Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Oncogenic Fusion Protein EWS-FLI1 is a Network Hub that Regulates Alternative Splicing

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Garrett Graham

PROVIDER: E-GEOD-65941 | 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.

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.

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Oncogenic Fusion Protein EWS-FLI1 is a Network Hub that Regulates Alternative Splicing

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