pevonedistat and Esophageal-Neoplasms

Inhibition of the protein neddylation process by the small-molecule inhibitor MLN4924 has been recently indicated as a promising direction for cancer treatment. However, the knowledge of all biological consequences of MLN4924 for cancer cells is still incomplete. Here, we report that MLN4924 inhibits tumor necrosis factor-alpha (TNF-α)-induced matrix metalloproteinase 9 (MMP9)-driven cell migration. Using real-time polymerase chain reaction (PCR) and gelatin zymography, we found that MLN4924 inhibited expression and activity of MMP9 at the messenger RNA (mRNA) and protein levels in both resting cells and cells stimulated with TNF-α, and this inhibition was closely related to impaired cell migration. We also revealed that MLN4924, similar to TNF-α, induced phosphorylation of inhibitor of nuclear factor kappa B-alpha (IκB-α). However, contrary to TNF-α, MLN4924 did not induce IκB-α degradation in treated cells. In coimmunoprecipitation experiments, nuclear IκB-α which formed complexes with nuclear factor kappa B p65 subunit (NFκB/p65) was found to be highly phosphorylated at Ser32 in the cells treated with MLN4924, but not in the cells treated with TNF-α alone. Moreover, in the presence of MLN4924, nuclear NFκB/p65 complexes were found to be enriched in c-Jun and cyclin dependent kinase inhibitor 1 A (CDKN1A/p21) proteins. In these cells, NFκB/p65 was unable to bind to the MMP9 gene promoter, which was confirmed by the chromatin immunoprecipitation (ChIP) assay. Taken together, our findings identified MLN4924 as a suppressor of TNF-α-induced MMP9-driven cell migration in esophageal squamous cell carcinoma (ESCC), likely acting by affecting the nuclear ubiquitin-proteasome system that governs NFκB/p65 complex formation and its DNA binding activity in regard to the MMP9 promoter, suggesting that inhibition of neddylation might be a new therapeutic strategy to prevent invasion/metastasis in ESCC patients.

Topics: Cell Line, Tumor; Cell Movement; Cyclopentanes; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 9; NEDD8 Protein; Neoplasm Invasiveness; NF-KappaB Inhibitor alpha; Phosphorylation; Promoter Regions, Genetic; Protein Processing, Post-Translational; Pyrimidines; Transcription Factor RelA; Tumor Necrosis Factor-alpha; Ubiquitin-Activating Enzymes

Protein neddylation plays a tumor-promoting role in esophageal cancer. Our previous study demonstrated that neddylation inhibition induced the accumulation of ATF4 to promote apoptosis in esophageal cancer cells. However, it is completely unknown whether neddylation inhibition could induce autophagy in esophageal cancer cells and affect the expression of other members of ATF/CREB subfamily, such as ATF3.. The expression of relevant proteins of NF-κB/Catalase/ATF3 pathway after neddylation inhibition was determined by immunoblotting analysis and downregulated by siRNA silencing for mechanistic studies. ROS generation upon MLN4924 treatment was determined by H2-DCFDA staining. The proliferation inhibition induced by MLN4924 was evaluated by ATPLite assay and apoptosis was evaluated by Annexin V /PI double staining.. For the first time, we reported that MLN4924, a specific inhibitor of Nedd8-activating enzyme, promoted the expression of ATF3 to induce autophagy in esophageal cancer. Mechanistically, MLN4924 inhibited the activity of CRLs and induced the accumulation of its substrate IκBα to block NF-κB activation and Catalase expression. As a result, MLN4924 activated ATF3-induced protective autophagy, thereby inhibiting MLN4924-induced apoptosis, which could be alleviated by ATF3 silencing.. In our study, we elucidates a novel mechanism of NF-κB/Catalase/ATF3 pathway in MLN4924-induced protective autophagy in esophageal cancer cells, which provides a sound rationale and molecular basis for combinational anti-ESCC therapy with knockdown ATF3 and neddylation inhibitor (e.g. MLN4924). Video abstract.

Topics: Activating Transcription Factor 3; Apoptosis; Autophagy; Catalase; Cell Line, Tumor; Cyclopentanes; Enzyme Inhibitors; Esophageal Neoplasms; Humans; NF-kappa B; Pyrimidines; Signal Transduction; Ubiquitin-Activating Enzymes

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.. 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.. 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.. Our findings highlight a critical role of ATF4-CHOP-DR5 axis-mediated extrinsic apoptosis in neddylation-targeted 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. Clin Cancer Res; 22(16); 4145-57. ©2016 AACR.

Topics: Activating Transcription Factor 4; Animals; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Biomarkers; Caspase 8; Cell Line, Tumor; Cyclopentanes; Disease Models, Animal; Esophageal Neoplasms; Gene Silencing; Humans; Mice; Models, Biological; Prognosis; Pyrimidines; Receptors, TNF-Related Apoptosis-Inducing Ligand; RNA Interference; Signal Transduction; Transcription Factor CHOP; Xenograft Model Antitumor Assays