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MicroRNA expression profiling of Ewing sarcoma cell lines

ABSTRACT: Ewing’s sarcoma family tumors (ESFT) are the second most common bone malignancy in children and young adults, characterized by the expression of a unique chromosomal translocation, that in 85% of cases lead to the expression of the EWS-FLI-1 fusion protein. ESW-FLI-1 functions as an aberrant transcription factor that can both induce and suppress its target genes. We have recently demonstrated that microRNA (miRNA) 145 is a direct EWS-FLI-1 target implicated in ESFT development. Here, we use global miRNA array profiling to show that ESFT display a distinct miRNA signature characterized by the induction or repression of a limited number of miRNAs, including the oncogenic and tumor suppressor miRNA 17-92 and let-7 families, respectively. We demonstrate that repression of the let-7 family member let-7a may be due to direct binding of EWS-FLI-1 to the let-7a promoter and that it participates in the tumorigenic potential of ESFT cells in vivo. The mechanism whereby let-7a repression enhances ESFT growth is shown to be the induction of its target gene HMGA2, as overexpression of let-7a or repression of HMGA2 blocked ESFT cell tumorigenicity. Consistent with these observations, systemic delivery of synthetic let-7a that restored its expression in tumor cells and decreased HMGA2 expression inhibited ESFT growth in vivo. Our observations provide evidence that deregulation of let-7a target gene expression participates in ESFT development and identify let-7a as a promising new therapeutic target for one of the most aggressive pediatric malignancies.

OTHER RELATED OMICS DATASETS IN: PRJNA140303

ORGANISM(S): Mus musculus Rattus norvegicus synthetic construct Homo sapiens

PROVIDER: GSE29085 | GEO | 2011/12/27

SECONDARY ACCESSION(S): PRJNA140303

REPOSITORIES: GEO

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Gene expression analysis of human pediatric mesenchymal stem cells (hpMSCs) upon expression of EWS-FLI-1

Project description:Cancer stem cells (CSCs) display plasticity and self-renewal properties reminiscent of normal tissue stem cells, but the events responsible for their emergence remain obscure. We recently identified CSCs in Ewing sarcoma family tumors (ESFTs) and showed that they retain mesenchymal stem cell (MSC) plasticity. In the present study, we addressed the mechanisms that underlie ESFT CSC development. We show that the EWS-FLI-1 fusion gene, associated with 85%-90% of ESFTs and believed to initiate their pathogenesis, induces expression of the embryonic stem cell (ESC) genes OCT4, SOX2, and NANOG in human pediatric MSCs (hpMSCs) but not in their adult counterparts. Moreover, under appropriate culture conditions, hpMSCs expressing EWS-FLI-1 generate a cell subpopulation displaying ESFT CSC features in vitro. We further demonstrate that induction of the ESFT CSC phenotype is the result of the combined effect of EWS-FLI-1 on its target gene expression and repression of microRNA-145 (miRNA145) promoter activity. Finally, we provide evidence that EWS-FLI-1 and miRNA-145 function in a mutually repressive feedback loop and identify their common target gene, SOX2, in addition to miRNA145 itself, as key players in ESFT cell differentiation and tumorigenicity. Our observations provide insight for the first time into the mechanisms whereby a single oncogene can reprogram primary cells to display a CSC phenotype. 4 samples of hpMSCs expressing EWS-FLI-1 vs. 4 matched samples of empty-vector-infected cells.

2011-08-04 | E-GEOD-31215 | biostudies-arrayexpress

Gene expression analysis of human pediatric mesenchymal stem cells (hpMSCs) upon expression of EWS-FLI-1

2011-08-05 | GSE31215 | GEO

RNA immunoprecipitation (RIP)-Chip analysis for EWS-bound mRNA

Project description:Although EWS/FLI-1 fusion protein is responsible for most Ewing’s sarcoma family tumors (ESFT), the function of native EWS remains largely unknown. Here, we first showed that EWS repressed protein expression in a tethering assay. mRNAs bound to EWS were determined by RNA-immunoprecipitation Chip assay, and one of them, proline-rich Akt substrate of 40 kDa (PRAS40) mRNA, directly interacted with EWS. The inhibitor of AKT, API-2, repressed ESFT cell proliferation. We demonstrate that EWS negatively regulated PRAS40 protein expression through binding to PRAS40 3’UTR. Furthermore, PRAS40 knockdown inhibited the proliferation and metastatic potential of ESFT cells.

2012-01-16 | GSE29313 | GEO

RNA immunoprecipitation (RIP)-Chip analysis for EWS-bound mRNA

Project description:Although EWS/FLI-1 fusion protein is responsible for most EwingM-bM-^@M-^Ys sarcoma family tumors (ESFT), the function of native EWS remains largely unknown. Here, we first showed that EWS repressed protein expression in a tethering assay. mRNAs bound to EWS were determined by RNA-immunoprecipitation Chip assay, and one of them, proline-rich Akt substrate of 40 kDa (PRAS40) mRNA, directly interacted with EWS. The inhibitor of AKT, API-2, repressed ESFT cell proliferation. We demonstrate that EWS negatively regulated PRAS40 protein expression through binding to PRAS40 3M-bM-^@M-^YUTR. Furthermore, PRAS40 knockdown inhibited the proliferation and metastatic potential of ESFT cells. Cytoplasmic lysates or whole cell lysates were prepared from HeLa S3 cells transfected with pFLAG-EWS , and incubated with anti-FLAG M2 Affinity Gel (Sigma) at 4M-BM-0C for 2 h. RNAs from lysates and immunoprecipitates were analysed using GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix).

2012-01-16 | E-GEOD-29313 | biostudies-arrayexpress

RUNX3 Facilitates Growth of Ewing Sarcoma Cells

Project description:Ewing sarcoma is an aggressive pediatric small round cell tumor that predominantly occurs in bone. Approximately 85% of Ewing sarcomas harbor the EWS/FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS/FLI interacts with numerous lineage-essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS/FLI binds the osteogenic transcription factor RUNX2 and prevents osteoblast differentiation. In this study, we investigated the role of another Runt-domain protein, RUNX3, in Ewing sarcoma. RUNX3 participates in mesenchymal-derived bone formation and is a context dependent tumor suppressor and oncogene. RUNX3 was detected in all Ewing sarcoma cells examined, whereas RUNX2 was detected in only 73% of specimens. Like RUNX2, RUNX3 binds to EWS/FLI via its Runt domain. EWS/FLI prevented RUNX3 from activating the transcription of a RUNX-responsive reporter, p6OSE2. Stable suppression of RUNX3 expression in the Ewing sarcoma cell line A673 delayed colony growth in anchorage independent soft agar assays and reversed expression of EWS/FLI-responsive genes. These results demonstrate an important role for RUNX3 in Ewing sarcoma. RNA-seq to compare transcriptiome of control A673 ewing sarcoma cells stably expression a non-target or RUNX3 shRNA

2014-05-01 | E-GEOD-56900 | biostudies-arrayexpress

LIN28B underlies the pathogenesis and therapeutic sensitivity of a subclass of Ewing sarcoma

Project description:Ewing sarcoma (EwS) is a pediatric bone malignancy associated with high mortality despite aggressive multimodal therapy. Targeting EWS-FLI-1, the fusion protein responsible for EwS pathogenesis, or its principal downstream target gene products have not produced effective therapeutic options, fuelling the quest for alternative approaches. Here, we show that the oncofoetal RNA-binding protein Lin28B is expressed in a subset of EwS in which it directly binds and safeguards EWS-FLI-1 transcripts. Depletion and pharmacologic inhibition of Lin28B in primary patient-derived EwS cultures led not only to a decrease in EWS-FLI-1 expression but also to the deconstruction of its direct transcriptional network, abrogating EwS cell self-renewal and tumorigenicity. Our observations demonstrate that Lin28B provides indispensable support for EWS-FLI-1 to sustain the emergence of a EwS subset in which it also constitutes a promising therapeutic target.

2020-04-30 | GSE122632 | GEO

MicroRNA expression profiling of Ewing sarcoma cancer stem cells

Project description:We have recently demonstrated that human paediatric mesenchymal stem cells can be reprogrammed toward a Ewing’s sarcoma family tumor (ESFT) cancer stem cell (CSC) phenotype by mechanisms that implicate microRNAs (miRNAs). Here, we show that the miRNA profile of ESFT CSC is shared by embryonic stem cells and CSC from divergent tumor types. We also provide evidence that the miRNA profile of ESFT CSC is the result of reversible disruption of TARBP2-dependent miRNA maturation. Restoration of TARBP2 activity and systemic delivery of synthetic forms of either of two of its targets, miRNA-143 or miRNA-145, inhibited ESFT CSC clonogenicity and tumor growth in vivo. Our observations suggest that CSC self-renewal and tumor initiation may depend on deregulation of TARBP2-dependent miRNA expression. 2 samples of primary Ewing sarcomas, divided into CD133+ and CD133- fractions. One sample of EWS-FLI1 expressing human pediatric mesenchymal stem cells, divided into CD133+ and CD133- fractions. One sample of STA-ET-8.2 cells, divided into CD133+ and CD133- fractions.

2012-06-19 | E-GEOD-30513 | biostudies-arrayexpress

Identification of two types of GGAA microsatellites and their roles in EWS/FLI binding and gene regulation in Ewing sarcoma

Project description:Ewing sarcoma is a bone malignancy of children and young adults, frequently harboring the EWS/FLI t(11;22)(q24;q12) chromosomal translocation. The resulting fusion protein is an aberrant transcription factor that uses highly repetitive GGAA-containing elements (microsatellites) to activate and repress thousands of target genes mediating oncogenesis. However, the mechanisms of EWS/FLI interaction with microsatellites and regulation of target genes expression is not clearly understood. Here, we profile genome-wide protein binding and gene expression. Using a combination of unbiased genome-wide computational and experimental analysis, we define GGAA-microsatellites in a Ewing sarcoma context. Our study identifies two distinct classes of GGAA-microsatellites and demonstrates that EWS/FLI responsiveness is dependent on microsatellite length. At close range (within 5 kb) “promoter-like” microsatellites, EWS/FLI binding and subsequent target genes activation is highly dependent on the number of GGAA-motifs. “Enhancer-like” microsatellites demonstrate a positive correlation with length-dependent EWS/FLI binding, but minimal correlation for activated and none for repressed target genes. Our data suggest that EWS/FLI binds to “promoter-like” and “enhancer-like” microsatellites to mediate activation and repression of target genes through different regulatory mechanisms. Such characterization contributes valuable insight to EWS/FLI transcription factor biology and clarifies the role of GGAA-microsatellites on a global genomic scale. This may provide a unique perspective on the role of non-coding DNA in cancer susceptibility and therapeutic development.

2017-11-09 | GSE99959 | GEO

Transcriptomic analysis to functionally map the intrinsically disordered domain of EWS/FLI [Experiment 2]

Project description:The purpose of this study was to define unique classes of EWS/FLI target genes and the distinct set of structural features present in the EWS domain required for target gene regulation at different EWS/FLI response elements. This study reports the first genome-wide transcriptomic description of a partially-functional EWS/FLI mutant . We report new structure-function dependencies across different classes of response element and tie these dependencies to the ability of EWS/FLI to transform cells.

2019-02-21 | GSE122536 | GEO

Transcriptomic analysis to functionally map the intrinsically disordered domain of EWS/FLI [Experiment 1]

2019-02-21 | GSE122535 | GEO

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