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

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Abstract

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.

Research highlights

► 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.

Introduction

Ewing’s family tumors are malignancies that share histological features as well as a recurrent and specific t(11;22) chromosomal translocation [1]. This translocation results in a chimeric transcript encoding the N-terminal domain of the RNA-binding protein EWS and the DNA-binding domain of the ETS family transcription factor Fli1 [2]. The resultant EWS-Fli1 chimeric fusion protein is known to affect both gene transcription and RNA splicing [3], [4].

EWS-Fli11 has been proposed to be an oncogenic fusion protein based on its ability to transform cells. While a number of EWS-Fli1 target genes have been identified by ectopic expression of EWS-Fli1 in non-Ewing’s cells [5], [6], [7], [8], several studies have indicated that EWS-FLi1 target genes identified in non-Ewing’s cells do not overlap with those in Ewing’s cells [9], [10]. To study how EWS-Fli1 influences gene expression in the genetic background of Ewing’s sarcoma, we and others have examined the effects of EWS-Fli1 knockdown by siRNA in actual Ewing’s cell lines [4], [11], [12]. We previously found that knockdown of EWS-Fli1 in Ewing’s cells leads to growth arrest and reduced invasiveness [4]. More recently we have shown that EWS-Fli1 abolishes cellular senescence in Ewing’s sarcoma cells by suppressing the functions of retinoblastoma protein [13].

To gain further insight into the oncogenic mechanisms of EWS-Fli1 fusion protein, in this manuscript we carried out microarray analysis of RNA samples from Ewing’s sarcoma cells transfected with synthetic siRNAs against EWS-Fli1. Analysis of our microarray data revealed that EWS-Fli1 affects many genes including repression of the FOXO1 gene in Ewing’s sarcoma cells. While synthetic siRNAs represent a convenient approach in the knockdown of EWS-Fli1, they also have severe limitations such as a relatively short duration of robust siRNA knockdown and a need to transfect fresh cells for each experiment. To achieve sustained and reversible siRNA knockdown of EWS-Fli1, we utilized the pSLIK (single lentivector for inducible knockdown) platform in Ewing’s sarcoma cells to conditionally turn on production of siRNA against EWS-Fli1. Using this inducible siRNA system in Ewing’s sarcoma cells, we have found that doxycycline-induced siRNA knockdown of EWS-Fli1 in Ewing’s sarcoma cells is accompanied by an increase in FOXO1 expression and a decrease in proliferation. Through chromatin immunoprecipitation (CHIP) assay, we showed that EWS-Fli1 binds directly to the promoter region of FOXO1. Taken together, these findings suggest that FOXO1 is a downstream target of EWS-Fli1, and that the tumor suppressor activity of FOXO1 is likely silenced by the oncogenic EWS-Fli1 fusion protein during tumorigenesis.

Section snippets

Cell culture

Human Ewing’s sarcoma cell lines A673, SK-ES and RD-ES were obtained from ATCC and maintained in RPMI, McCoy’s 5A and Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal bovine serum, respectively.

siRNA transfection and DNA microarray analysis

SK-ES cells (2 × 106) in 0.1 ml PBS were electroporated with 4 μl of siRNA and plated onto 4 wells in a collagen-coated 6-well plate. Later (48 h), total RNA was prepared for DNA microarray at the University of Washington Center for Expression Array. Target labeling and hybridization to

Transient knockdown of EWS-Fli1 by synthetic siRNA affects gene expression in Ewing’s sarcoma cells

In earlier studies, we and others have found that genes affected by EWS-Fli1 and its related fusion proteins vary greatly in cells that were not derived from the tumors [9], [10]. Therefore, bona fide EWS-Fli1 target genes must be identified in the genetic background of Ewing’s sarcoma cells but not in surrogate cells such as NIH3T3. To this end, we were able to achieve a specific siRNA knockdown of EWS-Fli1 in SK-ES Ewing’s sarcoma cells, whereas a non-specific siRNA against the firefly

Discussion

In addition to synthetic siRNA, we previously also used an adenoviral vector to deliver siRNA and achieved near complete depletion of EWS-Fli1 for a sustained period of time in infected Ewing cells [13]. However, siRNA knockdown via adenoviral vector is irreversible and fresh infection has to be carried out for each new experiment. In this study, we have established a conditional lentiviral siRNA system to reversibly knockdown EWS-Fli1 in several Ewing cell lines. This doxycycline inducible

Acknowledgments

This work was supported by Public Health Service Grant RO1 AR051455 (to L.Y.) and by a Veterans Affairs Merit Review Award (to H.A.C.).

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