Abstract 903: Metformin antitumoral activity is exclusively mediated by the membrane functional expression of the Chloride Intracellular Channel 1 in glioblastoma stem cells
Authors: Verduci Ivan , Cianci Francesca , Cazzoli Riccardo , Cannavale Gaetano , Castiglione Stefania , Ranucci Matteo , Palloni Luca , Balboni Beatrice , Faletti Stefania , Girotto Stefania , Pelicci Giuliana , Minucci Saverio , Fantin Alessandro , Florio Tullio , Mazzanti Michele 
Published: June 2022

Abstract
Glioblastoma (GB) is the deadliest brain tumor, representing a major clinical challenge in the field. Despite standard of care together with cutting-edge strategies, life expectancy stands invariantly poor. Tumor heterogeneity and its high-grade of invasiveness make it difficult to be surgically resected, and recurrence is inevitable. There is scientific consensus that tumor relapse originates from progenitor/stem-like cells known as glioblastoma stem cells (GSCs) displaying properties of self-renewal and multi-lineage differentiation that contribute to tumor mass heterogeneity. Thus, GSCs represent the target of newly designed therapies. In recent years, it has been highlighted the anti-cancer properties of metformin, the first-line drug in Type-2 diabetes (T2D) treatment. Metformin assumption in T2D patients has been positively linked with a decrease in the risk of several cancer types and cancer-related mortality, including glioblastoma. Additionally, numerous studies demonstrated metformin's anticancer properties against GSCs. Despite metformin's mechanism of action on cancer cells has not been clarified yet, its effect has been proposed to target the oxidative phosphorylation (OXPHOS) pathway. Considering that GSCs have been reported to shuttle between glycolysis and OXPHOS energetic pathways, their metabolic plasticity should prevent the antiproliferative effect of metformin. However, metformin-treated GSCs show a phenotype superimposable to that caused by the blockage of the transmembrane form of the chloride intracellular channel 1 (tmCLIC1). tmCLIC1 involvement in the progression of GB has been assessed deeply in vitro as well as in vivo. In addition, its peculiar localization and enrichment on GSCs' membrane renders it a valuable pharmacological target for GB.
Here, we show that in glioblastoma, the transmembrane form of Chloride Intracellular Channel 1 (tmCLIC1) works as a privileged and unique metformin membrane receptor. Nuclear magnetic resonance experiments on live cells confirm the binding to tmCLIC1. Metformin impairs tmCLIC1 activity by a specific binding on a single amino acid, the arginine29. Its mutation demonstrates that metformin-depending tmCLIC1 block is prevented, failing to reduce glioblastoma cells proliferation in 2D and 3D models as well. Moreover, in vivo experiments on zebrafish embryos closely mirror the behavior observed in vitro. Our results demonstrate that tmCLIC1 is essential for metformin antineoplastic effect in glioblastoma and prove a direct binding between the drug and its target. Considering tmCLIC1 contribution to glioblastoma progression, the present work paves the way for future investigations intended to develop a strategy aimed to enhance metformin-tmCLIC1 interaction. Ivan Verduci was supported by a AIRC fellowship for Italy.
Citation Format: Ivan Verduci, Francesca Cianci, Riccardo Cazzoli, Gaetano Cannavale, Stefania Castiglione, Matteo Ranucci, Luca Palloni, Beatrice Balboni, Stefania Faletti, Stefania Girotto, Giuliana Pelicci, Saverio Minucci, Alessandro Fantin, Tullio Florio, Michele Mazzanti. Metformin antitumoral activity is exclusively mediated by the membrane functional expression of the Chloride Intracellular Channel 1 in glioblastoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 903.