ROS, PH AND CHLORIDE CURRENTS AS A VIRTUOUS (VICIOUS?) LOOP IN CELL CYCLE PROGRESSION OF GLIOBLASTOMA CANCER STEM CELLS

The intracellular chloride channel 1 (CLIC1) is a peculiar metamorphic protein, belonging to a still partially unexplored family of chloride channels, that shuttles between a cytoplasmic and a transmembrane form, the latter able to form a chloride selective ion channel. Different factors regulate this membrane insertion, in particular an increase in the oxidative level and a modification in the pH. CLIC1 has been found to be overexpressed in different tumors, among the others in Glioblastoma Multiforme (GBM). GBM is the most lethal, aggressive and diffuse brain tumor. One of the clinical challenges of GBM treatment is to hit selectively its cancer stem cells (CSCs) that are responsible for tumor origin, progression and recurrence. CLIC1 protein, in its transmembrane form, has a pivotal role in the tumorigenic potential, proliferation and self-renewal of CSCs isolated from grade IV human GBM. CLIC1 could represent a suitable pharmacological target as the protein, physiologically located in the cytoplasm, is highly expressed in the plasma membrane only of glioblastoma CSCs enriched cultures. In a work published last year from our laboratory we have shown that blocking CLIC1 ionic current impairs specifically proliferation of CSCs and tumor development; moreover, we demonstrated a partial but significant arrest of cells in G1 phase after CLIC1 functional inhibition. Our experiments further demonstrate the great potential of CLIC1 as a pharmaceutical target since the functional expression of CLIC1 protein as a chloride ion channel occurs selectively in CSCs compared to Mesenchimal Stem Cells. My thesis work has been concentrated in the direction of uncovering the mechanism that regulates the protein expression in the plasma membrane of GBM cancer stem cells. My results have shown that CLIC1 membrane ionic current is differently tuned during the cell division process and its activity is fundamental for the progression of the cell cycle since the inhibition of CLIC1 transmembrane ionic flow causes a drastic reduction in the transition between G1 and S phase. Electrophysiology experiments showed that the chloride conductance mediated by CLIC1 in CSCs is increased at specific time points after the release from G1 synchronization of the cells. This tuning is regulated by an increase in the internal pH of CSCs that occurs during the progression of G1 phase of the cell cycle. Moreover, the last experiment set that I performed showed a regulation of CLIC1 chloride conductance during G1 phase also by Reactive Oxygen Species (ROS) production by NADPH oxidase. Since both acute and chronic inhibition of CLIC1 channel activity with the specific inhibitor IAA94 leads to alteration in both internal pH and ROS levels I speculate a feed-forward mechanisms that links all these three elements that work synchronously to allow the progression through the cell cycle.