PTEN suppresses hyaluronic acid-induced matrix metalloproteinase-9 expression in U87MG glioblastoma cells through focal adhesion kinase dephosphorylation

Glioblastoma is a severe type of primary brain tumor and its invasion is strongly correlated with the secretion of matrix metalloproteinases (MMPs). To investigate a role of PTEN, a tumor suppressor gene, in the regulation of hyaluronic acid (HA)-induced invasion of glioma cells, we examined the secretion of MMP-9 in various glioma cells with or without a functional PTEN gene. The secretion of MMP-9 in glioma cells lacking functional PTEN (U87MG, U251MG, and U373MG) was induced by HA, although not in wildtype (wt)-PTEN-harboring cells (LN229, LN18, and LN428). In addition, stable expression of wt-PTEN into U87MG cells significantly decreased the secretion of HA-induced MMP-9 and basal levels of MMP-2, inhibiting the activation of focal adhesion kinase and extracellular signal-regulated kinase 1/2, whereas the secretion levels of the tissue inhibitor of metalloproteinase-1 and -2 were increased, finally resulting in the inhibition of invasion by HA in vitro. Ectopic expressions of adenoviral (Ad)-wt-PTEN and -lipid phosphatase-deficient (G129E)-PTEN, but not both protein and -lipid phosphatase-deficient (C124S)-PTEN, reduced MMP-9 secretion and invasion by HA. These results were also confirmed by expressions of Ad-wt-PTEN and Ad-G129E-PTEN in other glioblastoma cells lacking functional PTEN, U251MG, and U373MG. These findings strongly suggest the possibility that PTEN may block HA-induced MMP-9 secretion and invasion through its protein phosphatase activity.     

Tenascin-C stimulates glioma cell invasion through matrix metalloproteinase-12

The capacity of glioma cells to invade extensively within the central nervous system is a major cause of the high morbidity rate of primary malignant brain tumors. Glioma cell invasion involves the attachment of tumor cells to extracellular matrix (ECM), degradation of ECM components, and subsequent penetration into adjacent brain structures. These processes are accomplished in part by matrix metalloproteinases (MMP) within a three-dimensional milieu of the brain parenchyma. As the majority of studies have used a two-dimensional monolayer culture system, we have used a three-dimensional matrix of collagen type I gel to address glioma-secreted proteases, ECM, and invasiveness of glioma cells. We show that in a three-dimensional collagen type I matrix, the presence of tenascin-C, commonly elevated in high-grade gliomas, increased the invasiveness of glioma cells. The tenascin-C-mediated invasiveness was blocked by metalloproteinase inhibitors, but this did not involve the gelatinases (MMP-2 and MMP-9) commonly implicated in two-dimensional glioma growth. A thorough analysis of 21 MMPs and six members of a disintegrin and metalloproteinase domain showed that MMP-12 was increased in gliomas by tenascin-C in three-dimensional matrix. Furthermore, examinations of resected specimens revealed high MMP-12 levels in the high-grade glioblastoma multiforme tumors. Finally, a function-blocking antibody as well as small interfering RNA to MMP-12 attenuated the tenascin-C-stimulated glioma invasion. These results identify a new factor, MMP-12, in regulating glioma invasiveness through interaction with tenascin-C.     

Suppression of invasion in human U87 glioma cells by adenovirus-mediated co-transfer of TIMP-2 and PTEN gene

TIMPs and PTEN are known to be inhibitors of the invasive activities of malignant glioma. But there has been no literature reported concerning the effect of combined gene transfer of these two genes on invasiveness of glioma. This study was designed to evaluate the effect of adenovirus-mediated in vitro gene transfer of tissue inhibitor of metalloproteinases-2 (TIMP-2) and phosphatase and tensin homology deleted on chromosome ten (PTEN) on invasion of human U87 glioma cells. The mRNA and protein expressions of TIMP-2 and PTEN in U87 cells infected with AdTIMP-2 and AdPTEN were determined by RT-PCR and Western blot, respectively. The relative activity of MMP-2 and MMP-9 were measured by Gelatin zymogram and invasion of U87 in vitro were detected using Boyden chamber. The number of invasion cell of U87, U87 infected with Ad-gal, AdPTEN, AdTIMP-2 and AdPTEN/TIMP-2 was 55.63+/-13.27, 48.27+/-14.75, 35.27+/-10.94, 27.37+/-12.81, and 19.17+/-5.45, respectively. In vitro invasiveness of glioma cells was significantly inhibited by infection with AdTIMP-2 and/or AdPTEN, which was not consistent with the change of MMPs activity. And in the combinated group, the inhibition effect was more remarkable than in single group. Our studies suggest that adenovirus-mediated combined TIMP-2 and PTEN gene therapy is possibly useful for anti-invasion therapy of malignant glioma.     

Expression of matrix metalloproteinases in human glioma cell lines in the presence of IL-10

Matrix metalloproteinases have been implicated to play a vital role in glioma invasion as they degrade extracellular matrix to facilitate the subsequent migration of tumor cells into the surrounding brain tissue. The cytokine Interleukin-10 (IL-10) was detected recently in glial tumors in vivo. Expression of specific IL-10 mRNA as well as blood serum levels of IL-10 in glioma patients increased with malignancy suggesting a functional role of IL-10 in glioma progression. Moreover, glioma cell migration in vitro was enhanced in the presence of IL-10. We therefore investigated the expression of the matrix metalloproteinases (MMPs) stromelysin-1 (MMP-3), 72-kDa collagenase (MMP-2), 92-kDa collagenase (MMP-9), matrilysin (MMP-7) and the human macrophage metalloelastase (MMP-12). In addition, a possible relation between exposure of glioma cells to IL-10 and invasiveness of these cells due to MMP expression was analyzed. Experiments with Matrigel coated Boyden chambers revealed a pronounced dose dependent effect of IL-10 on glioma invasiveness. The synthetic MMP-inhibitor Marimastat markedly reduced cell invasion in the Boyden chambers confirming the significance of MMPs in the process of invasion. Subsequently, the expression level of MMPs and the serine protease uPA was investigated in 7 glioma cell lines (U373, GaMG, U251, GHE, SNB19, U138 and D54) by RT-PCR. In all but one cell line no enhancement of MMP expression by IL-10 was detected. Matrilysin in U373 cells was the only protease found to be upregulated in the presence of IL-10 dependent on cell density. The present data suggest that IL-10 related effects on the invasive properties of the cell lines are not directly mediated by an upregulation of matrix metalloproteinase expression.     

PTEN gene transfer in human malignant glioma: sensitization to irradiation and CD95L-induced apoptosis.

The tumor suppressor gene PTEN (MMAC1, TEP1) encodes a dual-specificity phosphatase and is considered a progression-associated target of genetic alterations in human gliomas. Recently, it has been reported that the introduction of wild type PTEN into glioma cells containing endogenous mutant PTEN alleles (U87MG, LN-308), but not in those which retain wild-type PTEN (LN-18, LN-229), causes growth suppression and inhibits cellular migration, spreading and focal adhesion. Here, we show that PTEN gene transfer has no effect on the chemosensitivity of the four cell lines. Further, a correlational analysis of the endogenous PTEN status of 12 human glioma cell lines with their sensitivity to seven different cancer chemotherapy drugs reveals no link between PTEN and chemosensitivity. In contrast, ectopic expression of wild type PTEN, but not the PTEN(G129R) mutant, in PTEN-mutant gliomas markedly sensitizes these cells to irradiation and to CD95-ligand (CD95L)-induced apoptosis. PTEN-mediated facilitation of CD95L-induced apoptosis is associated with enhanced CD95L-evoked caspase 3 activity. Protein kinase B (PKB/Akt), previously shown to inhibit CD95L-induced apoptosis in nonglial COS7 cells, is inactivated by dephosphorylation. Interestingly, both PTEN-mutant U87MG and PTEN-wild-type LN-229 cells contain phosphorylated PKB constitutively. Wild-type PTEN gene transfer promotes dephosphorylation of PKB specifically in U87MG cells but not in LN-229 cells. Sensitization of U87MG cells to CD95L-apoptosis by wild-type PTEN is blocked by insulin-like growth factor-1 (IGF-1). The protection by IGF-1 is inhibited by the phosphoinositide 3-OH (PI 3) kinase inhibitor, wortmannin. Although PKB is a down-stream target of PI 3 kinase, the protection by IGF-1 was not associated with the reconstitution of PKB phosphorylation. Thus, PTEN may sensitize human malignant glioma cells to CD95L-induced apoptosis in a PI 3 kinase-dependent manner that may not require PKB phosphorylation.     

The PTEN lipid phosphatase domain is not required to inhibit invasion of glioma cells.

The tumor suppressor PTEN negatively controls the phosphoinositide 3-kinase pathway for cell survival by dephosphorylating the phospholipid substrates phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. PTEN has been proposed to dephosphorylate focal adhesion kinase and is implicated in the regulation of cell spreading and motility. We analyzed the role of PTEN in invasion using the two highly infiltrative glioma cell lines U87MG (which lacks functional PTEN) and LN229 (wild-type PTEN). After constitutive overexpression of wild-type and phosphatase-deficient (C124S) PTEN, we found significant inhibition of invasion (50-70%) independent of the PTEN status of the cell and of the catalytic core domain of PTEN. Although wild-type but not mutant (C124S) PTEN decreased PKB/Akt phosphorylation and induced a stellate morphology in U87MG cells, an accompanying reduction of focal adhesion kinase phosphorylation was not seen. We conclude that phosphatase-independent domains of PTEN markedly reduced the invasive potential of glioma cells, defining a structural role for PTEN that regulates cell motility distinct of the PKB/Akt pathway.     

Mechanisms of action of rapamycin in gliomas

Rapamycin has previously been shown to be efficacious against intracerebral glioma xenografts and to act in a cytostatic manner against gliomas. However, very little is known about the mechanism of action of rapamycin. The purpose of our study was to further investigate the in vitro and in vivo mechanisms of action of rapamycin, to elucidate molecular end points that may be applicable for investigation in a clinical trial, and to examine potential mechanisms of treatment failure. In the phosphatase and tensin homolog deleted from chromosome 10 (PTEN)-null glioma cell lines U-87 and D-54, but not the oligodendroglioma cell line HOG (PTEN null), doses of rapamycin at the IC50 resulted in accumulation of cells in G1, with a corresponding decrease in the fraction of cells traversing the S phase as early as 24 h after dosing. All glioma cell lines tested had markedly diminished production of vascular endothelial growth factor (VEGF) when cultured with rapamycin, even at doses below the IC50. After 48 h of exposure to rapamycin, the glioma cell lines (but not HOG cells) showed downregulation of the membrane type-1 matrix metalloproteinase (MMP) invasion molecule. In U-87 cells, MMP-2 was downregulated, and in D-54 cells, both MMP-2 and MMP-9 were downregulated after treatment with rapamycin. Treatment of established subcutaneous U-87 xenografts in vivo resulted in marked tumor regression (P < 0.05). Immunohistochemical studies of subcutaneous U-87 tumors demonstrated diminished production of VEGF in mice treated with rapamycin. Gelatin zymography showed marked reduction of MMP-2 in the mice with subcutaneous U-87 xenografts that were treated with rapamycin as compared with controls treated with phosphatebuffered saline. In contrast, treatment of established intracerebral U-87 xenografts did not result in increased median survival despite inhibition of the Akt pathway within the tumors. Also, in contrast with our findings for subcutaneous tumors, immunohistochemistry and quantitative Western blot analysis results for intracerebral U-87 xenografts indicated that there is not significant VEGF production, which suggests possible deferential regulation of the hypoxia-inducible factor 1alpha in the intracerebral compartment. These findings demonstrate that the complex operational mechanisms of rapamycin against gliomas include cytostasis, anti-VEGF, and anti-invasion activity, but these are dependent on the in vivo location of the tumor and have implications for the design of a clinical trial.     

ELMO1 and Dock180, a bipartite Rac1 guanine nucleotide exchange factor, promote human glioma cell invasion

A distinct feature of malignant gliomas is the intrinsic ability of single tumor cells to disperse throughout the brain, contributing to the failure of existing therapies to alter the progression and recurrence of these deadly brain tumors. Regrettably, the mechanisms underlying the inherent invasiveness of glioma cells are poorly understood. Here, we report for the first time that engulfment and cell motility 1 (ELMO1) and dedicator of cytokinesis 1 (Dock180), a bipartite Rac1 guanine nucleotide exchange factor (GEF), are evidently linked to the invasive phenotype of glioma cells. Immunohistochemical analysis of primary human glioma specimens showed high expression levels of ELMO1 and Dock180 in actively invading tumor cells in the invasive areas, but not in the central regions of these tumors. Elevated expression of ELMO1 and Dock180 was also found in various human glioma cell lines compared with normal human astrocytes. Inhibition of endogenous ELMO1 and Dock180 expression significantly impeded glioma cell invasion in vitro and in brain tissue slices with a concomitant reduction in Rac1 activation. Conversely, exogenous expression of ELMO1 and Dock180 in glioma cells with low level endogenous expression increased their migratory and invasive capacity in vitro and in brain tissue. These data suggest that the bipartite GEF, ELMO1 and Dock180, play an important role in promoting cancer cell invasion and could be potential therapeutic targets for the treatment of diffuse malignant gliomas.     

Adenovirus-mediated expression of antisense MMP-9 in glioma cells inhibits tumor growth and invasion

Matrix metalloproteinase 9 (MMP-9) is known to play a major role in cell migration and invasion in both physiological and pathological processes. Our previous work has shown that increased MMP-9 levels are associated with human glioma tumor progression. In this study, we evaluated the ability of an adenovirus containing a 528 bp cDNA sequence in antisense orientation to the 5' end of the human MMP-9 gene (Ad-MMP-9AS) to inhibit the invasiveness and migratory capacity of the human glioblastoma cell line SBN19 in in vitro and in vivo models. Infection of glioma cells with Ad-MMP-9AS reduced MMP-9 enzyme activity by approximately 90% compared with mock- or Ad-CMV-infected cells. Migration and invasion of glioblastoma cells infected with Ad-MMP-9AS were significantly inhibited relative to Ad-CMV-infected controls in spheroid and Matrigel assays. Intracranial injections of SNB19 cells infected with Ad-MMP-9AS did not produce tumors in nude mice. However, injecting the Ad-MMP-9AS construct into subcutaneous U87MG tumors in nude mice caused regression of tumor growth. These results support the theory that adenoviral-mediated delivery of the MMP-9 gene in the antisense orientation has therapeutic potential for treating gliomas.     

Independent motile microplast formation correlates with glioma cell invasiveness

Diffuse brain invasion co ntributes to the poor prognosis for patients with gliomas. Analyzing glioma cell migration in vitro, we have demonstrated the spontaneous shedding of anucleate cell fragments that separate from glioma cell bodies and maintain viability from hours to days. Unlike previously described cell fragments that are released from cells as diffusible vectors, glioma cell fragments are independently motile. We used computerized time-lapse microscopy to characterize the formation of these independent motile microplasts (IMMPs) in human cell cultures derived from the most highly invasive glial tumor, glioblastoma. IMMPs were larger than previously described cell fragments, ranging in size from approximately 2% to nearly half of the area of their parent cells. Complex cell-like behaviors—including establishment of polarity, extension of lamellipodia and filopodia, and change in direction of movement—remained intact in IMMPs. The average direction and velocity of the IMMPs were indistinguishable from those of their parent cells. IMMPs formed at a significantly higher rate in glioma cell lines rendered more invasive by overexpression of invasion-related genes than in vector-transfected controls. The correlation with cell invasiveness indicates that IMMP formation may be related to the cell-invasive phenotype. Further investigation will determine whether IMMPs represent a novel addition to the growing list of viable cell fragments with biological relevance. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Inhibition of cell invasion by indomethacin on glioma cell lines: <Emphasis Type="Italic">in vitro</Emphasis> study

Malignant glioma invasion into the surrounding brain tissue is still a major problem for any therapeutical methods. Matrix metalloproteinases (MMPs) have been implicated as important factors in this pathological process. In this study, one of the non-steroidal anti-inflammatory drugs (NSAIDs) indomethacin was employed to investigate the effect of inhibition of cell invasion mediated by MMP-2 and MMP-9 in human malignant glioma cell lines, A172, U87MG, U251MG, and U373MG in vitro. MTT assay was firstly examined to determine non-cytotoxic dose range, then gelatin zymography, matrigel invasion assay, migration assay and MMP-2 activity assay for 24 h exposure in indomethacin were employed to assess the inhibitory effect of indomethacin. MTT assay revealed that dose with 0, 50, and 500 M/ml were non-cytotoxic. Zymography demonstrated: (a) expression of MMP-2 and MMP-9 activity was downregulated along with elevated dose of indomethacin. (b) MMP-2 activity that changed from pro-MMP-2 to active form of MMP-2 in supernatants of cell lines could not be inhibited by indomethacin. Invasion assay disclosed that the number of invading cells through the matrigel were significantly decreased in a dose dependent manner. Migration assay indicated indomethacin did not affect cells migration. MMP-2 activity assay showed the total and active MMP-2 secretion was suppressed by 500 M/ml of indomethacin. Our present study is the first report on inhibitive effect of indomethacin mediated by MMP-2 and MMP-9 in invasion assay of glioma cell lines. The current study suggested that non-cytotoxic level of indomethacin was able to reduce the cell invasion of malignant gliomas mediated by MMP-2 and MMP-9, but it did not affected on cell motility. It also lowered down the activity of MMP-2 and MMP-9, and could reduce of MMP-2 secretion of cell lines. Thus, high concentration of indomethacin within non-cytotoxic dose might offer a new therapeutic strategy to impair cell invasion of gliomas.     

The phosphorylation of EphB2 receptor regulates migration and invasion of human glioma cells

Eph receptor tyrosine kinases and their ligands, ephrins, mediate neurodevelopmental processes such as boundary formation, axon guidance, vasculogenesis, and cell migration. We determined the expression profiles of the Eph family members in five glioma cell lines under migrating and nonmigrating conditions. EphB2 mRNA was overexpressed in all five during migration (1.2-2.8-fold). We found abundant EphB2 protein as well as strong phosphorylation of EphB2 in migrating U87 cells. Confocal imaging showed EphB2 localized in lamellipodia of motile U87 cells. Treatment with ephrin-B1/Fc chimera stimulated migration and invasion of U87, whereas treatment with a blocking EphB2 antibody significantly inhibited migration and invasion. Forced expression of EphB2 in U251 cells stimulated cell migration and invasion and diminished adhesion concomitant with the tyrosine phosphorylation of EphB2. U251 stably transfected with EphB2 showed more scattered and more pronounced invasive growth in an ex vivo rat brain slice. In human brain tumor specimens, EphB2 expression was higher in glioblastomas than in low-grade astrocytomas or normal brain; patterns of phosphorylated EphB2 matched the expression levels. Laser capture microdissection of invading glioblastoma cells revealed elevated EphB2 mRNA (1.5-3.5-fold) in 7 of 7 biopsy specimens. Immunohistochemistry demonstrated EphB2 localization primarily in glioblastoma cells (56 of 62 cases) and not in normal brain. This is the first demonstration that migrating glioblastoma cells overexpress EphB2 in vitro and in vivo; glioma migration and invasion are promoted by activation of EphB2 or inhibited by blocking EphB2. Dysregulation of EphB2 expression or function may underlie glioma invasion.     

Silencing of WNK2 is associated with upregulation of MMP2 and JNK in gliomas

Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade extracellular matrix (ECM), thus assisting invasion. Upregulation of MMPs, frequently reported in gliomas, is associated with aggressive behavior. WNK2 is a tumor suppressor gene expressed in normal brain, and silenced by promoter methylation in gliomas. Patients without WNK2 exhibited poor prognosis, and its downregulation was associated with increased glioma cell invasion.Here we showed that MMP2 expression and activity are increased in glioma cell lines that do not express WNK2. Also, WNK2 inhibited JNK, a process associated with decreasing levels of MMP2. Thus, WNK2 promoter methylation and silencing in gliomas is associated with increased JNK activation and MMP2 expression and activity, thus explaining in part tumor cell invasion potential.     

Rictor regulates MMP-9 activity and invasion through Raf-1-MEK-ERK signaling pathway in glioma cells

Glioblastoma multiforme (GBM) is the most common and highly aggressive type of primary brain tumor. Tumor-associated macrophages (TAMs) secrete TNF-α that activates important survival pathways including Akt (PKB)/mTOR network. The mammalian target of rapamycin (mTOR) network functions downstream of PI3K/Akt pathway to regulate cell growth, proliferation and survival. mTOR exists in two distinct complexes-mTORC1 and mTORC2 that differ in their components and sensitivity to rapamycin. The rapamycin-insensitive complex (mTORC2) consists of mTOR, mLST8, Rictor, mSin1 and Protor and regulates the actin cytoskeleton in addition to activating Akt (protein kinase B). The present study aimed to investigate the role of Rictor-a core component of mTORC2 in regulating proliferation, survival, and invasion in gliomas. siRNA-mediated loss of Rictor function in human glioma cell lines, LN18 and LN229 and in primary GBM cells resulted in elevated expression and activity of MMP-9 and significant increase in the invasive potential of these cells. Mechanistic studies revealed that the activation of Raf-1-MEK-ERK pathway was essential for induction of MMP-9 activity and enhanced invasion. Interestingly, ablation of Rictor did not affect TNF-α-induced MMP-9 activity and invasiveness suggesting that TNF-α in the microenvironment of tumor might overrule the function of Rictor as a negative regulator of MMP-9 and invasion. Silencing Rictor had no effect on the survival or proliferation in the cell lines in the presence or absence of TNF-α. Our findings identify a role for Rictor in bridging two major pathways-Akt (PKB)/mTOR and Raf-1-MEK-ERK in regulating MMP-9 activity and invasion of glioma tumor cells.     

Inhibition of ADAM17 reduces hypoxia-induced brain tumor cell invasiveness

The membrane-anchored metalloproteinase tumor necrosis factor-alpha-converting enzyme (TACE/a disintegrin and metalloproteinase [ADAM] 17) is key in proteolytic ectodomain shedding of several membrane-bound growth factors, cytokines and receptors. The expression and activity of ADAM17 increases under some pathological conditions including stroke, and promotes neural progenitor cell migration and contributes to stroke-induced neurogenesis. Hypoxia initiates cellular invasive processes that occur under both physiological and pathological conditions such as invasion and metastasis of some tumors. In the present study, we sought to elucidate whether ADAM17 contributes to brain tumor invasion. To this end, we examined the role of ADAM17 in the invasiveness of two different brain tumor cell lines, 9L rat gliosarcoma and U87 human glioma, under normoxic and hypoxic conditions. Additionally, we tested the effects of ADAM17 suppression on in vitro tumor cell invasion by means of ADAM17 proteolytic inhibitors and specific small interfering RNA. We found that tumor cells upregulated ADAM17 expression under hypoxia, and that ADAM17 activity correlated with increased tumor cell invasion. Conversely, suppression of ADAM17 proteolysis decreased invasiveness induced by hypoxia in 9L and U87 cells. Furthermore, the contribution of ADAM17 to tumor invasion was independent of matrix metalloproteinase (MMP)-2 and MMP-9 activity. ADAM17 was also found to activate the epidermal growth factor/phosphoinositide-3 kinase/serine/threonine kinase signal transduction pathway. Our data suggest that hypoxia-induced ADAM17 contributes to glioma cell invasiveness through activation of the EGFR signal pathway.