TARGETING GLIOBLASTOMA STEM CELLS VIA INHIBITION OF PI3K/AKT PATHWAY ALONE AND IN COMBINATION WITH AUTOPHAGY BLOCKADE

BACKGROUND: Glioblastoma Multiforme (GBM) is the most aggressive form of malignant primary brain tumor. A subset of glioblastoma cells, known as glioblastoma stem-like cells (GSCs), are chemotherapy and radiotherapy-resistent and are hypothesized to be responsible for tumor formation, maintenance, and recurrence. The PI3K/AKT pathway is overactive in GBM and has been shown to play a role in cancer stem cell maintenance. Therefore we sought to determine the efficacy of drugs targeting the AKT pathway in multiple patient-derived GSC lines. METHODS: GSC lines were generated from patient's tumors, propagated in neurosphere media and analyzed for stem cell markers by immunocytochemistry. Effect of growth factor receptor inhibitors (erlotinib, gefetinib, sunitinib), the intracellular PI3K/AKT pathway inhibitors (GDC-0941, OSU-03012, perifosine, NVP-BEZ235, KU-0063794 and everolimus) and autophagy inhibitor (chloroquine) on cell viability was determined with MTS assay. Caspase 3/7 activity was determined using CellEvent caspase reagent. The effect of the inhibitors on cell signaling pathways was evaluated by western blot analysis. RESULTS: Growth factor receptor inhibition failed to induce significant reduction in cell viability. Intracellular inhibitors produced variable results except for dual PI3K/mTOR inhibitor NVB-BEZ235 which induced approximately 50% reduction in cell viability. While the inhibition of autophagy with chloroquine significantly increased inhibitor induced cell death in some cell lines, combined PDK1 and autophagy inhibition induced robust caspase activity and significantly reduced viability in all six GSCs examined to an average of 6.1 + 2.7% viability compared to non-treated controls. CONCLUSION: The AKT/PI3K pathway is overactive in GSCs and may contribute to stem cell maintenance as well as inhibition of apoptosis. Our data indicates that inhibiting autophagy concommitantly with PI3K/AKT pathway inhibition can potentiate their effect. Furthermore combined chloroquine and OSU-03012 resulted in a substantial decrease cell viability accross multiple GSC lines and may represent an adjuvant therapy for a disease with minimal survival.