EWS-Fli1 fusion gene as a diagnostic and therapeutic molecule for Ewing's sarcoma and PNET

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The translocation t(11;22)(q24:q12) is a specific chromosomal abnormality detected in Ewing's sarcoma (ES) and Primitive Neuroectodermal Tumor (PNET). The translocation results in an EWS-Fli1 fusion gene, made up of the 5' half of the EWS gene on chromosome 22 fused to the 3' half of the Fli1 gene on chromosome 11. Recent studies have evaluated transforming potentials of the fusion gene products acting as an aberrant transcription factor. However, the biological significance of EWS-Fli1 is still unknown. Using a competitive PCR technique, we have found that there might be a correlation between the expression levels of the EWS-Fli1 fusion gene and the proliferative activities of ES/PNET cells. When the EWS-Fli1 expression was inhibited by an antisense oligonucleotide (AS) against the fusion gene, the growth of the ES/PNET cells was significantly reduced both in vitro and in vivo. The flow cytometry analysis indicated that the growth inhibition of the cells by AS was mediated by G1 arres … More t in the cell cycle progression. In the present study, we examined the effects of AS on the cell cycle regulatory factors. We found that G1 cyclins including cyclin D1 and cyclin E were upregulated by EWS-Fli1, whereas CDK inhibitors, p21 and p27, were downregulated. The luciferase analysis and gel shift assay revealed that EWS-Fli1 directly bound to the Ets consensus sequences in p21 gene promoter and inhibited its activity. Histone deacetylase inhibitors (HDACI) which are known to induce p21 expression in cancer cells inhibited ES/PNET cell growth via induction of p21 expression. The low level expression of p27 in ES/PNET specimen had strong correlation with worse clinical prognosis of the patients. Introduction of p27 expression vector markedly inhibited the growth of ES/PNET cells. We also used cDNA microarray analysis to identify target genes of EWS-Fli1 in G1-S cell cycle regulation. The gene expression analysis showed that 12 genes were significantly up-regulated after the AS treatment, whereas 6 genes including E2F1 were down-regulated by the treatment. Among these possible EWS-Fli1-target genes, we focused on E2F1 since it play central role in the regulation of G1-S transition. Western blot and RT-PCR analyses confirmed that E2F1 expression in ES/PNET cells was significantly inhibited by AS treatment. When E2F-decoy oligonucleotides were transfected into ES/PNET cells, growth of the cells was significantly inhibited to less than 40 % of control and mutant-decoy treated cells. These results suggest that cyclin D1, cyclin E, p21, p27 and E2F1 might be the target genes of EWS-Fli1 chimeric transcription factor. Inhibition of EWS-Fli1 downstream signals might lead to the molecular target therapy for the treatment of ES/PNET patients. Less