An inducible ectopic expression system of EWSR1-FLI1 as a tool for understanding Ewing sarcoma oncogenesis

Abstract

The presence of the chimeric EWSR1-FLI1 oncoprotein is the main and initiating event defining Ewing sarcoma (ES). The dysregulation of epigenomic and proteomic homeostasis induced by the oncoprotein contributes to a wide variety of events involved in oncogenesis and tumor progression. Attempts at studying the effects of EWSR1-FLI1 in non-tumor cells to understand the mechanisms underlying sarcomagenesis have been unsuccessful to date, as ectopic expression of EWSR1-FLI1 blocks cell cycle progression and induces apoptosis in the tested cell lines. Therefore, it is essential to find a permissive cell type for EWSR1-FLI1 expression that allows its endogenous molecular functions to be studied. Here we have demonstrated that HeLa cell lines are permissive to EWSR1-FLI1 ectopic expression, and that our model substantially recapitulates the endogenous activity of the EWSR1-FLI1 fusion protein. This model could contribute to better understanding ES sarcomagenesis by helping to understand the molecular mechanisms induced by the EWSR1-FLI1 oncoprotein.

Fig 1. EWSR1-FLI1 expression in a HeLa Tet-On model.

A. Schematic diagram of the retroviral vector used in the study. B, C. Western blot analysis showing the inducible expression of EWSR1-FLI1 in two HeLa Tet-On EF clones (#3.10 and #3.15) using the FLI1 and FLAG antibodies in the presence of different concentrations of doxycycline at different time points. The RDES and A673 ES cell lines were used as controls for FLI expression. D. Proliferation of parental HeLa3G cells (control) and HeLa Tet-On EF #3.10 and #3.15 clones in the presence or absence of doxycycline. Values are mean ± s.d. of three biological independent replicates. Statistical tests: 2-way ANOVA multiple comparisons. E. Doxycycline does not affect the morphology of parental cells or the HeLa Tet-On EWSR1-FLI1 models (#3.10 and #3.15). The cell lines were grown in medium with or (for controls) without doxycycline (1 μg/ml) for 18 days. Phase contrast shows 20× and 40× magnifications.

Fig 2. The HeLa model expresses the aberrant transcription factor EWSR1-FLI1.

A. EWSR1-FLI1 gene fusion expression analysis in parental HeLa3G cells (control) and two different clones (HeLa Tet-On EF #3.10 and #3.15) in the presence or absence of doxycycline at different time points. B–D. EWSR1-FLI1 up- and downregulated target genes are shown after 24 h and 18 days of doxycycline induction. Values are given as mean ± s.d. of three biological independent replicates. Statistical tests: significant analysis of multiple t-test; ***, <0.001; **, 0.01;*, 0.05; NS, not significant.

Fig 3. Ectopic EWSR1-FLI1 interacts with specific DNA and protein-binding sites.

A. EWSR1-FLI1 immunoprecipitation using anti-FLAG antibody in parental HeLa3G cells (control) and two different clones (HeLa Tet-On EF #3.10 and #3.15) in the presence or absence of doxycycline. B. Binding of EWSR1-FLI1 to the CAV1 enhancer and the promoters of the CAV1, EZH2 and AURKB genes. qPCR analysis of EWSR1-FLI1 ChIP signals for HeLa Tet-On EF #3.15 clone. C. Co-immunoprecipitation of EWSR1-FLI1 3×FLAG C-terminal and LSD1 protein in HeLa Tet-On EF #3.15 clone.

Fig 4. HeLa Tet-On EWSR1-FLI1 gene expression patterns correlated with different published models.

A. Venn diagram analysis of differentially expressed genes between HeLa Tet-On EWSR1-FLI1, hMSC_EWSR1-FLI1 and the ES cell line shEWSR1-FLI1. B. Venn diagrams showing the overlap between upregulated genes in the HeLa model and downregulated genes in ES cell lines shEWSR1-FLI1 (and vice versa). C. Overlapping genes between hMSC_EWSR1-FLI1 and shEWSR1-FLI1 ES cell line models are shown in analogous Venn diagrams. D. MSigDB analysis of genes that were significantly and at least 1.3-fold repressed or induced by EWSR1-FLI1 ectopic expression in HeLa cells shows marked overlap with EWSR1-FLI1 target genes (top). The enrichment plots with the best enrichment score are shown as |NES| (bottom). NES, the normalized enrichment score.