Characterization of the tumor suppressor function of PAX6 in glioblastoma: Regulation of tumor invasion and angiogenesis

Glioblastoma multiforme (GBM) is the most common and most deadly brain tumor which is characterized by its aggressive angiogenic and invasive nature. The transcription factor PAX6 has been reported to suppress the tumorigenecity of GBM cells, and its expression in human GBM tumors is a prognostic predictor of longer patient survival. However, the mechanisms behind these functions are not clear. Since a reverse correlation between PAX6 expression and the expression of the pro-invasive enzyme matrix metalloproteinase-2 ( MMP2) has previously been shown in human GBM, PAX6 regulation of MMP2 expression and glioma cell invasion was studied. Both stable and transient adenoviral-mediated over-expression of PAX6 within GBM cells in vitro caused a significant decrease in MMP2 expression. In addition, PAX6 was shown to inhibit GBM cell invasiveness, which correlated with MMP2 protein and mRNA levels within the cells. Luciferase assays displayed the ability of PAX6 to suppress MMP2 promoter activity, and EMSAs showed that PAX6 directly binds to a 245bp region in the MMP2 promoter. Furthermore, chromatin immunoprecipitation assays confirmed this binding in vivo. To further characterize possible mechanisms of GBM suppression by PAX6, PAX6's effect on the expression of the angiogenic vascular endothelial growth factor (VEGF) was studied. Results show that PAX6 suppressed VEGF expression in several GBM cell lines grown in vitro and in vivo. Luciferase assays suggested that PAX6's regulation of VEGF expression occurs through regulation of VEGF promoter activity. Since the tumor suppressor PTEN has been reported to suppress VEGF expression, the regulation of VEGF expression by both PAX6 and/or PTEN was examined. These studies elucidated a synergistic suppression of VEGF expression when both tumor suppressors were present. Co-immunoprecipitation studies revealed that PAX6 and PTEN interact with one another, however, PAX6 did not affect PTEN-mediated regulation of the PI3K/AKT pathway, through which VEGF is a target. Thus PAX6-mediated regulation of VEGF is independent of PTEN. Overall, data from this study further support the role of PAX6 in suppressing GBM and provides several mechanisms underlying this suppression---via suppressing cell invasiveness through directly suppressing MMP2 expression, by affecting angiogenesis via suppressing VEGF expression, and through enhancing PTEN-mediated suppression signals.