Epidemiological and preclinical studies propose that metformin, a first-line drug for type-2 diabetes, exerts direct antitumor activity. Although several clinical trials are ongoing, the molecular mechanisms of this effect are unknown.Here we show that chloride intracellular channel-1 (CLIC1) is a direct target of metformin in human glioblastoma cells. Metformin exposure induces antiproliferative effects in cancer stem cell-enriched cultures, isolated from three individual WHO grade IV human glioblastomas. These effects phenocopy metformin-mediated inhibition of a chloride current specifically dependent on CLIC1 functional activity. CLIC1 ion channel is preferentially active during the G1-S transition via transient membrane insertion. Metformin inhibition of CLIC1 activity induces G1 arrest of glioblastoma stem cells. This effect was time-dependent, and prolonged treatments caused antiproliferative effects also for low, clinically significant, metformin concentrations. Furthermore, substitution of Arg29 in the putative CLIC1 pore region impairs metformin modulation of channel activity.The lack of drugs affecting cancer stem cell viability is the main cause of therapy failure and tumor relapse. We identified CLIC1 not only as a modulator of cell cycle progression in human glioblastoma stem cells but also as the main target of metformin's antiproliferative activity, paving the way for novel and needed pharmacological approaches to glioblastoma treatment.

In recent decades, growing scientific evidence supports the role of ion channels in the development of different cancers. Both potassium selective pores and chloride permeabilities are considered the most active channels during tumorigenesis. High rate of proliferation, active migration, and invasiveness into non-neoplastic tissues are specific properties of neoplastic transformation. All these actions require partial or total involvement of chloride channel activity. In this context, this class of membrane proteins could represent valuable therapeutic targets for the treatment of resistant tumors. However, this encouraging premise has not so far produced any valid new channel-targeted antitumoral molecule for cancer treatment. Problematic for drug design targeting ion channels is their vital role in normal cells for essential physiological functions. By targeting these membrane proteins involved in pathological conditions, it is inevitable to cause relevant side effects in healthy organs. In light of this, a new protein family, the chloride intracellular channels (CLICs), could be a promising class of therapeutic targets for its intrinsic individualities: CLIC1 and CLIC4, in particular, not only are overexpressed in specific tumor types or their corresponding stroma but also change localization and function from hydrophilic cytosolic to integral transmembrane proteins as active ionic channels or signal transducers during cell cycle progression in certain cases. These changes in intracellular localization, tissue compartments, and channel function, uniquely associated with malignant transformation, may offer a unique target for cancer therapy, likely able to spare normal cells. This article is part of a special issue itled "Membrane Channels and Transporters in Cancers."

BackgroundChloride channels are physiologically involved in cell division and motility. Chloride intracellular channel 1 (CLIC1) is overexpressed in a variety of human solid tumors compared with normal tissues, suggesting a potential involvement of CLIC1 in the regulation of tumorigenesis. This led us to investigate the role of CLIC1 in gliomagenesis.MethodsWe used the neurosphere system to isolate stem/progenitor cells from human glioblastomas (GBMs). CLIC1 targeting in GBM neurospheres was achieved by both lentiviral-mediated short-hairpin RNA transduction and CLIC1 antibody treatment, and its effect on stem-like properties was analyzed in vitro by proliferation and clonogenic assays and in vivo by orthotopic injection in immunocompromised mice. Channel activity was studied by perforated patch clamp technique. Differences in expression were analyzed by analysis of variance with Tamhane’s multiple comparison test. Kaplan–Meier analyses and log-rank test were used to assess survival. All statistical tests were two-sided.ResultsCLIC1 was statistically significantly overexpressed in GBMs compared with normal brain tissues (P < .001) with a better survival of patients with CLIC1 low-expressing tumors (CLIC1low vs CLIC1high survival: χ2 = 74.35; degrees of freedom = 1; log-rank P < .001). CLIC1 was variably expressed in patient-derived GBM neurospheres and was found enriched in the stem/progenitor compartment. CLIC1 silencing reduced proliferative (P < .01), clonogenic (P < .01), and tumorigenic capacity (P < .05) of stem/progenitor cells. The reduction of CLIC1 chloride currents with a specific CLIC1 antibody mirrored the biological effects of CLIC1 silencing in GBM patient–derived neurospheres.ConclusionsReduced gliomagenesis after CLIC1 targeting in tumoral stem/progenitor cells and the finding that CLIC1 expression is inversely associated with patient survival suggest CLIC1 as a potential target and prognostic biomarker.