Irradiation-induced angiogenesis is associated with an MMP-9-miR-494-syndecan-1 regulatory loop in medulloblastoma cells.

Matrix metalloproteinase-9 (MMP-9) represents one of the most prominent proteins associated with tumorigenesis and is a modulator of the tumor microenvironment during angiogenesis. Recently, syndecan-1 (SDC1), a transmembrane heparan sulfate-bearing proteoglycan, was also speculated to have a critical role in contributing to angiogenesis when associated with MMP-9. However, the mechanism behind their synergistic regulation is not fully understood. In the current study, we report for the first time that ionizing radiation (IR)-induced MMP-9 enhances SDC1 shedding, corroborating to tube-inducing ability of medulloblastoma (MB) cells. Furthermore, we observed that the tumor angiogenesis is associated with higher MMP-9-SDC1 interactions on both the cell surface and extracellular medium. Our results also revealed the existence of a novel regulatory mechanism where MMP-9 drives the suppression of miR-494, resulting in enhanced SDC1 shedding and angiogenesis. From the in situ hybridization analysis, we found that MMP-9-specific shRNA (shMMP-9) treatment of mouse intracranial tumors resulted in elevated expression of miR-494. This negative correlation between MMP-9 and miR-494 levels was observed to be dependent on the methylation status of a miR-494 promoter-associated CpG island region (-186 to -20), which was confirmed by bisulfite-sequencing and methylation-specific PCR (MSP) analysis. Further, validation of MMP-9 and SDC1 3'-untranslated region (3'-UTR) targets with luciferase reporter assay provided a more favorable result for miR-494-mediated regulation of SDC1 but not of MMP-9, suggesting that the 3'-UTR of SDC1 mRNA is a direct target of miR-494. Overall, our results indicate that angiogenesis induced by radiotherapy is associated with an MMP-9-miR-494-SDC1 regulatory loop and that MMP-9-SDC1 activity creates a negative feedback loop by regulating the expression of miR-494.

uPAR and cathepsin B shRNA impedes TGF-ß1-driven proliferation and invasion of meningioma cells in a XIAP-dependent pathway.

Overexpression of transforming growth factor ß1 (TGF-ß1) has been linked to immune suppression, tumor angiogenesis, tumor cell migration, tumor cell survival, and tumor cell invasion in many cancers. In the present study, we found abundant expression of TGF-ß1 in the microenvironment of four different pathological types of meningioma tumors. TGF-ß1 induced invasion in malignant meningioma cells with an associated upregulation of urokinase-type plasminogen activator (uPA), uPAR, cathepsin B, and MMP-9, and this increase in proliferation was coupled with the expression of anti-apoptotic and pro-survival signaling molecules. In addition to the intense immunoreactivity of meningioma tumors to X-linked inhibitor to apoptosis (XIAP), its knockdown abolished the TGF-ß1-induced proliferation of these cells. The stimulation of XIAP expression and the activation of pSMAD-2 is mediated by phosphatidylinositol 3-kinase (PI3K)- and MEK-dependent pathways, and the addition of anti-TGF-ß1 antibodies prevented their expression with a consequent decrease in invasion. Bicistronic shRNA constructs targeting uPAR and cathepsin B (pUC) quenched TGF-ß1-driven invasion and survival of meningioma cells by downregulation of XIAP and pSMAD-2 expression. Animal models with intracranial tumors showed elevated levels of TGF-ß1, XIAP and pSMAD-2, and pUC treatment prevented this increased expression. Thus, targeted silencing of TGF-ß1-induced signaling by pUC in meningioma would provide new treatment approaches for management of meningioma.

MMP-2 mediates mesenchymal stem cell tropism towards medulloblastoma tumors.

Matrix metalloproteinases (MMPs) are a family of proteinases known to have a role in cell migration. In the present study, we evaluated the role of MMP-2 on tropism of human umbilical cord blood-derived stem cells (hUCBSCs) in a human medulloblastoma tumor model. Consequences of MMP-2 inhibition on stem cell tropism towards medulloblastoma were studied in terms of stem cell migration by using cell culture inserts, transwell chamber assay, western blotting for MMP-2 and migratory molecules, and immunohistochemistry. Conditioned medium from Daoy/D283 cells infected with adenoviral vector encoding MMP-2 small interfering RNA (siRNA) (Ad-MMP-2 si)-reduced stem cell migration as compared with conditioned medium from mock and scrambled vector (Ad-SV) infected cells. In addition, MMP-2 inhibition in the tumor cells decreased the expression of stromal cell-derived factor 1 (SDF1) in the tumor-conditioned medium, which results in impaired SDF1/CXCR4 signaling leading to decreased stem cell tropism towards the tumor cells. We further show that MMP-2 inhibition in the tumor cells repressed stem cell tropism towards medulloblastoma tumors in vivo. In summary, we conclude that hUCBSCs can integrate into human medulloblastoma after local delivery and that MMP-2 expression by the tumor cells mediates this response through the SDF1/CXCR4 axis.