PO-083 Suppressing the accumulation of cancer stem cells (CSC) using HDAC inhibitor reverses HNSCC chemoresistance by targeting NFKB signalling

Abstract

IntroductionThe vacuolar ATPase (V-ATPase) is a multisubunit proton pump acting in multiple processes in eukaryotic cells. Alteration of V-ATPase activity is associated to a wide range of human diseases including cancer. We have recently showed that V-ATPase expression has a central role in glioma stem cells (GSC) maintenance. Therefore we aimed to get insights into the signalling associated with V-ATPase expression in GSC.Material and methodsSignalling survey was performed using phospho-specific antibodies and the Cancer 10-pathway Reporter Luciferase Kit. GSC were treated with Ammonium chloride (NH4Cl) (10–50 mM), Bafilomycin A1 (BafA1) (10 nM) and the ERK inhibitor PD98059 (10 uM) Cell Invasion through collagen matrix and sphere formation were evaluated after 48 hour of drugs treatment, while cell cycle, apoptosis and ROS production were evaluated by flow cytometry after 24 hour. Mitochondrial depolarization and activity were evaluated by flow cytometry after TMRE staining and western blot, respectively. Autophagy was analysed by western blot using an antibody to p62. All experiments were performed using primary GSC cultures with high and low levels of V-ATPase G1 subunit (V1G1HIGH and V1G1LOW; n=3 each).Results and discussionsThe MAPK/Erk pathway was significantly upregulated in V1G1HIGH GSC and V-ATPase impairment by BafA1 reduced Erk phosphorylation, besides decreasing lysosomal acidification. Therefore we investigated if this effect was specific for the pump activity or if it was related to lysosomal dysfunction or to MAPK/Erk signalling.The comparison of the three drugs revealed that only BafA1 treatment induced cells death, reduced clonogenicity and invasion ability and decreased the phosphorylation level of proteins involved in proliferation and pro-apoptotic processes. Moreover BafA1 was the only drug that, at not lethal dosage, impaired cell cycle progression.This effect was associated with an increase in ROS production and mitochondrial depolarization and using a ROS inhibitor the effects of BafA1 were reverted.ConclusionTaken together these results indicate that the V-ATPase play a central role in GSC viability that goes beyond lysosomal activity or ERK phosphorylation. Further studies are needed to elucidate the roles of the proton pump in GSC and to target this molecule for innovative anti-cancer strategies.