Abstract

BackgroundEwing 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).MethodsMice 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.ResultsPrimary 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.ConclusionLoss 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.

Highlights

  • Ewing sarcoma is a malignancy of primitive cells, possibly of mesenchymal origin

  • We present in vitro and in vivo data that support a synergistic effect between Stag2 inhibition, EWS-FLI1 expression, and p53 mutation in the transformation of primary Mesenchymal stem cell (MSC)

  • Inhibition of Stag2 in MSCs bearing the genotype p53−/− EWS-FLI1 Starting with an EWS-FLI1 transgenic mouse bearing a construct that allows for conditional expression [26], we crossed to p53−/− null mice to obtain EWS-FLI1 p53−/− mice in a pure C57/Bl6 background

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Summary

Introduction

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. We hypothesize that loss of Stag cooperates with EWS-FLI1 in generating sarcomas derived from murine mesenchymal stem cells (MSCs). Stag 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 Stag inhibition (p < .001). Stag knockdown reduced survival of mice in Kaplan-Meier analysis (p < .001) It increased MSC migration and invasion in vitro but did not affect proliferation rate or aneuploidy. Ewing sarcoma is a malignancy of primitive cells that arises typically in young adolescents and adults [1, 2]. Its mutational inactivation or loss of expression have been documented in a variety of solid and hematologic malignancies, including glioblastoma, lymphomas, colorectal, prostate, urothelial bladder cancers, and Ewing sarcoma [14, 17, 19,20,21,22,23,24]

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