Abstract 1927: Tumor suppressor PTEN regulates cancer stem cells of glioblastoma multiforme: identification of signaling pathways as targets of therapy

Abstract

Abstract Glioblastoma Multiforme (GBM), a WHO defined grade IV astrocytoma, is a uniformly fatal CNS malignancy. The high rate of recurrence is attributed to the presence of its untreatable cancer stem cells (CSC). Phosphatase and tensin homologue on chromosome 10 (PTEN), a tumor suppressor is a central negative regulator of PI3K/AKT signaling axis that controls multiple cellular functions, including cell growth, survival and proliferation. Recent evidence suggests that PTEN loss is associated with increased CSC in GBM. Furthermore, downstream from PTEN/PI3K/AKT is mTOR, an atypical serine threonine kinase that exists in two multiprotein complexes (mTORC1 and mTORC2), which regulates growth and dissemination of GBM. The molecular mechanism involved in regulation of CSC by PTEN and its interacting pathways remains elusive. The aim of this study is to elucidate the role of PTEN in regulation and maintenance of CSC in GBM, and define the potential downstream targets of therapy. In order to achieve our goals, we evaluated the presence of activated AKT/mTOR, and stem cell marker nestin in surgically resected GBM tumors. GBM cell line U87 (PTEN mutated) was transfected with PTEN plasmids (wild type and 3 phosphorylated mutants) to evaluate its role in CSC self-renewal. Pharmacological treatments included an inhibitor of EGFR, gefitinib (Iressa), with or without inhibitors of PI3K (LY294002; LY) or inhibitors of mTORC1 (rapamycin) or mTORC1/2 (PP242). Expression of mTOR and stem cell markers were assessed using IHC or IF analysis, and protein expression was determined via Western Blotting. Functional analysis included self-renewal, drug withdrawal, differentiation, proliferation and migration. Results demonstrated that a significant number of GBM tumors expressed stem cell markers nestin as well as pAKT and pmTOR. Wild type PTEN expressing CSC had decreased self-renewal compared to PTEN expressing mutant CSCs. PI3K inhibitor, LY, with gefitinib was most effective in suppressing neurosphere formation. Rebound following withdrawal of inhibitors showed a significant upregulation in self-renewal of CSC in cells treated with gefitinib and either LY or PP242. These combinations also significantly suppressed CSC proliferation. mTOR suppression altered the expression of stem cell markers nestin and SOX2. In conclusion, the findings of this study demonstrate that PTEN/PI3K/mTOR signaling forms a complex network that regulates and maintains GBM CSC; this also underscores the combined suppression of interacting signaling pathways, such as EGFR, in targeting GBM CSC. Note: This abstract was not presented at the meeting. Citation Format: Michael LaBagnara, Keith Lambert, Sudeepta Sridhara, Michael Tobias, Raj Murali, Meena Jhanwar-Uniyal. Tumor suppressor PTEN regulates cancer stem cells of glioblastoma multiforme: identification of signaling pathways as targets of therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1927. doi:10.1158/1538-7445.AM2014-1927