The online version of this article (doi: https://doi.org/10.1186/s12885-017-3942-9) contains supplementary material, which is available to authorized users.
Protein neddylation is a post-translational modification by a covalent conjugation with the neural precursor cell expressed, developmentally downregulated 8 (NEDD8). Although this process has been reported to participate in diverse cellular signaling, little is known about its role in cancer cell migration. Given a recent proteomics report showing that NEDD8 is downregulated in prostate cancer tissues versus normal prostate tissues, we tested the possibility that neddylation plays a role in cancer evolution, and then tried to identify target proteins of the neddylation.
The neddylation process was inhibited by transfecting cancer cells with NEDD8-targeting siRNAs or by treating the cells with a NAE1 inhibitor MLN4924. Cell migration was evaluated by an in vitro wound-healing assay and a Transwell migration assay. His/NEDD8-conjugated proteins were pulled down with nickel-affinity beads under a denaturing condition, and identified by Western blotting. All data were processed using the Microsoft Excel program and analyzed statistically by two-sided, unpaired Student’s t-test.
Caveolin-1, which plays a critical role in cell migration, was identified to be conjugated with NEDD8. When the neddylation was inhibited, the phosphorylation of caveolin-1 at Tyr14 was augmented in PC3 and U373MG cells, thereby leading to increased cell migration. Such consequences by neddylation inhibition were abolished in the presence of a Src family kinase inhibitor PP2.
NEDD8 seems to inhibit the Src-mediated phosphorylation of caveolin-1 by modifying the structure of caveolin-1 protein, which blocks the migration of cancer cells. Although the neddylation process is currently regarded as an emerging target for cancer therapy, our results suggest the possibility that the inhibition of neddylation could facilitate cancer invasion or metastasis at least in some types of cancers.
Additional file 1: MLN4924 selectively inhibits NEDD8 activating enzyme (NAE) in the range of 0.25–0.5 μM. To examine the possible inhibition of the related enzymes ubiquitin-activating enzyme (UAE) and SUMO-activating enzyme (SAE), PC3 and U373MG cells were treated with 0.25 μM and 0.5 μM of MLN4924 for 24 h, respectively. Total modified forms of proteins by NEDD8, ubiquitin, and SUMO in cell lysates were analyzed by Western blotting with indicated antibodies. The doses of MLN4924 applied in our experiments (PC3: 0.25 μM, U373MG: 0.5 μM) inhibited neddylation without affecting ubiquitination and sumoylation. (PPTX 73 kb)12885_2017_3942_MOESM1_ESM.pptx
Additional file 2: N-terminal myc tagged caveolin-1 failed to be covalently conjugated with NEDD8. Ni-NTA-binding assay was performed in HEK293T cells co-expressing His-NEDD8 and myc-caveolin-1. Neddylated proteins were pulled down with Ni-NTA beads under a denaturing condition, and subjected to Western blotting with the indicated antibodies. (PPTX 18756 kb)12885_2017_3942_MOESM2_ESM.pptx
Additional file 3: Phosphorylated caveolin-1 is essential for MLN4924-induced cell migration. Scratch-based wound healing assays were performed for 24 h in PC3 (A) and U373MG (B) cells which were depleted of caveolin-1 using siRNA (#1 and #2, respectively) and si-control in the presence of MLN4924 (0.25 μM and 0.5 μM) or DMSO (top). The migration areas were calculated using ImageJ at just below. Proteins in cells lysates were analyzed by Western blotting (middle). The efficiency of the caveolin-1 knock-down and magnitude of the phosphorylation of caveolin-1 was quantified based upon the relative level of β-tubulin (bottom). Each bar represents the means + standard deviation of results from three independent experiments. * denotes P < 0.05 and n, s, does P > 0.05 between the indicated groups. Scale bar = 200 μm. (PPTX 12560 kb)12885_2017_3942_MOESM3_ESM.pptx
Additional file 4: Neddylation inhibition enhances the Src-mediated phosphorylation of caveolin-1. Scratch-based wound healing assays were performed for 24 h in PC3 (A) and U373MG (B) cells which were depleted of NEDD8 using siRNA #2 and si-control in the absence or presence 10 μM PP2 (top). The migration areas were calculated using ImageJ at just below. Proteins in cells lysates were analyzed by Western blotting (middle). The level of the phosphorylation of caveolin-1 was quantified based upon the relative level of β-tubulin (bottom). Each bar represents the means + standard deviation of results from three independent experiments. * denotes P < 0.05 between the indicated groups. Scale bar = 200 μm. (PPTX 21157 kb)12885_2017_3942_MOESM4_ESM.pptx
Additional file 5: PP2 can affect multiple cellular responses involving migration. A. Scratch-based wound healing assays were performed for 24 h in the vehicle (A) and si-control (C) of PC3 and U373MG cells which were treated with 10 μM PP2 (top). The migration areas were calculated using ImageJ at just below. Protein levels in cells lysates were analyzed by Western blotting. The PP2 mediated inhibition of the phosphorylation of caveolin-1 was quantified based upon the relative level of β-tubulin (bottom). Transwell migration assays were performed in the vehicle (B) and si-control (D) of PC3 and U373MG cells treated with 10 μM PP2 (left), and migrated cells were counted (right). Each bar represents the means + standard deviation of results from three independent experiments. * denotes P < 0.05 between the indicated groups. Scale bar = 200 μm. (PPTX 24837 kb)12885_2017_3942_MOESM5_ESM.pptx
Li H, Zhu H, Liu Y, He F, Xie P, Zhang L. Itch promotes the neddylation of JunB and regulates JunB-dependenttranscription. Cell Signal. 2016;28:1186–95.
Savage K, Lambros MB, Robertson D, Jones RL, Jones C, Mackay A, et al. Caveolin 1 is overexpressed and amplified in a subset of basal-like and metaplastic breast carcinomas: a morphologic, ultrastructural, immunohistochemical, and in situ hybridization analysis. Clin Cancer Res. 2007;13:90–101. CrossRefPubMed
Jaffray EG, Hay RT. Detection of modification by ubiquitin-like proteins. Methods 2006;38:35-38.
Webb DJ, Zhang H, Horwitz AF. Cell migration: an overview. Methods Mol Biol. 2005;294:3–11. PubMed
Ridley AJ. Rho GTPases and cell migration. J Cell Sci. 2001;114:2713–22. PubMed
Li L, Wang M, Yu G, Chen P, Li H, Wei D, et al. Overactivated neddylation pathway as a therapeutic target in lung cancer. J Natl Cancer Inst. 2014;106:dju083. PubMed
- Inhibition of neddylation facilitates cell migration through enhanced phosphorylation of caveolin-1 in PC3 and U373MG cells
Sung Yeon Park
- BioMed Central
Neu im Fachgebiet Onkologie
e.Med Kampagnen-Visual, Mail Icon II