Background Cancer cells use specific V-ATPase subunits to activate oncogenic pathways. Therefore, we investigated V-ATPase deregulation in aggressive gliomas and associated signaling. Methods V-ATPase genes expression and associated pathways were analyzed in different series of glioma available from public databases, as well as in patients' cohort. Activation of pathways was analyzed at gene and protein expression levels. A genetic model of glioma in Drosophila melanogaster and mice with GBM patients-derived orthotopic xenografts were used as in vivo models of disease. Findings GBM and recurrent gliomas display a specific V-ATPase signature. Such signature resolves the heterogeneous class of IDH-wild type lower-grade gliomas, identifying the patients with worse prognosis independently from clinical and molecular features (p = 0·03, by Cox proportional-hazards model). In vivo , V-ATPase subunits deregulation significantly impacts tumor growth and proliferation. At the molecular level, GBM-like V-ATPase expression correlates with upregulation of Homeobox genes. Interpretation Our data identify a V-ATPase signature that accompanies glioma aggressiveness and suggest new entry points for glioma stratification and follow-up. Fund This work was supported by Fondazione Cariplo (2014–1148 to VV), Fondazione IRCCS Ca' Granda, and Fondazione INGM Grant in Molecular Medicine 2014 (to VV).
Background The V-ATPase proton pump controls acidification of intra and extra-cellular milieu in both physiological and pathological conditions. We previously showed that some V-ATPase subunits are enriched in glioma stem cells and in patients with poor survival. In this study, we investigated how expression of a GBM-like V-ATPase pump influences the non-neoplastic brain microenvironment. Methods Large oncosome (LO) vesicles were isolated from primary glioblastoma (GBM) neurospheres, or from patient sera, and co-cultured with primary neoplastic or non-neoplastic brain cells. LO transcript and protein contents were analyzed by qPCR, immunoblotting and immunogold staining. Activation of pathways in recipient cells was determined at gene and protein expression levels. V-ATPase activity was impaired by Bafilomycin A1 or gene silencing. Findings GBM neurospheres influence their non-neoplastic microenvironment by delivering the V-ATPase subunit V1G1 and the homeobox genes HOXA7, HOXA10, and POU3F2 to recipient cells via LO. LOs reprogram recipient cells to proliferate, grow as spheres and to migrate. Moreover, LOs are particularly abundant in the circulation of GBM patients with short survival time. Finally, impairment of V-ATPase reduces LOs activity. Interpretation We identified a novel mechanism adopted by glioma stem cells to promote disease progression via LO-mediated reprogramming of their microenvironment. Our data provide preliminary evidence for future development of LO-based liquid biopsies and suggest a novel potential strategy to contrast glioma progression. Fund This work was supported by Fondazione Cariplo (2014-1148 to VV) and by the Italian Minister of Health-Ricerca Corrente program 2017 (to SF).
Glioblastoma (GBM), a very aggressive and incurable tumor, often results from constitutive activation of EGFR (epidermal growth factor receptor) and of phosphoinositide 3-kinase (PI3K). To understand the role of autophagy in the pathogenesis of glial tumors in vivo, we used an established Drosophila melanogaster model of glioma based on overexpression in larval glial cells of an active human EGFR and of the PI3K homolog Pi3K92E/Dp110. Interestingly, the resulting hyperplastic glia express high levels of key components of the lysosomal-autophagic compartment, including vacuolar-type H + -ATPase (V-ATPase) subunits and ref( 2)P (refractory to Sigma P), the Drosophila homolog of SQSTM1/ p62. However, cellular clearance of autophagic cargoes appears inhibited upstream of autophagosome formation. Remarkably, downregulation of subunits of V-ATPase, of Pdk1, or of the Tor (Target of rapamycin) complex 1 (TORC1) component raptor prevents overgrowth and normalize ref(2)P levels. In addition, downregulation of the V-ATPase subunit VhaPPA1-1 reduces Akt and Tor-dependent signaling and restores clearance. Consistent with evidence in flies, neurospheres from patients with high V-ATPase subunit expression show inhibition of autophagy. Altogether, our data suggest that autophagy is repressed during glial tumorigenesis and that V-ATPase and MTORC1 components acting at lysosomes could represent therapeutic targets against GBM.
MLL-rearranged leukemias are among the leukemic subtypes with poorest survival, and treatment options have barely improved over the last decades. Furthermore, despite increasing molecular understanding of the mechanisms behind these hematopoietic malignancies, this knowledge has had poor translation into the clinic. Identification of novel treatment methods is hampered by the lack of relevant in vivo models that allow for rapid identification of actionable drug targets and small molecule inhibitors. Here, we report a Drosophila melanogaster model system to explore the pathways affected in MLL-rearranged leukemia. We show that expression of the human leukemic oncogene MLL-AF4 in the Drosophila hematopoietic system resulted in increased levels of circulating hemocytes and an enlargement of the larval hematopoietic organ, the lymph gland. Strikingly, depletion of Drosophila orthologs of known interactors of MLL-AF4, such as DOT1L, rescued the leukemic phenotype. In agreement, treatment with small-molecule inhibitors of DOT1L also prevented the MLL-AF4-induced leukemia-like phenotype. Taken together, this model provides an in vivo system to unravel the genetic interactors involved in leukemogenesis and offers a strategy for a prompt identification of potential therapeutic options for treatment of MLL-rearranged leukemia.
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