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Etude des voies de signalisation associées aux effets du peptide vasoactif urotensine II sur la migration et l'adhésion des cellules tumorales gliales

Abstract : Chemotaxis is a guiding mechanism including a first processing phase through diffusion of a gradient concentration of specific mediators, and a second detection phase of this chemical gradient (defined as a chemoattractant or chemorepellant signal), by cell or organisms, and the adaptation of the movement direction. Although chemotaxis and directional migration finally represent universal mechanisms, and that detailed involved processes are widely studied, they constitute a major challenge for research exhibiting a number of physiopathological applications from embryogenesis, tissue repair and regeneration, inflammation to cancer. The development of cerebral glial tumors required specific features as tumoral cell invasion, neoangiogenesis and chemoattraction of proangiogenic macrophage cells. Despite intense research in neuro-oncology, the survival median of patients with high-grade tumors such as glioblastoma is only 15 months. Thus, G protein-coupled receptors (GPCRs) and their ligands expressed in glioblastoma and relaying effects on migration, invasion and angiogenesis, represent potential therapeutic targets. GPCRs are indeed able to recruit heterotrimeric G proteins and tyrosine kinase receptors (RTKs) likely through the formation of macromolecular complexes involving GPCR C-terminal cytosolic domains. Among major chemotaxis players, GPCR ligands, i.e. chemokines, lipids and vasoactive peptides, stimulate migration/invasion of various cell types, including tumor cells. Urotensin II (UII) and its paralog urotensin II-related peptide (URP) are the endogenous ligands of a GPCR named UT and are considered as most potent vasoactive factors characterized to date. The work performed in the team had previously shown the expression of UT in rat cortical astrocytes, in human astrocytes and in a high grade glioma cell line, and a specific role of UII in astrocyte proliferation/survival. These data suggested that the urotensinergic system would play a major role in astrocyte activity in physiological and pathophysiological conditions, and contribute to the development of high grade glioma. In this context, the aim of my thesis work was to determine the role of the urotensinergic system in the development of brain tumors of glial origin through in vitro investigation of signaling events relayed by UT. This research project aimed to i) investigate urotensinergic system expression in glioma, determine the effects of UII on proliferation, migration and adhesion of glial tumor cells and characterize the signaling pathways involved in these effects using pharmacological and molecular tools, ii) identify, by using two-hybrid strategy, protein partners of the C-terminal cytosolic domain of UT, potentially involved in transduction pathways activated by UII and iii) identify by means of a phosphoproteomics approach, a set of proteins phosphorylated and dephosphorylated in a glioblastoma cell line exposed to the UII peptide. In the first part of my work, we showed the expression of UT and UII in human astrocytoma and glioblastoma cell lines, in tumor explants from patients as well as in glioblastoma tissue sections obtained in collaboration with the anatomocytopathology department of the Rouen University Hospital. In fresh glioblastoma tumor explants and glioma cell lines, we demonstrated that UT was not coupled to the classical IP3/Ca2+pathway ang failed to modify proliferation, cell cycle progression as wel as ERK1/2 and AKT pathways. We have established for the first time that a gradient of low UII concentration induced chemotaxic migration of glioma cells. This effect was concentration-dependent and blocked by peptidic (urantide) and non-peptidic (palosuran) UT antagonists. Pharmacological agents, i.e. Gi/o (PTX), PI3K (LY294002), and ROCK (Y27632) inhibitors, and molecular tools (UT mutated in the G protein interacting motif ERY or siRNA anti Gα13), highlighted that UII-mediating migration is mainly relayed by the G13/Rho/ROCK cascade but also involved the Gi/o/PI3K pathway. Conversely, a high homogeneous concentration of UII inhibited cell motility and stimulated cell-matrix adhesions likely through actin polymerization, formation and maturation of focal adhesions. Glioma cell-matrix adhesion was shown to be mainly mediated by Gi/o and partially involved PI3K. In conclusion, these results suggest that UT behaves like a professional chemotaxic receptor activating a signaling switch between the directional cell migration and adhesion in response to a gradient or a uniform concentration of UII respectively and thus should play a role during high-grade glioma development. In the second part of this work, by means of the two-hybrid system, we aimed to characterize protein partners of the UT C-terminal domain. Thus, we identified 8 proteins including filamin A, an actin binding protein classically known to be involved in cell adhesion and migration. We showed that this large and multidomain protein interacted with the 332-351 C-terminal fragment of the human UT, through its 16 to 19 domains. We obtained some encouraging data confirming the interaction between UT and filamin A by GST-pull down, but our co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) experiments have to be reproduced with modifications in the strategy. By using cell lines deficient for filamin A (M2) or stably expressing filamin A (A7), we determined that this multifunctional protein is important for the dimerization of UT and the subcellular localization of the receptor. By immunocytochemistry, we observed that UT is colocalized with filamin A at the lamellipodia in A7 cells, or is mainly localized at the plasma membrane in M2 cells. After exposure to the agonist, UT appeared translocated to the plasma membrane in A7 whereas internalized (punctiform cytoplasmic labeling) in M2 cells. In both cell types (M2 and A7), UII stimulated cell migration and actin polymerization with a more pronounced effect on cells lacking filamin A. By flow cytometry and immunocytochemistry, we finally detected high filamin A expression in glioma cell lines U87 and SW1088 and in glioblastoma explants from patients. In these tumor cells, filamin A was colocalized with UT in protrusions and in cells exhibiting a fusiform morphology, and UII led to the dephosphorylation (Ser 2151) and cleavage of filamin A. Taken together, these results suggest that filamin A favors the formation of oligomeric structures, the cytosolic localization of UT, the translocation of UT at the plasma membrane and plays a role in cell migration. One hypothesis is that translocation of UT oligomers to the plasma membrane maybe subsequent to the activation of a subpopulation of mobile membrane UT, relays within lipid rafts, signaling pathways of cell adhesion. To identify the cellular actors of migration and adhesion, effectors of the urotensinergic system, we conducted a phosphoproteomics analysis of glioma cell line U87 in the absence or the presence of UII. This study, conducted in collaboration with the Proteomic platform PISSARO (Philipe Chan, Pascal Cosette, Julie Hardouin, Antoine Obry) allowed us to identify 13 and 32 proteins whose levels of phosphorylation and dephosphorylation were increased. Among them, 13 including filamin A, filamin C, LIMK or talin are involved in cell migration and/or adhesion. Validation by Western blot and the more detailed study of the identified proteins, will allow us to better understand the complexity of UT signaling pathways relaying mechanisms of migration and adhesion. Thus, we aim to initiate the development of urotensinergic ligands that would only activate adhesion pathways of glial tumor cells. Such ligands, promoting cell-matrix and cell-cell adhesions would confine the tumor mass by inhibiting invasion, neoangiogenesis, and chemoattraction of proangiogenic macrophages.
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Céline Lecointre. Etude des voies de signalisation associées aux effets du peptide vasoactif urotensine II sur la migration et l'adhésion des cellules tumorales gliales. Sciences du Vivant [q-bio]. université de Rouen, 2013. Français. ⟨tel-02415633⟩

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