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

Abstract

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 down-regulation enhances pro-migratory and stemness properties of oval cells by promoting an epithelial-mesenchymal-like process

“…Considering that migration is necessary for the expansion of oval cells to repair the chronically damaged liver, and that C3G regulates cell adhesion, migration and invasion 21 , 23 , 26 , we evaluated the effect of C3G down-regulation on these cellular processes. The analysis of cell migration using wound healing assays (Fig.…”

“…Previous data from the literature have demonstrated the relevance of TGF-β-induced EMT 8 for oval cell pro-regenerative capacity upon liver damage. C3G down-regulation is known to promote the acquisition of a more mesenchymal phenotype in HCC and glioblastoma cells 23 , 26 . Hence, we analyzed the potential induction of an EMT process in oval cells by C3G knock-down as a mechanism to enhance migration and invasion.…”

“…To determine the mechanism responsible for this reduced HGF/MET signaling in oval cells with C3G down-regulation, we search for a potential alteration in MET membrane localization and/or recycling as C3G facilitates recycling and membrane localization of EGFR in glioblastoma (GBM) cells 26 . However, we did not find significant changes in the levels of MET in the surface of shC3G oval cells (Fig.…”

“…Cells seeded on 2% gelatin-coated glass coverslips were fixed with 4% PFA for 20 min. F-actin was stained with rhodamine-conjugated phalloidin (Sigma Aldrich#P1951) as described 26 . Images were analyzed by confocal microscopy.…”

Glioblastoma Multiforme Selective Nanomedicines for Improved Anti-Cancer Treatments

“…Further evidence has also linked CSV to tumour growth, evidenced by its upregulation in various cancer types ranging from oral cancer, breast cancer, colon cancer, prostate cancer etc [53][54][55][56][57]. The presence of CSV in brain cancer and model cell lines has also been shown for numerous tumours in the CNS; however, the amounts of CSV are highly dependent on cell type, patient, and also treatment regimens [58][59][60][61]. While CSV is often upregulated in GBM, it has also been shown to be expressed at high levels in the brain endothelium cells, suggesting that these ligands could help in both BBB crossing and specifically targeting GBM cells [61][62][63][64].…”