Inhibited Glioblastoma Cell Invasion Research Articles

SMAGP knockdown inhibits the malignant phenotypes of glioblastoma cells by inactivating the PI3K/Akt pathway

Small trans-membrane and glycosylated protein (SMAGP), a novel small trans-membrane glycoprotein, is reported to be upregulated in multiple cancers and involved in tumor development. However, little is known about its role in the development of glioblastoma (GBM). GEPIA database was used to analyze SMAGP expression and evaluate the prognostic value of SMAGP in GBM. GO and KEGG pathway enrichment analyses were used to predict the biological functions and pathways of SMAGP and 948 SMAGP-correlated genes using DAVID database. Cell viability, colony formation ability, apoptosis, and invasion were evaluated by MTT, colony formation assay, flow cytometry analysis, and Transwell invasion assay, respectively. Western blot was applied to detect the expression of SMAGP, matrix metalloproteinase (MMP)-2, and MMP-9 and analyze the changes of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling. Results showed that SMAGP was upregulated and correlated with poor prognosis in GBM. Functional annotation analysis revealed that SMAGP and 948 SMAGP-correlated genes were primarily associated with cell adhesion and PI3K/Akt pathway. SMAGP interference inhibited cell viability and colony formation ability and promoted apoptosis in GBM cells. Moreover, SMAGP interference inhibited GBM cell invasion and suppressed MMP-2 and MMP-9 expression. Additionally, SMAGP silencing inhibited the PI3K/Akt pathway in GBM cells. Overexpression of Akt abolished the effects of SMAGP knockdown on the malignant phenotypes of GBM cells. In conclusion, SMAGP silencing inhibited the malignant phenotypes of GBM cells by inactivating the PI3K/Akt pathway.

TROY signals through JAK1-STAT3 to promote glioblastoma cell migration and resistance

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and carries a discouraging prognosis. Its aggressive and highly infiltrative nature renders the current standard treatment of maximal surgical resection, radiation, and chemotherapy relatively ineffective. Identifying the signaling pathways that regulate GBM migration/invasion and resistance is required to develop more effective therapeutic regimens to treat GBM. Expression of TROY, an orphan receptor of the TNF receptor superfamily, increases with glial tumor grade, inversely correlates with patient overall survival, stimulates GBM cell invasion in vitro and in vivo, and increases resistance to temozolomide and radiation therapy. Conversely, silencing TROY expression inhibits GBM cell invasion, increases sensitivity to temozolomide, and prolongs survival in a preclinical intracranial xenograft model. Here, we have identified for the first time that TROY interacts with JAK1. Increased TROY expression increases JAK1 phosphorylation. In addition, increased TROY expression promotes STAT3 phosphorylation and STAT3 transcriptional activity that is dependent upon JAK1. TROY-mediated activation of STAT3 is independent of its ability to stimulate activity of NF-κB. Inhibition of JAK1 activity by ruxolitinib or knockdown of JAK1 expression by siRNA significantly inhibits TROY-induced STAT3 activation, GBM cell migration, and decreases resistance to temozolomide. Taken together, our data indicate that the TROY signaling complex may represent a potential therapeutic target with the distinctive capacity to exert effects on multiple pathways mediating GBM cell invasion and resistance.

Irisin, an exercise myokine, potently suppresses tumor proliferation, invasion, and growth in glioma.

Glioblastoma multiforme is the most common and aggressive glial tumor with poor prognosis. Importantly, effective treatment options for glioblastoma are unmet needs. Obesity and low physical activity have been linked with a high risk of cancer, and exercise is related to delayed cancer development and progression. Epidemiological studies have revealed a correlation between exercise and the survival rate of patients with glioblastoma. Nevertheless, the mechanisms by which exercise exerts its anticancer effects in glioblastoma remain unclear. Here, we found that irisin, an exercise-induced myokine, induced G2 /M cell cycle arrest and increased p21 levels in glioblastoma cells, leading to the inhibition of cell proliferation. In addition, irisin inhibited glioblastoma cell invasion by upregulating TFPI-2 and even reversed the aggressive tumor phenotype promoted by co-cultivation with cancer-associated adipocytes. Furthermore, irisin retarded xenograft glioblastoma tumor growth, and radiolabeled irisin demonstrated specific tumor-targeting capability in vivo. Therefore, this study identified one potential molecular mechanism by which exercise prevents cancer progression via irisin. Intriguingly, irisin has the potential to be developed as a molecular imaging and therapeutic anticancer agent.

A Novel Signaling Complex between TROY and EGFR Mediates Glioblastoma Cell Invasion.

Glioblastoma is the most frequent primary brain tumor in adults and a highly lethal malignancy with a median survival of about 15 months. The aggressive invasion of the surrounding normal brain makes complete surgical resection impossible, increases the resistance to radiation and chemotherapy, and assures tumor recurrence. Thus, there is an urgent need to develop innovative therapeutics to target the invasive tumor cells for improved treatment outcomes of this disease. Expression of TROY (TNFRSF19), a member of the tumor necrosis factor (TNF) receptor family, increases with increasing glial tumor grade and inversely correlates with patient survival. Increased expression of TROY stimulates glioblastoma cell invasion in vitro and in vivo and increases resistance to temozolomide and radiation therapy. Conversely, silencing TROY expression inhibits glioblastoma cell invasion, increases temozolomide sensitivity, and prolongs survival in an intracranial xenograft model. Here, a novel complex is identified between TROY and EGFR, which is mediated predominantly by the cysteine-rich CRD3 domain of TROY. Glioblastoma tumors with elevated TROY expression have a statistically positive correlation with increased EGFR expression. TROY expression significantly increases the capacity of EGF to stimulate glioblastoma cell invasion, whereas depletion of TROY expression blocks EGF stimulation of glioblastoma cell invasion. Mechanistically, TROY expression modulates EGFR signaling by facilitating EGFR activation and delaying EGFR receptor internalization. Moreover, the association of EGFR with TROY increases TROY-induced NF-κB activation. These findings substantiate a critical role for the TROY-EGFR complex in regulation of glioblastoma cell invasion.Implications: The TROY-EGFR signaling complex emerges as a potential therapeutic target to inhibit glioblastoma cell invasion. Mol Cancer Res; 16(2); 322-32. ©2017 AACR.

In Silico prediction of the molecular basis of ClTx and AaCTx interaction with matrix metalloproteinase-2 (MMP-2) to inhibit glioma cell invasion

Glioblastoma is the deadliest type of brain cancer. Treatment could target the Matrix metalloproteinase-2 (MMP-2), which is known to be involved in the invasion process of glioblastoma cells. But current available inhibitors are not selective to MMP-2 due to their interaction with the catalytic binding site, which is highly conserved in all MMPs structures. Interestingly, members of the chloride channel blocker scorpion toxins, such as chlorotoxin (ClTx) and AaCTx, inhibit glioblastoma cell invasion and show a promising therapeutic potential. Indeed, it has been shown that CITx inhibits selectively MMP-2 and was also able to cross the blood brain and tissue barriers. Although ClTx and AaCTx show high sequence similarity, AaCTx is ten times less active than ClTx. By using molecular modeling, molecular dynamics and MM-PB(GB)SA free energy estimation, we present the first computational study reporting the interaction mode of ClTx/AaCTx with MMP-2. We found that the two peptides probably act on an exosite of MMP-2 comprising mainly residues from the collagen binding domain, a feature that could be exploited to enhance the selectivity toward MMP-2. van der Waals and hydrophobic forces are the primary mediators of this interaction. The N- and C-termini of the two peptides harbor the key residues of the interaction spread across a conserved amino acid patch. In particular, F6 contributes mostly to the binding free energy in ClTx. We also suggest that the lack of the C-terminal arginine and the residues P10 and R24, might be responsible for altering the activity of AaCTx toward glioblastoma cells compared to ClTx.

MicroRNA-181c inhibits glioblastoma cell invasion, migration and mesenchymal transition by targeting TGF-β pathway

MicroRNAs (miRNAs) are small non-coding RNAs frequently dysregulated in human malignancies. In this study, we found that miR-181c was down-regulated both in glioblastoma tissues and cell lines. We also annotated 566 TCGA miRNA expression profiles and found that patients with high microRNA-181c (miR-181c)-expressing tumors had significantly longer OS and PFS. Overexpression of miR-181c evidently inhibited glioblastoma cell line T98G migration and invasion. Further, the expression of E-cadherin was significantly upregulated and that of N-cadherin and vimentin was significantly down-regulated. We also found that miR-181c overexpression inhibited TGF-β signaling by down-regulating TGFBR1, TGFBR2 and TGFBRAP1 expression. Overall, our study found that miR-181c plays a key role in glioblastoma cell invasion, migration and mesenchymal transition suggesting potential therapeutic applications.

Propentofylline inhibits glioblastoma cell invasion and survival by targeting the TROY signaling pathway.

Glioblastoma (GBM) is the most common primary tumor of the CNS and carries a dismal prognosis. The aggressive invasion of GBM cells into the surrounding normal brain makes complete resection impossible, significantly increases resistance to the standard therapy regimen, and virtually assures tumor recurrence. Median survival for newly diagnosed GBM is 14.6months and declines to 8months for patients with recurrent GBM. New therapeutic strategies that target the molecular drivers of invasion are required for improved clinical outcome. We have demonstrated that TROY (TNFRSF19), a member of the TNFR super-family, plays an important role in GBM invasion and resistance. Knockdown of TROY expression inhibits GBM cell invasion, increases sensitivity to temozolomide, and prolongs survival in an intracranial xenograft model. Propentofylline (PPF), an atypical synthetic methylxanthine compound, has been extensively studied in Phase II and Phase III clinical trials for Alzheimer's disease and vascular dementia where it has demonstrated blood-brain permeability and minimal adverse side effects. Here we showed that PPF decreased GBM cell expression of TROY, inhibited glioma cell invasion, and sensitized GBM cells to TMZ. Mechanistically, PPF decreased glioma cell invasion by modulating TROY expression and downstream signaling, including AKT, NF-κB, and Rac1 activation. Thus, PPF may provide a pharmacologic approach to target TROY, inhibit cell invasion, and reduce therapeutic resistance in GBM.

Neuropeptides of the VIP family inhibit glioblastoma cell invasion.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are neuropeptides acting through VPAC1, VPAC2 and PAC1 receptors (referred here as the VIP-receptor system). In the central nervous system, VIP and PACAP are involved in neurogenesis, cell differentiation and migration, suggesting that they could be implicated in the development of glioblastoma (GBM). The infiltrative nature of GBM remains a major problem for the therapy of these tumors. We previously demonstrated that the VIP-receptor system regulated cell migration of the human cell lines M059J and M059K, derived from a single human GBM. Here, we evaluated the involvement of the VIP-receptor system in GBM cell invasion. In Matrigel invasion assays, M059K cells that express more the VIP-receptor system than M059J cells were less invasive. Invasion assays performed in the presence of agonists, antagonists or anti-PACAP antibodies as well as experiments with transfected M059J cells overexpressing the VPAC1 receptor indicated that the more the VIP-receptor system was expressed and activated, the less the cells were able to invade. Western immunoblotting experiments revealed that the VIP-receptor system inactivated the signaling protein AKT. Invasion assays carried out in the presence of an AKT inhibitor demonstrated the involvement of this signaling kinase in the regulation of cell invasion by the VIP-receptor system in M059K cells. The inhibition by VIP of invasion and AKT was also observed in U87 cells. In conclusion, VIP and PACAP act as anti-invasive factors in different GBM cell lines, a function mediated by VPAC1 inhibition of AKT signaling in M059K cells.

CB-11 * DIVERSE ROLES FOR RhoA AND RhoC IN GLIOBLASTOMA

A number of Rho family members have been shown to control tumor cell proliferation, survival and invasion. Rhophilin, an effector of both RhoA and RhoC, is amplified in glioblastoma and its expression strongly correlates with decreased patient survival. Importantly, rhophilin 2 has recently also been shown to be a critical driver gene of the mesenchymal phenotype in human glioblastoma. However, the cellular functions of RhoA and RhoC in glioblastoma largely remain to be characterized. Here we show that depletion of RhoA, but not RhoC, significantly inhibits glioblastoma cell proliferation. Conversely, depletion of RhoC, but not RhoA, significantly inhibits glioblastoma cell invasion into ex vivo brain slices. In line with this, depletion of RhoC, but not RhoA, also inhibits nuclear squeezing through a 3 mm pore size filter. In addition, we found that depletion of either RhoA or RhoC inhibit glioblastoma cell survival in clonogenic assays. Thus, RhoA and RhoC differentially regulate distinct aspects of the malignant behavior of glioblastoma. We also have identified PDZ-RhoGEF as a critical activator of both RhoA and RhoC in glioblastoma cells, as depletion of this guanine nucleotide exchange factor strongly diminishes serum-stimulated activation of both GTPases. In addition, consistent with the results of depleting either RhoA or RhoC, we showed that depletion of PDZ-RhoGEF, significantly inhibits glioblastoma cell proliferation, invasion and clonogenicity. We also showed that depletion of PDZ-RhoGEF significantly enhances mouse survival in a patient-derived xenograft model.

Netrin-1 induced activation of Notch signaling mediates glioblastoma cell invasion

Glioblastoma multiforme is an aggressively invasive human brain cancer, which lacks effective treatment. The axonal guidance protein, netrin-1, is overexpressed in glioblastoma tumor biopsies. In Matrigel invasion assays we observed that experimental overexpression of netrin-1 increased cell invasiveness and its downregulation decreased invasiveness. Using tandem affinity purification and mass spectrometry protein identification we found that netrin-1 forms a complex with both Notch2 and Jagged1. Recombinant netrin-1 colocalized with Jagged1 and Notch2 at the cell surface and was also present in the intracellular vesicles with Jagged1, but not with Notch2. Netrin-1 activated Notch signaling and subsequent glioblastoma cell invasion. Interestingly, the recombinant central domain of netrin-1 counteracted the effects of the full-length netrin-1: it inhibited glioblastoma cell invasion and Notch activation by retaining the Notch signaling complex at the cell surface. This finding may give rise to therapeutic applications. These results reveal a new mechanism leading to glioblastoma cell invasion, in which netrin-1 activates Notch signaling.

Abstract LB-516: PDZ-RhoGEF promotes glioblastoma cell invasion, proliferation and survival

Abstract Invasion of glioblastoma cells into the surrounding brain parenchyma is a critical parameter of this disease as it hampers complete surgical resection of the tumor mass. Glioblastoma tumors are highly proliferative and resistant to radio- and chemo-therapy, thereby leading to tumor recurrence. Rho family members have been shown to control both the invasive and proliferative behavior of tumor cells. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs). In this study we focused on PDZ-RhoGEF, as its role in glioblastoma is not known. It has been shown that invading glioblastoma cells need contractile force to squeeze their nucleus through narrow spaces that are characteristic of the brain parenchyma (Beadle et al, Mol. Biol. Cell (2008) 19: 3357). We found that depletion of PDZ-RhoGEF in U87 and SNB19 glioblastoma cells strongly inhibits cell migration across a small (3 μm) pore size filter, a process which necessitates nuclear squeezing. PDZ-RhoGEF depletion also inhibits cell migration across a larger (8 μm) pore size filter, albeit to a lesser extent than in the squeezing assay. Interestingly, cells lacking PDZ-RhoGEF display multi-lobed lamellae, suggesting a defect in cell polarization. In addition, PDZ-RhoGEF depletion strongly inhibits glioblastoma cell invasion into ex vivo brain slices. In vitro studies have demonstrated that PDZ-RhoGEF can act on both RhoA and RhoC. The roles of these GTPases in the invasive behavior of glioblastoma have not been reported to date. Interestingly, we found that depletion of RhoC strongly inhibits brain slice invasion and nuclear squeezing, whereas depletion of RhoA has no significant effect on these functions. Thus, taken together, these data suggest that PDZ-RhoGEF largely acts by activating RhoC to stimulate glioma cell invasion. Our preliminary data also indicate that PDZ-RhoGEF mediates glioma cell proliferation and survival. In conclusion, our findings suggest that PDZ-RhoGEF is a potential therapeutic target for glioma therapy. 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-516. doi:1538-7445.AM2012-LB-516

Combination of taxol and Bcl‐2 siRNA induces apoptosis in human glioblastoma cells and inhibits invasion, angiogenesis and tumour growth

Taxol is a powerful chemotherapeutic agent that binds to microtubules to prevent tumour cell division. However, a traditional high dose of taxol may also induce apoptosis in normal cells. The anti-apoptotic molecule Bcl-2 is up-regulated in tumour cells to prevent apoptosis. We designed this study to determine whether use of a low dose of taxol and anti-apoptotic Bcl-2 gene silencing would effectively induce apoptosis in human glioblastoma U251MG cells and also inhibit invasion, angiogenesis and intracranial as well as subcutaneous tumour growth. We treated the cells with either 100 nM taxol or transfected with a plasmid vector expressing Bcl-2 siRNA or both agents together for 72 h. Knockdown of Bcl-2 potentiated efficacy of taxol for cell death. Fluorescence-activated cell sorting analysis, double immunofluorescent staining and TUNEL assay demonstrated apoptosis in about 70% of the cells after treatment with the combination of taxol and Bcl-2 siRNA. In vitro Matrigel invasion assay demonstrated dramatic decrease in glioblastoma cell invasion and in vivo angiogenesis assay showed complete inhibition of neovascularization in athymic nude mice after treatment with the combination. Further, treatment with the combination of taxol and Bcl-2 siRNA caused suppression of intracranial tumour growth and subcutaneous solid tumour development. In conclusion, our results indicate that the combination of taxol and Bcl-2 siRNA effectively induces apoptosis and inhibits glioblastoma cell invasion, angiogenesis and intracranial as well as subcutaneous tumour growth. Therefore, the combination of a low dose of taxol and Bcl-2 siRNA is a promising therapeutic strategy for controlling the aggressive growth of human glioblastoma.

Sulindac and its metabolites inhibit invasion of glioblastoma cells via down‐regulation of Akt/PKB and MMP‐2

Non-steroidal anti-inflammatory drug (NSAID), sulindac has chemopreventive and anti-tumorigenic properties, however, the molecular mechanism of this inhibitory action has not been clearly defined. The Akt/protein kinase B, serine/threonine kinase is well known as an important mediator of many cell survival signaling pathways. In the present study, we demonstrate that down-regulation of Akt is a major effect of anti-invasiveness property of sulindac and its metabolites in glioblastoma cells. Myristoylated Akt (MyrAkt) transfected U87MG glioblastoma cells showed increase invasiveness, whereas DN-Akt transfected cells showed decrease invasiveness indicating that Akt potently promoted glioblastoma cell invasion. MMP-2 promoter and enzyme activity were up-regulated in Akt kinase activity dependent manner. Sulindac and its metabolites down-regulated Akt phosphorylation, inhibited MMP-2 production, and significantly inhibited invasiveness of human glioblastoma cells. In addition, sulindac and LY294002, a selective inhibitor of phosphoinositide 3-kinase (PI3K), synergistically inhibited the invasion of glioblastoma cells. Furthermore, only celecoxib showed Akt phosphorylation reduction and an anti-invasivness in glioblastoma cells, whereas aspirin, ketoprofen, ketorolac, and naproxen did not. In conclusion, our results provide evidence that down-regulation of Akt pathway and MMP-2 may be one of the mechanisms by which sulindac and its metabolites inhibit glioblastoma cell invasion.

The cyclooxygenase inhibitor indomethacin modulates gene expression and represses the extracellular matrix protein laminin γ1 in human glioblastoma cells

The induction of cyclooxygenase-2 (COX-2) expression is associated with more aggressive gliomas and poor survival. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit COX activity and have antitumorigenic properties. In this report, our initial aim was to determine if indomethacin would alter gene expression as measured by suppression subtractive hybridization (SSH). Three up-regulated and four down-regulated genes by indomethacin treatment were identified. Laminin γ1, an extracellular matrix molecule, was the most significantly repressed gene. The repression of laminin γ1 by indomethacin was confirmed by Northern and Western blot analyses and occurred in a concentration- and time-dependent manner at the protein level. Among several NSAIDs tested, only sulindac sulfide and indomethacin suppressed laminin γ1 protein expression, and this repression was observed in both COX-expressing and -deficient cell lines, suggesting that laminin γ1 repression by COX inhibitors was independent of COX. Indomethacin, at a concentration that represses laminin γ1, inhibited glioblastoma cell invasion that was partially restored with additional human laminin protein containing γ1 chain. The repression of laminin γ1 by NSAIDs may be related to attenuation of invasion of brain tumors. These findings are important in understanding the chemopreventive activity of some NSAIDs and could be relevant for designing therapeutic strategies against glioblastoma.

Downregulation of the urokinase-type plasminogen activator receptor through inhibition of translation by antisense oligonucleotide suppresses invasion of human glioblastoma cells.

We previously showed that downregulation of the urokinase-type plasminogen activator receptor (uPAR) in the SNB19 human glioblastoma cell line by the stable transfection of a plasmid expressing a 300 bp antisense sequence to the 5' end of the uPAR gene produced a decrease in the amount of target mRNA. In a more recent study, we found that adenovirus-mediated transduction (Ad-uPAR) of the same uPAR antisense gene construct in SNB19 cells also downregulated uPAR protein levels. We report here that Ad-uPAR-transfected SNB19 cells produced the same amounts of target uPAR mRNA but significantly less protein by in vitro translation and by in situ [35S] labeling compared to Ad-CMV vector-transfected and mock-transfected cells. This antisense construct also inhibited glioblastoma cell invasion confirming previous results. We conclude that downregulation of uPAR by this antisense gene construct results from inhibition of protein translation.