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.
Glioblastoma is the most common primary brain tumor in adults.1 Patients diagnosed with glioblastoma have a median survival of <1 year with generally poor responses to all therapeutic modalities. The existence of cancer stem cells responsible for tumor initiation and development has been demonstrated in a variety of tumors.2 The discovery of glioblastoma stem cells (GSCs) was made by applying techniques for cell culture and analysis of normal neural stem cells (NSCs) to brain tumor cell populations.3 Glioblastoma cells cultured in serum-free media supplemented with epidermal growth factor and basic fibroblast growth factor form spheres and maintain stem cell-like properties and tumorigenicity, but when grown under serum-containing culture conditions, glioblastoma cells undergo irreversible differentiation and lose their tumorigenicity.4 Therefore, the mechanisms of maintaining the stem-like properties of GSCs have been studied extensively.
D-type cyclins are known to have critical roles in cell cycle progression.5 Three D-type cyclins, cyclin D1, D2 and D3, are encoded by distinct genes, but show significant amino-acid similarity. Among these, cyclin D1 was first discovered and has been studied most extensively. D-type cyclins associate with partner cyclin-dependent kinases, CDK4 and CDK6, and promote phosphorylation and subsequent inactivation of the retinoblastoma tumor suppressor gene product, RB and RB-related proteins. This causes the release or de-repression of the E2F transcription factors and allows cells to enter the S phase. Dysregulation of the G1/S transition appears to be a common event in tumorigenesis.6 Indeed, alterations in important components of the RB pathway are frequently observed in a variety of tumors, including glioblastoma.6, 7, 8 In this study, we investigate the role of cyclin D2 in cell cycle progression and the tumorigenicity of GSCs.
Predominant expression of cyclin D2 in GSCs
We cultured GSCs isolated from four patients, GB1–3 and 5, under serum-free conditions that favor NSC growth.4 Our DNA array analysis revealed that GB1 and GB2, 3 and 5 belong to the mesenchymal and proneural groups, respectively9 (Supplementary Figure 1A). In vitro differentiation was induced in medium containing fetal bovine serum.4 Differentiated GB1–3 cells proliferated more rapidly than their parental undifferentiated cells did (data not shown). By contrast, proliferation of GB5 cells was inhibited in medium containing serum (Supplementary Figure 2). Consistent with these properties, immunoblotting analysis using anti-RB antibodies revealed that RB was highly phosphorylated in differentiated GB1–3 cells and undifferentiated GB5 cells (Figure 1a). These differences may reflect the properties associated with the primary tumors from which they were derived.
To study the molecular mechanisms of cell cycle regulation in GSCs, lysates from glioblastoma cells were subjected to immunoblotting analysis with antibodies to various cyclins. We found that cyclin D2 was abundantly expressed in undifferentiated GB2, 3 and 5 cells, but was barely expressed in differentiated GB2, 3 and 5 cells, which had been cultured in serum-containing medium (Figure 1a). By contrast, cyclin D1 expression was higher in differentiated than in undifferentiated glioblastoma cells. The expression levels of cyclin D3 did not change significantly. In GB1 cells, cyclin D2 expression was not detected in either the undifferentiated or differentiated state. This may be a common feature in the mesenchymal subtype of glioblastoma (Supplementary Figure 1B). Reverse transcription–polymerase chain reaction (RT–PCR) analysis also revealed that cyclin D2 messenger RNA (mRNA) was predominantly expressed in undifferentiated GB2, 3 and 5 cells (Figure 1b).
GSC-specific expression of cyclin D2
To further investigate the expression pattern of cyclin D2, we focused our analyses on GB2, as cyclin D2 is highly expressed and its expression is dramatically reduced by serum-induced differentiation in these cells. Time-course experiments showed that following serum addition, undifferentiated GB2 cells became attached to the bottom of the dish and formed a monolayer (Supplementary Figure 3A), as reported previously.4 The expression levels of cyclin D2 was significantly decreased within one day after serum addition and was completely downregulated in late-passage cells (>10 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 (>10 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 astrocytomas (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 phase (Figure 3a, left panel). Consistent with this result, knockdown of cyclin D2 led to an increase in the amount of the hypophosphorylated form of RB, suggesting that the activity of CDK4 and/or CDK6 was suppressed and cells were arrested in the G1 phase of the cell cycle (Figure 3b, left panel). Silencing of cyclin D2 expression also caused a reduction in the level of cyclin B1, the expression of which is known to be low in the G1 phase (Figure 3b, left panel). In addition, knockdown of cyclin D2 resulted in a slight decrease in the expression levels of E2F1 and E2F2. We also observed that ectopic expression of cyclin D1 or D3 partially rescued the reduction in RB expression and phosphorylation caused by knockdown of cyclin D2. Thus, a certain amount of D-type cyclins may be required for cell cycle progression of 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 medium (Figures 3a and b, right panels). In addition, although amplification of the cdk4 locus has been detected at a higher frequency than that of the cdk6 locus,7 we found that both CDK4 and CDK6 are responsible for phosphorylation of RB in undifferentiated GB2 cells (Supplementary Figures 7A and B). These results suggest that the cyclin D2–CDK4/6 complexes have an important role in cell cycle progression of undifferentiated, but not of differentiated, GSCs.
Critical role of cyclin D2 in the tumorigenicity of GSCs
To clarify the significance of cyclin D2 expression in the tumorigenicity of GSCs, we transplanted GB2 cells into the frontal lobe of immunocompromised mice. As reported previously,4 all mice transplanted with undifferentiated GB2 cells developed tumors and died within 2 months, whereas mice transplanted with differentiated GB2 cells survived over 5 months (Figure 4a). Histopathological analyses of tumor xenografts demonstrated that undifferentiated GB2 cells formed a highly invasive tumor spreading across the hemispheres, which represents an important feature of human glioblastoma (Figure 4b). RT–PCR analysis using human-specific primers demonstrated that the xenograft tumor still maintained the predominant expression of cyclin D2 (Figure 4c and Supplementary Figure 8A). Consistent with this observation, when mice were transplanted with undifferentiated GB2 cells in which cyclin D2 expression was stably repressed by lentivirus-delivered short hairpin RNAs (shRNAs) (Supplementary Figures 8B and C), they survived significantly longer than those transplanted with undifferentiated GB2 cells infected with control lentivirus (Figure 4a). By contrast, overexpression of cyclin D2 did not restore CD133 and nestin expression, as well as tumorigenicity of differentiated GB2 cells (Supplementary Figures 9A-C). These results suggest that cyclin D2 has a critical role in the tumorigenicity of GSCs.
A number of molecular studies have identified critical genetic events in glioblastoma, including the following: dysregulation of growth factor signaling; activation of the phosphatidylinositol-3-OH kinase pathway; and inactivation of the p53 and RB pathways.7, 8 Among those three core pathways, the RB pathway is obviously the most important for the regulation of G1/S progression.6 Actually, 78% of glioblastomas are shown to harbor RB pathway aberrations, such as deletion of the cdkn2a/cdkn2b locus, amplification of the cdk4 locus and deletion or inactivating mutations in RB1 (Cancer Genome Atlas Research Network7). Importantly, amplification of the cyclin D2 locus is also reported.7 In this study, we have shown that cyclin D2 is the most abundantly expressed cyclin in GSCs among the three D-type cyclins. Moreover, suppression of cyclin D2 expression by RNA interference caused G1 arrest in vitro and growth retardation of GSCs xenografted into immunocompromised mice in vivo. Altogether, these data suggest the critical role of cyclin D2 in cell cycle progression and the tumorigenicity of GSCs.
Tumor cells in culture are valuable for studying the mechanisms of tumorigenesis. Growth media containing serum have been used for maintaining a variety of cancer cells, including glioblastoma. However, it has been shown that serum causes irreversible differentiation of GSCs.4 Differentiated GSCs have gene expression profiles that are different from those of their parental GSCs and NSCs, and are neither clonogenic nor tumorigenic.4 Our study shows that cyclin D2 expression is silenced when GSCs are cultured in the presence of serum. We also found that cyclin D1 expression is enhanced during serum-induced differentiation of GSCs. These results explain why commonly used glioblastoma cell lines abundantly express cyclin D1, but not cyclin D2.
It has been shown that GSCs and NSCs share similar properties such as the potential for self-renewal and differentiation.12 Intriguingly, cyclin D2 has been reported to be the only D-type cyclin expressed in adult mouse NSCs.13, 14 Thus, it is interesting to speculate that the predominant expression of cyclin D2 in GSCs may be the reflection of the property associated with adult NSCs. It is possible that transcription factors that are important for the maintenance of the stem cell state may also be involved in cyclin D2 expression. It may also be possible that DNA methylation and/or mRNA stabilization by alternative cleavage- and polyadenylation-mediated shortening of 3′-UTR are involved in the alteration in cyclin D2 expression.15
It is important to define reliable markers that are expressed in cancer stem cells. Overexpression of cyclin D1 has been implicated in the pathogenesis of various human cancers.5, 6, 16 However, our results raise the possibility that cyclin D2, rather than cyclin D1, could be a novel prognostic marker for glioblastoma. Hence, it is intriguing to perform univariate and multivariate analyses to compare the expression levels of cyclin D2 with tumorigenic capacity and tumor invasiveness.
As cancer stem cells are considered to be responsible for tumor initiation and development, GSCs may be promising targets for the therapy of glioblastoma. We therefore speculate that inhibitors that block the expression of cyclin D2 could have a growth inhibitory effect on GSCs.
Materials and methods
Tumor specimens and primary tumor cultures
Following informed consent, tumor samples classified as primary glioblastoma were obtained from patients undergoing surgical treatment at the University of Tokyo Hospital as approved by the Institutional Review Board. Tumors were washed, and mechanically and enzymatically dissociated into single cells. Tumor cells were cultured in Neurobasal medium (Life Technologies, Carlsbad, CA, USA) containing B27 supplement minus vitamin A (Life Technologies), epidermal growth factor and basic fibroblast growth factor (20 ng/ml each; Wako Pure Chemical Industries, Osaka, Japan). For in vitro differentiation, tumor cells were cultured in Dulbecco’s modified Eagle’s medium/F-12 medium (Life Technologies) containing 10% fetal bovine serum. Cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA).
Mouse monoclonal antibodies to cyclin B1, E2F1 and α-tubulin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal antibodies to cyclin D1, D2, D3 and RB were from BD Biosciences (Billerica, MA, USA). Mouse monoclonal antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Millipore (Bedford, MA, USA). Rabbit polyclonal antibodies to E2F2 and green fluorescence protein (GFP) were from Santa Cruz Biotechnology. Rabbit pAbs to phospho-RB S780, S795 and S807/811 were from Cell Signaling Technology (Danvers, MA, USA).
Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 0.1% Triton X-100, 1 mM dithiothreitol, 1 mM EDTA, 50 mM NaF, 0.1 mM Na3VO4 and protease inhibitors). Lysates were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore). The membrane was subjected to immunoblot analysis using horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin G (GE Healthcare, Pittsburgh, PA, USA) or sheep anti-mouse immunoglobulin G (GE Healthcare) as a secondary antibody. Visualization was performed using the Enhanced Chemiluminescence Plus Western Blotting Detection System (GE Healthcare) and LAS-4000EPUVmini Luminescent Image Analyzer (GE Healthcare).
Total RNA was extracted using NucleoSpin RNA Clean-up kit (Takara Bio Inc., Shiga, Japan) and reverse-transcribed into cDNA using ReverTra Ace qPCR RT Kit (Toyobo Life Science, Osaka, Japan). Real-time PCR was performed using LightCycler480 SYBR Green I Master and a LightCycler480 Instrument (Roche, Indianapolis, IN, USA). The results were normalized with the detected value for GAPDH or ACTB. Primers used in RT–PCR were as follows: GAPDH forward (5′-IndexTermGCACCGTCAAGGCTGAGAAC-3′), GAPDH reverse (5′-IndexTermTGGTGAAGACGCCAGTGGA-3′); CD133 forward (5′-IndexTermAGTGGCATCGTGCAAACCTG-3′), CD133 reverse (5′-IndexTermCTCCGAATCCATTCGACGATAGTA-3′); nestin forward (5′-IndexTermGAGGTGGCCACGTACAGG-3′), nestin reverse (5′-IndexTermAAGCTGAGGGAAGTCTTGGA-3′); cyclin D1 forward (5′-IndexTermTGTCCTACTACCGCCTCACA-3′), cyclin D1 reverse (5′-IndexTermCAGGGCTTCGATCTGCTC-3′); cyclin D2 forward (5′-IndexTermGGACATCCAACCCTACATGC-3′), cyclin D2 reverse (5′-IndexTermCGCACTTCTGTTCCTCACAG-3′); and cyclin D3 forward (5′-IndexTermGCTTACTGGATGCTGGAGGTA-3′), cyclin D3 reverse (5′-IndexTermAAGACAGGTAGCGATCCAGGT-3′). Human-specific primers were as follows: ACTB forward (5′-IndexTermCGTCACCAACTGGGACGACA-3′), ACTB reverse (5′-IndexTermCTTCTCGCGGTTGGCCTTGG-3′); cyclin D1 forward (5′-IndexTermACTACCGCCTCACACGCTTC-3′), cyclin D1 reverse (5′-IndexTermCTTGACTCCAGCAGGGCTTC-3′); cyclin D2 forward (5′-IndexTermATCACCAACACAGACGTGGA-3′), cyclin D2 reverse (5′-IndexTermTGCAGGCTATTGAGGAGCA-3′); and cyclin D3 forward (5′-IndexTermTACACCGACCACGCTGTCT-3′), cyclin D3 reverse (5′-IndexTermGAAGGCCAGGAAATCATGTG-3′). Mouse-specific primers were as follows: ACTB forward (5′-IndexTermGGATGCAGAAGGAGATTACTGC-3′), ACTB reverse (5′-IndexTermCCACCGATCCACACAGAGCA-3′).
The stealth siRNA oligonucleotide sequences were 5′-IndexTermCCACAGAUGUGAAGUUCAUUUCCAA-3′ (cyclin D1), 5′-IndexTermUGCUCCUCAAUAGCCUGCAGCAGUA-3′ (cyclin D2#1), 5′-IndexTermUGACGGAUCCAAGUCGGAGGAUGAA-3′ (cyclin D2#2), 5′-IndexTermAACUACCUGGAUCGCUACCUGUCUU-3′ (cyclin D3), 5′-IndexTermGGGAGAUCAAGGUAACCCUGGUGUU-3′ (CDK4) and 5′-IndexTermACCGAGUAGUGCAUCGCGAUCUAAA-3′ (CDK6) (Life Technologies). Negative control stealth siRNA with medium GC content was purchased from Life Technologies. The silencer select siRNA oligonucleotide sequences were 5′-IndexTermGGACCUUCGUAGCAUUGCATT-3′ (E2F1) and 5′-IndexTermAGACAGUGAUUGCCGUCAATT-3′ (E2F2) (Life Technologies). Negative control silencer select siRNA was purchased from Life Technologies. Transfection of siRNA was performed using Lipofectamine RNAiMAX transfection reagent (Life Technologies). shRNAs targeting cyclin D2 were designed to harbor the same target sequences.
Cells were trypsinized, fixed in 70% ethanol and then stained with propidium iodide (Sigma, St Louis, MO, USA). Cells were passed through a FACSCalibur instrument (BD Biosciences) and the data were analyzed using the ModFit LT software (Verity Software House, Topsham, ME, USA).
Lentiviral vector CS-RfA-CG harboring an shRNA driven by the H1 promoter or CSII-CMV-RfA-IRES2-Venus harboring a cDNA driven by the CMV promoter was transfected with the packaging vectors pCAG-HIV-gp and pCMV-VSV-G-RSV-Rev into 293FT cells using Lipofectamine 2000 Transfection Reagent (Life Technologies). All plasmids were kindly provided by H Miyoshi (RIKEN BioResource Center, Ibaraki, Japan). Viral supernatant was purified by ultracentrifugation at 25 000 r.p.m. for 90 min (SW28 rotor, Beckman Coulter, Brea, CA, USA). Infection efficiency was monitored by GFP expression as it is driven by the CMV promoter.
At 1 week after lentivirus infection, 1 × 104 cells were injected stereotactically into the right frontal lobe of 5-week-old nude mice (BALB/cAJcl-nu/nu; CLEA Japn Inc., Tokyo, Japan), following administration of general anesthesia (n=6). The injection coordinates were 2 mm to the right of the midline, 1 mm anterior to the coronal suture and 3 mm deep. Mice were monitored for 6 months. Survival of mice was evaluated by Kaplan–Meier analysis. P-value was calculated using a log-rank test. Tumors were histologically analyzed after hematoxylin and eosin staining. Tumor distribution was analyzed by GFP immunostaining. All animal experimental protocols were performed in accordance with the politics of the Animal Ethics Committee of the University of Tokyo.
Samples were fixed in 3.7% buffered formalin, dehydrated and embedded in paraffin. Sections (6 μm) were rehydrated, and endogenous peroxidases were blocked by incubation in 0.3% H2O2 for 30 min. The primary antibody was detected using the VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA, USA). Slides were lightly counterstained with hematoxylin.
The expression profiles of undifferentiated GB1–3 and 5 cells were generated on the Affymetrix GeneChip HG-U133 Plus 2.0 microarray platform (Affymetrix, Santa Clara, CA, USA). The expression profiles of 15 GSCs were taken from the Gene Expression Omnibus database GSE7181 and GSE8049 (Lottaz et al.9).Data were analyzed using the software program GenePattern. The expression profiles of D-type cyclins in various histological grades of glioma were taken from GSE4290 (Sun et al.17). Data were analyzed using the software program R. P-value was calculated using a pairwise Wilcoxon’s test. The expression profiles of D-type cyclins in patient glioblastomas, glioma cell lines and normal NSCs were taken from GSE4536 (Lee et al.4).
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This work was supported by Research Program of Innovative Cell Biology by Innovative Technology (Integrated Systems Analysis of Cellular Oncogenic Signaling Networks), Grants-in-Aid for Scientific Research on Innovative Areas (Integrative Research on Cancer Microenvironment Network), Takeda Science Foundation and in part by Global COE Program (Integrative Life Science Based on the Study of Biosignaling Mechanisms), MEXT, Japan.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Oncogene website
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Cite this article
Koyama-Nasu, R., Nasu-Nishimura, Y., Todo, T. et al. The critical role of cyclin D2 in cell cycle progression and tumorigenicity of glioblastoma stem cells. Oncogene 32, 3840–3845 (2013). https://doi.org/10.1038/onc.2012.399
- cancer stem cells
- cell cycle
- cyclin D2
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