Platelet C3G: a key player in vesicle exocytosis, spreading and clot retraction.

C3G is a Rap1 GEF that plays a pivotal role in platelet-mediated processes such as angiogenesis, tumor growth, and metastasis by modulating the platelet secretome. Here, we explore the mechanisms through which C3G governs platelet secretion. For this, we utilized animal models featuring either overexpression or deletion of C3G in platelets, as well as PC12 cell clones expressing C3G mutants. We found that C3G specifically regulates α-granule secretion via PKCδ, but it does not affect δ-granules or lysosomes. C3G activated RalA through a GEF-dependent mechanism, facilitating vesicle docking, while interfering with the formation of the trans-SNARE complex, thereby restricting vesicle fusion. Furthermore, C3G promotes the formation of lamellipodia during platelet spreading on specific substrates by enhancing actin polymerization via Src and Rac1-Arp2/3 pathways, but not Rap1. Consequently, C3G deletion in platelets favored kiss-and-run exocytosis. C3G also controlled granule secretion in PC12 cells, including pore formation. Additionally, C3G-deficient platelets exhibited reduced phosphatidylserine exposure, resulting in decreased thrombin generation, which along with defective actin polymerization and spreading, led to impaired clot retraction. In summary, platelet C3G plays a dual role by facilitating platelet spreading and clot retraction through the promotion of outside-in signaling while concurrently downregulating α-granule secretion by restricting granule fusion.

New functions of C3G in platelet biology: Contribution to ischemia-induced angiogenesis, tumor metastasis and TPO clearance.

C3G is a Rap1 guanine nucleotide exchange factor that controls platelet activation, aggregation, and the release of α-granule content. Transgenic expression of C3G in platelets produces a net proangiogenic secretome through the retention of thrombospondin-1. In a physiological context, C3G also promotes megakaryocyte maturation and proplatelet formation, but without affecting mature platelet production. The aim of this work is to investigate whether C3G is involved in pathological megakaryopoiesis, as well as its specific role in platelet mediated angiogenesis and tumor metastasis. Using megakaryocyte-specific C3G knockout and transgenic mouse models, we found that both C3G overexpression and deletion promoted platelet-mediated angiogenesis, induced by tumor cell implantation or hindlimb ischemia, through differential release of proangiogenic and antiangiogenic factors. However, only C3G deletion resulted in a higher recruitment of hemangiocytes from the bone marrow. In addition, C3G null expression enhanced thrombopoietin (TPO)-induced platelet production, associated with reduced TPO plasma levels. Moreover, after 5-fluorouracil-induced platelet depletion and rebound, C3G knockout mice showed a defective return to homeostatic platelet levels, indicating impaired platelet turnover. Mechanistically, C3G promotes c-Mpl ubiquitination by inducing Src-mediated c-Cbl phosphorylation and participates in c-Mpl degradation via the proteasome and lysosome systems, affecting TPO internalization. We also unveiled a positive role of platelet C3G in tumor cell-induced platelet aggregation, which facilitated metastatic cell homing and adhesion. Overall, these findings revealed that C3G plays a crucial role in platelet-mediated angiogenesis and metastasis, as well as in platelet level modulation in response to pathogenic stimuli.

C3G downregulation induces the acquisition of a mesenchymal phenotype that enhances aggressiveness of glioblastoma cells.

Glioblastoma (GBM) is the most aggressive tumor from the central nervous system (CNS). The current lack of efficient therapies makes essential to find new treatment strategies. C3G, a guanine nucleotide exchange factor for some Ras proteins, plays a dual role in cancer, but its function in GBM remains unknown. Database analyses revealed a reduced C3G mRNA expression in GBM patient samples. C3G protein levels were also decreased in a panel of human GBM cell lines as compared to astrocytes. Based on this, we characterized C3G function in GBM using in vitro and in vivo human GBM models. We report here that C3G downregulation promoted the acquisition of a more mesenchymal phenotype that enhanced the migratory and invasive capacity of GBM cells. This facilitates foci formation in anchorage-dependent and -independent growth assays and the generation of larger tumors in xenografts and chick chorioallantoic membrane (CAM) assays, but with a lower cell density, as proliferation was reduced. Mechanistically, C3G knock-down impairs EGFR signaling by reducing cell surface EGFR through recycling inhibition, while upregulating the activation of several other receptor tyrosine kinases (RTKs) that might promote invasion. In particular, FGF2, likely acting through FGFR1, promoted invasion of C3G-silenced GBM cells. Moreover, ERKs mediate this invasiveness, both in response to FGF2- and serum-induced chemoattraction. In conclusion, our data show the distinct dependency of GBM tumors on C3G for EGF/EGFR signaling versus other RTKs, suggesting that assessing C3G levels may discriminate GBM patient responders to different RTK inhibition protocols. Hence, patients with a low C3G expression might not respond to EGFR inhibitors.