The SFRP Family of WNT Inhibitors Function As Novel Tumor Suppressor Genes Epigenetically Silenced in Medulloblastoma

Oncogene (2010) 29, 3017–3024 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc SHORT COMMUNICATION The SFRP family of WNT inhibitors function as novel tumor suppressor genes epigenetically silenced in medulloblastoma PN Kongkham1,2, PA Northcott1,3, SE Croul4, CA Smith1,2, MD Taylor1,3 and JT Rutka1,2 1Division of Neurosurgery, Arthur and Sonia Labatt Brain Tumor Research Centre, Toronto, Ontario, Canada; 2Program in Cell Biology, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada; 3Program in Developmental and Stem Cell Biology, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada and 4Department of Pathology, University Health Network, University of Toronto, Toronto, Ontario, Canada Medulloblastoma (MB) is the most common malignant signaling is crucial for normal cerebellar development, pediatric brain tumor. Dysregulation of WNT signaling as transgenic mice with reduced WNT signaling exhibit occurs in up to 20% of cases. Using a genome-wide cerebellar aplasia (McMahon and Bradley, 1990). approach, we identified the secreted frizzled-related Turcot syndrome patients with germline adenomatous protein 1, 2 and 3 (SFRP1, SFRP2 and SFRP3) family polyposis coli (APC) gene mutation and resultant of WNT inhibitors as putative tumor suppressor genes increased WNT signaling have an increased risk of silenced by promoter region methylation in MB. SFRP1, MB (Hamilton et al., 1995). Furthermore, mutations SFRP2 and SFRP3 expression increased after 5-aza-20- affecting WNT signaling members (APC, CTNNB1, deoxycytidine treatment. SFRP1, SFRP2 and SFRP3 AXIN1 and AXIN2) are seen in 15–20% of sporadic MB methylation was identified in 23.5, 3.9 and 15.7% of cases (Eberhart et al., 2000; Baeza et al., 2003; primary MB specimens, respectively, by methylation- Thompson et al., 2006; Koch et al., 2007). specific PCR. Stable SFRP1, SFRP2 and SFRP3 Recent gene expression profiling studies show that expression reduced phospho-DVL2 levels and hindered human MBs may be divided into distinct molecular MB cell proliferation and colony formation in soft agar subgroups including one characterized by expression of in vitro. In 60% of primary tumors, SFRP1 was expressed markers of canonical WNT signaling such as WIF1, at levels twofold lower than that in normal cerebellum. DKK2, LEF1, KREMEN1 and AXIN2 (Thompson SFRP1 expression impaired tumor formation in vivo in et al., 2006; Kool et al., 2008; Northcott et al., 2009a). flank and orthotopic intracerebellar xenograft models and Most tumors in the WNT subgroup have monosomy 6, conferred a significant survival advantage (Po0.0001). activating CTNNB1 mutations, and carry a more We identify for the first time tumor suppressor gene favorable prognosis compared with other MB sub- function of SFRP genes in MB, and suggest that loss of groups (Eberhart et al., 2000; Ellison et al., 2005; Kool WNT pathway inhibition due to SFRP gene silencing is an et al., 2008; Northcott et al., 2009a). additional mechanism that may contribute to excessive Recently, we used a microarray-based genome-wide WNT signaling in this disease. epigenetic approach to identify novel tumor suppressor Oncogene (2010) 29, 3017–3024; doi:10.1038/onc.2010.32; genes silenced by promoter methylation in MB (Kongk- published online 8 March 2010 ham et al., 2008). Among candidates identified were members of the secreted frizzled-related protein (SFRP) Keywords: medulloblastoma; WNT signaling; epige- family of WNT inhibitors, including SFRP1, SFRP2 netics; methylation; tumor suppressor gene and SFRP3. Functionally, SFRP genes can limit canonical and noncanonical WNT signaling, binding WNT ligand and sequestering it away from Fz receptors (Bovolenta et al., 2008). SFRPs have been identified as tumor suppressor genes whose expression is reduced in a Introduction variety of malignancies (Dahl et al., 2007). Only SFRP1 has been examined in the context of MB previously. Medulloblastoma (MB) is the most common malignant SFRP1 expression has been shown to increase after posterior fossa tumor. Dysregulation of developmental treatment of the D283 cell line with the histone signaling pathways, including WNT signaling, is known deacetylase inhibitor, trichostatin, but the functional to have a major role in MB pathogenesis. WNT significance of this was not examined (Vibhakar et al., 2007). A second study looking at SFRP1 in MB failed to Correspondence: Dr JT Rutka, The Division of Neurosurgery, Arthur identify promoter methylation, which may have been in and Sonia Labatt Brain Tumor Research Centre, Suite 1503, The part due to the limited number of tumors examined Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, (Chang et al., 2005). Canada M5G 1X8. E-mail: [email protected] Here, we show for the first time that SFRP1, SFRP2 Received 1 September 2009; revised 15 November 2009; accepted 3 and SFRP3 promoter region methylation is associated January 2010; published online 8 March 2010 with gene silencing in MB cell lines and primary tumors. SFRP gene silencing in medulloblastoma PN Kongkham et al 3018 Expression of SFRP1, SFRP2 or SFRP3 in MB cells reduced phospho-DVL2 in both the D283 and ONS76 reduces their proliferative capacity and anchorage- cell lines compared with controls (Figure 2a and Supple- independent growth in vitro. SFRP1, SFRP2 or SFRP3 mentary Figure 5). Stable re-expression of SFRP1-3 in expression limits WNT signaling, as evidenced by D283 cells did not alter the levels of activated b-catenin reduced levels of phospho-DVL2. SFRP1 expression (Supplementary Figure 8). Expression of SFRP1, SFRP2 also limits tumor burden and prolongs overall survival and SFRP3 also decreased D283 and ONS76 cell proli- in vivo in an intracranial xenograft model of MB. feration, as measured by MTS (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assay (Figure 2b and Supplementary Figure 6). Finally, SFRP1, SFRP2 and SFRP3 expression significantly impaired D283 capacity for Results and discussion anchorage-independent growth (Figure 2c and Supplemen- tary Figure 7). SFRP1, SFRP2 and SFRP3 silencing is associated with promoter region methylation Increased expression of SFRP1, SFRP2 and SFRP3 0 MB subgroup-specific expression of SFRP1 after 5-aza-2 -deoxycytidine (5-aza-dC) treatment in MB Using Affymetrix Genechip Human Exon Arrays cell lines was validated using quantitative real-time (Affymetrix, Santa Clara, CA, USA) to assess gene PCR. SFRP1 expression increased in the ONS76, expression in a large cohort of 103 primary MBs, 7 MB UW228, UW426, D425, D458 and MED8A cell lines cell lines, 9 fetal and 5 adult cerebellar samples, we after treatment (Figure 1a). SFRP2 expression increased observed SFRP1 expression levels to be more than in the UW228 cell line, and SFRP3 in the DAOY, D283 twofold lower in 60% (62/103) of primary MBs and MED8A cell lines (Supplementary Figure 1). compared with normal cerebellum. Unsupervised hier- Bisulfite sequencing confirmed promoter region methyl- archical clustering based on 1450 differentially expressed ation for SFRP1, SFRP2 and SFRP3 in MB cell lines genes divided primary tumors into four molecular compared with normal fetal and adult cerebellum subgroups (WNT, Sonic hedgehog (SHH) homolog, (Figure 1b and Supplementary Figures 2 and 3). Methyla- Group C and Group D) as described by our group and tion-specific PCR identified methylation of the SFRP1, others (Thompson et al., 2006; Kool et al., 2008; SFRP2 and SFRP3 promoter regions in 12/51 (23.5%), 2/ Northcott et al., 2009a). Interestingly, SFRP1 expres- 51 (3.9%) and 8/51 (15.7%) primary MB specimens, sion was reduced in WNT, Group C and Group D MB respectively (Figure 1c and Supplementary Figure 4). subgroups in comparison with the SHH group of Where sufficient mRNA was available, SFRP1 expression tumors (Figure 3a). was correlated with SFRP1 promoter methylation status A common feature of WNT subgroup tumors is the by quantitative real-time PCR. Samples identified as having presence of downstream activating CTNNB1 mutations. aberrant promoter region methylation by methylation- One might question the functional significance of specific PCR showed low levels of SFRP1 gene expression reduced SFRP1 expression in the context of downstream compared with normal cerebellum (Figure 1d). activating mutations. However, it has been shown in the context of colorectal cancer that loss of SFRP gene SFRP1, SFRP2 and SFRP3 re-expression reduces WNT expression in the presence of downstream mutations of signaling, cell proliferation and anchorage-independent APC or activating CTNNB1 mutations function in a growth synergistic fashion, and that expression of SFRPs can To determine the functional significance of SFRP1, reduce WNT signaling even in the presence of down- SFRP2 and SFRP3 silencing in MB, we stably expressed stream activating mutations (Suzuki et al., 2004). each gene individually in the D283 and ONS76 MB cell Interestingly, SFRP1 expression was also reduced in lines and assessed the effect on WNT signaling, cell Group C and Group D tumors that do not possess proliferation and colony formation in soft agar com- canonical WNT gene expression signatures (Figure 3a). pared with both empty vector and an EGFP-expressing This suggests the possibility that lack of SFRP1 control stable cell lines (Figure 2 and Supplementary expression in these tumors may influence MB pathogen- Figures 5–8). esis through dysregulation of noncanonical WNT SFRPs exert their effect upstream at the level

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