Epidermal Growth Factor Receptor-Mediated Regulation of Urokinase Plasminogen Activator Expression and Glioblastoma Invasion via C-SRC/MAPK/AP-1 Signaling Pathways

One of the major pathophysiological features of malignant astrocytomas is their ability to infiltrate surrounding brain tissue. The epidermal growth factor receptor (EGFR) and proteases are known to be overexpressed in glioblastomas (GBMs), but the interaction between the activation of the EGFR and urokinase plasminogen activator (uPA) in promoting astrocytic tumor invasion has not been fully elucidated. Here, we characterized the signal transduction pathway(s) by which EGF regulates uPA expression and promotes astrocytoma invasion. We show that EGFR activation and constitutively active EGFR vIII in GBM cell lines upregulate uPA expression. Small-molecule inhibitors of mitogen-activated protein kinase, tyrosine kinase, and small interfering RNA targeting c-Src blocked uPA upregulation. Similarly, mutations in the activator protein 1 binding site of the uPA promoter reduced EGF-induced increases in uPA promoter activity. Treatment of GBM cells with EGF increased in vitro cell invasion, and the invasive phenotype was attenuated by gene silencing of uPA using small interfering RNA and short hairpin RNA. In addition, uPA knockdown clones formed smaller well-circumscribed tumors than nontarget U1242 control cells in a xenograft GBM mouse model in vivo. In summary, these results suggest that c-Src, mitogen-activated protein kinase, and a composite activator protein 1 on the uPA promoter are responsible for EGF-induced uPA expression and GBM invasion

EGFR- and EGFR VIII–Mediated Regulation of Urokinase Promotes Astrocytic Tumor Invasion Via the C-SRC/MEK/AP-1 Signaling Pathways

One of the major pathophysiological features of malignant astrocy-tomas is their ability to diffusely infiltrate the surrounding brain tissue. Although it is known that the epidermal growth factor receptor (EGFR) is amplified or overexpressed in primary glioblastomas and that malignant gliomas express higher levels of urokinase-type plasminogen activator (uPA) than normal brain tissue, little is known about the possible interaction between the activation of EGFR or EGFR VIII and uPA and its role in promoting astrocytic tumor invasion. In this study, we characterized the signal transduction pathway by which EGF regulates uPA expression and promotes astrocytoma invasion. Our data showed that the treatment of glioblastoma cell lines with EGF upregulates the expression and activity of uPA in a time-dependent manner. Similarly, the expression of the EGFR VIII mutant receptor also induced high uPA expression levels. The increase in uPA protein by EGF or EGFR VIII was abrogated by the MEK inhibitor UO 126, the tyrosine kinase inhibitor AG 1478, the small interfering RNA (siRNA) targeting c-Src, and the c-Src inhibitor PP2. Also, EGF-increased uPA promoter activity was abrogated by mutations in the AP-1 sites. Furthermore, treatment with UO 126 attenuated the promoter activity, while the phosphatidylinositol 3-kinase inhibitor LY294002 did not affect the EGF-induced increase in promoter activity. Treatment with EGF increased the extent of in vitro invasion as determined by the Boyden chamber assay, and invasion was attenuated by UO 126, siRNA, and short hairpin RNA (shRNA) directed against uPA. In addition, uPA knockdown cells formed fewer colonies on soft agar than wild-type cells and formed smaller, well-circumscribed tumors than parent U1242 cells in a xeno-graft glioblastoma multiforme mouse model. In summary, we conclude that EGF requires EGFR kinase activity, mitogen-activated protein kinase, and AP-1-dependent pathways to induce uPA expression and to promote glioblastoma invasion

EGFR- and EGFR VIII–Mediated Regulation of Urokinase Promotes Astrocytic Tumor Invasion Via the C-SRC/MEK/AP-1 Signaling Pathways

One of the major pathophysiological features of malignant astrocy-tomas is their ability to diffusely infiltrate the surrounding brain tissue. Although it is known that the epidermal growth factor receptor (EGFR) is amplified or overexpressed in primary glioblastomas and that malignant gliomas express higher levels of urokinase-type plasminogen activator (uPA) than normal brain tissue, little is known about the possible interaction between the activation of EGFR or EGFR VIII and uPA and its role in promoting astrocytic tumor invasion. In this study, we characterized the signal transduction pathway by which EGF regulates uPA expression and promotes astrocytoma invasion. Our data showed that the treatment of glioblastoma cell lines with EGF upregulates the expression and activity of uPA in a time-dependent manner. Similarly, the expression of the EGFR VIII mutant receptor also induced high uPA expression levels. The increase in uPA protein by EGF or EGFR VIII was abrogated by the MEK inhibitor UO 126, the tyrosine kinase inhibitor AG 1478, the small interfering RNA (siRNA) targeting c-Src, and the c-Src inhibitor PP2. Also, EGF-increased uPA promoter activity was abrogated by mutations in the AP-1 sites. Furthermore, treatment with UO 126 attenuated the promoter activity, while the phosphatidylinositol 3-kinase inhibitor LY294002 did not affect the EGF-induced increase in promoter activity. Treatment with EGF increased the extent of in vitro invasion as determined by the Boyden chamber assay, and invasion was attenuated by UO 126, siRNA, and short hairpin RNA (shRNA) directed against uPA. In addition, uPA knockdown cells formed fewer colonies on soft agar than wild-type cells and formed smaller, well-circumscribed tumors than parent U1242 cells in a xeno-graft glioblastoma multiforme mouse model. In summary, we conclude that EGF requires EGFR kinase activity, mitogen-activated protein kinase, and AP-1-dependent pathways to induce uPA expression and to promote glioblastoma invasion

H-Ras Increases Urokinase Expression and Cell Invasion in Genetically Modified Human Astrocytes through Ras/Raf/MEK signaling pathway

Previous study reported that the activation of Ras pathway cooperated with E6/E7-mediated inactivation of p53/pRb to transform immortalized normal human astrocytes (NHA/hTERT) into intracranial tumors strongly resembling human astrocytomas. The mechanism of how H-Ras contributes to astrocytoma formation is unclear. Using genetically modified NHA cells (E6/E7/hTERT and E6/E7/hTERT/Ras cells) as models, we investigated the mechanism of Ras-induced tumorigenesis. The overexpression of constitutively active H-RasV12 in E6/E7/hTERT cells robustly increased the levels of urokinase plasminogen activator (uPA) mRNA, protein, activity and invasive capacity of the E6/E7/hTERT/Ras cells. However, the expressions of MMP-9 and MMP-2 did not significantly change in the E6/E7/hTERT and E6/E7/hTERT/Ras cells. Furthermore, E6/E7/hTERT/Ras cells also displayed higher level of uPA activity and were more invasive than E6/E7/hTERT cells in 3D culture, and formed an intracranial tumor mass in a NOD-SCID mouse model. uPA specific inhibitor (B428) and uPA neutralizing antibody decreased uPA activity and invasion in E6/E7/hTERT/Ras cells. uPA-deficient U-1242 glioblastoma cells were less invasive in vitro and exhibited reduced tumor growth and infiltration into normal brain in xenograft mouse model. Inhibitors of Ras (FTA), Raf (Bay 54−9085) and MEK (UO126), but not of phosphatidylinositol 3-kinase (PI3K) (LY294002) and of protein kinase C (BIM) pathways, inhibited uPA activity and cell invasion. Our results suggest that H-Ras increased uPA expression and activity via the Ras/Raf/MEK signaling pathway leading to enhanced cell invasion and this may contribute to increased invasive growth properties of astrocytomas

Protein Kinase C-α–Mediated Regulation of Low-Density Lipoprotein Receptor–Related Protein and Urokinase Increases Astrocytoma Invasion

Aggressive and infiltrative invasion is one of the hallmarks of glioblastoma. Low-density lipoprotein receptor–related protein (LRP) is expressed by glioblastoma, but the role of this receptor in astrocytic tumor invasion remains poorly understood. We show that activation of protein kinase C-α (PKC-α) phosphorylated and down-regulated LRP expression. Pretreatment of tumor cells with PKC inhibitors, phosphoinositide 3-kinase (PI3K) inhibitor, PKC-α small interfering RNA (siRNA), and short hairpin RNA abrogated phorbol 12-myristate 13-acetate–induced down-regulation of LRP and inhibited astrocytic tumor invasion in vitro. In xenograft glioblastoma mouse model and in vitro transmembrane invasion assay, LRP-deficient cells, which secreted high levels of urokinase-type plasminogen activator (uPA), invaded extensively the surrounding normal brain tissue, whereas the LRP-overexpressing and uPA-deficient cells did not invade into the surrounding normal brain. siRNA, targeted against uPA in LRP-deficient clones, attenuated their invasive potential. Taken together, our results strongly suggest the involvement of PKC-α/PI3K signaling pathways in the regulation of LRP-mediated astrocytoma invasion. Thus, a strategy of combining small molecule inhibitors of PKC-α and PI3K could provide a new treatment paradigm for glioblastomas

An Extensive Invasive Intracranial Human Glioblastoma Xenograft Model : Role of High Level Matrix Metalloproteinase 9

The lack of an intracranial human glioma model that recapitulates the extensive invasive and hypervascular features of glioblastoma (GBM) is a major hurdle for testing novel therapeutic approaches against GBM and studying the mechanism of GBM invasive growth. We characterized a high matrix metalloproteinase-9 (MMP-9) expressing U1242 MG intracranial xenograft mouse model that exhibited extensive individual cells and cell clusters in a perivascular and subpial cellular infiltrative pattern, geographic necrosis and infiltrating tumor-induced vascular proliferation closely resembling the human GBM phenotype. MMP-9 silencing cells with short hairpin RNA dramatically blocked the cellular infiltrative pattern, hypervascularity, and cell proliferation in vivo, and decreased cell invasion, colony formation, and cell motility in vitro, indicating that a high level of MMP-9 plays an essential role in extensive infiltration and hypervascularity in the xenograft model. Moreover, epidermal growth factor (EGF) failed to stimulate MMP-9 expression, cell invasion, and colony formation in MMP-9-silenced clones. An EGF receptor (EGFR) kinase inhibitor, a RasN17 dominant-negative construct, MEK and PI3K inhibitors significantly blocked EGF/EGFR-stimulated MMP-9, cell invasion, and colony formation in U1242 MG cells, suggesting that MMP-9 is involved in EGFR/Ras/MEK and PI3K/AKT signaling pathway-mediated cell invasion and anchorage-independent growth in U1242 MG cells. Our data indicate that the U1242 MG xenograft model is valuable for studying GBM extensive invasion and angiogenesis as well as testing anti-invasive and anti-angiogenic therapeutic approaches