Sphingosine-1-phosphate stimulates motility and invasiveness of human glioblastoma multiforme cells

Sphingosine-1-phosphate (S1P) is a bioactive lipid which is a potent mitogen for glioblastoma multiforme cells. Here we show that S1P also potently enhances the in vitro motility of glioblastoma cells by signaling through receptors coupled to G(i/o) proteins. Moreover, S1P also enhanced in vitro invasion of glioblastoma cells through Matrigel. S1P had no effect on matrix metalloproteinase secretion but did enhance glioblastoma cell adhesion. S1P is present at high levels in brain tissue. Thus it is possible that autocrine or paracrine signaling by S1P through its G protein-coupled receptors enhances both glioma cell proliferation and invasiveness.     

Targeting of surface alpha-enolase inhibits the invasiveness of pancreatic cancer cells

Pancreatic Ductal Adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness and resistance to treatment. We have previously demonstrated that most PDAC patients have circulating antibodies against the glycolytic enzyme alpha-enolase (ENO1), which correlates with a better response to therapy and survival. ENO1 is a metabolic enzyme, also expressed on the cell surface where it acts as a plasminogen receptor. ENO1 play a crucial role in cell invasion and metastasis by promoting plasminogen activation into plasmin, a serine-protease involved in extracellular matrix degradation. The aim of this study was to investigate the role of ENO1 in PDAC cell invasion. We observed that ENO1 was expressed on the cell surface of most PDAC cell lines. Mouse anti-human ENO1 monoclonal antibodies inhibited plasminogen-dependent invasion of human PDAC cells, and their metastatic spreading in immunosuppressed mice was inhibited. Notably, a single administration of Adeno-Associated Virus (AAV)-expressing cDNA coding for 72/1 anti-ENO1 mAb reduced the number of lung metastases in immunosuppressed mice injected with PDAC cells. Overall, these data indicate that ENO1 is involved in PDAC cell invasion, and that administration of an anti-ENO1 mAb can be exploited as a novel therapeutic option to increase the survival of metastatic PDAC patients.     

Decursinol angelate blocks transmigration and inflammatory activation of cancer cells through inhibition of PI3K,ERK and NF-κB activation

Inflammation is known to be closely associated with the development of cancer. Decursinol angelate (DA), a coumarin compound isolated from Angelica gigas and related compounds have been shown to possess potent anti-inflammatory activities. However, little is known about their effects on the inflammatory processes associated with cancer. In this study, the anti-inflammatory effect of DA was evaluated in cancer cell lines with respect to cellular invasion through the extracellular matrix (ECM) and the expression of pro-inflammatory mediators such as cytokine, cell adhesion molecules and matrix metalloproteinase (MMP)-9. DA inhibited the invasion of fibrosarcoma cell line, HT1080 and breast cancer cell line, MDA-MB-231 in the Matrigel invasion assay. DA-mediated suppression of cancer cell invasion was accomplished by suppression of PI3K activity known to be associated with cytoskeletal rearrangement related to cellular migration. DA also suppressed the adhesion of cancer cells to ECM mediated by down-regulation of β₁-integrin expression levels. Furthermore, DA inhibited the expression of pro-inflammatory cytokines and MMP-9 through suppression of PI3K, ERK and NF-κB activation. These results demonstrate that DA suppresses invasion and inflammatory activation of cancer cells through modulation of PI3K/AKT, ERK and NF-κB. These anti-inflammatory activities of DA may contribute to its anti-cancer activity.     

Identification of integrins α6 and β7 as c‐Jun‐ and transformation‐relevant genes in highly invasive fibrosarcoma cells

Understanding the mechanisms of tumor cell invasion is essential for our attempts to prevent cancer deaths. We screened by DNAmicroarrays the c‐Jun‐ and transformation‐related gene expression changes in S‐adenosylmethionine decarboxylase (AdoMetDC)‐overexpressing mouse fibroblasts that are highly invasive in vivo, and their derivatives expressing a tetracycline‐inducible dominant‐negative mutant of c‐Jun (TAM67) or c‐Jun shRNA. Among the small set of target genes detected were integrins α6 and β7, cathepsin L and thymosin β4, all upregulated in the AdoMetDC‐transformed cells and downregulated upon reversal of transformation by TAM67 or c‐Jun shRNA. The upregulation of integrin α6 subunit, pairing with integrin β1, endowed the transformed cells with the capability to attach to basement membrane laminin and to spread. Further, inhibition of integrin α6 or β1 function with neutralizing antibodies blocked the invasiveness of AdoMetDC‐transformants and human HT‐1080 fibrosarcoma cells in three‐dimensional Matrigel. Moreover, immunohistochemical analyses showed strong integrin α6 staining in high‐grade human fibrosarcomas. Our data show that c‐Jun can regulate all three key steps of invasion: cell adhesion (integrin α6), basement membrane/extracellular matrix degradation (cathepsin L) and cell migration (thymosin β4). In addition, this is the first study to associate integrin β7, known as a leukocyte‐specific integrin binding to endothelial/epithelial cell adhesion molecules, with the transformed phenotype in cells of nonleukocyte origin. As tumor cell invasion is a prerequisite for metastasis, the observed critical role of integrin α6β1 in fibrosarcoma cell invasion/spreading allures testing antagonists to integrin α6β1, alone or combined with inhibitors of cathepsin L and thymosin β4, as chemotherapeutic agents. © 2009 UICC     

Human bladder cancer invasion model using rat bladder in vitro and its use to test mechanisms and therapeutic inhibitors of invasion

As well as being a passive support, the extracellular matrix also regulates key biological processes such as invasion, differentiation and angiogenesis. We have therefore developed an in vitro model of bladder cancer invasion using de-epithelialized rat bladder to allow for tumour cell-extracellular matrix interactions. Onto this we have seeded a panel of human bladder cancer cell lines (RT4, RT112, 253J and EJ28 (T24)) representing progression from well to poorly differentiated phenotypes and used as models of superficial to invasive bladder cancer. The better differentiated cell lines RT4 and RT112 reproducibly grew as stratified epithelium, whereas poorly differentiated EJ28 cells invaded across a broad front. Invasion was not simply related to proliferation rate, measured either as doubling time on plastic (non-invasive 253J and invasive EJ28 having the same doubling time) or by Ki-67 proliferation index within the model. We used the model to test the ability of 4 compounds that interfere with tumour cell-extracellular matrix interactions (suramin, N-acetylcysteine and the urokinase plasminogen activator pathway antagonists A5 compound and monoclonal antibody Mab 3936) to inhibit invasion. At non-toxic concentrations, all significantly inhibited invasion (P< 0.05), although to varying degrees, suramin and A5 almost completely and N-acetylcysteine the least. In conclusion, this model shows the urokinase system is important for bladder invasion and can be used to investigate other mechanisms of bladder cancer invasion and also for the testing of intravesical drugs.     

Upregulation of tissue inhibitor of matrix metalloproteinases-1 confers the anti-invasive action of cisplatin on human cancer cells

Cancer cell invasion is one of the crucial events in local spreading, growth and metastasis of tumors. The present study investigates the mechanism underlying the anti-invasive action of the chemotherapeutic cisplatin. In human cervical carcinoma cells (HeLa), cisplatin caused a time- and concentration-dependent suppression of cell invasion through Matrigel. Inhibition of invasion was accompanied by upregulation of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), whereas levels of matrix metalloproteinase-2 (MMP-2), MMP-9 and TIMP-2 remained unchanged. Cisplatin's effects on TIMP-1 expression and invasion were associated with phosphorylations of p38 and p42/44 mitogen-activated protein kinases and were abrogated by specific inhibitors of both pathways. The impact of TIMP-1 in the anti-invasive action of cisplatin was proven by transfecting cells with small interfering RNA targeting TIMP-1, which completely reversed suppression of invasion by cisplatin. A functional relevance of TIMP-1 upregulation was substantiated by findings showing a concentration-dependent inhibition of Matrigel invasion by recombinant TIMP-1. The essential role of TIMP-1 in the anti-invasive action of cisplatin was confirmed using another human cervical carcinoma cell line (C33A) and human lung carcinoma cells (A549). Altogether, our data demonstrate a hitherto unknown mechanism by which cisplatin exerts its antimetastatic properties on highly invasive cancer cells.     

Histone deacetylase inhibitor, apicidin, inhibits human ovarian cancer cell migration via class II histone deacetylase 4 silencing

This study examined the molecular mechanisms of apicidin in the modulation of human ovarian cancer SKOV-3 cells invasion and migration. Apicidin markedly decreased histone deacetylase 4 (HDAC4) expression and blocked cell migration and invasion. Cell migration was inhibited via down-regulation of matrix metalloproteinase-2 (MMP-2) and up-regulation of RECK in the HDAC4-blocked SKOV-3 cells. Apicidin significantly suppressed the binding of HDAC4 to Sp1 binding elements of the RECK promoter via repression of HDAC4. In an in vivo model, apicidin suppressed the growth of transplanted SKOV-3 cells by down-regulating HDAC4 and MMP-2. Apicidin may potentially be used as an anti-cancer agent for inhibition of cancer cell migration and invasion through the repression of MMP-2 which is related to the reduction of HDAC4.     

Stromal cell-derived factor-1alpha promotes melanoma cell invasion across basement membranes involving stimulation of membrane-type 1 matrix metalloproteinase and Rho GTPase activities

Tissue invasion by tumor cells involves their migration across basement membranes through activation of extracellular matrix degradation and cell motility mechanisms. Chemokines binding to their receptors provide chemotactic cues guiding cells to specific tissues and organs; they therefore could potentially participate in tumor cell dissemination. Melanoma cells express CXCR4, the receptor for the chemokine stromal cell-derived factor-1alpha (SDF-1alpha). Using Matrigel as a model, we show that SDF-1alpha promotes invasion of melanoma cells across basement membranes. Stimulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) activity by SDF-1alpha was necessary for invasion, involving at least up-regulation in the expression of this metalloproteinase, as detected in the highly metastatic BLM melanoma cell line. Moreover, SDF-1alpha triggered the activation of the GTPases RhoA, Rac1, and Cdc42 on BLM cells, and expression of dominant-negative forms of RhoA and Rac1, but not Cdc42, substantially impaired the invasion of transfectants in response to SDF-1alpha, as well as the increase in MT1-MMP expression. Furthermore, CXCR4 expression on melanoma cells was notably augmented by transforming growth factor-beta1, a Matrigel component, whereas anti-transforming growth factor-beta antibodies inhibited increases in CXCR4 expression and melanoma cell invasion toward SDF-1alpha. The identification of SDF-1alpha as a potential stimulatory molecule for MT1-MMP as well as for RhoA and Rac1 activities during melanoma cell invasion, associated with an up-regulation in CXCR4 expression by interaction with basement membrane factors, could contribute to better knowledge of mechanisms stimulating melanoma cell dissemination.     

Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling

Background  

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are bioactive lipid signaling molecules implicated in tumor dissemination. Membrane-type matrix metalloproteinase 1 (MT1-MMP) is a membrane-tethered collagenase thought to be involved in tumor invasion via extracellular matrix degradation. In this study, we investigated the molecular requirements for LPA- and S1P-regulated tumor cell migration in two dimensions (2D) and invasion of three-dimensional (3D) collagen matrices and, in particular, evaluated the role of MT1-MMP in this process.     

Stimulation of haptotaxis and migration of tumor cells by serum spreading factor

A central feature of tumor metastasis is the migration of malignant cells through interstitial tissues and vascular structures as they spread throughout the body. Various components of the extracellular matrix and of basement membranes, consisting of genetically distinct collagens, proteoglycans, and noncollagenous glycoproteins, are known to modulate certain aspects of cell behavior, including cell movement. Serum spreading factor is a glycoprotein component of human serum that is also found in interstitial tissues. Two native forms are seen in human serum, a Mr 65,000 and a Mr 75,000 component. Spreading factor promotes substratum attachment and spreading of diverse cell types, including epithelial and fibroblastic cells, and will affect the growth rate and differentiation of cells in serum-free culture media. Serum spreading factor was shown to promote the directed migration of the following tumor cell lines in modified Boyden chamber assays: murine melanoma K-1735 (clones M2, M4, and 16); human breast carcinoma MCF-7; and human fibrosarcoma HT-1080. The stimulation of movement occurred over a concentration range of 0.5 to 50 micrograms of serum spreading factor per ml with a maximum response between 5 and 10 micrograms/ml. The maximal response varied with the cell line and ranged from 5- to 50-fold greater migration than control. A monoclonal antibody to spreading factor, previously shown to inhibit the attachment and spreading-promoting activity, abrogated this migration response. Experiments using filters that were precoated with spreading factor indicated that cells could migrate on an insolubilized layer of this protein by haptotaxis. Tumor cell migration to spreading factor in vitro suggests a possible role for this protein in the phenotypic behavior of metastatic cells.     

Bone marrow endothelial cells increase the invasiveness of human multiple myeloma cells through upregulation of MMP-9: evidence for a role of hepatocyte growth factor.

The migration of multiple myeloma (MM) cells from the circulation into the bone marrow (BM) implicates that they must have the capacity to cross the BM endothelium including the subendothelial basement membrane. In this study, human CD138+ MM cells were immunomagnetically isolated from BM samples of MM patients and their invasion through Matrigel, that is, a reconstituted basement membrane, was determined. We demonstrated that primary MM cells have the capacity to transmigrate through basement membrane and that this invasiveness was considerably increased when assessed on Matrigel filters coated with BM endothelial cells (EC) (4LHBMEC line) (transendothelial invasion). The isolated MM cells were shown by zymography to secrete matrix metalloproteinase (MMP)-9 and anti-MMP-9 antibodies inhibited transendothelial invasion, indicating that MMP-9 is involved in this process. BM EC were found to increase the MMP-9 secretion in MM cells, indicating that EC enhance MM cell invasion through stimulation of MMP-9 secretion. BM EC were found to produce hepatocyte growth factor (HGF), and this cytokine also stimulated MMP-9 secretion in MM cells, while anti-HGF antibodies significantly inhibited EC-stimulated MM cell invasion. In summary, our findings provide evidence that MM cell-BM EC interactions enhance the invasion of human MM cells through stimulation of MMP-9 secretion.     

Tamoxifen, 17beta-oestradiol and the calmodulin antagonist J8 inhibit human melanoma cell invasion through fibronectin.

Invasion through stromal extracellular matrix (ECM) is part of the complex, multistep process of tumour cell invasion and metastasis. Our group has previously demonstrated that calcium and calmodulin are important in another step in the metastatic cascade - that of attachment of cells to ECM. Interestingly, the non-steroidal anti-oestrogen tamoxifen (which also has calmodulin antagonist activity), used in the treatment of breast cancer and now in metastatic cutaneous melanoma, can inhibit the attachment of normal and neoplastic cells to ECM. In this study, we investigated whether such drugs, known to inhibit cell attachment, could also subsequently reduce their invasion through a layer of human fibronectin. We examined the ability of the specific calmodulin antagonist J8, tamoxifen and its two major metabolites, N-desmethyltamoxifen (N-des) and 4-hydroxytamoxifen (4-OH), as well as the pure anti-oestrogen ICI 182,780 and 17beta-oestradiol to inhibit invasion of the human cutaneous melanoma cell line, A375-SM, uveal melanoma cells and uveal melanocytes. A375-SM cells and uveal melanoma cells showed a high level of invasion (15.2% and 33.7% respectively) compared with melanocytes (around 5%) under the experimental conditions used. Submicromolar concentrations of N-des, tamoxifen, J8 and 17beta-oestradiol significantly reduced the invasiveness of the A375-SM cell line. The uveal melanoma cells also showed similar inhibition, although at higher concentrations of these agents. 4-OH and ICI 182, 780 had little or no effect on invasion of A375-SM cells (these were not tested on uveal melanoma cells). All cells used in this study were found to be negative for type I nuclear oestrogen receptors, reinforcing the possibility that tamoxifen and 17beta-oestradiol can act via mechanisms unrelated to binding to classical oestrogen receptors to inhibit tumour cell invasion.     

Effects of Epidermal Growth Factor on Invasiveness through the Extracellular Matrix in High- and Low-metastatic Clones of RCT Sarcoma in vitro

We investigated the invasiveness of tumor cells through the extracellular matrix and the influence of epidermal growth factor (EGF) on tumor cell invasion using in vitro systems in high-[RCT(+)] and low-metastatic [RCT(–)] clones established from poorly differentiated murine RCT sarcoma in C3H/He mice. In the invasion assay using a filter coated with reconstituted basement membrane (Matrigel) in a Boyden chamber, RCT(+) cells were more invasive than RCT(-) cells. The attachment of RCT(+) cells to extracellular matrix components and the degradation of type IV collagen by the cells were significantly greater than with RCT(–) cells. However, there was no significant difference in the migration of cells to the extracellular matrix components between cultured RCT(+) and RCT(-) cells. These findings suggested that the different invasiveness of these clone cells was associated with the difference in the ability of attachment to and degradation of the matrix. The level of laminin receptor expression in RCT(+) cells was about four-fold that in RCT(–) cells and laminin stimulated the type IV collagenolytic activity of RCT(+) cells, suggesting that RCT(+) cell attachment to laminin via laminin receptor on the cell surface induced the production of type IV collagenase by the tumor cells. EGF did not affect the invasiveness of RCT(–) cells. In RCT(+) cells, EGF stimulated the invasiveness through Matrigel, the attachment to extracellular matrix components and the degradation of type IV collagen through high-affinity EGF receptors (EGFR), with Kt of p M order, while the migration to the matrix was not influenced by EGF. These findings suggest that the stimulatory effect of EGF on invasion is related to the acceleration of cell adhesion, and the degradative cascade of the extracellular matrix and high-affinity EGFRs play an important role in the effect of EGF on in vitro invasiveness in this tumor.     

Curcumin inhibits tongue carcinoma cells migration and invasion through downregulation of matrix metallopeptidase 10

Squamous cell carcinoma (SCC) of tongue is an aggressive head and neck cancer with high propensity of regional spreading and invasion. Tongue carcinoma cells treated with curcumin, the major curcuminoid of the turmeric, demonstrated reduction in adhesion, migration, and invasion ability. High-throughput microarray analysis indicated that curcumin treatment suppressed matrix metallopeptidase 10 (MMP10) expression. MMP10 is overexpressed in tongue carcinoma tissues in comparison with the normal epithelia. Curcumin treatment on tongue carcinoma cell lines suppressed MMP10 expression at both mRNA and protein levels. Our results suggested that curcumin is a promising inhibitor to tongue cancer cells migration and invasion.     

Cell surface heparan sulfate proteoglycans control adhesion and invasion of breast carcinoma cells

Background

Cell surface proteoglycans interact with numerous regulators of cell behavior through their glycosaminoglycan chains. The syndecan family of transmembrane proteoglycans are virtually ubiquitous cell surface receptors that are implicated in the progression of some tumors, including breast carcinoma. This may derive from their regulation of cell adhesion, but roles for specific syndecans are unresolved.

Methods

The MDA-MB231 human breast carcinoma cell line was exposed to exogenous glycosaminoglycans and changes in cell behavior monitored by western blotting, immunocytochemistry, invasion and collagen degradation assays. Selected receptors including PAR-1 and syndecans were depleted by siRNA treatments to assess cell morphology and behavior. Immunohistochemistry for syndecan-2 and its interacting partner, caveolin-2 was performed on human breast tumor tissue arrays. Two-tailed paired t-test and one-way ANOVA with Tukey’s post-hoc test were used in the analysis of data.

Results

MDA-MB231 cells were shown to be highly sensitive to exogenous heparan sulfate or heparin, promoting increased spreading, focal adhesion and adherens junction formation with concomitantly reduced invasion and matrix degradation. The molecular basis for this effect was revealed to have two components. First, thrombin inhibition contributed to enhanced cell adhesion and reduced invasion. Second, a specific loss of cell surface syndecan-2 was noted. The ensuing junction formation was dependent on syndecan-4, whose role in promoting actin cytoskeletal organization is known. Syndecan-2 interacts with, and may regulate, caveolin-2. Depletion of either molecule had the same adhesion-promoting influence, along with reduced invasion, confirming a role for this complex in maintaining the invasive phenotype of mammary carcinoma cells. Finally, both syndecan-2 and caveolin-2 were upregulated in tissue arrays from breast cancer patients compared to normal mammary tissue. Moreover their expression levels were correlated in triple negative breast cancers.

Conclusion

Cell surface proteoglycans, notably syndecan-2, may be important regulators of breast carcinoma progression through regulation of cytoskeleton, cell adhesion and invasion.     

MCAM is a novel metastasis marker and regulates spreading, apoptosis and invasion of ovarian cancer cells

Melanoma cell adhesion molecule (MCAM) is a cell adhesion molecule that is abnormally expressed in a variety of tumours and is closely associated with tumour metastasis. The role of MCAM in ovarian cancer development has not been fully studied. In this study, through immunohistochemical staining of ovarian cancer tissue samples and RNA interference to silence MCAM in ovarian cancer cells, we examined the impact of MCAM on the biological functions of ovarian cancer cells and attempted to reveal the role of MCAM in ovarian cancer development. Our results showed that MCAM expression was particularly high in metastatic ovarian cancers compared with other pathological types of ovarian epithelial tissues. After MCAM silencing in the MCAM high-expression ovarian cancer cell line SKOV-3, the cell apoptosis was increased, whereas the cell spreading and invasion were significantly reduced, which may be related with dysregulation of small RhoGTPase (RhoA and Cdc42).These results suggest that MCAM expression in ovarian cancer is highly correlated with the metastatic potential of the cancer. MCAM is likely to participate in the regulation of the Rho signalling pathway to protect ovarian cancer cells from apoptosis and promote their malignant invasion and metastasis. Therefore, MCAM can be used not only as a molecular marker to determine the prognosis of ovarian cancer but also as a therapeutic target in metastatic ovarian cancer.     

Acid ceramidase-mediated production of sphingosine 1-phosphate promotes prostate cancer invasion through upregulation of cathepsin B

Invasiveness is one of the key features of aggressive prostate cancer; however, our understanding of the precise mechanisms effecting invasion remains limited. The ceramide hydrolyzing enzyme acid ceramidase (AC), overexpressed in most prostate tumors, causes an aggressive and invasive phenotype through downstream effectors that have not yet been well characterized. Here, we demonstrate that AC, through generation of sphingosine-1-phosphate (S1P), promotes Ets1 nuclear expression and binding to the promoter region of matrix-degrading protease cathepsin B. Through confocal microscopy and flow cytometry, we found that AC overexpression promotes pericellular localization of cathepsin B and its translocation to the outer leaflet of the cell membrane. AC overexpressing cells have an increased abundance of cathepsin B-enriched invasive structures and enhanced ability to invade through a collagen matrix, but not in the presence of an inhibitor of cathepsin B. In human prostate tissues, AC and cathepsin B overexpression were strongly associated and may relate to poor outcome. These results demonstrate a novel pathway by which AC, through S1P, promotes an invasive phenotype in prostate cancer by causing overexpression and secretion of cathepsin B through activation and nuclear expression of Ets1. As prostate cancer prognosis is dramatically worse when invasion has occurred, this study provides critical insight into the progression toward lethal prostate cancer.     

Tumor Cell Attachment to Lammin Promotes Degradation of the Extracellular Matrix and Cell Migration in High-metastatic Clone Cells of RCT Sarcoma in vitro

We investigated the roles of extracellular matrix proteins, iaminin and fibronectin, in promoting invasiveness through the extracellular matrix in high-metastatic [RCT(+)] clone cells established from poorly differentiated murine RCT sarcoma in C3H/He mice. Laminin stimulated the type IV collagenolytic activity of RCT(+) cells. After more than 6 h of incubation, the type IV collagenolysis of the cell-conditioned medium was significantly higher in laminin-treated groups compared with the control. The migration activity of RCT(+) cells was stimulated by laminin. However, fibronectin did not influence the type IV collagenolysis or cell migration in this clone cell. The amino acid sequence YIGSR, which is derived from laminin, inhibited the laminin-mediated cell attachment and the laminin-promoted type IV collagenolysis, as well as cell migration of RCT(+) cells. RGD derived from fibronectin did not influence the cell attachment to laminin or Matrigel in this clone. In the invasion assay employing a Matrigel coated filter in a Boyden chamber, YIGSR showed greater inhibition of invasion through the Matrigel than did RGD with RCT(+) cells. YIGSR might inhibit the promoted-matrix degradation and cell migration in response to the cell attachment to laminin by competing with laminin for binding to cell surface laminin receptor. We suggest that laminin-mediated cell attachment to the extracellular matrix may play a role in promoting the matrix degradation and cell migration during metastatic cascades.     

Altered actin cytoskeleton and inhibition of matrix metalloproteinase expression by vanadate and phenylarsine oxide, inhibitors of phosphotyrosine phosphatases: modulation of migration and invasion of human malignant glioma cells

Cell-matrix interactions exert a profound influence on cell function and behavior. Our earlier observations suggested that disruption of the actin cytoskeleton results in the inhibition of phorbol ester-induced matrix metalloproteinase (MMP)-9 expression. In this study, to understand the role of protein tyrosine phosphatases in matrix metalloproteinase-9 expression, we treated glioblastoma cells with vanadate and phenylarsine oxide (PAO), which are inhibitors of protein tyrosine phosphatases. Vanadate and PAO inhibited expression of phorbol ester-induced MMP-9 as well as constitutive expression of matrix metalloproteinase-2 in a dose- and time-dependent fashion. An assay of the activity of phosphotyrosine phosphatase (PTPase) indicated that vanadate-treated cells had reduced PTPase activity compared with that of untreated controls. Vanadate and PAO also inhibited actin polymerization, cell spreading, migration, and invasion of glioma cells. Furthermore, elevated levels of protein tyrosine phosphorylation were observed in vanadate- and PAO-treated cells in both a concentration- and time-dependent fashion and were seen to have an inverse correlation with focal adhesion kinase protein expression. These results suggest that vanadate and PAO inhibited migration and invasion of glioma cells by their effect on the cytoskeleton and inhibition of MMP expression.     

Altered in vitro spreading and cytoskeletal organization in human glioma cells by downregulation of urokinase receptor

The interaction of urokinase-type plasminogen activator (uPA) with its cell-surface receptor (uPAR) is implicated in diverse biological processes such as cell migration, tissue remodeling, and tumor cell invasion. Recent studies indicated that uPAR can act as an extracellular matrix receptor during cell adhesion. Recently, we showed that transfection of the humanglioma cell line SNB19 with antisense uPAR resulted in downregulation of uPAR at both the mRNA and protein levels. In this study, we used SNB19 to determine how the presence or absence of uPAR promotes cellspreading and associated changes in cell morphology. Microscopic analysis of cell spreading revealed that antisense uPAR–transfected cells were larger, remained round, and did not spread efficiently over extracellular matrix substrate type IV collagen and fibronectin, unlike parental SNB19 cells, which were smaller and spindle shaped. Biochemical studies showed that antisense uPAR–transfected cells, in addition to not spreading, exhibited increased expression of α3β1 integrin but not α5β1 integrin. However, we could not find a change in the expression of extracellular matrix components or altered growth rate in these cells. Furthermore, despite the increased α3β1 integrin expression, antisenseuPAR–transfected cells failed to form an organized actin cytoskeleton when plated on type IV collagen or fibronectin, unlike parental SNB19 cells, which displayed an organized cytoskeleton. These findings show that the absence of uPAR in human glioma cells leads to morphological changes associated with decreased spreading and a disorganized cytoskeleton resulting in altered cell morphology, suggesting that coordinated expression of uPAR and integrin may be involved in spreading of antisense uPAR–transfected glioma cells. Mol. Carcinog. 20:355–365, 1997. © 1997 Wiley-Liss, Inc.