Cancer Institute, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, China.
School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, 450001, China.
Cell Commun Signal. 2020 May 12;18(1):72. doi: 10.1186/s12964-020-00576-z.
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.
蛋白 neddylation 在食管癌中发挥促瘤作用。我们之前的研究表明，neddylation 抑制诱导 ATF4 积累，从而促进食管癌细胞凋亡。然而，完全不清楚 neddylation 抑制是否会诱导食管癌细胞自噬，并影响 ATF/CREB 亚家族的其他成员（如 ATF3）的表达。
通过免疫印迹分析确定 neddylation 抑制后 NF-κB/Catalase/ATF3 通路相关蛋白的表达，并通过 siRNA 沉默进行机制研究。用 H2-DCFDA 染色法测定 MLN4924 处理后 ROS 的产生。用 ATPLite 测定法评估 MLN4924 诱导的增殖抑制，用 Annexin V/PI 双染色法评估凋亡。
我们首次报道，Nedd8 激活酶的特异性抑制剂 MLN4924 促进 ATF3 的表达，诱导食管癌自噬。在机制上，MLN4924 抑制了 CRLs 的活性，并诱导其底物 IκBα的积累，从而阻断 NF-κB 的激活和 Catalase 的表达。结果，MLN4924 激活了 ATF3 诱导的保护性自噬，从而抑制了 MLN4924 诱导的凋亡，而 ATF3 沉默可以减轻这种抑制。
在本研究中，我们阐明了 NF-κB/Catalase/ATF3 通路在 MLN4924 诱导的食管癌细胞保护性自噬中的新机制，为下调 ATF3 和 neddylation 抑制剂（如 MLN4924）联合抗 ESCC 治疗提供了合理的依据和分子基础。
