EWS/FLI-1 silencing and gene profiling of Ewing cells reveal downstream oncogenic pathways and a crucial role for repression of insulin-like growth factor binding protein 3.
Ewing tumors are characterized by abnormal transcription factors resulting from the oncogenic fusion of EWS with members of the ETS family, most commonly FLI-1. RNA interference targeted to the junction between EWS and FLI-1 sequences was used to inactivate the EWS/FLI-1 fusion gene in Ewing cells and to explore the resulting phenotype and alteration of the gene expression profile. Loss of expression of EWS/FLI-1 resulted in the complete arrest of growth and was associated with a dramatic increase in the number of apoptotic cells. Gene profiling of Ewing cells in which the EWS/FLI-1 fusion gene had been inactivated identified downstream targets which could be grouped in two major functional clusters related to extracellular matrix structure or remodeling and regulation of signal transduction pathways. Among these targets, the insulin-like growth factor binding protein 3 gene (IGFBP-3), a major regulator of insulin-like growth factor 1 (IGF-1) proliferation and survival signaling, was strongly induced upon treating Ewing cells with EWS/FLI-1-specific small interfering RNAs. We show that EWS/FLI-1 can bind the IGFBP-3 promoter in vitro and in vivo and can repress its activity. Moreover, IGFBP-3 silencing can partially rescue the apoptotic phenotype caused by EWS/FLI-1 inactivation. Finally, IGFBP-3-induced Ewing cell apoptosis relies on both IGF-1-dependent and -independent pathways. These findings therefore identify the repression of IGFBP-3 as a key event in the development of Ewing's sarcoma.
Laboratoire de Pathologie Moléculaire des Cancers, INSERM U509, Section de Recherche, Institut Curie, 75248 Paris, France.
Alexandre
A
Prieur
Franck
F
Tirode
Pinchas
P
Cohen
Olivier
O
Delattre
D017209:Apoptosis / Q000502:physiology; D019677:Artificial Gene Fusion; D001483:Base Sequence; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000378:metabolism*; D051789:Proto-Oncogene Protein c-fli-1; D034622:RNA Interference; D034802:RNA-Binding Protein EWS; D012512:Sarcoma, Ewing / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
C106031:EWS-FLI fusion protein; D018972:Insulin-Like Growth Factor Binding Protein 3; D015514:Oncogene Proteins, Fusion; D051789:Proto-Oncogene Protein c-fli-1; D034802:RNA-Binding Protein EWS; D014157:Transcription Factors