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1.
Oncogene ; 39(4): 849-861, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31562394

RESUMO

In vitro models represent a critical tool in cancer research to study tumor biology and to evaluate new treatment options. Unfortunately, there are no effective preclinical models available that represent Wilms tumor (WT) - the most common pediatric renal tumor. Especially the high-risk blastemal WT subtype is not represented by the few primary cell lines established until now. Here, we describe a new 3D approach for in vitro cultivation of blastemal WT cells, where primary cultures grown in suspension as spheroids could be propagated long-term. Besides blastemal cultures, we could generate spheroids representing epithelial and stromal WT. Spheroid cultures were analyzed by immunohistochemistry in comparison to corresponding tumor sections and were further characterized by RNA sequencing. Histological appearance of spheroids resembled the original tumor and they expressed marker genes characteristic of early renal development and blastemal WT elements. The cultures were amenable to genetic manipulation and they formed xenograft tumors, which resemble the primary human tumor. This collection of WT spheroids that carry different genetic drivers forms a long-sought tool for drug testing and in vitro modeling.

2.
Int J Cancer ; 145(4): 941-951, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-30694527

RESUMO

Two percent of patients with Wilms tumors have a positive family history. In many of these cases the genetic cause remains unresolved. By applying germline exome sequencing in two families with two affected individuals with Wilms tumors, we identified truncating mutations in TRIM28. Subsequent mutational screening of germline and tumor DNA of 269 children affected by Wilms tumor was performed, and revealed seven additional individuals with germline truncating mutations, and one individual with a somatic truncating mutation in TRIM28. TRIM28 encodes a complex scaffold protein involved in many different processes, including gene silencing, DNA repair and maintenance of genomic integrity. Expression studies on mRNA and protein level showed reduction of TRIM28, confirming a loss-of-function effect of the mutations identified. The tumors showed an epithelial-type histology that stained negative for TRIM28 by immunohistochemistry. The tumors were bilateral in six patients, and 10/11 tumors are accompanied by perilobar nephrogenic rests. Exome sequencing on eight tumor DNA samples from six individuals showed loss-of-heterozygosity (LOH) of the TRIM28-locus by mitotic recombination in seven tumors, suggesting that TRIM28 functions as a tumor suppressor gene in Wilms tumor development. Additionally, the tumors showed very few mutations in known Wilms tumor driver genes, suggesting that loss of TRIM28 is the main driver of tumorigenesis. In conclusion, we identified heterozygous germline truncating mutations in TRIM28 in 11 children with mainly epithelial-type Wilms tumors, which become homozygous in tumor tissue. These data establish TRIM28 as a novel Wilms tumor predisposition gene, acting as a tumor suppressor gene by LOH.


Assuntos
Haploinsuficiência/genética , Proteína 28 com Motivo Tripartido/genética , Tumor de Wilms/genética , Carcinogênese/genética , Pré-Escolar , DNA de Neoplasias/genética , Feminino , Genes do Tumor de Wilms/fisiologia , Predisposição Genética para Doença/genética , Genótipo , Mutação em Linhagem Germinativa/genética , Heterozigoto , Humanos , Lactente , Neoplasias Renais/genética , Mutação com Perda de Função/genética , Perda de Heterozigosidade/genética , Masculino , Sequenciamento Completo do Exoma/métodos
3.
Int J Cancer ; 144(6): 1432-1443, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30155889

RESUMO

Wilms tumors are the most common type of pediatric kidney tumors. While the overall prognosis for patients is favorable, especially tumors that exhibit a blastemal subtype after preoperative chemotherapy have a poor prognosis. For an improved risk assessment and therapy stratification, it is essential to identify the driving factors that are distinctive for this aggressive subtype. In our study, we compared gene expression profiles of 33 tumor biopsies (17 blastemal and 16 other tumors) after neoadjuvant chemotherapy. The analysis of this dataset using the Regulator Gene Association Enrichment algorithm successfully identified several biomarkers and associated molecular mechanisms that distinguish between blastemal and nonblastemal Wilms tumors. Specifically, regulators involved in embryonic development and epigenetic processes like chromatin remodeling and histone modification play an essential role in blastemal tumors. In this context, we especially identified TCF3 as the central regulatory element. Furthermore, the comparison of ChIP-Seq data of Wilms tumor cell cultures from a blastemal mouse xenograft and a stromal tumor provided further evidence that the chromatin states of blastemal cells share characteristics with embryonic stem cells that are not present in the stromal tumor cell line. These stem-cell like characteristics could potentially add to the increased malignancy and chemoresistance of the blastemal subtype. Along with TCF3, we detected several additional biomarkers that are distinctive for blastemal Wilms tumors after neoadjuvant chemotherapy and that may provide leads for new therapeutic regimens.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/patologia , Células-Tronco Neoplásicas/patologia , Tumor de Wilms/patologia , Adolescente , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Biópsia , Criança , Pré-Escolar , Conjuntos de Dados como Assunto , Feminino , Perfilação da Expressão Gênica , Humanos , Lactente , Rim/citologia , Rim/patologia , Rim/cirurgia , Neoplasias Renais/genética , Neoplasias Renais/terapia , Masculino , Camundongos , Terapia Neoadjuvante/métodos , Nefrectomia , Cultura Primária de Células , Células Tumorais Cultivadas , Tumor de Wilms/genética , Tumor de Wilms/terapia
4.
Nat Commun ; 9(1): 2378, 2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29915264

RESUMO

Soft tissue tumors of infancy encompass an overlapping spectrum of diseases that pose unique diagnostic and clinical challenges. We studied genomes and transcriptomes of cryptogenic congenital mesoblastic nephroma (CMN), and extended our findings to five anatomically or histologically related soft tissue tumors: infantile fibrosarcoma (IFS), nephroblastomatosis, Wilms tumor, malignant rhabdoid tumor, and clear cell sarcoma of the kidney. A key finding is recurrent mutation of EGFR in CMN by internal tandem duplication of the kinase domain, thus delineating CMN from other childhood renal tumors. Furthermore, we identify BRAF intragenic rearrangements in CMN and IFS. Collectively these findings reveal novel diagnostic markers and therapeutic strategies and highlight a prominent role of isolated intragenic rearrangements as drivers of infant tumors.


Assuntos
Fibrossarcoma/genética , Genes erbB-1 , Neoplasias Renais/genética , Nefroma Mesoblástico/genética , Proteínas Proto-Oncogênicas B-raf/genética , Feminino , Rearranjo Gênico , Humanos , Lactente , Recém-Nascido , Masculino
6.
Bioinformatics ; 34(20): 3503-3510, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-29741575

RESUMO

Motivation: Transcriptional regulators play a major role in most biological processes. Alterations in their activities are associated with a variety of diseases and in particular with tumor development and progression. Hence, it is important to assess the effects of deregulated regulators on pathological processes. Results: Here, we present REGulator-Gene Association Enrichment (REGGAE), a novel method for the identification of key transcriptional regulators that have a significant effect on the expression of a given set of genes, e.g. genes that are differentially expressed between two sample groups. REGGAE uses a Kolmogorov-Smirnov-like test statistic that implicitly combines associations between regulators and their target genes with an enrichment approach to prioritize the influence of transcriptional regulators. We evaluated our method in two different application scenarios, which demonstrate that REGGAE is well suited for uncovering the influence of transcriptional regulators and is a valuable tool for the elucidation of complex regulatory mechanisms. Availability and implementation: REGGAE is freely available at https://regulatortrail.bioinf.uni-sb.de. Supplementary information: Supplementary data are available at Bioinformatics online.


Assuntos
Regulação da Expressão Gênica , Neoplasias/genética , Transcrição Genética , Feminino , Humanos , Probabilidade , Software
7.
Nature ; 555(7696): 321-327, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29489754

RESUMO

Pan-cancer analyses that examine commonalities and differences among various cancer types have emerged as a powerful way to obtain novel insights into cancer biology. Here we present a comprehensive analysis of genetic alterations in a pan-cancer cohort including 961 tumours from children, adolescents, and young adults, comprising 24 distinct molecular types of cancer. Using a standardized workflow, we identified marked differences in terms of mutation frequency and significantly mutated genes in comparison to previously analysed adult cancers. Genetic alterations in 149 putative cancer driver genes separate the tumours into two classes: small mutation and structural/copy-number variant (correlating with germline variants). Structural variants, hyperdiploidy, and chromothripsis are linked to TP53 mutation status and mutational signatures. Our data suggest that 7-8% of the children in this cohort carry an unambiguous predisposing germline variant and that nearly 50% of paediatric neoplasms harbour a potentially druggable event, which is highly relevant for the design of future clinical trials.


Assuntos
Genoma Humano/genética , Genômica , Mutação/genética , Neoplasias/classificação , Neoplasias/genética , Adolescente , Adulto , Criança , Cromotripsia , Estudos de Coortes , Variações do Número de Cópias de DNA/genética , Diploide , Predisposição Genética para Doença/genética , Mutação em Linhagem Germinativa/genética , Humanos , Terapia de Alvo Molecular , Taxa de Mutação , Neoplasias/tratamento farmacológico , Proteína Supressora de Tumor p53/genética , Adulto Jovem
8.
J Pathol Clin Res ; 3(4): 234-248, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29085664

RESUMO

TP53 mutations have been associated with anaplasia in Wilms tumour, which conveys a high risk for relapse and fatal outcome. Nevertheless, TP53 alterations have been reported in no more than 60% of anaplastic tumours, and recent data have suggested their presence in tumours that do not fulfil the criteria for anaplasia, questioning the clinical utility of TP53 analysis. Therefore, we characterized the TP53 status in 84 fatal cases of Wilms tumour, irrespective of histological subtype. We identified TP53 alterations in at least 90% of fatal cases of anaplastic Wilms tumour, and even more when diffuse anaplasia was present, indicating a very strong if not absolute coupling between anaplasia and deregulation of p53 function. Unfortunately, TP53 mutations do not provide additional predictive value in anaplastic tumours since the same mutation rate was found in a cohort of non-fatal anaplastic tumours. When classified according to tumour stage, patients with stage I diffuse anaplastic tumours still had a high chance of survival (87%), but this rate dropped to 26% for stages II-IV. Thus, volume of anaplasia or possible spread may turn out to be critical parameters. Importantly, among non-anaplastic fatal tumours, 26% had TP53 alterations, indicating that TP53 screening may identify additional cases at risk. Several of these non-anaplastic tumours fulfilled some criteria for anaplasia, for example nuclear unrest, suggesting that such partial phenotypes should be under special scrutiny to enhance detection of high-risk tumours via TP53 screening. A major drawback is that these alterations are secondary changes that occur only later in tumour development, leading to striking intratumour heterogeneity that requires multiple biopsies and analysis guided by histological criteria. In conclusion, we found a very close correlation between histological signs of anaplasia and TP53 alterations. The latter may precede development of anaplasia and thereby provide diagnostic value pointing towards aggressive disease.

9.
J Clin Oncol ; 34(26): 3195-203, 2016 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-27432915

RESUMO

PURPOSE: Wilms tumor (WT) is the most common pediatric renal tumor. Treatment planning under International Society of Paediatric Oncology (SIOP) protocols is based on staging and histologic assessment of response to preoperative chemotherapy. Despite high overall survival (OS), many relapses occur in patients without specific risk factors, and many successfully treated patients are exposed to treatments with significant risks of late effects. To investigate whether molecular biomarkers could improve risk stratification, we assessed 1q status and other potential copy number biomarkers in a large WT series. MATERIALS AND METHODS: WT nephrectomy samples from 586 SIOP WT 2001 patients were analyzed using a multiplex ligation-dependent probe amplification (MLPA) assay that measured the copy number of 1q and other regions of interest. RESULTS: One hundred sixty-seven (28%) of 586 WTs had 1q gain. Five-year event-free survival (EFS) was 75.0% in patients with 1q gain (95% CI, 68.5% to 82.0%) and 88.2% in patients without gain (95% CI, 85.0% to 91.4%). OS was 88.4% with gain (95% CI, 83.5% to 93.6%) and 94.4% without gain (95% CI, 92.1% to 96.7%). In univariable analysis, 1q gain was associated with poorer EFS (P < .001; hazard ratio, 2.33) and OS (P = .01; hazard ratio, 2.16). The association of 1q gain with poorer EFS retained significance in multivariable analysis adjusted for 1p and 16q loss, sex, stage, age, and histologic risk group. Gain of 1q remained associated with poorer EFS in tumor subsets limited to either intermediate-risk localized disease or nonanaplastic localized disease. Other notable aberrations associated with poorer EFS included MYCN gain and TP53 loss. CONCLUSION: Gain of 1q is a potentially valuable prognostic biomarker in WT, in addition to histologic response to preoperative chemotherapy and tumor stage.


Assuntos
Biomarcadores Tumorais/genética , Aberrações Cromossômicas , Cromossomos Humanos Par 1 , Variações do Número de Cópias de DNA , Terapia Neoadjuvante , Nefrectomia , Tumor de Wilms/terapia , Quimioterapia Adjuvante , Intervalo Livre de Doença , Feminino , Predisposição Genética para Doença , Humanos , Estimativa de Kaplan-Meier , Modelos Logísticos , Masculino , Análise Multivariada , Terapia Neoadjuvante/efeitos adversos , Terapia Neoadjuvante/mortalidade , Estadiamento de Neoplasias , Nefrectomia/efeitos adversos , Nefrectomia/mortalidade , Fenótipo , Modelos de Riscos Proporcionais , Estudos Prospectivos , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Tumor de Wilms/genética , Tumor de Wilms/mortalidade , Tumor de Wilms/patologia
10.
Oncotarget ; 6(9): 7232-43, 2015 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-25749049

RESUMO

Genomic gain of the proto-oncogene transcription factor gene MYCN is associated with poor prognosis in several childhood cancers. Here we present a comprehensive copy number analysis of MYCN in Wilms tumour (WT), demonstrating that gain of this gene is associated with anaplasia and with poorer relapse-free and overall survival, independent of histology. Using whole exome and gene-specific sequencing, together with methylation and expression profiling, we show that MYCN is targeted by other mechanisms, including a recurrent somatic mutation, P44L, and specific DNA hypomethylation events associated with MYCN overexpression in tumours with high risk histologies. We describe parallel evolution of genomic copy number gain and point mutation of MYCN in the contralateral tumours of a remarkable bilateral case in which independent contralateral mutations of TP53 also evolve over time. We report a second bilateral case in which MYCN gain is a germline aberration. Our results suggest a significant role for MYCN dysregulation in the molecular biology of Wilms tumour. We conclude that MYCN gain is prognostically significant, and suggest that the novel P44L somatic variant is likely to be an activating mutation.


Assuntos
Regulação Neoplásica da Expressão Gênica , Genes myc , Neoplasias Renais/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Tumor de Wilms/metabolismo , Metilação de DNA , Análise Mutacional de DNA , Intervalo Livre de Doença , Exoma , Dosagem de Genes , Perfilação da Expressão Gênica , Genes p53 , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/genética , Mutação , Recidiva Local de Neoplasia/genética , Análise de Sequência com Séries de Oligonucleotídeos , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Prognóstico , Proteína Supressora de Tumor p53/metabolismo , Tumor de Wilms/genética
11.
Cancer Cell ; 27(2): 298-311, 2015 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-25670083

RESUMO

Blastemal histology in chemotherapy-treated pediatric Wilms tumors (nephroblastoma) is associated with adverse prognosis. To uncover the underlying tumor biology and find therapeutic leads for this subgroup, we analyzed 58 blastemal type Wilms tumors by exome and transcriptome sequencing and validated our findings in a large replication cohort. Recurrent mutations included a hotspot mutation (Q177R) in the homeo-domain of SIX1 and SIX2 in tumors with high proliferative potential (18.1% of blastemal cases); mutations in the DROSHA/DGCR8 microprocessor genes (18.2% of blastemal cases); mutations in DICER1 and DIS3L2; and alterations in IGF2, MYCN, and TP53, the latter being strongly associated with dismal outcome. DROSHA and DGCR8 mutations strongly altered miRNA expression patterns in tumors, which was functionally validated in cell lines expressing mutant DROSHA.


Assuntos
Proteínas de Homeodomínio/genética , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Tumor de Wilms/genética , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/biossíntese , Mutação , Proteínas de Neoplasias/biossíntese , Transcriptoma , Tumor de Wilms/patologia
12.
Int J Cancer ; 131(3): 673-82, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-21913182

RESUMO

Wilms Tumor (WT) is the most common renal childhood tumor. Recently, we reported a cDNA microarray expression pattern that varied between WTs with different risk histology. Since the Societé Internationale d'Oncologie Pédiatrique (SIOP) in Europe initiates treatment without a histological confirmation, it is important to identify blood-born markers that indicate WT development. In a multicenter study, we established an autoantibody signature by using an array with 1,827 recombinant E. coli clones. This array was screened with sera of patients with WT recruited by SIOP or the Children's Oncology Group (COG). We report an extended number of antigens that are reactive with autoantibodies present in sera from patients with WT. We established an autoantibody signature that separates untreated patients with WT recruited in SIOP from non-WT controls with a specificity of 0.83 and a sensitivity of 0.82 at standard deviations of 0.02 and 0.04, respectively. Likewise, patients recruited in the COG in the United States were separated from the controls with an accuracy of 0.83 at a standard deviation of 0.02. Proteins that were most significant include zinc finger proteins (e.g., ZFP 346), ribosomal proteins and the protein fascin that has been associated with various types of cancer including renal cell carcinoma. Our study provides first evidence for autoantibody signatures for WTs and suggests that these may be most informative before chemotherapy. We present the first multicenter study of autoantibody signatures in patients with WT. We established an autoantibody signature that separates patients with WT from controls.


Assuntos
Anticorpos Antineoplásicos/sangue , Autoanticorpos/sangue , Proteínas de Transporte/sangue , Proteínas de Ligação a DNA/sangue , Neoplasias Renais/diagnóstico , Proteínas dos Microfilamentos/sangue , Proteínas de Ligação a RNA/sangue , Proteínas Ribossômicas/sangue , Tumor de Wilms/diagnóstico , Tumor de Wilms/imunologia , Adolescente , Anticorpos Antineoplásicos/imunologia , Autoanticorpos/imunologia , Biomarcadores Tumorais/sangue , Proteínas de Transporte/imunologia , Criança , Pré-Escolar , Proteínas de Ligação a DNA/imunologia , Feminino , Humanos , Neoplasias Renais/sangue , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/imunologia , Masculino , Proteínas dos Microfilamentos/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Análise Serial de Proteínas , Proteínas de Ligação a RNA/imunologia , Proteínas Ribossômicas/imunologia , Sensibilidade e Especificidade , Resultado do Tratamento , Tumor de Wilms/sangue , Tumor de Wilms/tratamento farmacológico
13.
Genes Chromosomes Cancer ; 51(1): 92-104, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22034155

RESUMO

Functional analysis of gene candidates and testing of novel therapeutics in Wilms tumors (WT) has been hampered by the lack of in vitro model systems. WT are characterized by a spectrum of histological appearances, but published cell lines are mostly derived from rare anaplastic variants or even non-WT. There has been some success in establishing primary cultures, but these are often poorly characterized or only derived from less frequent WT1 mutant tumors. We report the generation of a set of primary WT-cell cultures using a simple cultivation protocol. Our cultures could be established after preoperative chemotherapy and irrespective of histological subtypes or genetic alterations. The presence of tumor-specific genetic alterations validates these cultures as being tumor-derived. Genetic characterization is of utmost importance as some cultures with similar morphological appearance lacked such alterations and either represent clonal variants or normal cells. By immunohistochemistry, the cells are either epithelial or more mesenchymal, and the latter exhibiting a longer life span with 30 or more passages before undergoing senescence. This may be related to WT being embryonal tumors with a strong differentiation potential that may prevail in vitro. Telomeres progressively shorten with cultivation, but their length does not predict lifespan. hTERT transfection may partly allow establishment of immortalized lines, because 2/7 cultures avoid senescence even in later passages. Importantly, these cells can be efficiently manipulated by transfection, making them a useful model system for in vitro testing.


Assuntos
Cultura Primária de Células/métodos , Tumor de Wilms/genética , Tumor de Wilms/metabolismo , Proliferação de Células , Senescência Celular , Criança , Pré-Escolar , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Lactente , Masculino , Mutação , Telomerase/genética , Telomerase/metabolismo , Transfecção , Células Tumorais Cultivadas , Tumor de Wilms/patologia
14.
Mol Cancer ; 10: 136, 2011 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-22067876

RESUMO

BACKGROUND: Wilms tumor (WT) is one of the most common malignancies in childhood. With current therapy protocols up to 90% of patients can be cured, but there is still a need to improve therapy for patients with aggressive WT and to reduce treatment intensity where possible. Prior data suggested a deregulation of the retinoic acid (RA) signaling pathway in high-risk WT, but its mode of action remained unclear. RESULTS: The association of retinoid signaling and clinical parameters could be validated in a large independent tumor set, but its relevance in primary nephrectomy tumors from very young children may be different. Reduced RA pathway activity and MYCN overexpression were found in high risk tumors as opposed to tumors with low/intermediate risk, suggesting a beneficial impact of RA especially on advanced WT. To search for possible modes of action of retinoids as novel therapeutic options, primary tumor cell cultures were treated in vitro with all-trans-RA (ATRA), 9cis-RA, fenretinide and combinations of retinoids and a histone deacetylase (HDAC) inhibitor. Genes deregulated in high risk tumors showed opposite changes upon treatment suggesting a positive effect of retinoids. 6/7 primary cultures tested reduced proliferation, irrespective of prior RA signaling levels. The only variant culture was derived from mesoblastic nephroma, a distinct childhood kidney neoplasm. Retinoid/HDAC inhibitor combinations provided no synergistic effect. ATRA and 9cis-RA induced morphological changes suggestive of differentiation, while fenretinide induced apoptosis in several cultures tested. Microarray analysis of ATRA treated WT cells revealed differential expression of many genes involved in extracellular matrix formation and osteogenic, neuronal or muscle differentiation. The effects documented appear to be reversible upon drug withdrawal, however. CONCLUSIONS: Altered retinoic acid signaling has been validated especially in high risk Wilms tumors. In vitro testing of primary tumor cultures provided clear evidence of a potential utility of retinoids in Wilms tumor treatment based on the analysis of gene expression, proliferation, differentiation and apoptosis.


Assuntos
Tretinoína/fisiologia , Tumor de Wilms/genética , Antineoplásicos/farmacologia , Apoptose , Diferenciação Celular , Expressão Gênica , Humanos , Proteína Proto-Oncogênica N-Myc , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Tretinoína/farmacologia , Células Tumorais Cultivadas , Tumor de Wilms/tratamento farmacológico , Tumor de Wilms/patologia
15.
Clin Cancer Res ; 16(7): 2036-45, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-20332316

RESUMO

PURPOSE: Wilms' tumor (WT), the most common pediatric renal malignancy, is associated with mutations in several well-characterized genes, most notably WT1, CTNNB1, WTX, and TP53. However, the majority of cases do not harbor mutations in these genes. We hypothesized that additional drivers of tumor behavior would be contained within areas of consistent genomic copy number change, especially those associated with the WT risk groups defined by the International Society of Paediatric Oncology (SIOP). EXPERIMENTAL DESIGN: We analyzed high-resolution (Affymetrix 250K single nucleotide polymorphism array) genomic copy number profiles of over 100 tumors from selected risk groups treated under the SIOP protocols, further characterizing genes of interest by sequencing, Multiplex Ligation-dependent Probe Amplification, or fluorescence in situ hybridization. RESULTS: We identified FBXW7, an E3 ubiquitin ligase component, as a novel Wilms' tumor gene, mutated or deleted in approximately 4% of tumors examined. Strikingly, 3 of 14 (21%) of tumors with epithelial type histology after neoadjuvant chemotherapy had FBXW7 aberrations, whereas a fourth WT patient had germline mutations in both FBXW7 and WT1. We also showed that MYCN copy number gain, detected in 9 of 104 (8.7%) of cases, is relatively common in WT and significantly more so in tumors of the high risk diffuse anaplastic subtype (6 of 19, 32%). CONCLUSIONS: Because MYCN is itself a target of FBXW7-mediated ubiquitination and degradation, these results suggest that a common pathway is dysregulated by different mechanisms in various WT subtypes. Emerging therapies that target MYCN, which is amplified in several other pediatric cancers, may therefore be of value in high risk Wilms' tumor.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas F-Box/genética , Dosagem de Genes , Neoplasias Renais/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Ubiquitina-Proteína Ligases/genética , Tumor de Wilms/genética , Criança , Cromossomos Humanos Par 2 , Proteína 7 com Repetições F-Box-WD , Feminino , Amplificação de Genes , Deleção de Genes , Dosagem de Genes/fisiologia , Genes do Tumor de Wilms , Humanos , Neoplasias Renais/classificação , Neoplasias Renais/patologia , Mães , Mutação , Proteína Proto-Oncogênica N-Myc , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Tumor de Wilms/classificação , Tumor de Wilms/patologia
16.
Clin Cancer Res ; 15(19): 5985-92, 2009 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-19789318

RESUMO

PURPOSE: Wilms' tumor is a childhood cancer of the kidney with an incidence of approximately 1 in 10,000. Cooccurrence of Wilms' tumor with 2q37 deletion syndrome, an uncommon constitutional chromosome abnormality, has been reported previously in three children. Given these are independently rare clinical entities, we hypothesized that 2q37 harbors a tumor suppressor gene important in Wilms' tumor pathogenesis. EXPERIMENTAL DESIGN: To test this, we performed loss of heterozygosity analysis in a panel of 226 sporadic Wilms' tumor samples and mutation analysis of candidate genes. RESULTS: Loss of heterozygosity was present in at least 4% of cases. Two tumors harbored homozygous deletions at 2q37.1, supporting the presence of a tumor suppressor gene that follows a classic two-hit model. However, no other evidence of second mutations was found, suggesting that heterozygous deletion alone may be sufficient to promote tumorigenesis in concert with other genomic abnormalities. We show that miR-562, a microRNA within the candidate region, is expressed only in kidney and colon and regulates EYA1, a critical gene for renal development. miR-562 expression is reduced in Wilms' tumor and may contribute to tumorigenesis by deregulating EYA1. Two other candidate regions were localized at 2q37.3 and 2qter, but available data from patients with constitutional deletions suggest that these probably do not confer a high risk for Wilms' tumor. CONCLUSIONS: Our data support the presence of a tumor suppressor gene at 2q37.1 and suggest that, in individuals with constitutional 2q37 deletions, any increased risk for developing Wilms' tumor likely correlates with deletions encompassing 2q37.1.


Assuntos
Cromossomos Humanos Par 2 , Neoplasias Renais/genética , Perda de Heterozigosidade , MicroRNAs/fisiologia , Tumor de Wilms/genética , Células Cultivadas , Cromossomos Humanos Par 2/genética , Análise Mutacional de DNA , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Perda de Heterozigosidade/genética , MicroRNAs/genética , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo Genético , Proteínas Tirosina Fosfatases/genética
17.
Genes Chromosomes Cancer ; 48(12): 1102-11, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19760609

RESUMO

Wilms tumor (WT) is one of the most common solid tumors in childhood. Mutations in WT1 and CTNNB1 are well established as causal alterations in about 10-15% of cases. Recently, WTX (WT gene on the X-chromosome), a gene implicated in WNT signaling, has been identified as a third WT gene. We determined the mutation status of WTX, CTNNB1, and WT1 in a large set of 429 tumors. Genomic WTX alterations were identified in 17% of WTs, equally distributed between males and females. Analysis of 104 WT samples for WTX point mutations revealed a rate of only 2%. An additional 11.5% of tumor samples lacked expression of WTX mRNA. These WTX alterations can occur in parallel to WT1 or CTNNB1 mutations. However, we could not find a significant correlation between WTX deletion status or expression level and clinical parameters suggesting that WTX mutations apparently have little direct impact on tumor behavior and presentation. Incomplete deletions of WTX in several cases suggested heterogeneity in tumors. In a small number of cases, we could analyze separate tumor fragments or microdissected regions with different histology of tumors with heterozygous point mutations. Despite complete allele losses at other sites in the genome, we detected varying degrees of WTX mutation. This suggests that WTX alteration is not an essential and early mutation needed to drive tumorigenesis, but rather a later event that may affect only a fraction of cells with unclear clinical relevance.


Assuntos
Neoplasias Renais/genética , Proteínas Supressoras de Tumor/genética , Tumor de Wilms/genética , Proteínas Adaptadoras de Transdução de Sinal , Feminino , Humanos , Técnicas Imunoenzimáticas , Neoplasias Renais/metabolismo , Masculino , Mutação Puntual/genética , Reação em Cadeia da Polimerase , Prognóstico , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Proteínas WT1/genética , Proteínas WT1/metabolismo , Tumor de Wilms/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
18.
Genes Chromosomes Cancer ; 47(5): 386-95, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18260125

RESUMO

Current treatment protocols for Wilms tumor achieve 90% cure rates, but relapse risk and side effects from therapy remain challenging. Over the last decade, numerous markers have been proposed for classification and/or prediction of outcome. However, cohort sizes were quite variable and often small. We now provide a large-scale reassessment by real-time RT-PCR of 40 markers in 102 Wilms tumors followed by validation of potentially relevant markers in an independent set of 74 tumors. In the first data set, individual comparison with clinical data combined with adjustment for multiple testing and multivariate analysis revealed potentially relevant alteration of CA9, DKK1, EGR1, HEY2, MYC, MYCN, TERT, TOP2A, TRIM22, and VEGF expression in association with CTNNB1 mutation status, histological risk, response to chemotherapy, metastasis, relapse, or mortality. To further validate these data, potentially relevant genes for specific outcomes were reanalyzed in a second, independent tumor set. Here, univariate analysis confirmed the association of HEY2 with high-risk tumors and of TRIM22 with mortality. Even where significance levels could not be reached, the direction and extent of differential expression were generally reproducible. Multivariate analysis verified a weak correlation of TOP2A expression with metastasis and of TRIM22 with fatal outcome. Although we could corroborate only some of the previously reported associations of expression changes with clinical parameters, our results indicate that real-time RT-PCR analysis can facilitate further classification of Wilms tumor and prediction of outcome to adjust treatment accordingly. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.


Assuntos
Marcadores Genéticos , Prognóstico , Tumor de Wilms/genética , Perfilação da Expressão Gênica , Humanos , Análise Multivariada , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tumor de Wilms/patologia
19.
Int J Med Microbiol ; 298(1-2): 127-34, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17897883

RESUMO

Cardiovascular diseases are the major cause of human death and have been linked to many different risk factors. Among them, coxsackievirus B3 (CVB3), as a member of the enterovirus group, is one of the most important infectious agents of virus-induced myocarditis. Despite the fact that the molecular structure of this pathogen has been characterized very precisely, there is no virus-specific preventive or therapeutic procedure against CVB3-induced heart disease in clinical use today. A promising approach to prevent CVB3-caused myocarditis represents the mutation of the viral genome in a way that coding sequences of cytokines are integrated into the viral RNA. Recombinant cytokine-expressing CVB3 variants were established to increase the local cytokine concentration and to modulate TH1-/TH2-specific immune responses. Especially protective against CVB3-induced murine myocarditis is the application of an interferon-gamma (IFN-gamma)-expressing recombinant coxsackievirus variant. The local and simultaneous expression of an immuno-relevant cytokine by the virus itself induces a strong and long-lasting immune response which protects laboratory animals against lethal infections.


Assuntos
Infecções por Coxsackievirus/imunologia , Infecções por Coxsackievirus/terapia , Enterovirus Humano B/imunologia , Miocardite/imunologia , Miocardite/terapia , Vacinas Virais/imunologia , Animais , Infecções por Coxsackievirus/prevenção & controle , Infecções por Coxsackievirus/virologia , Enterovirus Humano B/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Miocardite/prevenção & controle , Miocardite/virologia , Pâncreas/imunologia , Pâncreas/virologia , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/uso terapêutico , Vacinas Virais/uso terapêutico
20.
J Pept Sci ; 11(7): 390-400, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15635669

RESUMO

Linear and cyclic phosphopeptides related to the pY2267 binding site of the epithelial receptor tyrosine kinase Ros have been synthesized as ligands for the amino-terminal SH2 (src homology) domain of protein tyrosine phosphatase SHP-1. The synthesis was accomplished by Fmoc-based solid-phase methodology using side-chain unprotected phosphotyrosine for the linear and mono-benzyl protected phosphotyrosine for the cyclic peptides. According to molecular modelling, the incorporation of a glycine residue between Lys (position pY-1 relative to phosphotyrosine) and Asp or Glu (position pY+2) was recommended for the cyclic candidates. The preparation of these peptides was successfully performed by the incorporation of a Fmoc-Xxx(Gly-OAll)-OH (Xxx = Asp, Glu) dipeptide building block that was prepared in solution prior to SPPS. The cyclization was achieved with PyBOP following Alloc/OAll-deprotection. This study demonstrates the usefulness of allyl-type protecting groups for the generation of side-chain cyclized phosphopeptides. Alloc/OAll-deprotection and cyclization are compatible with phosphorylated tyrosine.


Assuntos
Peptídeos Cíclicos/síntese química , Fosfopeptídeos/síntese química , Proteínas Tirosina Fosfatases/metabolismo , Cromatografia Líquida de Alta Pressão , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Espectrometria de Massas , Peptídeos Cíclicos/metabolismo , Fosfopeptídeos/metabolismo , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 6
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