PO-165 Effect of notch pathway and Skp2 inhibition in cell viability/proliferation of medulloblastoma cells

Abstract

IntroductionMedulloblastoma (MB) is an embryonic neuroepithelial tumour and important embryonic development pathways, such as WNT, Sonic Hedgehog (SHH) and Notch, are dysregulated on this cancer. The Notch pathway is important to cell differentiation and proliferation and it has a role in initiation and progression of MB regulating downstream effectors. Skp2 gene is a transcriptional target of Notch pathway and it has been associated with protumorigenic properties in some cancers, however, there is no study in MB. Therefore, the aim of this study was to investigate the biological role of Notch/Skp2 axis in MB.Material and methodsNOTCH1, 2, 3 and 4, as well as, their ligands, DLL1, 3 and 4, JAG1 and JAG2, and SKP2 gene expression levels were analysed in MB samples, subdivided in the molecular subgroups, WTN, SHH, group 3 and 4, comparing with cerebellum samples, from public databases (Robinson et al. 2012; Kool et al. 2008; Pfister et al. 2000 and Roth et al. 2006) using R2: Genomics Analysis and Visualisation Platform. NOTCH 1 and SKP2 gene expression level were analysed in Uw402, Uw473, ONS-76 and DAOY MB cell lines. DAOY, ONS-76 and Uw2282 cell lines were submitted to the Notch pathway inhibitor (gamma secretase inhibitor, DAPT), and to the SKP2 inhibitor, SZL-P1-41, at 2, 4, 8, 16 uM concentrations, and after 48 hour the cell viability was analysed. The cell cycle was also analysed after treatment with DAPT in DAOY cell line after 48 hour.Results and discussionsSKP2 was overexpressed in the WNT and SHH when compared to other groups and the same pattern was found for NOTCH2 and DLL3 in the database analysis. All MB cell lines expressed SKP2 and NOTCH1 genes and in the general the SKP2 expression was higher than NOTCH1 in all cell lines. After DAPT treatment the cell viability was reduced in DAOY, ONS-76 and Uw2282 cell lines after 48 hour. In the cell cycle analysis there was a decrease of cells on S-phase after Notch pathway inhibition in the DAOY cell line.ConclusionAs conclusions from these results, we can suggest that the Notch pathway/SKP2 axis activation occur mainly in WNT and SHH MB subgroups through interactions among NOTCH2 receptor, DLL3 ligand and SKP2 and that Notch pathway and SKP2 inhibition cause a decrease in MB cell viability and/or proliferation, with Notch pathway inhibition also decreasing the percentage of cells on S-phase cell cycle which may explain the decrease in cell proliferation. These are preliminary results but may suggest Notch/SKP2 axis as a possible new molecular targeted therapy for MB.