EWS/ATF1 expression induces sarcomas from neural crest–derived cells in mice
Kazunari Yamada, … , Akira Hara, Yasuhiro Yamada
Kazunari Yamada, … , Akira Hara, Yasuhiro Yamada
Published January 2, 2013
Citation Information: J Clin Invest. 2013;123(2):600-610. https://doi.org/10.1172/JCI63572.
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Research Article Oncology

EWS/ATF1 expression induces sarcomas from neural crest–derived cells in mice

Abstract

Clear cell sarcoma (CCS) is an aggressive soft tissue malignant tumor characterized by a unique t(12;22) translocation that leads to the expression of a chimeric EWS/ATF1 fusion gene. However, little is known about the mechanisms underlying the involvement of EWS/ATF1 in CCS development. In addition, the cellular origins of CCS have not been determined. Here, we generated EWS/ATF1-inducible mice and examined the effects of EWS/ATF1 expression in adult somatic cells. We found that forced expression of EWS/ATF1 resulted in the development of EWS/ATF1-dependent sarcomas in mice. The histology of EWS/ATF1-induced sarcomas resembled that of CCS, and EWS/ATF1-induced tumor cells expressed CCS markers, including S100, SOX10, and MITF. Lineage-tracing experiments indicated that neural crest–derived cells were subject to EWS/ATF1-driven transformation. EWS/ATF1 directly induced Fos in an ERK-independent manner. Treatment of human and EWS/ATF1-induced CCS tumor cells with FOS-targeted siRNA attenuated proliferation. These findings demonstrated that FOS mediates the growth of EWS/ATF1-associated sarcomas and suggest that FOS is a potential therapeutic target in human CCS.

Authors

Kazunari Yamada, Takatoshi Ohno, Hitomi Aoki, Katsunori Semi, Akira Watanabe, Hiroshi Moritake, Shunichi Shiozawa, Takahiro Kunisada, Yukiko Kobayashi, Junya Toguchida, Katsuji Shimizu, Akira Hara, Yasuhiro Yamada

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Figure 5

Fos is a direct target of EWS/ATF1.

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Fos is a direct target of EWS/ATF1. (A) Real-time RT-PCR analysis of G12...
(A) Real-time RT-PCR analysis of G1297 cells revealed significant upregulation of both EWS/ATF1 and Fos 3 hours after doxycycline exposure. (B) Relative expression of EWS/ATF1 and Fos in 4 EWS/ATF1-induced tumors from 4 independent mice. NIH3T3 cells served as a control. Transcript levels were normalized to β-actin. Data are mean ± SD (n = 3). (C) Fos induction by EWS/ATF1 was independent of the ERK pathway. Serum-starved MEFs and G1297 cells were stimulated with 30% FBS for the indicated times. Cells were also treated with 10 μM of the MEK inhibitor U0126. Whereas ERK1/2 inhibition by U0126 decreased Fos in MEFs, U0126 failed to suppress Fos expression in G1297 cells. NT, not treated. (D) Mouse Fos promoter–luciferase reporter constructs and pRL-SV40 vector (as an internal control) were cotransfected in G1297 cells treated with or without 0.2 μg/ml doxycycline. Luciferase activity of each construct was normalized to internal control activity. Data are mean ± SD (n = 3). (E) ChIP-PCR analysis was performed for the Fos promoter region containing CRE or the negative control cis element using HA-tag antibody or IgG as nonimmune immunoprecipitation, respectively. EWS/ATF1 was enriched at the CRE element of the Fos promoter in G1297 cells after treatment with 0.2 μg/ml doxycycline. Data (mean ± SD) were quantified by qRT-PCR and expressed as percent of input DNA.