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. 2021 Mar 18;19(4):504-517.
doi: 10.20892/j.issn.2095-3941.2020.0484. Online ahead of print.

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

Jingrong Xian #  1   2   3   4 , Shiwen Wang #  1   2   3   4 , Yanyu Jiang  2 , Lihui Li  2 , Lili Cai  2 , Ping Chen  5 , Yue Liu  1   2   3   4 , Xiaofei Zeng  6 , Guoan Chen  6 , Chen Ding  7   8 , Robert M Hoffman  9   10 , Lijun Jia  2 , Hu Zhao  1   3   4 , Yanmei Zhang  1   3   4
Affiliations

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

Jingrong Xian et al. Cancer Biol Med. .

Abstract

Objective: The hyperactivated neddylation pathway plays an important role in tumorigenesis and is emerging as a promising anticancer target. We aimed to study whether NEDD8 (neural precursor cell expressed, developmentally down-regulated 8) might serve 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 and tissue arrays. NEDD8-knockdown ESCC cells generated with the CRISPR/Cas9 system were used to explore the anticancer effects and mechanisms. Quantitative proteomic analysis was used to examine the variations in NEDD8 knockdown-induced biological pathways. The cell cycle and apoptosis were assessed with fluorescence activated cell sorting. A subcutaneous-transplantation mouse tumor 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 associated with poorer overall patient survival (mRNA level: P = 0.028, protein level: P = 0.026, log-rank test). Downregulation of NEDD8 significantly suppressed tumor growth both in vitro and in vivo. Quantitative proteomic analysis revealed that downregulation of NEDD8 induced cell cycle arrest, DNA damage, and apoptosis in ESCC cells. Mechanistic studies demonstrated that NEDD8 knockdown led to the accumulation of cullin-RING E3 ubiquitin ligases (CRLs) substrates through inactivation of CRLs, thus suppressing the malignant phenotype by inducing cell cycle arrest and apoptosis in ESCC. Rescue experiments demonstrated that the 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 NEDD8 as a potential target for ESCC therapy.

Keywords: NEDD8; anticancer target; apoptosis; cullin-RING E3 ubiquitin ligases; esophageal squamous cell carcinoma.

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Conflict of interest statement

No potential conflicts of interest are disclosed.

Figures

Figure 1
Figure 1
Overexpression of NEDD8 is predictive of poor overall survival in patients with ESCC. (A) The mRNA expression level of NEDD8 was higher in esophageal carcinoma than in normal esophageal tissues. N0, N1, N2, and N3 denote nodal metastasis status. (B) The mRNA level of NEDD8 was higher in ESCC and EAC than in normal esophageal tissues. (C) Kaplan-Meier curve analysis was performed to determine overall survival in patients with ESCC according to the NEDD8 mRNA expression data in the TCGA RNA-Seq database. (D) The correlation between NEDD8 and neddylation enzymes (NAE and UBC12) was analyzed in ESCC with the Spearman test. (E, F) A human ESCC tissue array was immunohistochemically stained with antibody specific to NEDD8 (E), and the difference in expression of NEDD8 in the ESCC tissues was calculated according to the histologic evaluation of tumors and adjacent normal tissues (F). Scale bar for 10× images, 500 μm; Scale bar for 200× images, 25 μm. (G) Kaplan-Meier curves based on NEDD8 protein expression in patients with ESCC. **P < 0.01, ***P < 0.001, n.s. = not significant.
Figure 2
Figure 2
NEDD8 knockdown suppresses the malignant phenotype of ESCC cells. (A) Global protein neddylation levels were suppressed in NEDD8-knockdown Kyse450 and EC1 cells. Immunoblotting was used to analyze the global protein neddylation levels after NEDD8 knockdown, with β-actin as a loading control. (B) Cell viability analysis with ATP-Lite assays after NEDD8 knockdown. (C, D) Colony formation in NEDD8-knockdown Kyse450 and EC1 cells, determined by crystal violet staining and counting. Representative images are shown. (E, F) NEDD8-knockdown Kyse450 and EC1 cells were used to determine the migration and invasion abilities, as described in the Materials and methods. Representative images are shown; scale bar = 200 μm. Average values with standard deviations of triplicate experiments are shown. NC, negative control; KD, NEDD8 knockdown; **P < 0.01, ***P < 0.001.
Figure 3
Figure 3
NEDD8 knockdown results in G2 phase cell cycle arrest. (A, B) Gene ontology (GO) analysis based on proteomics analysis was used to determine upregulated processes. (C, D) Downregulated biological processes were shown. (E–G) PI staining and FACS analysis were used to analyze the cell cycle profiles after NEDD8 depletion in Kyse450 and EC1 cells. NEDD8 knockdown induced ESCC cells arrest in G2 phase (presented in blue in E). The percentage of each cell cycle phase is shown in F and G.
Figure 4
Figure 4
NEDD8 knockdown blocks the degradation of CRL substrates. (A) NEDD8 knockdown suppressed neddylation of cullin1, 2, 3, 4A, 4B, and 5 in Kyse450 and EC1 cells. (B) NEDD8 knockdown induced the accumulation of p27, p21, and Wee1, and a decrease in p-H3 in 2 ESCC cell lines. (C, D) The half-lives of p27, p21, and Wee1 were prolonged after NEDD8 knockdown in Kyse450 and EC1 cells. Cells were treated with CHX to block protein synthesis for the indicated times and then subjected to immunoblotting of p27, p21, and Wee1, with β-actin as a loading control. The protein levels of p27, p21, and Wee1 were quantified in comparison with β-actin, through intensity calculations in Image J software. NC, Negative control; KD, NEDD8 knockdown.
Figure 5
Figure 5
NEDD8 knockdown triggers DR5-dependent apoptosis in ESCC cells. (A) The expression levels of the DNA damage response proteins CDT1, ORC1, and phosphorylated/total H2AX in NEDD8-knockdown Kyse450 and EC1 cells were determined by immunoblotting, with β-actin as a loading control. (B) NEDD8 knockdown triggered apoptosis of ESCC cells, as determined by Annexin V–FITC/PI double-staining analysis. (C) NEDD8 knockdown induced accumulation of ATF4, CHOP, DR5, NOXA, cleaved-caspase 8, cleaved-caspase 3, and cleaved-PARP, as assessed by immunoblotting. (D) Downregulation of DR5 rescued apoptotic induction in NEDD8-knockdown Kyse450 cells. NEDD8-knockdown Kyse450 cells transfected with siControl or siDR5 and then subjected to Annexin V–FITC/PI double-staining analysis. (E) NOXA knockdown had no rescue effect on apoptotic induction in NEDD8-knockdown Kyse450 cells. siControl or siNOXA were transfected into Kyse450 cells, which were subjected to Annexin V-FITC/PI double-staining analysis. The cartogram showed no significance between 2 matching groups. (F) Kyse450 cells with siControl or siDR5 were subjected to immunoblotting for cleaved-caspase 8, cleaved-caspase 3, cleaved-PARP, and DR5, with β-actin as a loading control. (G) Kyse450 cells with siControl or siNOXA were subjected to immunoblotting of cleaved-PARP and NOXA, with β-actin as a loading control. Average values with standard deviations of triplicate experiments are shown. NC, negative control, KD, NEDD8 knockdown; *P < 0.05, ***P < 0.001, n.s. = not significant.
Figure 6
Figure 6
NEDD8 knockdown suppresses ESCC tumor growth in vivo. The subcutaneous-transplantation tumor model was established by using NEDD8-knockdown EC1 cells. (A) Tumor size was measured with calipers at the indicated time points and converted to a tumor growth curve. (B) Tumor tissues were harvested and photographed on the day of sacrifice. Scale bar = 1 cm. (C) The tumor weight was measured. (D) Proteins extracted from tumor tissues were analyzed by immunoblotting against cullin2, p27, p21, Wee1, and NEDD8, with β-actin as a loading control. (E) Working model in which NEDD8 knockdown suppresses the tumor growth of ESCC. NC, negative control, KD, NEDD8 knockdown; ***P < 0.001.
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