Published OnlineFirst June 13, 2012; DOI: 10.1158/1078-0432.CCR-12-0399 Clinical Cancer Human Cancer Biology Research EZH2-Regulated DAB2IP Is a Medulloblastoma Tumor Suppressor and a Positive Marker for Survival Michiel Smits1, Sjoerd van Rijn1, Esther Hulleman1, Dennis Biesmans1, Dannis G. van Vuurden1, Marcel Kool4, Christine Haberler5, Eleonora Aronica2, W. Peter Vandertop1,3, David P. Noske1, and Thomas Wurdinger€ 1,6 Abstract 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; 1–11. Ó2012 AACR. Introduction 4 subtypes: WNT, SHH, group 3, and group 4, which differ Brain tumors are the most common form of solid tumors regarding histology and clinical outcome (4) and are in children of which medulloblastoma is the most frequent believed to derive from the deregulation of various signaling malignant variant, accounting for 20% of cases (1). Treat- pathways in brain development, such as the WNT-pathway ment modalities consist of surgery, radiotherapy, and che- and sonic hedgehog (SHH) signaling pathways. Overacti- motherapy and result in a 5-year survival rate of 40% in vation of these pathways leads to a loss of cell-cycle control high-risk patients and 80% to 90% in low-risk patients (2). and a dysfunctional apoptosis program, allowing for con- Approximately 30% of patients however remain incurable tinued growth and tumorigenesis, predominantly in the and current intensive treatment protocols cause significant cerebellum (5). adverse long-term effects (3). Medulloblastomas comprise 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 Authors' Affiliations: 1Neuro-oncology Research Group, Departments of GDP (6). DAB2IP acts as a putative tumor suppressor Neurosurgery and Pediatric Oncology/Hematology, Cancer Center gene and is downregulated by epigenetic modification in Amsterdam, VU University Medical Center; 2Department of (Neuro)Pathol- ogy, Academic Medical Center, University of Amsterdam; 3Neurosurgical multiple aggressive cancers. In prostate cancer, DAB2IP Center Amsterdam, Academic Medical Center, Amsterdam, The Nether- expression was shown to be repressed by promoter meth- 4 lands; Department of Molecular Genetics, German Cancer Research ylation and histone modification (7), whereas in breast Center (DKFZ), Heidelberg, Germany; 5Institut Curie, Laboratoire de Genet- ique et Biologie des Cancers, Paris, France; and 6Molecular Neurogenetics cancer (8), lung cancer (9), and gastrointestinal tumors Unit, Departments of Neurology and Radiology, Massachusetts General (10), aberrant promoter hypermethylation was shown to Hospital and Neuroscience Program, Harvard Medical School, Boston, Massachusetts downregulate DAB2IP. Moreover, it was shown in pros- tate cancer that downregulation of DAB2IP expression Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). results in resistance to ionizing radiation (11), it initiates epithelial-to-mesenchymal transition (12) and promotes Corresponding Author: Thomas Wurdinger,€ VU University Medical Cen- ter, CCA Room 3.60, De Boelelaan 1117, Amsterdam 1081 HV, The tumor growth and metastasis (13). In addition, DAB2IP is Netherlands. Phone: 31-204447909; Fax: 31-20-4442126; E-mail: involved in TNFa-induced apoptosis in prostate cancer [email protected] cells by suppressing the ASK1-JNK and PI3-AKT pathway doi: 10.1158/1078-0432.CCR-12-0399 (14), and in endothelial cells via the ASK1-JNK pathway Ó2012 American Association for Cancer Research. (15). www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst June 13, 2012; DOI: 10.1158/1078-0432.CCR-12-0399 Smits et al. was based on 108 cases for which expression and survival Translational Relevance data were available. Patient and tumor characteristics are Medulloblastoma is the most common malignant presented in Table 1. In brief, all samples were snap frozen pediatric brain tumor. Current treatment modalities in the institutional pathology departments immediately result in a 5-year survival rate of 40% in high-risk upon arrival. All samples were reviewed by experienced patients and 80% to 90% in standard risk patients. neuropathologists and examined for tumor content. Total Approximately 30% of patients however remain incur- RNA was extracted using TRIzol (Invitrogen). Gene expres- able and current intensive treatment protocols cause sion profiles were obtained by Affymetrix HG-U133 Plus 2.0 significant adverse long-term effects such as impaired arrays. Gene expression data were normalized using the neurologic function and endocrine dysfunction. GCRMA procedure. Informed consent and detailed meth- Currently, the staging for medulloblastoma between ods are described elsewhere (23, 24). high-risk and standard-risk disease is based on clinical Immunohistochemistry was conducted on a largely inde- parameters and histologic subtypes. However, it is sug- pendent medulloblastoma tissue microarray (TMA) cohort gested that including molecular markers in the risk with tumors from 87 patients obtained from the files of the stratification could improve survival and decrease treat- Department of Neuropathology of the Academic Medical ment-related toxicity. Here we show that high expression Center (University of Amsterdam). Subgroup information of DAB2IP in medulloblastoma is associated with a was obtained by immunohistochemistry using antibodies favorable prognosis, independent of metastatic stage. for the subgroup-specific protein markers b-catenin (WNT), This suggests DAB2IP expression inhibits tumor growth DKK1 (WNT), SFRP1 (SHH), NPR3 (Group 3), and KCNA1 and further research in its use as a potentially important (Group 4). Information on gender, age at diagnosis, his- prognostic factor and/or therapeutic target may contrib- tology, metastatic stage at diagnosis, and survival are pre- ute to improvements in the future treatment of patients sented in Table 2. The mean follow-up time of survivors in with medulloblastoma. 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 Apoptosis is a programmed variant of cell death common obtained from surgical specimens after informed consent to all human cells. Defects in the apoptosis program result and approval by the Medical Ethical Committee of the VU in an imbalance in the rate of cell proliferation and the rate University Medical Center. of cell death thereby contributing to tumor growth and treatment resistance. Essential steps in the apoptotic mech- Survival analysis anism are inactivated in medulloblastoma cells, resulting in Overall survival was calculated from the time of diagnosis resistance to apoptosis. A recent in vivo study showed that to the patient’s last follow-up or death. Survival of patients cerebellar stem cells can give rise to medulloblastomas was analyzed using Kaplan–Meier survival curves, and the when having acquired an impaired apoptosis mechanism log-rank test was used to examine the statistical significance. (16). Consequently, many of the apoptosis mediators are P-values < 0.05 were considered significant. Prognostic generally considered tumor suppressors (17). impact of covariates on survival was evaluated on the basis Here, we describe a comprehensive meta-analysis of gene of hazard ratios from Cox’s proportional hazards regression expression studies of mouse and human medulloblastoma model. Multivariate Cox’s proportional hazards regression (18–23) identifying multiple medulloblastoma tumor sup- models were used to estimate effects of additional the pressor candidates, including DAB2IP. We found DAB2IP covariates age, metastatic stage, and histology. expression to be strongly downregulated in human medul- loblastoma cells and in primary human medulloblastoma Cells tissues. We show that DAB2IP downregulation is—at least Human D283-med, D556-med (Dr Darrell Bigner, Duke partially—caused by EZH2-mediated