The potential therapeutic benefit of targeting S-phase kinase-associated protein 2 (SKP2) in neuroblastoma

Abstract

S-phase kinase-associated protein 2 (SKP2) is the substrate recognition subunit of the SCF E3 ubiquitin ligase complex which monitors the G1/S transition of the cell cycle. SKP2 is a positive regulator of cell cycle progression targeting tumour suppressor proteins for degradation, primarily the cyclin-dependent kinase inhibitor p27KIP1. An oncogenic protein, SKP2 is frequently overexpressed in human cancers and contributes to malignant progression. SKP2 has previously been identified as a possible MYCN target gene in neuroblastoma and based on these reports it is hypothesised that SKP2 is a potential therapeutic target in MYCN amplified disease. In this study a positive correlation between MYCN expression and SKP2 mRNA expression was shown in the SHEP-Tet21N MYCN-regulatable cell line and in a panel of MYCN amplified and non-amplified neuroblastoma cell lines. In chromatin immunoprecipitation and reporter gene assays, MYCN bound directly to E-box DNA binding motifs within the SKP2 promoter, and induced transcriptional activity which was decreased by the removal of MYCN and mutation of the E-boxes. SKP2 knockdown induced cell cycle arrest and apoptosis in non-MYCN amplified neuroblastoma cell lines independent of the p53 pathway. The G1 arrest induced was rescued in-part by the knockdown of p27KIP1 confirming the importance of the SKP2/p27 axis in cell cycle progression in neuroblastoma. Structure-activity relationship analysis identified a sub-set of putative SKP2 inhibitors which inhibited growth and suppressed SKP2-mediated p27 degradation in HeLa cells. Additionally, treatment of the MYCN regulatable SHEP-Tet21N cell line with commercially available direct or indirect modulators of SKP2 activity identified a MYCN-dependent sensitivity. In conclusion these data show that SKP2 is a direct transcriptional target of MYCN and suggests that SKP2 is a potential therapeutic target in neuroblastoma.