Abstract 1028: Anti-angiogenic activity of MLN4924 suppresses the growth of urothelial carcinoma via enhancing vascular normalization
Authors: Huang Kuo-How , Pu Yeong-Shiau , Tsai Yu-Chieh , Kuo Kuan-Lin , Ho I-Lin , Hsieh Ju-Ton , Shih Tsung-Hsien 
Published: May 2015

Abstract
Introduction:
Cullin-RING ligases (CRLs), the cullin-scaffold RING-finger domain containing E3, needs Nedd8 modification, neddylation, for the complete E3 activity, which promote the ubiquitination of specific substrate's degradation and regulate numerous biological processes. MLN4924, a neddylation inhibitor, was repoted to interfere in the neddylation of cullins and subsequently inactivate CRLs, leading to cell cycle arrest and apoptosis of tumors. Thus, we hereby present the detailed mechanism regarding the anti-angiogenesis of MLN4924 on suppressing the growth of urothelial carcinoma (UC) via inducing vascular normalization.
Materials & methods:
Human umbilical vein endothelial cells (HUVECs) were used to investigate the anti-angiogenic functions of MLN4924 in vitro, including WST-1 assay for evaluating proliferation, Transwell with or without Matrigel-coating for testing invasion and migration, gelatin zymography for assaying the matrix metalloproteinase activity, propidium iodide (PI) staining for investigating cell cycle progression, PI and annexin V co-staining for exploring cell death, Matrigel tube formation for examining angiogenic activity, and Western blotting for checking protein levels in cell cycle, apoptosis, and angiogenesis-associated signaling pathways. Besides, Matrigel plug assay was used to studying angiogenesis in vivo, and UC xenograft model was applied to research the tumor growth and tumor vascularity.
Results:
Our results showed that MLN4924 suppressed HUVEC's proliferation, migration, invasion, MMP 2 activation, and angiogenic tube formation. MLN4924 decreased angiogenic receptor VEGFR 2 and Tie2 expressions and inhibited VEGFR 2, ERK1/2, STAT 1 and 3, and AKT phosphorylations. MLN4924 induced the G2/M cell cycle arrest, which increased p21 and p27 accumulations and p53 phosphorylation but diminished histone 3 phosphorylation. Additionally, MLN4924 enhanced PARP, caspase -3, and -7 cleavages, which caused apoptosis. Furthermore, MLN4924 inhibited VEGF-induced angiogenesis in Matrigel-plug assay and repressed UC growth. CD31 and α-SMA immuno-staining was used for revealing the tumor vascularity, which showed that the microvessel density had no significantly difference between MLN4924-treated tumors and control tumors. However, the higher maturation vasculatures were found in MLN4924-treated tumors than control tumors.
Conclusion:
Our data suggest that MLN4924 inhibits the angiogenic phenotype of endothelial cells for enhancing tumor vascular normalization, which alleviates the hypoxic stress for slowing tumor growth. In addition, due to MLN4924 resulting in vascular normalization, MLN4924 may be able to synergize with conventional chemotherapies for treating UC. Therefore, exploring the function of MLN4924 in angiogenesis may result in a new perspective on targeting tumors and angiogenesis-dependent diseases.
Note: This abstract was not presented at the meeting.
Citation Format: Kuo-How Huang, Yeong-Shiau Pu, Yu-Chieh Tsai, Kuan-Lin Kuo, I-Lin Ho, Ju-Ton Hsieh, Tsung-Hsien Shih. Anti-angiogenic activity of MLN4924 suppresses the growth of urothelial carcinoma via enhancing vascular normalization. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1028. doi:10.1158/1538-7445.AM2014-1028