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RNA-based therapeutics are emerging as innovative options for cancer treatment, with microRNAs being attractive targets for therapy development. We previously implicated microRNA-642a-5p (miR-642a-5p) as a tumor suppressor in prostate cancer (PCa), and here we characterize its mode of action, using 22Rv1 PCa cells. In an in vivo xenograft tumor model, miR-642a-5p induced a significant decrease in tumor growth, compared to negative control. Using RNA-Sequencing, we identified gene targets of miR-642a-5p which were enriched for gene sets controlling cell cycle; downregulated genes included Wilms Tumor 1 gene (WT1), NUAK1, RASSF3 and SKP2; and upregulated genes included IGFBP3 and GPS2. Analysis of PCa patient datasets showed a higher expression of WT1, NUAK1, RASSF3 and SKP2; and a lower expression of GPS2 and IGFBP3 in PCa tissue compared to non-malignant prostate tissue. We confirmed the prostatic oncogene WT1, as a direct target of miR-642a-5p, and treatment of 22Rv1 and LNCaP PCa cells with WT1 siRNA or a small molecule inhibitor of WT1 reduced cell proliferation. Taken together, these data provide insight into the molecular mechanisms by which miR-642a-5p acts as a tumor suppressor in PCa, an effect partially mediated by regulating genes involved in cell cycle control; and restoration of miR-642-5p in PCa could represent a novel therapeutic approach., (© 2021. The Author(s).)

Acute myeloid leukemia (AML) is caused by recurrent mutations in members of the gene regulatory and signaling machinery that control hematopoietic progenitor cell growth and differentiation. Here, we show that the transcription factor WT1 forms a major node in the rewired mutation-specific gene regulatory networks of multiple AML subtypes. WT1 is frequently either mutated or upregulated in AML, and its expression is predictive for relapse. The WT1 protein exists as multiple isoforms. For two main AML subtypes, we demonstrate that these isoforms exhibit differential patterns of binding and support contrasting biological activities, including enhanced proliferation. We also show that WT1 responds to oncogenic signaling and is part of a signaling-responsive transcription factor hub that controls AML growth. WT1 therefore plays a central and widespread role in AML biology., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Dysregulated alternative splicing plays a prominent role in all hallmarks of cancer. The splice factor kinase SRPK1 drives the activity of oncogenic splice factors such as SRSF1. SRSF1 in turn promotes the expression of splice isoforms that favour tumour growth, including proangiogenic VEGF. Knockdown (with siRNA) or chemical inhibition (using SPHINX) of SRPK1 in K562 leukemia and PC3 prostate cancer cell lines reduced cell proliferation, invasion and migration. In glomerular podocytes, the Wilms tumour suppressor zinc-finger transcription factor WT1 represses SRPK1 transcription. Here we show that in cancer cells WT1 activates SRPK1 transcription, unless a canonical WT1 binding site adjacent to the transcription start site is mutated. The ability of WT1 to activate SRPK1 transcription was reversed by the transcriptional corepressor BASP1, and both WT1 and BASP1 co-precipitated with the SRPK1 promoter. BASP1 significantly increased the expression of the antiangiogenic VEGF 165 b splice isoform. We propose that by upregulating SRPK1 transcription WT1 can direct an alternative splicing landscape that facilitates tumour growth., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We previously reported that the transcription factor Wilms tumor 1 ( WT1 ) regulates the expression of insulin-like growth factor-binding protein-1 ( IGFBP-1 ) and prolactin ( PRL ) during decidualization of human endometrial stromal cells (ESCs). However, other roles of WT1 in decidualization remain to be fully clarified. Here, we investigated how WT1 regulates the physiological functions of human ESCs during decidualization. We incubated ESCs isolated from proliferative-phase endometrium with cAMP to induce decidualization, knocked down WT1 with siRNA, and generated three types of treatments (nontreated cells, cAMP-treated cells, and cAMP-treated + WT1-knockdown cells). To identify WT1-regulated genes, we used gene microarrays and compared the transcriptome data obtained among these three treatments. We observed that WT1 up-regulates 121 genes during decidualization, including several genes involved in lipid transport. The WT1 knockdown inhibited lipid accumulation (LA) in the cAMP-induced ESCs. To examine the mechanisms by which WT1 regulates LA, we focused on very low-density lipoprotein receptor ( VLDLR ), which is involved in lipoprotein uptake. We found that cAMP up-regulates VLDLR and that the WT1 knockdown inhibits it. Results of ChIP assays revealed that cAMP increases the recruitment of WT1 to the promoter region of the VLDLR gene, indicating that WT1 regulates VLDLR expression. Moreover, VLDLR knockdown inhibited cAMP-induced LA, and VLDLR overexpression reverted the suppression of LA caused by the WT1 knockdown. Taken together, our results indicate that WT1 enhances lipid storage by up-regulating VLDLR expression in human ESCs during decidualization., (© 2020 Tamura et al.)

Wilm's tumour-1 (WT1) is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and enhances metastasis. Deubiquitination stabilizes target proteins, and inhibiting deubiquitination facilitates the degradation of target proteins. However, whether inhibiting deubiquitination of WT1 facilitates its degradation and presents anti-cancer ability in PDAC is unknown. Here, we found that deubiquitinase inhibitor degrasyn rapidly induced the degradation of endogenous and exogenous WT1 through enhancing ubiquitination of WT1 followed by the up-regulation of E-cadherin. Knockdown of WT1 by short hairpin RNAs (shRNAs) inhibited metastasis and overexpression of WT1 partially prevented degrasyn-induced anti-metastasis activity, suggesting that degrasyn presents anti-metastasis activity partially through degrading WT1 protein. We further identified that USP5 deubiquitinated WT1 and stabilized its expression. The higher expressions of USP5 and WT1 are associated with tumour metastasis. More importantly, degrasyn inhibited the activity of USP5 and overexpression of USP5 partially prevented degrasyn-induced degradation of WT1 protein, suggesting that degrasyn degraded WT1 protein through inhibiting the activity of USP5. Finally, degrasyn reduced the tumorigenicity in a xenograft mouse model and reduced the metastasis in vivo. Our results indicate that degrasyn presents strong anti-cancer activity through USP5-WT1-E-cadherin signalling in PDAC. Therefore, degrasyn holds promise as cancer therapeutic agent in PDAC with high expressions of USP5 and WT1., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: Podocyte injury is the hallmark of proteinuric kidney diseases, such as FSGS and minimal change disease, and destabilization of the podocyte's actin cytoskeleton contributes to podocyte dysfunction in many of these conditions. Although agents, such as glucocorticoids and cyclosporin, stabilize the actin cytoskeleton, systemic toxicity hinders chronic use. We previously showed that loss of the kidney-enriched zinc finger transcription factor Krüppel-like factor 15 (KLF15) increases susceptibility to proteinuric kidney disease and attenuates the salutary effects of retinoic acid and glucocorticoids in the podocyte., Methods: We induced podocyte-specific KLF15 in two proteinuric murine models, HIV-1 transgenic ( Tg26 ) mice and adriamycin (ADR)-induced nephropathy, and used RNA sequencing of isolated glomeruli and subsequent enrichment analysis to investigate pathways mediated by podocyte-specific KLF15 in Tg26 mice. We also explored in cultured human podocytes the potential mediating role of Wilms Tumor 1 (WT1), a transcription factor critical for podocyte differentiation., Results: In Tg26 mice, inducing podocyte-specific KLF15 attenuated podocyte injury, glomerulosclerosis, tubulointerstitial fibrosis, and inflammation, while improving renal function and overall survival; it also attenuated podocyte injury in ADR-treated mice. Enrichment analysis of RNA sequencing from the Tg26 mouse model shows that KLF15 induction activates pathways involved in stabilization of actin cytoskeleton, focal adhesion, and podocyte differentiation. Transcription factor enrichment analysis, with further experimental validation, suggests that KLF15 activity is in part mediated by WT1., Conclusions: Inducing podocyte-specific KLF15 attenuates kidney injury by directly and indirectly upregulating genes critical for podocyte differentiation, suggesting that KLF15 induction might be a potential strategy for treating proteinuric kidney disease., (Copyright © 2018 by the American Society of Nephrology.)

Background/aims: IL-1β is an important mediator of "inflammation-cancer" transformation through IL-1β/NF-κB/COX-2/HIF-1α signaling pathway, whereas certain portion of patients with lung adenocarcinoma (LUAD) still suffer from rapid tumor progression in clinical practice, indicating the occurrence of potential bypass., Methods: Real-time polymerase chain reaction was applied to examine the expressions of mir-144-3p, WT1, NF-κB, COX2 and HIF-1α at the mRNA level in 127 LUAD samples and corresponding adjacent tissues. miR-144-3p mimic and antagormiR were used to trigger activation and suppression of miR-144-3p in A549 cells, respectively. MTT assay and Western blotting analysis were carried out to evaluate the cell proliferation. Stable clones with over-expression or knockdown of WT1 were generated with plasmid or shRNA by lentiviral vector technology in H1568 and H1650 NSCLC cell lines, respectively. Dual luciferase reporter assay was performed to validate the effect of miR-144-3p on WT1D. Xenograft model was established for in vivo experiment, and TCGA data were extracted for validation., Results: miR-144-3p could suppress the WT1D expression at the post-transcriptional level, hence regulating cell proliferation in LUAD. WT1 and COX-2 were independent prognostic factors of LUAD patients. In addition, inhibition of IL-1β/miR-144-3p/WT1D and IL-1β/NF-κB/COX-2/HIF-1α pathways using miR-144-3p mimic and Celecoxib, respectively, displayed synergistic suppressive effect on cell proliferation in LUAD., Conclusion: A de novo IL-1β/miR-144-3p/WT1D axis was involved in proliferative regulation of LUAD. Moreover, simultaneous blockade of both IL-1β/miR-144-3p/WT1D and IL-1β/NF-κB/COX-2/ HIF-1α pathways might have synergistic suppressive effect on cell proliferation in LUAD., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

The WT1 gene is an important oncogene, and its overexpression is considered as an effective target for anticancer therapy. Regulation of its gene transcription is one way for WT1-targeting drug design. Recently, in silico analysis of some oncogene promoters like WT1 showed some guanine-rich regions with the ability to form G-quadruplex structures. Ligands like 5,10,15,20-tetra(N-methyl-4-pyridyl)-porphine (TMPyP4) have predominant effect on G-quadruplex stabilization. The aim of this study was to understand the effect of TMPyP4 on WT1 gene transcription via stabilization of promoter G-quadruplexes. We examined the formation of new G-quadruplex motifs in WT1 promoter in the presence of TMPyP4. In order to understand the nature of its interaction with WT1 promoter quadruplexes, differential pulse voltammetry (DPV), circular dichroism (CD), polyacrylamide gel electrophoresis, electrophoretic mobility shift assay (EMSA), polymerase chain reaction (PCR) stop assays, and quantitative RT-PCR were performed. According to the results, the WT1 promoter can form stable intramolecular parallel G-quadruplexes. In addition, after 48 and 96 h of incubation, 100 μM TMPyP4 reduced the WT1 transcription to 9 and 0.4 %, respectively, compare to control. We report that ligand-mediated stabilization of G-quadruplexes within the WT1 promoter can silence WT1 expression. This study might offer the basis for the reasonable design and improvement of new porphyrin derivatives as effective anti-leukemia agents for cancer therapy.

Increased visceral fat, rather than subcutaneous fat, during the onset of obesity is associated with a higher risk of developing metabolic diseases. The inherent adipogenic properties of human adipose-derived stem cells (ASCs) from visceral depots are compromised compared with those of ASCs from subcutaneous depots, but little is known about the underlying mechanisms. Using ontological analysis of global gene expression studies, we demonstrate that many genes involved in retinoic acid (RA) synthesis or regulated by RA are differentially expressed in human tissues and ASCs from subcutaneous and visceral fat. The endogenous level of RA is higher in visceral ASCs; this is associated with upregulation of the RA synthesis gene through the visceral-specific developmental factor WT1. Excessive RA-mediated activity impedes the adipogenic capability of ASCs at early but not late stages of adipogenesis, which can be reversed by antagonism of RA receptors or knockdown of WT1. Our results reveal the developmental origin of adipocytic properties and the pathophysiological contributions of visceral fat depots., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

The Wilms' tumor gene WT1 consists of 10 exons and encodes a zinc finger transcription factor. There are four major WT1 isoforms resulting from alternative splicing at two sites, exon 5 (17AA) and exon 9 (KTS). All major WT1 isoforms are overexpressed in leukemia and solid tumors and play oncogenic roles such as inhibition of apoptosis, and promotion of cell proliferation, migration and invasion. In the present study, a novel alternatively spliced WT1 isoform that had an extended exon 4 (designated as exon 4a) with an additional 153 bp (designated as 4a sequence) at the 3' end was identified and designated as an Ex4a(+)WT1 isoform. The insertion of exon 4a resulted in the introduction of premature translational stop codons in the reading frame in exon 4a and production of C-terminal truncated WT1 proteins lacking zinc finger DNA-binding domain. Overexpression of the truncated Ex4a(+)WT1 isoform inhibited the major WT1-mediated transcriptional activation of anti-apoptotic Bcl-xL gene promoter and induced mitochondrial damage and apoptosis. Conversely, suppression of the Ex4a(+)WT1 isoform by Ex4a-specific siRNA attenuated apoptosis. These results indicated that the Ex4a(+)WT1 isoform exerted dominant negative effects on anti-apoptotic function of major WT1 isoforms. Ex4a(+)WT1 isoform was endogenously expressed as a minor isoform in myeloid leukemia and solid tumor cells and increased regardless of decrease in major WT1 isoforms during apoptosis, suggesting the dominant negative effects on anti-apoptotic function of major WT1 isoforms. These results indicated that Ex4a(+)WT1 isoform had an important physiological function that regulated oncogenic function of major WT1 isoforms.

Angiogenesis, activation of metastasis and avoidance of immune destruction are important for cancer progression. These biological capabilities are, apart from cancer cells, mediated by different cell types, including endothelial, haematopoietic progenitor and myeloid-derived suppressor cells. We show here that all these cell types frequently express the Wilms' tumour suppressor Wt1, which transcriptionally controls expression of Pecam-1 (CD31) and c-kit (CD117). Inducible conditional knockout of Wt1 in endothelial, haematopoietic and myeloid-derived suppressor cells is sufficient to cause regression of tumour vascularization and an enhanced immune response, leading to decreased metastasis, regression of established tumours and enhanced survival. Thus, Wt1 is an important regulator of cancer growth via modulation of tumour vascularization, immune response and metastasis formation.

Wilms' tumor gene 1 (WT1) plays complex roles in tumorigenesis, acting as tumor suppressor gene or an oncogene depending on the cellular context. WT1 expression has been variably reported in both benign and malignant peripheral nerve sheath tumors (MPNSTs) by means of immunohistochemistry. The aim of the present study was to characterize its potential pathogenetic role in these relatively uncommon malignant tumors. Firstly, immunohistochemical analyses in MPNST sNF96.2 cell line showed strong WT1 staining in nuclear and perinuclear areas of neoplastic cells. Thus, we investigated the effects of silencing WT1 by RNA interference. Through Western Blot analysis and proliferation assay we found that WT1 knockdown leads to the reduction of cell growth in a time- and dose-dependent manner. siWT1 inhibited proliferation of sNF96.2 cell lines likely by influencing cell cycle progression through a decrease in the protein levels of cyclin D1 and inhibition of Akt phosphorylation compared to the control cells. These results indicate that WT1 knockdown attenuates the biological behavior of MPNST cells by decreasing Akt activity, demonstrating that WT1 is involved in the development and progression of MPNSTs. Thus, WT1 is suggested to serve as a potential therapeutic target for MPNSTs.

Steroidogenic factor 1 (SF1; Ad4BP/NR5A1) plays key roles in gonadal development. Initially, the Sf1 gene is expressed in mouse fetal gonads of both sexes, but later is up-regulated in testes and down-regulated in ovaries. While Sf1 expression is activated and maintained by Wilms tumor 1 (WT1) and LIM homeobox 9 (LHX9), the mechanism of sex-specific regulation remains unclear. We hypothesized that Sf1 is repressed by the transcription factor Forkhead box L2 (FOXL2) during ovarian development. In an in vitro system (TM3 cells), up-regulation of Sf1 by the WT1 splice variant WT1-KTS was antagonized by FOXL2, as determined by quantitative RT-PCR. Using reporter assays, we localized the Sf1 proximal promoter region involved in this antagonism to a 674-bp interval. A conserved FOXL2 binding site was identified in this interval by in vitro chromatin immunoprecipitation. Introducing mutations into this site abolished negative regulation by FOXL2 in reporter assays. Finally, in Foxl2-null mice, Sf1 expression was increased 2-fold relative to wild-type XX fetal gonads. Our results support the hypothesis that FOXL2 negatively regulates Sf1 expression by antagonizing WT1-KTS during early ovarian development in mice.

Background: Wilms' tumor 1 (WT1) gene has different functional properties depending on the isoform type. This gene correlates with cell proliferation in various types of cancer. Here, we investigated the expression of WT1 isoforms in breast cancer tissues, and focused on the oncogenic role through estrogen receptor-alpha (ER-α) and human epidermal growth factor receptor 2 (HER2)., Materials and Methods: Expression of WT1(17AA+) and (17AA-) was investigated in adjacent normal breast and breast cancer using Reverse transcription-polymerase chain reaction and western blotting. The correlation of WT1 isoforms with HER2 and ER-α was examined using MCF-7 cells stably-overexpressing WT1s and siRNA against WT1 gene., Results: The expression of WT(17AA-) was significantly found in adjacent normal breast tissues. A mixture of WT1(17AA+) and WT1(17AA-) were highly expressed in breast carcinoma tissues. MCF-7 cells overexpressing WT1+/+ and WT1+/- represented strong expression of ER-α and HER2. Moreover, the silencing of WT1+/+ and WT1+/- resulted in a decrease of both ER-α and HER2 and led to a decrease of cell numbers., Conclusion: Our results suggest that WT1(17AA+) was exhibited dominantly in breast carcinoma tissues. WT1+/+ and WT1+/- correlated with the high expression of ER-α and HER2, leading to cell proliferation and might be involved in cancer development and progression.

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., (© 2013 UICC.)

The prognosis for patients with glioblastoma is very poor, despite intensive treatment, including surgery and chemoradiotherapy. Wilms' tumor 1 (WT1) is expressed in most glioblastoma samples, and immunotherapy targeting WT1 has proven to be effective in recurrent glioblastoma. However, the functional roles of WT1 in glioblastoma are not clear. To examine the functional roles of WT1 in glioblastoma, glioblastoma cell lines with reduced WT1 expression were generated using short hairpin RNA(shRNA)-expressing lentivirus. Proliferation of WT1-knockdown glioblastoma cells was significantly slower than control cells with high WT1 expression. In addition, apoptosis was increased in WT1-knockdown glioblastoma cells. Furthermore, WT1-knockdown glioblastoma cells, and control glioblastoma cells were intra-cranially injected into immunodeficient mice. In vivo tumor growth of WT1-knockdown glioblastoma cells was significantly reduced compared to control glioblastoma cells. These results show that WT1 is involved in glioblastoma cell proliferation and apoptosis and that this protein has oncogenic roles in glioblastoma.

Introduction: The Wilms' tumor gene (WT1) has been identified as an oncogene in many malignant diseases, and aberrant WT1 expression has been linked to development, progression, and prognosis of non-small-cell lung cancer (NSCLC). We sought to investigate the underlying mechanism of WT1 and metastasis in NSCLC., Methods: Real-time polymerase chain reaction was applied to detect WT1 and CDH1 mRNA in 159 NSCLC samples and corresponding adjacent tissues. Stable clones with overexpression and knockdown of WT1 were generated with plasmid and shRNA via lentivirus technology in H1568 and H1650 NSCLC cell lines. Wound-healing assay, transwell assays, and polymerase chain reaction array were carried out for invasion evaluation. Dual luciferase reporter assay was performed to validate the effect of WT1 on CDH1., Results: The level of the WT1 mRNA was negatively correlated with that of E-cadherin (CDH1) and associated with pathological stage, metastasis, and survival rate of 159 NSCLC patients. A series of genes were regulated by WT1, and WT1 could suppress CDH1 transcription via direct binding to its promoter and may enhance the invasive ability of H1568 and H1650 NSCLC cell lines., Conclusions: WT1 expression was correlated with clinical stage, metastasis, and survival rate in 159 NSCLC patients. Via direct binding to the promoter, WT1 could suppress CDH1 and promote NSCLC invasion.

Wilms tumor gene WT1 encodes a zinc finger-containing transcription factor which is required for renal development. Mutations in WT1 are observed in 20% of Wilms tumors (a pediatric kidney cancer), but the in vivo WT1 targets and associated molecular pathways involved in the etiology of Wilms tumor are still elusive. To identify WT1 targets we performed genome-wide comprehensive expression profiling using Affymetrix Gene Chip Mouse Genome 430 2.0 Arrays, comparing E13.5 mouse kidneys in which Wt1 had been somatically ablated with littermate controls. We identified Usp18 as the most differentially expressed gene in mutant kidney. Using tetracycline inducible cells we demonstrated a repressive effect of WT1 on USP18 expression. Conversely, knockdown of WT1 led to the upregulation of Usp18. Furthermore, direct binding of WT1 to the Usp18 promoter was demonstrated by ChIP assay. Overexpression of USP18 in murine and human cell lines resulted in cell proliferation. Additionally, Usp18 upregulation was observed in a mouse model of Wilms tumor. Taken together our data demonstrate that Usp18 is a transcriptional target of WT1 and suggest that increased expression of USP18 following WT1 loss contributes to Wilms tumorigenesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aim: Recent studies have reported that double-stranded RNA (dsRNA) can activate gene expression by targeting promoter sequence in a process termed RNA activation. The present study was conducted to evaluate the potential of WT1 induction by small activating RNA targeting the WT1 promoter (dsWT1) in the treatment of hepatocellular carcinoma., Methods: The human hepatocellular carcinoma cell line HepG2 was transfected with dsRNA by liposomes. The expression of mRNA and protein in cells were investigated using real-time reverse real-time quantitative PCR and Western blot, respectively. Cell viability and clonogenicity were determined by MTT assay and clonogenicity assay, respectively. Cell apoptosis was evaluated by flow-cytometric analysis., Results: Expressions of WT1 mRNA and protein in dsWT1 treated HepG2 cells were significantly elevated. Inhibition of cell viability by dsWT1 was dose-dependent and time-dependent. Reduction of the number and size of colonies formed were found in dsWT1 treated cells. dsWT1 induced significant apoptosis in HepG2 cells. The decreased anti-apoptotic protein Bcl-2 and elevated pro-apoptotic protein Bak expression were detected in dsWT1 treated cells. The level of pro-caspase-3 remarkably decreased and cleaved caspase-3 and PARP fragment were also detected in dsWT1 treated cells., Conclusion: These data show that RNAa-mediated overexpression of WT1 may have therapeutic potential in the treatment of hepatocellular carcinoma., (© 2012 Qin et al; licensee BioMed Central Ltd.)

Podocytes help form the glomerular blood filtration barrier in the kidney and their injury or loss leads to renal disease. The Wilms' tumor suppressor-1 (Wt1) and the FoxC1/2 transcription factors, as well as Notch signaling, have been implicated as important regulators of podocyte fate. It is not known whether these factors work in parallel or sequentially on different gene targets, or as higher-order transcriptional complexes on common genes. Here, we use the zebrafish to demonstrate that embryos treated with morpholinos against wt1a, foxc1a, or the Notch transcriptional mediator rbpj develop fewer podocytes, as determined by wt1b, hey1 and nephrin expression, while embryos deficient in any two of these factors completely lack podocytes. From GST-pull-downs and co-immunoprecipitation experiments we show that Wt1a, Foxc1a, and Rbpj can physically interact with each other, whereas only Rbpj binds to the Notch intracellular domain (NICD). In transactivation assays, combinations of Wt1, FoxC1/2, and NICD synergistically induce the Hey1 promoter, and have additive or repressive effects on the Podocalyxin promoter, depending on dosage. Taken together, these data suggest that Wt1, FoxC1/2, and Notch signaling converge on common target genes where they physically interact to regulate a podocyte-specific gene program. These findings further our understanding of the transcriptional circuitry responsible for podocyte formation and differentiation during kidney development., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: To study the effect of WT1 antisense oligodeoxynucleotide (ASODN) transfection on the proliferation and apoptosis of SKOV3 cells., Methods: There were four groups in our study: normal control group, WT1 ASODN group, WT1 SODN group and lipofectamine group. Cell apoptosis was observed by flow cytometry. The effect of WT1 ASODN on cell proliferation was assayed by the MTT method. RT-PCR and Western blot were used to detect the expression level of WT1 mRNA and protein., Results: The growth of the ovarian cancer cell line SKOV3 became significantly slower and its activity was reduced after being transfected by WT1 ASODN, with the inhibition rate of 49.48%. WT1 antisense phosphorothioate oligonucleotides did not only inhibit cell proliferation, arrest cell cycle at G0-G1 checkpoint and induce apoptosis in SKOV3 ovarian carcinoma cells, but also downregulated WT1 mRNA and protein expression, which contributed to the apoptosis (p < 0.05)., Conclusion: WT1 antisense phosphorothioate oligonucleotides could both inhibit the proliferation and induce the apoptosis in SKOV3 ovarin carcinoma cell lines. Antisense oligonucleotides of WT1 may potentially help with the gene therapy of ovarian carcinoma.

We investigated the prognostic significance of early peripheral blast clearance as assessed by WT1 transcript reduction during the first days of standard induction therapy in 57 adult patients with acute myeloid leukemia (AML). Quantification of WT1 transcript by real-time quantitative PCR in peripheral blood on days 1 and 5 of treatment was performed. WT1 ratio was defined as the ratio of copy number measured on day 1 and on day 5. The median WT1 ratio was greater in patients attaining CR as compared to non-responders (11.68 vs. 2.14, respectively; P=0.0006). Furthermore, DFS and OS were significantly longer in patients displaying a WT1 ratio greater than 5.82 (i.e. the median value of whole cohort) than in patients with WT1 ratio of 5.82 or under (P=0.024 and P<0.001, respectively). These data suggest that early decrease of WT1 copy number in peripheral blood predicts for better outcome and should be considered in the management of AML patients.

Store-operated calcium entry (SOCE) is a key evolutionarily conserved process whereby decreases in endoplasmic reticulum Ca(2+) content lead to the influx of Ca(2+) across the plasma membrane. How this process is regulated in specific tumor cell types is poorly understood. In an effort to address this concern, we obtained and tested primary Wilms tumor cells, finding no detectable SOCE in this cell type. Analysis of the expression levels of STIM1 and ORAI1 (the molecular mediators of SOC) revealed poor STIM1 expression. Analysis of the STIM1 promoter using the TESS search system (University of Pennsylvania) revealed four putative response elements to the zinc-finger proteins WT1 (Wilms tumor suppressor 1) and EGR1 (early growth response 1). Either overexpression of WT1 or knockdown of EGR1 resulted in loss of STIM1 expression and a resultant decrease in SOCE. Furthermore, examination of Egr1 knock-out animals revealed loss of STIM1 expression in multiple tissues. Finally, using chromatin immunoprecipitation, we reveal direct binding of both WT1 and EGR1 to putative response elements located within 500 bp of the transcriptional start site of STIM1. Considering that WT1 and EGR1 are well described oncogenes and tumor suppressors, these observations may reveal new mechanisms responsible for distinct Ca(2+) signals in cancer cells.

Wilms' tumor gene WT1 is overexpressed in leukemia and various types of solid tumors and plays an important role in leukemogenesis and tumorigenesis. We tested apoptosis-inducing ability of short hairpin RNAs targeting exon 5 (shWTE5), exon10 (shWTE10) and 3'UTR (shWT3U) of the WT1 gene. Among the three WT1-shRNAs, since shWTE5 most effectively induced apoptosis, its ability as an apoptosis-inducing agent was intensively examined. shWTE5 induced mitochondrial damage and resultant apoptosis in five WT1-expressing solid cancer cells originated from gastric (AZ-521), lung (LU99B), ovarian (TYKnuCPr) cancers, fibrosarcoma (HT-1080) and glioblastoma (A172). Moreover, shWTE5 significantly enhanced apoptosis induced by chemotherapeutic agents, doxorubicin (DOX) and etoposide (ETP), or by death ligand TRAIL in all of the four solid tumor cells examined (HT-1080, LU99B, TYK and A172). Transduction of one each of WT1 isoforms with exon 5 [17AA(+)KTS(+) and 17AA(+)KTS(-)] prevented mitochondrial damage induced by ETP or TRAIL and inhibited apoptosis. These results showed that shWTE5 induced apoptosis through the suppression of the WT1 isoform with exon 5. Furthermore, shWTE5 increased expression of proapoptotic Bak and Bax proteins and decreased antiapoptotic Bcl-xL and Bcl-2 proteins in WT1-expressing HT-1080 cells, indicating that WT1 isoforms with exon 5 might play an antiapoptotic role through regulation of Bcl-2 family genes in solid tumor cells. The results presented here demonstrated that WT1-shRNA targeting exon 5 should serve as a potent anti-cancer agent for various types of solid tumors.

Progesterone regulates the proliferation and differentiation of normal mammary epithelium. In breast cancer cells, progesterone and its synthetic analogs, progestins, induce long-term growth inhibition and differentiation. However, the mechanisms responsible are not fully understood. When T-47D breast cancer cells were treated with the synthetic progestin ORG 2058 (16alpha-ethoxy-21-hydroxy-19-norpregn-4-en-3,20-dione), all isoforms of Wilms' tumor protein 1 (Wt1) mRNA and protein were rapidly downregulated. We reasoned that the decrease in Wt1 levels may contribute to the long-term antiproliferative and differentiative effects of progestins as proliferation and differentiation are known end points of Wt1 action. Consistent with this idea, Wt1 small interfering RNA led to a decrease in S phase and cyclin D1 levels, and increased Oil-Red-O staining, indicating increased lipogenesis. Conversely, overexpression of Wt1 attenuated the decrease in S phase induced by ORG 2058 at 48-96 h. This was accompanied by the sustained expression of cyclin D1 despite progestin treatment, and increased levels of retinoblastoma (Rb) phosphorylation at sites targeted by cyclin D1-Cdk4 (Ser249/Thr252). Wt1 overexpression also attenuated the ORG 2058-mediated increase in fatty acid synthase levels and reduced lipogenesis. Thus, Wt1 downregulation was sufficient to mimic the effects of progestin and was necessary for complete progestin-mediated proliferative arrest and subsequent differentiation. Furthermore, Wt1 overexpression modulated the effects of progestins but not anti-estrogens or androgens. These results indicate that Wt1 is an important early target of progestins that regulates both proliferation and differentiation in breast cancer cells.

Matrix metalloproteinase-9 (MMP-9) is released by human lung epithelial cells (LEC) in conditions such as asthma and chronic obstructive pulmonary disease and expression of MMP-9 correlates with the severity of these disorders. MMP-9 production has been reported to be regulated by a NO/soluble guanylate cyclase-dependent pathway. Transcriptional regulation of this enzyme, however, is poorly understood. Using phylogenetic analysis, we observed a highly conserved sequence in the 5' flanking region of the MMP-9 gene containing binding sites for the transcription factor Wilms tumor 1 (WT1). We confirmed the presence of WT1 in human LEC and that treatment with TNF or a mixture containing LPS, PMA, and IFN-gamma resulted in translocation of WT1 from the nucleus to the cytosol. This translocation coincided with increased expression of MMP-9 and could be blocked by inhibitors of the NO/soluble guanylate cyclase pathway. WT1 knockdown using small-interfering RNA up-regulated MMP-9 expression in the presence of the NO synthase inhibitor 1400W. Using either WT1 pulldown with probes for the conserved region of the MMP-9 promoter or chromatin immunoprecipitation, we confirmed WT1 binding to the MMP-9 promoter. These findings indicate WT1 is a repressor of MMP-9, regulated by a NO-mediated pathway in human LEC. To our knowledge, this is the first report of WT1 regulating MMP-9 expression. Further study is needed to determine whether clinical conditions exhibiting tissue remodeling, such as asthma and/or chronic obstructive pulmonary disease, demonstrate reduced levels of WT1 or its repressor activity.

Recent advances in molecular genetics have increased knowledge regarding the mechanisms leading to myelodysplastic syndrome (MDS), secondary acute myeloid leukemia (AML), and therapy-induced MDS. Many genetic defects underlying MDS and AML have been identified thereby allowing the development of new molecular-targeted therapies. Several new classes of drugs have shown promise in early clinical trials and may probably alter the standard of care of these patients in the near future. Among these new drugs are farnesyltransferase inhibitors and receptor tyrosine kinase inhibitors including FLT3 and VEGF inhibitors. These agents have been tested in patients with solid tumors and hematologic malignancies such as AML and MDS. Most of the studies in MDS are still in early stages of development. The DNA hypomethylating compounds azacytidine and decitabine may reduce hypermethylation and induce re-expression of key tumor suppressor genes in MDS. Biochemical compounds with histone deacetylase inhibitory activity, such as valproic acid (VPA), have been tested as antineoplastic agents. Finally, new vaccination strategies are developing in MDS patients based on the identification of MDS-associated antigens. Future therapies will attempt to resolve cytopenias in MDS, eliminate malignant clones, and allow differentiation by attacking specific mechanisms of the disease.

WT1 was originally identified as an inactivated gene in Wilms tumor, a childhood kidney cancer. Alternative splicing of the WT1 transcript generates four major protein isoforms, each having different functional properties. Here we characterized a short transcript originating from a second promoter located within intron 1 of WT1. This 2.3-kb sWT1 transcript encodes a protein of approximately 35-37 kDa that retains intact DNA-binding and transactivation domains but lacks the 147 amino acids at the N terminus required for transcriptional repression. We found sWT1 to be a more potent transcriptional activator than WT1 for cyclin E and insulin-like growth factor 1 receptor promoters, which are normally repressed by WT1. The expression patterns of the sWT1 and WT1 transcripts differed slightly in various organs; we found sWT1 protein in tissue samples from adult testis and fetal kidney, with low-level expression in adult kidney as well. The sWT1 transcript, but not the full-length transcript, was over-expressed in the leukemia samples tested. sWT1-specific small interfering RNA retarded the proliferation of leukemia cell line K562 in vitro. Finally, sWT1 cooperated with Ras in transforming primary fibroblasts in vitro. Further studies are needed to clarify the oncogenic behavior of this isoform and to determine the mechanism underlying its up-regulation in leukemia and other forms of cancer.

Background: The objective of the current study was to verify the ability to predict response to imatinib therapy using in vitro assays to evaluate the inhibition of Wilms tumor gene (WT1) expression and colony growth after samples obtained from patients with chronic myelogenous leukemia (CML) before the start of treatment were subjected to short-term incubation with imatinib., Methods: WT1 transcript levels and colony growth in bone marrow (BM) samples from 23 patients with CML that was later identified as being responsive to imatinib and from 13 patients with CML that was later identified as not being responsive to imatinib were evaluated after incubation of these samples with imatinib at a concentration of 1 microM for 18 hours. In addition, real-time quantitative polymerase chain reaction (RQ-PCR) analysis of WT1 expression was performed during follow-up, and the results were analyzed for associations with cytogenetic response and with BCR/ABL transcript levels as determined using RQ-PCR analysis., Results: Before treatment, it was found that WT1 expression was elevated in BM samples obtained from all patients with CML. WT1 expression and colony growth were reduced significantly after an 18-hour incubation with imatinib in samples obtained from patients who were later identified as responders to treatment, but not in samples obtained from patients who did not experience responses to treatment. Inhibition of WT1 expression in vitro was associated with inhibition of imatinib-induced BCR-ABL tyrosine kinase activity, a finding that also has been made in studies involving certain Philadelphia chromosome (Ph)-positive and Ph-negative cell lines., Conclusions: Inhibition of WT1 transcript levels after a short period of in vitro exposure of pretherapy BM samples to imatinib was correlated with inhibition of colony growth and may represent the basis for an easy test that is capable of predicting the sensitivity of CML to treatment with imatinib for individual patients., (Copyright 2004 American Cancer Society.)

Among clinicians, initial awareness of the Wilms' tumor gene was limited mostly to pediatric oncologists. Almost a decade ago, overexpression of Wilms' tumor 1 (WT1) was observed in adult acute leukemia. Subsequent studies indicated that WT1 overexpression occurs in most cases of acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia (CML), and myelodysplastic syndrome (MDS). Limited tissue expression of WT1 in adults suggests that WT1 can be a target for leukemia/MDS therapy. WT1 expression in stem/progenitor cells remains unsettled. However, lack of progenitor cell suppression by WT1 antisense or WT1-specific cytotoxic T cells provide some assurance that WT1 expression in progenitor cells is minimal or absent. Immunotherapy-based WT1 approaches are furthest along in preclinical development. WT1-specific cytotoxic lymphocytes can be generated from normals and leukemic patients. In mice, WT1 vaccines elicit specific immune responses without evidence of tissue damage. In this paper, we review studies validating the immunogenicity of WT1 and propose that leukemia and MDS may be a good clinical model to test the efficacy of a WT1 vaccine.