Gene expression of distinct histological components of Wilms tumor analyzed under the perspective of kidney development
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ABSTRACT: Gene expression analyses through cDNA microarray of unique Wilms tumor (WT) histological components, blastema (BL), epithelia (EP) and stroma (ST), from different patients were performed and compared with non-neoplastic mature and pool of fetal kidney (FK). We used a customized cDNA array containing 4 608 human genes and demonstrated that BL had over representation of genes with similar expression behavior to the earliest stage of normal renal development. Moreover, since WT is a result of loss of developmental control and gain of tumorigenic potential in a successive way, herein we identified genes whose expression level is altered during the kidney development and also in WT and classified as WT-kidney development set. From this set, a smaller group of 36 differentially expressed genes was derived, which was enriched by genes involved in signal transduction, such as APC, BZRP, MET, PLAU, GPR35 and TRADD. An over representation of genes belonging to the WNT signaling pathway were observed. Immunostaining assays of APC and beta-catenin were carried out in 108 specimens showing differential labeling localization in WT. Altogether our data show molecular evidences confirming the recapitulation of embryonic kidney by WT components and strongly suggest that the WNT signaling pathway plays a crucial role in Wilms tumorigenesis. Keywords: Wilms tumor, cDNA microarray, Histological components, WNT signaling pathway
Project description:Wilms tumour (WT), a childhood kidney cancer with embryonal origins, has been extensively characterised for genetic and epigenetic alterations, but a proportion of WTs still lack identifiable abnormalities. To uncover DNA methylation changes critical for WT pathogenesis, we compared the epigenome of fetal kidney with two WT cell lines, using methyl-CpG immunoprecipitation. We filtered our results to remove common cancer-associated epigenetic changes, and to enrich for genes involved in early kidney development. This identified four candidate genes that were hypermethylated in WT cell lines compared to fetal kidney, of which ESRP2 (epithelial splicing regulatory protein 2), was the most promising gene for further study. ESRP2 was commonly repressed by DNA methylation early in WT development (in nephrogenic rests) and could be reactivated by DNA methyltransferase inhibition in WT cell lines. When ESRP2 was expressed in WT cell lines, it acted as an inhibitor of cellular proliferation in vitro and in vivo it suppressed tumour growth of orthotopic xenografts in nude mice. RNA-seq of the ESRP2-expressing WT cell lines identified several novel splicing targets, in addition to well-characterised targets of ESRP2. One of these targets, LEF1, is a component of the Wnt signalling pathway that is essential for kidney development and commonly disrupted in WT. We propose a model in which the Wnt pathway can be disrupted in early kidney development to generate WT, either by genetic abnormalities such as WT1 mutations, or by epigenetic defects, such as ESRP2 methylation. The microarray data in this deposition identified ESRP2 as a commonly hypermethylated gene in Wilms tumour.
Project description:Wilms tumor (WT), a childhood kidney cancer with embryonal origins, has been extensively characterised for genetic and epigenetic alterations, but a proportion of WTs still lack identifiable abnormalities. To uncover DNA methylation changes critical for WT pathogenesis, we compared the epigenome of fetal kidney with two WT cell lines, using methyl-CpG immunoprecipitation. We filtered our results to remove common cancer-associated epigenetic changes, and to enrich for genes involved in early kidney development. This identified four candidate genes that were hypermethylated in WT cell lines compared to fetal kidney, of which ESRP2 (epithelial splicing regulatory protein 2), was the most promising gene for further study. ESRP2 was commonly repressed by DNA methylation early in WT development (in nephrogenic rests) and could be reactivated by DNA methyltransferase inhibition in WT cell lines. When ESRP2 was expressed in WT cell lines, it acted as an inhibitor of cellular proliferation in vitro and in vivo it suppressed tumor growth of orthotopic xenografts in nude mice. RNA-seq of the ESRP2-expressing WT cell lines identified several novel splicing targets, in addition to well-characterised targets of ESRP2. One of these targets, LEF1, is a component of the Wnt signalling pathway that is essential for kidney development and commonly disrupted in WT. We propose a model in which the Wnt pathway can be disrupted in early kidney development to generate WT, either by genetic abnormalities such as WT1 mutations, or by epigenetic defects, such as ESRP2 methylation.
Project description:Almost all colorectal cancers (CRC) present with mutations in the Apc gene, leading to unrestrained Wnt activation and the initiation of tumour development. We previously reported the competitive benefit of Apc-mutant intestinal stem cells (ISCs) within the crypt, however, the mechanism by which they outcompete their wild type (WT) neighbours remained elusive. Here, we studied the effect of Apc-mutants using an in vitro culture system of WT (Lgr5-CreErt2) and Apc-/- (Lgr5-CreErt2;Apcfl/fl) organoids . The expression patterns of WT and Apc-/- organoids revealed significant upregulation in specific secreted Wnt antagonists Notum, Wif1 and Dkk2. Furthermore, we studied the effect of the secreted antagonist by treating WT organoids with either WT or Apc-/- conditioned medium (CM) for 48 hours and observed a significant decrease in stem cell markers and an increase in goblet cell markers. We report that Apc-mutants act as bona fide supercompetitors by secreting Wnt antagonists that result in active differentiation of WT ISCs.
Project description:Wilms tumor (nephroblastoma) is a pediatric kidney tumor that arises from renal progenitor cells. Since the blastemal type is associated with adverse prognosis, we characterized such Wilms tumors by exome and transcriptome analysis. We detected novel, recurrent somatic mutations affecting the SIX1/2 – SALL1 pathway implicated in kidney development, the DROSHA/DGCR8 microprocessor genes as well as alterations in MYCN and TP53, the latter being strongly associated with dismal outcome. The DROSHA mutations impair the RNase III domains, while DGCR8 exhibits stereotypic E518K mutations in the RNA binding domain - both may skew miRNA representation. SIX1 and SIX2 mutations affect a single hotspot (Q177R) in the homeodomain indicative of a dominant effect. In larger cohorts, these mutations cluster in blastemal and chemotherapy-induced regressive tumors that likely derive from blastemal cells and these are characterized by generally higher SIX1/2 expression. These findings broaden the spectrum of human cancer genes and may open new avenues for stratification and therapeutic leads for Wilms tumors. 53 Wilms tumor samples were selected for RNA extraction and hybridization on Affymetrix Affymetrix Human Genome U133 Plus 2.0 Arrays.
Project description:Wilms tumor (nephroblastoma) is a pediatric kidney tumor that arises from renal progenitor cells. Since the blastemal type is associated with adverse prognosis, we characterized such Wilms tumors by exome and transcriptome analysis. We detected novel, recurrent somatic mutations affecting the SIX1/2 – SALL1 pathway implicated in kidney development, the DROSHA/DGCR8 microprocessor genes as well as alterations in MYCN and TP53, the latter being strongly associated with dismal outcome. The DROSHA mutations impair the RNase III domains, while DGCR8 exhibits stereotypic E518K mutations in the RNA binding domain - both may skew miRNA representation. SIX1 and SIX2 mutations affect a single hotspot (Q177R) in the homeodomain indicative of a dominant effect. In larger cohorts, these mutations cluster in blastemal and chemotherapy-induced regressive tumors that likely derive from blastemal cells and these are characterized by generally higher SIX1/2 expression. These findings broaden the spectrum of human cancer genes and may open new avenues for stratification and therapeutic leads for Wilms tumors.
Project description:We performed CRISPR screens on both a sub-library and a genome-wide scale in human intestinal organoids to discover cancer driver genes. We investigated the Wnt and the TGFB pathway and used both WT, APC-mutant and APC-TP53-mutant organoids.
Project description:We performed CRISPR screens on both a sub-library and a genome-wide scale in human intestinal organoids to discover cancer driver genes. We investigated the Wnt and the TGFB pathway and used both WT, APC-mutant and APC-TP53-mutant organoids.
Project description:We performed CRISPR screens on both a sub-library and a genome-wide scale in human intestinal organoids to discover cancer driver genes. We investigated the Wnt and the TGFB pathway and used both WT, APC-mutant and APC-TP53-mutant organoids.
Project description:We performed CRISPR screens on both a sub-library and a genome-wide scale in human intestinal organoids to discover cancer driver genes. We investigated the Wnt and the TGFB pathway and used both WT, APC-mutant and APC-TP53-mutant organoids.