Canonical WNT Signalling Determines Lineage Specificity in Wilms Tumour
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Oncogene (2009) 28, 1063–1075 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE Canonical WNT signalling determines lineage specificity in Wilms tumour R Fukuzawa, MR Anaka, RJ Weeks, IM Morison and AE Reeve Cancer Genetics Laboratory, Department of Biochemistry, University of Otago, Dunedin, New Zealand Wilms tumours (WTs) have two distinct types of histology to translocation of the protein to the nucleus (Clevers, with or without ectopic mesenchymal elements, suggesting 2006). Nuclear accumulation of b-catenin is therefore a that WTs arise from either the mesenchymal or epithelial surrogate marker for activation of the WNT-signalling nephrogenic lineages. Regardless of the presence or pathway in many types of cancers (Clevers, 2006). absence of CTNNB1 mutations, nuclear accumulation of Wilms tumour (WT) is an embryonal tumour that b-catenin is often observed in WTs with ectopic mesench- recapitulates kidney development. WT typically has a ymal elements. Here, we addressed the relationship triphasic histology, which consists of varying propor- between the WNT-signalling pathway and lineage in tions of blastemal, epithelial and stromal elements WTs by examining CTNNB1 and WT1 mutations, (Beckwith et al., 1990). The stromal tissues occasionally nuclear accumulation of b-catenin, tumour histology and contain ectopic mesenchymal elements such as rhabdo- gene expression profiles. In addition, we screened for myoblasts, chondrocytes and adipocytes. Remarkably, mutations in WTX, which has been proposed to be a the presence or absence of ectopic mesenchymal negative regulator of the canonical WNT-signalling elements in WT histology is associated with specific pathway. Unsupervised clustering analysis identified two precursor lesions, intralobar and perilobar nephrogenic classes of tumours: mesenchymal lineage WNT-dependent rests (ILNR and PLNR), respectively (Beckwith et al., tumours, and epithelial lineage WNT-independent tu- 1990). The different localization of the nephrogenic rests mours. In contrast to the mesenchymal lineage specificity (ILNR in the centre and PLNR in the periphery of the of CTNNB1 mutations, WTX mutations were surprisingly renal lobe) indicates that ILNR and PLNR occur in the observed in both lineages. WTX-mutant WTs with ectopic early and late kidney development, respectively (Beck- mesenchymal elements had nuclear accumulation of with et al., 1990). As ILNR-derived tumours have b-catenin, upregulation of WNT target genes and an ectopic mesenchymal elements and PLNR-derived association with CTNNB1 mutations in exon 7 or 8. tumours do not, the distinction of the associated WT However, epithelial lineage WTs with WTX mutations histology suggests that ILNR have the potential for had no indications of active WNT signalling, suggesting mesenchymal differentiation including renal epithelial that the involvement of WTX in the WNT-signalling development, whereas PLNR have a limited potential pathway may be lineage dependent, and that WTX may toward nephrogenic epithelial differentiation. Therefore, have an alternative function to its role in the canonical the timing of kidney developmental errors determines WNT-signalling pathway. distinct types of nephrogenic rests containing progenitor Oncogene (2009) 28, 1063–1075; doi:10.1038/onc.2008.455; cells that are committed to different lineages (Fukuzawa published online 12 January 2009 et al., 2008). The WNT-signalling pathway is known to be involved Keywords: WTX; cancer stem cell; nephrogenic rest; in a subset of WT (Koesters et al., 1999, 2003; Maiti mesenchymal stem cell; multiplex ligation-dependent et al., 2000; Li et al. 2004; Fukuzawa et al., 2004a). probe amplification These tumours typically have WT1 mutations and/or CTNNB1 mutations (Koesters et al., 1999; Maiti et al., 2000; Li et al., 2004; Fukuzawa et al., 2004a). WT1 mutations are strongly associated with ILNR (Beckwith, Introduction 1998) that often develop into WTs with ectopic mesenchymal elements (Beckwith et al., 1990; Beckwith, WNT signalling plays a role in embryogenesis and 1998). A series of immunohistochemical studies of carcinogenesis (Clevers, 2006). The most well-character- b-catenin in WT have shown that nuclear accumulation ized WNT pathway is the canonical b-catenin/Tcf- of b-catenin is predominantly observed in mesenchymal mediated pathway, which stabilizes b-catenin and leads cells irrespective of CTNNB1 mutations (Koesters et al., 2003; Li et al., 2004; Fukuzawa et al., 2004a, 2008; Zirn et al., 2006). These observations suggest a relationship Correspondence: Dr R Fukuzawa, Cancer Genetics Laboratory, between the mesenchymal lineage in WT and active Department of Biochemistry, University of Otago, PO Box 56, WNT signalling. Dunedin, New Zealand. E-mail: [email protected] The WTX gene is located in the chromosome band Received 29 July 2008; revised 3 November 2008; accepted 10 November Xq11.1 and encodes a protein of 1135 amino acids. 2008; published online 12 January 2009 WTX (also called AMER1) has been reported to be WNT signalling and lineage specificity in Wilms tumour R Fukuzawa et al 1064 deleted or mutated in 7–29% of WTs (Rivera et al., 2003; Fukuzawa et al., 2004a; Li et al., 2004; Fukuzawa 2007; Perotti et al., 2008; Ruteshouser et al., 2008). et al., 2007). Nuclear immunostaining of b-catenin was WTX appears to act as an unusual tumour suppressor detected predominantly in the undifferentiated mesench- gene, in which it is inactivated by one-hit mutational ymal cells and sometimes in the blastemal cells in 17 events: mutations in the single X chromosome in tumours (T2–4, 6–14, 16–20) (Table 1a). tumours from males and the active X chromosome in tumours from females cause inactivation of this gene (Rivera et al., 2007; Perotti et al., 2008; Ruteshouser Wilms tumours with mesenchymal differentiation have et al., 2008). Recently, WTX was found to form a active WNT signalling complex with AXIN, APC (adenomatous polyposis coli) Unsupervised hierarchical clustering analysis classified and b-catenin in the cytoplasm (Grohmann et al., 2007; WTs into two major groups, according to the presence Major et al., 2007). WTX was shown to antagonize or absence of ectopic mesenchymal elements (myogen- WNT/b-catenin signalling, as WTX interacted directly esis, chondrogenesis) (Figure 1a). The first group with b-catenin to promote its ubiquitination and (Cluster A) consisted of 15 tumours with ectopic degradation (Major et al., 2007). Although two studies mesenchymal elements, of which 9 contained ILNR. have found that mutations in WTX and CTNNB1 were Twelve tumours were analysable by IHC, of which 10 mutually exclusive in WTs (Rivera et al., 2007; Perotti tumours had CTNNB1 mutations and 7tumours had et al., 2008), one study showed that WTX mutations WT1 mutations (Figure 1a). Remarkably, all 12 partially overlapped with CTNNB1 mutations in WTs tumours were found to have nuclear accumulation of (Ruteshouser et al., 2008). However, the majority of the b-catenin (Figure 1a). Significantly, nuclear accumula- CTNNB1 mutations in tumours with WTX alterations tion of b-catenin and/or CTNNB1 mutations were were found in exon 7or 8, which unlike exon 3 exclusively observed in this cluster, while neither nuclear mutations, do not affect the b-catenin serine threonine accumulation of b-catenin nor CTNNB1 mutations was phosporylation-binding site and therefore may not lead found in cluster B that had no ectopic mesenchymal to constitutive b-catenin activation. Mutations in elements (Figure 1a). Therefore, cluster A was consid- CTNNB1 and WTX were therefore proposed to be not ered to be WNT-dependent. functionally redundant (Ruteshouser et al., 2008), To examine the relationship between the lineage supporting the notion that b-catenin and WTX act in specificity and activated WNT signalling, we compared a common pathway. Alternatively, WTX has been expression profiles between the two groups. Molecular independently reported as an APC-interacting protein features in cluster A characterized the ability to controlling the subcellular distribution of APC between differentiate into multiple mesenchymal lineages. Espe- the cell membrane and the microtubule of epithelial cells cially, a sequential myogenic differentiation signature (Grohmann et al., 2007), suggesting another tumour was prominent (Table 2a). Transcription factors (PAX3, suppressor function of WTX independent of WNT EYA2, MEOX2, LBX1) that are expressed in the signalling. paraxial mesoderm and contribute to the specification Given the importance of WTX in WNT signalling, we of myogenesis were present (Ridgeway and Skerjanc, investigated the relationship between tumour lineages in 2001). Myogenic regulatory transcription factors WT and WNT-signalling activation, including the (MYF5, MYOD1, MYF6, MYOG) and a cofactor involvement of WTX in the WNT-signalling pathway. (MEF2C), which are subsequently required for myogen- The predominance of b-catenin nuclear accumulation in esis, were also significantly upregulated. CDH15 is WT mesenchymal cells suggested that aberrant WNT- expressed in myoblasts and myotube-forming cells. signalling activation is a consequence of the tumour- Transcription factors (TBX3, MSX1, SOX8)anda initiating lineage, independent of an association with notch pathway ligand (JAG2) that determine limb WT1 mutations. We also examined whether WTX patterning originating from the mesoderm were upre- mutations are restricted to the WNT-dependent me- gulated. Other categories characterizing