The Blockage Of Autophagic Response Enhances The Anticancer Effect Of Neddylation Inhibitor MLN4924 In Liver Cancer

Liver cancer is one of most common malignancies, ranking 6th cancer incidence and 2nd leading cause of cancer death worldwide in 2012. Both incidence and mortality of liver cancer in China account for about 50% of that in the world, and are still on rising. Although the level of diagnosis and treatment of liver cancer have greatly improved, lack of knowledge and targets about the disease hampers the advance of the liver cancer treatment. To date, only one molecular targeted drug has been approved by FDA for liver cancer therapy. The initiation and development of liver cancer is a complicated process, which involves a set of molecular and signal pathway alterations. Thus, it has become a strategy to screen and identify the key molecule and signal pathway for liver cancer research and therapy.As a novel protein post-translational modification, neddylation is a process of adding NEDD8 to substrates in an ubiquitin-like reaction. Through the process, neddylation regulates the conformation, stability and localization of substrates, thus modulates a series of bilogical processes, including cell cycle, proliferation and apoptosis. As a potent inhibitor of neddylation pathway, MLN4924 binds to NEDD8 and forms MLN4924-NEDD8 adduct, which competitively prevents the activity of NAE, leading to the inactivation of neddylation pathway. Due to its potent anticancer effect and well-tolerance, MLN4924 has been advanced in phase â… /â…¡ clinical trial for the treatment of hematological malignancy and solid tumors.In mechanism, MLN4924 elicts anticancer effect through the induction of the accumulation of subsbrates of CRL, which in turn triggers the aberrant regulation of signal pathway, resulting in cell death. Previous studies showed that MLN4924 induces apoptosis in a set of human cancer cells, including liver cancer cells,but it remains elusive how apoptosis was initiated and mediated. Recently, we found that MLN4924 induces the accumulation of Deptor, a substrate of CRL, which subsequently triggers autophagic response through Deptor-mTOR axis. Autophagy serves as a protective role through its anti-apoptotic capability, which may in turn induce drug resistance of liver cancer,as well as attenuating the anticancer efficacy of MLN4924, suggesting blokage of autophagic response may potentiate anticancer effect of MLN4924.To test and verify the hypothesis above, autophagy inhibitors and genetic manipulation were adminstrated to block autophagic flux. And we found that:1) blockage of autophagic response through pharmaceutical chemicals or genetic manipulation enhanced the apoptosis induction and proliferation of MLN4924.2) In vitro, the augment apoptosis induced by chloroquine together with MLN4924 was mediated by NOXA. The combination of MLN4924 with chloroquine induced the accumulation and up-regulation of NOXA, while knockdown of NOXA through siRNA significantly attenuated the apoptosis induction and growth inhibition of MLN4924 combined with chloroquine.3) The up-regulation of NOXA during the combination of MLN4924 with chloroquine was partially due to the accumulation of CRL substrates, including CDT1 and ORC1, which in turn triggered DNA damage and subsequent up-regulation of NOXA. Knockdown of CDT1 and ORC1 can partially rescue NOXA-dependent apoptosis and proliferation inhibition. On the other hand, the combination of MLN4924 with chloroquine induced the formation of high level ROS, which in turn promoted the transactivation of NOXA. The application of ROS scavenger, NAC effectively down-regulated the mRNA of NOXA, meanwhile largely abrogated the apoptosis induction and proliferation inhibition of MLN4924 and chloroquine.4) In vivo, autophagy inhibitor chloroquine enhanced the anticancer effect of MLN4924 in orthotopic xenograft model of liver cancer. Furthermore, the combination of chloroquine with MLN4924 also induced the significant up-regulation of NOXA and c-Caspase 3 in mouse liver cancer tissue through immunohistochemistry, indicating synergistic apoptosis induction, which was consistent to in vitro results.In sum, blockage of autophagic response enhances anticancer effect of MLN4924, which is primarily mediated through NOXA-dependent apoptosis, offering rational support for the clinical application of MLN4924.