Ewing’s sarcoma precursors are highly enriched in embryonic osteochondrogenic progenitors
Miwa Tanaka, … , Jun Kanno, Takuro Nakamura
Miwa Tanaka, … , Jun Kanno, Takuro Nakamura
Published June 9, 2014
Citation Information: J Clin Invest. 2014;124(7):3061-3074. https://doi.org/10.1172/JCI72399.
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Research Article Oncology

Ewing’s sarcoma precursors are highly enriched in embryonic osteochondrogenic progenitors

Abstract

Ewing’s sarcoma is a highly malignant bone tumor found in children and adolescents, and the origin of this malignancy is not well understood. Here, we introduced a Ewing’s sarcoma–associated genetic fusion of the genes encoding the RNA-binding protein EWS and the transcription factor ETS (EWS-ETS) into a fraction of cells enriched for osteochondrogenic progenitors derived from the embryonic superficial zone (eSZ) of long bones collected from late gestational murine embryos. EWS-ETS fusions efficiently induced Ewing’s sarcoma–like small round cell sarcoma formation by these cells. Analysis of the eSZ revealed a fraction of a precursor cells that express growth/differentiation factor 5 (Gdf5), the transcription factor Erg, and parathyroid hormone-like hormone (Pthlh), and selection of the Pthlh-positive fraction alone further enhanced EWS-ETS–dependent tumor induction. Genes downstream of the EWS-ETS fusion protein were quite transcriptionally active in eSZ cells, especially in regions in which the chromatin structure of the ETS-responsive locus was open. Inhibition of β-catenin, poly (ADP-ribose) polymerase 1 (PARP1), or enhancer of zeste homolog 2 (EZH2) suppressed cell growth in a murine model of Ewing’s sarcoma, suggesting the utility of the current system as a preclinical model. These results indicate that eSZ cells are highly enriched in precursors to Ewing’s sarcoma and provide clues to the histogenesis of Ewing’s sarcoma in bone.

Authors

Miwa Tanaka, Yukari Yamazaki, Yohei Kanno, Katsuhide Igarashi, Ken-ichi Aisaki, Jun Kanno, Takuro Nakamura

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Figure 6

Modulation of gene expression and growth suppression of tumor cells by gene silencing.

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Modulation of gene expression and growth suppression of tumor cells by g...
(A) GSEA of eSZ and eGP cells with EWS-FLI1 (left and central panels) and between eSZ/EWS-FLI1 and eGP, eSZ, and eGP/EWS-FLI1 (right) resulted in enrichment of the WNT/β-catenin pathway, the EGF pathway, and receptor tyrosine kinase activities. (B) Real-time quantitative RT-PCR for Dkk2, Dkk1, Wif1, Prkcb1, Flt4, and Musk in eSZ or eGP cells with/without EWS-FLI1 at 0 or 48 hours after introduction. The mean ± SEM of 3 independent experiments are shown. (C) Inhibition of cell proliferation by knockdown of EWS-FLI1 and genes of the pathways specified in A. Relative growth of tumor cells 48 hours after siRNA treatment was calculated by comparing each cell number to cells treated with control siRNA. The symbols of siRNA used are indicated. Dkk1 was tested as a negative control. Gene knockdown was confirmed by immunoblotting (Fli1, Catnb, Ezh2, and Prkcb1) or RT-PCR (Dkk2 and Igf1). The experiment was repeated 3 times, and representative results are shown. (D) Effect of MAPK pathway inhibition on tumor growth. Erk phosphorylation was inhibited by a MEK1/2 inhibitor U0126 (10 μM) (top), and tumor proliferation was inhibited in a dose-dependent manner 48 hours after treatment (bottom). The mean ± SEM of 3 independent experiments are shown. *P < 0.01; **P < 0.02.