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. 2017 Feb 15;23(4):1104-1116.
doi: 10.1158/1078-0432.CCR-16-1585. Epub 2016 Sep 2.

Neddylation E2 UBE2F Promotes the Survival of Lung Cancer Cells by Activating CRL5 to Degrade NOXA via the K11 Linkage

Weihua Zhou  1 , Jie Xu  1 , Haomin Li  2   3 , Ming Xu  4 , Zhijian J Chen  4   5 , Wenyi Wei  6 , Zhenqiang Pan  7 , Yi Sun  8   2   9
Affiliations

Neddylation E2 UBE2F Promotes the Survival of Lung Cancer Cells by Activating CRL5 to Degrade NOXA via the K11 Linkage (VSports app下载)

Weihua Zhou et al. Clin Cancer Res. .

Abstract

Purpose: Recent studies have shown that the process of protein neddylation was abnormally activated in several human cancers. However, it is unknown whether and how UBE2F, a less characterized neddylation E2, regulates lung cancer cell survival, and whether and how NOXA, a proapoptotic protein, is ubiquitylated and degraded by which E3 and via which ubiquitin linkage. Experimental Design: Methods of immunohistochemistry and immunoblotting were utilized to examine UBE2F protein expression. The biological functions of UBE2F were evaluated by in vitro cell culture and in vivo xenograft models. The in vivo complex formation among UBE2F-SAG-CUL5-NOXA was measured by a pulldown assay. Polyubiquitylation of NOXA was evaluated by in vivo and in vitro ubiquitylation assays. Results: UBE2F is overexpressed in non-small cell lung cancer (NSCLC) and predicts poor patient survival. While UBE2F overexpression promotes lung cancer growth both in vitro and in vivo, UBE2F knockdown selectively inhibits tumor growth. By promoting CUL5 neddylation, UBE2F/SAG/CUL5 tri-complex activates CRL5 (Cullin-RING-ligase-5) to ubiquitylate NOXA via a novel K11, but not K48, linkage for targeted proteasomal degradation VSports手机版. CRL5 inactivation or forced expression of K11R ubiquitin mutant caused NOXA accumulation to induce apoptosis, which is rescued by NOXA knockdown. Notably, NOXA knockdown rescues the UBE2F silencing effect, indicating a causal role of NOXA in this process. In lung cancer tissues, high levels of UBE2F and CUL5 correlate with a low level of NOXA and poor patient survival. Conclusions: By ubiquitylating and degrading NOXA through activating CRL5, UBE2F selectively promotes lung cancer cell survival and could, therefore, serve as a novel cancer target. Clin Cancer Res; 23(4); 1104-16. ©2016 AACR. .

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Conflict of interest statement

Conflict of interest: All the authors declare no conflict of interest.

Figures

Figure 1
Figure 1. UBE2F high expression in human lung tumor tissues associates with poor patient survival and UBE2F overexpression promotes the growth of lung cancer cells both in vitro and in vivo
(A) UBE2F staining in lung tumor tissues: LUAD (lung adenocarcinoma) and LUSC (lung squamous carcinoma) tissue microarrays were stained by using Abs against UBE2F (Abcam). (B-D) Kaplan-Meier curves to show association between the levels of UBE2F expression and overall survival of patients with NSCLC (B), LUAD (C), and LUSC (D). (E) Expression of UBE2F: indicated plasmids were transfected into H358 cells individually, followed by G418 selection for 2 weeks. The stable clones were pooled and subjected to IB. (F&G) Effects of UBE2F on cell growth and survival: H358 stable clones were seeded into 96-well plates, followed by ATPlite assay after incubation for various time points (F); or seeded into 60-mm dishes at 800 cells per dish in duplicate, and incubated at 37°C for 14 days, followed by 0.05% methylene blue staining and colony counting (G). Shown is mean ± SD from three independent experiments (*, p<0.05; **, p<0.01); (H&I) Effects of UBE2F on tumor growth in xenograft models: 5 × 106 of H358 stable clones were inoculated subcutaneously in both flanks of nude mice, with 5 mice in each group. The tumor growth was monitored up to 60 days and growth curve plotted (H). Tumor tissues were weighed and photographed at 60 days (I). Student's t test was used to compare each experimental group with the control group. Shown are mean ± SEM, *, p<0.05; **, p<0.01; (J&K) IHC staining of mouse tumor tissues: tumor tissues were fixed, sectioned, and stained. Scale bars: 100 μM. Positively stained cells were counted out of a total of 500 cells on average from 3 independent tumors derived from 3 mice per group. Shown are mean ± SD, *, p<0.05; **, p<0.01.
Figure 2
Figure 2. UBE2F silencing selectively inhibits growth of lung cancer cells by inducing apoptosis
(A) Cell growth assay: after UBE2F siRNA silencing, cells were seeded into 96-well plates at 3,000 per well in triplicate and measured by ATPlite assay over periods up to 120 hrs. Shown is mean ± SD from three experiments (*, p<0.05); (B&C) Clonogenic survival assay. Cells after UBE2F siRNA silencing were seeded into 60-mm dishes at 800 cells (C; H358) or 600 cells (D; A427) per dish in duplicate, and incubated at 37°C for 14 days, followed by 0.05% methylene blue staining and colony counting. Results are representative of three independent experiments (mean ± SD; **, p<0.01); (D&E) UBE2F knockdown induces apoptosis in lung cancer cells: cells were transfected with si-Cont and si-UBE2F for 48 hrs, followed by IB (E) and DNA fragmentation (F). Results are representative of three independent experiments.
Figure 3
Figure 3. UBE2F negatively regulates NOXA level in lung cancer cells
(A) NOXA is increased upon silencing of CRL5 components. H358 cells were transfected with indicated siRNAs for 48 hrs. Cells were then harvested for IB. (B-E) Regulation of Cullins, known CUL5 substrates, and NOXA by UBE2F: H358 stable clones (B&D), expressing wild-type or enzymatic dead UBE2F mutant, along with pcDNA control, were harvested for IB. H358 (C&E) cells were transfected with si-Cont or si-UBE2F-1/-2 and harvested 48 hrs later for IB. (F-I) Measurement of NOXA T1/2: lung cancer cells were either transfected with indicated plasmids (F&G) or siRNAs (H&I). Cells were then switched to fresh medium (10% FBS) containing cycloheximide (CHX) and incubated for indicated time periods before being harvested for IB (F&H). The band density was quantified using ImageJ software and plotted (G&I).
Figure 4
Figure 4. CRL5 facilitates poly-ubiquitylation of NOXA via the K11-linkage
(A) UBE2F, CUL5, SAG, and NOXA bind to each other. H358 cells were transfected with FLAG-CUL5 or FLAG-CUL1, and lysates were immunoprecipitated using FLAG-tagged beads or IgG control, followed by IB to detect endogenous proteins as indicated. The 10% of the extracts used for the input. (B) UBE2F/SAG specifically promotes CUL5 neddylation using an in vitro neddylation assay. APP-BP1/UBA3, NEDD8, co-purified full-length-SAG/truncated-CUL5 (398–780) proteins, and UBE2F or UBE2M, were incubated in a reaction mixture with or without addition of MLN4924 for 10 min at room temperature, followed by IB by using CUL5 Ab. (C&D) CRL5 promotes poly-ubiquitylation of NOXA via the K11-linked chain using an in vivo ubiquitylation assay. 293 cells were co-transfected with pcDNA3, FLAG-NOXA (substrate), E3 complex (FLAG-CUL5; FLAG-SAG; HA-UBE2F), and wild type His-HA-tagged ubiquitin or various ubiquitin mutants, as indicated. Whole cell extracts and Ni-NTA affinity purified fractions were analyzed by IB with anti-NOXA antibody (Mouse; Millipore). *: non-specific band. (E&F) CRL5 promotes poly-ubiquitylation of NOXA via K11-linked chain, using an in vitro ubiquitylation assay. The 293 cells were co-transfected with FLAG-CUL5 and FLAG-SAG (F-CUL5/-SAG), or FLAG-CUL1 and -RBX1 (F-CUL1/-RBX1). Cells were then collected 48 hrs post transfection and lysed, followed by pull-down with FLAG-tagged beads. FLAG-tagged CUL5/SAG or CUL1/RBX1, which serve as the E3 complex, were then incubated in a reaction mixture containing ATP, ubiquitin, E1, E2 (UBE2S and UBCH10), and substrate (purified NOXA protein), followed by ubiquitylation assay. *: non-specific band.
Figure 5
Figure 5. NOXA is selectively accumulated and responsible for the apoptotic phenotype in tetracycline-induced U2OS-shUb-Ub (K11R) cells
(A) NOXA is specifically accumulated in the cells with tetracycline-induced replacement of endogenous Ub with Ub (K11R): U2OS-shUb-Ub (WT), -Ub (K11R), -Ub (K48R), and -Ub (K63R) cells were treated with or without tetracycline (1 mg/mL) for 4 days before cell lysates were prepared for IB. The ratio of the intensity of the bands corresponded to NOXA, p21, p27, Bim and β-actin, were shown in the bottom. Results are representative of three different experiments. (B&C) Enhanced apoptosis was detected in the U2OS-shUb-Ub (K11R) cells upon tetracycline treatments. Cells were seeded in duplicate and treated with or without tetracycline (1 mg/mL) for 4 days and then collected for IB (B) and DNA fragmentation assay (C). Results are representative of three different experiments. (D-F) NOXA knockdown reverses the growth suppression and apoptotic induction in U2OS-shUb-Ub (K11R) cells. U2OS-shUb-Ub (K11R) cells were seeded in duplicate and treated with or without tetracycline (1 mg/mL) for 3 days, and then transfected with LT-Cont and LT-NOXA, followed by ATPlite assay 72 hrs later (D), IB (E) and DNA fragmentation assay (F). (G-I) NOXA knockdown rescues apoptotic phenotypes induced by UBE2F silencing. H358 cells were transfected with si-Cont and si-UBE2F first, and then with LT-Cont and LT-NOXA, and incubated for 72 hrs. Cells were split and seeded for ATPlite assay (G), clonogenic assay (H), and IB (I). Shown are mean ± SD; **, p<0.01. Results are all representative of three different experiments.
Figure 6
Figure 6. Expression of UBE2F, CUL5, and NOXA in lung cancer tissues and their association with patient survival
(A) UBE2F, CUL5, and NOXA staining in lung tumor tissues: LUAD (lung adenocarcinoma) and LUSC (lung squamous carcinoma) tissue microarrays were stained by using Abs against UBE2F (Abcam), CUL5 (Sata cruz) and NOXA (Millipore). (B&C) Kaplan-Meier estimation of overall survival between the levels of CUL5 (E), NOXA (F) and overall survival of patients with high UBE2F expression. (D) A working model. UBE2M-RBX1 neddylates CUL1-4 to activate CRL1-4 for substrate ubiquitylation via the K48-linkage, followed by degradation, while UBE2F-SAG neddylates CUL5 to activate CRL5 for NOXA ubiquitylation via the K11-linkage and subsequent degradation, leading to cell survival. Thus, targeting CRL5 would cause NOXA accumulation to induce apoptosis and inhibit tumorigenesis.
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