WT1 Silencing Research Articles

Cardiomyocyte-Specific Wt1 Is Involved in Cardiac Metabolism and Response to Damage.

The Wilms tumor suppressor gene (Wt1) encodes a C2H2-type zinc-finger transcription factor that participates in transcriptional regulation, RNA metabolism, and protein-protein interactions. WT1 is involved in the development of several organs, including the kidneys and gonads, heart, spleen, adrenal glands, liver, diaphragm, and neuronal system. We previously provided evidence of transient WT1 expression in about 25% of cardiomyocytes of mouse embryos. Conditional deletion of Wt1 in the cardiac troponin T lineage caused abnormal cardiac development. A low expression of WT1 has also been reported in adult cardiomyocytes. Therefore, we aimed to explore its function in cardiac homeostasis and in the response to pharmacologically induced damage. Silencing of Wt1 in cultured neonatal murine cardiomyocytes provoked alterations in mitochondrial membrane potential and changes in the expression of genes related to calcium homeostasis. Ablation of WT1 in adult cardiomyocytes by crossing αMHCMerCreMer mice with homozygous WT1-floxed mice induced hypertrophy, interstitial fibrosis, altered metabolism, and mitochondrial dysfunction. In addition, conditional deletion of WT1 in adult cardiomyocytes increased doxorubicin-induced damage. These findings suggest a novel role of WT1 in myocardial physiology and protection against damage.

Abstract 2707: Therapeutic effects of WT1 silencing by respiratory administration of liposomal siRNA in a melanoma murine model

Abstract Introduction: Cancer is considered among the main causes of death worldwide and the number of cases keeps rising every year, as there is not an effective treatment to offer for most of them. The lungs represent a frequent target of primary and metastatic tumors due to his high oxygenation and large air spaces to grow. Melanoma is the most aggressive type of skin cancer showing a high affinity to metastasize into the lungs, being the overexpression of the WT1 protein associated with the development of this cancer, as well as several other types. Methods: In this study, we evaluated the effects of siRNA silencing the WT1 protein in both, in vitro in the B16F10 melanoma cell line, and in vivo in a murine model of pulmonary metastatic melanoma implementing a novel delivery strategy of neutral DOPC liposomal-siRNA system administrated via inhalation. Results: In vitro studies showed an effective silencing of WT1 protein (86%) in the siRNAsWT1 treated cells when compared with controls, resulting in a loss of viability (40%) and proliferation (49%) of the cells, induced G1 cell-cycle arrest (11%), a restriction of migration (87%) and invasion (100%) capacities, as well as an activation of apoptosis (12%). In vivo, we tested our delivery system obtaining an efficient biodistribution in lungs, kidney, liver and spleen. The respiratory administration of LWT1siRNA after two weeks of treatment resulted in silencing of WT1 protein (59%), achieving an important antitumor activity with a 44% reduction in tumor weight. In the survival study 80% of the L-WT1siRNA treated mice were alive after all the controls mice had died, delaying the death almost a week at the end of the study. Conclusions: This work demonstrates the efficacy of the neutral DOPC liposomal-siRNA delivery system by respiratory administration as a novel therapy to reduce the malignancy of pulmonary tumors and to increase survivability by silencing specific cancer biomarkers as WT1. Citation Format: Martin Ramos-Gonzalez, Eduardo Vázquez-Garza, Gerardo García-Rivas, Cristian Rodríguez-Aguayo, Arturo Chávez-Reyes. Therapeutic effects of WT1 silencing by respiratory administration of liposomal siRNA in a melanoma murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2707.

WT1 regulates HOXB9 gene expression in a bidirectional way

The homeoboxB9 (HOXB9) gene is necessary for specification of the anterior-posterior body axis during embryonic development and expressed in various types of cancer.Here we show that the Wilms tumor transcription factor WT1 regulates the HOXB9 gene in a bidirectional manner. Silencing of WT1 activates HOXB9 in Wt1 expressing renal cell adenocarcinoma-derived 786-0 cells, mesonephric M15 cells and ex vivo cultured murine embryonic kidneys. In contrast, HOXB9 expression in U2OS osteosarcoma and human embryonic kidney (HEK) 293 cells, which lack endogenous WT1, is enhanced by overexpression of WT1. Consistently, Hoxb9 promoter activity is stimulated by WT1 in transiently transfected U2OS and HEK293 cells, but inhibited in M15 cells with CRISPR/Cas9-mediated Wt1 deletion. Electrophoretic mobility shift assay and chromatin immunoprecipitation demonstrate binding of WT1 to the HOXB9 promoter in WT1-overexpressing U2OS cells and M15 cells. BASP1, a transcriptional co-repressor of WT1, is associated with the HOXB9 promoter in the chromatin of these cell lines. Co-transfection of U2OS and HEK293 cells with BASP1 plus WT1 prevents the stimulatory effect of WT1 on the HOXB9 promoter.Our findings identify HOXB9 as a novel downstream target gene of WT1. Depending on the endogenous expression of WT1, forced changes in WT1 can either stimulate or repress HOXB9, and the inhibitory effect of WT1 on transcription of HOXB9 involves BASP1. Consistent with inhibition of Hoxb9 expression by WT1, both transcripts are distributed in an almost non-overlapping pattern in embryonic mouse kidneys. Regulation of HOXB9 expression by WT1 might become relevant during kidney development and cancer progression.

KDM6B Inhibition Has Anti-Tumor Activity in Anaplastic Wilms Tumor Cells Associated with WT1 And MYCN Pathway Silencing

KDM6B Inhibition Has Anti-Tumor Activity in Anaplastic Wilms Tumor Cells Associated with WT1 And MYCN Pathway Silencing

MiR193a Modulation and Podocyte Phenotype

Apolipoprotein L1 (APOL1)-miR193a axis has been reported to play a role in the maintenance of podocyte homeostasis. In the present study, we analyzed transcription factors relevant to miR193a in human podocytes and their effects on podocytes’ molecular phenotype. The motif scan of the miR193a gene provided information about transcription factors, including YY1, WT1, Sox2, and VDR-RXR heterodimer, which could potentially bind to the miR193a promoter region to regulate miR193a expression. All structure models of these transcription factors and the tertiary structures of the miR193a promoter region were generated and refined using computational tools. The DNA-protein complexes of the miR193a promoter region and transcription factors were created using a docking approach. To determine the modulatory role of miR193a on APOL1 mRNA, the structural components of APOL1 3’ UTR and miR193a-5p were studied. Molecular Dynamic (MD) simulations validated interactions between miR193a and YY1/WT1/Sox2/VDR/APOL1 3′ UTR region. Undifferentiated podocytes (UPDs) displayed enhanced miR193a, YY1, and Sox2 but attenuated WT1, VDR, and APOL1 expressions, whereas differentiated podocytes (DPDs) exhibited attenuated miR193a, YY1, and Sox2 but increased WT1, VDR, APOL1 expressions. Inhibition of miR193a in UPDs enhanced the expression of APOL1 as well as of podocyte molecular markers; on the other hand, DPD-transfected with miR193a plasmid showed downing of APOL1 as well as podocyte molecular markers suggesting a causal relationship between miR193a and podocyte molecular markers. Silencing of YY1 and Sox2 in UPDs decreased the expression of miR193a but increased the expression of VDR, and CD2AP (a marker of DPDs); in contrast, silencing of WT1 and VDR in DPDs enhanced the expression of miR193a, YY1, and Sox2. Since miR193a-downing by Vitamin D receptor (VDR) agonist not only enhanced the mRNA expression of APOL1 but also of podocyte differentiating markers, suggest that down-regulation of miR193a could be used to enhance the expression of podocyte differentiating markers as a therapeutic strategy.

The Wilms tumor protein WT1 stimulates transcription of the gene encoding insulin-like growth factor binding protein 5 (IGFBP5)

Insulin-like growth factor (IGF) binding proteins (IGFBPs) constitute a family of six secreted proteins that regulate the signaling of insulin-like growth factors (IGFs). IGFBP5 is the most conserved family member in vertebrates and the major IGF binding protein in bone. IGFBP5 is required for normal development of the musculoskeletal system, and various types of cancer frequently express high levels of IGFP5. Here we identify the gene encoding IGFBP5 as a novel downstream target of the Wilms tumor protein WT1. IGFBP5 and WT1 are expressed in an overlapping pattern in the condensing metanephric mesenchyme of embryonic murine kidneys. Down-regulation of WT1 by transfection with antisense vivo-morpholino significantly decreased Igfbp5 transcripts in murine embryonic kidney explants. Likewise, silencing of Wt1 in a mouse mesonephros-derived cell line reduced Igfbp5 mRNA levels by approximately 80%. Conversely, induction of the WT1(−KTS) isoform, whose role as transcriptional regulator has been firmly established, significantly increased IGFBP5 mRNA and protein levels in osteosarcoma cells. IGFBP5 expression was not significantly changed by WT1(+KTS) protein, which exhibits lower DNA binding affinity than the WT1(−KTS) isoform and has a presumed role in post-transcriptional gene regulation. Luciferase reporter constructs harboring 0.8 and 1.6 kilobases of the murine Igfbp5 promoter, respectively, were stimulated approximately 5-fold by co-transfection of WT1(−KTS). The WT1(+KTS) variant had no significant effect on IGFBP5 promoter activity. Binding of WT1(−KTS), but not of WT1(+KTS) protein, to the IGFBP5 promoter in human osteosarcoma cells was proven by chromatin immunoprecipitation (ChIP) and confirmed by electrophoretic mobility shift assay. These findings demonstrate that WT1 activates transcription of the IGFBP5 gene with possible implications for kidney development and bone (patho)physiology.

Repression of CMIP transcription by WT1 is relevant to podocyte health

The WT1 (Wilm's tumor suppressor) gene is expressed throughout life in podocytes and is essential for the functional integrity of the glomerular filtration barrier. Wehave previously shown that CMIP (C-Maf inducing protein) is overproduced in podocyte diseases and alters intracellular signaling. Here we isolated the proximal region of the human CMIP promoter and showed by chromatin immunoprecipitation assays and electrophoretic-mobility shift that Wilm's tumor protein (WT1) bound to 2 WT1 response elements, located at positions -290/-274 and -57/-41 relative to transcription start site. Unlike the human CMIP gene, only one Wt1 response element was identified inthe mouse Cmip proximal promoter located at position -217/-206. Luciferase reporter assays indicated that WT1 dose-dependently inhibited the transcriptional induction of the CMIP promoter. Transfection of decoy oligonucleotides mimicking the WT1 response elements prevented the inhibition of WT1 on CMIP promoter activity. Furthermore, WT1 silencing promoted Cmip expression. In line with these findings, the abundance of Cmip was early and significantly increased at the transcript and protein level in podocytes displaying a primary defect in Wt1, including Denys-Drash syndrome and Frasier syndrome. Thus, WT1 is a major repressor of the CMIP gene in physiological situations, while conditional deletion of CMIP in the developing kidney did not affect the development of mature glomeruli.

DNA Hydroxymethylation Profiling Reveals that WT1 Mutations Result in Loss of TET2 Function in Acute Myeloid Leukemia

Somatic mutations in IDH1/IDH2 and TET2 result in impaired TET2-mediated conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). The observation that WT1 inactivating mutations anticorrelate with TET2/IDH1/IDH2 mutations in acute myeloid leukemia (AML) led us to hypothesize that WT1 mutations may impact TET2 function. WT1 mutant AML patients have reduced 5hmC levels similar to TET2/IDH1/IDH2 mutant AML. These mutations are characterized by convergent, site-specific alterations in DNA hydroxymethylation, which drive differential gene expression more than alterations inDNA promoter methylation. WT1 overexpression increases global levels of 5hmC, and WT1 silencing reduced 5hmC levels. WT1 physically interacts with TET2 and TET3, and WT1 loss of function results in a similar hematopoietic differentiation phenotype as observed with TET2 deficiency. These data provide a role for WT1 in regulating DNA hydroxymethylation and suggest that TET2 IDH1/IDH2 and WT1 mutations define an AML subtype defined by dysregulated DNA hydroxymethylation.

Transcriptional Regulation by the Wilms Tumor Protein, Wt1, Suggests a Role of the Metalloproteinase Adamts16 in Murine Genitourinary Development

ADAMTS16 (a disintegrin and metalloproteinase with thrombospondin motifs) is a secreted mammalian metalloproteinase with unknown function. We report here that murine Adamts16 is co-expressed with the Wilms tumor protein, Wt1, in the developing glomeruli of embryonic kidneys. Adamts16 mRNA levels were significantly reduced upon transfection of embryonic murine kidney explants with Wt1 antisense vivo-morpholinos. Antisense knockdown of Adamts16 inhibited branching morphogenesis in kidney organ cultures. Adamts16 was detected by in situ mRNA hybridization and/or immunohistochemistry also in embryonic gonads and in spermatids and granulosa cells of adult testes and ovaries, respectively. Silencing of Wt1 by transfection with antisense vivo-morpholinos significantly increased Adamts16 mRNA in cultured embryonic XY gonads (11.5 and 12.5 days postconception), and reduced Adamts16 transcripts in XX gonads (12.5 and 13.5 days postconception). Three predicted Wt1 consensus motifs could be identified in the promoter and the 5'-untranslated region of the murine Adamts16 gene. Binding of Wt1 protein to these elements was verified by EMSA and ChIP. A firefly luciferase reporter gene under control of the Adamts16 promoter was activated ∼8-fold by transient co-transfection of human granulosa cells with a Wt1 expression construct. Gradual shortening of the 5'-flanking sequence successively reduced and eventually abrogated Adamts16 promoter activation by Wt1. These findings demonstrate that Wt1 differentially regulates the Adamts16 gene in XX and XY embryonic gonads. It is suggested that Adamts16 acts immediately downstream of Wt1 during murine urogenital development. We propose that Adamts16 is involved in branching morphogenesis of the kidneys in mice.

The Wilms’ tumor gene (WT1) regulates E-cadherin expression and migration of prostate cancer cells

BackgroundOne key step in the development of prostate cancer (PCa) metastasis is the loss of E-cadherin expression associated with increased cellular motility and tumor invasion. This loss of E-cadherin expression is also required during normal embryogenesis and similar transcriptional repressors have been identified in both processes. We have previously reported the presence of one such transcription factor, WT1 in high Gleason grade prostate tumor tissues, and its absence in non-neoplastic or benign prostatic hyperplasia tissues.ResultsTo better understand the effect of WT1 on E-cadherin expression and migration of PCa cells we quantified WT1 and E-cadherin mRNA levels in normal prostate epithelial and PCa cell lines with varying migratory potential. In WT1 transfected cells E-cadherin transcript levels were decreased, while they were increased in siWT1-RNA transfected PCa cells, suggesting that elevated WT1 expression was sufficient to dampen E-cadherin levels and potentially enhance migratory ability. To delineate the mechanism of WT1-mediated repression of E-cadherin, potential WT1 binding sites were tested in vitro and in vivo binding of WT1 to the E-cadherin promoter in the chromatin of LNCaP and PC3 cells was assessed by Chromatin Immunoprecipitation. The effect of WT1 binding was measured in reporter assays; in PC3 and DU145 cells WT1 decreased the activity of the proximal E-cadherin promoter. Using site-directed mutagenesis, a newly identified WT1 binding site located 146 bp from the transcription start site was shown to be required for this repression by WT1. Transwell migration and wound healing assays revealed that in LNCaP cells with low migratory potential, over-expression of WT1 was sufficient to enhance migration, conversely, in the highly migratory PC3 cells silencing of WT1 decreased migration.ConclusionsThese findings suggested that WT1 expression in high grade prostate cancer may contribute to migration and metastasis. Thus, in prostate cancer WT1 may function as a novel oncogene facilitating development of the lethal metastatic phenotype.

Abstract 1303: The Wilms’ tumor gene, WT1, regulates E-cadherin expression and migration of prostate cancer cells.

Abstract Carcinoma of the prostate is one of the most common malignancies and second leading cause of death among American men. Even though patients with localized disease have high survival rates, patients with detectable metastasis have a median survival of only 12-15 months. One key step in the metastatic process is the loss of E-cadherin expression associated with tumor invasion. However the regulation of E-cadherin gene expression in prostate cancer (PrCa) cells is not well understood. Recently, the zinc finger transcription factor, WT1 was shown to regulate E-cadherin in NIH 3T3 cells and epicardial cells. We have previously reported the presence of WT1 protein in high grade prostate tumor sections, with little or no WT1 staining in non-neoplastic or benign prostatic hyperplasia (BPH) tissues. However, the significance of WT1 expression in prostate cancer has not been established. The objective of this study was to determine whether WT1 might contribute to increase migration of PrCa cells by repressing E-cadherin expression. Using quantitative real-time PCR (QRTPCR), WT1 and E-cadherin mRNA levels were measured and found to be inversely related in PrCa cells. Similarly, QRTPCR results showed a decrease in E-cadherin mRNA in GFP/WT1 transfected PrCa cells, while E-cadherin levels were increased in PrCa cells with suppressed WT1 expression. Titration of WT1 levels also affected the migration of PrCa cells in wound healing and transwell migration assays. Silencing of WT1 by siWT1 RNA decreased the migratory potential of PC3 cells and the overexpression of GFP/WT1 increased the migration of LNCaP cells. A bioinformatics approach was used to identify potential WT1 binding sites in E-cadherin promoter, then direct in vivo binding of WT1 to the E-cadherin promoter in the chromatin of PrCa cells was confirmed by Chromatin Immunoprecipitation. Transcriptional control was quantified by co-transfection of a GFP/WT1 expression construct with E-cadherin promoter constructs. Results showed that WT1 decreased the activity of the proximal E-cadherin promoter in PC3 cells and DU145 cells. Using site directed mutagenesis, a newly identified WT1 binding site located 146 bp from the transcription start site was shown to be required for the repression of the E-cadherin proximal promoter by WT1. Overall these results demonstrate that WT1 transcriptionally down-regulates E-cadherin expression and thereby increases migration of PrCa cells. This finding suggests that the biological significance of WT1 expression in high grade prostate cancer lies in its contribution to cell migration and potentially to the promotion of metastasis. Supported by NIHR15CA11360 and the Ohio Board of Regents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1303. doi:1538-7445.AM2012-1303

WT1 silencing by RNAi synergizes with chemotherapeutic agents and induces chemosensitization to doxorubicin and cisplatin in B16F10 murine melanoma cells

The Wilm's tumor gene (WT1), encoding a transcription factor that modulates the expression of certain genes that are involved in proliferation and apoptosis, is overexpressed in numerous solid tumors. WT1 is important for cell proliferation and in the diagnosis of melanoma. The objectives of this study were to investigate whether WT1 silencing is capable of synergizing with chemotherapeutic agents and whether this silencing is capable of sensitizing cancer cells to doxorubicin and cisplatin in the B16F10 murine melanoma cell line. In the present study, B16F10 cells were simultaneously treated with median lethal doses (LD50s) of WT1-1 or WT1-2 small hairpin RNAs (shRNAs) and chemotherapeutic agents. A total of 24 h post-transfection, a [3-(4,5-dimethylthiazol-2yl)-2,5- diphenyl tetrazolium bromide assay] MTT assay was performed. To determine whether shRNA interference (shRNAi) is capable of sensitizing B16F10 cells to chemotherapeutic agents, cells were transfected with an LD50 of each of the recombinant plasmids, treated with varying concentrations of doxorubicin or cisplatin 24 h post-transfection, and analyzed 48 h later for inhibition of cell proliferation using the MTT assay. We observed that WT1-RNAi and the two chemotherapeutic agents acted synergistically to inhibit B16F10 cell proliferation. The greatest inhibition of cell proliferation was observed with the WT1-2/cisplatin (91%) and WT1-1/cisplatin combinations (85%). WT1 silencing using shRNAi induced the chemosensitization of cells to doxorubicin and cisplatin, with the greatest inhibition (85%) of cell proliferation being observed in the cells treated with the WT1-2/cisplatin 6ng/µl combination. Our results provide direct evidence that WT1 gene silencing has a synergistic effect with chemotherapeutic drugs and sensitizes B16F10 melanoma cells to doxorubicin and cisplatin. This suggests that these combination strategies are potentially utilized in melanoma therapy.

MiR-15a and miR-16-1 inhibit the proliferation of leukemic cells by down-regulating WT1 protein level

BackgroundmiR-15a and miR-16-1(miR-15a/16-1) have been implicated as tumor suppressors in chronic lymphocytic leukemia, multiple myeloma, and acute myeloid leukemic cells. However the mechanism of inhibiting the proliferation of leukemic cells is poorly understood.MethodsK562 and HL-60 cells were transfected with pRS-15/16 or pRS-E, cell growth were measured by CCK-8 assay and direct cell count. Meanwhile WT1 protein and mRNA level were measured by Western blotting and quantitative real-time PCR.ResultsIn this study we found that over-expression of miR-15a/16-1 significantly inhibited K562 and HL-60 cells proliferation. Enforced expression of miR-15a/16-1 in K562 and HL-60 cells significantly reduced the protein level of WT1 but not affected the mRNA level. However enforced expression of miR-15a/16-1 can not reduce the activity of a luciferase reporter carrying the 3'-untranslated region(3'UTR) of WT1. Silencing of WT1 by specific siRNA suppressed leukemic cells proliferation resembling that of miR-15a/16-1 over-expression. Anti-miR-15a/16-1 oligonucleotides (AMO) reversed the expression of WT1 in K562 and HL-60 cells. Finally, we found a significant inverse correlation between miR-15a or miR-16-1 expression and WT1 protein levels in primary acute myeloid leukemia (AML) blasts and normal controls.ConclusionsThese data suggest that miR-15a/16-1 may function as a tumor suppressor to regulate leukemic cell proliferation potentially by down-regulating the WT1 oncogene. However WT1 is not directly targeted by miR-15a/16-1 through miRNA-mRNA base pairing, therefore more study are required to understand the mechanism by which miR-15a/16-1 downregulate WT1.

Myocardial ischemia and hypoxia exert opposite effects on the expression of amiloride binding protein 1 (Abp1)

The polyamine putrescine is required for normal cell growth and division. Putrescine formation is enhanced during myocardial ischemia presumably due to increased ornithine decarboxylase activity. The goal of this study was to assess the role of the diamine oxidase Abp1 in regulating putrescine degradation in the ischemic rat heart. At 24 h, 3 and 6 weeks after myocardial infarction Abp1 mRNA was significantly reduced compared to sham‐operation. Inhibition of Abp1 by amiloride prevented the in vitro differentiation of C2C12 myoblasts. Exposure of mice at 8% O2 (6 h) increased Abp1 mRNA in the hearts 11‐fold (P<0.02, n=5). Abp1 mRNA was also increased in several cell lines at 1% O2 (24 h). Reporter gene assays revealed an approx. 3,5‐fold stimulation of the Abp1 promoter in transfected C2C12 myoblasts at 1% O2. Promoter activity was also significantly enhanced by the Wilms' tumor transcription factor (Wt1), which is activated in hypoxia and controls myocardial blood vessel formation. Abp1 transcripts were reduced 60% in the hearts of Wt1‐deficient vs. wild‐type murine embryos (P<0.05, n=6). Silencing of Wt1 by RNAi reduced the Abp1 mRNA in M15 cells by more than 60%. In conclusion, myocardial ischemia and hypoxia exert opposite effects on Abp1 expression. A cooperative regulation of the Abp1 promoter by Wt1 and hypoxia may contribute to adequate putrescine levels in the developing and ischemic heart.

WT1: a weak spot in KRAS-induced transformation

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.

Wilms’ tumor protein Wt1 regulates the Interleukin-10 (IL-10) gene

We identified the Wilms’ tumor protein, Wt1, as a novel transcriptional activator of the immunosuppressant cytokine interleukin-10 (IL-10). Silencing of Wt1 by RNA interference reduced IL-10 mRNA levels by approximately 90%. IL-10 transcripts were increased more than 15-fold upon forced expression of Wt1. Electrophoretic mobility shift assay and chromatin immunoprecipitation revealed a cis-element that was responsible for activation of the IL-10 promoter by Wt1 in murine macrophages. Mutation of the Wt1 binding motif abrogated stimulation of the IL-10 promoter by tumor necrosis factor-α (TNFα). These results suggest a novel immune regulatory function of Wt1 in controlling IL-10 gene expression.

Effect of WT1 gene silencing on the tumorigenicity of human glioblastoma multiforme cells

Wilms tumor 1 (WT1) is overexpressed in many human cancers, including glioblastoma multiforme (GBM). In another study, the authors showed that transient WT1 silencing increases the radiosensitivity of glioma cells. Studies of nonglioma cell lines have demonstrated that WT1 promotes cell proliferation and survival; however, this ability has not been rigorously analyzed in human GBM. The authors tested the efficacy of 2 sequences of short hairpin RNA (shRNA) directed against WT1 in U251MG human GBM cells and found that 1 sequence was capable of stably silencing WT1 expression. They then evaluated the effect of WT1 silencing on cellular proliferation, invasion, and in vivo tumor formation. Stable WT1-shRNA expression significantly decreased the proliferation of U251MG cells in vitro as demonstrated by both an adenosine 5'-triphosphate-based viability assay and tritiated thymidine uptake. Furthermore, stable WT1 silencing caused significantly slower growth after the subcutaneous inoculation of tumor cells in the flanks of athymic nude mice and was associated with an increased latency period. Data in this study provide proof of the principle that downregulation of WT1 causes decreased tumorigenicity of a GBM cell line in vitro and in vivo and suggest that WT1 is a promising target for novel molecular GBM therapies, perhaps in combination with standard treatment modalities.

The Wilms' tumour suppressor WT1 is involved in endothelial cell proliferation and migration: expression in tumour vessels in vivo

Vascularization is an important step in tumour growth. Although a variety of molecules, for example, VEGF, ETS-1 or nestin have been implicated in tumour angiogenesis, the molecular mechanisms of vessel formation are not fully characterized. We showed that the Wilms' tumour suppressor WT1 activates nestin during development. Here we tested whether WT1 might also be involved in tumour angiogenesis. Endothelial WT1 expression was detected in 95% of 113 tumours of different origin. To analyse the function of WT1 in endothelial cells, we used an RNAi approach in vitro and showed that inhibition of WT1 reduces cell proliferation, migration and endothelial tube formation. On a molecular level, WT1 silencing diminished expression of the ETS-1 transcription factor. WT1 and ETS-1 shared an overlapping expression in tumour endothelia. The ETS-1 promoter was stimulated approximately 10-fold by transient co-transfection of a WT1 expression construct and WT1 bound to the ETS-1 promoter in chromatin immunoprecipitation and electrophoretic mobility shift assays. Deletion of the identified WT1-binding site abolished stimulation of the ETS-1 promoter by WT1. These findings suggest that transcriptional activation of ETS-1 by the Wilms' tumour suppressor WT1 is a crucial step in tumour vascularization via regulation of endothelial cell proliferation and migration.

Interferons Downregulate the Expression of WT1 in Myeloid Leukemia through the JAK/STAT Pathway and This Potentiates Chemotherapy Induced Apoptosis.

Introduction: Expression of the WT1 gene is tightly regulated during normal hematopoietic development, and dysregulation of WT1 expression can lead to leukemia. However, relatively little is known about how the WT1 gene is regulated. Whereas WT1 overexpression blocks myeloid progenitor differentiation, the interferons (IFN, both type I and type II) have been shown to promote this differentiation, which underlies their clinical use in the myeloproliferative disorders. We hypothesized that the effect of IFN on myelopoeisis was via regulation of WT1 expression.Materials and Methods: We examined the effect of interferon-(both α and γ) treatment on WT1 mRNA expression on the CML cell line K562 and the AML cell line KG1. We studied the gene expression by RT-PCR and the protein expression by western blotting and flow cytometry by methods previously described. WT1 gene was knocked down using shRNA transfected by standard lipofectamine method. Cell viability was measured by annexin V staining by FACS.Results: Interferon (α and γ) treatment resulted in downregulation of WT1 mRNA expression indicating a common IFN-induced mechanism(s). Importantly, downregulation of WT1 was also associated with downregulation of downstream WT1 targets, such as the bcl-2 and c-myc. Blocking STAT1 phosphorylation by epigallocatechin (EGCG) completely abrogated this effect of IFNg on WT1 protein expression. These findings also suggested that this regulation was primarily at the transcription level, which lead us to initially examine the WT1 promoter. Analysis of the promoter region revealed no canonical STAT1 binding sites; however IFN-induced STAT1 signaling transcriptionally regulates the expression of the transcription factor IRF-1. We first analyzed the WT1 promoter (Transcription Element Search Software, www.cbil.upenn.edu/tess) for possible IRF-1 binding sites and identified a putative site at -222 to - 240 of transcription start site. The actual interaction was confirmed by Electromobility gel shift assay (EMSA) using oligonucleotides derived from the putative sequence. Treatment with IFNg substantially increased the amount of shifted probe, consistent with increased oligonucleotide binding by IRF-1 protein. IFNg treatment alone had little effect on cell viability. However, treatment with IFNg or INFa resulted in significant sensitization of both K562 and KG1 to chemotherapy and significantly increases apoptosis in both cell lines. Since it is possible that IFN-mediated sensitization is due to one of the myriad of interferon responses we specifically down regulated WT1 expression by shRNA in K 562 cells. The chosen shRNA clone alone induced some apoptosis but the combination of WT1 silencing plus etoposide markedly increased cell death.Conclusions: In summary our data strongly support the hypothesis that downregulation of WT1 expression impairs the survival of leukemic blasts, and sensitizes these cells to chemotherapy-induced apoptosis. The data also demonstrate that WT1 expression can be modulated by clinically available agents (INFa), and point to potentially new strategies to target AML. Ongoing studies in primary AML cells will not only confirm the findings above but also lay the foundation for therapeutic targeting of a key protein necessary for leukemia cell survival in the face of cytotoxic therapy.

The Wilms’ tumor suppressor WT1 is associated with melanoma proliferation

Expression of the Wilms' tumor suppressor WT1 has been demonstrated in a variety of tumors and tumor cell lines, e.g., in breast cancer and melanoma cell lines. Its role is controversial, with evidence for both tumor-promoting and tumor-suppressing activities. In this paper, we show that WT1 is expressed in malignant melanoma in >80% of the tumor cells, but not in normal skin or benign melanocytic nevi in vivo. We detected an unusual shift of WT1 isoforms towards WT1(+17AA/+KTS) in melanoma. WT1 shared an overlapping expression with proliferating nuclear cell antigen and with Nestin and Zyxin, which are involved in melanoma cell proliferation. To investigate whether WT1 is directly involved in melanoma cell proliferation, we made use of an RNAi approach in vitro. WT1 silencing significantly reduced the expression of Nestin and Zyxin and resulted in inhibition of melanoma cell proliferation as determined by a reduced BrdU incorporation. These findings suggest a direct role of WT1 in melanoma proliferation, which might be mediated via Nestin and Zyxin. Furthermore, expression of WT1 in vivo clearly discriminates between benign acquired nevi and malignant melanomas and appears to be correlated with melanocytic atypia and malignancy.