Abstract 1974: C3G down-regulation in glioblastoma induces a pro-invasive and glycolytic phenotype, accompanied by RTKs dysregulation
Authors: Manzano Sara , Herranz Óscar , Bragado Paloma , Jáuregui Patricia , Rodrigo María , Sequera Celia , Baquero Cristina , Palao Nerea , Rubio Ignacio , Gutierrez-Uzquiza Álvaro , Guerrero Carmen , Porras Almudena 
Published: July 2021

Abstract
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.