Malignant transformation of cells resulting from enhanced proliferation and aberrant differentiation is often accompanied by changes in transient receptor potential vanilloid (TRPV) channels expression. In gliomas, recent evidence indicates that TRPV type 2 (TRPV2) negatively controls glioma cell survival and proliferation. In addition, cannabinoids, the ligands of both cannabinoid and TRPV2 receptors, promote glioblastoma stem‐like cells (GSCs) differentiation and inhibit gliomagenesis. Herein, we provide evidence on the expression ofTRPV2in human GSCs and that GSCs differentiation reduces nestin and progressively increases both the glial fibrillary acidic protein (GFAP) and TRPV2 expression. Therefore, we evaluated the role of TRPV2 cation channel in GSC lines differentiation. Treatment of GSC lines with the TRPV antagonist Ruthenium Red, with ethylene glycol‐bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid or knockdown ofTRPV2gene during differentiation, decreases GFAP and class III beta‐tubulin (β_III‐tubulin) expression; conversely, phorbol‐12‐myristate‐13‐acetate stimulates GSCs proliferation, reduces TRPV2 expression and partially reverts astroglial differentiation. In addition, forcedTRPV2expression in GSC lines by stableTRPV2transfection increases GFAP and β_III‐tubulin expression and parallelly reduces proliferation. Finally,TRPV2overexpression inhibits GSCs proliferation in a xenograft mouse model, as shown by reduced tumor diameter and mitotic index, and promotes the differentiation of GSCs toward a more mature glial phenotype. Overall, our results demonstrate that TRPV2 promotesin vitroandin vivoGSCs differentiation and inhibits their proliferation. Better understanding of the molecular mechanisms that regulate the balance between proliferation and differentiation of GSCs would lead to more specific and efficacious pharmacological approaches.

Glioblastoma (GBM) is the most common and deadly adult brain tumor. Despite aggressive surgery, radiation, and chemotherapy, the life expectancy of patients diagnosed with GBM is ∼14 months. The extremely aggressive nature of GBM results from glioblastoma stem-like cells (GSCs) that sustain GBM growth, survive intensive chemotherapy, and give rise to tumor recurrence. There is accumulating evidence revealing that GSC resilience is because of concomitant activation of multiple survival pathways. In order to decode the signal transduction networks responsible for the malignant properties of GSCs, we analyzed a collection of GSC lines using a dual, but complementary, experimental approach, that is, reverse-phase protein microarrays (RPPMs) and kinase inhibitor library screening. We treated GSCs in vitro with clinically relevant concentrations of temozolomide (TMZ) and performed RPPM to detect changes in phosphorylation patterns that could be associated with resistance. In addition, we screened GSCs in vitro with a library of protein and lipid kinase inhibitors to identify specific targets involved in GSC survival and proliferation. We show that GSCs are relatively insensitive to TMZ treatment in terms of pathway activation and, although displaying heterogeneous individual phospho-proteomic profiles, most GSCs are resistant to specific inhibition of the major signaling pathways involved in cell survival and proliferation. However, simultaneous multipathway inhibition by the staurosporin derivative UCN-01 results in remarkable inhibition of GSC growth in vitro. The activity of UCN-01 on GSCs was confirmed in two in vivo models of GBM growth. Finally, we used RPPM to study the molecular and functional effects of UCN-01 and demonstrated that the sensitivity to UCN-01 correlates with activation of survival signals mediated by PDK1 and the DNA damage response initiated by CHK1. Taken together, our results suggest that a combined inhibition of PDK1 and CHK1 represents a potentially effective therapeutic approach to reduce the growth of human GBM.

Cytochrome c release from mitochondria promotes apoptosome formation and caspase activation. The question as to whether mitochondrial permeabilization kills cells via a caspase-independent pathway when caspase activation is prevented is still open. Here we report that proneural cells of embryonic origin, when induced to die but rescued by apoptosome inactivation are deprived of cytosolic cytochrome c through proteasomal degradation. We also show that, in this context, those cells keep generating ATP by glycolysis for a long period of time and that they keep their mitochondria in a depolarized state that can be reverted. Moreover, under these conditions, such apoptosome-deficient cells activate a Beclin 1-dependent autophagy pathway to sustain glycolytic-dependent ATP production. Our findings contribute to elucidating what the point-of-no-return in apoptosis is. They also help in clarifying the issue of survival of apoptosome-deficient proneural cells under stress conditions. Unraveling this issue could be highly relevant for pharmacological intervention and for therapies based on neural stem cell transfer in the treatment of neurological disorders.