Targeting of EWS/FLI-1 by RNA interference attenuates the tumor phenotype of Ewing's sarcoma cells in vitro.
The defining cytogenetic abnormality of Ewing's sarcoma is the presence of a balanced t(11;22) translocation expressing the EWS/FLI-1 chimeric fusion protein. The effect of EWS/FLI-1 appears to be dominant negative since over-expression of EWS does not overcome the sarcoma phenotype. Previous studies have shown that EWS/FLI-1 as well as related sarcoma fusion proteins are necessary and sufficient to induce transformation both in vitro and in vivo. In this study we report that synthetic small interfering RNA (siRNA) specifically suppresses EWS/FLI-1 fusion gene expression in SK-ES Ewing's sarcoma cells. Knockdown of the EWS/FLI-1 fusion protein is correlated with decreased cell proliferation and increased apoptosis. We demonstrate that Ewing's sarcoma tumors as well as Ewing's sarcoma cell lines predominantly express the CXCR4 chemokine receptor. Using an in vitro invasion assay, the SDF-1 ligand of CXCR4 was shown to be a potent stimulus of invasion by SK-ES cells. Knockdown of EWS/FLI-1 by RNA interference abrogates the invasiveness of SK-ES cells. These experiments suggest that targeted silencing of the EWS/FLI-1 fusion gene by siRNA represents a promising strategy to study the loss of EWS/FLI-1 protein in Ewing's sarcoma cells of otherwise identical genetic background.
Chansky
Howard A
HA
Department of Orthopedics and Sports Medicine, School of Medicine, University of Washington, 1660 S. Columbian Way, ORT112, Seattle, WA 98108, USA. chansky@u.washington.edu
Barahmand-Pour
Fariba
F
Mei
Qi
Q
Kahn-Farooqi
Waqqar
W
Zielinska-Kwiatkowska
Anna
A
Blackburn
Michael
M
Chansky
Kari
K
Conrad
Ernest U
EU
Bruckner
James D
JD
Greenlee
Theodore K
TK
Yang
Liu
L
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D020868:Gene Silencing / Q000187:drug effects; D016153:Genes, Suppressor / Q000502:physiology; D006801:Humans; D015514:Oncogene Proteins, Fusion / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D051789:Proto-Oncogene Protein c-fli-1; D034741:RNA, Small Interfering / Q000494:pharmacology*; D034802:RNA-Binding Protein EWS; D019718:Receptors, CXCR4 / Q000378:metabolism; D012512:Sarcoma, Ewing / Q000188:drug therapy* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D000970:Antineoplastic Agents; C106031:EWS-FLI fusion protein; D015514:Oncogene Proteins, Fusion; D051789:Proto-Oncogene Protein c-fli-1; D034741:RNA, Small Interfering; D034802:RNA-Binding Protein EWS; D019718:Receptors, CXCR4; D014157:Transcription Factors