Oncogene (2008) 27, 1478–1488 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ONCOGENOMICS Deregulated Wnt/b-catenin program in high-risk neuroblastomas without MYCN amplification X Liu1, P Mazanek1, V Dam1, Q Wang1, H Zhao2, R Guo2, J Jagannathan1, A Cnaan2, JM Maris1,3 and MD Hogarty1,3 1Division of Oncology, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 2Department of Biostatistics and Epidemiology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA and 3Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Neuroblastoma (NB) is a frequently lethal tumor of Introduction childhood. MYCN amplification accounts for the aggres- sive phenotype in a subset while the majority have no Neuroblastoma (NB) is a childhood embryonal malig- consistently identified molecular aberration but frequently nancy arising in the peripheral sympathetic nervous express MYC at high levels. We hypothesized that acti- system. Half of all children with NB present with features vated Wnt/b-catenin (CTNNB1) signaling might account that define their tumorsashigh riskwith poor overall for this as MYC is a b-catenin transcriptional target and survival despite intensive therapy (Matthay et al., 1999). multiple embryonal and neural crest malignancies have A subset of these tumors are characterized by high-level oncogenic alterations in this pathway. NB cell lines without genomic amplification of the MYCN proto-oncogene MYCN amplification express higher levels of MYC and (Matthay et al., 1999) but the remainder have no b-catenin (with aberrant nuclear localization) than MYCN- consistently identified aberration to account for their amplified cell lines. Evidence for aberrant b-catenin–TCF aggressive phenotype. transcriptional activity was demonstrated using expression MycN isa member of the Myc family of genes( MYC, profiles from 73 primary NBs. Findings included increased MYCN, MYCL) that play a central role in diverse WNT ligands (WNT1, WNT6, WNT7A, WNT10B), cellular processes (Grandori et al., 2000) and their DVL1 and TCF7 expression in high-risk NBs without deregulated expression frequently contributes to neo- MYCN amplification, consistent with canonical b-catenin plasia (Cole and McMahon, 1999). NB-derived cell lines signaling. More directly, Patterns of Gene Expression and without MYCN amplification generally express MYC Gene Set Enrichment Analyses demonstrated b-catenin rather than MYCN, often at higher levelsthan normal target genes (for example, MYC, PPARD, NRCAM, tissues (Sadee et al., 1987). MYC amplification (in CD44, TCF7) as coordinately upregulated in high-risk NBs contrast to MYCN) israre in NB (Kohl et al., 1983) yet without MYCN amplification in comparison to high-risk alternative mechanisms of MYC deregulation have not MYCN-amplified or intermediate-risk NBs, supporting been elucidated. pathway activation in this subset. Thus, high-risk NBs A recurring paradigm in embryonal oncogenesis is the without MYCN amplification may deregulate MYC and aberrant appropriation of developmental programs. other oncogenic genes via altered b-catenin signaling Indeed, activation of the Notch, Sonic hedgehog and providing a potential candidate pathway for therapeutic Wnt/b-catenin (CTNNB1) developmental programs inhibition. contribute to malignant transformation (Taipale and Oncogene (2008) 27, 1478–1488; doi:10.1038/sj.onc.1210769; Beachy, 2001; Allenspach et al., 2002; Giles et al., 2003). published online 27 August 2007 Wnt/b-catenin signaling may be of particular relevance to NBs, which arise from migratory neural crest-derived Keywords: neuroblastoma; CTNNB1/b-catenin; MYC; neuroblasts, as this program mediates neural crest cell ONCOGENOMICS MYCN; embryonal cancer fate and neural stem-cell expansion (Chenn and Walsh, 2002; Zechner et al., 2003; Lee et al., 2004). Further, Wnt/b-catenin signaling is aberrantly activated in multi- ple embryonal and neural crest-derived malignancies (Giles et al., 2003). Activation of the canonical Wnt/b-catenin pathway leads to transactivation of target genes, which may play a direct role in tumorigenesis (Goss and Groden, 2000; Correspondence: Dr MD Hogarty, Division of Oncology/The Moon et al., 2002). During the nonactivated state, Children’sHospitalof Philadelphia, 9 North ARC (902C), 3516 b-catenin is sequestered in the cytosol by a complex that Civic Center Boulevard, Philadelphia, PA 19104-4318, USA. E-mail: [email protected] includesAPC, GSK3B, Axin proteinsand others.This Received 25 September 2006; revised 27 June 2007; accepted 1 August structure facilitates phosphorylation of b-catenin target- 2007; published online 27 August 2007 ing it for proteasomal degradation (Goss and Groden, Deregulated b-catenin in neuroblastoma XLiuet al 1479 2000) and b-catenin targetsremain actively repressed. intragenic deletion that hinders b-catenin degradation Canonical pathway activation hinders b-catenin phos- and leadsto canonical signaling(He et al., 1998). phorylation resulting in enhanced protein stability and NB cell lineswithout MYCN amplification express nuclear translocation, where it forms a heterodimeric b-catenin at high levelswith aberrant nuclear localization. transcription factor with proteins of the TCF/LEF Subcellular protein fractions were assessed for expres- family and transactivates target genes that include MYC sion of b-catenin (Figures1c and d). NALM-6 and PB1 (He et al., 1998), CCND1 and others(Miller et al., 1999; controls demonstrate modest b-catenin expression limited Hecht and Kemler, 2000). to the cytosol. HepG2 cells express both wild-type and Malignant cellsmay activate the canonical pathway mutant b-catenin, with the stabilized truncated b-catenin through gain-of-function mutationsin b-catenin, by in higher abundance and enriched in the nuclear fraction inactivation of componentsof the scaffolding complex (positive control for deregulated b-catenin). (Giles et al., 2003), or by autocrine Wnt/receptor signaling NB cellswith MYCN amplification express b-catenin (Bafico et al., 2004). We hypothesized that deregulated comparable to that of the wild-type protein in HepG2. b-catenin signaling may occur in high-risk NBs without The majority iscytosolic, with a minor nuclear contri- MYCN amplification resulting in transactivation of MYC bution (o30% of total). In contrast, MYCN single-copy and other target genes to induce an aggressive phenotype. cell linesexpresshigher levelsof b-catenin overall aswell asa higher amount in the nucleus(similar to that for the mutant allele in HepG2). The absence of significant Results nuclear b-tubulin argues against cytosolic spillover contributing to artifactual localization (see Supplemen- There isa reciprocal relationship between MYC and tary Figure S1). MYCN expression in NB-derived cell lines. Cell lines Immunofluorescence cytology confirmed aberrant with MYCN gene amplification expressed high levels of nuclear localization (Figure 2a). HepG2 cellsdemon- MYCN and undetectable MYC (Figures1a and b). In strated higher b-catenin expression with strong nuclear contrast, NB cell lines without MYCN amplification localization in B25% of cells(arrowhead). The MYCN- expressed MYC in the absence of detectable MYCN amplified cell linesshow b-catenin largely restricted to with the exception of the NBL-S cell line. Thisunique the cytosol (IMR5 shown). In contrast, SK-N-AS and nonamplified cell line hasa prolonged MycN protein LA-N-6 demonstrated higher b-catenin expression with half-life (Cohn et al., 1990). Of note, control HepG2 moderate and marked homogeneousnuclear localiza- cellsexpresshighlevelsof MYC due to a hemizygous tion, respectively. In addition to validating localization, Figure 1 Reciprocal MYCN and MYC expression (a) in NB at both the mRNA (relative to GAPDH) and protein level. Note marked HepG2 MYC expression secondary to deregulated b-catenin. Immunoblot (IB) validation of Myc protein expression (b) wasdone using fractionated protein from the nuclear (N) and cytosolic (C) compartments. (c)IBofb-catenin and tubulin. Wild-type (wt) and truncated b-catenin (*) are depicted. A single representative experiment is shown. (d) Histogram of expression and localization by densitometry for three replicate experiments (as in (c), with cytosolic (black portion) and nuclear (gray portion) b-catenin detected by densitometry). Wt and mutant alleles for HepG2 are shown separately. Replicates are from independent protein fractionations. Oncogene Deregulated b-catenin in neuroblastoma XLiuet al 1480 No activating b-catenin mutationsare identified in NB cell linesor primary NBs.No aberrantly migrating b-catenin bands to suggest truncating deletions were present in NB (Figure 1c). Single-strand conformational polymorphism (SSCP) was used to analyse the critical b-catenin regulatory region in NB cell lines( N ¼ 12) and primary NBs( N ¼ 26) and no aberrantly migrating bandswere detected (Table 1). The HepG2 cell line hasa hemizygousmutant b-catenin allele with an exon 3 deletion. Since the SSCP primersamplify within exon 3, HepG2 ishemizygousfor thisPCR product and the banding pattern appearswild type. To exclude similar larger deletions in NB cells, we used primers within exons2 and 4 to amplify a larger genomic fragment encompassing the entire regulatory domain. No internal deletionswere identified in NB while HepG2 demon- strated the internal deletion (data not shown). Despite aberrant nuclear b-catenin, NB-derived cell linesdid not exhibit enhanced b-catenin–TCF transcrip-