Synergistic inhibition of autophagy and neddylation pathways as a novel therapeutic approach for targeting liver cancer.

Ping Chen,Chunjie Li,Yongfu Pan,Meng Yang,Ping Zhang,Yiwei Chu,Lijun Jia,Lak Shin Jeong,Robert M Hoffman,Yanan Jiang,Tao Hu,Dongping Wei,Pei Li,Yupei Liang,Hui Qi,Ziming Dong

doi:10.18632/oncotarget.3282

Abstract

Liver cancer is the second-most frequent cause of cancer death in the world and is highly treatment resistant. We reported previously that inhibition of neddylation pathway with specific NAE inhibitor MLN4924, suppressed the malignant phenotypes of liver cancer. However, during the process, MLN4924 induces pro-survival autophagy as a mechanism of drug resistance. Here, we report that blockage of autophagy with clinically-available autophagy inhibitors (e.g. chloroquine) significantly enhanced the efficacy of MLN4924 on liver cancer cells by triggering apoptosis. Mechanistically, chloroquine enhanced MLN4924-induced up-regulation of pro-apoptotic proteins (e.g. NOXA) and down-regulation of anti-apoptotic proteins. Importantly, the down-regulation of NOXA expression via siRNA silencing substantially attenuated apoptosis of liver cancer cells. Further mechanistic studies revealed that blockage of autophagy augmented MLN4924-induced DNA damage and reactive oxygen species (ROS) generation. The elimination of DNA damage or blockage of ROS production significantly reduced the expression of NOXA, and thereby attenuated apoptosis and reduced growth inhibition of liver cancer cells. Moreover, blockage of autophagy enhanced the efficacy of MLN4924 in an orthotopic model of human liver cancer, with induction of NOXA and apoptosis in tumor tissues. These findings provide important preclinical evidence for clinical investigation of synergistic inhibition of neddylation and autophagy in liver cancer.

Highlights

Liver cancer is one of the most common and deadly human malignancies, leading the second cause of cancerrelated death worldwide [1]
We found that autophagy-inhibiting agents sensitized liver cancer cells to MLN4924 both in vitro and in vivo by inducing NOXA-dependent apoptosis
Since MLN4924 treatment induces pro-survival autophagy in cancer cells [20, 29], we reasoned that blockage of this protective autophagic response would enhance the effect of MLN4924 on liver cancer growth

Summary

Introduction

Liver cancer is one of the most common and deadly human malignancies, leading the second cause of cancerrelated death worldwide [1]. Post-translational protein neddylation is a process that adds the ubiquitin-like molecule NEDD8 to substrates and regulates their conformation, stability, localization and function [8,9,10]. This process is catalyzed by a cascade comprising the NEDD8-activating enzyme E1 (NAE), NEDD8-conjugating enzyme E2, and substrate-specific NEDD8-E3 ligases [8,9,10]. NEDD8 conjugation to cullins induces conformational changes and activation of CRL to regulate the turnover of diverse specific CRL substrates with fundamental roles in carcinogenesis and tumor progression. Up-regulation of NEDD8-catalyzing enzymes (E1/E2/E3) and/or global protein neddylation in human cancer further highlights the important role of neddylation in cancer [15,16,17,18,19]

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Conclusion