FOXO1 is a direct target of EWS-Fli1 oncogenic fusion protein in Ewing’s sarcoma cells

• Published in: Biochemical and biophysical research communications 2010-11, Vol.402 (1), p.129-134
• Main Authors: Yang, Liu, Hu, Hsien-Ming, Zielinska-Kwiatkowska, Anna, Chansky, Howard A.
• Format: Article
• Language: English
• Subjects: 60 APPLIED LIFE SCIENCES; Bone Neoplasms - genetics; Cell Line, Tumor; DNA; Doxycycline - pharmacology; Ewing’s sarcoma; EWS-Fli1; Forkhead Box Protein O1; Forkhead Transcription Factors - genetics; FOXO1; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; GENES; Humans; Inducible siRNA; INHIBITION; Oncogene Proteins, Fusion - genetics; Oncogene Proteins, Fusion - metabolism; Oncogenic fusion protein; Promoter Regions, Genetic; PROMOTERS; PROTEINS; Proto-Oncogene Protein c-fli-1 - genetics; Proto-Oncogene Protein c-fli-1 - metabolism; RNA; RNA interference; RNA, Small Interfering - genetics; RNA-Binding Protein EWS; Sarcoma, Ewing - genetics; SARCOMAS; Suppression, Genetic; TRANSCRIPTION; TRANSFORMATIONS; TRANSLOCATION; Tumor suppressor gene
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2010.09.129

► Inducible and reversible siRNA knockdown of an oncogenic fusion protein such as EWS-Fli1 is feasible and more advantageous than other siRNA methods. ► The tumor suppressor gene FOXO1 is a new EWS-Fli1 target. ► While trans-activators are known for the FOXO1 gene, there has been no report on negative regulators of FOXO1 transcription. ► This study provides first evidence that the EWS-Fli1 oncogenic fusion protein can function as a transcriptional repressor of the FOXO1 gene. Ewing’s family tumors are characterized by a specific t(11;22) chromosomal translocation that results in the formation of EWS-Fli1 oncogenic fusion protein. To investigate the effects of EWS-Fli1 on gene expression, we carried out DNA microarray analysis after specific knockdown of EWS-Fli1 through transfection of synthetic siRNAs. EWS-Fli1 knockdown increased expression of genes such as DKK1 and p57 that are known to be repressed by EWS-Fli1 fusion protein. Among other potential EWS-Fli1 targets identified by our microarray analysis, we have focused on the FOXO1 gene since it encodes a potential tumor suppressor and has not been previously reported in Ewing’s cells. To better understand how EWS-Fli1 affects FOXO1 expression, we have established a doxycycline-inducible siRNA system to achieve stable and reversible knockdown of EWS-Fli1 in Ewing’s sarcoma cells. Here we show that FOXO1 expression in Ewing’s cells has an inverse relationship with EWS-Fli1 protein level, and FOXO1 promoter activity is increased after doxycycline-induced EWS-Fli1 knockdown. In addition, we have found that direct binding of EWS-Fli1 to FOXO1 promoter is attenuated after doxycycline-induced siRNA knockdown of the fusion protein. Together, these results suggest that suppression of FOXO1 function by EWS-Fli1 fusion protein may contribute to cellular transformation in Ewing’s family tumors.