FURTHER INSIGHT INTO V-ATPASE ROLE IN GLIOMA STEM CELLS

V-ATPase is a proton pump mainly localized on lysosomes and on plasma membrane of specialized cells. It is responsible of proton translocation and acidification of intra- and extra-cellular environment. Our group demonstrated that the over-expression of the subunit G1 (V1G1) is involved in the maintenance of the stem cell niche in glioblastoma (GBM) and correlates with poor prognosis in GBM patients. In this work we aimed to elucidate the role of V-ATPase in GBM stem cells from a functional perspective. We demonstrated that neurospheres (NS) with higher levels of V1G1 subunit (High-V1G1), compared with NS with lower levels of V1G1 (Low-V1G1), were characterized by increased clonogenicity in vitro and in vivo, invasiveness, lysosomal acidification and ERK pathway activation. Specific inhibition of V-ATPase activity, by Bafilomycin (BafA1), but not of ERK or other lysosomal drugs, induced reactive oxygen species (ROS)-mediated apoptosis only in High-V1G1 NS. In addition, BafA1 treatment affected mitochondria homeostasis only in High-V1G1 NS. Preliminary experiments suggested that a V-ATPase pump might be localized on the mitochondria or it could mediate direct contacts between mitochondria and lysosomes thus causing an imbalance of charges (proton flux) when perturbed. Finally, High-V1G1 and Low-V1G1 NS differed in terms of metabolic behaviours: preferential use of glycolysis by Low-V1G1 NS opposite to use of oxidative metabolism in High-V1G1 NS. V-ATPase block by BafA1 in High-V1G1 NS shifted their metabolism to that of Low-V1G1 NS. On the other hand, the autophagic pathway, that is directly connected with lysosomal function, was blocked by BafA1 only in Low-V1G1 NS. These phenotypes were not modulated by ERK or lysosomal acidification inhibitors alone, indicating a specific role for V-ATPase proton pump in modulating them. Taken together, these results indicate that V-ATPase is crucial for GBM stem cells viability through different mechanisms that include bioenergetics sensing and requiring, mitochondrial homeostasis and ERK signalling activity.