Oncoprotein EWS-FLI1 Activity Is Enhanced by RNA Helicase A

Abstract

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 anchorageindependent 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)

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

“…S3). RHA, previously identified as a direct partner of EWS-FLI1 (27) was confirmed using a RHA-specific antibody (SI Appendix, Table S2 and Fig. S3).…”

“…Proteins that could be analyzed using commercially available antibodies were evaluated by coimmunoprecipitation (co-IP) with EWS-FLI1 using nuclear lysate from TC32 cells, a well-characterized ES cell line. An anti-FLI1 antibody was used for EWS-FLI1 co-IP, as ES cells do not express wild-type (WT) FLI1 (27). Of the spliceosomal proteins validated by co-IP, 12 of 13 identified in an EWS-FLI1 complex were confirmed (SI Appendix, Table S2 and Fig.…”

Ewing sarcoma EWS protein regulates midzone formation by recruiting Aurora B kinase to the midzone

“…2,[8][9][10][11][12][13][14] The interaction between RNA helicase A and EWS/ FLI1 enhances EWS/FLI1-dependent transcriptional activity. 15,16 EWS/FLI1 also regulates a subgroup of miRNAs that specifically regulate the IGF pathway. 17 Ewing sarcoma patient cells display high rates of aneuploidy.…”

Oncogenic role of DDX3 in breast cancer biogenesis

“…In addition, DDX5 has been shown to upregulate the expression of cyclin D1 along with c-myc genes, which promoted cell proliferation in a series of mammalian cell lines (Yang et al, 2007). Along these lines, the activity of oncoprotein EWS-FLI1 has been shown to be enhanced by RNA helicase H in the Ewing's sarcoma family of tumors (Toretsky et al, 2006). The X-linked DEAD box RNA helicase, DDX3 has been shown to be upregulated in hepatocellular carcinoma tissues and through its interaction with hepatitis C virus core protein, may be a contributing factor to hepatitis C virus-mediated carcinogenic processes (Owsianka and Patel, 1999;Huang et al, 2004).…”