The effects of expression of the EWS-FLI1 oncoprotein on alternative splicing
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE318194 | GEO | 2026/06/26
REPOSITORIES: GEO
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Project description:Ewing sarcoma is driven by fusion proteins containing a low complexity (LC) domain that is intrinsically disordered and a powerful transcriptional regulator. The most common fusion protein found in Ewing sarcoma, EWS-FLI1, takes its LC domain from the RNA-binding protein EWSR1 (Ewing Sarcoma RNA-binding protein 1) and a DNA-binding domain from the transcription factor FLI1 (Friend Leukemia Virus Integration 1). The LC domain in EWS-FLI1 can bind RNA polymerase II (RNA Pol II) and can self-assemble through a process known as phase separation. The ability of EWSR1 and related RNA-binding proteins to assemble into ribonucleoprotein granules in cells has been intensely studied but the role of phase separation in EWS-FLI1 activity is less understood. We investigated the overlapping functions of EWSR1 and EWS-FLI1 in controlling gene expression and tumorigenic cell growth in Ewing sarcoma, and our results suggested that these proteins function closely together. We then studied the nature of interactions among EWS-FLI1, EWSR1, and RNA Pol II. We observed EWSR1 and RNA Pol II to be present in protein granules in cells. We then identified protein granules in cells associated with the fusion protein, EWS-FLI1. The tyrosine residues in the LC domain are required for the abilities of EWS-FLI1 to bind its partners, EWSR1 and RNA Pol II, and to incorporate into protein granules. These data suggest that interactions among EWS-FLI1, RNA Pol II, and EWSR1 in Ewing sarcoma can occur in the context of a molecular scaffold found within protein granules in the cell.
2020-07-23 | GSE154944 | GEO
Project description:Ewing sarcoma (EWS) is a malignant pediatric bone cancer. Most Ewing sarcomas are driven by EWS-FLI1 oncogenic transcription factor that plays roles in transcriptional regulation, DNA damage response, cell cycle checkpoint control, and alternative splicing. USP1, a deubiquitylase which regulates DNA damage and replication stress responses, is overexpressed at both the mRNA and protein levels in EWS cell lines compared to human mesenchymal stem cells, the EWS cell of origin. The functional significance of high USP1 expression in Ewing sarcoma is not known. Here, we identify USP1 as a transcriptional target of EWS-FLI1 and a key regulator of EWS cell survival. We show that EWS-FLI1 knockdown decreases USP1 mRNA and protein levels. ChIP and ChIP-seq analyses show EWS-FLI1 occupancy on the USP1 promoter. Importantly, USP1 knockdown or inhibition arrests EWS cell growth and induces cell death by apoptosis. We observe destabilization of Survivin (also known as BIRC5 or IAP4) and activation of caspases-3 and -7 following USP1 knockdown or inhibition in the absence of external DNA damage stimuli. Notably, EWS cells display hypersensitivity to combinatorial treatment of doxorubicin or etoposide, EWS standard of care drugs, and USP1 inhibitor compared to single agents alone. Together, our study demonstrates that USP1 is regulated by EWS-FLI1, the USP1-Survivin axis promotes EWS cell survival, and USP1 inhibition sensitizes EWS cells to standard of care chemotherapy.
2024-01-26 | PXD041033 | Pride
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: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
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:Pediatric malignancies frequently harbor chromosomal translocations that induce expression of fusion oncoproteins. The EWSR1-FLI1 fusion oncoprotein acts as a neomorphic transcription factor and is the dominant genetic driver of Ewing’s sarcoma. However, interrogation of the mechanisms by which EWSR1-FLI1 drives tumorigenesis has been limited by a lack of model systems to precisely and selectively control its expression in patient-derived cell lines and xenografts. Here, we report the generation of a panel of patient-derived EWS cell lines in which inducible protein degrons have been engineered into the endogenous EWSR1-FLI1 locus. These systems enable precise control of EWSR1-FLI1 expression and the timing of oncoprotein depletion. We establish that complete suppression of EWSR1-FLI1 leads to a reversible cell cycle arrest at the G1-S phase transition, and we identify a core set of transcripts downstream of EWSR1-FLI1 across multiple cell lines and degron systems. Additionally, depletion of EWSR1-FLI1 using this system potently suppresses tumor growth in xenograft models validating efforts to directly target EWSR1-FLI1 in Ewing’s sarcoma.
2025-01-20 | GSE270570 | GEO
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.
2011-11-01 | GSE31185 | GEO
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: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.
2011-11-01 | GSE31186 | GEO
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.
2012-02-23 | GSE36007 | GEO