Abstract 5158: Differentiation of glioblastoma stem-like cells induced by retinoic acid

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Abstract We investigated the molecular and cellular responses of glioblastoma stem-like cells (GBM-SCs) to all-trans retinoic acid (RA)-induced differentiation. Four GBM-SC lines were used. Under neurosphere culture conditions, GBM-SCs grow as non-adherent neurospheres. RA (10 μmol/l) treatment for 48 h induced cells to adhere to culture surfaces and generate processes. RA inhibited G1/G0 to S transition, decreased cyclin D1 expression (50%) and increased p27 expression (2-fold). Immunofluorescence staining, western blot and flow cytometry analyses showed that RA induced the expression of differentiation markers Tuj1 and GFAP and inhibited nestin expression. RA treatment reduced the number of cells expressing CD133 from 44% to 10%. We measured the effects of RA on neurosphere number and size. RA treatment dramatically decreased (∼90%) the number of neurospheres > 50 um diameter. Clonogenic assay showed that RA reduced the colony formation ability of GBM-SCs in soft agar. Microarray analysis was performed to determine the global gene expression profile of RA-induced GBM-SC differentiation. Data were analyzed using Significance Analysis of Microarrays (SAM) and the false discovery rate was set at 20%. Approximately 350 genes were altered after RA treatment for 48h; ∼20% were down-regulated and 80% were up-regulated. We selected 18 candidates and validated with real-time PCR. All 18 candidates exhibited the same pattern of up- or down-regulation by RA. Western blot analysis for some candidate gene products also confirmed the alteration by RA at the protein level. A web-based (portal.genego.com) molecular pathway analysis of RA-induced gene expression profiling identified several pathways altered during RA-induced differentiation process. The most prominent of them was Notch signaling. Notch pathway members Delta-like 1 (DLL1), Notch 1, NCID, NEXT and Notch 1 precursor were found to be down-regulated by RA. Only Delta-like 4 (DLL4) was up-regulated by RA. We also found that cyclin D, cyclin D1, PP2A and SMAD3, gene products involved in G1-S transition, were down-regulated by RA. This is consistence with our findings that RA induces cell cycle arrest. Numerous mechanisms involved in cell adhesion, cell-matrix interaction and cytoskeleton remodeling were also affected by RA. This analysis provides guidelines to further explore pathways and mechanisms during RA-induced GBM-SC differentiation. Our findings may be applicable to identify new molecular target applicable to differentiation therapies for glioblastomas. This will also stimulate the development of GBM stem cell based pre-clinical experimental models with the potential to improve mechanistic and pre-clinical therapeutic research. 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 5158.