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| Status |
Public on Mar 31, 2015 |
| Title |
Oncogenic Fusion Protein EWS-FLI1 is a Network Hub that Regulates Alternative Splicing |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
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.
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| Overall design |
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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| Contributor(s) |
Selvanathan SP, Graham GT, Toretsky JA |
| Citation(s) |
25737553 |
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| Submission date |
Feb 13, 2015 |
| Last update date |
Apr 16, 2015 |
| Contact name |
Garrett Thomas Graham |
| E-mail(s) |
gtg9@georgetown.edu
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| Phone |
8148822017
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| Organization name |
Georgetown University Medical Center
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| Department |
Oncology
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| Street address |
3970 Reservoir Rd NW
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| City |
Washington |
| State/province |
DC |
| ZIP/Postal code |
20007 |
| Country |
USA |
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| Platforms (1) |
| GPL5188 |
[HuEx-1_0-st] Affymetrix Human Exon 1.0 ST Array [probe set (exon) version] |
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| Samples (18)
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| GSM1611519 |
TC32 wild-type cells expressing EWS-FLI1 (+EF), biological rep #1 |
| GSM1611520 |
TC32 wild-type cells expressing EWS-FLI1 (+EF), biological rep #2 |
| GSM1611521 |
TC32 wild-type cells expressing EWS-FLI1 (+EF), biological rep #3 |
| GSM1611522 |
TC32 cells with shRNA reduction of EWS-FLI1 (-EF), biological rep #1 |
| GSM1611523 |
TC32 cells with shRNA reduction of EWS-FLI1 (-EF), biological rep #2 |
| GSM1611524 |
TC32 cells with shRNA reduction of EWS-FLI1 (-EF), biological rep #3 |
| GSM1611525 |
A673i wild-type cells expressing EWS-FLI1 (+EF), biological rep #1 |
| GSM1611526 |
A673i wild-type cells expressing EWS-FLI1 (+EF), biological rep #2 |
| GSM1611527 |
A673i wild-type cells expressing EWS-FLI1 (+EF), biological rep #3 |
| GSM1611528 |
A673i doxycycline-inducible shRNA reduction of EWS-FLI1 (-EF), biological rep #1 |
| GSM1611529 |
A673i doxycycline-inducible shRNA reduction of EWS-FLI1 (-EF), biological rep #2 |
| GSM1611530 |
A673i doxycycline-inducible shRNA reduction of EWS-FLI1 (-EF), biological rep #3 |
| GSM1611531 |
hMSC wild-type cells not expressing EWS-FLI1 (-EF), biological rep #1 |
| GSM1611532 |
hMSC wild-type cells not expressing EWS-FLI1 (-EF), biological rep #2 |
| GSM1611533 |
hMSC wild-type cells not expressing EWS-FLI1 (-EF), biological rep #3 |
| GSM1611534 |
hMSC cells expressing EWS-FLI1 (+EF), biological rep #1 |
| GSM1611535 |
hMSC cells expressing EWS-FLI1 (+EF), biological rep #2 |
| GSM1611536 |
hMSC cells expressing EWS-FLI1 (+EF), biological rep #3 |
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| Relations |
| BioProject |
PRJNA275445 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE65941_RAW.tar |
426.9 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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