WT1-AS

Gene Summary

Gene:WT1-AS; WT1 antisense RNA
Aliases: WIT1, WIT-1, WT1AS, WT1-AS1
Location:11p13
Summary:This gene is located upstream of the Wilms tumor 1 (WT1) gene; these two genes are bi-directionally transcribed from the same promoter region. This gene is imprinted in kidney, with preferential expression from the paternal allele. Imprinting defects at chromosome 11p13 may contribute to tumorigenesis. [provided by RefSeq, May 2014]
Databases:OMIM, HGNC, Ensembl, GeneCard, Gene
Source:NCBIAccessed: 31 August, 2019

Ontology:

What does this gene/protein do?
WT1-AS is implicated in:
- biological_process
- cellular_component
- molecular_function
Data from Gene Ontology via CGAP

Cancer Overview

Research Indicators

Publications Per Year (1994-2019)
Graph generated 31 August 2019 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Residual Disease
  • Gene Expression Regulation
  • Western Blotting
  • Cancer Gene Expression Regulation
  • Transcription Factors
  • Leukaemia
  • DNA-Binding Proteins
  • Kidney
  • DNA Methylation
  • Ovarian Cancer
  • Transfection
  • Neoplasm Proteins
  • Childhood Cancer
  • WT1
  • Cell Proliferation
  • Recurrence
  • Adolescents
  • Wilms Tumor Genes
  • ras Proteins
  • Tumor Suppressor Gene
  • Molecular Sequence Data
  • Acute Myeloid Leukaemia
  • Base Sequence
  • Cell Differentiation
  • p53 Protein
  • Cancer RNA
  • Biomarkers, Tumor
  • Promoter Regions
  • Immunohistochemistry
  • Infant
  • Kidney Cancer
  • Mutation
  • Chromosome 11
  • RTPCR
  • Messenger RNA
  • Protein Isoforms
  • Gene Expression
  • Gene Expression Profiling
  • Apoptosis
Tag cloud generated 31 August, 2019 using data from PubMed, MeSH and CancerIndex

Specific Cancers (5)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: WT1-AS (cancer-related)

Krueger K, Catanese L, Sciesielski LK, et al.
Deletion of an intronic HIF-2α binding site suppresses hypoxia-induced WT1 expression.
Biochim Biophys Acta Gene Regul Mech. 2019; 1862(1):71-83 [PubMed] Related Publications
Hypoxia-inducible factors (HIFs) play a key role in the adaptation to low oxygen by interacting with hypoxia response elements (HREs) in the genome. Cellular levels of the HIF-2α transcription factor subunit influence the histopathology and clinical outcome of neuroblastoma, a malignant childhood tumor of the sympathetic ganglia. Expression of the Wilms tumor gene, WT1, marks a group of high-risk neuroblastoma. Here, we identify WT1 as a downstream target of HIF-2α in Kelly neuroblastoma cells. In chromatin immunoprecipitation assays, HIF-2α bound to a HRE in intron 3 of the WT1 gene, but not to another predicted HIF binding site (HBS) in the first intron. The identified element conferred oxygen sensitivity to otherwise hypoxia-resistant WT1 and SV40 promoter constructs. Deletion of the HBS in the intronic HRE by genome editing abolished WT1 expression in hypoxic neuroblastoma cells. Physical interaction between the HRE and the WT1 promoter in normoxic and hypoxic Kelly cells was shown by chromosome conformation capture assays. These findings demonstrate that binding of HIF-2α to an oxygen-sensitive enhancer in intron 3 stimulates transcription of the WT1 gene in neuroblastoma cells by hypoxia-independent chromatin looping. This novel regulatory mechanism may have implications for the biology and prognosis of neuroblastoma.

Yang K, Lu XF, Luo PC, Zhang J
Identification of Six Potentially Long Noncoding RNAs as Biomarkers Involved Competitive Endogenous RNA in Clear Cell Renal Cell Carcinoma.
Biomed Res Int. 2018; 2018:9303486 [PubMed] Free Access to Full Article Related Publications

Wang X, Han L, Zhou L, et al.
Prediction of candidate RNA signatures for recurrent ovarian cancer prognosis by the construction of an integrated competing endogenous RNA network.
Oncol Rep. 2018; 40(5):2659-2673 [PubMed] Free Access to Full Article Related Publications
Tumor recurrence hinders treatment of ovarian cancer. The present study aimed to identify potential biomarkers for ovarian cancer recurrence prognosis and explore relevant mechanisms. RNA‑sequencing of data from the TCGA database and GSE17260 dataset was carried out. Samples of the data were grouped according to tumor recurrence information. Following data normalization, differentially expressed genes/micro RNAs (miRNAs)/long non‑coding (lncRNAs) (DEGs/DEMs/DELs) were selected between recurrent and non‑recurrent samples. Their correlations with clinical information were analyzed to identify prognostic RNAs. A support vector machine classifier was used to find the optimal gene set with feature genes that could conclusively distinguish different samples. A protein‑protein interaction (PPI) network was established for DEGs using relevant protein databases. An integrated 'lncRNA/miRNA/mRNA' competing endogenous RNA (ceRNA) network was constructed to reveal potential regulatory relationships among different RNAs. We identified 36 feature genes (e.g. TP53 and RBPMS) for the classification of recurrent and non‑recurrent ovarian cancer samples. Prediction with this gene set had a high accuracy (91.8%). Three DELs (WT1‑AS, NBR2 and ZNF883) were highly associated with the prognosis of recurrent ovarian cancer. Predominant DEMs with their targets were hsa‑miR‑375 (target: RBPMS), hsa‑miR‑141 (target: RBPMS), and hsa‑miR‑27b (target: TP53). Highlighted interactions in the ceRNA network were 'WT1‑AS‑hsa‑miR‑375‑RBPMS' and 'WT1‑AS‑-hsa‑miR‑27b‑TP53'. TP53, RBPMS, hsa‑miR‑375, hsa‑miR‑141, hsa‑miR‑27b, and WT1‑AS may be biomarkers for recurrent ovarian cancer. The interactions of 'WT1‑AS‑hsa‑-miR‑375‑RBPMS' and 'WT1‑AS‑hsa‑miR‑27b‑TP53' may be potential regulatory mechanisms during cancer recurrence.

Buglyó G, Beyer D, Biró S, Oláh É
The Wilms' tumour 1 gene as a factor in non-syndromic hypospadias: evidence and controversy.
Pathology. 2018; 50(4):377-381 [PubMed] Related Publications
Hypospadias is one of the most frequent congenital anomalies of the male external genitalia. Its pathogenesis is due to largely unknown or poorly understood genetic factors and is further complicated by environmental-intrauterine-risk factors. One of the genes currently in focus by molecular biologists and clinicians studying syndromic forms of hypospadias is the Wilms' tumour 1 (WT1) gene. There is controversy over whether WT1 defects are also responsible for isolated hypospadias. In this review, we briefly cover the role of WT1 as a transcription factor and discuss proposed pathogenic pathways leading to hypospadias, outlining possible directions for research. We assess available evidence on the gene's mutations and polymorphisms recently suggested in the background of the disease, and examine the putative role of WT1-associated proteins. We also review relevant aspects of genome-wide association studies carried out so far, and raise some points to consider in future efforts.

Wang LP, Zhao YN, Sun X, Gao RL
Effects of bufalin on up-regulating methylation of Wilm's tumor 1 gene in human erythroid leukemic cells.
Chin J Integr Med. 2017; 23(4):288-294 [PubMed] Related Publications
OBJECTIVE: To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm' tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells.
METHODS: The HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively.
RESULTS: The bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC
CONCLUSIONS: Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G

Yang S, Zhang Y, Zhao X, et al.
microRNA-361 targets Wilms' tumor 1 to inhibit the growth, migration and invasion of non-small-cell lung cancer cells.
Mol Med Rep. 2016; 14(6):5415-5421 [PubMed] Related Publications
The expression and functions of microRNA-361 (miR-361) have been studied in various human cancers. However, its expression and role in non‑small‑cell lung cancer (NSCLC) remains unclear. In the present study, the expression levels of miR‑361 in NSCLC tissues and cell lines were determined using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). In addition, the effect of miR‑361 on the proliferation, migration and invasion of NSCLC cells was assessed. Furthermore, a dual‑Luciferase reporter assay, RT‑qPCR and western blotting were performed to investigate whether miR‑361 directly targeted the 3' untranslated region of Wilms' tumor 1 (WT1). The results of the present study revealed that miR‑361 was downregulated in NSCLC tissues and cell lines. Enforced expression of miR‑361 suppressed the proliferation, migration and invasion of NSCLC cells. WT1 was identified as a direct target gene of miR‑361 in NSCLC. Furthermore, knockdown of WT1 had similar effects to miR‑361 overexpression in NSCLC cells. The present study provided novel insights into the molecular mechanism underlying the rapid growth and metastasis of NSCLC, and identified the association between miR‑361 and WT1 as a potential therapeutic target for the treatment of NSCLC.

Taube ET, Denkert C, Sehouli J, et al.
Wilms tumor protein 1 (WT1)-- not only a diagnostic but also a prognostic marker in high-grade serous ovarian carcinoma.
Gynecol Oncol. 2016; 140(3):494-502 [PubMed] Related Publications
AIMS: Wilms tumor protein 1 (WT1) expression is used in gynecological pathology as a diagnostic marker of serous differentiation, and is frequently co-expressed with ER-α. Early phase studies on WT1 vaccine in gynecological cancers are ongoing. In this study we aimed to determine the prognostic value of WT1 in high-grade serous ovarian carcinoma.
METHODS: WT1 protein expression was determined by immunohistochemistry in a cohort of 207 primary high-grade serous ovarian carcinomas. WT1 mRNA expression was evaluated in a cohort of 1137 ovarian carcinomas from publically available gene expression datasets.
RESULTS: High WT1 expression was a significant positive prognostic factor in primary high-grade serous ovarian carcinoma regarding overall survival (OS, p=0.008) and progression free survival (PFS, p=0.015), which was independent of age, stage, and residual tumor (OS: p=0.024, PFS: p=0.047). The prognostic significance of immunohistochemical WT1 expression could be reproduced in an independent cohort of 72 patients. On the mRNA level the prognostic significance was validated in silico in publically available gene expression datasets including TCGA data (OS: p=0.002, PFS: p=0.011). WT1 expression was significantly linked to ER-α expression (p=0.001), and tumors that co-expressed both markers (WT1+/ER-α+) had a longer survival time than tumors of all other marker combinations (OS: p=0.002, PFS: p=0.013).
CONCLUSION: We present WT1 as a robust prognostic marker in high-grade serous ovarian carcinoma, which adds prognostic information to ER-α. This should be kept in mind when WT1 is used as a biomarker in the context of WT1-targeting therapies.

Lv L, Chen G, Zhou J, et al.
WT1-AS promotes cell apoptosis in hepatocellular carcinoma through down-regulating of WT1.
J Exp Clin Cancer Res. 2015; 34:119 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: The antisense of the tumor suppressor gene WT1 (WT1-AS) is a long non-coding RNA. The role of WT1-AS in the development of hepatocellular carcinoma (HCC) has not yet been elucidated.
METHODS: Quantitative real-time PCR and western blot analyses were used to measure levels of WT1-AS and its related genes in tumor and corresponding adjacent tumor tissues of HCC patients. The effect on HCC cell proliferation and apoptosis was assessed by EdU incorporation assays and PI-Annexin-V staining, respectively. ShRNA and dual-luciferase assays were used to investigate the regulatory relationship between WT1-AS and WT1 in cell lines.
RESULTS: WT1-AS expression correlated negatively with WT1 expression in HCC tumor tissue. Kaplan-Meier curve analysis revealed that WT1-AS expression is a reliable indicator of HCC prognosis. The downregulation of WT1 expression by WT1-AS promoted cell apoptosis by suppressing the JAK/STAT3 signaling pathway. Bioinformatics analysis showed that WT1-AS downregulates WT1 by binding to the TATA region of the WT1 promotor. WT1-AS was also able to reverse WT1-mediated resistance to Dox based chemotherapy in HCC cells.
CONCLUSIONS: WT1-AS downregulates WT1 expression in HCC tumors and promotes apoptosis by binding to the promoter region of WT1. Our findings suggest that WT1-AS may function as a tumor suppressor in HCC by reversing the oncogenic effects of WT1.

Du T, Zhang B, Zhang S, et al.
Decreased expression of long non-coding RNA WT1-AS promotes cell proliferation and invasion in gastric cancer.
Biochim Biophys Acta. 2016; 1862(1):12-9 [PubMed] Related Publications
Tumor recurrence and metastasis remain the major obstacles for the successful treatment of patients diagnosed with gastric cancer. In recent years, long non-coding RNAs (lncRNAs) have been considered as key regulators of tumor behavior. In this study, we investigated the expression and biological role of a newly-identified cancer-related lncRNA, WT1-AS. We found that WT1-AS expression was significantly down-regulated in tumor tissues compared to matched adjacent non-tumor tissues. The WT1-AS expression level was also associated with tumor size and the clinicopathological stage. Cell proliferation, migration, and invasion were inhibited, and the proportion of G0/G1 cells increased when WT1-AS was ectopically-expressed in gastric cancer cells. Furthermore, ectopic expression of WT1-AS was demonstrated to inhibit tumor growth and metastasis in vivo. Finally, we found that WT1-AS overexpression could decrease ERK protein phosphorylation. Our study indicates that WT1-AS is significantly down-regulated in gastric cancers and may be correlated with tumor progression.

Zhang R, Yang JY, Sun HQ, et al.
Comparison of minimal residual disease (MRD) monitoring by WT1 quantification between childhood acute myeloid leukemia and acute lymphoblastic leukemia.
Eur Rev Med Pharmacol Sci. 2015; 19(14):2679-88 [PubMed] Related Publications
OBJECTIVE: Wilms tumor gene 1 (WT1) has been identified as an independent risk prognostic factor in acute leukemia. However, there exists a controversy that WT1 as a marker for minimal residual disease (MRD) monitoring in acute leukemias. We detected WT1-RNA transcript level to estimate the diagnostic value of monitoring MRD in childhood acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).
PATIENTS AND METHODS: WT1 mRNA expression levels were detected by real-time quantitative reverse transcriptase PCR (qRT-PCR) in bone marrow (BM) samples from 107 childhood ALL and 35 childhood AML at diagnosis. MRD was consecutively performed after induction and consolidation (early intensification in ALL) chemotherapy. Receiver operating characteristics (ROC) analysis and the largest areas under the curve (AUC) were applied to define optimal threshold value of MRD level. Sensitivity, specificity, positive likelihood ratio (+LR) and negative likelihood ratio (-LR) were used to evaluate diagnostic power for MRD. Relapse free survival (RFS) was evaluated by the Kaplan-Meier statistical method.
RESULTS: The largest areas under the curve (AUC), specificity, +LR and -LR showed higher accuracy in childhood AML than ALL. Compared the diagnostic parameters, the post-induction time wasn't good enough to show the better time than post-consolidation time for MRD assessment in AML. The threshold was set at 150 WT1 copies/104 ABL copies as the optimal cut-off value of MRD level post induction in childhood AML. MRD+ (WT1>150) children had increased the risk of relapse with poor prognosis, showing lower RFS than MRD- group (p=0.01). However, the threshold 70 WT1 copies/104 ABL copies post induction in childhood ALL did not show clinical significance for predicting prognosis (p=0.056).
CONCLUSIONS: WT1 gene will be a useful marker for monitoring MRD to predict relapse in childhood AML. But it did not show good enough to monitor MRD in childhood ALL.

Khalili S, Rahbar MR, Dezfulian MH, Jahangiri A
In silico analyses of Wilms׳ tumor protein to designing a novel multi-epitope DNA vaccine against cancer.
J Theor Biol. 2015; 379:66-78 [PubMed] Related Publications
Predefined and pre-weighted objective criteria and essential role of Wilms׳ tumor wild type gene (WT1) for maintaining transformed features of cancer cells confirm the high potency of WT1 as a valuable cancer antigen. The antigen was at the top of the ranking among 75 representative cancer antigens. In the present study, an in silico approach was launched to characterized novel CTL epitopes and design a novel multi-epitope DNA vaccine to elicit a desirable immune response against cancers over expressing WT1. Forty-four novel epitopes were described. A multi-epitope construct was designed based on predicted epitopes which is 310 residues in length. The vaccine candidate designed here displays acceptable population coverage (>65%) in different ethnicities as well as high probability of eliciting WT1 antibodies which both are pertinent goals in the context of appropriate multi-epitope vaccines. Various in silico analyses indicate that final vaccine is a qualified immunotherapy candidate capable of eliciting both CD4+ and CD8+ T cell responses.

Stirzaker C, Zotenko E, Song JZ, et al.
Methylome sequencing in triple-negative breast cancer reveals distinct methylation clusters with prognostic value.
Nat Commun. 2015; 6:5899 [PubMed] Related Publications
Epigenetic alterations in the cancer methylome are common in breast cancer and provide novel options for tumour stratification. Here, we perform whole-genome methylation capture sequencing on small amounts of DNA isolated from formalin-fixed, paraffin-embedded tissue from triple-negative breast cancer (TNBC) and matched normal samples. We identify differentially methylated regions (DMRs) enriched with promoters associated with transcription factor binding sites and DNA hypersensitive sites. Importantly, we stratify TNBCs into three distinct methylation clusters associated with better or worse prognosis and identify 17 DMRs that show a strong association with overall survival, including DMRs located in the Wilms tumour 1 (WT1) gene, bi-directional-promoter and antisense WT1-AS. Our data reveal that coordinated hypermethylation can occur in oestrogen receptor-negative disease, and that characterizing the epigenetic framework provides a potential signature to stratify TNBCs. Together, our findings demonstrate the feasibility of profiling the cancer methylome with limited archival tissue to identify regulatory regions associated with cancer.

Niskakoski A, Kaur S, Staff S, et al.
Epigenetic analysis of sporadic and Lynch-associated ovarian cancers reveals histology-specific patterns of DNA methylation.
Epigenetics. 2014; 9(12):1577-87 [PubMed] Free Access to Full Article Related Publications
Diagnosis and treatment of epithelial ovarian cancer is challenging due to the poor understanding of the pathogenesis of the disease. Our aim was to investigate epigenetic mechanisms in ovarian tumorigenesis and, especially, whether tumors with different histological subtypes or hereditary background (Lynch syndrome) exhibit differential susceptibility to epigenetic inactivation of growth regulatory genes. Gene candidates for epigenetic regulation were identified from the literature and by expression profiling of ovarian and endometrial cancer cell lines treated with demethylating agents. Thirteen genes were chosen for methylation-specific multiplex ligation-dependent probe amplification assays on 104 (85 sporadic and 19 Lynch syndrome-associated) ovarian carcinomas. Increased methylation (i.e., hypermethylation) of variable degree was characteristic of ovarian carcinomas relative to the corresponding normal tissues, and hypermethylation was consistently more prominent in non-serous than serous tumors for individual genes and gene sets investigated. Lynch syndrome-associated clear cell carcinomas showed the highest frequencies of hypermethylation. Among endometrioid ovarian carcinomas, lower levels of promoter methylation of RSK4, SPARC, and HOXA9 were significantly associated with higher tumor grade; thus, the methylation patterns showed a shift to the direction of high-grade serous tumors. In conclusion, we provide evidence of a frequent epigenetic inactivation of RSK4, SPARC, PROM1, HOXA10, HOXA9, WT1-AS, SFRP2, SFRP5, OPCML, and MIR34B in the development of non-serous ovarian carcinomas of Lynch and sporadic origin, as compared to serous tumors. Our findings shed light on the role of epigenetic mechanisms in ovarian tumorigenesis and identify potential targets for translational applications.

Messina C, Candoni A, Carrabba MG, et al.
Wilms' tumor gene 1 transcript levels in leukapheresis of peripheral blood hematopoietic cells predict relapse risk in patients autografted for acute myeloid leukemia.
Biol Blood Marrow Transplant. 2014; 20(10):1586-91 [PubMed] Related Publications
Autologous hematopoietic stem cell transplantation (ASCT) is a curative option alternative to allogeneic transplantation for patients with acute myeloid leukemia (AML). Relapse after ASCT can be due to contamination with leukemic blasts of autologous peripheral blood stem cells (PBSCs) collected by leukapheresis (LK). Identification and quantification of a minimal residual disease (MRD) marker in PBSCs could be relevant in determining the relapse risk after ASCT. High levels of the WT1 gene transcript in bone marrow of AML patients after treatment completion predict disease relapse. We evaluated WT1 transcript levels in autologous PBSC from LK used for ASCT in 30 consecutive AML patients in complete remission (CR) and established a correlation with clinical outcome. At diagnosis, all patients had WT1 overexpression. All patients were in morphological and genetic CR at the time of PBSC collection and before ASCT. Real-time quantitative PCR of WT1 was performed in samples of each LK, using TaqMan technology on RNA from mononucleated cells. The median WT1 transcript level in the PBSC graft (WT1-LK) of patients who relapsed was significantly higher than of those who did not relapse after transplantation (P <.0001). We defined a cut-off level of 80 WT1-LK copies/ABL 10e4 copies to discriminate between positive and negative PBSC grafts. The cut-off level was strongly associated with disease recurrence, DFS and OS. Our study represents the largest series of patients evaluating WT1 as a marker of MRD in PBSC LK products using a completely standardized real-time WT1-reverse transcriptase-PCR based assay. These data, if confirmed by prospective study, will help to determine an individual patient's adapted postremission allocation strategy.

Toska E, Roberts SG
Mechanisms of transcriptional regulation by WT1 (Wilms' tumour 1).
Biochem J. 2014; 461(1):15-32 [PubMed] Related Publications
The WT1 (Wilms' tumour 1) gene encodes a zinc finger transcription factor and RNA-binding protein that direct the development of several organs and tissues. WT1 manifests both tumour suppressor and oncogenic activities, but the reasons behind these opposing functions are still not clear. As a transcriptional regulator, WT1 can either activate or repress numerous target genes resulting in disparate biological effects such as growth, differentiation and apoptosis. The complex nature of WT1 is exemplified by a plethora of isoforms, post-translational modifications and multiple binding partners. How WT1 achieves specificity to regulate a large number of target genes involved in diverse physiological processes is the focus of the present review. We discuss the wealth of the growing molecular information that defines our current understanding of the versatility and utility of WT1 as a master regulator of organ development, a tumour suppressor and an oncogene.

Busch M, Schwindt H, Brandt A, et al.
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.
Hum Mol Genet. 2014; 23(15):3958-74 [PubMed] Free Access to Full Article Related Publications
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 genome-wide gene expression analysis. We also expressed wild-type and mutant WT1 proteins in human mesenchymal stem cells and established gene expression profiles. A detailed analysis of gene expression data enabled us to classify the WT1 mutations as gain-of-function mutations. The mutant WT1(Wilms2) and WT1(Wilms3) 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 tumour cell proliferation. p53 negatively regulates the activity of a large number of these genes that 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. We show that mutant WT1 can physically interact with p53. Together the findings show for the first time that mutant WT1 proteins have a gain-of-function and act as oncogenes for Wilms tumour development by regulating Wilms tumour cell proliferation.

Rauscher J, Beschorner R, Gierke M, et al.
WT1 expression increases with malignancy and indicates unfavourable outcome in astrocytoma.
J Clin Pathol. 2014; 67(7):556-61 [PubMed] Related Publications
AIMS: The zinc finger transcription factor WT1 is expressed in astrocytic neoplasms and therefore is a potential target of immunotherapy in brain tumours. Our aim was to further elucidate the role of WT1 as a diagnostic and prognostic marker in neuropathology, particularly as to the differentiation of astrocytoma from oligodendroglioma as well as to the dependency of WT1 expression on clinically relevant parameters.
METHODS: 829 evaluable brain tumour samples were investigated by WT1 immunohistochemistry on full tissue routine slides, consisting of 442 glioblastomas, 303 astrocytomas, 41 oligodendrogliomas and 43 oligoastrocytomas. In addition public WT1 gene expression data of 351 gliomas were analysed.
RESULTS: Our data show that WT1 expression in diffuse astrocytic tumours increases with WHO tumour grade and is associated with older age, absence of IDH1 mutation but not related to O(6)- methyl guanine methyl transferase (MGMT) promoter methylation status. Univariable, but not multivariable survival analysis indicates that WT1 expression is associated with worse outcome in patients with diffuse astrocytoma but not glioblastoma.
CONCLUSIONS: The significant WT1 expression differences between diffuse astrocytomas, oligoastrocytomas and oligodendrogliomas, which are also present in the Repository for Molecular Brain Neoplasia Data, National Cancer Institute (REMBRANDT, 2005, http://rembrandt.nci.nih.gov) gene database set, provide a rationale for use of WT1 as part of a routine immunohistochemistry panel.

Rather MI, Swamy S, Gopinath KS, Kumar A
Transcriptional repression of tumor suppressor CDC73, encoding an RNA polymerase II interactor, by Wilms tumor 1 protein (WT1) promotes cell proliferation: implication for cancer therapeutics.
J Biol Chem. 2014; 289(2):968-76 [PubMed] Free Access to Full Article Related Publications
The Wilms tumor 1 gene (WT1) can either repress or induce the expression of genes. Inconsistent with its tumor suppressor role, elevated WT1 levels have been observed in leukemia and solid tumors. WT1 has also been suggested to act as an oncogene by inducing the expression of MYC and BCL-2. However, these are only the correlational studies, and no functional study has been performed to date. Consistent with its tumor suppressor role, CDC73 binds to RNA polymerase II as part of a PAF1 transcriptional regulatory complex and causes transcriptional repression of oncogenes MYC and CCND1. It also represses β-catenin-mediated transcription. Based on the reduced level of CDC73 in oral squamous cell carcinoma (OSCC) samples in the absence of loss-of-heterozygosity, promoter methylation, and mutations, we speculated that an inhibitory transcription factor is regulating its expression. The bioinformatics analysis predicted WT1 as an inhibitory transcription factor to regulate the CDC73 level. Our results showed that overexpression of WT1 decreased CDC73 levels and promoted proliferation of OSCC cells. ChIP and EMSA results demonstrated binding of WT1 to the CDC73 promoter. The 5-azacytidine treatment of OSCC cells led to an up-regulation of WT1 with a concomitant down-regulation of CDC73, further suggesting regulation of CDC73 by WT1. Exogenous CDC73 attenuated the protumorigenic activity of WT1 by apoptosis induction. An inverse correlation between expression levels of CDC73 and WT1 was observed in OSCC samples. These observations indicated that WT1 functions as an oncogene by repressing the expression of CDC73 in OSCC. We suggest that targeting WT1 could be a therapeutic strategy for cancer, including OSCC.

Israyelyan A, La Rosa C, Tsai W, et al.
Detection and preliminary characterization of CD8+T lymphocytes specific for Wilms' tumor antigen in patients with non-Hodgkin lymphoma.
Leuk Lymphoma. 2013; 54(11):2490-9 [PubMed] Free Access to Full Article Related Publications
Wilms' tumor antigen (WT1) is overexpressed in many different solid tumors and hematologic malignancies. However, little is known about WT1 expression or WT1-specific immune responses in patients with non-Hodgkin lymphoma (NHL). In a cross-sectional survey study, we investigated the immune recognition of WT1 by patients with NHL. Utilizing a WT1 overlapping peptide library, we discovered that a large percentage of patients with NHL of all grades maintain WT1-specific T cells. Ex vivo frequencies of these T cells measured from unfractionated samples by the CD137 activation marker assay were high in many patients (some > 1% CD8+). Using standard in vitro techniques we discovered that they were cytotoxic to WT1 peptide library-loaded T2 cells and WT1 antigen-primed autologous Epstein-Barr virus-transformed B cell lines (EBV-LCLs) and expressed interferon gamma (IFN-γ). In addition, we detected WT1 mRNA transcripts in diseased lymph node tissues of patients with NHL utilizing real-time quantitative polymerase chain reaction (RT-qPCR) technology. These results are the first example of strong T cell reactivity against WT1 in patients with NHL which also demonstrate strong cytotoxicity against peptide-loaded tumor cells. The potential for developing WT1 as a target for immunotherapy in NHL deserves further exploration.

Coosemans A, Van Calster B, Verbist G, et al.
Wilms tumor gene 1 (WT1) is a prognostic marker in high-grade uterine sarcoma.
Int J Gynecol Cancer. 2011; 21(2):302-8 [PubMed] Related Publications
INTRODUCTION: Wilms tumor gene 1 (WT1) contributes to uterine sarcoma tumor biology. In this study, we aimed to clarify the prognostic value of WT1.
METHODS: A retrospective clinical and histopathological review of 71 women with high-grade uterine sarcoma (leiomyosarcoma [n = 24], undifferentiated sarcoma [n = 9]), and carcinosarcoma (n = 38) was performed. Patients were followed up for at least 12 months. Wilms tumor gene 1 expression was determined by immunohistochemistry. Data on recurrence (progression-free survival) and overall survival (OS) were available for all patients. Univariate and multivariate analyses of WT1 expression were carried out using Kaplan-Meier curves and Cox regression, respectively.
RESULTS: Forty-nine (69%) tumors were WT1 positive. Forty-seven (66%) patients died of the disease, with a median OS time of 22 months. Wilms tumor gene 1 was a predictor of survival in the univariate analysis: the hazard ratio of WT1 positivity was 2.44 (95% confidence interval, 1.34-4.71) for progression-free survival and 2.48 (95% confidence interval, 1.26-4.90) for OS. Multivariate analysis including stage, age, tumor size, and sarcoma subtype identified only stage and WT1 positivity as independent prognostic markers for survival.
CONCLUSIONS: The identification of WT1 as a prognostic marker confirms its role in high-grade uterine sarcoma and carcinosarcoma tumor biology.

Vicent S, Chen R, Sayles LC, et al.
Wilms tumor 1 (WT1) regulates KRAS-driven oncogenesis and senescence in mouse and human models.
J Clin Invest. 2010; 120(11):3940-52 [PubMed] Free Access to Full Article Related Publications
KRAS is one of the most frequently mutated human oncogenes. In some settings, oncogenic KRAS can trigger cellular senescence, whereas in others it produces hyperproliferation. Elucidating the mechanisms regulating these 2 drastically distinct outcomes would help identify novel therapeutic approaches in RAS-driven cancers. Using a combination of functional genomics and mouse genetics, we identified a role for the transcription factor Wilms tumor 1 (WT1) as a critical regulator of senescence and proliferation downstream of oncogenic KRAS signaling. Deletion or suppression of Wt1 led to senescence of mouse primary cells expressing physiological levels of oncogenic Kras but had no effect on wild-type cells, and Wt1 loss decreased tumor burden in a mouse model of Kras-driven lung cancer. In human lung cancer cell lines dependent on oncogenic KRAS, WT1 loss decreased proliferation and induced senescence. Furthermore, WT1 inactivation defined a gene expression signature that was prognostic of survival only in lung cancer patients exhibiting evidence of oncogenic KRAS activation. These findings reveal an unexpected role for WT1 as a key regulator of the genetic network of oncogenic KRAS and provide important insight into the mechanisms that regulate proliferation or senescence in response to oncogenic signals.

Licciulli S, Kissil JL
WT1: a weak spot in KRAS-induced transformation.
J Clin Invest. 2010; 120(11):3804-7 [PubMed] Free Access to Full Article Related Publications
Activating mutations in the Ras alleles are found frequently in tumors, making the proteins they encode highly attractive candidate therapeutic targets. However, Ras proteins have proven difficult to target directly. Recent approaches have therefore focused on identifying indirect targets to inhibit Ras-induced oncogenesis. For example, RNAi-based negative selection screens to identify genes that when silenced in concert with activating Ras mutations are incompatible with cellular proliferation, a concept known as synthetic lethality. In this issue of the JCI, Vicent et al. report on the identification of Wilms tumor 1 (Wt1) as a Kras synthetic-lethal gene in a mouse model of lung adenocarcinoma. Silencing of Wt1 in cells expressing an endogenous allele of activated Kras triggers senescence in vitro and has an impact on tumor progression in vivo. These findings are of significant interest given previous studies suggesting that the ability of oncogenic Kras to induce senescence versus proliferation depends on its levels of expression.

Jahn JE, Best DH, Coleman WB
Exogenous expression of synaptotagmin XIII suppresses the neoplastic phenotype of a rat liver tumor cell line through molecular pathways related to mesenchymal to epithelial transition.
Exp Mol Pathol. 2010; 89(3):209-16 [PubMed] Related Publications
The molecular pathogenesis of hepatocellular carcinoma is well-studied but not completely understood. We utilized a microcell-hybrid model of tumor suppression in rat liver tumor cells to facilitate the identification of liver tumor suppressor genes located on human chromosome 11. These investigations confirmed a liver tumor suppressor locus at human 11p11.2, identified Wt1 as a potential effector of 11p11.2-mediated tumor suppression, and subsequently identified human SYT13 as a strong candidate for the 11p11.2 liver tumor suppressor gene. In the studies presented here, we introduced SYT13 into the GN6TF rat liver tumor cell line to characterize a functional role for SYT13 in this model system. Transfected clones expressing an SDS-resistant dimer form of the SYT13 protein displayed induction of Wt1 gene expression and a significant attenuation of the neoplastic phenotype exhibited by the parental tumor cell line. Saturation densities and anchorage-independent growth of SYT13 dimer-positive cell lines were reduced in vitro, and tumorigenicity was significantly decreased or ablated in syngeneic host rats in vivo. In addition, restoration of the contact-inhibited, epithelioid morphology observed in normal liver epithelial cells accompanied ectopic expression of the SYT13 protein dimer, suggesting that SYT13 may be mediating an epithelial differentiation coordinate with tumor suppression in these cells. Accordingly, the expression of E-cadherin (Cdh1) mRNA was increased >100-fold in SYT13-dimer-positive cell lines and the Cdh1 transcriptional repressor Snail was decreased >3-fold in these cells compared to the parental tumor cells. These studies combine to suggest that SYT13 is a liver tumor suppressor gene and that its function may be mediated through pathways implicated in mesenchymal to epithelial transition.

Bansal H, Bansal S, Rao M, et al.
Heat shock protein 90 regulates the expression of Wilms tumor 1 protein in myeloid leukemias.
Blood. 2010; 116(22):4591-9 [PubMed] Free Access to Full Article Related Publications
The aberrant overexpression of Wilms tumor 1 (WT1) in myeloid leukemia plays an important role in blast cell survival and resistance to chemotherapy. High expression of WT1 is also associated with relapse and shortened disease-free survival in patients. However, the mechanisms by which WT1 expression is regulated in leukemia remain unclear. Here, we report that heat shock protein 90 (Hsp90), which plays a critical role in the folding and maturation of several oncogenic proteins, associates with WT1 protein and stabilizes its expression. Pharmacologic inhibition of Hsp90 resulted in ubiquitination and subsequent proteasome-dependant degradation of WT1. RNAi-mediated silencing of WT1 reduced the survival of leukemia cells and increased the sensitivity of these cells to chemotherapy and Hsp90 inhibition. Furthermore, Hsp90 inhibitors 17-AAG [17-(allylamino)-17-demethoxygeldanamycin] and STA-9090 significantly reduced the growth of myeloid leukemia xenografts in vivo and effectively down-regulated the expression of WT1 and its downstream target proteins, c-Myc and Bcl-2. Collectively, our studies identify WT1 as a novel Hsp90 client and support the crucial role for the WT1-Hsp90 interaction in maintaining leukemia cell survival. These findings have significant implications for developing effective therapies for myeloid leukemias and offer a strategy to inhibit the oncogenic functions of WT1 by clinically available Hsp90 inhibitors.

Essafi A, Hastie ND
WT1 the oncogene: a tale of death and HtrA.
Mol Cell. 2010; 37(2):153-5 [PubMed] Related Publications
Here, Hartkamp et al. (2010) identify WT1 as a novel bona fide substrate of the HtrA2/Omi mitochondrial protease and show that this reaction modulates WT1 antiapoptotic activity under cytotoxic stress. This supports an oncogenic function for WT1, with implications for novel chemotherapeutic avenues.

Michiels JF, Perrin C, Leccia N, et al.
PPARbeta activation inhibits melanoma cell proliferation involving repression of the Wilms' tumour suppressor WT1.
Pflugers Arch. 2010; 459(5):689-703 [PubMed] Free Access to Full Article Related Publications
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that strongly influence molecular signalling in normal and cancer cells. Although increasing evidence suggests a role of PPARs in skin carcinogenesis, only expression of PPARgamma has been investigated in human melanoma tissues. Activation of PPARalpha has been shown to inhibit the metastatic potential, whereas stimulation of PPARgamma decreased melanoma cell proliferation. We show here that the third member of the PPAR family, PPARbeta/delta is expressed in human melanoma samples. Specific pharmacological activation of PPARbeta using GW0742 or GW501516 in low concentrations inhibits proliferation of human and murine melanoma cells. Inhibition of proliferation is accompanied by decreased expression of the Wilms' tumour suppressor 1 (WT1), which is implicated in melanoma proliferation. We demonstrate that PPARbeta directly represses WT1 as (1) PPARbeta activation represses WT1 promoter activity; (2) in chromatin immunoprecipitation and electrophoretic mobility shift assays, we identified a binding element for PPARbeta in the WT1 promoter; (3) deletion of this binding element abolishes repression by PPARbeta and (4) the WT1 downstream molecules nestin and zyxin are down-regulated upon PPARbeta activation. Our findings elucidate a novel mechanism of signalling by ligands of PPARbeta, which leads to suppression of melanoma cell growth through direct repression of WT1.

Madore J, Ren F, Filali-Mouhim A, et al.
Characterization of the molecular differences between ovarian endometrioid carcinoma and ovarian serous carcinoma.
J Pathol. 2010; 220(3):392-400 [PubMed] Related Publications
The histopathological diagnosis of high-grade endometrioid and serous carcinoma of the ovary is poorly reproducible under the current morphology based classification system, especially for anaplastic, high-grade tumours. The transcription factor Wilms' tumour-1 (WT1) is differentially expressed among the gynaecological epithelia from which epithelial ovarian cancers (EOCs) are believed to originate. In EOCs, WT1 protein is observed in the majority of serous carcinomas and in up to 30% of endometrioid carcinomas. It is unclear whether the latter is a reflection of the actual incidence of WT1 protein expression in endometrioid carcinomas, or whether a significant number of high-grade serous carcinomas have been misclassified as endometrioid carcinoma. Several genetic aberrations are reported to occur in EOCs. These include mutation of the TP53 gene, aberrant activation of beta-catenin signalling and loss of PTEN protein expression, among others. It is unclear whether these aberrations are histotype-specific. The aim of this study was to better define the molecular characteristics of serous and endometrioid carcinomas in an attempt to address the problems with the current histopathological classification methods. Gene expression profiles were analysed to identify reproducible gene expression phenotypes for endometrioid and serous carcinomas. Tissue microarrays (TMA) were used to assess the incidence of TP53, beta-catenin and PTEN aberrations in order to correlate their occurrence with WT1 as an immunohistochemistry based biomarker of serous histotype. It was found that nuclear WT1 protein expression can identify misclassified high-grade endometrioid carcinomas and these tumours should be reassigned to serous histotype. Although low-grade endometrioid carcinomas rarely progress to high-grade carcinomas, a combined WT1-negative, TP53-positive immunophenotype may identify an uncommon high-grade subtype of ovarian endometrioid carcinoma. GEO database: array data accession number GSE6008.

Glienke W, Maute L, Wicht J, Bergmann L
Wilms' tumour gene 1 (WT1) as a target in curcumin treatment of pancreatic cancer cells.
Eur J Cancer. 2009; 45(5):874-80 [PubMed] Related Publications
The transcription factor WT1 plays an important role in cellular proliferation and survival of various cancer cells, and is frequently expressed in pancreatic cancer. Curcumin has been shown to be a potentially effective agent in pancreatic cancer. In this context, the purpose of this study was to determine the role of WT1 in a curcumin-treated pancreatic cancer cell line. To study the effect of curcumin on the expression of WT1, we incubated the pancreatic cancer cell line PANC-1 with different amounts of curcumin. The expression of WT1 on mRNA and protein level was measured with real-time RT-PCR and Western blot analysis. The incubation of the pancreatic cancer cell line PANC-1 with curcumin resulted in an inhibition of cellular proliferation as measured with MTT assay. The expression of WT1 on mRNA and protein level was significantly down-regulated in a concentration-dependent manner after treatment with curcumin. The WT1 mRNA levels were decreased by 20%, 25%, 40%, 78% and 88% in response to 10, 20, 30, 40 and 50 microM curcumin. The use of small inhibitory RNA (siRNA) targeting WT1 down-regulated the expression of WT1 about 90%. Combined treatment with curcumin and siRNA targeting WT1 resulted in a significant inhibition of cell proliferation compared to curcumin-treated cells alone. In conclusion, WT1 is involved in cellular proliferation of PANC-1 cells. Targeting WT1 gene expression with siRNA may enhance the efficacy of curcumin to inhibit cell proliferation.

Li H, Smolen GA, Beers LF, et al.
Adenosine transporter ENT4 is a direct target of EWS/WT1 translocation product and is highly expressed in desmoplastic small round cell tumor.
PLoS One. 2008; 3(6):e2353 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Desmoplastic Small Round Cell Tumor (DSRCT) is a highly aggressive malignancy that affects mainly adolescents and young adults. A defining characteristic of DSRCT is a specific chromosomal translocation, t(11;22)(p13;q12), that fuses EWS with WT1, leading to a production of two isoforms of chimeric transcription factor, EWS/WT1(-KTS) and EWS/WT1(+KTS). The chimeric proteins are thought to play critical roles in various stages of oncogenesis through aberrant transcription of different genes, but only a few of these genes have been identified.
METHODOLOGY/PRINCIPAL FINDINGS: We report the identification of a new target of EWS/WT1, ENT4 (equilibrative nucleoside transporter 4) which encodes a pH-dependent adenosine transporter. ENT4 is transcriptionally activated by both isoforms of EWS/WT1 as evidenced by promoter-reporter and chromatin immunoprecipitation (ChIP) analyses. Furthermore, ENT4 is highly and specifically expressed in primary tumors of DSRCT as well as in a DSRCT cell line, JN-DSRCT-1. Treatment of JN-DSRCT-1 cells with adenosine analogs, such as 2-chloro-2'-deoxyadenosine (2-CdA), resulted in an increased cytotoxic response in dose- and pH-dependent manner.
CONCLUSIONS/SIGNIFICANCE: Our detailed analyses of a novel target of EWS/WT1 in DSRCT reveal an insight into the oncogenic mechanism of EWS-fusion chromosomal translocation gene products and provide a new marker for DSRCT. Furthermore, identification of ENT4 as a highly expressed transcript in DSRCT may represent an attractive pathway for targeting chemotherapeutic drugs into DSRCT.

Klisovic RB, Stock W, Cataland S, et al.
A phase I biological study of MG98, an oligodeoxynucleotide antisense to DNA methyltransferase 1, in patients with high-risk myelodysplasia and acute myeloid leukemia.
Clin Cancer Res. 2008; 14(8):2444-9 [PubMed] Related Publications
PURPOSE: Epigenetic silencing via aberrant promoter DNA hypermethylation of normal genes has been described as a leukemogenic mechanism in myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). We hypothesized that MG98, an oligonucleotide antisense to DNA methyltransferase 1 (DNMT1), could reverse malignant phenotypes by down-regulating DNMT1 and inducing reexpression of hypermethylated genes. This phase I study was conducted to determine a biologically effective dose and describe the safety of MG98 in MDS/AML.
EXPERIMENTAL DESIGN: Twenty-three patients with MDS (n = 11) and AML (n = 12) were enrolled. Biologically effective dose was defined as the dose at which > or =50% of patients experienced >50% reduction in DNMT1 expression with acceptable toxicity. Escalating doses of MG98 were administered according to two schedules (2-hour i.v. bolus followed by 5-day continuous i.v. infusion every 14 days, or 14-day continuous i.v. infusion every 21 days).
RESULTS: DNMT1 down-regulation was observed in 8 patients. However, biologically effective dose was not reached. Reexpression of target genes (P15, WIT1, and ER) was observed in 12 patients but did not correlate with DNMT1 down-regulation. Escalation was stopped due to dose-limiting toxicities (bone pain, nausea, and fever). No objective clinical response was observed. Disease stabilization occurred in 6 (26%) patients.
CONCLUSIONS: No pharmacodynamic or clinical activity was observed at MG98 doses and schedules administered. Despite this, pursuing DNMT1 down-regulation remains a sound approach for targeting aberrant epigenetics in AML/MDS. Future studies with different formulation and/or doses and schedules will be required to ensure efficient MG98 intracellular uptake and fully evaluate its therapeutic potential.

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