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Purpose: Targeting the protein neddylation pathway has become an attractive anticancer strategy; however, the role of death receptor-mediated extrinsic apoptosis during treatment remained to be determined.Experimental Design: The activation of extrinsic apoptosis and its role in MLN4924 treatment of human esophageal squamous cell carcinoma (ESCC) were evaluated both in vitro and in vivo. The expression of the components of extrinsic apoptotic pathway was determined by immunoblotting analysis and downregulated by siRNA silencing for mechanistic studies.Results: Pharmaceutical or genetic inactivation of neddylation pathway induced death receptor 5 (DR5)-mediated apoptosis and led to the suppression of ESCC in murine models. Mechanistically, neddylation inhibition stabilized activating transcription factor 4 (ATF4), a Cullin-Ring E3 ubiquitin ligases (CRL) substrate. Transcription factor CHOP was subsequently transactivated by ATF4 and further induced the expression of DR5 to activate caspase-8 and induce extrinsic apoptosis. Moreover, the entire neddylation pathway was hyperactivated in ESCC and was negatively associated with patient overall survival.Conclusions: Our findings highlight a critical role of ATF4-CHOP-DR5 axis-mediated extrinsic apoptosis in neddylationtargeted cancer therapy and support the clinical investigation of neddylation inhibitors (e.g., MLN4924) for the treatment of ESCC, a currently treatment-resistant disease with neddylation hyperactivation.

Objectives: To investigate the occupational and genetic risk factors inducing lumbar disc degeneration in a Chinese population, and to explore their synergistic interactions. Methods: A case-control study involving 178 low back pain patients with lumbar disc degeneration and 284 controls was carried out. Five types of work-related factors were investigated using questionnaires. Polymerase chain reaction and restriction fragments length polymorphism was used to detect the polymorphisms of MMP-3 (matrix metalloproteinase-3)(rs731236), VDR-Taq (vitamin D receptor-Taq) and VDR-Apa (vitamin D receptor-Apa)(rs35068180). Rothman's synergy index was used to measure the synergistic interactions between gene polymorphisms and occupational risk factors. Results: Family history of lumbar disc diseases, back injury history, whole-body vibration, bending/twisting, heavy physical workload, age, mutation alleles 5A of MMP-3 and A of VDR-Apa were significantly associated with lumbar disc degeneration (OR=12. 70, 11.79, 8.96, 5.46, 1.05, 1.96 and 1.70, respectively, p<0.05). Synergistic interactions existed between the mutation allele 5A of MMP-3 and wholebody vibration exposure, the mutation allele 5A of MMP-3 and bending/twisting, and the mutation allele A of VDR-Apa and bending/twisting (SI=13.27, 2.91, 2.35, respectively, p<0.05). Conclusions: The results of this study suggest that gene-occupation interaction might play a certain role in exaggerating lumbar disc degeneration. There is a possibility that subjects who carry mutation alleles 5A of MMP-3 and/or A of VDRApa are more vulnerable to lumbar disc degeneration when they are exposed to whole-body vibration and/or bending/twisting under ergonomic loads. It is estimated that 15% to 20% of adults have back pain during a single year, and 50% to 80% experience at least one episode of back pain during their lifetime 1) . Jin 2) reported that the incidence of low-back pain (LBP) was as high as 50% in an investigation of one population of Chinese workers. Lumbar disc degeneration is considered to be a primary cause of LBP 3,4) . The pathogenesis of lumbar disc degeneration includes radial fissures, rim tears in the annulus, and loss of water content in the nucleus pulpous and annulus. These changes are suspected of underlying many back pain symptoms.The etiology of lumbar disc degeneration is complex, and recent research on the causes of disc degeneration have shed light upon a better understanding of the risk factors contributing to disc degeneration. Magora 5) suggested that occupational factors contributing to the acceleration of spinal degeneration included heavy physical loads, manual materials handling consisting of lifting, bending and twisting, prolonged sitting, sustained non-neutral work postures, and vehicle driving. Videman 6) confirmed that occupational exposures are viewed as the primary source of the mechanical factors

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.

Background The neddylation pathway is overactivated in human cancers. Inhibition of neddylation pathway has emerged as an attractive anticancer strategy. The mechanisms underlying neddylation overactivation in cancer remain elusive. MLN4924/Pevonedistat, a first-in-class NEDD8-activating enzyme (NAE, E1) inhibitor, exerts significant anti-tumor effects, but its mutagenic resistance remains unresolved. Methods The expression of NEDD8-conjugating enzyme UBC12/UBE2M (E2) and NEDD8 were estimated by bioinformatics analysis and western blot in human lung cancer cell lines. The malignant phenotypes of lung cancer cells were evaluated both in vitro and in vivo upon UBC12 knockdown. Cell-cycle arrest was evaluated by quantitative proteomic analysis and propidium iodide stain and fluorescence - activated cell sorting (FACS). The growth of MLN4924 - resistant H1299 cells was also evaluated upon UBC12 knockdown. Findings The mRNA level of UBC12 in lung cancer tissues was much higher than that in normal lung tissues, increased with disease deterioration, and positively correlated with NEDD8 expression. Moreover, the overexpression of UBC12 significantly enhanced protein neddylation modification whereas the downregulation of UBC12 reduced neddylation modification of target proteins. Functionally, neddylation inactivation by UBC12 knockdown suppressed the malignant phenotypes of lung cancer cells both in vitro and in vivo . The quantitative proteomic analysis and cell cycle profiling showed that UBC12 knockdown disturbed cell cycle progression by triggering G 2 phase cell-cycle arrest. Further mechanistical studies revealed that UBC12 knockdown inhibited Cullin neddylation, led to the inactivation of CRL E3 ligases and induced the accumulation of tumor-suppressive CRL substrates (p21, p27 and Wee1) to induce cell cycle arrest and suppress the malignant phenotypes of lung cancer cells. Finally, UBC12 knockdown effectively inhibited the growth of MLN4924-resistant lung cancer cells. Interpretation These findings highlight a crucial role of UBC12 in fine-tuned regulation of neddylation activation status and validate UBC12 as an attractive alternative anticancer target against neddylation pathway. Fund Chinese Minister of Science and Technology grant (2016YFA0501800), National Natural Science Foundation of China (Grant Nos. 81401893, 81625018, 81820108022, 81772470, 81572340 and 81602072), Innovation Program of Shanghai Municipal Education Commission (2019-01-07-00-10-E00056), Program of Shanghai Academic/Technology Research Leader (18XD1403800), National Thirteenth Five-Year Science and Technology Major Special Project for New Drug and Development (2017ZX09304001). The funders had no role in study design, data collection, data analysis, interpretation, writing of the...