Analysis of the mechanisms of the proteolysis of p27 in Ewing's sarcoma and its clinical application.

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The translocation t(11;22)(q24:q12) is a specific chromosomal abnormality detected in Ewing's sarcoma (ES). The translocation results in an EWS-Fli1 fusion gene. 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. We have found that there is a correlation between the expression levels of the EWS-Fli1 fusion gene and the proliferative activities of ES cells, and that EWS-Fli1 inhibited the expression of p21 and p27 whereas induced the expression of cyclin E and D1. Among these target factors involved in the G1/S transition in the cell cycle, p27 showed the strongest correlation with the prognosis of the patients with ES. In the present study, we aimed to investigate the mechanisms by which EWS-Fli1 inhibits the expression of p27 in ES. When the expression of EWS-Fli1 was inhibited by siRNA, the expression of a p27-specific ubiquitin-ligase, Skp2, was also inhibited. The expression level of Skp2 was paralleled with that of EWS-Fli1 in ES cells. EWS-Fli1 enhanced the ubiquitination of p27 protein. The challenge of proteasome inhibitor to ES cells significantly increased the expression of p27 and inhibited the growth of ES cells both in vitro and in vivo. On the other hand, the proteasome inhibitor could not inhibit the growth of the drug-resistant ES cells. The co-treatment with the efflux pump inhibitor and the proteasome inhibitor efficiently inhibited the growth of the drug-resistant ES cells. These results suggest that the EWS-Fli1 might increase the expression of Skp2 resulting in the ubiquitination and degradation of p27 protein in ES cells. The proteasome inhibitor might be a promising reagent for novel molecular targeted therapy for ES.