1 and metformin or related molecules, enhancing their therapeutic efficacy.