Functional In Vitro Assessment of VEGFA/NOTCH2 Signaling Pathway and pRB Proteasomal Degradation and the Clinical Relevance of Mucolipin TRPML2 Overexpression in Glioblastoma Patients

Abstract

Glioblastoma (GBM) is the most malignant glioma with an extremely poor prognosis. It is characterized by high vascularization and its growth depends on the formation of new blood vessels. We have previously demonstrated that TRPML2 mucolipin channel expression increases with the glioma pathological grade. Herein by ddPCR and Western blot we found that the silencing of TRPML2 inhibits expression of the VEGFA/Notch2 angiogenic pathway. Moreover, the VEGFA/Notch2 expression increased in T98 and U251 cells stimulated with the TRPML2 agonist, ML2-SA1, or by enforced-TRPML2 levels. In addition, changes in TRPML2 expression or ML2-SA1-induced stimulation, affected Notch2 activation and VEGFA release. An increased invasion capability, associated with a reduced VEGF/VEGFR2 expression and increased vimentin and CD44 epithelial-mesenchymal transition markers in siTRPML2, but not in enforced-TRPML2 or ML2-SA1-stimulated glioma cells, was demonstrated. Furthermore, an increased sensitivity to Doxorubicin cytotoxicity was demonstrated in siTRPML2, whereas ML2-SA1-treated GBM cells were more resistant. The role of proteasome in Cathepsin B-dependent and -independent pRB degradation in siTRPML2 compared with siGLO cells was studied. Finally, through Kaplan-Meier analysis, we found that high TRPML2 mRNA expression strongly correlates with short survival in GBM patients, supporting TRPML2 as a negative prognostic factor in GBM patients.

TRPML2 Mucolipin Channels Drive the Response of Glioma Stem Cells to Temozolomide and Affect the Overall Survival in Glioblastoma Patients

“…The expression profiling of several TRP ion channels, including the TRPC1, TRPM4 TRPML1 and TRPML2 genes, has been reported to predict the clinical outcome in cancer patients [ 19 , 25 , 26 ]. Thus, the correlation between TRPML2 mRNA expression and OS and PFS was evaluated.…”

“…Moreover, it inhibits the VEGF-A/Notch2 angiogenic pathway, whereas TRPML2 overexpression or its activation increases the VEGF-A/Notch2 expression. In addition, an increased invasion capability, epithelial–mesenchymal transition markers expression, and sensitivity to doxorubicin in silenced TRPML2 and a shorter OS in TRPML2 overexpressing GBM patients was reported [ 19 ]. Finally, overexpression of TRPML2 as well as the complete loss of this channel have been associated with worse OS and prognosis, whereas lower TRPML2 expression shows a protective effect in GBM patients [ 18 , 20 ].…”

Coexpression of TRPML1 and TRPML2 Mucolipin Channels Affects the Survival of Glioblastoma Patients

“…Thus, DN GBM cells compared to siTRPML2 or siTRPML1 T98 and U251 cells showed an increased growth capability, mesenchymal stem-like phenotype, augmented STAT3 proliferative level and increased migration/invasion capability, compared to wild-type control cells. Finally, given the role of the VEGFA/NOTCH2 signaling pathway in TRPML2-positive GBM cells [ 23 ], a reduced VEGFA/B and NOTCH2 mRNA expression was detected in DN siTRPML2/TRPML1 GBM cells.…”

“…Cell invasion was evaluated via a transwell assay using the transwell chambers (BD Biosciences, Franklin Lakes, NJ, USA) as previously described [ 23 ]. Briefly, a total of 750 μL cell culture medium supplemented with 10% FBS was added in the lower chamber.…”

“…In regard to the expression and role of TRPML channels in gliomas, we previously reported that TRPML2 silencing inhibits cell viability and proliferation [ 22 ] and increases the invasion [ 23 ] in T98 and U251 GBM cells; activation of TRPML1 channels by the MK6-83 agonist inhibits cell viability and induces apoptosis in T98 and U251 cells [ 24 ] and silencing of TRPML1 promotes the survival and invasion capability in T98 and U251 cells by affecting the autophagy and cell senescence processes [ 25 ]. Finally, concordantly with the Human Protein Atlas showing no TRPML3 mRNA expression in GBM biopsies, no TRPML3 transcripts were evidenced in the T98 and U251 cell lines [ 24 ].…”