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Oncoprotein EWS-FLI1 Activity Is Enhanced by RNA Helicase A

¹ Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia; ² Department of Pathology, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts; ³ Division of Hematology/Oncology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; ⁴ Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut; and ⁵ Genetics of Development and Disease Branch, National Institute of Diabetes & Digestive & Kidney Diseases, NIH, Bethesda, Maryland

Requests for reprints: Jeffrey A. Toretsky, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Research Building, Room W316, 3970 Reservoir Road Northwest, Washington D.C. 20057-1469. Phone: 202-687-8655; Fax: 202-687-1434; E-mail: jat42{at}georgetown.edu.

RNA helicase A (RHA), a member of the DEXH box helicase family of proteins, is an integral component of protein complexes that regulate transcription and splicing. The EWS-FLI1 oncoprotein is expressed as a result of the chromosomal translocation t(11;22) that occurs in patients with the Ewing's sarcoma family of tumors (ESFT). Using phage display library screening, we identified an EWS-FLI1 binding peptide containing homology to RHA. ESFT cell lines and patient tumors highly expressed RHA. GST pull-down and ELISA assays showed that EWS-FLI1 specifically bound RHA fragment amino acids 630 to 1020, which contains the peptide region discovered by phage display. Endogenous RHA was identified in a protein complex with EWS-FLI1 in ESFT cell lines. Chromatin immunoprecipitation experiments showed both EWS-FLI1 and RHA bound to EWS-FLI1 target gene promoters. RHA stimulated the transcriptional activity of EWS-FLI1 regulated promoters, including Id2, in ESFT cells. In addition, RHA expression in mouse embryonic fibroblast cells stably transfected with EWS-FLI1 enhanced the anchorage-independent phenotype above that with EWS-FLI1 alone. These results suggest that RHA interacts with EWS-FLI1 as a transcriptional cofactor to enhance its function. (Cancer Res 2006; 66(11): 5574-81)