These studies for the first time demonstrate that, like BM-MSC, neural crest-derived stem cells tolerate EWS-FLI1. Moreover functional studies suggest that the mechanism of oncogene tolerance in these cells is mechanistically linked, at least in part, to up regulation of BMI-1 and epigenetic repression of p16. In addition, gene expression profiling studies reveal a high degree of similarity between ESFT and hNCSC, confirming that activation and maintenance of the NCSC genetic program is an integral feature of ESFT pathogenesis.
Importantly, although tolerance of EWS-FLI1 and up regulation of polycomb proteins was found to be universal in all hNCSC-derived populations, some EWS-FLI1+ cells differentially avoided cellular senescence. These senescence-avoiding cells retained the ability to form neurospheres in non-adherent culture but did not form colonies in soft-agar nor subcutaneous tumors in immune deficient mice (not shown) again demonstrating that like primary human BM-MSC 
, other epigenetic and/or genetic events are necessary to achieve full malignant transformation of human neural crest cells. Technical limitations currently preclude expansion and study of EWS-FLI1 transduced hNCSC at the level of single cells. Therefore, although known to be of neural crest origin, the precise nature of the derivative cells that avoided senescence downstream of EWS-FLI1 activation remains unknown. Nevertheless, gene expression and functional studies support a neuro-mesenchymal stem cell phenotype.
EWS-FLI1 induces cell cycle arrest and death in primary human fibroblasts 
, and initiation of malignant transformation in human BM-MSC 
. This diversity in functional outcome is reflected in a marked difference in EWS-ETS target genes in disparate cell types 
, a difference which is again highlighted by our current study. Importantly, differences in different cellular contexts are particularly notable when comparing stem cells and established ESFT cell lines. These findings suggest that EWS-FLI1 targets are dynamic and may change during the transition from tumor initiation to tumor maintenance. Continued evolution of transcription factor targets is fundamental to cMYC-induced tumor progression 
and is no doubt also necessary for successful EWS-FLI1-mediated malignant transformation of primary cells to ESFT.
The mechanisms of EWS-FLI1-mediated gene repression are not yet clearly understood but are likely to be indirect 
. In normal stem cells, epigenetic repression of developmental genes is largely regulated by polycomb proteins which act together in multi-factorial complexes to modify histones, altering chromatin structure and inhibiting transcriptional activation 
. Deregulation of polycomb genes is pervasive in human cancer 
and over-expression of BMI-1 and EZH2 contribute to the tumorigenicity of established ESFT cells 
. In the current study we have shown that EWS-FLI1 activation leads to a rapid and profound up regulation of BMI-1 protein in hNC-MSC. Our observation that protein induction is more profound than transcriptional induction is consistent with there being a post-transcriptional component to BMI-1 regulation in these cells. Studies in glioma have shown that miRNA128 regulates BMI-1 mRNA and protein expression by directly targeting the 3′-UTR of BMI-1 mRNA 
. Whether miRNA128 contributes to BMI-1 regulation in ESFT remains to be elucidated. In addition, it has been proposed that in normal stem cells interactive feedback loops exist between PRC1 and PRC2 polycomb group complexes and that these feedback loops lead to altered translation and stability of individual PRC proteins, thereby affecting stem cell function 
. EWS-FLI1 induces EZH2
and we have found that knockdown of BMI-1 in neural crest cells also results in down-regulation of EZH2 (). Further studies are needed to establish the mechanism of BMI-1 upregulation ESFT and its relationship, if any, to EZH2
In contrast to BMI-1
is a known direct transcriptional target of EWS-FLI1 
and is one of 46 genes induced by EWS-FLI1 irrespective of cellular context (). Moreover, JARID2
, a recently characterized member of the Jumonji family that complexes with EZH2 to regulate polycomb-mediated gene repression in stem cells 
, was induced by EWS-FLI1 in both hNC-MSC and BM-MSC 
. Together these findings suggest that abnormal induction of polycomb proteins and subsequent deregulation of polycomb target gene expression might be critical early events in EWS-FLI1-induced transformation.
Successful transformation of primary human cells requires that innate tumor suppressor pathways be repressed. In some experimental models, oncogene-induced transformation has been shown to be dependent on BMI-1-mediated epigenetic repression of the p16/ARF-encoding CDKN2A 
. In addition, epigenetic silencing of p16 is an early event in the transformation of primary human mammary epithelial cells 
. Our data suggest that epigenetic repression of p16 by BMI-1 may also be an early initiating event in EWS-FLI1-induced malignant transformation. This is in contrast to other studies from our lab which showed that, in established ESFT, BMI-1 contributes to tumorigenicity by means that are largely independent of p16 repression 
. Thus, as discussed above in reference to EWS-FLI1, disparate molecular mechanisms may contribute to BMI-1's function as an oncogene during tumor initiation and tumor maintenance and we hypothesize that BMI-1 targets change and evolve during ESFT initiation and progression. Further studies are now required to define and compare BMI-1-bound promoters in normal stem cells, their downstream progeny and established tumors.
In summary, we have used a novel model of hESC-derived NCSC to investigate EWS-FLI1 biology and the cellular origins of ESFT. Our findings reveal that ESFT are genetically highly related to NCSC. In addition, neural crest-derived stem cells are permissive for EWS-FLI1 expression and susceptible to oncogene-induced immortalization, at least in part as a result of aberrant up regulation of BMI-1 and epigenetic repression of p16. Together these data support the hypothesis that at least some ESFT might arise from malignant transformation of neural crest-derived stem cells.