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 1

Development of murine Ewing’s sarcoma.

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Development of murine Ewing’s sarcoma. (A) Microdissection of mouse embr...
(A) Microdissection of mouse embryonic bone. The femur was lightly stained with methylene blue. Experimental strategy of the ex vivo model. Each cell type targeted with EWS-ETS was injected into nude mice or subjected to gene expression profiling. (B) Cumulative incidence (percentage) of small round cell tumors induced by eSZ, eGP, and eSyR cells expressing EWS-ETS or by eSZ with an empty vector and by embryonic mesenchymal cells of the trunk, head, and shaft expressing EWS-FLI1 or eSZ expressing EWS-CHOP or SYT-SSX1. P < 0.02 in eSZ/EWS-FLI1 vs. eGP, eSyR, or shaft; P < 0.04 in eSZ/EWS-FLI1 vs. trunk and head, log-rank test. (C) Tumors were observed as subcutaneous masses in recipient nude mice. (D) Histology of murine Ewing’s sarcoma. H&E staining, with low (left) and high (center) magnification. Pulmonary metastasis of murine Ewing’s sarcoma developed by tail vein injection of tumor cells (right). Scale bar: 100 μm. (E) Cre/loxP-mediated knockout of the EWS-FLI1 transgene. Ewing’s sarcoma developed by transplantation of eSZ cells transduced with the floxed EWS-FLI1 retrovirus (left). Sarcoma cells were then maintained in vitro and transduced with the pMSCV-Cre retrovirus. Cre expression suppressed colony formation (right). The experiment was repeated 3 times and the representative results, are shown (graph inset). Scale bar: 100 μm. *P < 0.01. The mean ± SEM of 3 independent experiments is shown.