Alveolar rhabdomyosarcoma (ARMS) is an aggressive soft tissue of the striated muscle lineage that occurs in children and young adults. Chromosomal analyses have identified a typical t(2;13) or variant t(1;13) translocation in this cancer. Molecular genetic studies have shown that these locations juxtapose the PAX3 or PAX7 locus with the FKHR locus to create chimeric genes that encode PAX3-FKHR or PAX7-FKHR fusion products. The wild-type genes encode members of the paired box and fork head transcription factor families, and the fusion products combine the PAX3/7 DNA binding domain and FKHR transactivation domain to create highly potent transcriptional activators. In addition to these functional changes, expression studies have demonstrated overexpression of the fusion products in ARMS cases. Furthermore, experiments in model cell culture systems have provided evidence that the fusion proteins can induce transformation, inhibit myogenic differentiation, and suppress apoptosis. These combined findings support the hypothesis that the translocations generate aberrant transcription factors that, when expressed above a critical threshold, inappropriately regulate transcription of a set of genes with PAX3/7 DNA binding sites and thereby induce oncogenic behavior through multiple phenotypic pathways. The current proposal will further explore this hypothesis and extend these findings by examining the transcriptional interactions of the wild-type and fusion proteins, defining the oncogenic functions of the fusion proteins in ARMS cells, and investigating downstream target genes. Transcriptional studies will examine the competitive and inhibitory effects of wild-type PAX3 and PAX7 on fusion protein transcriptional activity to consider a functional basis for the observed fusion gene overexpression pattern, identify interactions between wild-type and fusion proteins, and prepare functional inhibitors for phenotypic studies. These functional inhibitors as well as antisense expression constructs will be employed to alter the transcriptional function or expression of the fusion proteins in ARMS cells, and thereby investigate in vitro and in vivo oncogenic effects of these fusions in the relevant cellular environment. Finally, downstream target genes whose expression is altered by the PAX3/7-FKHR fusion proteins will be identified by assaying expression of candidate genes in tumor specimens and genetically modified tumor cell lines and employing a differential hybridization strategy to screen for additional candidate target genes.
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