The invasion of tumor cells into brain tissue is a pathologic hallmark of malignant gliomas and contributes to treatment failures. Diffuse glioblastomas contain numerous microglial cells, which enhance the progression of gliomas; however, factors responsible for invasionpromoting role of microglia are unknown. Transforming growth factor-b (TGF-b) can enhance tumor growth, invasion, angiogenesis and immunosuppression. Antagonizing TGF-b activity has been shown to inhibit tumor invasion in vitro and tumorigenicity, but a systemic inhibition or lack of TGF-b signaling results in acute inflammation and disruption of immune system homeostasis. We developed plasmid-transcribed small hairpin RNAs (shRNAs) to downregulate the TGF-b type II receptor (TbIIR) expression, which effectively inhibited cytokine-induced signaling pathways and transcriptional responses in transiently transfected human glioblastoma cells. Silencing of TbIIR abolished TGF-b-induced glioblastoma invasiveness and migratory responses in vitro. Moreover, tumorigenicity of glioblastoma cells stably expressing TbIIR shRNAs in nude mice was reduced by 50%. Microglia strongly enhanced glioma invasiveness in the co-culture system, but this invasion-promoting activity was lost in glioma cells stably expressing shTbRII, indicating a crucial role of microglia-derived TGF-b in tumor-host interactions. Our results demonstrate a successful targeting of TGF-b-dependent invasiveness and tumorigenicity of glioblastoma cells by RNAi-mediated gene silencing.

Glioblastoma (GBM) is the most aggressive form of brain cancer, with a dismal prognosis and extremely low percentage of survivors. Novel therapies are in dire need to improve the clinical management of these tumors and extend patient survival. Genetic therapies for GBM have been postulated and attempted for the past twenty years, with variable degrees of success in pre-clinical models and clinical trials. Here we review the most common approaches to treat GBM by gene therapy, including strategies to deliver tumor-suppressor genes, suicide genes, immunomodulatory cytokines to improve immune response, and conditionally-replicating oncolytic viruses. The review focuses on the strategies used for gene delivery, including the most common and widely used vehicles (i.e., replicating and non-replicating viruses) as well as novel therapeutic approaches such as stem cell-mediated therapy and nanotechnologies used for gene delivery. We present an overview of these strategies, their targets, different advantages, and challenges for success. Finally, we discuss the potential of gene therapy-based strategies to effectively attack such a complex genetic target as GBM, alone or in combination with conventional therapy.

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.

Tumor cell invasiveness is a critical challenge in the clinical management of glioma patients. In addition, there is accumulating evidence that current therapeutic modalities, including anti-angiogenic therapy and radiotherapy, can enhance glioma invasiveness. Glioma cell invasion is stimulated by both autocrine and paracrine factors that act on a large array of cell surface-bound receptors. Key signaling elements that mediate receptor-initiated signaling in the regulation of glioblastoma invasion are Rho family GTPases, including Rac, RhoA and Cdc42. These GTPases regulate cell morphology and actin dynamics and stimulate cell squeezing through the narrow extracellular spaces that are typical of the brain parenchyma. Transient attachment of cells to the extracellular matrix is also necessary for glioblastoma cell invasion. Interactions with extracellular matrix components are mediated by integrins that initiate diverse intracellular signalling pathways. Key signaling elements stimulated by integrins include PI3K, Akt, mTOR and MAP kinases. In order to detach from the tumor mass, glioma cells secrete proteolytic enzymes that cleave cell surface adhesion molecules, including CD44 and L1. Key proteases produced by glioma cells include uPA, ADAMs and MMPs. Increased understanding of the molecular mechanisms that control glioma cell invasion has led to the identification of molecular targets for therapeutic intervention in this devastating disease.

Human malignant glioblastomas are highly invasive tumors. Increased cell motility and degradation of the surrounding extracellular matrix are essential for tumor invasion. PI3K/Akt signaling pathway emerges as a common pathway regulating cellular proliferation, migration and invasion; however, its contribution to particular process and downstream cascades remain poorly defined. We have previously demonstrated that Cyclosporin A (CsA) affects glioblastoma invasion in organotypic brain slices and tumorigenicity in mice. Here we show that CsA impairs migration and invasion of human glioblastoma cells by downregulation of Akt phosphorylation. Interference with PI-3K/Akt signaling was crucial for CsA effect on invasion, because overexpression of constitutively active myr-Akt antagonized drug action. Furthermore, the drug was not effective in T98G glioblastoma cells with constitutively high level of phosphorylated Akt. CsA, comparably to pharmacological inhibitors of PI3K/Akt signaling (LY294002, A443654), reduced motility of glioblastoma cells, diminished MMP-2 gelatinolytic activity and MMP-2 and MT1-MMP expression. The latter effect was mimicked by overexpression of dominant negative Akt mutants. We demonstrate that CsA and LY294002 reduced MMP transcription partly via modulation of IκB phosphorylation and NFκB transcriptional activity. Those effects were not mediated by inhibition of calcineurin, a classical CsA target. Additionally, CsA reduced phosphorylation and activity of focal adhesion kinase that was associated with rapid morphological alterations, rearrangement of lamellipodia and impairment of MT1-MMP translocation to membrane protrusions. Our results document novel, Akt-dependent mechanisms of interference with motility/invasion of human glioblastoma cells: through a rapid modulation of cell adhesion and MT1-MMP translocation to membrane protrusions and delayed, partly NFκB-dependent, downregulation of MMP-2 and MT1-MMP expression. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

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Molecular profiling of glioblastomas has revealed the presence of key signaling hubs that contribute to tumor progression and acquisition of resistance. One of these main signaling mechanisms is the NF-κB pathway, which integrates multiple extracellular signals into transcriptional programs for tumor growth, invasion, and maintenance of the tumor-initiating population. We show here that an extracellular protein released by glioblastoma cells, fibulin-3, drives oncogenic NF-κB in the tumor and increases NF-κB activation in peritumoral astrocytes. Fibulin-3 expression correlates with a NF-κB-regulated “invasive signature” linked to poorer survival, being a possible tissue marker for regions of active tumor progression. Accordingly, fibulin-3 promotes glioblastoma invasion in a manner that requires NF-κB activation both in the tumor cells and their microenvironment. Mechanistically, we found that fibulin-3 activates the metalloprotease ADAM17 by competing with its endogenous inhibitor, TIMP3. This results in sustained release of soluble TNFα by ADAM17, which in turn activates TNF receptors and canonical NF-κB signaling. Taken together, our results underscore fibulin-3 as a novel extracellular signal with strong activating effect on NF-κB in malignant gliomas. Because fibulin-3 is produced de novo in these tumors and is absent from normal brain we propose that targeting the fibulin-3/NF-κB axis may provide a novel avenue to disrupt oncogenic NF-κB signaling in combination therapies for malignant brain tumors.

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