Abstract B39: Targeting cancer stem cells via inhibition of PI3K/AKT pathway alone and in combination with autophagy blockade
Authors: Graham Regina M. , Middleton Andrew , Benito Daniel A. , Uddin Raisa , Zhang Baoyu , Walters Winston , Bregy Amade , Vanni Steven , Komotar Ricardo J. 
Published: October 2015

Abstract
Background: Glioblastoma Multiforme (GBM) is the most aggressive form of malignant primary brain tumor. Despite an aggressive treatment regimen consisting of surgery, radiation and chemotherapy, individuals with GBM survive on average 15 months. 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 an important 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. In addition, we evaluated the cytotxicity of the most promising therapy on medulloblastoma and neuroblastoma stem cells.
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. Medulloblastoma and neuroblastoma stem cells were derived from Daoy and NB1691 cells respectively via propagation in neurosphere media.
Results: GSC's expressed nestin, CD133, GFAP, musashi, BMi1, and SOX2. In addition, transplantation of GSC's into nude mice generated tumors. Of 11 GSC lines examined, all 11 demonstrated activation of the AKT pathway as indicated by robust phosphorylation of AKT (S473) and BAD (S136) and 10/11 demonstrated phosphorylation of mTOR (S2481). 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 (100nM) which consistantly 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 (100±1.9%). Similarly the combination significantly reduced both DAOY and NB1691 stem cell viability. In contrast the combination had no effect on primary neuronal cultures.
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 cytotoxic effect. Furthermore combined chloroquine and OSU-03012 resulted in a substantial decrease cell viability accross multiple GSC lines and in medulloblastoma and neuroblastoma stem cells suggesting that this combination may represent a potential adjuvant therapy these cancers.
Citation Format: Regina M. Graham, Andrew Middleton, Daniel A. Benito, Raisa Uddin, Baoyu Zhang, Winston Walters, Amade Bregy, Steven Vanni, Ricardo J. Komotar. Targeting cancer stem cells via inhibition of PI3K/AKT pathway alone and in combination with autophagy blockade. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B39.