EWS-FLI1 inhibits TNFα-induced NFκB-dependent transcription in Ewing sarcoma cells

https://doi.org/10.1016/j.bbrc.2010.08.004Get rights and content

Abstract

Ewing sarcoma is primarily caused by a t(11;22) chromosomal translocation encoding the EWS-FLI1 fusion protein. To exert its oncogenic function, EWS-FLI1 acts as an aberrant transcription factor, broadly altering the gene expression profile of tumor cells. Nuclear factor-kappaB (NFκB) is a tightly regulated transcription factor controlling cell survival, proliferation and differentiation, as well as tumorigenesis. NFκB activity is very low in unstimulated Ewing sarcoma cells, but can be induced in response to tumor necrosis factor (TNF). We wondered whether NFκB activity could be modulated by EWS-FLI1 in Ewing sarcoma. Using a knockdown approach in Ewing sarcoma cells, we demonstrated that EWS-FLI1 has no influence on NFκB basal activity, but impairs TNF-induced NFκB-driven transcription, at least in part through inhibition of NFκB binding to DNA. We detected an in vivo physical interaction between the fusion protein and NFκB p65, which could mediate these effects. Our findings suggest that, besides directly controlling the activity of its primary target promoters, EWS-FLI1 can also indirectly influence gene expression in tumor cells by modulating the activity of key transcription factors such as NFκB.

Research highlights

► EWS-FLI1 interferes with TNF-induced activation of NFκB in Ewing sarcoma cells. ► EWS-FLI1 knockdown in Ewing sarcoma cells increases TNF-induced NFκB binding to DNA. ► EWS-FLI1 reduces TNF-stimulated NFκB-dependent transcriptional activation. ► Constitutive NFκB activity is not affected by EWS-FLI1. ► EWS-FLI1 physically interacts with NFκB p65 in vivo.

Introduction

Ewing sarcoma, the second most common bone tumor of children and young adults, is primarily associated with a t(11;22)(q24;q12) chromosomal translocation encoding the chimeric protein EWS-FLI1 [1]. Ectopic expression of EWS-FLI1 is sufficient to transform murine immortal NIH3T3 fibroblasts [2], and ongoing synthesis of the fusion protein is essential to maintain the tumoral phenotype of human Ewing sarcoma cells [3]. EWS-FLI1 is usually considered as an aberrant transcription factor, in which the N-terminal EWS activation domain (EAD)2 is fused to the C-terminal DNA-binding domain (DBD) of the ETS family member FLI1, both domains being required for oncogenesis [2]. Since EWS-FLI1 binds to DNA with the same specificity and affinity as native FLI1 [4], it might deregulate the expression of some of its numerous target genes in tumor cells by substituting for the activating factor FLI1, and providing either enhanced transcriptional stimulation, or transcriptional repression. For instance, the expression of IGFBP3, a proapoptotic gene downregulated in Ewing sarcoma, may be moderately activated by FLI1, and repressed by EWS-FLI1, through the same ETS-binding sites [5]. Nevertheless, mutagenesis studies have suggested that EWS-FLI1-mediated oncogenesis might occur via two distinct pathways, the one dependent on, the other independent of, a functional EWS-FLI1 DBD [6]. Because the coactivator cAMP-response element-binding (CREB)-binding protein (CBP) binds to the EAD [7], interference of the fusion protein with CBP recruitment by other transcription factors has been invoked to account for the inhibition of p53-driven transcription by EWS-ATF1 in soft-tissue clear-cell sarcoma [8] and, more recently, by EWS-FLI1 in Ewing sarcoma [9]. Also, part of EWS-FLI1 pleiotropic effects on the transcriptome of Ewing sarcoma cells may be due to indirect effects, as illustrated by the involvement of NKX2.2, which is upregulated by EWS-FLI1 and, in turn, represses the transcription of a fraction of EWS-FLI1-downregulated target genes [10].

In an earlier study, Javelaud et al. [11] observed that Ewing sarcoma cells are resistant to TNFα-induced apoptosis, due to NFκB activation, and suggested that the unusually high amplitude of the induction could rely upon lowered NFκB activity in unstimulated tumor cells. NFκB refers to a family of transcription factors regulating inflammation, immune response, development, cell proliferation and apoptosis. The five NFκB protein family members RelA/p65, RelB, c-Rel, NFκB1/p50, and NFκB2/p52 form homo- and heterodimeric DNA-binding complexes, the most common of which is p50:p65. In unstimulated cells, most NFκB dimers are retained in the cytosol through association with IκB family proteins. In response to canonical NFκB inducers, e.g. TNFα, a complex signaling cascade leads to IκB proteasomal degradation, allowing NFκB p50:p65 to translocate to the nucleus, gain access to its target genes and regulate their transcription. Various post-translational modifications of NFκB p65, including phosphorylation, and acetylation, further control its activity (reviewed in [12]). A negative effect of EWS-FLI1 on NFκB activity could result in extensive gene expression profile alterations in tumor cells, which, to our knowledge, has not been investigated yet. The aim of the present study was to test whether EWS-FLI1 exerts any modulatory effect on constitutive or TNFα-induced NFκB activity in Ewing sarcoma cells, as evaluated at the level of its general ability to specifically bind to DNA and to activate transcription.

Section snippets

Cells

A673 and HEK293T cells were obtained from the ATCC. All cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% [v/v] heat-inactivated fetal calf serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). For NIH3T3 and NIH3T3-EF cells (gift from J. Ghysdael, Orsay, France), newborn serum replaced fetal serum [13]. The establishment and culture conditions of the shA673-1C cell line have been described previously [14]. Recombinant human TNFα (1–9 × 108 U/mg)

EWS-FLI1 inhibits TNFα-induced NFκB DNA-binding activity in Ewing sarcoma cells

In the absence of the untransformed, healthy counterparts of Ewing tumor cells amenable to direct comparative studies, we chose to test whether EWS-FLI1 functionally interacts with NFκB by a knockdown approach in the A673 Ewing sarcoma cell line. A673 cells were transiently transfected with a small interfering RNA (siRNA) targeting the junction region of EWS-FLI1 type I mRNA (EF) or a control siRNA (CT) [5]. Nuclear and cytosolic extracts were separated following cell lysis performed before or

Discussion

Several mechanisms, which are not mutually exclusive, could be invoked to account for EWS-FLI1-mediated inhibition of TNFα-induced NFκB activation. Our results favor a nuclear mechanism of inhibition, since consistent differences in DNA binding were observed, together with a statistically significant decrease in transcriptional activation, without any marked reduction in the cytosolic or nuclear NFκB subunit levels. Furthermore, a physical interaction between nuclear EWS-FLI1 and p65, were it

Acknowledgments

We thank Gérald Peyroche for helpful discussion. This work was funded by the CNRS, the Ministère de l’Enseignement Supérieur et de la Recherche, NOE CONTICANET 6ème PCRDT, the INSERM and the Ligue Nationale contre le Cancer (O. Delattre: Equipe labellisée).

References (24)

  • W.A. May et al.

    Ewing sarcoma 11;22 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLI1 for transformation

    Proc. Natl. Acad. Sci. USA

    (1993)
  • M. Ouchida et al.

    Loss of tumorigenicity of Ewing’s sarcoma cells expressing antisense RNA to EWS-fusion transcripts

    Oncogene

    (1995)
  • Cited by (4)

    • Synthesis and anti-hepatitis B virus activity of C4 amide-substituted isosteviol derivatives

      2015, Bioorganic and Medicinal Chemistry
      Citation Excerpt :

      The mixture was separated on a 5% nondenatured polyacrylamide gel in 0.5× TBE (Tris–borate–EDTA) buffer at 150 V for 90 min, and the binding shift was detected from an autoradiograph. The oligonucleotide-probe for the DNA-binding assay comprised the NF-κB consensus binding site sequence: 5′-ACTATCATCCGGAAAGCCCCCAAAAGTCCC-3′.28 Data are expressed as the mean and standard deviation of the mean (SD) for three independent experiments.

    1

    Present address: Universités Montpellier 2 et 1, CNRS UMR5237, CRBM, 1919 route de Mende, F-34293 MONTPELLIER CEDEX 5, France.

    View full text