BACKGROUND Ewing sarcoma is a malignancy of primitive cells, possibly of mesenchymal origin. It is probable that genetic perturbations other than EWS-FLI1 cooperate with it to produce the tumor. Sequencing studies identified STAG2 mutations in approximately 15% of cases in humans. In the present study, we hypothesize that loss of Stag2 cooperates with EWS-FLI1 in generating sarcomas derived from murine mesenchymal stem cells (MSCs). METHODS Mice bearing an inducible EWS-FLI1 transgene were crossed to p53-/- mice in pure C57/Bl6 background. MSCs were derived from the bone marrow of the mice. EWS-FLI1 induction and Stag2 knockdown were achieved in vitro by adenovirus-Cre and shRNA-bearing pGIPZ lentiviral infection, respectively. The cells were then treated with ionizing radiation to 10 Gy. Anchorage independent growth in vitro was assessed by soft agar assays. Cellular migration and invasion were evaluated by transwell assays. Cells were injected with Matrigel intramuscularly into C57/Bl6 mice to test for tumor formation. RESULTS Primary murine MSCs with the genotype EWS-FLI1 p53-/- were resistant to transformation and did not form tumors in syngeneic mice without irradiation. Stag2 inhibition increased the efficiency and speed of sarcoma formation significantly in irradiated EWS-FLI1 p53-/- MSCs. The efficiency of tumor formation was 91% for cells in mice injected with Stag2-repressed cells and 22% for mice receiving cells without Stag2 inhibition (p < .001). Stag2 knockdown reduced survival of mice in Kaplan-Meier analysis (p < .001). It also increased MSC migration and invasion in vitro but did not affect proliferation rate or aneuploidy. CONCLUSION Loss of Stag2 has a synergistic effect with EWS-FLI1 in the production of sarcomas from murine MSCs, but the mechanism may not relate to increased proliferation or chromosomal instability. Primary murine MSCs are resistant to transformation, and the combination of p53 null mutation, EWS-FLI1, and Stag2 inhibition does not confer immediate conversion of MSCs to sarcomas. Irradiation is necessary in this model, suggesting that perturbations of other genes beside Stag2 and p53 are likely to be essential in the development of EWS-FLI1-driven sarcomas from MSCs.

中文翻译：

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Stag2的丢失与EWS-FLI1协同转化鼠间充质干细胞。

背景技术尤文氏肉瘤是原始细胞的恶性肿瘤，可能是间充质起源的。EWS-FLI1以外的遗传干扰很可能与之协同产生肿瘤。测序研究确定了大约15％的人类病例中的STAG2突变。在本研究中，我们假设Stag2的缺失与EWS-FLI1协同作用，产生了源自鼠间充质干细胞（MSCs）的肉瘤。方法将携带可诱导的EWS-FLI1转基因的小鼠与纯C57 / B16背景的p53-/-小鼠杂交。MSC来自小鼠的骨髓。EWS-FLI1诱导和Stag2敲低分别通过腺病毒-Cre和shRNA携带的pGIPZ慢病毒感染在体外实现。然后将细胞用电离辐射处理至10 Gy。通过软琼脂分析评估了体外不依赖于锚定的生长。通过transwell测定法评估细胞迁移和侵袭。将基质胶肌肉注射到C57 / B16小鼠中以测试肿瘤的形成。结果EWS-FLI1 p53-/-基因型的原代小鼠MSC对转化具有抗性，并且在未经辐射的同系小鼠中不形成肿瘤。在辐射的EWS-FLI1 p53-/-MSC中，Stag2抑制显着提高了肉瘤形成的效率和速度。对于注射了Stag2抑制细胞的小鼠而言，肿瘤形成的效率为91％，而接受无Stag2抑制作用的细胞则为22％（p <.001）。在Kaplan-Meier分析中，Stag2敲低会降低小鼠的存活率（p <.001）。它也增加了MSC在体外的迁移和侵袭，但不影响增殖速率或非整倍性。结论Stag2的丧失与EWS-FLI1在鼠类MSC肉瘤的产生中具有协同作用，但其机制可能与增殖增加或染色体不稳定有关。原代小鼠MSC对转化具有抗性，p53无效突变，EWS-FLI1和Stag2抑制的组合不能使MSC立即转化为肉瘤。在该模型中需要进行辐照，这表明除了Stag2和p53外，其他基因的扰动对于从MSC产生EWS-FLI1驱动的肉瘤而言可能也是必不可少的。但其机制可能与增殖增加或染色体不稳定性有关。原代小鼠MSC对转化具有抗性，p53无效突变，EWS-FLI1和Stag2抑制的组合不能使MSC立即转化为肉瘤。在该模型中需要进行辐照，这表明除了Stag2和p53外，其他基因的扰动对于从MSC产生EWS-FLI1驱动的肉瘤而言可能也是必不可少的。但其机制可能与增殖增加或染色体不稳定性有关。原代小鼠MSC对转化具有抗性，p53无效突变，EWS-FLI1和Stag2抑制的组合不能使MSC立即转化为肉瘤。在该模型中需要进行辐照，这表明除了Stag2和p53外，其他基因的扰动对于从MSC产生EWS-FLI1驱动的肉瘤而言可能也是必不可少的。