Background
Protein neddylation is a post-translational modification by a covalent binding of NEDD8 (neural precursor cell expressed developmentally downregulated 8) to proteins. Like the ubiquitination process, neddylation is proceeded in three sequential steps of E1–3. The first step is govern by the NEDD8-activating enzyme (NAE), which is composed of amyloid beta precursor protein-binding protein 1 (APPBP1) and ubiquitin-like modifier activating enzyme 3 (UBA3). The second and third steps are carried out by the NEDD8-conjugating enzyme (UBC12) and variable substrate-specific NEDD8-E3 ligases, respectively [
1,
2]. This results in the changes in protein stability and functionality. In case of transcription factors, the neddylation regulates gene expressions by modulating the transcriptional activities of its targets [
3‐
5].
Caveolin-1 is an integral membrane protein implicated in a wide variety of physiological functions [
6]. In cancer development, caveolin-1 has been reported to behave as a tumor suppressor in the early stage [
7]. By contrast, it is regarded as a tumor promoter because caveolin-1 overexpression facilitates cancer cell migration, invasion and metastasis, and also induces multiple resistances to anticancer agents [
8‐
11]. The
CAV1 gene is presented as two variants through alternative mRNA splicing, Cav1α and Cav1β [
12]. Of them, only Cav1α possesses the tyrosine-14 (Y14) residue that is phosphorylated by non-receptor tyrosine kinases, including the proto-oncogene Src [
13]. The phosphorylation at Y14 has been reported to promote the localization and stabilization of focal adhesion kinase which is essential for cell migration [
14,
15]. Accordingly, the Src-caveolin-1 pathway is believed to be critically involved in cancer cell migration.
Given a recent proteomics report showing that NEDD8 is downregulated in prostate cancer tissues versus normal prostate tissues [
16], the authors were encouraged to investigate the role of NEDD8 in prostate cancer promotion. Mechanistically, caveolin-1 was post-translationally modified by NEDD8 conjugation, which attenuated the Src-mediated phosphorylation of caveolin-1 at Y14. Consequently, the inhibition of caveolin-1 neddylation stimulated the migration of prostate cancer and glioblastoma cells. From these results, it is proposed that the neddylation of caveolin-1 stops cell migration at least in prostate cancer and glioblastoma by counteracting the Src-caveolin-1 pathway.
Methods
Antibodies and reagents
Antibodies against NEDD8 and Myc tag (Cell Signaling Technology, Danvers, MA), FLAG tag (Sigma-Aldrich, St. Louis, MO), caveolin-1 and Y14-phospho-caveolin-1 (BD Biosciences, San Jose, CA), β-Tubulin, SUMO-1, and ubiquitin (Santa Cruz Biotechnology, Dallas, TX, USA) were purchased from the indicated companies. 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl) pyrazolo [3,4-d] pyrimidine (PP2) was purchased from Calbiochem (San Diego, CA). MLN4924 was synthesized, as described previously [
17].
Cell culture
HEK293T, PC3, U373MG, and A549 cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea). HEK293T was maintained in DMEM. U373MG, PC3, A549 cells in RPMI. All media were supplemented with 10% fetal bovine serum (FBS).
Western blotting
Total cell lysates were prepared using 2× denaturing SDS sample buffer, subjected to SDS-PAGE, and transferred to an Immobilon-P membrane (Millipore, Bedford, MA). Membranes were blocked with 5% skim milk in TTBS for 1 h and then were incubated overnight at 4 °C with the primary antibody. Membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, and stained with the enhanced chemiluminescent-plus reagent (Thermo Fisher Scientific, Rockford, IL).
Transient transfection
For transient transfection, cells were transfected with siRNAs using Lipofectamine® RNAiMax™ (Invitrogen, Carlsbad, CA) or with plasmids using the calcium phosphate reagent. Transfected cells were stabilized for 48 h before subsequent experiments. The siRNA duplexes were synthesized by Integrated DNA Technologies (Hanam, South Korea), and their nucleotide sequences are as follows:
-
caveolin-1#1, 5′-CCUUCACUGUGACGAAAUACUGGTT-3′;
-
caveolin-1#2, 5’-GCAGUUGUACCAUGCAUUAAGAGCT-3′;
-
NEDD8#1, 5′-UCCUUGAUUCGCUCCACCUUGUCUGUG-3′;
-
NEDD8#2, 5’-UUCACUUUAAUUAGCAUCUUCUUCCCA-3′.
FLAG- and His-tagged plasmids was constructed as described previously [
3], and GFP-tagged caveolin-1 was kindly given by Dr. Sang Jeong Kim (Seoul National University, Seoul, South Korea) and Myc-tagged caveolin-1 was constructed by replacing GFP with myc tagging. GFP-tagged caveolin-1-K5R was generated by site directed mutagenesis.
Identification of NEDD8 conjugation
Identification of NEDD8 conjugation was performed and modified based on the description in Jaffray and Hay [
18]. After transfected with His-tagged NEDD8 or NEDD8ΔGG plasmid, cells were lysed in a denaturing buffer (6 M guanidine hydrochloride, 0.1 M Na
2HPO
4/NaH
2PO
4, 0.01 M Tris-HCl, pH 8.0, plus 10 mM imidazole and 10 mM β-mercaptoethanol). The lysates were mixed with Ni
2+-NTA agarose beads (Qiagen, Valencia, CA) and incubated for 4 h at room temperature using a rotator. The beads were successively washed for 5 min each with the following solutions: lysis buffer (pH 8.0), washing buffer (pH 8.0; 8 M urea, 0.1 M Na
2HPO
4/NaH
2PO
4, 0.01 M Tris-HCl, pH 8.0, plus 20 mM imidazole, and 10 mM β-mercaptoethanol), washing buffer (pH 6.3) plus 0.2% Triton X-100, and washing buffer (pH 6.3) plus 0.1% Triton X-100. Then, the beads were eluted with SDS sample buffer and analyzed by Western blotting.
Immunoprecipitation
For immunoprecipitation, cell lysates (1 mg of protein) were incubated with 5 μL of antibody for 2 h and then incubated with 10 μL of protein A/G-Sepharose® beads (GE Healthcare, Pittsburgh, PA) for 4 h at 4 °C. After washing, the immunoprecipitated proteins were eluted in SDS sample buffer and subjected to SDS-PAGE and Western blotting.
Wound healing assay
Cultured cells were grown in 12-well plates until they reached confluence. The medium was removed and the cells were washed with PBS three times before culturing was continued in serum-free medium for an additional 24 h. Then, a rectangular lesion was created in the monolayers with a pipette tip. Cells were washed at least three times with PBS to remove debris and then cultured in serum-free medium. After 24 h, three randomly selected fields at the lesion border were assessed under an inverted microscope. The area of migration was measured using the ImageJ software (National Institutes of Health, Bethesda, MD).
Transwell migration assay
Assays were performed in Boyden chambers (Transwell® Costar®; 6.5 mm diameter, 8 μm pore size) according to the manufacturer’s protocol. Briefly, the bottom sides of the inserts were coated with 0.5 mg/mL collagen. Cells (2.5–5 × 104), re-suspended in 100 μL serum-free medium containing the designated concentration of reagents, were plated in the top of each chamber insert and the bottom chambers were filled with 600 μL complete medium containing 10% FBS. Cells were allowed to migrate for 24 h. Stationary cells on the top surface of the inserts were scraped with a cotton swab, and the cells that migrated to the bottom side of the inserts were fixed with methanol, washed, and stained with 0.1% crystal violet in 2% methanol. Images were acquired using an inverted microscope, and the number of cells that migrated to four independent areas per filter was counted using the ImageJ software.
Statistical analysis
All data were analyzed using Microsoft Excel 2007 and expressed as the means and standard deviations (sd). Continuous variables were analyzed using Student’s t-tests if the data were normally distributed. All statistical tests were two-sided. P values <0.05 were considered to indicate statistical significance.
Discussion
In this study, NEDD8 knock-down enhanced cell migration in PC3 and U373MG cells. As caveolin-1 was identified to be conjugated with NEDD8, we hypothesized that the neddylation of caveolin-1 determines cancer cell migration. Although it was not regulated in the protein level by neddylation, caveolin-1 was functionally regulated by neddylation. Caveolin-1 is activated by being phosphorylated at the Y14 residue, which was found to be inhibited by the neddylation of caveolin-1. The inhibition of neddylation, which was achieved using NEDD8-targeting siRNAs or MLN4924, enhanced the Src-mediated Y14-phosphorylation of caveolin-1, thereby promoting the migration of PC3 and U373MG cells.
Several target proteins of neddylation have been identified; the most intensively-characterized substrate is the cullin family, a component of Really Interesting New Gene (RING) E3-ubiquitin ligases (cullin ring ligases, CRLs). Neddylation of cullins stimulates the activity of the ubiquitin E3 ligase, resulting in increased proteasomal degradation of proteins which include tumor suppressors, cell cycle regulators, components of the DNA replication machinery, and mediators of cellular stress [
2,
5,
22,
23]. In addition, neddylation has been reported to stabilize hypoxia-inducible factor 1-alpha (HIF-1α) and transforming growth factor-beta receptor II [
3,
24]. Neddylation is also involved in regulating the transcriptional activities of several substrates. For example, when cell-cycle-regulating transcription factor E2 is subjected to neddylation, the transcription factor is degraded and functionally repressed, leading to impaired cell growth [
25,
26]. The p53 tumor suppressor is another target of neddylation which is mediated by the mouse double minute 2 (Mdm2), and its transcriptional activity is repressed by neddylation [
27]. Moreover, neddylation has been shown to regulate cell movement. Renaudin et al. demonstrated that neddylation promotes the trafficking of C-X-C chemokine receptor type 5 to the plasma membrane and by doing so enhances cell migration [
28]. We here identified carveolin-1 as a new target of neddylation, and to our best knowledge, this is for the first time reported in this study.
Cell migration is a pivotal biological process required in processes such as embryonic development and tissue repair and regeneration; it is also involved in pathological conditions, including arthritis, atherosclerosis, and the metastasis of cancer cells [
29]. This event arises through the dynamic interplay of multiple cellular components associated with cell adhesion and movement. First, the microtubule organizing center is polarized towards the leading edge of the cell [
30]. Once oriented, the cell extends in the direction of migration either via broad (lamellipodia) or focused (filopodia) protrusions. Then, interactions with many molecules related to focal contacts and integrins allow the cell to migrate in the desired direction [
31‐
33]. Consistent with this, we observed that the lamellipodia at the edge of U373MG cells were extended more after treatment with MLN4924 or the depletion of NEDD8 compared with the control, which may facilitate dragging for movement (data not shown). In recent years, a strong connection has been established between caveolin-1 and cell migration/invasion. In particular, phosphorylation on Y14 of caveolin-1 appears to be required for cell migration. The caveolin-1 phosphorylation facilitates anchorage-independent growth by recruiting growth factor receptor-bound protein 7 (Grb7) [
34], integrin-dependent internalization of membrane micro-domains [
35], and activation of matrix metalloproteinases [
20]. Additionally, the caveolin-1 phosphorylation is involved in the localization and stabilization of focal adhesion kinase (FAK) essential for cell migration [
15]. Considering such roles of phosphorylated caveolin-1, it is not surprising that cancer cell migration is enhanced by inhibiting neddylation.
MLN4924, a first-in-class NAE inhibitor, has shown therapeutic efficacy in preclinical studies [
36]. In clinical trials, MLN4924 also showed a modest effect against acute myeloid leukemia [
37]. As it inactivates CRLs, MLN4924 accumulates tumor-suppressive CRL substrates, which induces genotoxic stress, cell cycle arrest, autophagy, apoptosis, and cell senescence [
38‐
42]. In addition to growth inhibition, MLN4924 has been reported to suppress cell migration in lung and urothelial carcinomas and to reduce cancer metastasis in animals [
43,
44]. However, Zhou et al. recently claimed that MLN4924 at a low concentration (30–100 nM) stimulated cancer cell proliferation, sphere formation, and tumorigenesis [
45]. Consistent with this report, we observed that even at moderate nanomolar concentrations (250 and 500 nM) MLN4924 accelerated cell migration at least in two cancer cell-lines. MLN4924 may affect cancer cell migration in different ways depending on cell contexts.
Acknowledgements
We would like to thank Uk-Il Ju for technical teaching and assistance about mammalian cell culture system.