Abstract 1278: Simultaneous downregulation of uPAR and cathepsin B inhibits angiogenesis and migration and induces caspase 8-mediated apoptosis in U87 stem cells and 4910 glioma xenograft stem cells

Abstract

Abstract Malignant gliomas are characterized by invasive and infiltrative behavior. Gliomas are considered to be heterogeneous bulk tumors that comprise differentiated and undifferentiated cells with self-renewal and partial differentiation capabilities. These glioma-initiating cells (GIC) are responsible for the initiation and recurrence of gliomas even after various treatments. As such, novel therapies should target GIC. Cathepsin B and urokinase-type plasminogen activator receptor (uPAR) are overexpressed in gliomas, including GIC. We isolated GIC from U87 (U87S) and 4910 (4910S) by sorting the cells for both CD133 and STRO-1 by FACS analysis. The stem cell nature of these cells was confirmed by neurosphere formation assay and western blot analysis for CD133 and STRO-1 expression. GIC cells expressed 2 to 3-fold increased levels of CD133 and STRO-1 as compared to glioma cells (U87N and 4910N). By isolating and utilizing GIC, we demonstrate that downregulation of uPAR (pU) and cathepsin B (pC) individually and simultaneously (pCU) lead to remarkable inhibition of angiogenesis and migration and activated caspase 8-mediated apoptosis in GIC and U87N and 4910N cells. The treatment retarded the expression of pro-angiogenic molecules in both stem cells and glioma cells but responded differently in terms of expression of certain molecules, such as angiogenin and TIMP-1. Similarly, an apoptotic antibody array revealed that stem cells and glioma cells respond differently to pCU treatment, but both sets of cells exhibited increased expression of pro-apoptotic molecules. This induction of apoptosis was also confirmed by cleavage of caspase 8, caspase 3 and PARP as well as TUNEL assay. Similarly, spheroid migration and matrigel invasion assays, revealed that downregulation of uPAR and cathepsin B inhibited cell migration by 70-75% and 45-50% and invasion by 75-80% and 45-50% in GIC and normal glioma cells, respectively. Untreated glioma stem cells were more migratory (∼30%) and invasive (30-40%) as compared to normal glioma cells. In vivo studies showed few to no tumor cells in brain sections of mice implanted with either GIC or normal glioma cells treated with pCU as determined by H&E staining. These results indicate the potential of RNAi-mediated downregulation of uPAR and cathepsin B in developing new therapeutics for GIC in particular and gliomas in general. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1278.