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.

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 is a guanine-nucleotide exchange factor (GEF) for Rap1, although it can act through GEF-independent mechanisms. C3G plays a dual role in cancer, acting as either a tumor suppressor or inducer depending on the tumor type/stage. It regulates different key aspects of the tumorigenic process such as invasion, apoptosis or proliferation. In colon carcinoma, C3G represses invasion through down-regulation of p38αMAPK activity and promotes tumor growth. We have now analyzed the role of C3G in glioblastoma (GBM), a tumor characterized by its aggressiveness and disseminative capacity. To do it, we have used different experimental approaches: permanent gene silencing, knock-out using CRISPR/Cas9 technology and transient overexpression in U87 cell line and patient-derived GBM cells. We found that C3G down-regulation enhances invasion through the induction of an epithelial/glial to mesenchymal transition-like process. C3G deficiency also facilitates foci generation in anchorage-dependent and independent growth assays, but with lower cell density as a consequence of a reduced proliferation. In in vivo analyses, cells with C3G knock-down generated larger tumors in xenografts and chick chorioallantoic membrane xenografts assays with increased angiogenesis and α-SMA+ fibroblasts, but a lower proliferation. Mechanistically, C3G down-regulation impairs EGF/EGFR signaling by decreasing EGFR cell membrane localization, leading to a reduction in EGF/EGFR-induced invasiveness. In contrast, C3G down-regulation promotes the activation of several receptor tyrosine kinases (RTKs) that might promote invasion. In particular, the enhanced FGF2/FGFR1 signaling increases invasion in C3G-silenced cells, through a mechanism likely dependent on ERKs. Moreover, a proteomic analysis revealed that C3G down-regulation increases the levels of different key glycolytic enzymes, such as aldolase, phosphoglycerate kinase, enolase and pyruvate kinase (PK). The activity of PK and lactate dehydrogenase as well as lactate release to the extracellular environment were also increased upon C3G silencing. In conclusion, our data demonstrate that C3G inhibits invasion of GBM cells, while increasing their proliferative capacity. Moreover, we show a distinct dependency on C3G for EGF/EGFR signaling versus other RTKs, suggesting that assessing C3G levels may discriminate GBM patient responders to different RTK inhibitors. In addition, our results indicate that C3G regulates not only GBM growth and invasiveness, but it also contributes to reprogram its glycolytic metabolism. Hence, low levels of C3G correlate with a more mesenchymal and glycolytic phenotype with enhanced aggressiveness and worse patient prognosis. This promising role of C3G as a key player in GBM should be further characterized to define its prognostic value and its potential relevance as a predictor of the response to therapy. Citation Format: Sara Manzano, Óscar Herranz, Paloma Bragado, Patricia Jáuregui, María Rodrigo, Celia Sequera, Cristina Baquero, Nerea Palao, Ignacio Rubio, Álvaro Gutierrez-Uzquiza, Carmen Guerrero, Almudena Porras. C3G down-regulation in glioblastoma induces a pro-invasive and glycolytic phenotype, accompanied by RTKs dysregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1974.