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UV (UVC, 10J/m2) light irradiation effect on Ewing Sarcoma cells

ABSTRACT: LAP-35 and SK-N_MC cells were treated with 10 J/m2 UV light versus untreated Alternative pre-mRNA processing plays a key role in the response to DNA damage as well as in neoplastic transformation. We found that two Ewing Sarcoma (ES) cell lines exhibit different sensitivity to UV light irradiation, with SK-N-MC cells being more sensitive than LAP-35 cells. RNA profiling during the response to low doses of UV light irradiation revealed genes differentially regulated between the two cell lines. In particular, UV light irradiation induced a novel isoform of the RNA helicase DHX9 which is targeted to nonsense-mediated decay (NMD) and therefore causes down-regulation of DHX9 in SK-N-MC cells, but not in LAP-35 cells. DHX9 protein forms a complex with RNA polymerase II (RNAPII) and EWS-FLI1 to enhance transcription, and we found that down-regulation of DHX9 by UV light irradiation in SK-N-MC cells impairs the recruitment of EWS-FLI1 to target genes and increases sensitivity to DNA damage. Notably, sensitivity of SK-N-MC cells to irradiation correlated with enhanced phosphorylation and decreased processivity of RNAPII upon irradiation, which in turn causes inclusion of the novel DHX9 exon in SK-N-MC cells exposed to UV light, an observation that could be recapitulated in LAP35 cells by pharmacological reduction of RNAPII processivity. Our data suggest that EWS-FLI1 oncogene activity could be targeted by modulation of DHX9 gene expression.

ORGANISM(S): Homo sapiens

PROVIDER: GSE59889 | GEO | 2015/10/20

SECONDARY ACCESSION(S): PRJNA256994

REPOSITORIES: GEO

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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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UV (UVC, 10J/m2) light irradiation effect on Ewing Sarcoma cells

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