EWS-FLI1 interferes with p53-dependent growth control in Ewing´s sarcoma by suppressing autostimulation of the NOTCH pathway Free

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The EWS-FLI1 fusion oncoprotein is the driving force in the pathogenesis of Ewing´s sarcoma family tumors (ESFT). It acts as an aberrant transcriptional regulator conferring tumorigenic properties to several transfected cell types including bone marrow mesenchymal progenitor cells, and is required for sustained growth of ESFT cells in-vitro and in-vivo. Silencing of EWS-FLI1 expression in ESFT cell lines leads to growth arrest and eventually to cell death or senescence. We have observed transcriptional stimulation of p21^CDKN1A expression in six wildtype p53 ESFT cell lines upon RNAi-mediated EWS-FLI1 silencing. This induction was p53 dependent. Among the genes upregulated by EWS-FLI1 suppression we identified the hairy enhancer of split (HES) family transcription factor gene HEY1, which has recently been shown to positively modulate p53 protein levels in human colon cancer HCT116 cells. In fact, silencing of EWS-FLI1 in ESFT cells resulted in p53 stabilization and Ser15 phosphorylation, which were inhibited by siRNA to HEY1. Since HES transcription factors are downstream effectors of NOTCH signalling, we investigated the expression and the activation status of NOTCH signalling components in ESFT cells before and after EWS-FLI1 knock-down by RNAi. A strong induction of the NOTCH ligand JAG1 and variable increases in the expression of NOTCH receptors 2 and 3 were consistently observed upon silencing of EWS-FLI1. Concomitantly, NOTCH pathway activation by gamma secretase dependent proteolytic receptor cleavage leading to nuclear translocation of the NOTCH intracellular domain (NICD) was detected. Modulation of JAG1 induction by specific siRNA and inhibition of NOTCH processing by either a chemical gamma secretase inhibitor or ectopic expression of the physiologic NOTCH inhibitory docking protein NUMB1 inhibited p53 and p21^CDKN1A induction. These results indicate that suppression of EWS-FLI1 expression reactivates NOTCH signalling in ESFT resulting in p53 dependent cell cycle arrest. Gene expression profiling of 27 primary tumors and 12 cell lines revealed consistent expression of NOTCH2 by ESFT. Since ectopic JAG1 expression sufficed to activate the p53 checkpoint via NOTCH pathway stimulation, we hypothesize that homing of ESFT cells to a NOTCH ligand-expressing compartment like the trabecular bone and the bone marrow may slow-down ESFT growth, possibly providing a molecular basis for dormant disease and late relapse.

Supported by the Austrian Genome Research Programme GEN-AU (grant 200.136/1-VI/1/2005 "GEN-AU-Ch.I.L.D."), the European commission (grant EC-FP6 STREP 503036 “PROTHETS”), and the Austrian Research Fund, grants 16067-B04 and 18046-B12.