Specific V-ATPase expression sub-classifies IDHwt lower-grade gliomas and impacts glioma growth in vivo

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\u2019 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\ub703, 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\u20131148 to VV), Fondazione IRCCS Ca\u2019 Granda, and Fondazione INGM Grant in Molecular Medicine 2014 (to VV)

A GBM-like V-ATPase signature directs cell-cell tumor signaling and reprogramming via large oncosomes

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)

Measurement of prompt hadron production ratios in pppp collisions at s=\sqrt{s} = 0.9 and 7 TeV

The charged-particle production ratios $\bar{p}/p$, $K^-/K^+$, $\pi^-/\pi^+$, $(p + \bar{p})/(\pi^+ + \pi^-)$, $(K^+ + K^-)/(\pi^+ + \pi^-)$ and $(p + \bar{p})/(K^+ + K^-)$ are measured with the LHCb detector using $0.3 {\rm nb^{-1}}$ of $pp$ collisions delivered by the LHC at $\sqrt{s} = 0.9$ TeV and $1.8 {\rm nb^{-1}}$ at $\sqrt{s} = 7$ TeV. The measurements are performed as a function of transverse momentum $p_{\rm T}$ and pseudorapidity $\eta$. The production ratios are compared to the predictions of several Monte Carlo generator settings, none of which are able to describe adequately all observables. The ratio $\bar{p}/p$ is also considered as a function of rapidity loss, $\Delta y \equiv y_{\rm beam} - y$, and is used to constrain models of baryon transport.Comment: Incorrect entries in Table 2 corrected. No consequences for rest of pape

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

V-ATPase controls tumor growth and autophagy in a Drosophila model of gliomagenesis

Glioblastoma (GBM), a very aggressive and incurable tumor, often results from constitutive activation of EGFR (epidermal growth factor receptor) and of PI3K (phosphoinositide 3-kinase). 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 Dp110. Interestingly, the resulting hyperplastic glia expresses high levels of ref(2)P (refractory to Sigma P), the Drosophila homolog of p62/SQSTM1. However, cellular clearance of autophagic cargoes appears inhibited upstream of autophagosome formation. Remarkably, downregulation of subunits of the vacuolar-H+-ATPase (V-ATPase) prevents overgrowth, reduces PI3K 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 VATPase could represent a therapeutic target against GBM

Interplay Between V-ATPase G1 and Small EV-miRNAs Modulates ERK1/2 Activation in GBM Stem Cells and Nonneoplastic Milieu

The ATP6V1G1 subunit (V1G1) of the vacuolar proton ATPase (V-ATPase) pump is crucial for glioma stem cells (GSC) maintenance and in vivo tumorigenicity. Moreover, V-ATPase reprograms the tumor microenvironment through acidification and release of extracellular vesicles (EV). Therefore, we investigated the role of V1G1 in GSC small EVs and their effects on primary brain cultures. To this end, small EVs were isolated from patients-derived GSCs grown as neurospheres (NS) with high (V1G1HIGH-NS) or low (V1G1LOW-NS) V1G1 expression and analyzed for V-ATPase subunits presence, miRNA contents, and cellular responses in recipient cultures. Our results show that NS-derived small EVs stimulate proliferation and motility of recipient cells, with small EV derived from V1G1HIGH-NS showing the most pronounced activity. This involved activation of ERK1/2 signaling, in a response reversed by V-ATPase inhibition in NS-producing small EV. The miRNA profile of V1G1HIGH-NS-derived small EVs differed significantly from that of V1G1LOW-NS, which included miRNAs predicted to target MAPK/ERK signaling. Mechanistically, forced expression of a MAPK-targeting pool of miRNAs in recipient cells suppressed MAPK/ERK pathway activation and blunted the prooncogenic effects of V1G1HIGH small EV. These findings propose that the GSC influences the brain milieu through a V1G1-coordinated EVs release of MAPK/ERK-targeting miRNAs. Interfering with V-ATPase activity could prevent ERK-dependent oncogenic reprogramming of the microenvironment, potentially hampering local GBM infiltration. IMPLICATIONS: Our data identify a novel molecular mechanism of gliomagenesis specific of the GBM stem cell niche, which coordinates a V-ATPase-dependent reprogramming of the brain microenvironment through the release of specialized EVs

The experimental facility for the Search for Hidden Particles at the CERN SPS

The Search for Hidden Particles (SHiP) Collaboration has shown that the CERN SPS accelerator with its 400 GeV/c proton beam offers a unique opportunity to explore the Hidden Sector [1-3]. The proposed experiment is an intensity frontier experiment which is capable of searching for hidden particles through both visible decays and through scattering signatures from recoil of electrons or nuclei. The high-intensity experimental facility developed by the SHiP Collaboration is based on a number of key features and developments which provide the possibility of probing a large part of the parameter space for a wide range of models with light long-lived super-weakly interacting particles with masses up to (10) GeV/c2 in an environment of extremely clean background conditions. This paper describes the proposal for the experimental facility together with the most important feasibility studies. The paper focuses on the challenging new ideas behind the beam extraction and beam delivery, the proton beam dump, and the suppression of beam-induced background

A facility to Search for Hidden Particles (SHiP) at the CERN SPS

A new general purpose fixed target facility is proposed at the CERN SPS accelerator which is aimed at exploring the domain of hidden particles and make measurements with tau neutrinos. Hidden particles are predicted by a large number of models beyond the Standard Model. The high intensity of the SPS 400~GeV beam allows probing a wide variety of models containing light long-lived exotic particles with masses below ${\cal O}$(10)~GeV/c$^2$, including very weakly interacting low-energy SUSY states. The experimental programme of the proposed facility is capable of being extended in the future, e.g. to include direct searches for Dark Matter and Lepton Flavour Violation