BRCA1, R-loops and Recombination defects in Ewing's sarcoma (DRIP-seq)
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE68845 | GEO | 2018/03/09
REPOSITORIES: GEO
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Project description:Ewing’s sarcoma family of tumors (ESFT) is an aggressive pediatric bone and soft tissue cancer. It is the prototypical example of mesenchymal tumors driven by a fusion oncogene involving the ewing sarcoma break point region 1 (EWSR1) gene, most frequently– EWS-FLI1. We have discovered that loss of EWSR1 leads to accumulation of R-loops, replication stress and impaired homologous recombination, recapitulating breast cancer 1, early onset (BRCA1) deficiency. EWS-FLI1 acts dominant negatively in ESFT to impart the same phenotypes. Further we demonstrate that in ESFT, BRCA1 predominantly associates with the elongating transcription machinery and is unavailable for DNA strand break repair. Gene expression profiling identified upregulated compensatory mechanisms in ESFT cells to process increased R-loops (RNASEH2 and FEN1) and replication stress (Fanconi Anemia). Taken together, our data identifies BRCA1 sequestration due to transcription stress as the mechanistic basis for ESFT chemosensitivity and suggests potential targets for the much lacking second-line therapy.
2018-03-09 | GSE68836 | GEO
Project description:Ewing sarcoma (EwS) is an aggressive pediatric bone and soft tissue cancer. It is driven by a fusion oncogene involving the EWSR1 gene, predominantly EWS-FLI1. EwS is exquisitely sensitive to genotoxic agents, but the molecular basis for this sensitivity is relatively underexplored. We have discovered that EwS displays alterations in damage-induced transcription regulation and an accumulation of R-loops. Consequently, we observe an increase in replication stress but impaired homologous recombination. We demonstrate an enriched interaction between BRCA1 and the elongating transcription machinery and suggest that this interaction may be the underlying cause for impaired recombination. Investigation into the unique transcription profile of EwS also revealed upregulated mechanisms (including RNASEH2, FEN1 and Fanconi Anemia pathway) to compensate for the increased EWS-FLI1 induced toxicity. Finally, we have uncovered a novel role for EWSR1 transcriptional response to damage, suppressing R-loops and promoting homologous recombination. Taken together, our findings improve the current understanding of wild-type EWSR1 function, elucidate the mechanistic basis for EwS chemosensitivity (including PARP1 inhibitors) and suggest potential targets for novel second-line therapy.
2018-03-09 | GSE87324 | GEO
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:EWS-FLI1 is a chimeric ETS transcription factor that is, due to a chromosomal rearrangement, specifically expressed in Ewing’s 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. Keywords: Ewing's Sarcoma, EWS-FLI1, RNAi knock down and controls
2009-04-30 | GSE14543 | GEO
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 family of tumors (ESFT) are aggressive bone and soft tissue tumors of unknown cellular origin. Most ESFT express EWS-FLI1, a chimeric protein which functions as a growth-promoting oncogene in ESFT but is toxic to most normal cells. A major difficulty in understanding EWS-FLI1 function has been the lack of an adequate model in which to study EWS-FLI1-induced transformation. Although the cell of origin of ESFT remains elusive, both mesenchymal (MSC) and neural crest (NCSC) have been implicated. We recently developed the tools to generate NCSC from human embryonic stem cells (hNCSC). In the current study we used this model to test the hypothesis that neural crest-derived stem cells are the cells of origin of ESFT and to evaluate the consequences of EWS-FLI1 expression on human neural crest biology. hNCSC transduced with an EWS-FLI1 lentivirus tolerated expression of the oncoprotein. Moreover, EWS-FLI1-transduced hNCSC continued to proliferate and maintain EWS-FLI1 expression in culture for several weeks after transduction. Affymetrix HuEx 1.0 expression profiling of hNCSC cells five days post-transduction with EWS-FLI1 demonstrated the expected induction and repression of well-established EWS-FLI1 targets and also identified numerous other novel EWS-FLI1-regulated genes that are likely to be cell-type and situation specific. In particular, the EWS-FLI1 repressive signature was found to be highly context dependent. Moreover, while control vector transduced cells displayed an MSC-like phenotype, EWS-FLI1-transduced cells maintained a NCSC-like phenotype and genetic profiling revealed reprogramming towards a more pluriopotent, neuroectodermal state. Finally, EWS-FLI1 expressing cells upregulated expression of the polycomb proteins BMI-1 and EZH2. These data implicate neural crest-derived cells in the origin of ESFT and suggest that EWS-FLI1 enables malignant transformation by inducing maintenance of a multipotent, NCSC-state through deregulation of polycomb genes.
2010-06-15 | GSE21511 | GEO
Project description:Ewing sarcoma family of tumors (ESFT) are aggressive bone and soft tissue tumors of unknown cellular origin. Most ESFT express EWS-FLI1, a chimeric protein which functions as a growth-promoting oncogene in ESFT but is toxic to most normal cells. A major difficulty in understanding EWS-FLI1 function has been the lack of an adequate model in which to study EWS-FLI1-induced transformation. Although the cell of origin of ESFT remains elusive, both mesenchymal (MSC) and neural crest (NCSC) have been implicated. We recently developed the tools to generate NCSC from human embryonic stem cells (hNCSC). In the current study we used this model to test the hypothesis that neural crest-derived stem cells are the cells of origin of ESFT and to evaluate the consequences of EWS-FLI1 expression on human neural crest biology. hNCSC transduced with an EWS-FLI1 lentivirus tolerated expression of the oncoprotein. Moreover, EWS-FLI1-transduced hNCSC continued to proliferate and maintain EWS-FLI1 expression in culture for several weeks after transduction. Affymetrix HuEx 1.0 expression profiling of hNCSC cells five days post-transduction with EWS-FLI1 demonstrated the expected induction and repression of well-established EWS-FLI1 targets and also identified numerous other novel EWS-FLI1-regulated genes that are likely to be cell-type and situation specific. In particular, the EWS-FLI1 repressive signature was found to be highly context dependent. Moreover, while control vector transduced cells displayed an MSC-like phenotype, EWS-FLI1-transduced cells maintained a NCSC-like phenotype and genetic profiling revealed reprogramming towards a more pluriopotent, neuroectodermal state. Finally, EWS-FLI1 expressing cells upregulated expression of the polycomb proteins BMI-1 and EZH2. These data implicate neural crest-derived cells in the origin of ESFT and suggest that EWS-FLI1 enables malignant transformation by inducing maintenance of a multipotent, NCSC-state through deregulation of polycomb genes. 3 replicate samples for 4 different stem cell populations were analyzed by HuEx arrays. The 4 sample types were adult human bone marrow-derived mesenchymal stem cells, human embryonic stem cell-derived nueral crest stem cells (hNCSC), control vector-transduced hNCSC-derived mesenchymal stem cells (NC-MSC), and EWS-FLI1-transduced hNCSC-derived mesenchymal stem cells (NC-MSC EF). Control and EWS-FLI1 transduced NC-MSC were isolated 5 days after lentiviral transduction. Transcript level expression data was compared among the different populations to determine differences and similarities between NCSC, BM-MSC and NC-MSC with/without EWS-FLI1 expression. These data were used to identify EWS-FLI1 targets in NC-MSC and to characterize the genetic changes that occur in NCSC as they generate NC-MSC progeny.
2010-06-23 | E-GEOD-21511 | biostudies-arrayexpress
Project description:Ewing sarcoma family of tumors (ESFT) is a uniquely human, highly aggressive bone and soft tissue malignancy peaking during adolescence characterized by EWSR1/ETS rearrangements. EWSR1/FLI1 expression is only tolerated in embryonic and mesenchymal stem cells (hESC and hMSC, respectively) that, although acquire ESFT transcriptional signatures, are devoid of tumorigenic potential. Here we report that ectopic EWSR1/FLI1 expression in hESC allows in vivo differentiation towards the three germ layers. In vitro, teratoma-derived hMSCs are devoid of transformation abilities. In vivo, these hMSCs led to the generation of T-cell lymphomas and ESFT-like tumors, composed of small-round blue cells. This new experimental approach constitutes a testing bench for the biology of translocation-dependent developmental tumors, from acute lymphoblastic leukemia to a variety of sarcomas.
2022-12-31 | GSE140328 | GEO
Project description:Ewing sarcoma family of tumors (ESFT) is a uniquely human, highly aggressive bone and soft tissue malignancy peaking during adolescence characterized by EWSR1/ETS rearrangements. EWSR1/FLI1 expression is only tolerated in embryonic and mesenchymal stem cells (hESC and hMSC, respectively) that, although acquire ESFT transcriptional signatures, are devoid of tumorigenic potential. Here we report that ectopic EWSR1/FLI1 expression in hESC allows in vivo differentiation towards the three germ layers. In vitro, teratoma-derived hMSCs are devoid of transformation abilities. In vivo, these hMSCs led to the generation of T-cell lymphomas and ESFT-like tumors, composed of small-round blue cells. This new experimental approach constitutes a testing bench for the biology of translocation-dependent developmental tumors, from acute lymphoblastic leukemia to a variety of sarcomas.
2022-12-31 | GSE141888 | GEO
Project description:Translocations of ETS transcription factors are driver mutations in diverse cancers. We investigated the genomic network of the ETS fusion EWS/FLI1 in Ewing's sarcoma (ESFT) as a model of ETS-driven tumorigenesis. ChIP-Seq and transcriptional analysis identified E2F3 as a principle co-factor of EWSFLI1 defining functionally distinct gene sets. While EWS/FLI1 binding independent of E2F3 predominantly associated with repressed differentiation genes, significant co-localization with E2F3 was discovered at proximal promoters of activated growth-related genes. Thus, EWS/FLI1 promotes oncogenesis by simultaneously perturbing differentiation state and augmenting the expression of genes co-regulated by E2F3. Integration of additional E2F3 and ERG localization data from prostate cancer containing TMPRSS2/ERG verified that the ETS-E2F module is also found in prostate cancer and may be of general relevance to ETS driven cancers. Timecourse with 6 timepoints of a doxicyclin inducible EWS-FLI1 knockdown in the A673 Ewing's Sarcoma celline
2013-07-01 | E-GEOD-27524 | biostudies-arrayexpress