Oncogene (2002) 21, 5566 – 5573 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc Changes in WT1 splicing are associated with a specific gene expression profile in Wilms’ tumour Dominique Baudry1, Marine Faussillon1, Marie-Odile Cabanis1, Muriel Rigolet1,6, Jean-Michel Zucker2, Catherine Patte3, Sabine Sarnacki4, Liliane Boccon-Gibod5, Claudine Junien1 and Ce´ cile Jeanpierre*,1 1INSERM U383, Hoˆpital Necker-Enfants Malades, Universite´ Rene´ Descartes, 75743 Paris Cedex 15, France; 2Institut Curie, 75248 Paris, France; 3Institut Gustave Roussy, 94805 Villejuif, France; 4Clinique Chirurgicale infantile, Hoˆpital Necker-Enfants Malades, 75743 Paris Cedex 15, France; 5Service d’Anatomie Pathologique, Hoˆpital Armand Trousseau, 75012 Paris, France Wilms’ tumour (WT) or nephroblastoma is the most accounting for 7% of cases. A family history of WT is frequent kidney cancer in children. In a previous study, observed in 2% of cases. This tumour may also arise in we reported alterations to WT1 transcription in 90% of association with rare malformation syndromes: WAGR WT tested, with decreased exon 5 +/7 isoform ratio syndrome (WT, aniridia, genitourinary malformations being the most frequent alteration (56% of WT). We and mental retardation), Denys – Drash syndrome now report an approach based on cDNA profiling of (DDS) and Beckwith – Wiedemann syndrome (BWS). tumour pools to identify genes likely to be dysregulated The WT1 gene, located at 11p13, encodes a zinc in association with a decreased WT1 exon 5 +/7 ratio. finger transcription factor involved in renal and gonad We compared the expression profiles of pools of tumours development (Hastie, 2001; Mrowka and Schedl, 2000). classified according to whether this isoform imbalance Two alternative splicing sites give rise to four major was present (five tumours) or not (four tumours), using isoforms. The alternative splicing of exon 5 results in Atlas Cancer cDNA expression arrays. Fourteen of 588 the inclusion or omission of 17 amino acids in the genes tested displayed specific up-regulation (CCND2, transcriptional regulation domain of the protein. The PCNA, N-MYC, E2F3, TOP2A, PAK1, DCC and second alternatively spliced region corresponds to three PCDH2) or down-regulation (VEGF, IGFBP5, TIMP3, amino acids (KTS) located between the third and ARHB, C-FOS and CD9) in the pool of tumours with fourth zinc fingers of the DNA-binding domain. The decreased exon 5 +/7 ratio. These results were four isoforms are produced in a constant ratio that is validated by RT – PCR analysis of four genes (CCND2, conserved between species, suggesting that they have PCNA, VEGF and IGFBP5). We extended the analysis non-overlapping functions. WT1 (-KTS) proteins of VEGF expression to 51 tumours by real-time RT – regulate in vitro the transcription of genes essential PCR and ascertained differential expression of this gene for kidney development, such as IGF2, PDGFA, associated with WT1 expression pattern. Moreover, our EGFR, PAX-2, and WT1 itself (reviewed in Menke results suggest that the VEGF expression level may be of et al., 1998). Other potential targets, such as amphire- prognosis relevance for relapsed patients. gulin and RbAP46, have been identified by expression Oncogene (2002) 21, 5566 – 5573. doi:10.1038/sj.onc. profiling (Guan et al., 1998; Lee et al., 1999). It is now 1205752 clearly established in vivo that the function of WT1 as a transcription regulator is modulated by post-transcrip- Keywords: Wilms’ tumour; expression profiling; WT1 tional modifications and by interaction with other splicing; prognosis proteins including SF1, p53, PAR4 and CBP (Nachti- gal et al., 1998; Maheswaran et al., 1993; Richard et al., 2001; Wang et al., 2001). Exon 5 encodes an Introduction additional regulator domain, with potential repressor and activator functions (Reddy et al., 1995; Wang et Wilms’ tumour (WT) or nephroblastoma is the most al., 1995). WT1 (+KTS) proteins have a low affinity frequent kidney cancer in children. It affects 1 in 10 000 for DNA and may be involved in RNA splicing children, usually around the age of 5 years. Most cases (Larsson et al., 1995; Davies et al., 1998). The presence are sporadic and unilateral, with bilateral tumours of both the WT1 (+KTS) and WT1 (-KTS) isoforms is essential for normal urogenital development (Barbaux et al., 1997; Hammes et al., 2001). Furthermore, the balance between WT1 (+exon 5) and WT1 (-exon 5) *Correspondence: C Jeanpierre; E-mail: [email protected] appears to be essential for the regulation of major 6Current address: UMR 7592 CNRS, Institut Jacques Monod, 75251 Paris Cedex 05, France cellular functions such as proliferation, differentiation Received 2 January 2002; revised 28 May 2002; accepted 7 June and apoptosis (Englert et al., 1997; Hewitt and 2002 Saunders, 1996; Kudoh et al., 1995; Mayo et al., 1999). Expression profiling of Wilms’ tumours D Baudry et al 5567 In Wilms’ tumours in patients with WAGR and in WT(UNB1), WT9 and WT53; (2) similar in Denys – Drash syndromes, a first germline event at the WT(NK-like) and NK; (3) different in WT(UNB1) WT1 locus (deletion or mutation) accounts for and NK; (4) different in WT(UNB1) and FK. Fourteen predisposition and is usually followed by a somatic genes met these criteria, among which six were down- mutation or loss of the normal allele (Jeanpierre et al., regulated and eight were up-regulated in WT(UNB1) 1998). In sporadic tumours, somatic mutations in WT1 (Figure 1). are rare. However, we recently described changes in WT1 transcription in 90% of cases (Baudry et al., Validation of cDNA pooling for identification of 2000). A disruption of exon 5 splicing, with a relative differentially expressed genes decrease in the amount of the WT1 (+exon 5) isoforms, was the most frequent alteration, observed We analysed two overexpressed genes (CCND2 and in 56% of the tumours. It was thought probable that PCNA) and two underexpressed genes (IGFBP5 and these changes would affect the regulation of genes VEGF), by semi-quantitative RT – PCR using pooled downstream in the WT1 pathway. Moreover, little is cDNAs and individual cDNAs (WT9, WT77, WT84, currently known about the genes and mechanisms that WT97, WT190, WT52, WT72, WT75, WT133, NK183, control WT1 expression and alternative splicing. NK186, NK188 and NK189) at the same time (Figure We recently reported preliminary data on expression 2a). Although different normalization parameters were profiling in Wilms’ tumours using cDNA macroarrays, used for the quantification of array hybridizations resulting in the identification of 153 genes differentially (global normalisation) and RT – PCR assays (HPRT as expressed in tumours and normal kidneys (Rigolet et a control gene), differences in expression pattern al., 2001). We now describe a more specific cDNA between pool WT(UNB1) and pool WT(NK-like) were macroarray approach, intended to identify genes likely consistent for all four genes tested. Moreover, the to be dysregulated in tumours with a decreased WT1 levels of expression of the four genes in the individual exon 5 +/7 isoform ratio. This approach was based tumours were consistent with levels of expression in the on cDNA profiling of pools of tumours classified corresponding pools (Figure 2b). according to whether isoform imbalance was present or not. First, we identified 14 genes that displayed specific VEGF expression in a series of 51 tumours dysregulation in the pool of tumours with imbalance. Second, we validated the pooling strategy for tumour We extended the analysis of VEGF expression to a profiling, by RT – PCR analysis of four genes. Third, series of 51 tumours, 13 normal kidneys and 2 foetal we extended the analysis of one of the down-regulated kidneys, using real-time RT – PCR. Normal and foetal genes, VEGF, to 51 tumours by real-time RT – PCR, kidney VEGF expression levels were evaluated as and showed that the level of expression of this gene 0.99+0.49 and 0.82+0.05 respectively, according to correlated with the pattern of WT1 expression. Fourth, R ratios (see Materials and methods). Thirty-seven we analysed the prognosis significance of VEGF tumours underexpressed VEGF (R50.5) and six expression level. tumours overexpressed VEGF (R41.5) (Table 1). Decreased expression in tumours was confirmed by comparing the level of expression in seven tumours and Results paired normal kidneys (Figure 3). The WT1 expression of the tumours was assessed in a previous study RNA profiling of pools of tumours with decreased and (Baudry et al., 2000). We compared the expression of normal WT1 exon 5 +/7 isoform ratios VEGF and WT1 and found that 29 out of 34 tumours We probed Atlas Human Cancer cDNA expression (85%) with a decreased WT1 exon 5 +/7 ratio or no arrays (Clontech), containing 588 genes involved in WT1 expression (no transcript or truncated protein) various aspects of malignancy and belonging to underexpressed VEGF. There was no difference in different functional classes, with cDNAs from: VEGF expression between tumours with an abnormal (1) tumours with a decreased exon 5 +/7 ratio and KTS +/7 ratio and tumours with a normal KTS a normal KTS +/7 ratio, either as a pool +/7 ratio. Furthermore, all three tumours with an (WT(UNB1)) or individually (WT9, WT53); (2) a pool increased WT1 exon 5 +/7 ratio (UNB2) expressed of tumours with exon 5 +/7 and KTS +/7 ratios VEGF at a higher level. There was no evidence of similar to those in normal kidneys (WT(NK-like)); (3) a correlation between the level of VEGF expression a pool of normal kidneys (NK) and (4) a pool of foetal and treatment or not by preoperative chemotherapy kidneys (FK). Comparison of the expression profiles of (Table 1). WT(UNB1) and WT(NK-like) pools of tumours Finally, we investigated the correlation between revealed 81 differentially expressed genes. We then VEGF expression in tumours and prognosis.