Dissection of the EWS-FLI1 oncogenic pathway by RNAi

The EWS-FLI1 chimeric gene product in Ewings sarcoma represents the prototype of a family of DNA-binding tumor specific fusion proteins resulting from chromosomal translocations involving TET family genes in human sarcomas and acute leukemia. Available data suggest that these proteins function as transcriptional regulators. In addition, protein interaction studies link the fusion proteins and their intact precursors to RNA processing and to DNA repair. However, the extent to which these activities contribute to the oncogenic properties of the translocation products remains elusive. Functional studies on EWS-FLI1 have so far been impeded by the lack of a relevant model system and by toxicity of the fusion protein in transgenic systems. As a paradigm for TET protein fusions we suggest to study the EWS-FLI1 pathway directly in the Ewings sarcoma cell by post-transcriptionally knocking-down the expression of EWS-FLI1 and its partner proteins hsRPB7, U1C and BARD1, and subsequently of putative up-stream (bFGFR, IGFR) and down-stream (BRCA1, CD99, PDGF-C) pathway components by means of RNA interference (RNAi). We will apply synthetic small interfering RNAs (siRNA) for transient and small hairpin RNAs (shRNA) expressed from RNA Pol III promoter driven expression plasmids for stable gene suppression. The consequences of the depletion from the indicated gene products will be studied and compared on the genotypic and functional level, including gene expression profiling, reporter gene and splicing assays, cell cycle analysis, apoptotic propensity, differentiation potential, and DNA repair capacity. We expect these studies to unravel the complex interrelations within the EWS-FLI1 pathway and to identify essential components and putative therapeutic targets. Since siRNA is discussed as a future smart drug in medicine, our results will contribute to increased knowledge about the therapeutic potential and mechanisms of resistance in the use of RNAi-based treatment strategies.

Ewings sarcoma is the second most frequent malignant bone tumor in children and young adults. It is characterized by a unique chromosomal rearrangement that leads to the fusion of two unrelated genes, EWS and FLI1, to form a chimeric gene with novel properties (EWS-FLI1). The presence of this chromosome aberration is always accompanied by high levels of a protein of unknown function, CD99, on the surface of the tumor cells, suggesting a functional link between these two diagnostic features of the disease. The tissue of origin for Ewings sarcoma is not known. When introduced into normal primary human cell types, EWS-FLI1 kills the cells by a mechanism involving a master regulatory protein of cell fate regulation, p53. Paradoxically, p53 is retained in more than 90% of Ewings sarcomas suggesting the presence of a protection mechanism that renders the tumor cells tolerant to EWS-FLI1. Therefore, the most relevant model for studying the oncogenic properties of the fusion gene remains the Ewings sarcoma cell itself. To assess the importance of the presence of EWS-FLI1 for sustained tumor cell growth, we chose to suppress its expression in 6 Ewings sarcoma cell lines exploiting an evolutionary conserved mechanism of gene regulation by small gene-specific double stranded RNAs, called RNA interference (RNAi). By comparing the profiles of overall gene expression in the presence and absence of EWS-FLI1 we assessed the molecular consequences of the chromosomal rearrangement for the tumor cells. In addition, we adopted a method, chromatin immunoprecipitation (ChIP), which enabled us to isolate genes directly bound by EWS-FLI1 within the tumor cells, in order to identify candidate mediators of its oncogenic function. Using a combination of these approaches we identified and studied in great detail a new mechanism by which EWS-FLI1 keeps p53 in check to allow for sustained Ewings sarcoma cell proliferation. It relies on the suppression of an important pathway in normal development and tissue formation, the NOTCH signalling pathway. Our results suggest that the enigmatic cell of origin for Ewings sarcoma is a resting cell with elevated basal p53 expression and active NOTCH signalling. Further, we identified 99 genes which appeared to be directly regulated by EWS-FLI1. The regulation of one of these genes, MK-STYX, was characterized in detail. In addition, we found that, although influenced by EWS-FLI1, high CD99 expression is a feature of Ewings sarcoma independently required for the pathogenesis of the disease. Down-regulation of CD99 resulted in a loss of the migratory and tumor forming ability of the tumor cells. Applying RNAi to CD99 we were able to identify a gene downstream of this protein and involved in its migration regulatory function. Thus, our study resulted in important new findings about the characteristics of the tissue of origin, the mechanism of growth regulation and metastasis in Ewings sarcoma.