Purpose: Medulloblastoma is the most common malignant brain tumor in children. Despite recent improvements, the molecular mechanisms driving medulloblastoma are not fully understood and further elucidation could provide cues to improve outcome prediction and therapeutic approaches.

Experimental Design: Here, we conducted a meta-analysis of mouse and human medulloblastoma gene expression data sets, to identify potential medulloblastoma tumor suppressor genes.

Results: We identified DAB2IP, a member of the RAS-GTPase–activating protein family (RAS GAP), and showed that DAB2IP expression is repressed in medulloblastoma by EZH2-induced trimethylation. Moreover, we observed that reduced DAB2IP expression correlates significantly with a poor overall survival of patients with medulloblastoma, independent of metastatic stage. Finally, we showed that ectopic DAB2IP expression enhances stress-induced apoptosis in medulloblastoma cells and that reduced expression of DAB2IP in medulloblastoma cells conveys resistance to irradiation-induced cell death.

Conclusion: These results suggest that repression of DAB2IP may at least partly protect medulloblastoma cells from apoptotic cell death. Moreover, DAB2IP may represent a molecular marker to distinguish patients with medulloblastoma at high risk from those with a longer survival prognosis. Clin Cancer Res; 18(15); 4048–58. ©2012 AACR.

Translational Relevance

Medulloblastoma is the most common malignant pediatric brain tumor. Current treatment modalities result in a 5-year survival rate of 40% in high-risk patients and 80% to 90% in standard risk patients. Approximately 30% of patients however remain incurable and current intensive treatment protocols cause significant adverse long-term effects such as impaired neurologic function and endocrine dysfunction. Currently, the staging for medulloblastoma between high-risk and standard-risk disease is based on clinical parameters and histologic subtypes. However, it is suggested that including molecular markers in the risk stratification could improve survival and decrease treatment-related toxicity. Here we show that high expression of DAB2IP in medulloblastoma is associated with a favorable prognosis, independent of metastatic stage. This suggests DAB2IP expression inhibits tumor growth and further research in its use as a potentially important prognostic factor and/or therapeutic target may contribute to improvements in the future treatment of patients with medulloblastoma.

Brain tumors are the most common form of solid tumors in children of which medulloblastoma is the most frequent malignant variant, accounting for 20% of cases (1). Treatment modalities consist of surgery, radiotherapy, and chemotherapy and result in a 5-year survival rate of 40% in high-risk patients and 80% to 90% in low-risk patients (2). Approximately 30% of patients however remain incurable and current intensive treatment protocols cause significant adverse long-term effects (3). Medulloblastomas comprise 4 subtypes: WNT, SHH, group 3, and group 4, which differ regarding histology and clinical outcome (4) and are believed to derive from the deregulation of various signaling pathways in brain development, such as the WNT-pathway and sonic hedgehog (SHH) signaling pathways. Overactivation of these pathways leads to a loss of cell-cycle control and a dysfunctional apoptosis program, allowing for continued growth and tumorigenesis, predominantly in the cerebellum (5).

DAB2IP—disabled homolog 2–interacting protein, located at chromosome 9q33.1-q33.3—is a member of the RAS-GTPase activating protein family (RAS GAP) that inactivates RAS by promoting conversion of GTP into GDP (6). DAB2IP acts as a putative tumor suppressor gene and is downregulated by epigenetic modification in multiple aggressive cancers. In prostate cancer, DAB2IP expression was shown to be repressed by promoter methylation and histone modification (7), whereas in breast cancer (8), lung cancer (9), and gastrointestinal tumors (10), aberrant promoter hypermethylation was shown to downregulate DAB2IP. Moreover, it was shown in prostate cancer that downregulation of DAB2IP expression results in resistance to ionizing radiation (11), it initiates epithelial-to-mesenchymal transition (12) and promotes tumor growth and metastasis (13). In addition, DAB2IP is involved in TNFα-induced apoptosis in prostate cancer cells by suppressing the ASK1-JNK and PI3-AKT pathway (14), and in endothelial cells via the ASK1-JNK pathway (15).

Apoptosis is a programmed variant of cell death common to all human cells. Defects in the apoptosis program result in an imbalance in the rate of cell proliferation and the rate of cell death thereby contributing to tumor growth and treatment resistance. Essential steps in the apoptotic mechanism are inactivated in medulloblastoma cells, resulting in resistance to apoptosis. A recent in vivo study showed that cerebellar stem cells can give rise to medulloblastomas when having acquired an impaired apoptosis mechanism (16). Consequently, many of the apoptosis mediators are generally considered tumor suppressors (17).

Here, we describe a comprehensive meta-analysis of gene expression studies of mouse and human medulloblastoma (18–23) identifying multiple medulloblastoma tumor suppressor candidates, including DAB2IP. We found DAB2IP expression to be strongly downregulated in human medulloblastoma cells and in primary human medulloblastoma tissues. We show that DAB2IP downregulation is—at least partially—caused by EZH2-mediated repression through histone methylation, conveying apoptosis resistance in immortalized neural precursor and medulloblastoma cells. Furthermore, we show that DAB2IP expression correlates significantly with the overall survival of patients with medulloblastoma, independent of metastatic stage.

Detailed protocols are in the Supplementary Data.

Biologic samples

Original data on tumor samples from 2 retrospective studies (23, 24) were used for this study. Survival analysis was based on 108 cases for which expression and survival data were available. Patient and tumor characteristics are presented in Table 1. In brief, all samples were snap frozen in the institutional pathology departments immediately upon arrival. All samples were reviewed by experienced neuropathologists and examined for tumor content. Total RNA was extracted using TRIzol (Invitrogen). Gene expression profiles were obtained by Affymetrix HG-U133 Plus 2.0 arrays. Gene expression data were normalized using the GCRMA procedure. Informed consent and detailed methods are described elsewhere (23, 24).

Table 1.

Patient/tumor characteristics of medulloblastoma series used for survival analysis

DAB2IP low, M0DAB2IP low, M+DAB2IP high, M0DAB2IP high, M+
Total no. of cases 57 24 20 
Gender     
 Male 39 16 11 
 Female 18 
Age at diagnosis     
 Average age 8.0 7.3 8.4 7.8 
 Median age 7.0 7.6 6.6 8.1 
 Age range 1.0–35.3 2.0–16.6 0.8–25.6 2.8–13.5 
Age groups     
 Infants (<4) 16 
 Children (4–16) 37 18 17 
 Adults (>16) 
Histology     
 Classic 37 21 16 
 Desmoplastic 13 
 Large cell/anaplastic 
 Nodular/desmoplastic 
Molecular subgroups     
 WNT 
 SHH 21 
 Group 3 12 10 
 Group 4 20 13 
DAB2IP low, M0DAB2IP low, M+DAB2IP high, M0DAB2IP high, M+
Total no. of cases 57 24 20 
Gender     
 Male 39 16 11 
 Female 18 
Age at diagnosis     
 Average age 8.0 7.3 8.4 7.8 
 Median age 7.0 7.6 6.6 8.1 
 Age range 1.0–35.3 2.0–16.6 0.8–25.6 2.8–13.5 
Age groups     
 Infants (<4) 16 
 Children (4–16) 37 18 17 
 Adults (>16) 
Histology     
 Classic 37 21 16 
 Desmoplastic 13 
 Large cell/anaplastic 
 Nodular/desmoplastic 
Molecular subgroups     
 WNT 
 SHH 21 
 Group 3 12 10 
 Group 4 20 13 

Immunohistochemistry was conducted on a largely independent medulloblastoma tissue microarray (TMA) cohort with tumors from 87 patients obtained from the files of the Department of Neuropathology of the Academic Medical Center (University of Amsterdam). Subgroup information was obtained by immunohistochemistry using antibodies for the subgroup-specific protein markers β-catenin (WNT), DKK1 (WNT), SFRP1 (SHH), NPR3 (Group 3), and KCNA1 (Group 4). Information on gender, age at diagnosis, histology, metastatic stage at diagnosis, and survival are presented in Table 2. The mean follow-up time of survivors in the TMA cohort was 6.2 years (range 0.1–19.4 years). Informed consent was obtained for the use of brain tissue and for access to medical records for research purposes. MB1 and MB2 primary human medulloblastoma tissues were obtained from surgical specimens after informed consent and approval by the Medical Ethical Committee of the VU University Medical Center.

Table 2.

Patient/tumor characteristics of medulloblastoma series on TMA

  
Total no. of cases 87 
Gender  
 Male 27 
 Female 12 
 Unknown 48 
Age at diagnosis  
 Average age 14.2 
 Median age 7.0 
 Age range 1–53 
Age groups  
 Infants (<4) 
 Children (4–16) 19 
 Adults (>16) 15 
 Unknown 46 
Histology  
 Classic 23 
 Large cell/anaplastic 
 Nodular/desmoplastic 
 Unknown 53 
Molecular subgroups  
 WNT 
 SHH 18 
 Group 3 14 
 Group 4 33 
 Unknown 15 
Metastatic stage  
 M0 35 
 M+ 
 Unknown 46 
Survival  
 Alive 61 
 Diseased 26 
  
Total no. of cases 87 
Gender  
 Male 27 
 Female 12 
 Unknown 48 
Age at diagnosis  
 Average age 14.2 
 Median age 7.0 
 Age range 1–53 
Age groups  
 Infants (<4) 
 Children (4–16) 19 
 Adults (>16) 15 
 Unknown 46 
Histology  
 Classic 23 
 Large cell/anaplastic 
 Nodular/desmoplastic 
 Unknown 53 
Molecular subgroups  
 WNT 
 SHH 18 
 Group 3 14 
 Group 4 33 
 Unknown 15 
Metastatic stage  
 M0 35 
 M+ 
 Unknown 46 
Survival  
 Alive 61 
 Diseased 26 

Survival analysis

Overall survival was calculated from the time of diagnosis to the patient's last follow-up or death. Survival of patients was analyzed using Kaplan–Meier survival curves, and the log-rank test was used to examine the statistical significance. P-values < 0.05 were considered significant. Prognostic impact of covariates on survival was evaluated on the basis of hazard ratios from Cox's proportional hazards regression model. Multivariate Cox's proportional hazards regression models were used to estimate effects of additional the covariates age, metastatic stage, and histology.

Cells

Human D283-med, D556-med (Dr Darrell Bigner, Duke University, NC, USA), Daoy (ATCC, Manassas, VA) and C17.2 murine cerebellar progenitor cells immortalized by v-myc (25) were cultured in DMEM containing 10% FBS and antibiotics.

Acumen proliferation assay

Cells were plated in 96-well plates (Greiner), fixed at 24, 48, 72, and 96 hours after plating using formaldehyde, stained with DAPI, and subsequently signal intensity was measured using an Acumen eX3 apparatus (TTP LabTech).

Apoptosis assay

Cells were plated in white opaque 96-well plates (Greiner) and treated with TNFα (Invitrogen). After 6 hours of treatment, caspase activity was measured using the Caspase-Glo 3/7 assay (Promega) according to the manufacturer's instructions. Fluorescence and luminescence read-out was conducted using a Tecan Infinite F200 Microplate Reader (Tecan Trading AG).

Clonogenic assay

Daoy cells were plated in 6-well plates at a density of 500 to 1,000 cells per well depending on the used dose of irradiation or TNFα concentration. Subsequently, cells were treated with increasing doses of irradiation or TNFα (10 ng/mL). After 10 to 14 days of culturing to allow colony formation, the colonies were fixed with 3.7% formaldehyde in PBS and stained with Giemsa solution. Groups consisting of 50 cells or more were defined as a colony. The colony counts using light microscopy were conducted independently by at least 2 investigators.

Meta-analysis of candidate tumor suppressor genes in medulloblastoma

To identify candidate tumor suppressor genes in medulloblastoma, we determined transcripts that were downregulated in medulloblastoma as compared with normal cerebellar precursor cells in 7 data sets comparing gene expression of normal cerebellar precursor cells and medulloblastoma in mice. We used mouse studies because that allowed for a comparison between medulloblastoma cells and proliferating progenitor cells. All data sets used mouse models that mimic the SHH-medulloblastoma subgroup (Supplementary Table S1A, GSE9299, GSE2426, GSE7212, GSE11859, GSE6463; refs. 18–22). This analysis yielded 56 genes that were significantly downregulated (>2-fold) in at least 5 of 7 mouse medulloblastoma data sets. Subsequently, to increase the relevance of our screen for human medulloblastoma, we compared the expression of the significantly downregulated mouse genes to a human medulloblastoma gene expression dataset (GSE10327; ref. 23) and an independent gene expression data set of normal human cerebellum (GSE3526; ref. 26). We established that 37 genes of our set of 56 genes were significantly downregulated in both mouse and human medulloblastoma (Supplementary Table S2A). In addition, we compared genes that were significantly downregulated in both mouse and human medulloblastoma to a composed list of designated tumor suppressor genes. This resulted in 2 candidate gene sets, one set of 37 highly deregulated genes that have not before been designated as tumor suppressors (Fig. 1A and Supplementary Table S2A), and one set of 26 genes that have been reported as a tumor suppressor before—mostly in cancers other than medulloblastoma (Fig. 1B and Supplementary Table S2B).

Figure 1.

Meta-analysis of potential tumor suppressor genes in medulloblastoma. A, downregulated genes in medulloblastoma versus normal cerebellar precursor cells in at least 5 of 7 mouse medulloblastoma data sets. The heatmap only represents transcripts that are also downregulated in human medulloblastoma versus normal cerebellum. B, representation of putative tumor suppressor genes downregulated in at least 3 of 7 mouse medulloblastoma data sets. The heatmap only represents transcripts that are also downregulated in human medulloblastoma versus normal cerebellum.

Figure 1.

Meta-analysis of potential tumor suppressor genes in medulloblastoma. A, downregulated genes in medulloblastoma versus normal cerebellar precursor cells in at least 5 of 7 mouse medulloblastoma data sets. The heatmap only represents transcripts that are also downregulated in human medulloblastoma versus normal cerebellum. B, representation of putative tumor suppressor genes downregulated in at least 3 of 7 mouse medulloblastoma data sets. The heatmap only represents transcripts that are also downregulated in human medulloblastoma versus normal cerebellum.

Close modal

DAB2IP is downregulated in medulloblastoma and is associated with poor clinical outcome

We focused on the downregulated medulloblastoma genes with designated tumor suppressor function (Fig. 1B) and analyzed by literature search to which of these genes a proapoptotic function could be attributed. This resulted in the emergence of DAB2IP as a potential proapoptotic tumor suppressor gene in medulloblastoma, because DAB2IP is the third most differentially expressed transcript in our analysis and literature search suggests its function in apoptosis regulation (14, 15, 27). To confirm that DAB2IP is downregulated in medulloblastoma, we first analyzed the DAB2IP expression levels in medulloblastoma cells. Quantitative real-time PCR (qRT-PCR) revealed that DAB2IP mRNA expression was downregulated in medulloblastoma cells D283-med, D556-med, Daoy and in primary human medulloblastoma cells (Fig. 2A), as compared with normal human cerebellar tissue. This was confirmed on protein level by DAB2IP Western blot analysis (Fig. 2B). To determine the correlation of DAB2IP downregulation to clinical outcome, DAB2IP expression was evaluated in mRNA expression data sets of human medulloblastoma (23, 24), and was correlated to survival (Fig. 2C). The clinical characteristics of the tumors/patients used in the survival analysis are summarized in Table 1. Kaplan–Meier analysis shows that low DAB2IP mRNA expression correlates significantly with a poor prognosis, as measured by a lower overall survival probability (P = 0.010). Multivariate Cox proportional-hazards analysis shows that DAB2IP expression can predict prognosis (HR, 3.0; 95% CI, 1.1–8.6, P = 0.036), independently of clinical variables such as age, metastatic stage, and histology. Medulloblastoma is known to comprise 4 subtypes: WNT, SHH, group 3, and group 4, which differ regarding histology, molecular biology, genetics, and clinical outcome (4). However, we did not find a significant correlation between DAB2IP expression and any specific subtype in our patient series (Supplementary Fig. S1). We also investigated the relation between DAB2IP expression and metastatic stage in our patient group. However, we did not find a significant correlation (Fig. 2D). Finally, to further analyze the effects of DAB2IP expression and metastatic stage on prognosis, patients were stratified on the basis of metastatic stage (Fig. 2D and Supplementary Fig. S2A and S2B). Again Kaplan–Meier analysis showed that low DAB2IP expression correlated with a poor prognosis (P = 0.055) both in the metastatic patient and nonmetastatic patient groups.

Figure 2.

DAB2IP is downregulated in medulloblastoma and is associated with poor clinical outcome. A, RNA, extracted from various medulloblastoma cell lines (D283-med, D556-med, Daoy) and nonneoplastic brain cell lines (left) and from 2 primary human medulloblastoma samples and 1 normal human cerebellum tissue sample (right) were analyzed by qRT-PCR for expression levels of DAB2IP. The data were normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in each sample. Error bars indicate SD. *, P < 0.05; ***, P < 0.001; t test. B, protein expression analysis of DAB2IP in various medulloblastoma and nonneoplastic brain cell lines. Numbers indicate relative DAB2IP protein expression normalized against β-actin expression. C, Kaplan–Meier analysis shows that individuals with medulloblastoma who have lower expression of DAB2IP have a significantly lower overall survival probability (P = 0.010). Cutoff for high and low expression is based on maximum likelihood. D, scatterplot of DAB2IP mRNA expression levels (arbitrary units) in patients based on metastatic stage M0/M1/M2-3-4 shows that average DAB2IP expression does not correlate with metastatic stage in medulloblastoma (left). Kaplan–Meier survival curves of (C) further stratified on the basis of metastatic stage (right).

Figure 2.

DAB2IP is downregulated in medulloblastoma and is associated with poor clinical outcome. A, RNA, extracted from various medulloblastoma cell lines (D283-med, D556-med, Daoy) and nonneoplastic brain cell lines (left) and from 2 primary human medulloblastoma samples and 1 normal human cerebellum tissue sample (right) were analyzed by qRT-PCR for expression levels of DAB2IP. The data were normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in each sample. Error bars indicate SD. *, P < 0.05; ***, P < 0.001; t test. B, protein expression analysis of DAB2IP in various medulloblastoma and nonneoplastic brain cell lines. Numbers indicate relative DAB2IP protein expression normalized against β-actin expression. C, Kaplan–Meier analysis shows that individuals with medulloblastoma who have lower expression of DAB2IP have a significantly lower overall survival probability (P = 0.010). Cutoff for high and low expression is based on maximum likelihood. D, scatterplot of DAB2IP mRNA expression levels (arbitrary units) in patients based on metastatic stage M0/M1/M2-3-4 shows that average DAB2IP expression does not correlate with metastatic stage in medulloblastoma (left). Kaplan–Meier survival curves of (C) further stratified on the basis of metastatic stage (right).

Close modal

DAB2IP and EZH2 are inversely expressed in medulloblastoma

Previously it was described that DAB2IP expression is epigenetically suppressed by EZH2, a member of the polycomb complex and a histone-methylating enzyme (28). Therefore, we determined the EZH2 expression levels in medulloblastoma cells and tissues and compared these to DAB2IP expression. First, DAB2IP and EZH2 expression levels were evaluated in the mRNA expression data set of 62 human medulloblastoma (9 WNT, 15 SHH, 11 group 3, and 27 group 4; ref. 23) and 9 normal cerebellum samples (26). As expected, the DAB2IP mRNA levels were significantly downregulated in the medulloblastoma samples as compared with normal human cerebellum (Fig. 3A). In contrast, EZH2 mRNA levels were significantly upregulated in the medulloblastoma samples as compared with normal human cerebellum (Fig. 3A). There was no significant difference in the expression of DAB2IP between the 4 subgroups of medulloblastoma, whereas the expression of EZH2 showed an increasing trend from the WNT and SHH subgroups to groups 3 and 4 (Fig. 3A). In addition, we compared DAB2IP and EZH2 expression in individual samples and found a negative correlation between DAB2IP and EZH2 mRNA expression (Supplementary Fig. S3A). This negative correlation was also found on protein level (Supplementary Fig. S3B). In parallel to the DAB2IP expression analysis in medulloblastoma cell lines and primary tissues (Fig. 2A), EZH2 expression analysis was conducted using the same samples. This showed an increased EZH2 expression in medulloblastoma cells as compared with normal cerebellum (Fig. 3B), again correlating inversely with DAB2IP expression in these samples. Finally, immunohistochemical analysis on TMAs, composed of 276 pediatric medulloblastoma tissues from 87 patients, showed that EZH2 expression was significantly overexpressed in 30 of 87 samples. In contrast, DAB2IP protein expression was not detectable in medulloblastoma samples (Fig. 3C). However, in 1 patient sample that included adjacent normal cerebellar tissue, the adjacent tissue stained negatively for EZH2 and positively for DAB2IP (Fig. 3C, bottom). The clinical characteristics of the tumors/patients are summarized in Table 2.

Figure 3.

DAB2IP and EZH2 are inversely expressed in medulloblastoma. A, microarray analysis of 62 pediatric/human medulloblastoma tissues (9 WNT, 15 SHH, 11 Group 3 and 27 Group 4) and 9 normal human cerebellum tissues. A, scatterplot of DAB2IP expression, bar represents mean (left). Scatterplot of EZH2 expression, bar represents mean (right). B, RNA extracted from various medulloblastoma cell lines (D283-med, D556-med, Daoy) and nonneoplastic brain cell lines (left), and 2 primary human medulloblastoma samples and nonneoplastic brain samples (right) was analyzed by qRT-PCR for expression levels of EZH2. The data were normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in each sample. Error bars indicate SD. *, P < 0.05; ***, P < 0.001; t test. C, representative immunohistochemical staining for EZH2 (left) and DAB2IP (right) in human medulloblastoma (MB) tissues. Left and right belong to the same tumor sample. Tumor samples are from different medulloblastoma subgroups. Bottom, tumor samples with adjacent normal cerebellar tissue that stains negatively for EZH2 and positively for DAB2IP—opposite to the tumor samples. Scale bar, 100 μm.

Figure 3.

DAB2IP and EZH2 are inversely expressed in medulloblastoma. A, microarray analysis of 62 pediatric/human medulloblastoma tissues (9 WNT, 15 SHH, 11 Group 3 and 27 Group 4) and 9 normal human cerebellum tissues. A, scatterplot of DAB2IP expression, bar represents mean (left). Scatterplot of EZH2 expression, bar represents mean (right). B, RNA extracted from various medulloblastoma cell lines (D283-med, D556-med, Daoy) and nonneoplastic brain cell lines (left), and 2 primary human medulloblastoma samples and nonneoplastic brain samples (right) was analyzed by qRT-PCR for expression levels of EZH2. The data were normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in each sample. Error bars indicate SD. *, P < 0.05; ***, P < 0.001; t test. C, representative immunohistochemical staining for EZH2 (left) and DAB2IP (right) in human medulloblastoma (MB) tissues. Left and right belong to the same tumor sample. Tumor samples are from different medulloblastoma subgroups. Bottom, tumor samples with adjacent normal cerebellar tissue that stains negatively for EZH2 and positively for DAB2IP—opposite to the tumor samples. Scale bar, 100 μm.

Close modal

Epigenetic modulation of DAB2IP expression in medulloblastoma

To determine whether DAB2IP expression in medulloblastoma is regulated by EZH2-mediated epigenetic histone modulation, we transfected medulloblastoma cells with siRNAs directed against EZH2 (siEZH2). At 96 hours after transfection, we observed significantly reduced EZH2 levels by Western blot analysis in medulloblastoma cells transfected with siEZH2 (Fig. 4A). In addition, EZH2-mediated histone 3 methylation at lysine 27 was reduced and DAB2IP protein levels increased in the siEZH2 transfected medulloblastoma cells. We also analyzed lysates from medulloblastoma cells treated with the S-adenosylhomocysteine hydrolase inhibitor DZNep, a potent inhibitor of EZH2 histone methyltransferase activity (29–31). Again, a reduction in EZH2 protein levels was observed with a delayed increase in DAB2IP levels (Fig. 4B). Besides histone methylation, DAB2IP expression can be altered by histone acetylation (7) and promoter DNA hypermethylation (7–10). It was previously shown that in various cancer types DAB2IP expression could be restored by treatment with the DNA hypomethylation agent 5-aza-2′-deoxycytidine (DAC). In the medulloblastoma cells used here, we could not detect an increase in DAB2IP expression after DAC treatment (Fig. 4C). However, additional treatment with the histone deacetylase inhibitor trichostatin A (TSA) did significantly increase DAB2IP expression (Fig. 4D). This suggests that histone modifications may play a more significant role in suppressing DAB2IP expression in medulloblastoma, as was described also in prostate cancer (7).

Figure 4.

Epigenetic modulation of DAB2IP expression in medulloblastoma cells. A, D556-med and D283-med protein expression analysis of DAB2IP and EZH2 and H3K27me3 at 48 and 96 hours after transfection with EZH2 siRNA or nonrelated siRNAs. β-Actin expression was used as normalization control. B, D556-med and D283-med protein expression analysis of DAB2IP and EZH2 at 48 and 96 hours after treatment with DZNep. C, effect of 5-aza-2′-deoxycytidine (DAC) treatment on DAB2IP mRNA expression in D556-med cells. D, effect of combined DAC and trichostatin-A (DAC-TSA) treatment on DAB2IP mRNA expression in D556-med cells. Error bars indicate SD. *, P < 0.05; ***P < 0.001; t test. CTRL, control.

Figure 4.

Epigenetic modulation of DAB2IP expression in medulloblastoma cells. A, D556-med and D283-med protein expression analysis of DAB2IP and EZH2 and H3K27me3 at 48 and 96 hours after transfection with EZH2 siRNA or nonrelated siRNAs. β-Actin expression was used as normalization control. B, D556-med and D283-med protein expression analysis of DAB2IP and EZH2 at 48 and 96 hours after treatment with DZNep. C, effect of 5-aza-2′-deoxycytidine (DAC) treatment on DAB2IP mRNA expression in D556-med cells. D, effect of combined DAC and trichostatin-A (DAC-TSA) treatment on DAB2IP mRNA expression in D556-med cells. Error bars indicate SD. *, P < 0.05; ***P < 0.001; t test. CTRL, control.

Close modal

DAB2IP promotes stress-induced apoptosis in medulloblastoma

To assess the functional role of DAB2IP in medulloblastoma, we examined the effects of DAB2IP modulation in human medulloblastoma cells and mouse neuronal precursor cells. Because DAB2IP was reported to enhance TNFα-induced apoptotic cell death in endothelial cells (15) and prostate cancer cells (14), we analyzed the effect of DAB2IP overexpression on TNFα-induced apoptotic cell death in medulloblastoma cells. Lentiviral vectors encoding for DAB2IP or LacZ were used to stably transduce Daoy medulloblastoma cells. Treatment with TNFα (100 ng/mL) resulted in a 2-fold increase in caspase activity in Daoy cells overexpressing DAB2IP, as compared with LacZ control cells. This increase was neutralized by simultaneous treatment with the caspase inhibitor z-VAD (20 μmol/L; Fig. 5A). In addition, we measured the cell proliferation rate of Daoy cells overexpressing DAB2IP or LacZ control. Daoy-DAB2IP cells showed a lower proliferation rate as compared with their controls. This result was confirmed in D283-med cells (data not shown). Treatment with a low dose of TNFα (10 ng/mL)—to induce mild cellular stress—impaired proliferation of the DAB2IP-overexpressing cells even further, whereas the proliferation rate of control cells was not significantly inhibited (Fig. 5B). Furthermore, DAB2IP overexpression also inhibited anchorage independent growth after treatment with a low dose of TNFα (10 ng/mL) in Daoy cells (Supplementary Fig. S4). Because ionizing radiation (IR) is an important treatment modality in medulloblastoma and downregulation of DAB2IP gene expression was related to resistance to IR in prostate cancer cells (11), we studied the effect of DAB2IP modulation on the clonogenic growth of medulloblastoma cells after IR. Daoy-DAB2IP and Daoy-LacZ cells were irradiated with doses of 0 to 5 Gy. DAB2IP overexpression showed an IR dose-dependent reduction in clonogenic survival as compared with LacZ control cells (Fig. 5C). Finally, to further study the effect of DAB2IP on TNFα-induced apoptosis, we used a shDAB2IP construct to transiently knockdown DAB2IP expression in C17.2 murine cerebellar progenitor cells immortalized by v-myc (25). Moderate knockdown of DAB2IP was confirmed by Western blot analysis. DAB2IP knockdown in these immortalized neural precursor cells significantly reduced TNFα-induced caspase activation (Fig. 5D), suggesting that DAB2IP has a proapoptotic function in these stressed neural precursor cells. In the absence of TNFα induced stress, caspase activation was similar as was observed for DAB2IP knockdown and C17.2 shCTRL cells (Fig. 5D).

Figure 5.

DAB2IP promotes stress-induced apoptosis in medulloblastoma cells. A, DAB2IP overexpression increased caspase activation in Daoy cells treated with TNFα 6 hours after treatment. Overexpression of DAB2IP in Daoy cells was confirmed by Western blotting. B, Acumen proliferation assay of Daoy cells overexpressing DAB2IP after treatment with or without a low dose of TNFα. C, colony formation of DAB2IP overexpressing Daoy cells, after exposure to increasing doses of IR. D, DAB2IP knockdown decreased caspase activation in C17.2 neural precursor cells treated with TNFα 6 hours after treatment. Knockdown of DAB2IP in C17.2 cells was confirmed by Western blot analysis. Error bars indicate SD. *, P < 0.05; ***, P < 0.001; t test.

Figure 5.

DAB2IP promotes stress-induced apoptosis in medulloblastoma cells. A, DAB2IP overexpression increased caspase activation in Daoy cells treated with TNFα 6 hours after treatment. Overexpression of DAB2IP in Daoy cells was confirmed by Western blotting. B, Acumen proliferation assay of Daoy cells overexpressing DAB2IP after treatment with or without a low dose of TNFα. C, colony formation of DAB2IP overexpressing Daoy cells, after exposure to increasing doses of IR. D, DAB2IP knockdown decreased caspase activation in C17.2 neural precursor cells treated with TNFα 6 hours after treatment. Knockdown of DAB2IP in C17.2 cells was confirmed by Western blot analysis. Error bars indicate SD. *, P < 0.05; ***, P < 0.001; t test.

Close modal

We conducted a meta-analysis of publicly available medulloblastoma gene expression data sets to identify potential medulloblastoma tumor suppressor genes. We identified DAB2IP to be strongly downregulated in medulloblastoma cells and in primary medulloblastoma tissues. Reduced DAB2IP expression was shown to correlate significantly with poor overall survival of patients with medulloblastoma, independent of clinical variables such as age, metastatic stage, and histology. Moreover, DAB2IP was shown to be regulated by histone modifications, including histone acetylation, and histone methylation by the polycomb group member EZH2. Finally, we showed that ectopic DAB2IP expression enhances stress-induced apoptotsis in medulloblastoma cells, and that reduced expression of DAB2IP in medulloblastoma conveys resistance to irradiation-induced cell death.

Medulloblastomas are known to comprise 4 subtypes: WNT, SHH, group 3, and group 4, which differ regarding histology, molecular biology, genetics, and clinical outcome (4). The studies used in our meta-analysis all used mouse models that mimic SHH-subgroup medulloblastoma (Supplementary Table S1A). While this paper was being revised, the first MYC-based medulloblastoma mouse models that mimic group 3 were generated (32, 33). The medulloblastomas generated by one of these MYC models (32) show a reduced DAB2IP expression in line with our results (Supplementary Fig. S5A). However, the medulloblastomas generated by the other model (33) do not show differential DAB2IP expression compared with the control tissue used in that study (Supplementary Fig. S5B). Recently, a mouse model that mimics WNT subgroup medulloblastoma was also described (34). However, the small number (3) of samples and the strong variation in their DAB2IP expression preclude making any meaningful comments on DAB2IP expression in this model (Supplementary Fig. S5C). Finally, our human medulloblastoma data set shows that DAB2IP expression is significantly reduced in all subgroups (Fig. 3A and Supplementary Fig. S1).

To increase the likelihood of identifying tumor suppressors in medulloblastoma, we compared the deregulated mRNA transcripts with a composed list of tumor suppressor genes. This list was established by interrogating publicly available gene ontology databases that included transcripts that have been tied to a tumor suppressor function. A number of well-known tumor suppressors in the context of medulloblastoma such as PTCH1, SUFU, APC, AXIN and TP53 (35–41) are not included in our candidate gene sets. This is likely to be caused by our stringent threshold and the various medulloblastoma mouse models used to gather the data sets. For instance, PTCH1 is down-regulated >2-fold in 2 of 7 medulloblastoma mouse model datasets, thereby not reaching our cutoff of deregulation in at least 3 of 7 data sets. However, in 2 additional data sets PTCH1 is downregulated ∼1.8-fold. Another well-known tumor suppressor, TP53, is downregulated >2-fold in 3 of 7 different mouse models datasets. However, TP53 is not differentially expressed between the human medulloblastoma and normal cerebellum data set, supporting reports that TP53 downregulating mutations in sporadic human medulloblastoma are not very common (37). In addition, transcripts can have inactivating mutations without being downregulated at mRNA level, and are therefore not detected using mRNA expression analysis. Similar observations have also been reported for PTCH1, AXIN2, and TP53 in various subgroups of medulloblastoma (23).

It was previously shown that DAB2IP expression can be silenced by promoter methylation and histone modification (7–10). Other reported mechanisms for DAB2IP inactivation include one case of a translocation that disrupts DAB2IP expression in acute myeloid leukemia (42), and a single nucleotide polymorphism in the DAB2IP gene in aggressive prostate cancer (43). LOH at DAB2IP was showed in 20% of cases in hepatocellular carcinoma (44). In addition, a common sequence variant within DAB2IP associates with the risk of abdominal aortic aneurysm (45) and recently a genetic variant of DAB2IP was also shown to be an independent risk factor for early onset of lung cancer (46). However, as far as we know in medulloblastoma no such mechanisms of DAB2IP deregulation have been reported yet. Here we show a significant negative correlation between overexpressed EZH2 and downregulated DAB2IP in human medulloblastoma samples. Moreover, we show that DAB2IP suppression in medulloblastoma cells can be at least partly reversed by EZH2 inhibition. Treatment with the DNA hypomethylation agent 5-aza-2′-deoxycytidine (DAC) did not affect DAB2IP expression in medulloblastoma cells. However, additional treatment with the histone deacetylase inhibitor trichostatin A (TSA) did significantly increase DAB2IP expression. This may suggest that histone modifications play a significant role in suppressing DAB2IP expression in medulloblastoma cells, however, we do not rule out that DAB2IP expression in medulloblastoma cells may also be impaired by additional mechanisms. Other transcripts in our list of potential medulloblastoma tumor suppressor genes may also be EZH2 targets, this is however only confirmed for CDH1 (47).

We found DAB2IP mRNA expression was reduced in medulloblastoma cell lines and primary tissues. DAB2IP protein levels were also reduced in medulloblastoma cell lines. Immunohistochemistry on the medulloblastoma TMA did not reveal DAB2IP expression in these samples. However, this does not necessarily mean that no protein is present at all. It may well be that small traces of DAB2IP are present in some of the medulloblastoma tissue samples, although undetectable by us in these experiments. Another explanation may be that posttranscriptional events account for the discrepancy between mRNA and protein levels.

Recently, 2 publications have related the loss of DAB2IP expression to increased epithelial-to-mesenchymal transition (EMT) and metastasis in prostate cancer. Xie and colleagues show that DAB2IP knockdown increases nuclear β-catenin accumulation and trans-activation of target genes involved in EMT by inhibiting GSK3β, indicating an inhibitory function of DAB2IP in WNT/β-catenin signaling (12). In the context of medulloblastoma, this seems paradoxical as metastasis is uncommon in the subgroup of human medulloblastoma in which WNT signaling is active (4). In our patient series, average DAB2IP expression was slightly higher in the group of WNT associated medulloblastomas (Fig. 3A), however this difference was nonsignificant. It would be of interest to determine the role of DAB2IP in WNT signaling in the context of medulloblastoma, because a major role in medulloblastoma oncogenesis has been attributed to WNT/β-catenin (48). Furthermore, it was reported that loss of DAB2IP expression induces the activation of Ras and NF-κB in prostate cancer, where Ras is presumed to play an essential role in primary tumor growth and NF-κB drives prostate cancer metastasis (13). Therefore, we investigated the relation between DAB2IP expression and metastatic stage. However, we were unable to show a significant correlation between DAB2IP expression and medulloblastoma metastases (Fig. 2E).

In a study of mammalian brain development, high levels of DAB2IP expression was found in the developing cerebellum, particularly in Purkinje cell precursors (49). Although there is no evidence that medulloblastoma arises directly from Purkinje cells, these cells play an important part in the development of the normal cerebellum. Purkinje cells generate SHH that projects on granule neuron precursor cells and stimulate their proliferation, before the granule neuron precursor cells migrate deeper into the forming cerebellum and differentiate further. A subgroup of medulloblastoma is believed to be derived from granule neuron precursor cells that fail to stop proliferating (3). We showed that DAB2IP knockdown in C17.2 neural precursor cells significantly reduced TNFα-induced caspase activation, suggesting that DAB2IP has a proapoptotic function in stressed neural precursor cells. However, because C17.2 cells are immortalized by overexpression of v-myc (25), the role of DAB2IP in altering apoptosis responses needs to be established in normal neural precursor cells.

We found a significant association between poor overall survival in patients with medulloblastoma and reduced DAB2IP mRNA levels. Interestingly, no significant difference in DAB2IP expression was observed between the group of WNT associated medulloblastoma—which has the best prognosis—and group 3 medulloblastoma—which has the worse prognosis (4). This association was observed in metastatic as well as in nonmetastatic patients with medulloblastoma, albeit at near significant levels. These results show that DAB2IP expression is a prognostic marker for medulloblastoma outcome and suggest that patients with nonmetastatic medulloblastoma with low DAB2IP expression may benefit from more aggressive treatment strategies. Recent studies have shown that medulloblastoma is a heterogeneous disease with diverse treatment outcome (23, 50). Currently staging for treatment is based on clinical parameters such as age, extent of surgical resection, presence of metastases, and histologic classification (51, 52). Various studies have suggested that this risk stratification could be improved by including molecular determinants (23, 50, 53–57). However, it remains to be investigated to what extent DAB2IP could contribute to the subclassification of medulloblastoma and whether it can aid as a prognostic factor in clinical practice.

In conclusion, we identified DAB2IP as a potential antiapoptotic tumor suppressor in medulloblastoma. Further research in its use as a potentially important prognostic factor and/or therapeutic target may contribute to improvements in the future treatment of patients with medulloblastoma.

No potential conflicts of interest were disclosed.

Conception and design: M. Smits, T. Würdinger

Development of methodology: M. Smits, T. Würdinger

Material supplied: E. Hulleman, D.G. van Vuurden, M. Kool, C. Haberler, E. Aronica

Conducted experiments: M. Smits, S. van Rijn, M. Kool

Analysis and interpretation of data: M. Smits, S. van Rijn, M. Kool, T. Würdinger

Writing of the manuscript: M. Smits, S. van Rijn, D. Biesmans, T. Würdinger

Review, and/or revision of the manuscript: E. Hulleman, W.P. Vandertop, D.P. Noske

Technical support: S. van Rijn, E. Hulleman, D. Biesmans, W.P. Vandertop, D. P. Noske

The authors thank O. Delattre for providing patient survival data, V.E. Marquez for providing DZNep, K. Cichowski for providing the DAB2IP overexpression construct, and J. T. Hsieh for providing a DAB2IP shRNA construct.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Crawford
JR
,
MacDonald
TJ
,
Packer
RJ
. 
Medulloblastoma in childhood: new biological advances
.
Lancet Neurol
2007
;
6
:
1073
85
.
2.
Smoll
NR
. 
Relative survival of childhood and adult medulloblastomas and primitive neuroectodermal tumors (PNETs)
.
Cancer
2012
;
118
:
1313
22
.
3.
Gilbertson
RJ
,
Ellison
DW
. 
The origins of medulloblastoma subtypes
.
Annu Rev Pathol
2008
;
3
:
341
65
.
4.
Taylor
MD
,
Northcott
PA
,
Korshunov
A
,
Remke
M
,
Cho
YJ
,
Clifford
SC
, et al
Molecular subgroups of medulloblastoma: the current consensus
.
Acta Neuropathol
2012
;
123
:
465
72
.
5.
Srivastava
VK
,
Nalbantoglu
J
. 
The cellular and developmental biology of medulloblastoma: current perspectives on experimental therapeutics
.
Cancer Biol Ther
2010
;
9
:
843
52
.
6.
Wang
Z
,
Tseng
CP
,
Pong
RC
,
Chen
H
,
McConnell
JD
,
Navone
N
, et al
The mechanism of growth-inhibitory effect of DOC-2/DAB2 in prostate cancer. Characterization of a novel GTPase-activating protein associated with N-terminal domain of DOC-2/DAB2
.
J Biol Chem
2002
;
277
:
12622
31
.
7.
Chen
H
,
Toyooka
S
,
Gazdar
AF
,
Hsieh
JT
. 
Epigenetic regulation of a novel tumor suppressor gene (hDAB2IP) in prostate cancer cell lines
.
J Biol Chem
2003
;
278
:
3121
30
.
8.
Dote
H
,
Toyooka
S
,
Tsukuda
K
,
Yano
M
,
Ouchida
M
,
Doihara
H
, et al
Aberrant promoter methylation in human DAB2 interactive protein (hDAB2IP) gene in breast cancer
.
Clin Cancer Res
2004
;
10
:
2082
9
.
9.
Yano
M
,
Toyooka
S
,
Tsukuda
K
,
Dote
H
,
Ouchida
M
,
Hanabata
T
, et al
Aberrant promoter methylation of human DAB2 interactive protein (hDAB2IP) gene in lung cancers
.
Int J Cancer
2005
;
113
:
59
66
.
10.
Dote
H
,
Toyooka
S
,
Tsukuda
K
,
Yano
M
,
Ota
T
,
Murakami
M
, et al
Aberrant promoter methylation in human DAB2 interactive protein (hDAB2IP) gene in gastrointestinal tumour
.
Br J Cancer
2005
;
92
:
1117
25
.
11.
Kong
Z
,
Xie
D
,
Boike
T
,
Raghavan
P
,
Burma
S
,
Chen
DJ
, et al
Downregulation of human DAB2IP gene expression in prostate cancer cells results in resistance to ionizing radiation
.
Cancer Res
2010
;
70
:
2829
39
.
12.
Xie
D
,
Gore
C
,
Liu
J
,
Pong
RC
,
Mason
R
,
Hao
G
, et al
Role of DAB2IP in modulating epithelial-to-mesenchymal transition and prostate cancer metastasis
.
Proc Natl Acad Sci U S A
2010
;
107
:
2485
90
.
13.
Min
J
,
Zaslavsky
A
,
Fedele
G
,
McLaughlin
SK
,
Reczek
EE
,
De
RT
, et al
An oncogene-tumor suppressor cascade drives metastatic prostate cancer by coordinately activating Ras and nuclear factor-kappaB
.
Nat Med
2010
;
16
:
286
94
.
14.
Xie
D
,
Gore
C
,
Zhou
J
,
Pong
RC
,
Zhang
H
,
Yu
L
, et al
DAB2IP coordinates both PI3K-Akt and ASK1 pathways for cell survival and apoptosis
.
Proc Natl Acad Sci U S A
2009
;
106
:
19878
83
.
15.
Zhang
R
,
He
X
,
Liu
W
,
Lu
M
,
Hsieh
JT
,
Min
W
. 
AIP1 mediates TNF-alpha-induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3
.
J Clin Invest
2003
;
111
:
1933
43
.
16.
Sutter
R
,
Shakhova
O
,
Bhagat
H
,
Behesti
H
,
Sutter
C
,
Penkar
S
, et al
Cerebellar stem cells act as medulloblastoma-initiating cells in a mouse model and a neural stem cell signature characterizes a subset of human medulloblastomas
.
Oncogene
2010
;
29
:
1845
56
.
17.
Sherr
CJ
. 
Principles of tumor suppression
.
Cell
2004
;
116
:
235
46
.
18.
Mao
J
,
Ligon
KL
,
Rakhlin
EY
,
Thayer
SP
,
Bronson
RT
,
Rowitch
D
, et al
A novel somatic mouse model to survey tumorigenic potential applied to the Hedgehog pathway
.
Cancer Res
2006
;
66
:
10171
8
.
19.
Oliver
TG
,
Read
TA
,
Kessler
JD
,
Mehmeti
A
,
Wells
JF
,
Huynh
TT
, et al
Loss of patched and disruption of granule cell development in a pre-neoplastic stage of medulloblastoma
.
Development
2005
;
132
:
2425
39
.
20.
Sasai
K
,
Romer
JT
,
Kimura
H
,
Eberhart
DE
,
Rice
DS
,
Curran
T
. 
Medulloblastomas derived from Cxcr6 mutant mice respond to treatment with a smoothened inhibitor
.
Cancer Res
2007
;
67
:
3871
7
.
21.
Schuller
U
,
Heine
VM
,
Mao
J
,
Kho
AT
,
Dillon
AK
,
Han
YG
, et al
Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Shh-induced medulloblastoma
.
Cancer Cell
2008
;
14
:
123
34
.
22.
Zindy
F
,
Uziel
T
,
Ayrault
O
,
Calabrese
C
,
Valentine
M
,
Rehg
JE
, et al
Genetic alterations in mouse medulloblastomas and generation of tumors de novo from primary cerebellar granule neuron precursors
.
Cancer Res
2007
;
67
:
2676
84
.
23.
Kool
M
,
Koster
J
,
Bunt
J
,
Hasselt
NE
,
Lakeman
A
,
van
SP
, et al
Integrated genomics identifies five medulloblastoma subtypes with distinct genetic profiles, pathway signatures and clinicopathological features
.
PLoS One
2008
;
3
:
e3088
.
24.
Fattet
S
,
Haberler
C
,
Legoix
P
,
Varlet
P
,
Lellouch-Tubiana
A
,
Lair
S
, et al
Beta-catenin status in paediatric medulloblastomas: correlation of immunohistochemical expression with mutational status, genetic profiles, and clinical characteristics
.
J Pathol
2009
;
218
:
86
94
.
25.
Snyder
EY
,
Deitcher
DL
,
Walsh
C
,
rnold-Aldea
S
,
Hartwieg
EA
,
Cepko
CL
. 
Multipotent neural cell lines can engraft and participate in development of mouse cerebellum
.
Cell
1992
;
68
:
33
51
.
26.
Roth
RB
,
Hevezi
P
,
Lee
J
,
Willhite
D
,
Lechner
SM
,
Foster
AC
, et al
Gene expression analyses reveal molecular relationships among 20 regions of the human CNS
.
Neurogenetics
2006
;
7
:
67
80
.
27.
Zhang
H
,
Zhang
H
,
Lin
Y
,
Li
J
,
Pober
JS
,
Min
W
. 
RIP1-mediated AIP1 phosphorylation at a 14-3-3-binding site is critical for tumor necrosis factor-induced ASK1-JNK/p38 activation
.
J Biol Chem
2007
;
282
:
14788
96
.
28.
Chen
H
,
Tu
SW
,
Hsieh
JT
. 
Down-regulation of human DAB2IP gene expression mediated by polycomb Ezh2 complex and histone deacetylase in prostate cancer
.
J Biol Chem
2005
;
280
:
22437
44
.
29.
Glazer
RI
,
Hartman
KD
,
Knode
MC
,
Richard
MM
,
Chiang
PK
,
Tseng
CK
, et al
3-Deazaneplanocin: a new and potent inhibitor of S-adenosylhomocysteine hydrolase and its effects on human promyelocytic leukemia cell line HL-60
.
Biochem Biophys Res Commun
1986
;
135
:
688
94
.
30.
Smits
M
,
Nilsson
J
,
Mir
SE
,
van der Stoop
PM
,
Hulleman
E
,
Niers
JM
, et al
miR-101 is down-regulated in glioblastoma resulting in EZH2-induced proliferation, migration, and angiogenesis
.
Oncotarget
2010
;
1
:
710
20
.
31.
Tan
J
,
Yang
X
,
Zhuang
L
,
Jiang
X
,
Chen
W
,
Lee
PL
, et al
Pharmacologic disruption of Polycomb-repressive complex 2-mediated gene repression selectively induces apoptosis in cancer cells
.
Genes Dev
2007
;
21
:
1050
63
.
32.
Kawauchi
D
,
Robinson
G
,
Uziel
T
,
Gibson
P
,
Rehg
J
,
Gao
C
, et al
A mouse model of the most aggressive subgroup of human medulloblastoma
.
Cancer Cell
2012
;
21
:
168
80
.
33.
Pei
Y
,
Moore
CE
,
Wang
J
,
Tewari
AK
,
Eroshkin
A
,
Cho
YJ
, et al
An animal model of MYC-driven medulloblastoma
.
Cancer Cell
2012
;
21
:
155
67
.
34.
Gibson
P
,
Tong
Y
,
Robinson
G
,
Thompson
MC
,
Currle
DS
,
Eden
C
, et al
Subtypes of medulloblastoma have distinct developmental origins
.
Nature
2010
;
468
:
1095
9
.
35.
Baeza
N
,
Masuoka
J
,
Kleihues
P
,
Ohgaki
H
. 
AXIN1 mutations but not deletions in cerebellar medulloblastomas
.
Oncogene
2003
;
22
:
632
6
.
36.
Huang
H
,
Mahler-Araujo
BM
,
Sankila
A
,
Chimelli
L
,
Yonekawa
Y
,
Kleihues
P
, et al
APC mutations in sporadic medulloblastomas
.
Am J Pathol
2000
;
156
:
433
7
.
37.
Huse
JT
,
Holland
EC
. 
Targeting brain cancer: advances in the molecular pathology of malignant glioma and medulloblastoma
.
Nat Rev Cancer
2010
;
10
:
319
31
.
38.
Kasper
M
,
Regl
G
,
Frischauf
AM
,
Aberger
F
. 
GLI transcription factors: mediators of oncogenic Hedgehog signalling
.
Eur J Cancer
2006
;
42
:
437
45
.
39.
Koch
A
,
Hrychyk
A
,
Hartmann
W
,
Waha
A
,
Mikeska
T
,
Waha
A
, et al
Mutations of the Wnt antagonist AXIN2 (Conductin) result in TCF-dependent transcription in medulloblastomas
.
Int J Cancer
2007
;
121
:
284
91
.
40.
Raffel
C
,
Jenkins
RB
,
Frederick
L
,
Hebrink
D
,
Alderete
B
,
Fults
DW
, et al
Sporadic medulloblastomas contain PTCH mutations
.
Cancer Res
1997
;
57
:
842
5
.
41.
Taylor
MD
,
Liu
L
,
Raffel
C
,
Hui
CC
,
Mainprize
TG
,
Zhang
X
, et al
Mutations in SUFU predispose to medulloblastoma
.
Nat Genet
2002
;
31
:
306
10
.
42.
von Bergh
AR
,
Wijers
PM
,
Groot
AJ
,
van Zelderen-Bhola
S
,
Falkenburg
JH
,
Kluin
PM
, et al
Identification of a novel RAS GTPase-activating protein (RASGAP) gene at 9q34 as an MLL fusion partner in a patient with de novo acute myeloid leukemia
.
Genes Chromosomes Cancer
2004
;
39
:
324
34
.
43.
Duggan
D
,
Zheng
SL
,
Knowlton
M
,
Benitez
D
,
Dimitrov
L
,
Wiklund
F
, et al
Two genome-wide association studies of aggressive prostate cancer implicate putative prostate tumor suppressor gene DAB2IP
.
J Natl Cancer Inst
2007
;
99
:
1836
44
.
44.
Calvisi
DF
,
Ladu
S
,
Conner
EA
,
Seo
D
,
Hsieh
JT
,
Factor
VM
, et al
Inactivation of Ras GTPase-activating proteins promotes unrestrained activity of wild-type Ras in human liver cancer
.
J Hepatol
2011
;
54
:
311
9
.
45.
Gretarsdottir
S
,
Baas
AF
,
Thorleifsson
G
,
Holm
H
,
den
HM
,
de Vries
JP
, et al
Genome-wide association study identifies a sequence variant within the DAB2IP gene conferring susceptibility to abdominal aortic aneurysm
.
Nat Genet
2010
;
42
:
692
7
.
46.
Yang
L
,
Li
Y
,
Ling
X
,
Liu
L
,
Liu
B
,
Xu
K
, et al
A common genetic variant (97906C>A) of DAB2IP/AIP1 is associated with an increased risk and early onset of lung cancer in Chinese males
.
PLoS One
2011
;
6
:
e26944
.
47.
Herranz
N
,
Pasini
D
,
Diaz
VM
,
Franci
C
,
Gutierrez
A
,
Dave
N
, et al
Polycomb complex 2 is required for E-cadherin repression by the Snail1 transcription factor
.
Mol Cell Biol
2008
;
28
:
4772
81
.
48.
Yokota
N
,
Nishizawa
S
,
Ohta
S
,
Date
H
,
Sugimura
H
,
Namba
H
, et al
Role of Wnt pathway in medulloblastoma oncogenesis
.
Int J Cancer
2002
;
101
:
198
201
.
49.
Homayouni
R
,
Magdaleno
S
,
Keshvara
L
,
Rice
DS
,
Curran
T
. 
Interaction of Disabled-1 and the GTPase activating protein Dab2IP in mouse brain
.
Brain Res Mol Brain Res
2003
;
115
:
121
9
.
50.
Ellison
DW
,
Kocak
M
,
Dalton
J
,
Megahed
H
,
Lusher
ME
,
Ryan
SL
, et al
Definition of disease-risk stratification groups in childhood medulloblastoma using combined clinical, pathologic, and molecular variables
.
J Clin Oncol
2011
;
29
:
1400
7
.
51.
Zeltzer
PM
,
Boyett
JM
,
Finlay
JL
,
Albright
AL
,
Rorke
LB
,
Milstein
JM
, et al
Metastasis stage, adjuvant treatment, and residual tumor are prognostic factors for medulloblastoma in children: conclusions from the Children's Cancer Group 921 randomized phase III study
.
J Clin Oncol
1999
;
17
:
832
45
.
52.
Giangaspero
F
,
Eberhart
CG
,
Haaspalo
H
,
Pietsch
T
,
Wiestler
OD
,
Ellison
DW
. 
Medulloblastoma
. In:
Louis
DN
,
Ohgaki
H
,
Wiestler
OD
,
Cavenee
WK
,
editors
. 
WHO classification of tumours of the central nervous system
.
Lyon, France
:
IARC Press
; 
2007
.
p.
132
40
.
53.
Cho
YJ
,
Tsherniak
A
,
Tamayo
P
,
Santagata
S
,
Ligon
A
,
Greulich
H
, et al
Integrative genomic analysis of medulloblastoma identifies a molecular subgroup that drives poor clinical outcome
.
J Clin Oncol
2011
;
29
:
1424
30
.
54.
Pfister
S
,
Remke
M
,
Benner
A
,
Mendrzyk
F
,
Toedt
G
,
Felsberg
J
, et al
Outcome prediction in pediatric medulloblastoma based on DNA copy-number aberrations of chromosomes 6q and 17q and the MYC and MYCN loci
.
J Clin Oncol
2009
;
27
:
1627
36
.
55.
Pomeroy
SL
,
Tamayo
P
,
Gaasenbeek
M
,
Sturla
LM
,
Angelo
M
,
McLaughlin
ME
, et al
Prediction of central nervous system embryonal tumour outcome based on gene expression
.
Nature
2002
;
415
:
436
42
.
56.
Remke
M
,
Hielscher
T
,
Korshunov
A
,
Northcott
PA
,
Bender
S
,
Kool
M
, et al
FSTL5 is a marker of poor prognosis in non-WNT/Non-SHH medulloblastoma
.
J Clin Oncol
2011
;
29
:
3852
61
.
57.
Poschl
J
,
Lorenz
A
,
Hartmann
W
,
von Bueren
AO
,
Kool
M
,
Li
S
, et al
Expression of BARHL1 in medulloblastoma is associated with prolonged survival in mice and humans
.
Oncogene
2011
;
30
:
4721
30
.

Supplementary data