Abstract 4239: Ectopic expression of EWS-FLI1 in human neural crest stem cells induces polycomb genes and maintenance of stemness

Abstract

Ewing sarcoma family of tumors (ESFT) are aggressive bone and soft tissue tumors of unknown cellular origin. Most ESFT express EWS-FLI1, a chimeric protein which functions as a growth-promoting oncogene in ESFT but is toxic to most normal cells. A major difficulty in understanding EWS-FLI1 function has been the lack of an adequate model in which to study EWS-FLI1-induced transformation. Although the cell of origin of ESFT remains elusive, both mesenchymal (MSC) and neural crest (NCSC) have been implicated. We recently developed the tools to generate NCSC from human embryonic stem cells (hNCSC). In the current study we have used this model to test the hypothesis that NCSC are the cells of origin of ESFT and to evaluate the consequences of EWS-FLI1 expression on human NCSC biology. ESFT demonstrate variable degrees of neuro-mesenchymal differentiation potential. Similarly, we have found that hNCSC can be induced to differentiate into neural, glial and mesenchymal progeny. Significantly, Affymetrix whole genome expression profiling of 32 primary ESFT, 11 normal adult tissues, bone marrow-derived MSC and hNCSC revealed that ESFT are more similar to hNCSC than any other normal tissue, including MSC, thus further implicating NCSC in the origin of ESFT. To evaluate the consequences of EWS-FLI1 on these multipotent stem cells, hNCSC were stably transduced with an EWS-FLI1 lentivirus. Unlike most normal cells, hNCSC tolerated expression of the oncoprotein. Moreover, EWS-FLI1-transduced hNCSC continued to proliferate and maintain EWS-FLI1 expression in culture for several weeks after transduction. Affymetrix HuEx 1.0 expression profiling of hNCSC cells five days post-transduction with EWS-FLI1 demonstrated the expected induction and repression of well-established EWS-FLI1 targets and identified numerous other novel EWS-FLI1-regulated genes that are likely to be cell-type and situation specific. In contrast to control vector-transduced cells, EWS-FLI1 transduced hNCSC reproducibly maintained expression of the NCSC markers p75 and HNK-1, even after transfer to differentiation-inducing conditions. In addition, expression of the polycomb genes BMI1 and EZH2 was consistently and significantly upregulated in EWS-FLI1-transduced cells. In keeping with persistent expression of these stem cell-associated genes, EWS-FLI1-expressing cells retained the ability to form neurospheres upon transfer to non-adherent conditions and also the ability to differentiate into neural and mesenchymal lineages several weeks following transduction. In contrast, after six weeks in differentiation media, control cells had upregulated p16 and had undergone senescence. Together these data implicate NCSC in the origin of ESFT and suggest that EWS-FLI1 enables malignant transformation by inducing maintenance of their multipotent, stem cell state through deregulation of polycomb genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4239.