Validation of NEDD8-conjugating enzyme UBC12 as a new therapeutic target in lung cancer

Abstract

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 G₂ 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 report.

Keywords: Anticancer target; Cell-cycle arrest; Lung cancer; Overcome drug resistance; UBC12.

Fig. 1

Overexpressed UBC12 correlated with global neddylation and predicted poor survival in lung cancer. a) UBC12 mRNA was lower in normal lung tissues as compared to tumors (p < .01, Hou's data). b) UBC12 mRNA was higher in poor differentiated tumor (p < .01, Shedden's data). (ADC, lung adenocarcinoma; LCC, large cell lung cancer; SCC, squamous cell lung cancer; Mod, Moderate). c) Kaplan–Meier curve was analyzed for overall survival rate of patients with lung adenocarcinoma according to the mRNA expression level of UBC12 (Shedden's data). d) and e) Kaplan–Meier curves were analyzed for overall survival rate of patients with lung adenocarcinomas (TCGA data) according to the mRNA expression level of NEDD8 (d) and UBC12 (e), respectively (p = .022 for NEDD8; p = .045 for UBC12, log-rank test). f) Correlation between neddylation enzymes and global protein neddylation was analyzed in lung cancer (Pearson chi-square test, TCGA data).

Fig. 2

Direct regulation of protein neddylation modification by UBC12. a) Immunoblotting was used to determine the expression levels of neddylation activating enzyme (NAE1 and UBA3), neddylation conjugating enzyme (UBC12/UBE2M) and the global protein neddylation. b) The expression levels of neddylation enzymes were further quantified by densitometric analysis using Image J software. The correlation was analyzed by GraphPad Prism5.0 software. c) Immunoblotting was used to analyze the neddylation levels of Cullin 1, 2, 3, 4a, 4b, 5 and global protein neddylation upon UBC12 overexpression in lung cancer cells PC9 and HCC827. d) Immunoblotting was used to analyze the neddylation levels of Cullin 1, 2, 3, 4a, 4b, 5 and global protein neddylation upon knocking down UBC12 in lung cancer cells A549 and H1299.

Fig. 3

Down regulation of UBC12 significantly suppressed the malignant phenotypes of lung cancer cells. a) Lung cancer cells were infected with lenti-guide-puro-control or lenti-guide-puro-UBC12, and subjected to cell proliferation analysis by ATPlite assay. The results were presented as mean value ± S.E. from three independent experiments (two-sided t-test). b and c) UBC12 knockdown reduced cologenic survival in H1299 and A549 cells (two-sided t-test). d and e) UBC12 knockdown inhibited the transwell ability in H1299 and A549 cells. These data are representative results of three independent experiments with similar trends (scale bar = 200 μm). Data represent means, and error bars are standard deviation (two-sided t-test).

Fig. 4

UBC12 knockdown induced G₂ cell-cycle arrest in lung cancer cells. a) A quantitative proteomic strategy based on mass spectra was used to identify the up- and down-regulated proteins upon UBC12 knockdown in A549 cells. KEGG enrichment analysis was used to reveal the changed biological pathways. b) 14 proteins involving in cell cycle were mapped the sub protein-protein interaction network. c) Lung cancer cells were infected with lenti-guide-puro-control or lenti-guide-puro-UBC12, and subjected to PI staining and FACS analysis. The percentage of cells at the G₂ -M phase and sub-G₁ phase was indicated. d) UBC12 knocking down decreased the expression levels of p-H3, but induced the accumulation of p21, p27 and Wee1. A549 and H1299 cells were infected with lenti-guide-puro-control or lenti-guide-puro-UBC12 and subjected to immunoblotting analysis for p21, p27, Wee1 and p-H3 with β-actin as a loading control. e) and f) Half-life of p21, p27 and Wee1 was extended upon down regulation of UBC12. CHX was used to block protein synthesis, and protein lysates were extracted and subjected to immunoblotting against p21, p27 and Wee1 in A549 and H1299 cells (e). The protein levels were quantified by densitometric analysis with Image J software (f).

Fig. 5

Targeting UBC12 suppressed cell growth and induced CRL substrates accumulation in MLN4924-resistant cells. a) IC50 of MLN4924-resistant H1299 cells (H1299-MR) increased to 2500 nM. Wild type H1299 (H1299-wt) cells were cultured in the media containing low dose of MLN4924 (330 nM) for 6 months. 1500 H1299-MR and H1299-wt cells were seeded in 96-well plates in triplicates and then treated with indicated dose of MLN4924 for 72 h and then lysed for ATPlite assay. b) H1299-wt and H1299-MR cells were treated with MLN4924 at the concentration of 1000 nM. Protein was extracted and subjected to immunoblotting against Cullin1–5 and CRLs substrates. c–e) UBC12 knocking down suppressed the viability of H1299-MR cells. H1299-MR cells were infected with lenti-guide-puro-control or lenti-guide-puro-UBC12. 1500 cells were seeded in 96-well plates in triplicates and cultured for 72 h and then lysed for ATPlite assay (c and d) (scale bar = 50 μm, two-sided t-test). 400 cells were seeded in 6 cm plates in triplicates and cultured for 10 days and then stained with crystal violet (e). f) UBC12 knocking down suppressed neddylation of Cullin1, Cullin 2, Cullin3, Cullin4a, Cullin4b, induced accumulation of CRLs substrates in H1299-MR cells. H1299-MR cells were infected with lenti-guide-puro-control or lenti-guide-puro-UBC12. Protein was extracted and subjected to immunoblotting against Cul1–5 and CRLs substrates. (MR, MLN4924 resistant). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Downregulation of UBC12 suppressed tumor growth and metastasis in vivo in nude mice. a–d) UBC12 knockdown inhibited tumor formation and growth in an A549 experimental subcutaneous xenograft tumor model in nude mice. 2*10⁶ A549 cells infected with lenti-guide-puro-control or lenti-guide-puro-UBC12 were injected subcutaneously and subjected to tumor growth analysis. Mice were sacrificed and photographed at 35th day after treatment (the end of study, n = 10) (a). Tumor tissues of mice were collected, photographed (b, <0.01 and c scale bar = 1 cm), and weighed (d; p < .05). e–f) Efficacy of silencing UBC12 on A549-GFP experimental lung metastasis. UBC12 knockdown inhibited tumor formation and growth in an experimental metastatic model in nude mice. 2*10⁶ A549 cells infected with lenti-guide-puro-control or lenti-guide-puro-UBC12 were injected through caudal vein and subjected to tumor growth analysis. Mice were sacrificed and photographed at 35th day after treatment (the end of study, n = 10). e) Mice were sacrificed at the end of study, and lung tissues were collected and imaged with a fluorescence imaging system. A representative lung per group was shown. Tumor nodules were indicated with arrows (scale bar = 2 mm). f) The number of tumor nodules on lung surfaces emitting green fluorescence was calculated (n = 10; 10 entire lungs from 10 mice per group) (two-sided t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)