PO-313 Inhibition of neddylation accelerates VEGF-activated VEGFR2-nitric oxide signalling for resulting in biphasic effects on angiogenesis and tumour growth

Abstract

IntroductionThe neddylation, a process of Nedd8 modification, is important for the complete activity of cullin-scaffold RING-finger E3 ligases (CRLs) which promote proteins’ ubiquitination and degradation. The inhibition of neddylation is reported to interfere with the neddylation of cullins for subsequently inactivating CRLs, leading to cell cycle arrest and apoptosis of tumours. This study investigated the effects of neddylation status on angiogenesis and tumour growth using in vitro and in vivo models.Material and methodsHuman umbilical vein endothelial cells (HUVECs) were used to examine the angiogenic effects of neddylation, including assays in viability, trans-migration, nitric oxide (NO) production, and angiogenic Matrigel tube formation. Further, protein profiles in neddylation, apoptosis, and angiogenesis-associated signalling pathways were verified in HUVECs by Western blotting. Additionally, dominant negative constructs of cullins were applied to investigate each cullin’s neddylation on VEGF-activated VEGFR2 degradation and signalling. Moreover, Matrigel-plug and xenograft tumour models were utilised to study tumour angiogenesis and growth.Results and discussionsNeddylation inhibition delayed vascular endothelial growth factor (VEGF)-activated VEGF receptor 2 (VEGFR2) degradation for sustaining VEGFR2 phosphorylations and its down-stream MAPKs/AKT-eNOS signalling, which accelerated the abnormal increment of NO, a biphasic pro- and anti-angiogenic factor, in HUVECs. Furthermore, neddylation inhibition exhibited biphasic effects on HUVECs’ viability, migration, and angiogenic tube formation. L-NAME, the NO inhibitor, notably blocked the proangiogenic activities and partly restored the anti-angiogenic and apoptotic activities of neddylation inhibition in HUVECs. Furthermore, we evidenced that the neddylation status of cullin 1 rather than other cullins was critically important in VEGF-activated VEGFR2 degradation and phosphorylations. Moreover, neddylation inhibitor displayed biphasic effects on angiogenic Matrigel-plug assay and on tumour angiogenesis and growth in xenograft tumour model.ConclusionNeddylation inhibitor could block the neddylation of cullin 1 through delaying and sustaining VEGF-activated VEGFR2 degradation and signalling, which accelerated the abnormal amount of NO production on mediating the biphasic effects of endothelial angiogenesis and tumour growth. Therefore, exploring the neddylation status in angiogenesis may result in a new perspective for targeting tumours and angiogenesis-dependent diseases.