Abstract 4306: Functional role of CD133 in glioblastoma multiforme

Abstract

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%). Such tumours are believed to be initiated and maintained by a subpopulation of cells, which resemble normal adult stem cells. Cancer stem cells (CSCs), may contribute to the chemo-/radio-resistance exhibited by these tumours and can be identified using the immunocytochemical marker CD133. This pentaspan membrane protein is associated with increased tumorigenicity, chemo-/radio-resistance and poor prognosis. In this study we investigated the functional role of CD133 in the progression of GBM. Methods: CD133-specific siRNA and siPORT-Amine transfection reagent were used to achieve transient knockdown of CD133 in GBM cell lines. Gene and protein expression were measured over time using real-time PCR and FACS, respectively. GBM cell lines were cultured in 1% oxygen to induce hypoxia. Transient knockdown of hypoxia-inducible factors (HIF) was also achieved using HIF-specific siRNA, in hypoxic conditions. Biological functions of CD133 were assessed by performing wound-healing assay to investigate migration; MTT to measure the rate of proliferation; neurosphere formation potential to assess tumorigenicity; and etoposide drug challenge to assess chemo-resistance. Results: CD133-specific siRNA successfully knocked down gene expression of CD133 (85% knockdown; p<0.0001 at Day 3 post transfection; n=3). Consequent downregulation in protein expression was also confirmed (p<0.05; n=1). CD133 knockdown was correlated to susceptibility to chemotherapeutic agents, migration, proliferation and tumorigenicity. Hypoxia upregulated CD133 expression by 4-fold (p<0.0001) compared to cells cultured in normoxia. Knockdown of hypoxia inducible factors resulted in the downregulation of CD133 in hypoxia, at 48hrs post transfection. In GBM cell line U251, HIF1-α and HIF2-α knockdown significantly downregulated CD133 gene expression (HIF1-α = 40% downregulation; HIF2-α = 60% downregulation; p<0.001); HIF2-α downregulated CD133 significantly more than HIF1-α (p<0.01), when compared to control negative siRNA. Conclusion: CD133 knockdown alters biological properties of GBM cells. Furthermore, hypoxia increases CD133 expression in GBM cells, mediated by HIF-1α and HIF-2α. This of particular importance in view of the hypoxic nature of GBMs, which suggests that hypoxia aids survival of cancer stem cells, rendering these tumours resistant to therapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 4306.