Downregulation Of WT1 Research Articles

Epicardial SMARCA4 deletion exacerbates cardiac injury in myocardial infarction and is related to the inhibition of epicardial epithelial-mesenchymal transition

The reactivated adult epicardium produces epicardium-derived cells (EPDCs) via epithelial-mesenchymal transition (EMT) to benefit the recovery of the heart after myocardial infarction (MI). SMARCA4 is the core catalytic subunit of the chromatin re-modeling complex, which has the potential to target some reactivated epicardial genes in MI. However, the effects of epicardial SMARCA4 on MI remain uncertain. This study found that SMARCA4 was activated over time in epicardial cells following MI, and some of activated cells belonged to downstream differentiation types of EPDCs. This study used tamoxifen to induce lineage tracing and SMARCA4 deletion from epicardial cells in Wt1-CreER;Smarca4fl/fl;Rosa26-RFP adult mice. Epicardial SMARCA4 deletion reduces the number of epicardial cells in adult mice, which was related to changes in the activation, proliferation, and apoptosis of epicardial cells. Epicardial SMARCA4 deletion reduced collagen deposition and angiogenesis in the infarcted area, exacerbated cardiac injury in MI. The exacerbation of cardiac injury was related to the inhibition of generation and differentiation of EPDCs. The alterations in EPDCs were associated with inhibited transition between E-CAD and N-CAD during the epicardial EMT, coupled with the down-regulation of WT1, SNAIL1, and PDGF signaling. In conclusion, this study suggests that Epicardial SMARCA4 plays a critical role in cardiac injury caused by MI, and its regulatory mechanism is related to epicardial EMT. Epicardial SMARCA4 holds potential as a novel molecular target for treating MI.

CMIP interacts with WT1 and targets it on the proteasome degradation pathway.

BackgroundThe Wilms tumor 1 suppressor gene, WT1, is expressed throughout life in podocytes and is essential for their function. Downregulation of WT1 has been reported in podocyte diseases but the underlying mechanisms remain unclear. Podocyte injury is the hallmark of idiopathic nephrotic syndrome (INS), the most frequent glomerular disease in children and young adults. An increase in the abundance of Cmaf‐inducing protein (CMIP) has been found to alter podocyte function, but it is not known whether CMIP affects WT1 expression.MethodsTranscriptional and post‐transcriptional regulation of WT1in the presence of CMIP was studied using transient transfection, mouse models, and siRNA handling.ResultsWe showed that overproduction of CMIP in the podocyte was consistently associated with a downregulation of WT1 according to two mechanisms. We found that CMIP prevented the NF‐kB‐mediated transcriptional activation of WT1. We demonstrated that CMIP interacts directly with WT1 through its leucine‐rich repeat domain. Overexpression of CMIP in the M15 cell line induced a downregulation of WT1, which was prevented by lactacystin, a potent proteasome inhibitor. We showed that CMIP exhibits an E3 ligase activity and targets WT1 to proteasome degradation. Intravenous injection of Cmip‐siRNA specifically prevented the repression of Wt1 in lipopolysaccharides‐induced proteinuria in mice.ConclusionsThese data suggest that CMIP is a repressor of WT1 and might be a critical player in the pathophysiology of some podocyte diseases. Because WT1 is required for podocyte integrity, CMIP could be considered a therapeutic target in podocyte diseases.

Abstract 1326: Second generation androgen receptor signaling inhibitor resistant neuroendocrine like prostate cancer: A mechanistic study

Abstract Background:Failure of castration resistant prostate cancer patients to Androgen Receptor Signaling Inhibitor (ARSI) poses a major challenge in terms of therapeutic development. The heterogeneous nature of prostate cancer makes it difficult to stratify the treatment modalities. A multitude of variable pathological and genetic alterations induced by systemic treatment adds further to the therapeutic complexity. Upon continued exposure to enzalutamide (ARSI), nearly 15-20% of castration resistant patients advance towards a highly aggressive neuroendocrine like phenotype with concomitant functional loss of RB1 and TP53 and persistent AR expression in some. Elucidation of the underlying mechanism of resistance among this aggressive subset of patients is of utmost importance to clinically stratify the treatment options and target these advanced neuroendocrine like patients, who currently lack specific treatment approaches. The following study aims to identify the molecular signature responsible for driving this enzalutamide resistant neuroendocrine like development. Objective:The primary objective of the study is to identify the molecular mechanism behind ARSI induced neuroendocrine like Prostate cancer development. Methods:We have developed two cell line model systems. C4-2 has been genetically modified to develop a simultaneous genetic loss of RB1 and TP53 (DKD). C4-2B has been kept on continued exposure to Enzalutamide to develop Enzalutamide Resistant (C4-2BER). C4-2BER maintains AR expression while DKD loses AR expression, resembling different stages in neuroendocrine like development. Using Western Blot and RT PCR analysis the neuroendocrine like background of these resistant cell line models has been characterized. We performed an RNA-Seq with C4-2B and its resistant model C4-2BER and further downstream analysis through gene ontology and GSEA were performed. Results:Model system characterization study revealed neuroendocrine like characteristics and gene expression. Genetic analysis revealed an upregulation of multiple genes in C4-2BER in comparison to its parental C4-2B, which is comparable to patient dataset available online. GSEA analysis revealed several neuronal biology related pathways to be prominently upregulated among C4-2BER. Further analysis revealed that expression of WT1, which binds near most of these upregulated genes, is downregulated in C4-2BER. Conclusions:The downregulation of WT1 is noted during progression from CRPC towards enzalutamide resistant neuroendocrine development in C4-2BER, which is in turn accompanied with various epigenetic changes. Understanding such pattern of changes and identifying the underlying molecular signature would help elucidate the targets behind treatment induced neuroendocrine characteristics from a therapeutic point of view. Citation Format: Sreyashi Bhattacharya, Ridwan Islam, Sanika Bodas, Navatha Polavaram, Kaustubh Datta, Samikshan Dutta. Second generation androgen receptor signaling inhibitor resistant neuroendocrine like prostate cancer: A mechanistic study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1326.

Expression of the Wilms' tumour gene and its association with PPARβ/δ in healthy skin and melanoma of horses.

The Wilms' tumour gene (WT1) has previously been described as an oncogene in several neoplasms of humans, including melanoma, and its expression increases cancer cell proliferation. Recent reports associate the expression of the PPARβ/δ gene (peroxisome proliferator-activated receptor beta/delta) with the downregulation of WT1 in human melanoma and murine melanoma cell lines. The aim of this work was to analyse the expression of WT1 and its association with PPARβ/δ in samples of healthy and melanoma-affected skin of horses by immunohistochemistry. WT1 protein expression was detected in healthy skin, mainly in the epidermis, hair follicle, sebaceous gland and sweat gland, while no expression was observed in equine melanoma tissues. Moreover, it was observed that PPARβ/δ has a basal expression in healthy skin and that it is overexpressed in melanoma. These results were confirmed by a densitometric analysis, where a significant increase of the WT1-positive area was observed in healthy skin (128.66 ± 19.84 pixels 106) compared with that observed in melanoma (1.94 ± 0.04 pixels 106). On the other hand, a positive area with an expression of PPARβ/δ in healthy skin (214.94 ± 11.85 pixels 106) was significantly decreased compared to melanoma (624.86 ± 181.93 pixels 106). These data suggest that there could be a regulation between WT1 and PPARβ/δ in this disease in horses.

C-mip displays E3 ligase activity and targets WT1 to proteasome dégradation

Introduction We show here, overproduction of C-mip was consistenly associated with a downregulation of WT1. C-mip interacts physically with WT1, and exhibits an E3 ligase activity, which targets WT1 to proteasome degradation. Description Overexpression of C-mip in M15 cell line induced a downregulation of WT1, which was prevented by lactacystin. We showed that C-mip exhibits an E3 ligase activity, which targets WT1 to proteasome degradation. Intravenous injection of c-mip-siRNA specifically prevented the repression of WT1 in LPS-induced proteinuria in mice. Methods (1) Transfected C-mip plasmid to M15 cell line, which expresses a high level of WT1, to see whether C-mip down-regulates the expression of WT1. (2) Cotransfected WT1 and C-mip expression plasmids into HEK cells, then the cells were incubated with lactacystin. (3) E3 ubiquitin ligase kit was used to determine whether C-mip could have an E3 ligase activity that drives ubiquitylation of WT1. (4) Knockdown of c-mip in vivo by RNA interference in LPS-treated mice. Results (1) WT1 abundance fell by 50% at 12 hours after transfection of C-mip plasmid to M15 cell line, shows C-mip destabilizes the WT1. (2) The abundance of WT1 protein was significantly reduced in the presence of C-mip, but coincubation with lactacystin inhibited WT1 decay, suggesting that C-mip targets WT1 to proteasome mediated degradation. (3) E3 ligase activity assay showed, C-mip stimulated the formation of polyubiquitin chains in a dose dependent manner, suggesting that C-mip exhibits an intrinsic ubiquitin E3 ligase activity. (4) In vivo c-mip siRNA treatment indicated knockdown of c-mip restores the expression of WT1 in LPS-treated mice. Conclusion (i) In vivo, in vitro overproduction of C-mip is associated with a downregulation of WT1; (ii) C-mip interacts directly with WT1 and displays E3 ligase activity, which promotes WT1 to proteasome-mediated degradation; (iii) Silencing endogenous c-mip expression with RNAi prevents the downregulation of WT1 in LPS-treated mice.

Wilms' tumor (WT1) (±KTS) variants decreases the progesterone secretion of bovine ovarian theca cells

Wilms' tumor gene WT1 encodes a nuclear transcriptional factor, which has been shown to regulate granulosa cell steroidogenesis in bovine; however, it is not known whether the functions of theca cells are regulated by WT1. Here, we determined the effects of this gene on theca cell proliferation, apoptosis, and steroidogenesis in vitro. In cultured bovine theca cells, the downregulation of WT1 increased the secretion of progesterone but had no effect on proliferation and apoptosis. WT1 includes the variants WT1(+KTS) and WT1(−KTS), which differ by 3 amino acids KTS (lysine, threonine, and serine). WT1(±KTS) upregulation increased the messenger RNA (mRNA) expression of STAR and CYP17A1 and decreased the progesterone secretion and CYP11A1 mRNA expression. In contrast to WT1(+KTS), WT1(−KTS) upregulation also decreased the mRNA expression of 3β-HSD. In both variants, WT1(−KTS) has more obvious effects. In conclusion, WT1 can decrease progesterone secretion, likely due in part to the inhibition of CYP11A1 and 3β-HSD.

Histone demethylase KDM6B regulates human podocyte differentiation in vitro.

Podocytes are terminally differentiated and highly specialized glomerular cells, which have an essential role as a filtration barrier against proteinuria. Histone methylation has been shown to influence cell development, but its role in podocyte differentiation is less understood. In this study, we first examined the expression pattern of histone demethylase KDM6B at different times of cultured human podocytes in vitro We found that the expression of KDM6B and podocyte differentiation markers WT1 and Nephrin are increased in the podocyte differentiation process. In cultured podocytes, KDM6B knockdown with siRNA impaired podocyte differentiation and led to expression down-regulation of WT1 and Nephrin. The treatment of podocytes with GSK-J4, a specific KDM6B inhibitor, can also obtain similar results. Overexpression of WT1 can rescue differentiated phenotype impaired by disruption of KDM6B ChIP (chromatin immunoprecipitation) assay further indicated that KDM6B can bind the promoter region of WT1 and reduce the histone H3K27 methylation. Podocytes in glomeruli from nephrotic patients exhibited increased KDM6B contents and reduced H3K27me3 levels. These data suggest a role for KDM6B as a regulator of podocyte differentiation, which is important for the understanding of podocyte function in kidney development and related diseases.

The Wilms tumor gene (WT1) (+/-KTS) isoforms regulate steroidogenesis by modulating the PI3K/AKT and ERK1/2 pathways in bovine granulosa cells†.

The Wilms tumor (WT) gene WT1 encodes the splicing variants WT1(+KTS) and WT1(-KTS). Recent data suggest that WT1 plays an important role in the development of mice follicles. However, the mechanism through which WT1 influences ovarian steroidogenesis remains unknown. This study identified WT1 and evaluated the impact of splicing variants WT1(+KTS) and WT1(-KTS) on steroidogenesis using adult bovine granulosa cells (GCs). Using RT-qPCR and western blotting, we found that the ratio between WT1(+KTS) and WT1(-KTS) was stabilized. WT1 expression, however, decreased gradually in bovine GCs in response to follicle enlargement or atresia. The downregulation of WT1 increased the secretion of basal and follicle-stimulating-hormone-induced progesterone (P4), but decreased the secretion of basal-induced estradiol (E2). This was associated with an increase in the expression of 3β-HSD, and a decrease in the expression of CYP19A1. In addition, WT1(-KTS) overexpression suppresses the secretion of E2 and P4 compared with WT1(+KTS) overexpression. This was associated with a decrease in the expression of CYP19A1, CYP11A1, and 3β-HSD in cultured bovine GCs. Of note, the downregulation of WT1 suppresses the phosphorylation levels of AKT and p-ERK1/2. However, WT1(-KTS) overexpression promotes the phosphorylation levels of AKT and suppresses p-ERK1/2 levels. LY294002 (AKT inhibitor) increases MKP3 mRNA expression levels but decreases the level of p-AKT and p-ERK1/2. Collectively, WT1 significantly suppresses the mRNA expression of CYP11A1 and 3β-HSD and the secretion of P4 in bovine GCs. Moreover, it regulates CYP19A1 mRNA expression and E2 secretion with complex networks, at least in part, by modulating AKT and ERK1/2 signaling. The effect of WT1(-KTS) was more pronounced than that exerted by WT1(+KTS).

The presence of human mesenchymal stem cells of renal origin in amniotic fluid increases with gestational time

BackgroundEstablished therapies for managing kidney dysfunction such as kidney dialysis and transplantation are limited due to the shortage of compatible donated organs and high costs. Stem cell-based therapies are currently under investigation as an alternative treatment option. As amniotic fluid is composed of fetal urine harboring mesenchymal stem cells (AF-MSCs), we hypothesized that third-trimester amniotic fluid could be a novel source of renal progenitor and differentiated cells.MethodsHuman third-trimester amniotic fluid cells (AFCs) were isolated and cultured in distinct media. These cells were characterized as renal progenitor cells with respect to cell morphology, cell surface marker expression, transcriptome and differentiation into chondrocytes, osteoblasts and adipocytes. To test for renal function, a comparative albumin endocytosis assay was performed using AF-MSCs and commercially available renal cells derived from kidney biopsies. Comparative transcriptome analyses of first, second and third trimester-derived AF-MSCs were conducted to monitor expression of renal-related genes.ResultsRegardless of the media used, AFCs showed expression of pluripotency-associated markers such as SSEA4, TRA-1-60, TRA-1-81 and C-Kit. They also express the mesenchymal marker Vimentin. Immunophenotyping confirmed that third-trimester AFCs are bona fide MSCs. AF-MSCs expressed the master renal progenitor markers SIX2 and CITED1, in addition to typical renal proteins such as PODXL, LHX1, BRN1 and PAX8. Albumin endocytosis assays demonstrated the functionality of AF-MSCs as renal cells. Additionally, upregulated expression of BMP7 and downregulation of WT1, CD133, SIX2 and C-Kit were observed upon activation of WNT signaling by treatment with the GSK-3 inhibitor CHIR99201. Transcriptome analysis and semiquantitative PCR revealed increasing expression levels of renal-specific genes (e.g., SALL1, HNF4B, SIX2) with gestational time. Moreover, AF-MSCs shared more genes with human kidney cells than with native MSCs and gene ontology terms revealed involvement of biological processes associated with kidney morphogenesis.ConclusionsThird-trimester amniotic fluid contains AF-MSCs of renal origin and this novel source of kidney progenitors may have enormous future potentials for disease modeling, renal repair and drug screening.

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.

Dysregulation of WTI (\u2212KTS) is Associated with the Kidney-Specific Effects of the LMX1B R246Q Mutation

Mutations in the LIM homeobox transcription factor 1-beta (LMX1B) are a cause of nail patellar syndrome, a condition characterized by skeletal changes, glaucoma and focal segmental glomerulosclerosis. Recently, a missense mutation (R246Q) in LMX1B was reported as a cause of glomerular pathologies without extra-renal manifestations, otherwise known as nail patella-like renal disease (NPLRD). We have identified two additional NPLRD families with the R246Q mutation, though the mechanisms by which LMX1BR246Q causes a renal-specific phenotype is unknown. In this study, using human podocyte cell lines overexpressing either myc-LMX1BWT or myc-LMX1BR246Q, we observed dominant negative and haploinsufficiency effects of the mutation on the expression of podocyte genes such as NPHS1, GLEPP1, and WT1. Specifically, we observed a novel LMX1BR246Q-mediated downregulation of WT1(−KTS) isoforms in podocytes. In conclusion, we have shown that the renal-specific phenotype associated with the LMX1BR246Q mutation may be due to a dominant negative effect on WT1(−KTS) isoforms that may cause a disruption of the WT1 (−KTS):(+KTS) isoform ratio and a decrease in the expression of podocyte genes. Full delineation of the LMX1B gene regulon is needed to define its role in maintenance of glomerular filtration barrier integrity.

In Vivo modeling of Resistance to PI3Kδ Inhibitor Treatment Using EµTCL1-Tg Tumor Transfer Model

Inhibitors of B-cell receptor (BCR) signaling have proven effective in the treatment of chronic lymphocytic leukemia (CLL). An important downstream mediator of BCR signaling is phosphoinositide 3-kinase delta (PI3Kd), which through activation of AKT, controls cell survival, growth and proliferation. Since it is expressed predominantly in cells of hematopoietic origin, PI3Kd is a promising target in CLL, and the novel PI3Kd inhibitor idelalisib is effective in treating relapsed/refractory CLL. However, a subset of patients relapse under therapy, and the mechanisms leading to resistance are not understood. To generate and study resistance mechanisms in vivo, a tool compound GS-649443 with favorable murine pharmacokinetic properties in vivo as compared to Idelalisib was used. GS-649443 is a highly specific and potent small molecule inhibitor of the PI3K isoform d, with an IC50 of 0.3nM and 86/420/4120 fold selectivity over PI3K isoforms g/b/a respectively. We performed an in vivo serial transfer and treatment scheme to generate specific resistance against GS-649443 using the transferable murine CLL clone TCL1-192(Chen SS et al., 2013). 5 million TCL1-192 cells were transferred by intravenous injection into the tail vein of CB17.SCID mice, followed by treatment with vehicle (N=12) or 5mg/kg GS-649443 (N=12) twice daily by oral gavage, starting from day 10. Analysis of the animals (N=6 vs. 6) at the death of the vehicle treated mice (11 days) showed a drastic reduction in spleen weight (median 0.650g vs. 0.345g, P=0.005), liver weight (median2.025g vs. 1.145g, P=0.0022) and WBC count (median220.4 cells/nl vs. 1.7 cells/nl, P=0.0078) as compared to vehicle treated mice. Also, GS-649443 treatment almost doubled the survival of the mice in comparison to vehicle treatment (Fig. 1, P=0.0011). To generate resistance against GS-649443 in vivo, TCL1-192 cells were kept under selection pressure using serial transfers and subsequent treatment. After three rounds of transfer and treatment, the mice treated with GS-649443 failed to respond and showed a survival identical to vehicle controls (Fig.1). Mutations acquired over time during the serial transfers in TCL1-192 cells from resistant mice were identified using whole exome sequencing. 64 treatment specific mutations were identified with a tumor variant allele frequency of ≥10%. Intriguingly, no single recurrent mutation that would likely contribute to drug resistance was identified. This is similar to the observation in patients refractory to idelalisib (abstract Ghia et al., submitted to ASH 2016). However, a subset of the genes such as Grb2, Mylk, Srfbp1, Ptk2, Cd44 and Prkd1 harboring mutations could be functionally grouped into integrin and extracellular matrix signaling. Consistent with this, all the resistant tumors showed a significant upregulation of genes involved in the integrin receptor complex such as talin 1 (P=0.004), talin 2 (P=0.004) and vinculin (P=0.009). RNAseq analysis for detecting changes in gene expression identified an upregulation of Igf1r expression in the resistant samples compared to vehicle treated samples (P The synergy between the receptor tyrosine kinases such as IGF1R and integrin mediated signaling is established in different cancers and a similar mechanism may play a role in compensating for the inhibition of BCR mediated PI3K/AKT signaling. In conclusion, resistance to a tool PI3Kd inhibitor is not mediated by unique recurrent mutations but by alterations in survival signaling with mutations playing a probable cooperative role. Disclosures Tausch:Celgene: Other: Travel support; Amgen: Other: Travel support; Gilead: Other: Travel support, Speakers Bureau. Yahiaoui:Gilead Sciences: Employment. Munugalavadla:Gilead Sciences: Employment, Equity Ownership. Tannheimer:Gilead Sciences: Employment. Stilgenbauer:Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding.

Down-regulation of Wt1 activates Wnt/β-catenin signaling through modulating endocytic route of LRP6 in podocyte dysfunction in vitro

Podocyte dysfunction plays important roles in the pathogenesis of chronic kidney disease, and Wt1 has long been considered to be a marker of podocyte, whereas its roles and mechanisms in podocyte injury are still unclear though Wt1 mutations are reported to be involved in the development of glomerular disease in human and mice. Here we show that down-regulation of Wt1 could induce podocyte dysfunction and apoptosis through activating Wnt/β-catenin signaling. Podocytes treated with adriamycin demonstrated decreased expression of Wt1, coupled with activated Wnt/β-catenin signaling in vitro. Reduced expression of Wt1 in podocytes transfected with Wt1 siRNA is correlated with activated Wnt/β-catenin signaling, increased podocyte apoptosis, as well as suppressed expression of nephrin. Blockade of Wnt/β-catenin signaling with Dickkopf-1 ameliorated podocyte injury and apoptosis induced by Wt1 siRNA. We also found that membrane LRP6 was increased dramatically in podocytes transfected with Wt1 siRNA compared with control siRNA, while no significant change was found in total LRP6. Caveolin- and clathrin-dependent endocytosis were both involved in the regulation of β-catenin signaling. And we found that down-regulation of Wt1 in podocytes mediates activation of Wnt/β-catenin signaling by recruiting LRP6 to the caveolin-mediated endocytosis route, thereby sequestering it from clathrin-dependent endocytosis. As a result, we concluded that Wt1 expression levels in podocytes regulate Wnt/β-catenin signaling through modulating the endocytic fate of LRP6, and this indicates a potential target for the therapy of CKD.

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

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

Early B-cell factor 1 is an essential transcription factor for postnatal glomerular maturation

The coordination of multiple cytokines and transcription factors with their downstream signaling pathways have been shown to be integral to nephron maturation. Here we present a completely novel role for the helix-loop-helix transcription factor Early B cell Factor 1 (Ebf1), originally identified for B cell maturation, for the proper maturation of glomerular cells from mesenchymal progenitors. The expression of Ebf1 was both spatially and temporally regulated within the developing cortex and glomeruli. Using Ebf1-null mice we then identified biochemical, metabolic, and histological abnormalities in renal development that arose in the absence of this transcription factor. In the Ebf1 knockout mice the developed kidneys show thinned cortices and reduced glomerular maturation. The glomeruli showed abnormal vascularization and severely effaced podocytes. The mice exhibited early albuminuria and elevated blood urea nitrogen levels. Moreover, the GFR was reduced over 66 percent and the expression of podocyte-derived VEGF-A was decreased compared to wild type control mice. Thus, Ebf1 has a significant and novel role in glomerular development, podocyte maturation, and the maintenance of kidney integrity and function.

Effects of WT1 gene downregulation on apoptosis in porcine fetal fibroblasts

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

Repeated Systemic Administration of Human Adipose-Derived Stem Cells Attenuates Overt Diabetic Nephropathy in Rats

Adipose-derived stem cells (ASCs) can alleviate acute kidney injury and promote kidney cell regeneration and repair. To investigate the role of ASCs in diabetic nephropathy (DN), Sprague-Dawley rats were made diabetic by intraperitoneal injection of streptozotocin (STZ) after uninephrectomy. After 12 weeks, proteinuria was well established. Five times of 5×10(6) human ASCs repeatedly injected through a tail vein at 4 weekly intervals. A reduction in proteinuria was not observed in diabetic rats until 24 weeks. However, urinary protein excretion was significantly suppressed at 28 weeks and persisted up to 32 weeks after STZ treatment. ASC treatment significantly attenuated glomerulus hypertrophy and tubular interstitial injury, and led to the downregulation of WT-1 and synaptopodin expression. CFSE labeled ASCs were injected into DN rats via the tail vein. Within 24 h after injection, the cells were detected in lung, spleen, and peritubular regions, but rarely in pancreas. Human Alu gene expression was detected in lung and spleen up to 4 weeks after ASCs injection. ASC treatment did not improve hyperglycemia or pancreatic damage. In vitro, recombinant human glial cell line-derived neurotrophic factor (GDNF) prevented podocyte injury by high glucose similarly to ASC-conditioned medium. After blocking GDNF in ASC-CM with neutralizing antibody, the therapeutic effect of ASC-CM was significantly decreased. ASCs cocultured with podocytes restored the downregulation of synaptopodin expression, which was weakened by GDNF-RNA interfering. These findings indicate that repeated intravenous ASC can reduce diabetic kidney damage in rats even at the progressive stage, and promote podocyte recovery via GDNF secretion.

The effects of the WT1 gene on apoptosis and development‐related gene expression in porcine kidney fibroblasts and swine testis cells

The Wilm's tumor 1 gene (WT1) encodes zinc finger proteins that function as tumor suppressors, and play important roles in the development of the genito-urinary system and other organs. Its precise function in the development of porcine tissues and organs, which is an attractive transplantation resource for certain human diseases, is still unclear. Here, we sought to define the role of WT1 in porcine kidney and testis tissues using porcine kidney fibroblasts (PKFs) and swine testis (ST) cells as in vitro models, both of which express WT1. The recombinant plasmids pLV3-WT1 shRNA and pIRES2 -WT1-EGFP were constructed to respectively down- and up-regulate the WT1 gene in porcine cells. The role of WT1 in cell proliferation was investigated by RT-PCR, immunocytochemical staining, apoptosis analysis, and Western blot. The pLV3-WT1 shRNA dramatically reduced WT1 expression at both the transcription and protein levels. The down-regulation of WT1 directly led to early cell apoptosis, and changes in Sf1, Sox9, and Gdnf gene expression in PKFs and ST cells. In contrast, up-regulation of WT1 gave no obvious phenotype in ST cells. Our results demonstrate that WT1 is essential for the survival of PKFs and ST cells because it regulates apoptosis- and development-related genes in the cells; however, no obvious effect was observed when WT1 was over-expressed.

Focal segmental glomerulosclerosis is induced by microRNA-193a and its downregulation of WT1

Focal segmental glomerulosclerosis (FSGS) is a frequent and severe glomerular disease characterized by destabilization of podocyte foot processes. We report that transgenic expression of the microRNA miR-193a in mice rapidly induces FSGS with extensive podocyte foot process effacement. Mechanistically, miR-193a inhibits the expression of the Wilms' tumor protein (WT1), a transcription factor and master regulator of podocyte differentiation and homeostasis. Decreased expression levels of WT1 lead to downregulation of its target genes PODXL (podocalyxin) and NPHS1 (nephrin), as well as several other genes crucial for the architecture of podocytes, initiating a catastrophic collapse of the entire podocyte-stabilizing system. We found upregulation of miR-193a in isolated glomeruli from individuals with FSGS compared to normal kidneys or individuals with other glomerular diseases. Thus, upregulation of miR-193a provides a new pathogenic mechanism for FSGS and is a potential therapeutic target.

AOSOP11 KI67 LEVEL IN HORMONE-RECEPTOR-POSITIVE BREAST CANCER: A RETROSPECTIVE REVIEW OF 8990 KOREAN WOMEN

s, 5th AOS and 9th Annual Conference of OOTR, 2013 / European Journal of Cancer 49S1 (2013) S1−S22 S5 Methods. RNA interference (RNAi) and RNA activation (RNAa) were used to down-regulate and up-regulate the expression of WT1, respectively. The impacts of WT1 on proliferation, cell cycle distribution, apoptosis, invasion, and migration of breast cancer cells were detected by CCK8, annexin V/PI, and Transwell methods, respectively. Results. The mRNA and protein expression of WT1 in MDAMB-321 cells were significantly higher than that in MCF-7, Bcap-37, and SKBR-3 cells. The WT1 small interfering RNA (siRNA) could effectively inhibit the WT1 expression of MDAMB-231 at 48h after transfection. Down-regulation of WT1 decreased cell proliferation, the proportion of cells in S and G2 phase as well as the invasion and migration ability, and increased the proportion of apoptotic cells and cells in G1 phase. WT1 double-stranded RNA (dsRNA) could increase the expression of WT1 in MCF-7 cells, and promoted the proliferation, invasion, and migration of MCF-7 cells. In addition, we also found that the inhibitor of differentiation 1 (Id1), a key molecule in cell proliferation and metastasis in breast cancer, was the target gene of WT1. Finally, we used transforming growth factor b1 (TGF-b1) to induce WT1 expression in MCF-7 cells with the concentration of 10ng/mL for 48h.After treatment,MCF-7 cells appeared fromepitheliallike to mesenchymal-like. Moreover, the expression of WT1 and Id1 in MCF-7 cells was increased, and the ability of invasion and migration were enhanced. Discussion. WT1 plays an oncogenic role in the progression of breast cancer, and promotes the proliferation, invasion, and migration of breast cancer cells partly through regulating Id1. Funding. This study was supported by NSFC (No. 81102030). The authors declared no conflicts of interest. AOSOP11 KI67 LEVEL IN HORMONE-RECEPTOR-POSITIVE BREAST CANCER: A RETROSPECTIVE REVIEW OF 8990 KOREAN WOMEN J. Kima *, W. Hana, Y.H. Parkb, S. Jungc, J. Roc, H. Moona, S. Ahna, J. Leea, M. Kima, D. Noha. aSeoul National University Hospital, Seoul, South Korea, bSamsung Medical Center, Seoul, South Korea, cNational Cancer Center, Seoul, South Korea Introduction. Breast cancer at a young age has poor prognosis and the number of young patients being diagnosed is increasing. Tumour proliferation marker Ki67 is known to be associated with prognosis and response to cancer therapy. This study aimed to determine the relationship between age and Ki67 in hormone-receptor-positive breast cancer. Methods.We retrospectively reviewed 8990 consecutive cases of hormone-receptor-positive invasive breast cancer among Korean women. Multicentre data for Ki67 level identified by immunohistochemistry (IHC) and age at diagnosis were analysed. Patients who had undergone neoadjuvant systemic therapy and who had in-situ carcinomas were excluded. Data were obtained from Seoul National University Hospital (SNUH), Samsung Medical Center (SMC), and National Cancer Center (NCC) after approval of the institutional review board from each institute. Results. Total 6222 cases from SNUH, 976 from SMC, and 1863 from NCC were included. The three datasets were analysed separately due to the variable IHC method in each institute. Mean ages were 49.30 years (range 20−86), 47.75 years (range 22−81), and 45.31 years (range 25−59) in SNUH, SMC, and NCC, respectively, and mean Ki67 levels were 4.66% (range 1– 100), 22.98% (range 1−97), and 14.58% (range 1−90). Ki67 levels were inversely correlated with age at diagnosis in all three datasets. Ki67 levels were significantly higher among patients aged younger than 40 years compared with 40 years and older (mean Ki67 5.97 vs 4.41, p<0.001; 28.60 vs 21.88, p<0.001; and 17.01 vs 14.03, p<0.001, in SNUH, SMC, and NCC, respectively). Mean ages were younger when Ki67 level was dichotomised into <10% versus 10%, <20% versus 20%, and <14% versus 14%, in SNUH, SMC, and NCC (p<0.001, p=0.03, and p<0.001, respectively). Discussion. Ki67 levels were significantly higher in hormonereceptor-positive breast cancer at a young age. This may be associated with the poor prognosis and poor response to endocrine therapy in young patients with this disease. However, due to the variability of assessing Ki67, careful interpretation should be made to achieve validity. Funding. None declared. The authors declared no conflicts of interest. AOSOP12 PRELIMINARY STUDY OF SPECTRAL CT IMAGING IN DIFFERENTIATING PULMONARY SQUAMOUS CELL CARCINOMA AND SMALL CELL CARCINOMA Y. Liua, J. Yub *, W. Looc, L. Chowc, A. Yipc. aThe Affiliated Hospital of Ningxia Medical University, Yinchuan, China, bNingxia People’s Hospital, Yinchuan, China, cOrganisation for Oncology and Translational Research and UNIMED Medical Institute, Hong