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The Critical Role of Cyclin D2 in Cell Cycle Progression and Tumorigenicity of Glioblastoma Stem Cells

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The Critical Role of Cyclin D2 in Cell Cycle Progression and Tumorigenicity of Glioblastoma Stem Cells Oncogene (2013) 32, 3840–3845 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE The critical role of cyclin D2 in cell cycle progression and tumorigenicity of glioblastoma stem cells R Koyama-Nasu1,4, Y Nasu-Nishimura1,4, T Todo2, Y Ino2, N Saito2, H Aburatani3, K Funato1, K Echizen1, H Sugano1, R Haruta1, M Matsui1, R Takahashi1, E Manabe1, T Oda1 and T Akiyama1 Cancer stem cells are believed to be responsible for tumor initiation and development. Much current research on human brain tumors is focused on the stem-like properties of glioblastoma stem cells (GSCs). However, little is known about the molecular mechanisms of cell cycle regulation that discriminate between GSCs and differentiated glioblastoma cells. Here we show that cyclin D2 is the cyclin that is predominantly expressed in GSCs and suppression of its expression by RNA interference causes G1 arrest in vitro and growth retardation of GSCs xenografted into immunocompromised mice in vivo. We also demonstrate that the expression of cyclin D2 is suppressed upon serum-induced differentiation similar to what was observed for the cancer stem cell marker CD133. Taken together, our results demonstrate that cyclin D2 has a critical role in cell cycle progression and the tumorigenicity of GSCs. Oncogene (2013) 32, 3840–3845; doi:10.1038/onc.2012.399; published online 10 September 2012 Keywords: cancer stem cells; cell cycle; cyclin D2; glioblastoma INTRODUCTION investigate the role of cyclin D2 in cell cycle progression and the Glioblastoma is the most common primary brain tumor in adults.1 tumorigenicity of GSCs. Patients diagnosed with glioblastoma have a median survival of o1 year with generally poor responses to all therapeutic modalities. The existence of cancer stem cells responsible for tumor initiation RESULTS and development has been demonstrated in a variety of tumors.2 Predominant expression of cyclin D2 in GSCs The discovery of glioblastoma stem cells (GSCs) was made by We cultured GSCs isolated from four patients, GB1–3 and 5, under applying techniques for cell culture and analysis of normal neural serum-free conditions that favor NSC growth.4 Our DNA array stem cells (NSCs) to brain tumor cell populations.3 Glioblastoma analysis revealed that GB1 and GB2, 3 and 5 belong to the cells cultured in serum-free media supplemented with epidermal mesenchymal and proneural groups, respectively9 (Supple- growth factor and basic fibroblast growth factor form spheres and mentary Figure 1A). In vitro differentiation was induced in medium maintain stem cell-like properties and tumorigenicity, but when containing fetal bovine serum.4 Differentiated GB1–3 cells grown under serum-containing culture conditions, glioblastoma proliferated more rapidly than their parental undifferentiated cells undergo irreversible differentiation and lose their cells did (data not shown). By contrast, proliferation of GB5 cells tumorigenicity.4 Therefore, the mechanisms of maintaining the was inhibited in medium containing serum (Supplementary stem-like properties of GSCs have been studied extensively. Figure 2). Consistent with these properties, immunoblotting D-type cyclins are known to have critical roles in cell cycle analysis using anti-RB antibodies revealed that RB was highly progression.5 Three D-type cyclins, cyclin D1, D2 and D3, are phosphorylated in differentiated GB1–3 cells and undifferentiated encoded by distinct genes, but show significant amino-acid GB5 cells (Figure 1a). These differences may reflect the properties similarity. Among these, cyclin D1 was first discovered and has associated with the primary tumors from which they were derived. been studied most extensively. D-type cyclins associate with To study the molecular mechanisms of cell cycle regulation in partner cyclin-dependent kinases, CDK4 and CDK6, and promote GSCs, lysates from glioblastoma cells were subjected to immuno- phosphorylation and subsequent inactivation of the blotting analysis with antibodies to various cyclins. We found that retinoblastoma tumor suppressor gene product, RB and RB- cyclin D2 was abundantly expressed in undifferentiated GB2, 3 related proteins. This causes the release or de-repression of the and 5 cells, but was barely expressed in differentiated GB2, 3 and 5 E2F transcription factors and allows cells to enter the S phase. cells, which had been cultured in serum-containing medium Dysregulation of the G1/S transition appears to be a common (Figure 1a). By contrast, cyclin D1 expression was higher in event in tumorigenesis.6 Indeed, alterations in important differentiated than in undifferentiated glioblastoma cells. components of the RB pathway are frequently observed in a The expression levels of cyclin D3 did not change significantly. variety of tumors, including glioblastoma.6–8 In this study, we In GB1 cells, cyclin D2 expression was not detected in either 1Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan; 2Department of Neurosurgery, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan and 3Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Meguro-ku, Tokyo, Japan. Correspondence: Professor T Akiyama, Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 110-0032, Japan. Email: [email protected] 4These authors contributed equally to this work. Received 22 February 2012; revised 29 June 2012; accepted 20 July 2012; published online 10 September 2012 Role of cyclin D2 in glioblastoma stem cells R Koyama-Nasu et al 3841 expression levels of cyclin D2 was significantly decreased within one day after serum addition and was completely downregulated in late-passage cells (410 passages), similar to what was observed for the cancer stem cell marker CD133 (Mizrak et al.10) and the NSC marker nestin11 (Figure 2a, left panel). By contrast, cyclin D1 mRNA was induced upon serum stimulation and highly expressed in late- passage cells. However, when differentiated GB2 cells (410 passages) were cultured in serum-free conditions, cells started to form nonadherent, multicellular spheres indistinguishable from undifferentiated GSCs (Supplementary Figure 3B). Nevertheless, the expression levels of cyclin D2 as well as CD133 and nestin were not fully restored by serum deprivation (Figure 2a, right panel). We also investigated the expression profiles of D-type cyclins in patient glioblastomas, commonly used glioma cell lines and normal human NSCs taken from a public microarray database (Figure 2b). As expected, almost all patient glioblastomas expressed substantial levels of cyclin D2, whereas no glioma cell line did. Cyclin D1 was highly expressed in the glioma cell lines. Moreover, the expression of cyclin D2 as well as CD133 and nestin was not restored by serum withdrawal in the glioma cell line T98G, U251 or U87 (Figure 2c). Furthermore, either overexpression of p21 or depletion of E2F1 and/or E2F2 did not lead to the restoration of cyclin D2 expression (Supplementary Figures 4A–D). These data are compatible with the notion that these cell lines have become differentiated under serum-containing conditions. Interestingly, cyclin D2 and D3, but not cyclin D1, were found to be expressed in human NSCs. Cyclin D3 and D1 showed a reciprocal expression pattern in the glioma cell lines. We next investigated the expression profiles of D-type cyclins in various histological grades of gliomas. The expression levels of cyclin D2, but not of cyclin D1 or D3, were found to be significantly upregulated in glioblastomas (grade IV) compared with astro- cytomas (grade II or III) and non-tumor tissues (Supplementary Figure 5 and Supplementary Table 1). Taken together, these data raise the possibility that cyclin D2 expression may be a common feature of GSCs. Important role of cyclin D2 in cell cycle progression of GSCs We next examined the role of cyclin D2 in cell cycle progression of GSCs using small interference RNA (siRNA). Flow-cytometric analyses of DNA content in undifferentiated GB2 cells showed that knockdown of cyclin D2, but not of cyclin D1 and/or D3, resulted in a significant increase in the fraction of cells in the G1 Figure 1. Predominant expression of cyclin D2 in GSCs. (a) Lysates phase (Figure 3a, left panel). Consistent with this result, knock- from undifferentiated (stem) or differentiated (diff) GB1–3 and 5 down of cyclin D2 led to an increase in the amount of the cells were immunoblotted with antibodies to the indicated proteins. hypophosphorylated form of RB, suggesting that the activity of The asterisk indicates a non-specific band. (b) The mRNA levels CDK4 and/or CDK6 was suppressed and cells were arrested in the of D-type cyclins in undifferentiated (stem) or differentiated (diff) G1 phase of the cell cycle (Figure 3b, left panel). Silencing of cyclin GB1–3 and 5 cells were evaluated by quantitative RT–PCR. Error D2 expression also caused a reduction in the level of cyclin B1, the bars represent the s.d. (n ¼ 3). expression of which is known to be low in the G1 phase (Figure 3b, left panel). In addition, knockdown of cyclin D2 the undifferentiated or differentiated state. This may be a resulted in a slight decrease in the expression levels of E2F1 and common feature in the mesenchymal subtype of glioblastoma E2F2. We also observed that ectopic expression of cyclin D1 or D3 (Supplementary Figure 1B). Reverse transcription–polymerase partially rescued the reduction in RB expression and phosphoryla- chain reaction (RT–PCR) analysis also revealed that cyclin D2 tion caused by knockdown of cyclin D2. Thus, a certain amount of messenger RNA (mRNA) was predominantly expressed in undif- D-type cyclins may be required for cell cycle progression of ferentiated GB2, 3 and 5 cells (Figure 1b). undifferentiated GB2 cells (Supplementary Figure 6). By contrast, knockdown of cyclin D1, but not of cyclin D2 or D3, induced G1 arrest of differentiated GB2 cells cultured in serum-containing GSC-specific expression of cyclin D2 medium (Figures 3a and b, right panels).
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