Overexpressed NEDD8 as a potential therapeutic target in esophageal squamous cell carcinoma

Objective: The hyperactivated neddylation pathway plays an important role in tumorigenesis and is emerging as a promisinganticancer target. We aimed to study whether NEDD8 (neural precursor cell expressed, developmentally down-regulated 8) mightserve as a therapeutic target in esophageal squamous cell carcinoma (ESCC). Methods: The clinical relevance of NEDD8 expression was evaluated by using The Cancer Genome Atlas (TCGA) database andtissue arrays. NEDD8-knockdown ESCC cells generated with the CRISPR/Cas9 system were used to explore the anticancer effectsand mechanisms. Quantitative proteomic analysis was used to examine the variations in NEDD8 knockdown-induced biologicalpathways. The cell cycle and apoptosis were assessed with fluorescence activated cell sorting. A subcutaneous-transplantation mousetumor model was established to investigate the anticancer potential of NEDD8 silencing in vivo. Results: NEDD8 was upregulated at both the mRNA and protein expression levels in ESCC, and NEDD8 overexpression was associatedwith poorer overall patient survival (mRNA level: P = 0.028, protein level: P = 0.026, log-rank test). Downregulation of NEDD8significantly suppressed tumor growth both in vitro and in vivo. Quantitative proteomic analysis revealed that downregulationof NEDD8 induced cell cycle arrest, DNA damage, and apoptosis in ESCC cells. Mechanistic studies demonstrated that NEDD8knockdown led to the accumulation of cullin-RING E3 ubiquitin ligases (CRLs) substrates through inactivation of CRLs, thussuppressing the malignant phenotype by inducing cell cycle arrest and apoptosis in ESCC. Rescue experiments demonstrated thatthe induction of apoptosis after NEDD8 silencing was attenuated by DR5 knockdown. Conclusions: Our study elucidated the anti-ESCC effects and underlying mechanisms of NEDD8 knockdown, and validated NEDD8as a potential target for ESCC therapy.