Abstract 3397: Validation of targets involved in supporting the stem cell niche in glioblastoma multiforme

Abstract

Introduction: Glioblastoma multiforme (GBM) is a cytologically malignant tumour of the central nervous system, associated with poor prognosis and fatal outcome (5 year survival, <6%). GBM cancer stem cells (CSCs), which can be identified using the immunocytochemical marker CD133, are associated with poor prognosis. The gastrin receptor, CCK2R, is also expressed in glioblastoma cell-lines and promotes cancer progression. In this study we investigated the functional role of these two molecules in GBM to elucidate any potential therapeutic benefits. Materials and methods: A panel of glioblastoma cells was grown either as monolayers, or, to provide a 3D in vitro tumour model, as neurospheres, which enrich for the cancer stem cell phenotype. Quantitative RT-PCR was used to quantify CD133 and CCK-2R gene expression over time in these cultures, and flow cytometry (FACS) to investigate expression at the protein level. CD133 and CCK-2R promoter reporters were constructed in pGL4 and promoter activity quantified using luciferase assays. Gene knockdown or specific inhibitors were used to investigate the role of these genes in glioblastoma cell tumorigenicity in vitro (using MTT, wound-healing, and neurosphere formation) and in an in vivo xenograft model. Results: CD133 and CCK-2R gene and protein expression were higher in the more tumorigenic glioblastoma cell-line, U251, compared with the less tumorigenic line, U373. For both markers, higher gene expression correlated with higher promoter activity and FACS demonstrated the presence of a small population of positively-stained cells. Expression of both genes was upregulated during neurosphere formation, which was accelerated in hypoxia. Knockdown of CD133 and CCK-2R inhibition in cell line U251 reduced neurosphere-forming potential (p<0.05). Knockdown of CD133 also significantly reduced migration (p<0.001) and increased susceptibility to the chemotherapeutic agent etoposide (p<0.05). Conclusions: CD133 and CCK-2R expression correlate with tumorigenic potential of GBM cells and are upregulated during neurosphere formation, which enriches for cancer stem cells. CD133 knockdown and CCK-2R inhibition reduced neurosphere formation. In addition, CD133 knockdown reduced GBM invasive capacity, and increased resistance to chemotherapeutic drugs. Therefore, use of CD133 and CCK-2R-targeted therapies, in combination with established standard of care may improve GBM patient outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3397. doi:10.1158/1538-7445.AM2011-3397