The "Ideal" Target's Network: Factors Modulating EWS-FLI1 Expression and Function in Ewing's Sarcoma

Abstract

EWS-FLI1 is a chimeric transcription factor that results from gene fusion as a consequence of chromosomal translocation exclusively in Ewing's sarcoma family tumors (ESFT). Recent genomic studies have identified a network of EWS-FLI1 regulated genes and functions. These studies mainly relied on experimentally modulating EWS-FLI1 expression in ESFT cell lines under standard in vitro growth conditions (21% oxygene, monolayer culture). In vivo, solid tumors grow as three-dimensional structures and are frequently hypoxic. We find that two-thirds of primary ESFT express hypoxia inducible factor HIF-1α consistent with a hypoxic state of these tumors. When culturing ESFT cell lines at 1% O2, a significant increase in the invasive capacity and anchorage-independent growth of ESFT cells was observed. Interestingly, hypoxia transiently upregulated EWS-FLI1 expression in a HIF-1α-dependent manner on a post-transcriptional level. Importantly, hypoxic growth conditions modulated the EWS-FLI1 transcriptional signature with both antagonistic and agonistic effects. A similar increase in EWS-FLI1 expression was observed upon reactivation of NOTCH signaling in ESFT cells. Studies functionally disabling the RISC complex suggest involvement of micro RNAs (miRNAs) into the post-transcriptional regulation of EWS-FLI1. We identified a miRNA that targets the FLI1 3'UTR. Interestingly, by pan-genomic micro RNA profiling of ESFT cells before and after transient or stable EWS-FLI1 knockdown this miRNA was also found to be suppressed by EWS-FLI1 in a feed-back loop. Together, these studies suggest that microenvironmental factors (i.e. hypoxia, NOTCH signaling) influence EWS-FLI1 expression and target gene regulation, which will have to be considered in the future selection of potential therapeutic targets for ESFT.