RESUMEN
Based on characterization of both genomic and expression status of WT1 and CTNNB1 (beta-catenin) in a series of 60 Wilms tumor samples, combined with genome-wide expression profiling of these tumors, normal mature and fetal kidney controls, we show that WT1/beta-catenin expression was a better classifier than WT1/CTNNB1 mutations. We present molecular data supporting that the WNT pathway is involved in both tumor classes, with and without WT1/beta-catenin alterations. In the tumor class with WT1/beta-catenin alterations, we identified overexpression of 14 previously unreported WNT target genes, including TWIST1. We show that the TWIST1 protein was specifically expressed in these tumors, where staining was restricted to the stromal, nuclear beta-catenin positive, component. By comparing the state of the WNT pathway in tumors without WT1/beta-catenin alterations and fetal kidneys we provide evidence that suggests that these tumors have a heightened level of pathway activation. We characterized mutations of the WNT pathway regulator gene WTX in 16% of this tumor class. Moreover, genome-transcriptome correlation analysis allowed us to identify three other WNT pathway regulator genes that could participate in the activation of the WNT pathway: BCL9 (1p36.2), CTNNBIP1 (1p36.2), and CBY1 (22q13.1). These genes thus represent new potential important actors in WT tumorigenesis.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Proteínas WT1/metabolismo , Tumor de Wilms/metabolismo , beta Catenina/metabolismo , Análisis por Conglomerados , Análisis Mutacional de ADN , Femenino , Humanos , Inmunohistoquímica , Quinasas Janus/genética , Quinasas Janus/metabolismo , Masculino , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteómica , Reproducibilidad de los Resultados , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo , Proteínas WT1/genética , Tumor de Wilms/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMEN
The novel continuous cell line WT-Pe.1 was established in vitro from Wilms tumor with histological features of diffuse anaplasia. The cultures grew as poorly differentiated epithelial-like cells with pleomorphic polygonal shapes and formation of typical monolayers. WT-Pe.1 cells were immunoreactive for cytokeratin, vimentin, laminin, villin, CD10, and CD24 proteins. Conventional cytogenetic analysis by RHG-banding revealed a hypotriploid karyotype with numerous abnormalities including ring chromosomes, double-minutes, homogeneous staining regions, radial structures, dicentrics, and several marker chromosomes. Comparative genomic hybridization analysis revealed DNA copy numbers losses on chromosome segments 1p, 3p, 6q, 9q34.1 approximately q34.3, 11q24 approximately q25, 14q12 approximately qter, 16q, 18q, and 22q11 approximately q13; gain of genomic material was localized on chromosome arms 1q, 4p, 6q, and 7p and the entire chromosome 12. With DNA from the original tumor, copy number losses were detected on chromosomes 1p, 14q, 16q, 17q, and 22q and gains were observed on 1q, 4p, 8q, 12p, 12q, and chromosome 14p. Copy number amplifications of distinct loci were found on 1q21.1 and 4p15.3, as well as an elevated copy number of cyclin D2 (CCND2) and cyclin D associated kinase (CDK4) genes on chromosome 12 (confirmed by fluorescence in situ hybridization).
Asunto(s)
Aberraciones Cromosómicas , Tumor de Wilms/genética , Animales , Línea Celular , Preescolar , Femenino , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Ratones , Ratones Desnudos , Fenotipo , Tumor de Wilms/patologíaRESUMEN
The expression status of the three cyclin D genes (CCND1, CCND2 and CCND3), the two cyclin D-dependent kinase genes (CDK4 and CDK6) and the p16(INK4a) gene was studied in a series of 47 Wilms' tumors, 16 normal mature kidneys and two fetal kidneys. We showed predominant overexpression of CCND2 and CDK4 compared to CCND1/D3 and CDK6 respectively. We found a specific correlation between relapse and CDK4 overexpression, but not CDK6 overexpression. We did not identify any methylation of the p16(INK4a) promoter. This suggests that dysregulation of CCND2 and CDK4 plays a specific role in WT tumorigenesis.
Asunto(s)
Quinasas Ciclina-Dependientes/genética , Ciclinas/genética , Neoplasias Renales/genética , Proteínas Proto-Oncogénicas/genética , Tumor de Wilms/genética , Ciclina D1/genética , Ciclina D2 , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Metilación de ADN , Humanos , Riñón/embriología , Riñón/enzimología , Regiones Promotoras GenéticasRESUMEN
Wilms' tumour (WT) or nephroblastoma is the most frequent kidney cancer in children. In a previous study, we reported alterations to WT1 transcription in 90% of WT tested, with decreased exon 5 +/- isoform ratio being the most frequent alteration (56% of WT). We now report an approach based on cDNA profiling of tumour pools to identify genes likely to be dysregulated in association with a decreased WT1 exon 5 +/- ratio. We compared the expression profiles of pools of tumours classified according to whether this isoform imbalance was present (five tumours) or not (four tumours), using Atlas Cancer cDNA expression arrays. Fourteen of 588 genes tested displayed specific up-regulation (CCND2, PCNA, N-MYC, E2F3, TOP2A, PAK1, DCC and PCDH2) or down-regulation (VEGF, IGFBP5, TIMP3, ARHB, C-FOS and CD9) in the pool of tumours with decreased exon 5 +/- ratio. These results were validated by RT-PCR analysis of four genes (CCND2, PCNA, VEGF and IGFBP5). We extended the analysis of VEGF expression to 51 tumours by real-time RT-PCR and ascertained differential expression of this gene associated with WT1 expression pattern. Moreover, our results suggest that the VEGF expression level may be of prognosis relevance for relapsed patients.