EWS/ATF1 expression induces sarcomas from neural crest–derived cells in mice
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
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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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 4

Establishment and analysis of tumor cell lines.

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Establishment and analysis of tumor cell lines. G1297 and G1169 cell lin...
G1297 and G1169 cell lines were established from 2 independent EWS/ATF1-induced tumors. (A) Morphology of the G1297 line after treatment without or with doxycycline (0 and 0.2 μg/ml, respectively). At concentrations above 0.1 μg/ml, small, round tumor cells grew rapidly, while dendritic fibroblast-like spindle cells were observed; tumor cell growth almost stopped at concentrations less than 0.05 μg/ml. Scale bars: 50 μm. (B) Effect of different levels of EWS/ATF1 on tumor cell growth. G1297 cells were cultured in different concentrations of doxycycline (0, 0.05, 0.1, and 0.2 μg/ml), and cell viability was determined by WST-8 assay. Data are mean ± SD (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001 vs. Dox 0.0 μg/ml. (C) Subcutaneous transplantation of 5.0 × 106 G1297 cells in immunocompromised mice resulted in tumor formation in mice treated with 50 μg/ml doxycycline (n = 8). Mean tumor volumes ± SEM are shown. ***P < 0.005. (D) Representative histology and HA immunostaining of tumors in nude mice. The tumor resembled the original sarcoma with EWS/ATF1 expression. Scale bars: 100 μm. (E) Doxycycline withdrawal led to rapid tumor regression. At 3 months after doxycycline withdrawal, no viable tumor cells were observed, and tumors were replaced by fibrous tissue. Widespread cell death was observed 4 days after doxycycline withdrawal. Scale bars: 200 μm.