Purpose
Glioblastoma (GBM) is the commonest brain tumour in adults. A sub-population of cells within these tumours, known as cancer stem cells (CSCs), is thought to mediate their chemo-/radiotherapy ...resistance. CD133 is a cell surface marker that is used to identify and isolate GBM CSCs. However, its functional significance, as well as the relevant microenvironment in which to study CD133, have so far remained unknown. Here, we examined the effect of hypoxia on the expression of CD133 and on that of the hypoxia-related factors HIF-1α and HIF-2α, and the potential functional significance of CD133 expression on the acquisition of chemo-resistance by GBM cells.
Methods
CD133, HIF-1α, HIF-2α, VEFG and (control) HPRT mRNA expression analyses were carried out on GBM cells (U251, U87 and SNB19; 2D or 3D cultures) under both normoxic and hypoxic conditions, using qRT-PCR. siRNA was used to downregulate CD133, HIF-1α and HIF-2α expression in the GBM cells, which was confirmed by flow cytometry and qRT-PCR, respectively. Drug sensitivity-related IC50 values were established using an Alamar Blue cell viability assay in conjunction with the Graphpad prism software tool.
Results
We found that the expression of CD133 was upregulated under hypoxic conditions in both the 2D and 3D GBM cell culture models. In addition, an increased resistance to cisplatin, temozolomide and etoposide was observed in the GBM cells cultured under hypoxic conditions compared to normoxic conditions. siRNA-mediated knockdown of either HIF-1α or HIF-2α resulted in a reduced CD133 expression, with HIF-2α having a more long-term effect. We also found that HIF-2α downregulation sensitized the GBM cells to cisplatin to a greater extent than HIF-1α, whereas CD133 knockdown had a more marked effect on cisplatin sensitisation than knockdown of either one of the HIFs, suggesting the existence of a HIF-independent cisplatin resistance mechanism mediated by CD133. This same mechanism does not seem to be involved in temozolomide resistance, since we found that HIF-1α downregulation, but not HIF-2α or CD133 downregulation, sensitized GBM cells to temozolomide.
Conclusions
From our data we conclude that the mechanisms underlying hypoxia-induced CD133-mediated cisplatin resistance may be instrumental for the design of new GBM treatment strategies.
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.
Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct ...adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3-4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents.
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
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