Re-activation of EWS-FLI1 suppressed FOXO1 expression as a novel therapeutic strategy for Ewing´s sarcoma
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Maximilian Kauer
PROVIDER: E-GEOD-37409 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based upon proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncogene with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate EWS-FLI1 upon post-transcriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis including CLK1, CASP3, PPFIBP1, and TERT validate as alternatively spliced by EWS-FLI1. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNPK, and PRPF6. Reduction of EWS-FLI1 produces an isoform of g-TERT that has increased telomerase activity compared to WT TERT. The small molecule YK-4-279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions including DDX5 and RNA helicase A (RHA) that alters RNA splicing ratios. As such, YK-4-279 validates the splicing mechanism of EWS-FLI1 showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells. Exon array analysis of 75 ES patient samples show similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing towards oncogenesis, and reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code. Alternative splicing of RNA allows a limited number of coding regions in the human genome to produce proteins with diverse functionality. Alternative splicing has also been implicated as an oncogenic process. Identifying aspects of cancer cells that differentiate them from non-cancer cells remains an ongoing challenge and our research suggests that alternatively spliced mRNA and subsequent protein isoforms will provide new anti-cancer targets. We determined that the key oncogene of Ewing sarcoma (ES), EWS-FLI1, regulates alternative splicing in multiple cell line models. These experiments establish oncogenic aspects of splicing which are specific to cancer cells and thereby illuminate potentially oncogenic splicing shifts as well as provide a useful stratification mechanism for ES patients. We analyzed three models of EWS-FLI1 using Affymetrix GeneChip Human Exon 1.0 ST microarray: (i) Ewing's sarcoma TC32 wild-type cells expressing EWS-FLI1, and TC32 cells where EWS-FLI1 was reduced with a lentiviral shRNA; (ii) A673i, which has a doxycycline-inducible shRNA to reduce EWS-FLI1 expression, and wild-type EWS-FLI1 to screen for alternative splicing as measured by exon-specific expression changes; and (iii) human mesenchymal stem cells (hMSC), a putative cell of origin of Ewing's sarcoma, exogenously expressing EWS-FLI1, and hMSC wild-type cells without EWS-FLI1. Three biological replicates were included for each condition. The Bioconductor package "oligo" in the R programming language was used for normalization and background correction. Analysis was carried out using only core probesets, as defined by the manufacturer.
2015-03-31 | E-GEOD-65941 | biostudies-arrayexpress
Project description:EWS-FLI1 is a chimeric ETS transcription factor that is, due to a chromosomal rearrangement, specifically expressed in Ewingâs sarcoma family tumors (ESFT) and is thought to be the initiating event in the development of the disease. Previous genomic profiling experiments have identified a number of EWS-FLI1 regulated genes and genes that discriminate ESFT from other sarcomas, but so far a comprehensive analysis of EWS-FLI1 dependent molecular functions characterizing this aggressive cancer is lacking. In this study a molecular function map of ESFT was constructed based on an integrative analysis of gene expression profiling experiments on a uniform microarray platform following EWS-FLI1 knockdown in a panel of five ESFT cell lines, and on gene expression data from the same platform of 59 primary ESFT tumors. Based on the assumption that EWS-FLI1 is the driving transcriptional force in ESFT pathogenesis, we predicted an inverse correlation of gene expression for EWS-FLI1 regulated genes between the putative tissue of origin and the cell lines under EWS-FLI1 knockdown conditions. Consistent with recent reports, mesenchymal progenitor cells (MPC) were found to fit this hypothesis best and were therefore used as the reference tissue for the construction of the molecular function map in ESFT. The interrelations of molecular pathways were visualized by measuring the similarity among annotated gene functions by gene sharing. The molecular function map highlighted distinct clusters of activities for EWS-FLI1 regulated genes in ESFT and revealed a striking difference between EWS-FLI1 up- and down-regulated genes: EWS-FLI1 induced genes mainly belong to cell cycle regulation, proliferation and response to DNA damage, while repressed genes were associated with differentiation and cell communication. This study revealed that EWS-FLI1 combines by distinct molecular mechanisms two important functions of cellular transformation in one protein, growth promotion and differentiation blockage. By taking MPC as a reference tissue a significant EWS-FLI1 signature was discovered in ESFT that only partially overlapped with previously published EWS-FLI1 dependent gene expression patterns, identifying a series of novel targets for the chimeric protein in ESFT. Our results may guide target selection for future ESFT specific therapies. Experiment Overall Design: EWS-FLI1 knock down was performed by expressing specific siRNAs against EWS-FLI1 as small hairpin (sh) RNAs from pSUPER-based retroviral expression constructs in 5 different Ewing's sarcoma cell lines (WE68, SK-N-MC, TC252, STA-ET-1, STA-ET-7.2). As a negative control in each cell line, pSTNeg (Ambion, Applied Biosystems, Brunn am Gebirge, Austria) encoding a scrambled shRNA with no significant similarity to human sequences was used.
2009-05-11 | E-GEOD-14543 | biostudies-arrayexpress
Project description:The fusion oncoprotein EWS-FLI1 arises from a t(11;22)(q24;q12) chromosomal translocation and causes Ewing's Sarcoma, a malignant bone tumor. The mechanism whereby EWS-FLI1 transforms cells is unknown. Somatic, mosaic expression of human EWS-FLI1 in zebrafish from the heat shock promoter [Tg(HSP:EWS-FLI1)] caused small round blue cell tumors (SRBCTs) similar to human Ewing's sarcoma. We performed microarray studies comparing zebrafish SRBCTs to another tumor type, zebrafish malignant peripheral nerve sheath tumors (MPNSTs). The results identify a conserved set of EWS-FLI1-regulated genes,and provide insight into the pathogenesis of Ewing's Sarcoma tumors. Zebrafish SRBCTs arising from somatic insertions of the EWS-FLI1 transgene were collected. MPNSTs from non-transgenic fish of the same genetic background were collected in parallel. RNA was prepared from all samples and hybridized to zebrafish-specific Affymetrix arrays.
2011-11-01 | E-GEOD-31186 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE31185: The human Ewing's Sarcoma oncoprotein EWS-FLI1 causes developmental defects in zebrafish embryos GSE31186: The human Ewing's Sarcoma oncoprotein EWS-FLI1 causes Ewing's-type tumors in zebrafish Refer to individual Series
2011-11-01 | E-GEOD-31217 | biostudies-arrayexpress
Project description:The fusion oncoprotein EWS-FLI1 arises from a t(11;22)(q24;q12) chromosomal translocation and causes Ewing's Sarcoma, a malignant bone tumor. The mechanism whereby EWS-FLI1 transforms cells is unknown. We made germline transgenic zebrafish expressing human EWS-FLI1 under the control of the heat shock promoter. Induction of EWS-FLI1 expression causes multiple defects in embryonic development. We compared gene expression in control and transgenic EWS-FLI1 zebrafish. The results identify a conserved set of EWS-FLI1-regulated genes, and provide insight into the pathogenesis of Ewing's Sarcoma tumors. We performed heat shock and isolated total RNA for microarray studies comparing wildtype AB strain zebrafish with transgenic zebrafish expressing human EWS-FLI1 [Tg(HSP:EWS-FLI1)]. RNA was biotin-lableled and hybridized to zebrafish-specific Affymetrix arrays.
2011-11-01 | E-GEOD-31185 | biostudies-arrayexpress
Project description:Using EWS-FLI and its parental transcription factor, FLI1, we created a unique experimental system to address questions regarding the genomic mechanisms by which chimeric transcription factors cause cancer. We found that in tumor cells, EWS-FLI targets regions of the genome distinct from FLI1, despite identical DNA-binding domains. In primary endothelial cells, however, EWS-FLI and FLI1 demonstrate similar targeting. To understand this mistargeting, we examined chromatin organization. Regions targeted by EWS-FLI are normally repressed and nucleosomal in primary endothelial cells. In tumor cells, however, bound regions are nucleosome-depleted and harbor the chromatin signature of enhancers. We next demonstrated that through chimerism, EWS-FLI acquired the ability to alter chromatin. Expression of EWS-FLI results in nucleosome depletion at targeted sites, whereas silencing of EWS-FLI in tumor cells restored nucleosome occupancy. Thus, the EWS-FLI chimera acquired chromatin-altering activity, leading to mistargeting, chromatin disruption, and ultimately transcriptional dysregulation. Examination of two transcription factors in two different cell types, as well as three histone methylation marks and FAIRE
2011-11-09 | E-GEOD-31838 | biostudies-arrayexpress
Project description:We have examined the biological effect of EWS/ETS in human MPCs using UET-13 cells that are obtained by prolonging the lifespan of human bone marrow stromal cells using the retroviral transgenes hTERT and E7. By exploiting tetracycline-inducible systems for expressing EWS/ETS (EWS/FLI1 and EWS/ERG), we investigated candidates for genes whose expression is regulated by EWS/ETS in human MPCs. Experiment Overall Design: A change of expression profiles in the presence of EWS/ETS using Affymetrix HG-U133 Plus 2.0 (GPL570).
2008-06-16 | E-GEOD-8665 | biostudies-arrayexpress
Project description:We show that EWS-FLI1, an aberrant transcription factor responsible for the pathogenesis of Ewing sarcoma, reprograms gene regulatory circuits by directly inducing or directly repressing enhancers. At GGAA repeats, which lack regulatory potential in other cell types and are not evolutionarily conserved, EWS- FLI1 multimers potently induce chromatin opening, recruit p300 and WDR5, and create de novo enhancers. GGAA repeat enhancers can loop to physically interact with target promoters, as demonstrated by chromosome conformation capture assays. Conversely, EWS-FLI1 inactivates conserved enhancers containing canonical ETS motifs by displacing wild-type ETS transcription factors and abrogating p300 recruitment. ChIP-seq for of 4 histone modifications (H3K27ac, H3K4me1, H3K4me3 and H3K27me3), FLI1, p300, WDR5, ELF1 and GABPA in primary Ewing sarcomas, Ewing sarcoma cell lines (A673 and SKMNC cells), and mesenchymal stem cells (MSC). EWS-FLI1 was knocked down in Ewing sarcoma cell lines with lentiviral shRNAs (shFLI1 and shGFP control). EWS-FLI1 was expressed in MSCs with lentiviral expression vectors (pLIV EWSFLI1 or pLIV empty vector control). * Raw data not provided for the MSC and Primary Ewing sarcoma samples. *
2014-10-30 | E-GEOD-61944 | biostudies-arrayexpress
Project description:We show that EWS-FLI1, an aberrant transcription factor responsible for the pathogenesis of Ewing sarcoma, reprograms gene regulatory circuits by directly inducing or directly repressing enhancers. At GGAA repeats, which lack regulatory potential in other cell types and are not evolutionarily conserved, EWS- FLI1 multimers potently induce chromatin opening, recruit p300 and WDR5, and create de novo enhancers. GGAA repeat enhancers can loop to physically interact with target promoters, as demonstrated by chromosome conformation capture assays. Conversely, EWS-FLI1 inactivates conserved enhancers containing canonical ETS motifs by displacing wild-type ETS transcription factors and abrogating p300 recruitment. Mesenchymal stem cells (MSCs) and a Ewing sarcoma cell line (SKNMC) were analyzed by ATAC-seq. EWS-FLI1 was expressed in MSCs using a lentiviral vector (pLIV EWSFLI1 or pLIV empty vector control). * Raw data not provided for the MSC samples. *
2014-10-30 | E-GEOD-61951 | biostudies-arrayexpress
Project description:The molecular hallmark of the Ewing family of tumors is the presence of balanced chromosomal translocations leading to the formation of chimerical transcription factors (i.e. EWS/FLI1) that play a pivotal role in the pathogenesis of Ewing tumors by deregulating gene expression. We have recently demonstrated that DAX1 (NR0B1), an orphan nuclear receptor which was not previously implicated in cancer, is induced by the EWS/FLI1 oncoprotein and is highly expressed in Ewing tumors, suggesting that DAX1 is a biologically relevant target of EWS/FLI1-mediated oncogenesis. In this work we demonstrate that DAX1 is a direct transcriptional target of the EWS/FLI1 oncoprotein through its binding to a GGA-rich region in the DAX1 promoter and show that DAX1 is a key player of EWS/FLI1-mediated oncogenesis. DAX1 silencing using an inducible model of RNA interference induces growth arrest in the A673 Ewing cell line and severely impairs its capability to grow in semisolid medium and form tumors in immunodeficient mice. Gene expression profile analysis demonstrated that about ten percent of the genes regulated by EWS/FLI1 in Ewing cells are DAX1 targets, confirming the importance of DAX1 in Ewing oncogenesis. These findings indicate that DAX1 is an important player in the pathogenesis of the Ewing family of tumors, identify new functions for DAX1 as a cell cycle progression regulator and open the possibility to new therapeutic approaches based on DAX1 function interference. A673 cells derived from Ewing sarcoma were genetically enginereed to express specific shRNAs against GFP (control), EWS/FLI1 and DAX1 upon doxycycline stimulation. Three independent clones and a polyclonal population from each enginereed cell were analyzed. Cells were stimulated with doxycycline for 72 hours to induce the expression of the corresponding shRNA and whole gene expression profile performed. Gene expression profile in A673 cells in which EWS/FLI1 or DAX1 were silenced were compared to the control cells.
2012-02-23 | E-GEOD-36007 | biostudies-arrayexpress