Salt-Inducible Kinase 1 is a potential therapeutic target in Desmoplastic Small Round Cell Tumor.

Desmoplastic Small Round Cell Tumor (DSRCT) is a rare and aggressive malignant cancer caused by a chromosomal translocation t(11;22)(p13;q12) that produces an oncogenic transcription factor, EWSR1-WT1. EWSR1-WT1 is essential for the initiation and progression of DSRCT. However, the precise mechanism by which EWSR1-WT1 drives DSRCT oncogenesis remains unresolved. Through our integrative gene expression analysis, we identified Salt Inducible Kinase 1 (SIK1) as a direct target of EWSR1-WT1. SIK1 as a member of the AMPK related kinase is involved in many biological processes. We showed that depletion of SIK1 causes inhibition of tumor cell growth, similar to the growth inhibition observed when EWSR1-WT1 is depleted. We further showed that silencing SIK1 leads to cessation of DNA replication in DSRCT cells and inhibition of tumor growth in vivo. Lastly, combined inhibition of SIK1 and CHEK1with small molecule inhibitors, YKL-05-099 and prexasertib, respectively, showed enhanced cytotoxicity in DSRCT cells compared to inhibition of either kinases alone. This work identified SIK1 as a new potential therapeutic target in DSRCT and the efficacy of SIK1 inhibition may be improved when combined with other intervention strategies.

Therapeutic Potential of NTRK3 Inhibition in Desmoplastic Small Round Cell Tumor.

PURPOSE: Desmoplastic small round cell tumor (DSRCT) is a highly lethal intra-abdominal sarcoma of adolescents and young adults. DSRCT harbors a t(11;22)(p13:q12) that generates the EWSR1-WT1 chimeric transcription factor, the key oncogenic driver of DSRCT. EWSR1-WT1 rewires global gene expression networks and activates aberrant expression of targets that together mediate oncogenesis. EWSR1-WT1 also activates a neural gene expression program. EXPERIMENTAL DESIGN: Among these neural markers, we found prominent expression of neurotrophic tyrosine kinase receptor 3 (NTRK3), a druggable receptor tyrosine kinase. We investigated the regulation of NTRK3 by EWSR1-WT1 and its potential as a therapeutic target in vitro and in vivo, the latter using novel patient-derived models of DSRCT. RESULTS: We found that EWSR1-WT1 binds upstream of NTRK3 and activates its transcription. NTRK3 mRNA is highly expressed in DSRCT compared with other major chimeric transcription factor-driven sarcomas and most DSRCTs are strongly immunoreactive for NTRK3 protein. Remarkably, expression of NTRK3 kinase domain mRNA in DSRCT is also higher than in cancers with NTRK3 fusions. Abrogation of NTRK3 expression by RNAi silencing reduces growth of DSRCT cells and pharmacologic targeting of NTRK3 with entrectinib is effective in both in vitro and in vivo models of DSRCT. CONCLUSIONS: Our results indicate that EWSR1-WT1 directly activates NTRK3 expression in DSRCT cells, which are dependent on its expression and activity for growth. Pharmacologic inhibition of NTRK3 by entrectinib significantly reduces growth of DSRCT cells both in vitro and in vivo, providing a rationale for clinical evaluation of NTRK3 as a therapeutic target in DSRCT.

EWSR1, a multifunctional protein, regulates cellular function and aging via genetic and epigenetic pathways.

Ewing's sarcoma (EWS) is a bone cancer arising predominantly in young children. EWSR1 (Ewing Sarcoma breakpoint region 1/EWS RNA binding protein 1) gene is ubiquitously expressed in most cell types, indicating it has diverse roles in various cellular processes and organ development. Recently, several studies have shown that missense mutations of EWSR1 genes are known to be associated with central nervous system disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Otherwise, EWSR1 plays epigenetic roles in gene expression, RNA processing, and cellular signal transduction. Interestingly, EWSR1 controls micro RNA (miRNA) levels via Drosha, leading to autophagy dysfunction and impaired dermal development. Ewsr1 deficiency also leads to premature senescence of blood cells and gamete cells with a high rate of apoptosis due to the abnormal meiosis. Despite these roles of EWSR1 in various cellular functions, the exact mechanisms are not yet understood. In this context, the current review overviews a large body of evidence and discusses on what EWSR1 genetic mutations are associated with brain diseases and on how EWSR1 modulates cellular function via the epigenetic pathway. This will provide a better understanding of bona fide roles of EWSR1 in aging and its association with brain disorders.

Genetic Ablation of EWS RNA Binding Protein 1 (EWSR1) Leads to Neuroanatomical Changes and Motor Dysfunction in Mice.

A recent study reveals that missense mutations of EWSR1 are associated with neurodegenerative disorders such as amyotrophic lateral sclerosis, but the function of wild-type (WT) EWSR1 in the central nervous system (CNS) is not known yet. Herein, we investigated the neuroanatomical and motor function changes in Ewsr1 knock out (KO) mice. First, we quantified neuronal nucleus size in the motor cortex, dorsal striatum and hippocampus of three different groups: WT, heterozygous Ewsr1 KO (+/-), and homozygous Ewsr1 KO (-/-) mice. The neuronal nucleus size was significantly smaller in the motor cortex and striatum of homozygous Ewsr1 KO (-/-) mice than that of WT. In addition, in the hippocampus, the neuronal nucleus size was significantly smaller in both heterozygous Ewsr1 KO (+/-) and homozygous Ewsr1 KO (-/-) mice. We then assessed motor function of Ewsr1 KO (-/-) and WT mice by a tail suspension test. Both forelimb and hindlimb movements were significantly increased in Ewsr1 KO (-/-) mice. Lastly, we performed immunohistochemistry to examine the expression of TH, DARPP-32, and phosphorylated (p)-DARPP-32 (Thr75) in the striatum and substantia nigra, which are associated with dopaminergic signaling. The immunoreactivity of TH and DARPP-32 was decreased in Ewsr1 KO (-/-) mice. Together, our results suggest that EWSR1 plays a significant role in neuronal morphology, dopaminergic signaling pathways, and motor function in the CNS of mice.

Integrated analysis of omics data using microRNA-target mRNA network and PPI network reveals regulation of Gnai1 function in the spinal cord of Ews/Ewsr1 KO mice.

BACKGROUND: Multifunctional transcription factor (TF) gene EWS/EWSR1 is involved in various cellular processes such as transcription regulation, noncoding RNA regulation, splicing regulation, genotoxic stress response, and cancer generation. Role of a TF gene can be effectively studied by measuring genome-wide gene expression, i.e., transcriptome, in an animal model of Ews/Ewsr1 knockout (KO). However, when a TF gene has complex multi-functions, conventional approaches such as differentially expressed genes (DEGs) analysis are not successful to characterize the role of the EWS gene. In this regard, network-based analyses that consider associations among genes are the most promising approach. METHODS: Networks are constructed and used to show associations among biological entities at various levels, thus different networks represent association at different levels. Taken together, in this paper, we report contributions on both computational and biological sides. RESULTS: Contribution on the computational side is to develop a novel computational framework that combines miRNA-gene network and protein-protein interaction network information to characterize the multifunctional role of EWS gene. On the biological side, we report that EWS regulates G-protein, Gnai1, in the spinal cord of Ews/Ewsr1 KO mice using the two biological network integrated analysis method. Neighbor proteins of Gnai1, G-protein complex subunits Gnb1, Gnb2 and Gnb4 were also down-regulated at their gene expression level. Interestingly, up-regulated genes, such as Rgs1 and Rgs19, are linked to the inhibition of Gnai1 activities. We further verified the altered expression of Gnai1 by qRT-PCR in Ews/Ewsr1 KO mice. CONCLUSIONS: Our integrated analysis of miRNA-transcriptome network and PPI network combined with qRT-PCR verifies that Gnai1 function is impaired in the spinal cord of Ews/Ewsr1 KO mice.

Intricate Transcriptional Networks of Classical Brown and Beige Fat Cells.

Brown adipocytes are a specialized cell type that is critical for adaptive thermogenesis, energy homeostasis, and metabolism. In response to cold, both classical brown fat and the newly identified "beige" or "brite" cells are activated by ß-adrenergic signaling and catabolize stored lipids and carbohydrates to produce heat via UCP1. Once thought to be non-existent in adults, recent studies have discovered active classical brown and beige fat cells in humans, thus reinvigorating interest in brown and beige adipocytes. This review will focus on the newly discovered transcription factors and microRNAs that specify and orchestrate the classical brown and beige fat cell development.

Uvrag targeting by Mir125a and Mir351 modulates autophagy associated with Ewsr1 deficiency.

The EWSR1 (EWS RNA-binding protein 1/Ewing Sarcoma Break Point Region 1) gene encodes a RNA/DNA binding protein that is ubiquitously expressed and involved in various cellular processes. EWSR1 deficiency leads to impairment of development and accelerated senescence but the mechanism is not known. Herein, we found that EWSR1 modulates the Uvrag (UV radiation resistance associated) gene at the post-transcription level. Interestingly, EWSR1 deficiency led to the activation of the DROSHA-mediated microprocessor complex and increased the level of Mir125a and Mir351, which directly target Uvrag. Moreover, the Mir125a- and Mir351-mediated reduction of Uvrag was associated with the inhibition of autophagy that was confirmed in ewsr1 knockout (KO) MEFs and ewsr1 KO mice. Taken together, our data indicate that EWSR1 is involved in the post-transcriptional regulation of Uvrag via a miRNA-dependent pathway, resulting in the deregulation of autophagy inhibition. The mechanism of Uvrag and autophagy regulation by EWSR1 provides new insights into the role of EWSR1 deficiency-related cellular dysfunction.

Inactivation of EWS reduces PGC-1α protein stability and mitochondrial homeostasis.

EWS (Ewing sarcoma) encodes an RNA/ssDNA binding protein that is frequently rearranged in a number of different cancers by chromosomal translocations. Physiologically, EWS has diverse and essential roles in various organ development and cellular processes. In this study, we uncovered a new role of EWS in mitochondrial homeostasis and energy metabolism. Loss of EWS leads to a significant decrease in mitochondria abundance and activity, which is caused by a rapid degradation of Peroxisome proliferator-activated receptor γ Coactivator (PGC-1α), a central regulator of mitochondria biogenesis, function, and cellular energy metabolism. EWS inactivation leads to increased ubiquitination and proteolysis of PGC-1α via proteasome pathway. Complementation of EWS in Ews-deficient cells restores PGC-1α and mitochondrial abundance. We found that expression of E3 ubiquitin ligase, FBXW7 (F-box/WD40 domain protein 7), is increased in the absence of Ews and depletion of Fbxw7 in Ews-null cells restores PGC-1α expression and mitochondrial density. Consistent with these findings, mitochondrial abundance and activity are significantly reduced in brown fat and skeletal muscles of Ews-deficient mice. Furthermore, expression of mitochondrial biogenesis, respiration and fatty acid ß-oxidation genes is significantly reduced in the liver of Ews-null mice. These results demonstrate a novel role of EWS in mitochondrial and cellular energy homeostasis by controlling PGC-1α protein stability, and further implicate altered mitochondrial and energy metabolism in cancers harboring the EWS translocation.

An essential role for Ewing sarcoma gene (EWS) in early white adipogenesis.

OBJECTIVE: White adipose tissue is important for mammalian energy homeostasis and metabolism. It was previously demonstrated that Ewing sarcoma gene (EWS) is essential for early classical brown fat lineage determination, but its role in white adipocyte differentiation is not known. METHODS: Mouse embryonic fibroblasts (MEFs) lacking Ews and shRNA-mediated silencing of Ews in 3T3L1 preadipocytes were used to investigate the role of EWS in adipogenesis. White fat differentiation was determined by analyzing the expression of key adipogenic genes and by Oil red O staining. RESULTS: Following adipogenic stimulation, Ews expression arose rapidly in 3T3L1 cells during early induction period. Ews-null MEFs and 3T3L1 cells with reduced Ews expression failed to undergo adipogenesis. This was accompanied by significant reduction in the expression of critical early adipogenic regulators, Bmp2, Bmp4 (bone morphogenic protein 2 and 4), Cebpß, and Cebpδ (CCAAT/enhancer binding protein ß and δ). Complementation of recombinant BMP2 or BMP4 partially rescued adipogenesis in Ews-depleted 3T3L1 cells. CONCLUSIONS: These results demonstrate that EWS is essential during the early steps of white adipocyte differentiation, at least in part through its regulation of BMP2 and BMP4 expression.

EWS-WT1 oncoprotein activates neuronal reprogramming factor ASCL1 and promotes neural differentiation.

The oncogenic fusion gene EWS-WT1 is the defining chromosomal translocation in desmoplastic small round-cell tumors (DSRCT), a rare but aggressive soft tissue sarcoma with a high rate of mortality. EWS-WT1 functions as an aberrant transcription factor that drives tumorigenesis, but the mechanistic basis for its pathogenic activity is not well understood. To address this question, we created a transgenic mouse strain that permits physiologic expression of EWS-WT1 under the native murine Ews promoter. EWS-WT1 expression led to a dramatic induction of many neuronal genes in embryonic fibroblasts and primary DSRCT, most notably the neural reprogramming factor ASCL1. Mechanistic analyses demonstrated that EWS-WT1 directly bound the proximal promoter of ASCL1, activating its transcription through multiple WT1-responsive elements. Conversely, EWS-WT1 silencing in DSRCT cells reduced ASCL1 expression and cell viability. Notably, exposure of DSRCT cells to neuronal induction media increased neural gene expression and induced neurite-like projections, both of which were abrogated by silencing EWS-WT1. Taken together, our findings reveal that EWS-WT1 can activate neural gene expression and direct partial neural differentiation via ASCL1, suggesting agents that promote neural differentiation might offer a novel therapeutic approach to treat DSRCT.

The tumor suppressor gene, RASSF1A, is essential for protection against inflammation -induced injury.

Ras association domain family protein 1A (RASSF1A) is a tumor suppressor gene silenced in cancer. Here we report that RASSF1A is a novel regulator of intestinal inflammation as Rassf1a(+/-) , Rassf1a(-/-) and an intestinal epithelial cell specific knockout mouse (Rassf1a (IEC-KO) ) rapidly became sick following dextran sulphate sodium (DSS) administration, a chemical inducer of colitis. Rassf1a knockout mice displayed clinical symptoms of inflammatory bowel disease including: increased intestinal permeability, enhanced cytokine/chemokine production, elevated nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) activity, elevated colonic cell death and epithelial cell injury. Furthermore, epithelial restitution/repair was inhibited in DSS-treated Rassf1a(-/-) mice with reduction of several makers of proliferation including Yes associated protein (YAP)-driven proliferation. Surprisingly, tyrosine phosphorylation of YAP was detected which coincided with increased nuclear p73 association, Bax-driven epithelial cell death and p53 accumulation resulting in enhanced apoptosis and poor survival of DSS-treated Rassf1a knockout mice. We can inhibit these events and promote the survival of DSS-treated Rassf1a knockout mice with intraperitoneal injection of the c-Abl and c-Abl related protein tyrosine kinase inhibitor, imatinib/gleevec. However, p53 accumulation was not inhibited by imatinib/gleevec in the Rassf1a(-/-) background which revealed the importance of p53-dependent cell death during intestinal inflammation. These observations suggest that tyrosine phosphorylation of YAP (to drive p73 association and up-regulation of pro-apoptotic genes such as Bax) and accumulation of p53 are consequences of inflammation-induced injury in DSS-treated Rassf1a(-/-) mice. Mechanistically, we can detect robust associations of RASSF1A with membrane proximal Toll-like receptor (TLR) components to suggest that RASSF1A may function to interfere and restrict TLR-driven activation of NFκB. Failure to restrict NFκB resulted in the inflammation-induced DNA damage driven tyrosine phosphorylation of YAP, subsequent p53 accumulation and loss of intestinal epithelial homeostasis.

A multifunctional protein, EWS, is essential for early brown fat lineage determination.

The recent surge in obesity has provided an impetus to better understand the mechanisms of adipogenesis, particularly in brown adipose tissue (BAT) because of its potential utilization for antiobesity therapy. Postnatal brown adipocytes arise from early muscle progenitors, but how brown fat lineage is determined is not completely understood. Here, we show that a multifunctional protein, Ewing Sarcoma (EWS), is essential for determining brown fat lineage during development. BATs from Ews null embryos and newborns are developmentally arrested. Ews mutant brown preadipocytes fail to differentiate due to loss of Bmp7 expression, a critical early brown adipogenic factor. We demonstrate that EWS, along with its binding partner Y-box binding protein 1 (YBX1), activates Bmp7 transcription. Depletion of either Ews or Ybx1 leads to loss of Bmp7 expression and brown adipogenesis. Remarkably, Ews null BATs and brown preadipocytes ectopically express myogenic genes. These results demonstrate that EWS is essential for early brown fat lineage determination.

The nucleotide sugar UDP-glucose mobilizes long-term repopulating primitive hematopoietic cells.

Hematopoietic stem progenitor cells (HSPCs) are present in very small numbers in the circulating blood in steady-state conditions. In response to stress or injury, HSPCs are primed to migrate out of their niche to peripheral blood. Mobilized HSPCs are now commonly used as stem cell sources due to faster engraftment and reduced risk of posttransplant infection. In this study, we demonstrated that a nucleotide sugar, UDP-glucose, which is released into extracellular fluids in response to stress, mediates HSPC mobilization. UDP-glucose-mobilized cells possessed the capacity to achieve long-term repopulation in lethally irradiated animals and the ability to differentiate into multi-lineage blood cells. Compared with G-CSF-mobilized cells, UDP-glucose-mobilized cells preferentially supported long-term repopulation and exhibited lymphoid-biased differentiation, suggesting that UDP-glucose triggers the mobilization of functionally distinct subsets of HSPCs. Furthermore, co-administration of UDP-glucose and G-CSF led to greater HSPC mobilization than G-CSF alone. Administration of the antioxidant agent NAC significantly reduced UDP-glucose-induced mobilization, coinciding with a reduction in RANKL and osteoclastogenesis. These findings provide direct evidence demonstrating a potential role for UDP-glucose in HSPC mobilization and may provide an attractive strategy to improve the yield of stem cells in poor-mobilizing allogeneic or autologous donors.

Mdm2 associates with Ras effector NORE1 to induce the degradation of oncoprotein HIPK1.

The Ras effector NORE1 is frequently silenced in primary adenocarcinomas, although the significance of this silencing for tumorigenesis is unclear. Here we show that NORE1 induces polyubiquitination and proteasomal degradation of oncoprotein HIPK1 by facilitating its interaction with the Mdm2 E3 ubiquitin ligase. Endogenous HIPK1 is stabilized in Nore1-deficient mouse embryonic fibroblasts, and depletion of HIPK1 in NORE1-silenced lung adenocarcinoma cells inhibits anchorage-independent cell growth and tumour formation in nude mice. These findings indicate that the control of HIPK1 stability by Mdm2-NORE1 has a major effect on cell behaviour, and epigenetic inactivation of NORE1 enables adenocarcinoma formation in vivo through HIPK1 stabilization.

Synergistic interactions between heregulin and peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist in breast cancer cells.

Here, we demonstrate that troglitazone (Rezulin), a peroxisome proliferator-activated receptor agonist, acted in synergy with heregulin to induce massive cell death in breast cancer cells. Although the combination of heregulin and troglitazone (HRG/TGZ) induced both apoptosis and necrosis, the main mode of cell death was caspase-independent and occurred via necrosis. This combination increased generation of superoxide in mitochondria, which in turn destabilized mitochondria potential. Pretreatment with N-acetyl-l-cysteine and catalase expression ameliorated cell death induced by the combination treatment, indicating a role of oxidative stress in mediating HRG/TGZ-induced cell death. Notably, pretreatment with pyruvate significantly prevented the cell death, suggesting a potential mechanistic link between metabolic stress and HRG/TGZ-induced cell death. The activation of the HRG signaling axis has been considered as a poor prognostic factor in breast cancer and confers resistance to gefitinib (Iressa) and tamoxifen. However, our data presented here paradoxically suggest that HRG expression can actually be beneficial when it comes to treating breast cancer with peroxisome proliferator-activated receptor-γ ligands. Taken together, the combination of HRG and TGZ may provide a basis for the development of a novel strategy in the treatment of apoptosis-resistant and/or hormone-refractory breast cancer.

Ewing sarcoma gene Ews regulates hematopoietic stem cell senescence.

The longevity of organisms is maintained by stem cells. If an organism loses the ability to maintain a balance between quiescence and differentiation in the stem/progenitor cell compartment due to aging and/or stress, this may result in death or age-associated diseases, including cancer. Ewing sarcoma is the most lethal bone tumor in young patients and arises from primitive stem cells. Here, we demonstrated that endogenous Ewing sarcoma gene (Ews) is indispensable for stem cell quiescence, and that the ablation of Ews promotes the early onset of senescence in hematopoietic stem progenitor cells. The phenotypic and functional changes in Ews-deficient stem cells were accompanied by an increase in senescence-associated ß-galactosidase staining and a marked induction of p16(INK4a) compared with wild-type counterparts. With its relevance to cancer and possibly aging, EWS is likely to play a significant role in maintaining the functional capacity of stem cells and may provide further insight into the complexity of Ewing sarcoma in the context of stem cells.

Tumor suppressor ras association domain family 5 (RASSF5/NORE1) mediates death receptor ligand-induced apoptosis.

Epigenetic silencing of RASSF (Ras association domain family) genes RASSF1 and RASSF5 (also called NORE1) by CpG hypermethylation is found frequently in many cancers. Although the physiological roles of RASSF1 have been studied in some detail, the exact functions of RASSF5 are not well understood. Here, we show that RASSF5 plays an important role in mediating apoptosis in response to death receptor ligands, TNF-α and TNF-related apoptosis-inducing ligand. Depletion of RASSF5 by siRNA significantly reduced TNF-α-mediated apoptosis, likely through its interaction with proapoptotic kinase MST1, a mammalian homolog of Hippo. Consistent with this, siRNA knockdown of MST1 also resulted in resistance to TNF-α-induced apoptosis. To further study the role of Rassf5 in vivo, we generated Rassf5-deficient mouse. Inactivation of Rassf5 in mouse embryonic fibroblasts (MEFs) resulted in resistance to TNF-α- and TNF-related apoptosis-inducing ligand-mediated apoptosis. Importantly, Rassf5-null mice were significantly more resistant to TNF-α-induced apoptosis and failed to activate Mst1. Loss of Rassf5 also resulted in spontaneous immortalization of MEFs at earlier passages than the control MEFs, and Rassf5-null immortalized MEFs, but not the immortalized wild type MEFs, were fully transformed by K-RasG12V. Together, our results demonstrate a direct role for RASSF5 in death receptor ligand-mediated apoptosis and provide further evidence for RASSF5 as a tumor suppressor.

SOX4 overexpression regulates the p53-mediated apoptosis in hepatocellular carcinoma: clinical implication and functional analysis in vitro.

BACKGROUND AND AIMS: The underlying molecular mechanisms of hepatocellular carcinoma (HCC) remain poorly understood due to its complex development process. The human T cell-specific transcription factor sex-determining region Y-related high-mobility group (HMG) box 4 (SOX4) has been linked to development and tumorigenesis. In this study, we characterized the roles of SOX4 in regulation of the p53 transcription activity and evaluated the expression patterns and prognostic value of the transcription factor SOX4 in HCC. METHODS: The expression levels of human SOX4 were examined in HCC samples obtained from 58 patients having curative partial hepatectomy. The interaction and effects of SOX4 on the p53 pathway were assessed in HCC cell lines. Luciferase reporter assay to examine p53-mediated transcription of target genes was performed. The association of SOX4 expression level with tumor recurrence and overall survival was evaluated. RESULTS: We showed that the HMG box domain of SOX4 interacted with p53, resulting in the inhibition of p53-mediated transcription by the Bax promoter. More importantly, SOX4 overexpression led to a significant repression of p53-induced Bax expression and subsequent repression of p53-mediated apoptosis induced by gamma-irradiation. In clinicopathological analysis, nuclear overexpression of SOX4 was observed in 37 out of 58 (63.8%) HCC samples, and this correlated with diminished risk of recurrence (P = 0.014) and improved overall survival time (P = 0.045) in HCC patients. CONCLUSION: These results suggest that SOX4 contributes to hepatocarcinogenesis by inhibiting p53-mediated apoptosis and that its overexpression might be a useful prognostic marker for survival after surgical resection.

A novel Wilms tumor 1 (WT1) target gene negatively regulates the WNT signaling pathway.

Mammalian kidney development requires the functions of the Wilms tumor gene WT1 and the WNT/beta-catenin signaling pathway. Recent studies have shown that WT1 negatively regulates WNT/beta-catenin signaling, but the molecular mechanisms by which WT1 inhibits WNT/beta-catenin signaling are not completely understood. In this study, we identified a gene, CXXC5, which we have renamed WID (WT1-induced Inhibitor of Dishevelled), as a novel WT1 transcriptional target that negatively regulates WNT/beta-catenin signaling. WT1 activates WID transcription through the upstream enhancer region. In the developing kidney, Wid and Wt1 are coexpressed in podocytes of maturing nephrons. Structure-function analysis demonstrated that WID interacts with Dishevelled via its C-terminal CXXC zinc finger and Dishevelled binding domains and potently inhibits WNT/beta-catenin signaling in vitro and in vivo. WID is evolutionarily conserved, and ablation of wid in zebrafish embryos with antisense morpholino oligonucleotides perturbs embryonic kidney development. Taken together, our results demonstrate that the WT1 negatively regulates WNT/beta-catenin pathway via its target gene WID and further suggest a role for WID in nephrogenesis.