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

// Ping Chen 1, 2, 3, 4 , Tao Hu 2, 4 , Yupei Liang 1 , Yanan Jiang 1, 3 , Yongfu Pan 1, 3 , Chunjie Li 1, 3 , Ping Zhang 1 , Dongping Wei 1 , Pei Li 2, 4 , Lak Shin Jeong 5 , Yiwei Chu 3 , Hui Qi 6, 7 , Meng Yang 6, 7 , Robert M. Hoffman 7, 8 , Ziming Dong 2, 4 , Lijun Jia 1 1 Cancer Institute, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China 2 College of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China 3 Department of Immunology, Shanghai Medical College, Fudan University, Shanghai, China 4 Collaborative Innovation Center of Henan Province for Cancer Chemoprevention, Zhengzhou, China 5 College of Pharmacy, Seoul National University, Seoul, Korea 6 AntiCancer Biotech Beijing Co. Ltd., Beijing, China 7 Anticancer, Inc., San Diego, CA, USA 8 Department of Surgery, University of California, San Diego, CA, USA Correspondence to: Lijun Jia, e-mail: ljjia@fudan.edu.cn Keywords: Neddylation, Autophagy, Apoptosis, MLN4924, Chloroquine Received: December 23, 2014 Accepted: February 05, 2015 Published: March 13, 2015 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.