Abstract 5503: Anti-proliferative role of MMP-9 and uPAR/cathepsin B knockdown in negative regulation of DNA repair mechanisms in human glioma xenografts

Abstract

Abstract Glioblastoma multiforme (GBM) is one of the most lethal forms of brain tumors. Efficient repair of DNA damage, especially double strand breaks (DSBs) and anti-apoptotic mechanisms associated with GBM, makes treating GBM challenging. In the present study, siRNA-mediated transcriptional silencing of MMP-9 and uPAR (MU) and MMP-9 and cathepsin B (MC) in 4910 and 5310 glioma xenograft cells had significant anti-proliferative effect as assessed by BrdU, MTT and clonogenic assays. FACS analysis revealed cells were in sub-G0-G1 phase after xenograft cells were treated with MU or MC. We also investigated the role of MU or MC knockdown in reducing DNA damage response (DDR), which eventually leads to apoptosis in glioma xenograft cells. RT-PCR and Western blot analyses revealed downregulation of DNA PKcs and Ku70/80 levels, which are responsible for non-homologous end joining (NHEJ) DNA repair. Elevated γH2AX protein expression and increased comet tail length indicated that the reduced NHEJ DNA repair led to accumulation of DSBs in MU and MC knocked down cells. The binding efficiency of Ku70 and Ku80 to DSB was significantly reduced in glioma cells treated with MU or MC. In addition, we noticed a reduced γH2AX and Ku70/Ku80 colocalization at DSBs in MU- and MC-treated xenograft cells. Moreover, MU and MC knockdown in these xenograft cells resulted in the downregulation of several other proteins involved in DNA damage response and the survival pathway (e.g., ATM, Rad 51, CHK2, EGFR, Akt, and ERK). Immunohistochemistry of the brain sections of xenografts injected into nude mice showed a reduced expression of DNA PKcs in animals treated with MU and MC siRNA. Taken together, our results clearly demonstrate that silencing via MU and MC not only negatively regulates DNA damage response, but also downregulates the proteins associated with the survival pathway. Based on these findings, it appears that silencing MU or MC in addition to radiation could be an effective strategy to treat radioresistant high-grade gliomas, where treatment usually fails due to the enhanced repair of DSBs induced by radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5503. doi:10.1158/1538-7445.AM2011-5503