Oncoprotein EWS-FLI1 activity is enhanced by RNA helicase A.

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.

[1]  Zhi-Ren Liu,et al.  Phosphorylations of DEAD Box p68 RNA Helicase Are Associated with Cancer Development and Cell Proliferation , 2005, Molecular Cancer Research.

[2]  A. Üren,et al.  PTPL1 is a direct transcriptional target of EWS-FLI1 and modulates Ewing's Sarcoma tumorigenesis , 2005, Oncogene.

[3]  Brian J. Wilson,et al.  The DEAD box protein p68: a novel transcriptional coactivator of the p53 tumour suppressor , 2005, The EMBO journal.

[4]  J. Toretsky,et al.  Recombinant EWS-FLI1 oncoprotein activates transcription. , 2004, Biochemistry.

[5]  T. Okamoto,et al.  RNA helicase A interacts with nuclear factor kappaB p65 and functions as a transcriptional coactivator. , 2004, European journal of biochemistry.

[6]  P. Meltzer,et al.  Soft tissue sarcomas of adults: state of the translational science. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[7]  M. Ladanyi,et al.  Beta-platelet-derived growth factor receptor mediates motility and growth of Ewing's sarcoma cells , 2003, Oncogene.

[8]  L. Starita,et al.  Overexpression of a protein fragment of RNA helicase A causes inhibition of endogenous BRCA1 function and defects in ploidy and cytokinesis in mammary epithelial cells , 2003, Oncogene.

[9]  S. Donaldson,et al.  Addition of ifosfamide and etoposide to standard chemotherapy for Ewing's sarcoma and primitive neuroectodermal tumor of bone. , 2003, The New England journal of medicine.

[10]  Akihiro Umezawa,et al.  Upregulation of Id2, an oncogenic helix-loop-helix protein, is mediated by the chimeric EWS/ets protein in Ewing sarcoma , 2003, Oncogene.

[11]  Yusuke Nakamura,et al.  The Id2 gene is a novel target of transcriptional activation by EWS-ETS fusion proteins in Ewing family tumors , 2002, Oncogene.

[12]  P. Hozák,et al.  The localization of nuclear DNA helicase II in different nuclear compartments is linked to transcription. , 2002, Experimental cell research.

[13]  H. Kovar,et al.  Interaction of the EWS NH2 terminus with BARD1 links the Ewing's sarcoma gene to a common tumor suppressor pathway. , 2002, Cancer research.

[14]  A. Orth,et al.  Large-scale analysis of the human and mouse transcriptomes , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[15]  B. Séraphin,et al.  The DEXD/H‐box RNA helicase RHII/Gu is a co‐factor for c‐Jun‐activated transcription , 2002, The EMBO journal.

[16]  Christopher T Denny,et al.  Biology of EWS/ETS fusions in Ewing's family tumors , 2001, Oncogene.

[17]  H. Kovar,et al.  The EWS protein is dispensable for Ewing tumor growth. , 2001, Cancer research.

[18]  B. Kay,et al.  Screening phage-displayed combinatorial peptide libraries. , 2001, Methods.

[19]  S. Baker,et al.  EWS/FLI Alters 5′-Splice Site Selection* , 2001, The Journal of Biological Chemistry.

[20]  M. Ladanyi,et al.  High-dose melphalan, etoposide, total-body irradiation, and autologous stem-cell reconstitution as consolidation therapy for high-risk Ewing's sarcoma does not improve prognosis. , 2001, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[21]  H. Chansky,et al.  Oncogenic TLS/ERG and EWS/Fli-1 fusion proteins inhibit RNA splicing mediated by YB-1 protein. , 2001, Cancer research.

[22]  S. Baker,et al.  The Splicing Factor U1C Represses EWS/FLI-mediated Transactivation* , 2000, The Journal of Biological Chemistry.

[23]  A. Üren,et al.  Secreted Frizzled-related Protein-1 Binds Directly to Wingless and Is a Biphasic Modulator of Wnt Signaling* , 2000, The Journal of Biological Chemistry.

[24]  P. Sorensen,et al.  Repression of the gene encoding the TGF-β type II receptor is a major target of the EWS-FLI1 oncoprotein , 1999, Nature Genetics.

[25]  J. Takahara,et al.  The inv(11)(p15q22) chromosome translocation of therapy-related myelodysplasia with NUP98-DDX10 and DDX10-NUP98 fusion transcripts. , 1999, International journal of hematology.

[26]  K. Manova,et al.  RNA helicase A is essential for normal gastrulation. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Erica A Golemis,et al.  Oncogenic EWS-Fli1 interacts with hsRPB7, a subunit of human RNA polymerase II , 1998, Oncogene.

[28]  J. Parvin,et al.  BRCA1 protein is linked to the RNA polymerase II holoenzyme complex via RNA helicase A , 1998, Nature Genetics.

[29]  Olivier Delattre,et al.  EWS, but Not EWS-FLI-1, Is Associated with Both TFIID and RNA Polymerase II: Interactions between Two Members of the TET Family, EWS and hTAFII68, and Subunits of TFIID and RNA Polymerase II Complexes , 1998, Molecular and Cellular Biology.

[30]  D. Leroith,et al.  The Insulin-like Growth Factor-I Receptor Is Required for EWS/FLI-1 Transformation of Fibroblasts* , 1997, The Journal of Biological Chemistry.

[31]  Jeffrey D. Parvin,et al.  RNA Helicase A Mediates Association of CBP with RNA Polymerase II , 1997, Cell.

[32]  K. Tanaka,et al.  EWS-Fli1 antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells. , 1997, The Journal of clinical investigation.

[33]  Y. Akao,et al.  The rck/p54 candidate proto-oncogene product is a 54-kilodalton D-E-A-D box protein differentially expressed in human and mouse tissues. , 1995, Cancer Research.

[34]  F. Grosse,et al.  Molecular Cloning of the Gene Encoding Nuclear DNA Helicase II. A BOVINE HOMOLOGUE OF HUMAN RNA HELICASE A AND DROSOPHILA Mle PROTEIN (*) , 1995, The Journal of Biological Chemistry.

[35]  J. Hurwitz,et al.  Human RNA helicase A is homologous to the maleless protein of Drosophila. , 1993, The Journal of biological chemistry.

[36]  M. Klemsz,et al.  Characterization of the ets oncogene family member, fli-1. , 1993, The Journal of biological chemistry.

[37]  G. Thomas,et al.  Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours , 1992, Nature.

[38]  M. Klemsz,et al.  The ETS-domain: a new DNA-binding motif that recognizes a purine-rich core DNA sequence. , 1990, Genes & development.

[39]  J. Hurwitz,et al.  Assemblage of the prespliceosome complex with separated fractions isolated from HeLa cells. , 1990, The Journal of biological chemistry.

[40]  J. Hurwitz,et al.  Isolation and characterization of two fractions from HeLa cells required for mRNA splicing in vitro. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[41]  N. Bhat,et al.  Inhibition of EWS-FLI-1 fusion protein with antisense oligodeoxynucleotides , 2004, Journal of Neuro-Oncology.

[42]  C. Bloomfield,et al.  Expression analyses identify MLL as a prominent target of 11q23 amplification and support an etiologic role for MLL gain of function in myeloid malignancies. , 2004, Blood.

[43]  Y. Arai,et al.  Fusion of the nucleoporin gene, NUP98, and the putative RNA helicase gene, DDX10, by inversion 11 (p15q22) chromosome translocation in a patient with etoposide-related myelodysplastic syndrome. , 2000, Internal medicine.

[44]  C. Denny,et al.  Biology of EWS/FLI and related fusion genes in Ewing's sarcoma and primitive neuroectodermal tumor. , 1997, Current topics in microbiology and immunology.