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Etude du rôle des protéines G dans les gliomes de haut grade : implication dans la migration et le phénotype mésenchymateux

Abstract : GBM is the most common (∼45% of all gliomas) and aggressive primary malignant brain tumor in adults. As described in this document's introduction, GBM highly heterogeneous phonotype associated with molecular signatures and gene expressions, but also with hypoxic and inflammatory microenvironmental conditions, contribute to frequent recurrence after complete resection-radio/chemotherapy, and explain the multiple therapeutic failures. Most of the current treatment options for GBM, although sometimes multimodal, the survival of GBM patients is not significantly improved, and the challenges to improve patient survival and quality of life remain enormous. Thus, the identification of differentially expressed factors that could better define the biological behavior of GBM, would provide a basis for the development of novel therapies and may be more effective. One of the characteristics of GBMs is their highly migratory and invasive properties, relayed mainly by chemotactic factors belonging to the hypoxic and inflammatory tumor microenvironment. G-protein coupled receptors (GPCRs) and their ligands, particularly the chemokines GPCRs, overexpressed in GBMs and stimulating migration, invasion and neoangiogenesis, play a key role in the development of GBM and the acquisition of an aggressive phenotype. In this context, our team demonstrated that UT, the receptor of urotensin II (UII), a pro-angiogenic and pro-inflammatory chemokine, as well as the well-known chemokine system SDF-1/CXCR4 are systematically co-expressed in GBMs particularly in vascular and perinecrotic areas and their expression are correlated with grade. We also demonstrated in vitro that UII/UT stimulate GBM cells chemotactic migration and invasion via activation of the pathways Gαi/PI3K and Gα13/Rho/ROCK, pathways that have previously been identified for the SDF-1α/CXCR4 system and other chemotactic GPCRs. In addition, a recent principal component analysis of TCGA (The Cancer Genome Atlas) database performed by Alexandre Mutel, PhD student in the team, has identified the expression signature of GPCRs in gliomas and particularly those which are overexpressed in mesenchymal GBM, among which many chemotactic GPCRs are included. Taking together, their redundant expression and signaling activity frequently associated with tumorigenesis, particularly in GBMs, raises the issue of studying signaling nodes common to all these GPCRs. These nodes, are primarily represented by heterotrimeric G proteins, composed of α, β and γ subunits, that couple these GPCRs relaying many intracellular secondary effectors, probably essentials in the regulation of GBM aggressiveness. In this context, the aim of my thesis work was to identify the main Ga, b and g subunits among the 31 G proteins expressed in human gliomas and those more specifically associated with the malignant grade, and the aggressiveness of GBMs and then to determine the role of one of these specific G proteins in GBM cells proliferation and invasion mechanisms. For that, we first analyzed the expression of the 31 subunits (15α, 5β and 11γ) of G proteins from the TCGA database and showed that the mRNA expression of Gαz, Gαi1, Gβ4, Gβ5 et Gγ3 are relatively low in GBMs while Gα12, Gα13, Gα15, Gαi2, Gαi3, Gβ2, Gγ5, Gγ11 and Gγ12 subunits, are particularly overexpressed in GBM and are associated with a poor prognosis in terms of recurrence and patient survival. We then confirmed by RT-qPCR the high expression of these interesting identified G proteins by analyzing their expression in 6 different glioma cell lines as well as in GBM patient biopsies obtained in collaboration with the pathology department of Rouen University Hospital (Collaboration Pr A. Laquerrère). TCGA database analysis associated with the clinical information of patients indicate that these subunits are all associated with a poor prognosis for glioma patient survival. Moreover, the downregulation of Gα15, Gαi3 or Gβ2 in U87 GBM cell lines significantly decrease the cells proliferation/survival, adhesion associated with migration. Thus, we propose these identified G proteins (Gα15, Gαi3, Gβ2, Gγ5, Gγ10, Gγ11 and Gγ12) as GBM malignancy biomarkers and they could contribute to the GBM aggressive phenotype. Among all the identified G proteins, we specifically focused on Gα15, an atypical and non-ubiquitous G protein long described to be specific to hematopoietic stem cell. The latter seems to be also expressed de novo in highgrade gliomas, more particularly in neural and mesenchymal GBMs. Thus, we investigated whether the expression of this Gα15 subunit in GBM cells, controls the mechanisms of proliferation/invasion and is a potential therapeutic target. To better analyze the function of Gα15, we used siRNA, shRNA or Crispr-Cas9 technology to stably inactivate GNA15 (Gα15-KO) gene in U87 and 8MG cell lines. From these cells, we have demonstrated that Gα15 silencing or knockout significantly decreases the number (nucleocounter counting) and viability of GBM cells (measure of cells metabolic activity determined by WST-1 cleavage). These findings demonstrated that Gα15 is an important G protein implicated in cell survival, without affecting the cell cycle, in the absence of GPCR activation. GBM is also characterized by its highly invasive behavior. This behavior in GBM is one of the hallmarks that contribute to tumor recurrence and poor prognosis of GBM. Tumor cell migration and invasion is a coordinated multi-step process including the dynamic reorganization of the actin cytoskeleton, membrane protrusions like lamellipodia, formation of adhesion complexes, cell body contraction, and tail detachment for mesenchymal migration. In this context we have demonstrated that the repression of Gα15 expression significantly decreases the number of migrated GBM cells in transwell migration and wound healing assays, while its overexpression has opposite effects. Interestingly, using videomicroscopy for timelapse recordings of cell motility, we observed that U87 control cells emits lamellipodia in the front of the cells to migrate whereas Gα15-KO clones failed to be polarized, exhibited a reduced trajectory length associated with average speed as well as cumulative distances significantly lowered compared with U87 control cells. Immunocytochemistry analysis revealed that the inactivation of Gα15 significantly reduces the number of focal adhesion complex (FA) rich in Phospho-paxillin and vinculin (two major proteins of FA), and impairs actin cytoskeleton remodeling (actin stress fibers polymerization) associated with a significant decrease of wellformed lamellipodia number. However, Gα15 overexpressing cells exhibit an exacerbation of both FA proteins and lamellipodia number. These observations show that Gα15 plays a major role in the regulation of migration/invasion and survival associated with GBMs growth. In order to identify the main Gα15-regulated signaling pathways to stimulate these processes, we analyzed the oncogenic signaling pathways by western blot and identified that Gα15 inactivation significantly inhibits the activation of ERK1/2 pathway, critically reduces the expression of STAT3, Akt and β-catenin. In addition, analysis of mRNA expression level of three main effectors of Gα15 (PLCβ1, PLCβ2 and TPR1) revealed only a significant decreased expression of PLCβ2 in Gα15-KO cells, suggesting a common mechanism between PLCβ2 and Gα15 in GBMs. In agreement with these data we hypothesized that Gα15 (1) stimulates at least ERK and STAT3 activation via PLCβ2/PKC pathway promoting high proliferation/survival of GBM cells, (2) stimulates PI3K/Akt pathway resulting in the expression and/or activation of NF-κB and β-catenin to regulate numerous aggressive gene expression, (3) probably regulates the actin polymerization through PLCβ2 and stimulates intracellular signaling pathways (ERK, Akt…) associated with adhesion protein paxillin and vinculin. To further understand whether gliomas aggressiveness regulated by Gα15 is associated to the regulation of mesenchymal transition, we analyzed by RT-qPCR or immunocytochemistry and demonstrated that Gα15 knockout in GBM cells is sufficient to significantly decrease (1) the mRNA expression of several mesenchymal transcription factors (TFs) including C/EBPβ, RUNX1, FOSL2, BHLHB2, ZEB1, N-cadherin membrane translocation, (2) pro-angiogenic markers (Angiogenin and VEGFA), (3) the expression of inflammatory marker SERPINA1 and a stemness TF KLF4. However, the mRNA expression of potential tumor repressors (DKK1, ZNF238) as well as the stemness gene SOX2 which is a key activator of DKK1 pathway, were significantly decreased in Gα15-KO cells. Collectively, these results highlight the essential role of Gα15 in the expression of oncogenic markers as well as in the acquisition of glioma mesenchymal phenotype, promoting the establishment of immunosuppressive and angiogenic microenvironment supporting GBM aggressiveness. Finally, to evaluate the importance of Gα15 in the growth and invasiveness of GBMs in vivo, orthotopic xenografts of U87 controls, two U87 Gα15-KO clones and U87 overexpressing Gα15 were performed in Nude mice, intrastrially. Our results show a significant improved survival of mice when Gα15 is knockout (prolongs the median survival to 60 and 84 days for KO 26-1 (p=0,0436) and 26-6 (p=0,0016) respectively vs 44 days for the control), and its overexpressing cells xenografted in Nude mice exhibit the poorest survival rates (50 days vs 61 days for the control, p = 0,0326). These results strongly suggest that Gα15 subunit is involved in GBM aggressiveness in vivo. In conclusion, our results show that Gα15 expression is sufficient to maintain the survival, to stimulate the migration of GBM cells, and to promote the expression or activation of factors involved in the mesenchymal transition, angiogenesis, stemness and inflammation status. Although additional experiments are needed to confirm the pathways and mechanisms associated with Gα15, these original works open important perspectives on the diagnostic potential of Gα15 for mesenchymal and inflammatory GBMs, and therapeutics to target the most invasive and resistant GBMs.
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Kleouforo Paul Dembele. Etude du rôle des protéines G dans les gliomes de haut grade : implication dans la migration et le phénotype mésenchymateux. Cancer. Université Rouen Normandie, 2019. Français. ⟨tel-02435413⟩

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