642. NDRG4 Is a Novel Therapeutic Target in Malignant Meningioma and Its Loss Induces Apoptosis

Abstract

NDRG4 is a member of the N-Myc downstream regulated gene family belonging to the alpha/beta hydrolase super family. The roles of NDRG4 in meningioma tumor development have not previously been evaluated. Effective local therapies include surgery and radiation, but there is a need for novel molecular targets to improve survival and reduce morbidity for meningioma patients. We have recently identified the NDRG4 protein as being overexpressed in aggressive meningiomas. We have demonstrated that NDRG4 downregulation results in decreased cell proliferation, migration and invasive properties. In follow up to our prior studies on the effects of NDRG4 gene silencing in IOMM-Lee and CH-157 MN meningioma cell lines; here we demonstrate that the predominant form of cell death is apoptosis, utilizing Annexin-V flow cytometry assay. We show that apoptosis caused by upregulation of p53, phosphorylation of p53 at ser 15, Bax activation and downregulation of Bcl-2 and BcL-xL along with cytochrome c release from mitochondria and execution of caspases after depletion of NDRG4. The proapoptotic effect of p53 was verified by the results in which a small molecule compound PFT-α, an inhibitor of p53 phosphorylation, is greatly protected against targeting NDRG4 induced apoptosis. Sub-cellular distribution of Bax and cytochrome c indicated mitochondrial-mediated apoptosis. In addition, we carried out the fluorescence cytohemcial analysis to confirm the mitochondrial-mediated apoptosis by changes in mitochondrial membrane potential (Ym), using JC-1 dye. These findings bring novel insight to the roles of NDRG4 in meningioma tumor cell progression. We hypothesized that NDRG4 has played crucial role in regulating the p53 by its molecular interactions. Binding of NDRG4 with p53 was confirmed by Immunoprecipitation and double Immunocolocalization was verified by confocal microscopy. In conclusion, we show that NDRG4 is predominantly a cytosolic protein expressed in response to cell proliferation and demonstrated the suppression of NDRG4 induces the apoptosis through p53 activated and mitochondrial mediated apoptotic pathway. This therapeutic strategy may have promise in the management of meningiomas.