Identification of the target genes of the EWS/NR4A3 fusion protein expressed in extraskeletal myxoid Chondrosarcoma
Extraskeletal myxoid chondrosarcoma (EMC) are soft tissue tumors occurring mainly in the extremities, most commonly the thigh and knee. In approximately 75% of these tumors, at (9;22) chromosome translocation is present. This translocation encodes a fusion protein named EWS/NR4A3 which consists of the amino-terminal domain of the EWS protein fused to the complete amino acid sequence of the NR4A3 nuclear receptor. In my laboratory we have shown that EWS/NR4A3 is a highly potent transcriptional activator, suggesting that one way in which it may contribute to tumorigenesis is by activating specific target genes. There are no cell lines established from EMC tumors, so to generate a human cellular model to study EWS/NR4A3 we have used a human bone marrow mesenchymal stem cell line. This cell line, named hTERT, has retained the ability to differentiate into osteoblasts and chondrocytes, does not form foci in soft agar, has a normal karyotype, has retained inhibition of proliferation by cell-cell contact, and does not induce tumors in nude mice. We have chosen this cell line because an extensive immunohistochemical study of EMC tumors has shown that they most likely consist of primitive mesenchymal cells. We have transfected the cells with an EWS/NR.4A3 expression vector and isolated several cell lines stably expressing EWS/NR4A3. These cell lines display a loss of inhibition of proliferation by cell-cell contact. Since loss of contact inhibition is one characteristic of tumor cells, our hypothesis is that this loss of contact inhibition is due to the activation of specific target genes by EWS/NR4A3 that may also play a role in the development of EMC tumors. To identify these genes, we will perform microarray analyses of the cell lines to identify genes over-expressed in the presence of EWS/NR4A3. We will compare those genes to a list of genes which we have previously found to be specifically over-expressed in EMC tumors expressing EWS/NR4A3. Genes over-expressed in both will be considered potential targets of the fusion protein. Future experiments will involve immunohistochemical analyses of EMC tumors to confirm expression of the corresponding proteins in tumor cells. On the long-term, these proteins could be targeted to treat patients with EMC tumors expressing the fusion protein.