Effects of increased Myc and Skp2 expression on ubiquitination of p27 in Ewing sarcoma cells

Ewing sarcoma remains partially uncontrollable even after treatment with chemotherapy, surgery, and radiation therapy due to its high malignancy. To explore genes that drive Ewing sarcoma cell proliferation, we conducted a comprehensive analysis of mRNA expression. Based on cDNA array results, we identified consistently elevated expression of Myc and S-phase kinase-associated protein 2 (Skp2) across all five Ewing sarcoma cell lines examined. Myc is a transcription factor linked to various tumor characteristics, including cell proliferation, resistance to apoptosis, and metastatic potential. Skp2 is a ubiquitin ligase that targets proteins with specific modifications. Knockdown (KD) using siRNAs specific for Myc and Skp2 resulted in reduced cell growth and an increased proportion of cells in the G0/G1 phase, indicating G1 arrest. In KD cells, we observed decreased CDK2 activity, increased p27 expression, and reduced expression of cyclin E (CCNE). The increased activity of the CCNE/CDK2 complex led to enhanced phosphorylation of p27 at Thr187, accelerating p27 degradation via Skp2-mediated ubiquitination. Concurrently, the CCNE/CDK2 complex promoted phosphorylation of Rb at Ser807/808, which is involved in E2F1 activation. In Ewing sarcoma cells, it has been clarified that the CCNE/CDK2 complex phosphorylates p27, and Skp2—highly expressed in these cells—facilitates p27 degradation. This mechanism was identified through a comprehensive expression analysis aimed at uncovering the drivers of cell cycle acceleration in Ewing sarcoma. The findings offer new insights into therapeutic strategies for this malignancy, which has seen little progress in treatment over several decades. This discovery holds the potential to transform the current landscape, as no effective molecularly targeted therapies have yet been developed.