Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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EWS/WT1 co-operates with the loss of p53 to exert its oncogenic effects and up-regulates canonical Wnt pathway signaling.

ABSTRACT: The t(11;22)(p13;q12) translocation is pathognomonic for the highly aggressive desmoplastic small round cell tumour (DSRCT). The translocation fuses exon 7 of the EWS gene to exon 8 of the WT1 gene (EWS/WT1). Two splice variants of EWS/WT1 exist, arising from the presence (+KTS) or absence (-KTS) of three amino acids: lysine, threonine and serine between zinc fingers 3 and 4. To investigate the oncogenic properties of both isoforms of EWS/WT1, we over-expressed EWS/WT1 in untransformed murine embryonic fibroblasts (MEFs). We demonstrate that neither isoform of EWS/WT1 is sufficient to transform wild type MEFs, however the oncogenic potential of both isoforms is unmasked by the loss of p53. Expression of EWS/WT1 in MEFs lacking at least one allele of p53 resulted in enhanced cell proliferation, clonogenic survival and anchorage-independent growth. In addition EWS/WT1 expression in wild type MEFs attenuated several p53-dependent responses, including cell cycle arrest after irradiation and daunorubicin induced apoptosis. We show that DSRCT commonly have copy number amplification of MDM2 and MDMX, suggesting loss of p53 function in the tumours. Expression of either isoform of EWS/WT1 in MEFs induced characteristic mRNA expression profiles, including up-regulation of canonical Wnt pathway signaling. This was validated in cell lines and in a series of DSCRT, which confirmed canonical Wnt pathway activation in the tumours. We show for the first time that both isoforms of EWS/WT1 have oncogenic potential and that in addition to co-operating with loss of p53 function can also further attenuate p53-mediated responses. In addition we provide the first link between EWS/WT1 and Wnt pathway signaling. These data provide novel insights into the function of the EWS/WT1 fusion protein which characterizes DSCRT. Four independently generated pools of wild type MEFs were infected with tetracycline repressible EWS/WT1+KTS, EWS/WT1-KTS or GFP, selected with hygromycin and protein expression confirmed. Four GFP, four KTS+ and four KTS- samples were hybridized to the Ilumina BeadChip.

ORGANISM(S): Mus musculus

SUBMITTER: Paul Ekert

PROVIDER: E-GEOD-42649 | 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. 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.

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EWS/WT1 co-operates with the loss of p53 to exert its oncogenic effects and up-regulates canonical Wnt pathway signaling.

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