Amanda Chan

The Guanine Nucleotide Exchange Factors Trio, Ect2, and Vav3 Mediate the Invasive Behavior of Glioblastoma

Malignant gliomas are characterized by their ability to invade normal brain tissue. We have previously shown that the small GTPase Rac1 plays a role in both migration and invasion in gliomas. Here, we aim to identify Rac-activating guanine nucleotide exchange factors (GEFs) that mediate glioblastoma invasiveness. Using a brain tumor expression database, we identified three GEFs, Trio, Ect2, and Vav3, that are expressed at higher levels in glioblastoma versus low-grade glioma. The expression of these GEFs is also associated with poor patient survival. Quantitative real-time polymerase chain reaction and immunohistochemical analyses on an independent set of tumors confirmed that these GEFs are overexpressed in glioblastoma as compared with either nonneoplastic brain or low-grade gliomas. In addition, depletion of Trio, Ect2, and Vav3 by siRNA oligonucleotides suppresses glioblastoma cell migration and invasion. Depletion of either Ect2 or Trio also reduces the rate of cell proliferation. These results suggest that targeting GEFs may present novel strategies for anti-invasive therapy for malignant gliomas.

The small GTPase RhoG mediates glioblastoma cell invasion.

BackgroundThe invasion of glioblastoma cells into regions of the normal brain is a critical factor that limits current therapies for malignant astrocytomas. Previous work has identified roles for the Rho family guanine nucleotide exchange factors Trio and Vav3 in glioblastoma invasion. Both Trio and Vav3 act on the small GTPase RhoG. We therefore examined the role of RhoG in the invasive behavior of glioblastoma cells.ResultsWe found that siRNA-mediated depletion of RhoG strongly inhibits invasion of glioblastoma cells through brain slices ex vivo. In addition, depletion of RhoG has a marginal effect on glioblastoma cell proliferation, but significantly inhibits glioblastoma cell survival in colony formation assays. We also observed that RhoG is activated by both HGF and EGF, two factors that are thought to be clinically relevant drivers of glioblastoma invasive behavior, and that RhoG is overexpressed in human glioblastoma tumors versus non-neoplastic brain. In search of a mechanism for the contribution of RhoG to the malignant behavior of glioblastoma cells, we found that depletion of RhoG strongly inhibits activation of the Rac1 GTPase by both HGF and EGF. In line with this observation, we also show that RhoG contributes to the formation of lamellipodia and invadopodia, two functions that have been shown to be Rac1-dependent.ConclusionsOur functional analysis of RhoG in the context of glioblastoma revealed a critical role for RhoG in tumor cell invasion and survival. These results suggest that targeting RhoG-mediated signaling presents a novel avenue for glioblastoma therapy.

Abstract LB-40: Guanine nucleotide exchange factors Dock7 and Ect2 mediate HGF/c-MET-induced glioblastoma cell invasion.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Glioblastoma multiforme (GBM) is the most common and lethal primary malignant brain tumor. It is an incurable disease with an average life expectancy of just 14 months following diagnosis. GBM morbidity is primarily due to its rapid growth, neovascularization and invasion throughout the brain. The angiogenesis inhibitor, bevacizumab, now approved for GBM treatment, has been shown to cause enhanced tumor cell invasion in part via c-MET (HGF-receptor) signaling. We have previously shown that several members of the Rho family of small GTPases, including Rac1, Rac3, RhoG and Cdc42, play central roles in GBM tumor cell invasion. Rho GTPases are activated by guanine nucleotide exchange factors (Rho GEFs). Herein we focus on identifying GEFs that act on Rac subfamily members and which are of functional importance in GBM. We found that Dock7 and Ect2, two GEFs that can activate Rac proteins, show elevated mRNA and protein expression in human GBM tissue in comparison with non-neoplastic brain. RNAi-mediated depletion of these GEFs in GBM cell lines resulted in decreased HGF- and serum-induced tumor cell invasion in 3-dimensional extracellular matrix in vitro and organotypic ex vivo brain slice invasion assays. GEF activity of Dock7 and Ect2 were shown to be increased by HGF using a pull-down with Rac1G15A, a nucleotide binding-deficient mutant of Rac1. Furthermore, Dock7 and Ect2 were shown to be required for HGF-induced Rac1 activation, lamellipodia formation and cell spreading. Dock7, but not Ect2, was shown to co-immunoprecipitate with c-MET and this interaction was HGF-dependent and required the c-MET adaptor protein GAB1. Dock7 and GAB1 were furthermore shown to co-immunoprecipitate in an HGF-dependent manner. GAB1 was shown to be required for HGF-induced Dock7 and Ect2 Rac1 GEF activities, Rac1 activation and HGF-induced GBM cell invasion. In summary, we have identified two GEFs that are of functional and clinical relevance in the development of the GBM pathologic phenotype. Dock7 and Ect 2 represent potential new drug targets for the management of GBM, especially in combination with an anti-angiogenic approach. Citation Format: David W. Murray, Sebastien Didier, Vincent M. Paulino, Amanda Chan, Nhan L. Tran, Annette T. Byrne, Marc H. Symons. Guanine nucleotide exchange factors Dock7 and Ect2 mediate HGF/c-MET-induced glioblastoma cell invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-40. doi:10.1158/1538-7445.AM2013-LB-40

Abstract LB-515: Guanine nucleotide exchange factor Dock7 expression is increased in human glioblastoma and mediates tumor cell invasion.

Glioblastoma multiforme (GBM), the most common and deadly adult brain malignancy, is an incurable disease with an average life expectancy of 14 months following diagnosis. GBM morbidity is primarily due to its rapid growth, neovascularization and invasion throughout the brain. Recently, anti-VEGF therapies approved in this indication have paradoxically been shown to stimulate the invasive behavior of GBM. Members of the Rho family of small GTPases have been established as important mediators of GBM cell invasion. These proteins are activated by guanine nucleotide exchange factors (GEFs). In the current study, we sought to identify GEFs that are up-regulated in high-grade glioma and which may be of functional importance in human GBM. In this study we have shown that that Dock7, a GEF that can mediate signaling of Rac1 and Cdc42 Rho GTPases is up-regulated in human GBM tissue in comparison with normal brain. We have further demonstrated that Dock7 silencing by RNA-interference inhibits invasion of U87 and SNB19 GBM cells into ex vivo brain slices. Dock7 silencing inhibits Hepatocyte Growth Factor (HGF)-induced cell invasion in a 3D in vitro assay. HGF is known to strongly stimulate GBM cell invasiveness and both HGF and its receptor c-MET have been shown to be up-regulated in high- versus low-grade gliomas. Interestingly, we have also shown that Dock7 binds to c-MET in an HGF-dependent manner and that this interaction requires the GAB1 adaptor protein. Dock7 silencing reduced HGF-induced activation of Rac1 and consistent with this, we have shown that Dock7 silencing results in the inhibition of HGF-induced lamellipodia formation in GBM cells. Finally Dock7 was shown to be activated in an HGF-dependant manner, using Rac1-G15A pull-down assay. In summary, our data suggests a role for Dock7 in GBM cell invasion thus, Dock7 may present a potential therapeutic target in the management of invasive GBM, in particular in the adjuvant setting in combination with anti-VEGF therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-515. doi:1538-7445.AM2012-LB-515

Abstract 3846: SGEF is over-expressed in advanced glial tumors and mediates glioma cell invasion and survival

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Glioblastoma multiforme (GBM) is the most malignant of all primary adult brain tumors, histopathologically characterized by the infiltrative capacity of glioma cells to diffuse into the surrounding normal brain. There are currently no anti-invasion therapies available, and thus the identification and functional understanding of genes that mediate malignant tumor cell dispersion could lead to the discovery of molecular targets which have the potential to respond to therapeutics. We have previously shown that several guanine nucleotide exchange factors for Rho family small GTPases are overexpressed in GBM tumors and play important roles in glioma invasion. Here we report a role for SGEF, a guanine nucleotide exchange factor (GEF) for RhoG, in mediating glioma invasion. SGEF mRNA expression increases in correlation with glioma grade and, within GBM tumors, levels of SGEF expression inversely correlate with patient survival. siRNA-mediated depletion of SGEF decreases in vitro glioma cell migration and ex vivo glioma cell invasion. In addition, genome-wide determination of NF-κB controlled genes in temozolomide-resistant primary GBM xenografts (GBM14-TMZ-R) revealed an increased occupancy of NF-κB on the SGEF gene promoter region via ChIP-on-chip analysis as compared to the parent primary line. In fact, increased phosphorylation of IkBa was detected in GBM-14-TMZ-R. Moreover, inhibition of NF-κB in GBM14-TMZ-R significantly decreases SGEF gene expression, and activation of the NF-κB pathway by TWEAK in GBM cells induces SGEF mRNA expression. Furthermore, siRNA-mediated depletion of SGEF expression enhances chemotherapy-induced cell death in glioma cells. Understanding the role of SGEF in promoting cell motility and chemotherapeutic resistance may direct the development of novel targeted therapeutics for GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3846. doi:10.1158/1538-7445.AM2011-3846

Abstract 5128: Identification of RhoGEFs within the TWEAK-R (Fn14)-Rac1 signaling pathway that mediate glioblastoma cell invasion

Glioblastoma multiforme (GBM) is the most malignant primary adult brain tumor. Despite efforts at surgical resection of the glioma mass, invasive cells are always left behind and the tumor will inevitably recur and kill the patient. As such, novel therapeutics targeting pro-invasive factors could improve neurological outcomes and survival for these patients. This requires a detailed understanding of the mechanisms driving glioma migration and invasion. We have been investigating whether the multifunctional cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) regulate glioma cell invasive activity. We have previously demonstrated that Fn14 expression was elevated in the invasive rim of GBM specimens and migrating glioma cells in vitro. The Fn14 signaling axis is known to drive glioma invasion via Rac1. We have previously reported that Ect2, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, including Rac1 and Cdc42, is overexpressed in GBM, and that overexpression of Ect2 correlates directly with tumor grade and inversely with patient survival. In this study we show that Ect2 regulates Rac1 activation downstream of Fn14. Depletion of Ect2 by siRNA duplexes abrogates Fn14-induced Rac1 activation and subsequent glioma cell migration and invasion. We also found that Cdc42 activation by TWEAK is directly mediated by Ect2. Interestingly, depletion of Cdc42 expression impairs TWEAK-induced Rac1 activation and also results in a significant reduction of glioma cell migration and invasion. This suggests that Cdc42 is, in part, important for Rac1 activation downstream of the TWEAK-Fn14 signaling pathway and argues that another GEF(s) may be involved in the Fn14-Rac1 signaling axis. Recently, we have identified Trio as an additional GEF that activates Rac1 downstream of Cdc42. It is also known that Trio expression correlates directly with brain tumor grade and inversely with patient survival. siRNA-mediated depletion of Trio inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation. Therefore, delineating the mechanisms of Fn14-RhoGEF-RhoGTPase signaling pathway, may lead to identification of novel targets that can serve as possible points of therapeutic intervention. (Supported by NIH R01-CA130940) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5128.