Downregulation of uPA inhibits migration and PI3k/Akt signaling in glioblastoma cells.

The ability of glioma cells to migrate great distances from a primary tumor mass is the primary cause of tumor recurrence. The urokinase-type plasminogen activator (uPA) is a serine protease that can initiate proteolytic cascades, which result in remodeling of extracellular matrix and basement membrane, allowing cells to move across and through these barriers. The binding between uPA and its receptor uPAR also mediates several signaling events that seem to contribute to the evolution of a migratory phenotype. In this study, we determined how the downregulation of uPA affects the signaling pathways leading to cell migration. Stably transfecting human glioblastoma cells with antisense uPA decreased the amount of cell-bound uPA and disrupted actin cytoskeleton formation and cell migration. The phosphatidylinositol 3-kinase (PI3k) and Akt signaling pathway has been suggested to mediate migration in various cancer cells. The antisense-uPA clones also had less phosphorylated PI3k and Akt than control cells, a finding associated with decreased cell migration, G2/M-phase arrest, and decreased clonogenic survival. Decreased activation of PI3k and the antiapoptotic factor Akt was not sufficient to induce apoptosis in the antisense-uPA clones, but staurosporine sensitized them to apoptosis to a greater extent than control cells. These results indicate that PI3k/Akt pathway is involved in the signaling cascade required to induce cell migration and that uPA has a direct role in regulating migration.

Modulation of invasive properties of human glioblastoma cells stably expressing amino-terminal fragment of urokinase-type plasminogen activator.

The binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR) on the surface of tumor cells is involved in the activation of proteolytic cascades responsible for the invasiveness of those cells. The diffuse, extensive infiltration of glioblastomas into the surrounding normal brain tissue is believed to rely on modifications of the proteolysis of extracellular matrix components; blocking the interaction between uPA and uPAR might be a suitable approach for inhibiting glioma tumorigenesis. We assessed how expression of an amino-terminal fragment (ATF) of uPA that contains binding site to uPAR affects the invasiveness of SNB19 human glioblastoma cells. SNB19 cells were transfected with an expression plasmid (pcDNA3-ATF) containing a cDNA sequence of ATF-uPA. The resulting ATF-uPA-expressing clones showed markedly less cell adhesion, spreading, and clonogenicity than did control cells. Endogenous ATF expression also significantly decreased the invasive capacity of transfected glioblastoma cells in Matrigel and spheroid-rat brain cell aggregate models. ATF-uPA transfectants were also markedly less invasive than parental SNB19 cells after injection into the brains of nude mice, suggesting that competitive inhibition of the uPA-uPAR interaction on SNB19 cells by means of transfection with ATF cDNA could be a useful therapeutic strategy for inhibiting tumor progression.

Downregulation of MMP-9 in ERK-mutated stable transfectants inhibits glioma invasion in vitro.

We previously showed that enhanced expression of MMP-9, an endopeptidase that digests basement-membrane type IV collagen, is related to tumor progression in vitro and in vivo; antisense-MMP-9 stably transfected clones were less invasive than untransfected parental cells and did not form tumors in nude mice. In this study, we examined the role of ERK-1 in the regulation of MMP-9 production and the invasive behavior of the human glioblastoma cell line SNB19, in which ERK1 is constitutively activated. SNB19 cells were stably transfected with mt-ERK, a vector encoding ERK-1 cDNA in which the conserved lysine at codon 71 was changed to arginine, thus impairing the catalytic efficiency of this enzyme. Gelatin zymography showed reduced levels of MMP-9 in the mt-ERK-transfected cell lines relative to those in vector-transfected and parental control cells. Reductions in MMP-9 protein mRNA levels were also detected in the mt-ERK-transfected cells by Western and Northern blotting. The mt-ERK-transfected cells were much less invasive than parental or vector control cells in a Matrigel invasion assay and in a spheroid coculture assay. Thus an ERK-dependent signaling pathway seems to regulate MMP-9 mediated glioma invasion in SNB19 cells; interfering with this pathway could be developed into a therapeutic approach, which aims at a reduction of cancer cell invasion.

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.

Suppression of glioma invasion and growth by adenovirus-mediated delivery of a bicistronic construct containing antisense uPAR and sense p16 gene sequences.

Our previous studies showed that the urokinase-type plasminogen activator receptor (uPAR) and the p16 tumor suppressor gene play a significant role in glioma invasion. We expected that downregulation of uPAR and overexpression of p16 using a bicistronic vector might cause a additive and cooperative effect in the suppression of glioma invasion and growth. The bicistronic construct (Ad-uPAR/p16)-infected glioblastoma cell lines had significantly lower levels of uPAR and higher levels of p16 than controls. Cell cycle analysis showed the bicistronic vector caused G0/G1 arrest of the cell cycle. In vitro glioblastoma cell growth and invasiveness were inhibited in Ad-uPAR/p16-infected cells compared with controls. Ad-uPAR/p16 suppressed the tumor growth of glioblastoma cell lines in an ex vivo intracerebral tumor model and an in vivo subcutaneous tumor model. Our results support the therapeutic potential of simultaneously targeting uPAR and p16 in the treatment of gliomas.

Glial cell-induced endothelial morphogenesis is inhibited by interfering with extracellular signal-regulated kinase signaling.

PURPOSE: Tumor vasculature provides the infrastructure by which malignant tissue can be nourished; therefore, targeting angiogenesis may be an effective means of treating cancer. We showed previously that SNB19 glioblastoma cells modulate bovine retinal endothelial cells in cocultures to form capillary-like network structures, that matrix metalloproteinase-9 (MMP-9) expression is critical for endothelial morphogenesis, and that MMP-9 expression in glioblastoma cells is regulated by extracellular signal-regulated kinase-1 (ERK-1). In the present study, we investigated whether interfering with the activation of this mitogen-activated protein (MAP) kinase would repress MMP-9 synthesis and inhibit capillary formation. EXPERIMENTAL DESIGN: Cocultures of bovine retinal endothelial and SNB19 cells were analyzed for MMP-9 secretion, and phospho- and total ERK levels. These cocultures were treated with PD98059, a specific inhibitor of MAP/ERK kinase 1, or transfected with dominant-negative ERK-1 mutant containing expression vector. Alterations in capillary-like structure formation, and actin cytoskeleton and secretion of vascular endothelial growth factor (VEGF), MMP-9, and tissue inhibitor of metalloproteinase-1 were determined by immunofluorescence, gelatin zymography, and Western blotting. RESULTS: We found that inhibition of the ERK-1/2 pathway with PD98059 abrogated glial cell-mediated capillary formation by the endothelial cells and reduced the levels of MMP-9 in the coculture. Strikingly, the abrogation of MAP kinase signaling by a dominant-negative ERK-1 mutant inhibited glial-induced capillary network formation by reducing VEGF levels and MMP-9 activity and increasing the levels of tissue inhibitor of metalloproteinase-1. Inhibition of ERK activity also disrupted the formation of the actin cytoskeleton, a prerequisite for endothelial cell migration. CONCLUSION: The mechanism underlying activation of ERK is involved in reorganization of the actin cytoskeleton, and induction of VEGF and MMP-9, thereby stimulating endothelial cell morphogenesis. These studies clearly provide experimental evidence that ERK inhibition diminishes glial-induced endothelial-cell morphogenesis; therefore, interfering with ERK signaling may be a viable approach to target angiogenesis.