Role Of WT1 Research Articles

WT1 to enhance proliferation and to impede apoptosis in KRAS-mutant NSCLC via targeting cMyc.

e22120 Background: A novel link between oncogenic KRAS signalling and WT1 was recently identified. We sought to investigate the role of WT1 and KRAS in proliferation and apoptosis. Methods: KRAS mu...

Wilms' tumor gene 1 regulates p63 and promotes cell proliferation in squamous cell carcinoma of the head and neck.

BackgroundWilms’ tumor gene 1 (WT1) can act as a suppressor or activator of tumourigenesis in different types of human malignancies. The role of WT1 in squamous cell carcinoma of the head and neck (SCCHN) is not clear. Overexpression of WT1 has been reported in SCCHN, suggesting a possible oncogenic role for WT1. In the present study we aimed at investigating the function of WT1 and its previously identified protein partners p63 and p53 in the SCCHN cell line FaDu.MethodsSilencing RNA (siRNA) technology was applied to knockdown of WT1, p63 and p53 in FaDu cells. Cell proliferation was detected using MTT assay. Chromatin immunoprecipitation (ChIP)/PCR analysis was performed to confirm the effect of WT1 on the p63 promoter. Protein co-immunoprecipitation (co-IP) was used to find protein interaction between WT1 and p53/p63. Microarray analysis was used to identify changes of gene expression in response to knockdown of either WT1 or p63. WT1 RNA level was detected using real-time quantitative PCR (RT-qPCR) in patients with SCCHN.ResultsWe found that WT1 and p63 promoted cell proliferation, while mutant p53 (R248L) possessed the ability to suppress cell proliferation. We reported a novel positive correlation between WT1 and p63 expression. Subsequently, p63 was identified as a WT1 target gene. Furthermore, expression of 18 genes involved in cell proliferation, cell cycle regulation and DNA replication was significantly altered by downregulation of WT1 and p63 expression. Several known WT1 and p63 target genes were affected by WT1 knockdown. Protein interaction was demonstrated between WT1 and p53 but not between WT1 and p63. Additionally, high WT1 mRNA levels were detected in SCCHN patient samples.ConclusionsOur findings suggest that WT1 and p63 act as oncogenes in SCCHN, affecting multiple genes involved in cancer cell growth.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1356-0) contains supplementary material, which is available to authorized users.

Cardiac endothelial cells express Wilms' tumor-1: Wt1 expression in the developing, adult and infarcted heart

Myocardial infarction is the leading cause of death worldwide. Due to their limited regenerative capacity lost cardiomyocytes are replaced by a non-contractile fibrotic scar tissue. The epicardial layer of the heart provides cardiac progenitor cells during development. Because this layer regains embryonic characteristics in the adult heart after cardiac injury, it could serve as a promising source for resident cardiac progenitor cells. Wilms' tumor-1 (Wt1) is associated with the activation and reactivation of the epicardium and therefore potentially important for the differentiation and regenerative capacity of the epicardium. To gain more insight into the regulation of Wt1 we examined the spatiotemporal expression pattern of Wt1 during murine development and after cardiac injury. Interestingly, we found that Wt1 is expressed in the majority of the cardiac endothelial cells within the myocardial ventricular layer of the developing heart from E12.5 onwards. In the adult heart only a subset of coronary endothelial cells remains positive for Wt1. After myocardial infarction Wt1 is temporally upregulated in the endothelial cells of the infarcted area and the border zone of the heart. In vitro experiments show that endothelial Wt1 expression can be induced by hypoxia. We show that Wt1 is associated with endothelial cell proliferation: Wt1 expression is higher in proliferating endothelial cells, Wt1 knockdown inhibits the proliferation of endothelial cells, and Wt1 regulates CyclinD1 expression. Finally, endothelial cells lacking Wt1 are not capable to establish a proper vascular network in vitro. Together, these results suggest a possible role for Wt1 in cardiac vessel formation in development and disease.

The level of bone marrow WT1 message is a useful marker to differentiate myelodysplastic syndromes with low blast percentage from cytopenia due to other reasons.

Myelodysplastic syndromes (MDS) are a group of hematological neoplasms associated with ineffective hematopoiesis and that transform to acute leukemia. Distinguishing MDS from other cytopenias is sometimes difficult even for trained hematologists. WT1, the gene mutated in Wilms' tumor, was found expressed in acute myeloid leukemia and MDS. The amount of WT1 in peripheral blood and bone marrow (BM) is low in low-risk MDS subtypes, and is high in high-risk MDS subtypes. However, the role of WT1 in the differential diagnosis between MDS and other diseases showing cytopenia has not been fully addressed. The present study evaluated whether WT1 expression level can assist in the differential diagnosis of MDS from other cytopenias. The amount of WT1 message was evaluated among 56 MDS patients and 47 patients with cytopenia for various other reasons (cytopenia VR) at the Nagasaki University Hospital. The level of WT1 was significantly related to the percentage of blasts in BM among MDS cases, and the type of French-American-British classification of MDS; refractory anemia (RA) cases showed significantly lower WT1 level than patients with RA with excess blasts. WT1 level was significantly related to the prognostic risk categories of MDS by the International Prognostic Scoring System (IPSS) and the revised IPSS. Although the blast percentage in the BM of RA and cytopenia VR were both less than 5%, there was a significant difference in the level of WT1 between MDS and cytopenia VR. WT1 might be a good marker to differentiate low blast percentage MDS and cytopenia VR.

Wilms' Tumor Protein Induces an Epithelial-Mesenchymal Hybrid Differentiation State in Clear Cell Renal Cell Carcinoma

The Wilms' tumor transcription factor (WT1) was originally classified as a tumor suppressor, but it is now known to also be associated with cancer progression and poor prognosis in several malignancies. WT1 plays an essential role in orchestrating a developmental process known as mesenchymal-to-epithelial transition (MET) during kidney development, but also induces the reverse process, epithelial-to-mesenchymal transition (EMT) during heart development. WT1 is not expressed in the adult kidney, but shows elevated expression in clear cell renal cell carcinoma (ccRCC). However, the role of WT1 in this disease has not been characterized. In this study, we demonstrate that WT1 is upregulated in ccRCC cells that are deficient in the expression of the von Hippel-Lindau tumor suppressor protein (VHL). We found that WT1 transcriptionally activated Snail, a master transcriptional repressor that is known to induce EMT. Although Snail represses E-cadherin and induces mesenchymal characteristics, we found partial maintenance of E-cadherin and associated epithelial characteristics in kidney cells and ccRCC cells that express WT1, since WT1 upregulates E-cadherin expression and competes with Snail repression. These findings support a novel paradigm in which WT1 induces an epithelial-mesenchymal hybrid transition (EMHT), characterized by Snail up-regulation with E-cadherin maintenance, a tumor cell differentiation state in which cancer cells keep both EMT and MET characteristics which may promote tumor cell plasticity and tumor progression.

Effects of WT1 gene downregulation on apoptosis in porcine fetal fibroblasts

Wilms' tumor gene 1 (WT1) is located on chromosome 11p13. Besides a role in the development of Wilms' tumor, specific mutations in the Zn finger region are found in Denys-Drash syndrome and Frasier syndrome, both characterized by urogenital abnormalities, sometimes in combination with Wilms' tumor. Our past study shows that WT1 is expressed in porcine kidney fibroblasts (PKFs) and swine testis cells (ST cells) and is essential for the maintenance of the development and survival of PKFs and ST cells. But we do not know whether WT1 gene was expressed in porcine fetal fibroblasts or not. To further explore whether WT1 was expressed in porcine fetal fibroblasts (PFFs) and its contribution to cell apoptosis, RT-PCR, immunocytochemical staining, and Western blot were used to detect the expression of WT1, the recombinant plasmids of pLV3-WT1 short hairpin ribonucleic acid (shRNA) were used to downregulate the WT1 gene in porcine fetal fibroblasts, and the role of WT1 in cell proliferation was examined by apoptosis analysis also. Our results indicated that WT1 was expressed in PFFs, the pLV3-WT1 shRNA dramatically reduced the expression of WT1, and downregulation of WT1 directly led to early cell apoptosis by downregulating the expression of antiapoptotic gene Bcl-2 and upregulating the expression of proapoptotic gene Bax in PFFs. Our results demonstrate that WT1 is also essential for the maintenance of the survival of PFFs.

The Wilms' tumor suppressor WT1 enhances CD95L expression and promotes activation‐induced cell death in leukemic T cells

The role of Wilms' tumor suppressor 1 (WT1) in leukemogenesis has been investigated mostly in acute (AML) and chronic (CML) myeloid leukemias. So far, its oncogenic role has been controversially discussed because both overexpression and inactivating mutations are found. A recent study on primary samples from patients with acute T-cell leukemia (T-ALL) revealed that most of them do not express WT1 proteins although they express WT1 mRNA. In our study, we investigated WT-1 expression in ten T-ALL cell lines established from leukemia/lymphoma patients. We show that consistent with the finding in primary T-ALL cells, most of the leukemic T-cell lines tested do not overexpress WT1 proteins. We found that leukemic T-cells overexpressing WT1 protein produce higher levels of CD95L and show elevated CD95L-mediated activation-induced cell death (AICD) compared to cells lacking or expressing low levels of WT1. Ectopic expression of WT1 in the WT1-nonexpressing leukemic T-cell line increases CD95L expression and elevates activation-induced apoptosis, whereas silencing WT1 expression in the WT1-overexpressing leukemic T-cell line by siRNA confers reduced CD95L expression and reduction in AICD. Chromatin immunoprecipitation and luciferase-promoter reporter analysis demonstrate that WT1 binds to and enhances CD95L promoter activity through the Egr-binding sites. Our study provides a new role of WT1 in regulation of CD95L-mediated cell death.

WT1 Promotes Cell Proliferation in Non-Small Cell Lung Cancer Cell Lines through Up-Regulating Cyclin D1 and p-pRb In Vitro and In Vivo

The Wilms’ tumor suppressor gene (WT1) has been identified as an oncogene in many malignant diseases such as leukaemia, breast cancer, mesothelioma and lung cancer. However, the role of WT1 in non-small-cell lung cancer (NSCLC) carcinogenesis remains unclear. In this study, we compared WT1 mRNA levels in NSCLC tissues with paired corresponding adjacent tissues and identified significantly higher expression in NSCLC specimens. Cell proliferation of three NSCLC cell lines positively correlated with WT1 expression; moreover, these associations were identified in both cell lines and a xenograft mouse model. Furthermore, we demonstrated that up-regulation of Cyclin D1 and the phosphorylated retinoblastoma protein (p-pRb) was mechanistically related to WT1 accelerating cells to S-phase. In conclusion, our findings demonstrated that WT1 is an oncogene and promotes NSCLC cell proliferation by up-regulating Cyclin D1 and p-pRb expression.

Abstract 775: Mutant Wilms' tumor gene 1 devoid of zinc-fingers promotes proliferation of human hematopoietic progenitor cells in vitro.

Abstract The transcription factor Wilms’ tumour gene 1 (WT1) is present in some primitive hematopoietic cells and is highly expressed in the majority of lymphoid and myeloid leukemias, suggesting a role for WT1 in leukemogenesis. Mutations within the WT1 gene are present in 10-15 % of patients with cytogenetically normal acute myeloid leukemias and may be an independent predictor of poor clinical outcome. The mutations are most often heterozygous frameshift mutations leading to premature termination of the reading frame and therefore deletion of the entire DNA-binding zinc-finger domain. We have investigated the cellular effects of mutant WT1 protein lacking all four zinc-fingers, WT1(delZ). We report that expression of WT1(delZ) prolongs the proliferation of human hematopoietic CD34+ progenitor cells in vitro. Moreover, WT1(delZ) transduced cells express erythroid markers, including hemoglobin and elevated STAT5 protein levels, in part independently of addition of erythropoietin. STAT5 was increased by a posttranscriptional mechanism. The erythroid phenotype was also observed at global gene expression level, where WT1(delZ) caused coordinated up-regulation of genes associated with erythroid maturation, as well as genes related to cell division, while monocytic and neutrophil genes were down modulated. Our results indicate that WT1(delZ) promotes cell proliferation and erythroid differentiation at the expense of monocytes and granulocytes. Citation Format: Karina Vidovic. Mutant Wilms' tumor gene 1 devoid of zinc-fingers promotes proliferation of human hematopoietic progenitor cells in vitro. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 775. doi:10.1158/1538-7445.AM2013-775

The Wilms’ tumor suppressor Wt1 regulates Coronin 1B expression in the epicardium

Coronin 1B has been shown to be critical for cell motility and various actin-dependent processes. To understand its role more extensively, the expression and transcriptional regulation of Coro1b gene during mouse development were explored. Coronin 1B is ubiquitously expressed in the whole embryo but nevertheless shows distinct expression pattern in developing heart. In addition to the localization in endocardium, Coronin 1B is specifically expressed in the endocardial cushion and epicardium where cardiac EMT processes take place as the heart develops. Promoter deletion analysis identified the positions between −1038 and −681 is important for Coro1b basal promoter activity. In addition to a correlation of Coronin 1B localization with Wt1 expression in the epicardium, we also identified putative Wt1 binding sequences within Coro1b promoter. Direct binding of Wt1 to GC-rich sequences within the Coro1b promoter is required for the regulation of Coro1b gene expression. In accordance with the motility defect found in Coronin 1B-knockdown cells, a modest decrease in expression of Coronin 1B in the remaining epicardium of Wt1EGFPCre/EGFPCre mutant embryos was observed. These findings seem to shed light on the role of Wt1 during cell migration and suggest that, at least in part, this involves transcriptional control of Coro1b gene expression.

The role of immunohistochemistry in the study of the newborn kidney

The identification of the different cell types involved in human nephrogenesis, when solely based on morphology, may lead to errors in its interpretation, given the complexity of the histological picture of the fetal and of the newborn kidney. In this study, the most recent works utilizing immunhistochemistry for the identification of the multiple cell types involved in human nephrogenesis are reviewed. The role of WT1, MUC1, Thymosin beta 10, Thymosin beta 4, CD10 and CD44 in the different phases of glomerulogenesis and of tubulogenesis is here described, with particular emphasis on their expression in the early phases of nephrogenesis. On the basis of our data, immunohistochemistry appears to be a useful tool in the study of human nephrogenesis, giving new data on the different steps of the differentiation of metanephric mesenchyme towards the multiple cell types characterizing the mature human kidney. Moreover, allowing a better knowledge of the protein products involved in the generation of new nephrons, immunohistochemistry could open new perspectives in the field of renal regenerating medicine, evidencing the factors able to prolong nephrogenesis after birth, helping us to reach our goal: allowing newborn kidneys to restore their nephron endowment, escaping susceptibility to hypertension and renal disease in adulthood.

Expression of four major WT1 splicing variants in acute and chronic myeloid leukemia patients analyzed by newly developed four real-time RT PCRs

Although the mechanism of action of leukemic oncogene Wilms' tumor gene 1 (WT1) remains unclear, WT1 has already been used in monitoring of patients with acute myeloid leukemia (AML) and it is being tested for immunotherapy. More detailed understanding of the role of WT1 in leukemia may improve its utilization. At least 36 isoforms may be produced. Four major variants denoted as -5/-KTS, -5/+KTS, +5/-KTS and +5/+KTS are produced by combining splicing of exon 5 and KTS sequence. In this study, we report applicability of newly developed real-time RT PCRs enabling for the first time full quantification of the four major WT1 splicing variants. Following careful optimization and testing of quantification reliability of four assays, we analyzed 34 samples of patients with AML and 12 samples of patients with chronic myeloid leukemia (CML) at the time of diagnosis. Analyses of five more CML patients provided insight into WT1 variants expression kinetics. We found predominance of +5/+KTS in both diagnoses. Comparison of WT1 variant expression in AML and CML patients' groups differing in response to therapy suggested possible importance of particular WT1 variant levels as markers of further disease course.

Possible Role of WT1 in a Human Fetus with Evolving Bronchial Atresia, Pulmonary Malformation and Renal Agenesis

The association of peripheral bronchial atresia and congenital pulmonary airway malformation (CPAM) has recently been recognised, but the pathology of the lesions evolving together has not been described. We present autopsy findings in a 20 week fetus showing areas of peripheral bronchial destruction and airway malformation consistent with developing CPAM in the right lung supporting a causal relationship between these lesions. This fetus also had congenital heart defect, bilateral renal agenesis and syndactyly. We identified another fetus from our autopsy files, with bilateral renal agenesis, similar right sided pulmonary malformation and cardiac defects. Similar bilateral renal agenesis and defects of the heart and lungs are found in wt1(-/-) mice and we have investigated the expression of WT1 in these fetuses. We hypothesise that the cardiac, liver, renal and possibly lung lesions in these two cases may arise due to mesenchymal defects consequent to WT1 misexpression and discuss evidence for this from the scientific literature. We used immunoperoxidase stains to analyse WT1 expression in autopsy hepatic tissue in both fetuses. We also investigated the expression of α-smooth muscle actin (α-SMA), a marker of activated hepatic stellate cells/myofibroblasts, and desmin in hepatic mesenchyme and compare these findings with control fetuses, without congenital malformations. We found reduced WT1 expression in hepatic mesothelium in both fetuses with malformations. There was also increased expression of α-SMA in liver perisinusoidal cells, as seen in the wt1(-/-) mouse model. We therefore propose that abnormality of WT1 signalling may be an underlying factor, as WT1 is expressed in coelomic lining cells from which mesenchyme is derived in many organs.

Abstract 5007: WT1 promotes tumor growth and vascularization by regulating matrix metalloproteinase 9 in Ewing's sarcoma xenografts

Abstract WT1, a zinc-finger transcription factor, is expressed in tumors of varied origins including sarcomas. High level WT1 expression often correlates with poor patient survival. The role of WT1 in sarcoma biology and the mechanism by which high WT1 expression confers a poor prognosis are both unclear. The aim of this study is to investigate the effect of WT1 expression on sarcoma growth and angiogenesis. We transfected the WT1-null Ewing's sarcoma cell line SK-ES-1 with one of two major WT1 isoforms, and used shRNA to silence WT1 expression in the WT1-positive Ewing's sarcoma cell line MHH-ES. Cells were injected into the flanks of immune deficient mice and tumor growth was monitored. Tumors arising fromWT1-expressing SK-ES-1 cells grew faster than control, while tumors arising from WT1-silenced MHH-ES cells grew more slowly than control. Because our laboratory and others have shown that WT1 regulates VEGF expression, we evaluated tumor vascularity using the endothelial cell marker CD31 and the pericyte marker αSMA. In xenografted SK-ES-1 cells, quantification of CD31 positive area in WT1 expressing tumors showed 60 -70 % more compared to the controls. In contrast, CD31 expression is abolished in tumors arising from WT1-silenced MHH-ES cells. To investigate the mechanism by which WT1 affects tumor growth and vascularity, we performed a gene expression profiling in SK-ES-1 cells expressing different isoforms of WT1. We identified a number of genes up- or downregulated by WT1, including matrix metalloproteinase-9 (MMP9). Luciferase reporter assays showed that WT1 can upregulate MMP9 promoter activity in transiently transfected NIH3T3 cells. Also, immunohistochemical analysis showed increased MMP9 expression in WT1-expressing tumors. Finally, imunohistochemical analysis of primary Ewing's sarcoma samples showed correlated expression of WT1, VEGF, and MMP9. Taken together, our data suggest that WT1 promotes tumor growth and angiogenesis, perhaps via upregulation of MMP9. Increased tumor angiogenesis may lead to more aggressive tumors and a worse outcome for patients, explaining the effect of WT1 on prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5007. doi:10.1158/1538-7445.AM2011-5007

Abstract 2169: The effect of WT1 on E-cadherin expression in prostate cancer cells

Abstract Prostate carcinoma is the most common malignancy and second leading cause of death among American men. The molecular mechanisms that control the progression of this type of cancer are still poorly understood. One of the genes expressed in prostate cancer epithelium is the zinc finger transcription factor Wilms’ Tumor 1, WT1. Previous studies in our lab have shown WT1 protein in a majority of high grade prostate tumor sections with little or no WT1 staining in non-neoplastic or benign prostatic hyperplasia (BPH) tissues. However, a mechanistic role for WT1 in prostate cancer has not been established. Recently, WT1 has been associated with the regulation of cell adhesion molecules such as E-cadherin in NIH 3T3 cells and epicardial cells. Loss of E-cadherin expression is frequently associated with increased cellular motility and tumor invasion. Several regulatory mechanisms controlling E-cadherin gene expression have been proposed in breast cancer cells, however the mechanisms of regulation of E-cadherin gene expression in prostate cancer cells are not yet understood. The objective of this study was to determine whether WT1 might regulate E-cadherin expression and contribute to cancer progression in prostate cancer cells. First, potential WT1 binding sites were identified in the promoter of the E-cadherin gene using a bioinformatics approach. Chromatin Immunoprecipitation (ChIP) showed direct in vivo binding of WT1 to the E-cadherin promoter in the chromatin of LNCaP and PC3 cells. The effect of transfection of prostate cancer cells with a GFP/WT1 expression construct was then tested by quantitative real-time PCR (QRT-PCR). QRT-PCR results showed a decrease in E-cadherin transcripts in GFP/WT1 transfected prostate cancer cells. Conversely, knockdown of WT1 mRNA in siWT1 transfected LNCaP cells, showed increased levels of E-cadherin mRNA. Moreover, co-transfection of WT1 expression construct with an E-cadherin promoter reporter construct showed that WT1 decreased the activity of the proximal E-cadherin promoter in PC3 cells. Overall these results demonstrated that WT1 modulated E-cadherin expression in prostate cancer cells and suggests a novel mechanism whereby WT1 may increase migration by decreasing E-cadherin levels, a novel role for WT1 mediated prostate cancer progression. This work was supported by NIHR15CA11360 and Ohio Board of Regents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2169. doi:10.1158/1538-7445.AM2011-2169

Abstract SY01-03: Comparative genomics as a tool for the identification of novel Kras synthetic lethal interactions

Abstract Gene expression profiling has been widely used as a tool to characterize genome-wide changes in expression level in various cancers. Expression profiling is generally used to identify specific genes with altered expression or to identify patterns that distinguish cancer subtypes. More recently, several approaches to use expression data to identify interactions between genes or build cancer-specific networks have been described. We have previously demonstrated that cross-species gene expression analysis can be used as a tool to identify oncogene-specific alterations in gene expression. Cross-species analysis is useful for this because it allows for filtering of noise inherent in human gene expression analysis. The identification of an oncogenic Kras-specific gene expression signature was accomplished using this approach. A central focus of our laboratory has been to use this and other oncogene-specific signatures to identify new critical downstream pathways necessary for oncogene function. Using several computational approaches, we identified several upstream transcriptional regulators of our previously identified oncogenic Kras signature. We hypothesized that some of the transcriptional regulators represent specific dependencies that are required for Kras to function as an oncogene. Thus, inhibition of the transcription factors could uncover novel synthetic lethal interactions specific for cells that express oncogenic Kras. Using an shRNA pooled library, we have confirmed one such synthetic lethal interaction betweeen oncogenic Kras and the Wilms-tumor 1 (Wt1), a gene known to function as a tumor suppressor in some contexts. Loss of Wt1 induces senescence in primary cells expressing oncogenic Kras. Human non-small cell lung cancer cells expressing oncogenic Kras also become senescent when Wt1 is knocked-down using shRNA. Thus, synthetic senescence is induced on Wt1 loss in oncogenic Kras expressing cells. A role for Wt1 in Kras-driven oncogenesis was further confirmed using a mouse model for lung cancer in which a mouse carrying a conditional Kras allele was crossed a mouse carrying an allelel that allows for conditional deletion of Wt1. The biological signficance of this intereation was further demonstrated by using a “Wt1 “high” or “Wt1 low” signature to classify human NSCLC gene expression profiles. This allowed us to determine that human tumor samples that express oncogenic Kras and express high levels of Wt1-regulated genes have a worse prognosis compared to samples expressing oncogenic Kras but having low low level of Wt1-regulated genes. In human NSCLC cell lines, loss of Wt1 leads to significant upregulation of the cell cycle regulator p21. Wt1 appears to regulate p21 levels posttranscriptionally, as p21 mRNA levels did not change. Further studies to elucidate the mechanisms leading to p21 upregulation upon Wt1 loss are underway and preliminary results will be discussed. To take these studies further, we began by generating a more refined Kras-specific signature using FACs-sorted tumor cells from the LSL-KrasG12D mouse model crossed to a fluorescent reporter. We have used this mouse-generated Kras signature for cross-species analysis using a much larger NSCLC dataset than was possible in our previously published studies. We then applied the ARACNE algorithm to identify transcription factors that regulate this Kras-signature. Using this approach, we have identified other transcription factors that are potential regulators of oncogenic Kras. The fact that Wt1 once again was identified as a regulator of oncogenic Kras using this novel method suggests the validity of this approach. Preliminary results testing the functional significance of these transcription factors for Kras-driven oncogenesis will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY01-03. doi:10.1158/1538-7445.AM2011-SY01-03

The podocyte as a target: cyclosporin A in the management of the nephrotic syndrome caused by WT1 mutations

Children with steroid-resistant nephrotic syndrome secondary to WT1-associated glomerulopathies (WT1-GP) were considered unresponsive to cyclosporin A (CsA). This assumption is challenged by the findings of recent studies. The patients of these studies had different types of WT1 mutations and varying clinical presentations. However, all of them were of young age and the favourable response to CsA might be the result of treatment at an early stage of the disease. The additional administration of angiotensin-converting enzyme inhibitors may have contributed to the positive outcome. We review recent data on the role of WT1 in the development of WT1-GP and discuss putative therapeutic targets explaining the therapeutic effect of CsA.

The role of WT1 gene in neuroblastoma

Background/Purpose The oncogenic properties of the Wilms' tumor gene ( WT1) have recently been reported in various malignancies. However, the role of WT1 in pediatric tumors is unclear. To elucidate the role of WT1 in the development of neuroblastoma (NB), we examined the WT1 expression in NB and the effect of WT1 suppression on NB cell proliferation. Methods We examined the expression of the WT1 protein in 20 NBs and 5 ganglioneuromas (GNs) by performing immunohistochemical analysis. We determined WT1 messenger RNA expression in 22 NBs, 5 GNs, and 4 NB cell lines by real-time reverse transcription polymerase chain reaction. We studied the effects of WT1 suppression on cell proliferation using small interfering RNA against WT1. Results Expression of WT1 was higher in mature ganglionic cells, and in the immunohistochemical analysis, the WT1 positivity for GNs was significantly higher than that for NBs ( P < .01). The level of WT1 messenger RNA expression did not correlate with histologic grade, clinical stage, and prognosis of the tumor. Knockdown of WT1 gene promoted the proliferation of NB69 cells ( P < .01). Conclusions The WT1 may govern cell differentiation and suppress cell proliferation in NB. The WT1 does not act as an oncogene, but it may participate in the maturation of NB.

Identifying a role for WT1 in pediatric leukemia.

e20005 Background: Leukemia is the most common malignancy in the pediatric age group. To understand the molecular basis of leukemogenesis we examined gene expression patterns. One of the genes expr...

Abstract 1237: WT1, Wnt signaling and E-cadherin in prostate cancer

Abstract Prostate carcinoma is the most common malignancy and second leading cause of death among American men. The molecular mechanisms that control the progression of this type of cancer are still poorly understood. Among the genes proposed to play a role in prostate cancer is the zinc finger transcription factor, WT1. Previous studies in our lab have shown WT1 protein in a majority of high grade prostate tumor sections with little or no WT1 staining in non-neoplastic or benign prostatic hyperplasia (BPH) tissues. However, a mechanistic role for WT1 in prostate cancer has not been established. Recently, WT1 has been associated with the Wnt signaling pathway and Beta catenin, the central molecule in the pathway. The cytoplasmic levels of Beta catenin can be regulated through the canonical Wnt signaling or through the intracellular adhesion complex where it binds to E-cadherin. Importantly, E-cadherin has previously been identified as a WT1 target gene in NIH 3T3 cells. The objective of this study was to determine whether WT1 might regulate genes that, in turn, regulate the levels of Beta catenin in prostate cancer cells. First potential WT1 binding sites were identified in the promoters of two candidate genes, GSK3 Beta and E-cadherin, using a bioinformatics approach. Then the effect of transfection of LNCaP prostate cancer cells with GFP-tagged WT1 expression constructs was tested by quantitative real time PCR (QRT-PCR). In vivo evidence of direct binding of these gene promoters by WT1 protein was obtained by Chromatin immunoprecipitation (ChIP) of GFP/WT1 transfected LNCaP cells. Using evolutionary conservation analysis we identified two WT1 binding sites in the E-cadherin promoter conserved among primate species and one that was conserved among primates, rodents and dog. QRT- PCR results showed a decrease in E-cadherin transcripts in GFP/WT1 transfected LNCaP cells, compared with untransfected cells. ChIP was used to demonstrate that this effect on E-cadherin expression was direct and mediated by DNA binding to the E-cadherin promoter in vivo. Overall these results suggest that WT1 modulates E-cadherin and the Wnt signaling pathway in LNCaP cells. These findings suggest a novel mechanism for proliferation and migration of prostate cancer cells and ultimately may contribute towards an improved cancer therapy. This work was supported by NIHR15CA11360 (GF) and GSS KSU grant(AB) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1237.