The inhibition of differentiation has been considered

The inhibition of differentiation has been considered to play a role in many types of tumor development through maintenance of the proliferating progenitor cell state. Previous studies demonstrated that the knockdown of EWS-FLI1 in Ewing sarcoma cell lines results in osteogenic, adipogenic and chondrogenic differentiation (Tirode et al., 2007). Similarly, in the present study, we found that EWS-FLI1-induced osteosarcomas exhibit robust osteogenic differentiation after the withdrawal of EWS-FLI1 expression, indicating that EWS-FLI1 expression inhibits osteogenic differentiation. Molecular mechanisms by which EWS-FLI1 expression blocks osteogenic differentiation have been proposed in previous studies. It was reported that EWS-FLI1 inhibits osteogenic differentiation in murine multipotent mesenchymal arginase inhibitor by binding to Runx2, an osteogenic transcription factor, and inhibiting its function (Li et al., 2010). Similarly, EWSR1 was shown to interact with SOX9, which is involved in chondrogenic differentiation in zebrafish (Merkes et al., 2015). However, we failed to detect a physical interaction between EWS-FLI1 and Runx2 or Sox9 in our osteosarcoma cells by immunoprecipitation (data not shown), suggesting that another mechanism may exist for the defective differentiation. Notably, Riggi et al. (2014) demonstrated that EWS-FLI1 expression causes the displacement of endogenous ETS transcription factors and p300 at the canonical ETS motifs in Ewing sarcoma cells. We found that EWS-FLI1 binds to the genome through the ETS motif in EWS-FLI1-dependent osteosarcoma cells. Given that the ETS family of transcription factors plays an important role in osteogenic differentiation as well as adipogenic and chondrogenic differentiation (Birsoy et al., 2011; Iwamoto et al., 2007; Raouf and Seth, 2000), the aberrantly occupied ETS motifs by EWS-FLI1 might inhibit ETS family-mediated differentiation, resulting in maintenance of the proliferating progenitor state. The majority of Ewing sarcomas arise in adolescence. Considering the young age at onset, it is suggested that Ewing sarcoma harbors few genetic abnormalities besides the EWS-FLI1 fusion gene. Indeed, recent genome-wide sequencing analyses revealed a paucity of somatic abnormalities (Crompton et al., 2014; Tirode et al., 2014). However, consistent with a number of previous studies, we failed to induce sarcomas by the sole expression of EWS-FLI1 in a variety of cell types in vivo, providing additional evidence that EWS-FLI1 expression is not sufficient for sarcoma development. Thus, we established iPSCs from EWS-FLI1-induced osteosarcoma cells, thereby harboring the same genetic abnormalities as the parental osteosarcoma cells. Interestingly, upon the induction of osteogenic differentiation, EWS-FLI1 expression turned sarcoma iPSC-derived osteogenic cells into sarcoma cells, whereas the expression was not sufficient for the transformation of those from control ESCs/iPSCs. It is noteworthy that sarcoma iPSCs showed an impairment of terminal osteogenic differentiation ability irrespective of EWS-FLI1 expression. Notably, we found that osteogenic lineage cells derived from sarcoma iPSCs exhibit higher proliferating activity compared with cells derived from control ESCs/iPSCs. Taken together, it is conceivable that the additive effect by both EWS-FLI1 expression and the defective differentiation properties of sarcoma iPSCs promotes sarcoma development by suppressing terminal differentiation and maintaining the proliferating progenitor state. The causative aberration of the impaired differentiation properties of sarcoma iPSCs remains unclear. Recently, Lee et al. (2015) established iPSCs from patients with Li-Fraumeni syndrome and demonstrated that mutant p53 causes defective osteoblastic differentiation. However, we failed to detect the Trp53 mutation in our sarcoma-derived iPSCs (Table S2), implying an alternative mechanism impairs osteogenic differentiation. Intriguingly, we observed that sarcoma iPSC teratomas sometimes exhibited impaired terminal differentiation of other lineages, which is also consistent with the fact that they lack the potential to make chimeric mice (Figure S5G). It is likely that a summation of extensive genetic abnormalities and epigenetic alterations is associated with the impaired differentiation of sarcoma iPSCs into multiple lineages. Further analysis is needed to determine the aberrations required for the sarcoma development associated with EWS-FLI1 expression.