Unknown,Transcriptomics,Genomics,Proteomics

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Classification of a frameshift/extended and a stop mutation in WT1 as gain of function mutations which activate cell cycle genes and promote Wilms tumor cell proliferation

ABSTRACT: The Wilms tumor 1 (WT1) gene encodes a zinc finger transcription factor important for normal kidney development. WT1 is a suppressor for Wilms tumor development and an oncogene for diverse malignant tumors. We recently established cell lines from primary Wilms tumors and identified the corresponding WT1 mutations (see GSE18058). To investigate the function of mutant WT1 proteins we performed WT1 knockdown experiments in primary Wilms tumor cell lines with a frameshift/extension (p.V432fsX87 = Wilms3) and a stop mutation (p.P362X = Wilms2) of WT1, followed by genome wide gene expression analysis. A detailed analysis of these gene expression data using MetaCore enabled us to classify the WT1 mutations as gain of function mutations. The mutant WT1Wilms2 and WT1Wilms3 proteins acquired an ability to modulate the expression of a highly significant number of genes from the G2/M phase of the cell cycle, and WT1 knockdown experiments showed that they are required for Wilms tumor cell proliferation. Data from the literature show that p53 negatively regulates the activity of a large number of these genes which are also part of a core proliferation cluster in diverse human cancers. Our data strongly suggest that mutant WT1 proteins facilitate expression of these cell cycle genes by antagonizing transcriptional repression mediated by p53. In light of this it is important to note that mutant WT1 has an ability to physically interact with the tumor suppressor p53. Together the findings show for the first time, that mutant WT1 proteins have a gain of function and act as oncogenes for Wilms tumor development by regulating Wilms tumor cell proliferation. siWT1 mediated knockdown in two Wilms tumor cell lines one and two days after transfection versus non-silencing controls

ORGANISM(S): Homo sapiens

SUBMITTER: Brigitte Royer-Pokora

PROVIDER: E-GEOD-54635 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Classification of a frameshift/extended and a stop mutation in WT1 as gain-of-function mutations that activate cell cycle genes and promote Wilms tumour cell proliferation.

Busch Maike M Schwindt Heinrich H Brandt Artur A Beier Manfred M Görldt Nicole N Romaniuk Paul P Toska Eneda E Roberts Stefan S Royer Hans-Dieter HD Royer-Pokora Brigitte B

Human molecular genetics 20140311 15

The WT1 gene encodes a zinc finger transcription factor important for normal kidney development. WT1 is a suppressor for Wilms tumour development and an oncogene for diverse malignant tumours. We recently established cell lines from primary Wilms tumours with different WT1 mutations. To investigate the function of mutant WT1 proteins, we performed WT1 knockdown experiments in cell lines with a frameshift/extension (p.V432fsX87 = Wilms3) and a stop mutation (p.P362X = Wilms2) of WT1, followed by ...[more]

PMID: 24619359

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Project description:The Wilms tumor-suppressor gene WT1, a key player in renal development, also has a crucial role in maintenance of the glomerulus in the mature kidney. However, molecular pathways orchestrated by WT1 in podocytes, where it is highly expressed, remain unknown. Their defects are thought to modify the cross-talk between podocytes and other glomerular cells and ultimately lead to glomerular sclerosis, as observed in diffuse mesangial sclerosis (DMS) a nephropathy associated with WT1 mutations. To identify podocyte WT1 targets, we generated a novel DMS mouse line, performed gene expression profiling in isolated glomeruli, and identified excellent candidates that may modify podocyte differentiation and growth factor signalling in glomeruli. Scel, encoding sciellin, a protein of the cornified envelope in the skin, and sulf1, encoding a 6-O endosulfatase, are shown to be expressed in wild type podocytes and to be strongly down-regulated in mutants. Co-expression of Wt1, Scel and Sulf1 was also found in a mesonephric cell line, and siRNA-mediated knockdown of WT1 decreased Scel and Sulf1 mRNAs and proteins. By ChIP we show that Scel and Sulf1 are direct WT1 targets. Cyp26a1, encoding an enzyme involved in the degradation of retinoic acid, is shown to be up-regulated in mutant podocytes. Cyp26a1 may play a role in the development of glomerular lesions but does not seem to be regulated by WT1. These results provide novel clues in our understanding of normal glomerular function and early events involved in glomerulosclerosis. Experiment Overall Design: Isolation of glomeruli from mutant (FVB-N4 Wt1+/R394W) and wild-type (FVB-N4 Wt1+/+) was performed after cardiac Dynabead perfusion. GeneChip analysis of glomeruli from 5 Wt1+/R394W mice and 5 Wt1+/+ littermates (N4-FVB) were performed independently. Animals were unweaned 27-day-old males. The Wt1+/R394W mice used were showing little albuminuria (<3 ug/ul on Coomassie blue stained SDS-PAGE gel) and no evidence of mesangial lesions by light microscopy.

Dataset Information

Classification of a frameshift/extended and a stop mutation in WT1 as gain of function mutations which activate cell cycle genes and promote Wilms tumor cell proliferation

Publications

Classification of a frameshift/extended and a stop mutation in WT1 as gain-of-function mutations that activate cell cycle genes and promote Wilms tumour cell proliferation.

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