Identification and functional analysis of EWS-WT1 targets in desmoplastic small round cell tumor

Desmoplastic small round cell tumor (DSRCT) is notable for its histology, in which nests of tumor cells are surrounded by a reactive stroma, and for the recurrent t(11;22)(p13;g12) translocation. This rearrangement fuses the transactivation domain of the Ewing sarcoma gene, EWS, to the DNA-binding domain of the Wilms tumor suppressor gene, WT1. The EWS-WT1 protein is a novel transcription factor, and identification of its target genes is crucial to understanding its role in DSRCT. To identify target genes of EWS-WT1, we examined the expression profiles of cells with regulated EWS-WT1 expression by hybridization to high-density oligonucleotide microarrays. Potential target genes were confirmed by Northern blot, their expression was determined in DSRCT primary tumors, and their functional relevance to DSRCT was explored.; The most strongly induced gene identified was the interleukin-2/15 receptor β-chain (IL-2/15Rβ). IL-2/15Rβ and its other receptor subunits are expressed in neoplastic cells of DSRCT samples, but not in the surrounding stroma. IL-2/15Rβ is directly activated by EWS-WT1, and EWS-WT1 binds the IL-2/15Rβ promoter at two sites required for optimal activity. Two potential ligands are expressed in the stroma of DSRCT, suggesting that the ligands are secreted by the stroma and act upon the receptor in tumor cells. We observed the expression of activated members of the Jak/STAT pathway in DSRCT tumor cells, suggesting that this pathway is activated in this tumor, potentially linking the EWS-WT1 translocation with a transformation pathway.; We also identified MLF1 as an EWS-WT1 target. MLF1 was initially identified due to its involvement in myelodysplastic syndrome and acute myeloid leukemia, in which the NPM gene is translocated to MLF1. MLF1 is expressed in the cytoplasm of DSRCT tumor cells, binds to 14-3-3, and forms cytoplasmic lattices when overexpressed. MLF1 provides cells with resistance to death induced by an apoptotic stimulus, and may have a role in differentiation.; It is evident that expression profiling by oligonucleotide microarrays can be used to identify targets of a chimeric transcription factor. Use of this strategy will allow for a better understanding of the pathways activated by EWS-WT1, enabling the design of more effective therapies for the treatment of DSRCT.