Purpose: Liver metastasis is the major and direct cause of death in patients with uveal melanoma (UM). There is no effective therapy for patients with metastatic UM. Improved treatments of hepatic metastatic patients with UM were urgently needed. Inspired by readily detectable key components in the neddylation pathway in UM cells, we aimed at exploring whether neddylation pathway was a therapeutic target for liver metastatic UM.

Experimental Design: Expression of key proteins in the neddylation pathway in UM was detected by Western blotting, real-time quantitative RT-PCR (qRT-PCR), and immunohistochemical staining. Cellular proliferation, apoptosis, cell cycle, migration, and cancer stem-like cells (CSCs) properties were examined upon treatment with MLN4924, a potent and selective NAE inhibitor. Antitumor activity and frequency of CSCs were determined by using a NOD-SCID mouse xenograft model. Liver metastasis was evaluated by use of a NOD-scid-IL2Rg−/− mouse model.

Results: NAE1 expression was readily detectable in UM. Inhibition of the neddylation pathway by MLN4924 repressed the CSCs properties in UM (capacities of tumorsphere formation and serially replating, aldehyde dehydrogenase-positive cells, and frequency of CSC) through Slug protein degradation. MLN4924 treatment disturbed the paracrine secretion of NF-κB-mediated VEGF-C and its dependent angiogenesis. The inhibitory effect of neddylation blockade on proliferation, which was confirmed by xenografted UM tumor in NOD-SCID mice, was involved in activation of ATM-Chk1-Cdc25C DNA damage response, and G2–M phase arrest. Neddylation inhibition profoundly inhibited hepatic metastasis in UM.

Conclusions: Our studies validate the neddylation pathway as a promising therapeutic target for the treatment of patients with hepatic metastasis of UM. Clin Cancer Res; 24(15); 3741–54. ©2017 AACR.

See related commentary by Yang et al., p. 3477

Translational Relevance

The clinical single site-specific organ metastasis pattern in certain types of cancer (e.g., hepatic metastasis in uveal melanoma, UM) may provide a study model to realize and circumvent metastasis. Most of stemness-related proteins are of short-life and sensitive to posttranslational modifications of proteins. We hypothesized that blocking the neddylation pathway may disturb homeostasis of proteins, which are essential for cancer stem–like cells (CSCs), and the microenvironment cytokine(s) in UM cells. We tested this hypothesis and found that MLN4924, a potent NEDD8-activating enzyme (NAE) inhibitor, repressed the CSC properties in UM through Slug protein degradation. MLN4924 treatment disturbed the paracrine secretion of nuclear factor-κB (NF-κB)-mediated vascular endothelial growth factor-C (VEGF-C) and its dependent angiogenesis. Notably, xenografted tumor experiments in mice revealed that MLN4924 abrogated growth and hepatic metastasis in UM. Our findings validate the neddylation pathway as a promising therapeutic target for the treatment of patients with hepatic metastasis of UM.

Metastasis is the major and direct cause of death in patients with cancer at the terminal stage. Unfortunately, there are no effective therapies for metastatic patients. The complicated metastasis process is principally composed of intravasation, circulation, extravasation, and colonization in the target organ (1). Intravasation is referred to as the process that the cancer cells in the primary tumor sites break out of physical barriers in the surrounding tissues, and enter the blood or lymphatic stream to become circulating tumor cells (CTCs; ref. 2). Only a small portion of CTCs that acquire stemness features can successfully escape anoikis, immune response, and shear stress (2). The CTCs undergo extravasation in which they leave from the bloodstream for distant organs and tissues, where they are called metastasis initiating cells (MICs). MICs interact with the microenvironment (niche) to establish their own niches for colonization and eventually form clinically overt metastatic foci. CTCs and MICs share many features (e.g., self-renewal, quiescence, and asymmetric division) with cancer stem-like cells (CSC; ref. 3).

In the clinic, patients with some types of cancer (e.g., breast cancer, lung cancer, and cutaneous melanoma) manifest metastasis at multiple different organ sites (4). Patients with certain other types of cancer [e.g., prostate cancer, pancreatic cancer, and uveal melanoma (UM)] manifest metastasis at a single organ site (e.g., prostate cancer to bone, pancreatic cancer, and UM to liver; ref. 4). The clinical single site-specific organ metastasis pattern in these types of cancer may provide a simplified realization window and study model to circumvent metastasis (4).

UM biologically distinct from cutaneous melanoma may provide a naturally clinical phenotype and typical example of organ-specifically liver metastasis (5). UM, the most common ocular malignancy in adults, usually originates from melanocytes of the choroid, ciliary body, and iris (6). Although successful treatment of the primary tumor with enucleation or radiotherapy can be achieved, half of the UM patients develop metastasis. Eighty-five percent of the patients with metastatic UM exhibit single liver metastasis (7). Little is known about the underlying mechanism of such liver-specific metastasis; there is no effective therapy for patients with metastatic UM, with a median survival of less than 12 months (8). Whole-genome sequencing has demonstrated that mutually exclusive gain-of-function mutations in GNAQ or GNA11 are found in 80% of UM, which may lead to activation of the mitogen-activated protein kinase (MAPK) pathway and transcriptional factor YAP pathway to promote growth and migration of UM cells (9, 10). However, no direct in vivo evidence supports that GNAQ/GNA11-MAPK and GNAQ/GNA11-YAP act as drivers in metastasis of UM.

Most of stemness-related proteins are of short-life with stability sensitive to posttranslational modifications (11). In addition, exocytosis, intracellular trafficking, and secretion of inflammatory cytokines are regulated by protein maturation and posttranslational modifications (12). Neural precursor cell expressed, developmentally downregulated 8 (NEDD8)-conjugation, also called neddylation, is first activated by an E1 enzyme [NEDD8-activating enzyme (NAE); a heterodimer consisting of NAE E1 subunit 1 (NAE1) and ubiquitin-like modifier activating enzyme 3 (UBA3)], transferred to an E2 enzyme (UBC12), and then conjugated to target substrates through cullin-RING ligases (CRL; ref. 13). Neddylation controls protein turnover of a number of CRL targets with essential roles in regulation of oncogenic transformation and pathogenesis. MLN4924 (pevonedistat), a potent and selective first-in-class NAE1 inhibitor (14), is currently in phase I clinical trials in some solid tumors and hematologic malignancies (15, 16).

We hypothesized that blocking the neddylation pathway might disturb the protein homeostasis between synthesis and degradation, which might in turn impact the stemness-related proteins to diminish CSCs, and decrease secretion of the microenvironment cytokine(s) in UM, both of which are fundamental for metastasis (12). The hypothesis was supported by the recent observation in a zebrafish xenograft model that MLN4924 inhibits migration and proliferation of UM cells (17) with the mechanism yet unknown. We tested the hypothesis to find that the neddylation blockade diminished organ-specifically hepatic metastasis by disrupting features of CSCs, niche secreting VEGF-C and its dependent angiogenesis in UM. The findings revealed that MLN4924 may be a promising agent for the treatment of UM patients with hepatic metastasis.

Cell culture

Genetic status of the known altered genes in human primary (92.1, Mel270) and metastatic (Omm1, Omm2.3) UM cells used in this study is summarized in Supplementary Table S1. 92.1 cells were established in Leiden University Medical Center, Leiden, the Netherlands (18). Mel270 and Omm2.3 cells originally derived from patients at the Bascom Palmer Eye Institute, University of Miami School of Medicine, Florida (19), and Omm1 cells were established by Luyten G at Rotterdam University Hospital, the Netherlands (20). These UM cell lines were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone) and 2 mmol/L L-glutamine (7, 21) and authenticated in HAKE Genetics Co., Ltd. by using short tandem repeat matching analysis at August 8, 2017. The cells were kept at 37°C in a humidified incubator with 5% CO2. No mycoplasma (ThermoFisher Scientific) contamination was detected.

Tumor samples of patients with UM

Human tissues from primary tumors were collected from enrolled patients (n = 45) with UM in The Sun Yat-sen University Zhongshan Ophthalmic Center during 2012–2015, after informed consent according to the institutional guidelines and the Declaration of Helsinki principles. The seventh edition of the American Joint Committee on Cancer tumor, node, metastasis (TNM7) classification for eye cancer was used (22, 23). Detailed information of UM patients is shown in Supplementary Table S2. The studies were approved by Institutional Review Board, The Sun Yat-sen University Zhongshan Ophthalmic Center.

Immunohistochemical (IHC) staining and evaluation of NAE1

IHC staining with anti-NAE1 antibody and quantification of NAE1 expression were performed as described (17, 21, 24), with details provided in the Supplementary information. The association between NAE1 expression and clinicopathologic features in UM patients is summarized in Supplementary Table S3.

Cell viability, colony-formation, apoptosis, cell cycle, Western blotting, real-time quantitative RT-PCR, luciferase activity, enzyme-linked immunosorbent analysis, retrovirus, and lentivirus infection

All above methods were performed as described previously reported (21, 25), with details provided in the Supplementary information.

Tumor xenograft experiments

Male NOD-SCID mice (4–6-week-old) purchased from Vital River Laboratory Animal Technology Co. and bred at the animal facility of Sun Yat-sen University were used for evaluation the in vivo antineoplastic efficacy of MLN4924 against UM as previously reported (21). After 4 weeks, the mice were randomly separated into two groups (n = 8 per group) and received treatment with vehicle (10% 2-hydroxypropyl-β-cyclodextrin) or MLN4924 (60 mg/kg/day, i.p.) for 14 days. The mice were euthanized, and tumor xenografts were immediately removed, recorded, fixed, and stored at −80°C.

Wound-healing scratch, migration and invasion, melanospheres formation, aldehyde dehydrogenase positive cells, limiting dilution assay in NOD-SCID mice, human umbilical vein endothelial cells tube formation, migration, and chicken chorioallantoic membrane (CAM) assay

The above methods were examined as previously reported (21, 24, 26), with details provided in the Supplementary information.

Liver metastasis mouse model

Twenty-four hours after Omm2.3-luciferase cells (1 × 106 in 50 μL PBS per mouse) were intrasplenically injected into the NOD-scid-IL2Rg−/− (NSI) mice (25, 27), the mice were randomly separated into two groups (n = 6 per group) and received treatment with vehicle or MLN4924 (90 mg/kg, bid, i.p.) 5 days on/2 days off for four cycles. Liver metastasis was detected by ex vivo bioluminescence imaging using the IVIS Lumina II (PerkinElmer) or counted the nodules on liver paraffin sections after Hematoxylin and eosin (H&E) staining. All animal studies were conducted with the approval of the Sun Yat-sen University Institutional Animal Care and Use Committee.

Statistical analysis

All experiments were performed three times, and results are reported as mean ± standard deviation (SD), unless otherwise stated. GraphPad Prism 5.0 was used for statistical analysis. Comparisons between two groups were analyzed by two-tailed Student t test and comparisons of multiple groups by one-way ANOVA with post hoc intergroup comparison with Tukey test. A P < 0.05 was considered statistically significant.

NAE1 expression is readily detectable in UM

We first examined certain key proteins involved in global NEDD8 conjunction in human UM cells. Western blotting results indicated that the levels of NAE1, UBA3, and UBC12 were higher in the human UM cells than those in ARPE-19 cells (Fig. 1A). Correspondingly, cullin1 was neddylated in human UM cells (Fig. 1A). Further, qRT-PCR analysis revealed that the mRNA levels of NAE1, UBA3, UBC12, and NEDD8 displayed 3.9- to 6.0-, 2.9- to 4.1-, 5.0- to 7.9-, and 4.7- to 7.0-fold increase in the UM cells compared with ARPE-19 cells, respectively (P < 0.01, Fig. 1B–E). These results suggest that the readily detectable status of these key proteins may occur at the transcriptional level.

Figure 1.

NAE1 expression is readily detectable in cells and specimens of human UM. A, Protein levels of NAE1, UBA3, UBC12, cullin1 neddylation, and global NEDD8 conjugation in human UM cells (e.g., Mel270, 92.1, Omm1, and Omm2.3) and human adult retinal pigmented epithelium (ARPE-19) cells were determined by Western blotting analysis. B–E, The mRNA levels of NAE1, UBA3, UBC12, and NEDD8 genes were analyzed by qRT-PCR. F, Representative IHC images of NAE1 expression in paraffin-embedded tissues from the patients with UM and adjacent normal tissues are shown. The protein expression of NAE1 was classified into four levels (negative, low, medium, and high). Brown: choroid pigment, Red: NAE1 staining. Scale bar: 200 μm (100×), 100 μm (200×). G, NAE1 expression was increased in UM specimens (n = 45) compared with adjacent normal tissues (n = 14). H, The percentage of NAE1 expression was 77.8% (35/45) in UM specimens. **, P < 0.01; ***, P < 0.0001, one-way ANOVA, post hoc intergroup comparisons, Tukey test.

Figure 1.

NAE1 expression is readily detectable in cells and specimens of human UM. A, Protein levels of NAE1, UBA3, UBC12, cullin1 neddylation, and global NEDD8 conjugation in human UM cells (e.g., Mel270, 92.1, Omm1, and Omm2.3) and human adult retinal pigmented epithelium (ARPE-19) cells were determined by Western blotting analysis. B–E, The mRNA levels of NAE1, UBA3, UBC12, and NEDD8 genes were analyzed by qRT-PCR. F, Representative IHC images of NAE1 expression in paraffin-embedded tissues from the patients with UM and adjacent normal tissues are shown. The protein expression of NAE1 was classified into four levels (negative, low, medium, and high). Brown: choroid pigment, Red: NAE1 staining. Scale bar: 200 μm (100×), 100 μm (200×). G, NAE1 expression was increased in UM specimens (n = 45) compared with adjacent normal tissues (n = 14). H, The percentage of NAE1 expression was 77.8% (35/45) in UM specimens. **, P < 0.01; ***, P < 0.0001, one-way ANOVA, post hoc intergroup comparisons, Tukey test.

Close modal

After verifying the specificity of antibody against NAE1 (Supplementary Fig. S1), we conducted IHC analysis of UM specimens. The results showed that no staining of NAE1 in the adjacent normal tissues was detected. In contrast, readily detectable expression of NAE1 scored from low to high was observed in 77.8% (35/45) patients with UM (Fig. 1F–H). Of importance, NAE1 expression was positively correlated to the largest basal diameter (P = 0.0373) and thickness (P = 0.0461) of the primary tumors we analyzed, two important independent predictors of UM metastatic death (28, 29) and the TNM stage (P = 0.0374; Supplementary Table S3).

MLN4924 specifically inhibits the neddylation pathway

To explore whether the neddylation pathway was a therapeutic target for UM, we first examined the specificity of MLN4924 against the neddylation pathway in UM cells. The results indicated that MLN4924, but not proteasome inhibitors MG132 and bortezomib, suppressed the global NEDD8 conjunction or cullin1 neddylation (Fig. 2A). Consequently, the substrates of CRLs (e.g., p21 and p27) were accumulated over all treatments; however, cyclin B1, a non-CRL substrate, was accumulated upon MG132 and bortezomib treatment but not upon MLN4924 treatment (Fig. 2A). Similarly, MLN4924 treatment increased the protein levels of the CRL substrates (e.g., NRF2, phospho-IκBα) in a concentration-dependent manner (Fig. 2B). MLN4924 treatment also decreased cullin1 neddylation in a time-dependent manner (Fig. 2C). Consistent with previous report (30), MLN4924 treatment inhibited 70% to 97% and 90% to 95% (P < 0.001) of the NF-κB-dependent reporter gene activity in Mel270 and Omm2.3 cells, respectively (Fig. 2D). These data suggest that MLN4924 specifically inhibits the neddylation pathway, leading to accumulation of CRL substrates.

Figure 2.

MLN4924, a potent and selective inhibitor of NAE, counteracts proliferation of UM cells. A, MLN4924 specifically inhibited the neddylation pathway. Human UM cells were treated with MLN4924 (1.0 μmol/L), MG132 (20.0 μmol/L), or Bort (bortezomib; 1.0 μmol/L) for 1 hour, global NEDD8 conjugation, cullin1 neddylation, and protein levels of p21, p27, or cyclin B1 were examined by Western blotting analysis. B, MLN4924 treatment impacted on the expression of CRL substrates. Human UM cells were treated various concentrations of MLN4924 for 48 hours, cullin1 neddylation and protein levels of CRL substrates were determined by Western blotting analysis. C, MLN4924 time-dependently inhibited cullin1 neddylation. D, MLN4924 dramatically inhibited the activity of NF-κB-dependent reporter gene in UM cells. Mel270 and Omm2.3 cells were transfected with NF-κB-TATA-Luc reporter construct (500 ng) and Renilla luciferase reporter construct (10 ng) for 24 hours, then treated with MLN4924 for 48 hours, luciferase activity was detected. ***, P < 0.0001, one-way ANOVA, post hoc intergroup comparisons, Tukey test. E, MLN4924 decreased cell viability of UM cells but not ARPE-19 cells. UM cells and ARPE-19 cells were treated with escalating concentrations of MLN4924 for 72 hours; cell viability was measured by MTS assay. Dose-response curves from 3 independent experiments are shown. F, MLN4924 concentration-dependently suppressed clonogenicity of UM cells in drug-free soft agar culture.

Figure 2.

MLN4924, a potent and selective inhibitor of NAE, counteracts proliferation of UM cells. A, MLN4924 specifically inhibited the neddylation pathway. Human UM cells were treated with MLN4924 (1.0 μmol/L), MG132 (20.0 μmol/L), or Bort (bortezomib; 1.0 μmol/L) for 1 hour, global NEDD8 conjugation, cullin1 neddylation, and protein levels of p21, p27, or cyclin B1 were examined by Western blotting analysis. B, MLN4924 treatment impacted on the expression of CRL substrates. Human UM cells were treated various concentrations of MLN4924 for 48 hours, cullin1 neddylation and protein levels of CRL substrates were determined by Western blotting analysis. C, MLN4924 time-dependently inhibited cullin1 neddylation. D, MLN4924 dramatically inhibited the activity of NF-κB-dependent reporter gene in UM cells. Mel270 and Omm2.3 cells were transfected with NF-κB-TATA-Luc reporter construct (500 ng) and Renilla luciferase reporter construct (10 ng) for 24 hours, then treated with MLN4924 for 48 hours, luciferase activity was detected. ***, P < 0.0001, one-way ANOVA, post hoc intergroup comparisons, Tukey test. E, MLN4924 decreased cell viability of UM cells but not ARPE-19 cells. UM cells and ARPE-19 cells were treated with escalating concentrations of MLN4924 for 72 hours; cell viability was measured by MTS assay. Dose-response curves from 3 independent experiments are shown. F, MLN4924 concentration-dependently suppressed clonogenicity of UM cells in drug-free soft agar culture.

Close modal

We next evaluated the effect of MLN4924 on cellular growth. MTS results showed that MLN4924 concentration-dependently inhibited the growth of UM cells (IC50 values: 433–890 nmol/L) but not of ARPE-19 cells (IC50 value: >10 μmol/L), hinting a therapeutic window in UM cells (Fig. 2E). MLN4924 concentration-dependently inhibited the clonogenicity of UM cells in soft agar, which can better reflect malignant behaviors of tumor cells (IC50 values: 93.8–134.4 nmol/L; Fig. 2F).

MLN4924 induces apoptosis in UM cells

We next ascertained the ability of MLN4924 to induce apoptosis in UM cells. MLN4924 increased the dead cell population in a concentration- (Fig. 3A) and time-dependent manner (Supplementary Fig. S2A) in UM cells. Western blotting analysis showed a concentration- (Fig. 3B) and time-dependent (Supplementary Fig. S2B) PARP cleavage and caspase-3 activation. Additionally, treatment with MLN4924 increased cytochrome c release from the mitochondria into the cytosol detected by immunoblotting (Fig. 3C) as well as the cell population with loss of mitochondrial potential (ΔΨm) detected by flow cytometry (Supplementary Fig. S2C). These data together suggest that MLN4924 induces mitochondrial damage and triggers intrinsic apoptosis pathway.

Figure 3.

MLN4924 induces apoptosis in human UM cells. A, UM cells were treated with escalating concentrations of MLN4924 for 48 hours, cell death was examined by flow cytometry after dual staining with Annexin V-FITC/propidium iodide (PI). Representative histograms (top) for 92.1 cells and quantitative analysis (bottom) from three independent experiments are shown. Data represent mean ± SEM. **, P < 0.01; ***, P < 0.0001, one-way ANOVA, post hoc intergroup comparisons, Tukey test. B, MLN4924 induced apoptosis-specific cleavage of PARP and caspase-3 activation in a concentration-dependent manner. UM cells were treated with increasing concentrations of MLN4924 for 48 hours, Western blotting analysis was performed with the specific antibodies, respectively. C, MLN4924 treatment induced release of cytochrome c into the cytosol. Cells were treated with MLN4924 (1.0 μmol/L) for the indicated durations, and the cytosolic fractions extracted with digitonin buffer were subjected to immunoblotting analysis for cytochrome c. D, Expression of apoptosis-related proteins was analyzed by Western blotting in UM cells treated with MLN4924 for 48 hours. E, Bcl-XL was overexpressed in tissues from 8 out of 80 patients with UM TCGA database (top); UM patients with higher Bcl-XL expression showed shorter overall survival (bottom). F and G, Ectopic Bcl-XL expression abrogated the MLN4924-induced apoptosis. Twenty-four hours after transfected with empty vector (pCMV6) or Bcl-XL (pCMV6-BCL2L1) constructs, Mel270 (left) and Omm2.3 (right) cells were exposed to the indicated concentrations of MLN4924 for 48 hours, followed by Western blotting of PARP, caspase-3 and Bcl-XL (F) and trypan blue exclusion assay (G), respectively. *, P < 0.05; **, P < 0.01; ***, P < 0.0001, Student t test.

Figure 3.

MLN4924 induces apoptosis in human UM cells. A, UM cells were treated with escalating concentrations of MLN4924 for 48 hours, cell death was examined by flow cytometry after dual staining with Annexin V-FITC/propidium iodide (PI). Representative histograms (top) for 92.1 cells and quantitative analysis (bottom) from three independent experiments are shown. Data represent mean ± SEM. **, P < 0.01; ***, P < 0.0001, one-way ANOVA, post hoc intergroup comparisons, Tukey test. B, MLN4924 induced apoptosis-specific cleavage of PARP and caspase-3 activation in a concentration-dependent manner. UM cells were treated with increasing concentrations of MLN4924 for 48 hours, Western blotting analysis was performed with the specific antibodies, respectively. C, MLN4924 treatment induced release of cytochrome c into the cytosol. Cells were treated with MLN4924 (1.0 μmol/L) for the indicated durations, and the cytosolic fractions extracted with digitonin buffer were subjected to immunoblotting analysis for cytochrome c. D, Expression of apoptosis-related proteins was analyzed by Western blotting in UM cells treated with MLN4924 for 48 hours. E, Bcl-XL was overexpressed in tissues from 8 out of 80 patients with UM TCGA database (top); UM patients with higher Bcl-XL expression showed shorter overall survival (bottom). F and G, Ectopic Bcl-XL expression abrogated the MLN4924-induced apoptosis. Twenty-four hours after transfected with empty vector (pCMV6) or Bcl-XL (pCMV6-BCL2L1) constructs, Mel270 (left) and Omm2.3 (right) cells were exposed to the indicated concentrations of MLN4924 for 48 hours, followed by Western blotting of PARP, caspase-3 and Bcl-XL (F) and trypan blue exclusion assay (G), respectively. *, P < 0.05; **, P < 0.01; ***, P < 0.0001, Student t test.

Close modal

MLN4924 disturbs balance of Bcl-2 family members in UM cells

To elucidate the mechanism of MLN4924-induced apoptosis, we evaluated the expression of apoptosis-related proteins. The results showed that the levels of pro-survival proteins survivin and Bcl-XL were decreased, whereas those of pro-apoptotic protein Bim were slightly increased (Fig. 3D). No change in Bcl-2, Bid, Bax, and Noxa was observed (Fig. 3D).

Because Bcl-XL (a known NF-κB target gene) was overexpressed in 10% of patients with UM extrapolated from The Cancer Genome Atlas (TCGA) database (http://www.cbioportal.org), which apparently correlated with poor overall survival (P = 0.0174; Fig. 3E). The mRNA levels of BCL2L1 gene were lowered by up to 63.9% and 56.9% in the MLN4924-treated Mel270 and Omm2.3 cells, respectively (Supplementary Fig. S2D). We next examined the role of Bcl-XL in MLN4924-induced apoptotic cell death in UM cells. Forced overexpression of Bcl-XL attenuated MLN4924-induced apoptosis in UM cells as reflected by caspase-3 activation (Fig. 3F) and trypan blue staining cells (19.3% decrease in Mel270, P < 0.01; 10.2% decrease in Omm2.3, P < 0.05; at 1.0 μmol/L treatment; Fig. 3G). Conversely, silencing Bcl-XL by siRNA duplexes potentiated the lethal effect of MLN4924 in UM cells (Supplementary Fig. S2E and F; increased dead cells by 25.6% and 27.9% in Mel270; 23.8% and 24.2% in Omm2.3, P < 0.01; at 1.0 μmol/L treatment).

Next, we evaluated the synergistic effect between MLN4924 and the conventional chemotherapeutic agent vinblastine in UM cells (17). MTS results showed that combinational treatment with MLN4924 and vinblastine synergistically (CI values <1) inhibited cell growth in Mel270 and Omm1 cells (Supplementary Fig. S3A), as evaluated by using the median-effect method of Chou and Talalay (31). Flow cytometer and Western blotting results suggest that combinational treatment with MLN4924 and vinblastine induced enhanced cell apoptosis as reflected by the increase of Annexin V+ cells, PARP cleavage, and caspase-3 activation, respectively (Supplementary Fig. S3B and C).

MLN4924 induces DNA damage response (DDR) predominantly by activating ATM in UM cells

Previous studies have shown that MLN4924 can induce DDR and cell cycle perturbation to inhibit cell growth in several types of cancer (32, 33). We first assessed the key components involved in DDR. Western blotting analysis showed that MLN4924-treated UM cells displayed an appreciably increased γ-H2AX, an indicative hallmark of DNA double strand breaks (DSB; Supplementary Fig. S4A). γ-H2AX not only indicated the existence of MLN4924-mediated DSBs but also reflected the activation of DDR. Indeed, the levels of p53 and phospho-p53 (S15) were increased by MLN4924 (Supplementary Fig. S4A).

Ataxia telangiectasia-mutated (ATM) kinase and ATM-Rad3-related (ATR) kinase function at the level of sensors and transducers of DNA damage signaling pathway in response to DSBs and single strand breaks (SSB), respectively (34). Our results showed that the phosphorylation of ATM was concentration-dependently increased in the MLN4924-treated UM cells (Supplementary Fig. S4A), suggesting a steady activation of ATM after MLN4924 treatment. A slight and nonconcentration-dependent increase in ATR phosphorylation was observed in the cells treated with certain lower concentrations of MLN4924 (Supplementary Fig. S4A). These findings together with the γ-H2AX change further hint that DSBs may be the major DNA damage form in response to neddylation inhibition.

We also detected DDR checkpoint kinase 1 (Chk1) and Chk2. The results revealed that the phosphorylation of both Chk1 and Chk2 was steadily increased by MLN4924 (Supplementary Fig. S4A). These data collectively indicate that neddylation inhibition induces DSBs and elicits DDR predominantly by ATM, Chk1, and Chk2.

Increased reactive oxygen species (ROS) generation is involved in MLN4924 action mechanism and may lead to DNA damage (35). To explore the reason that MLN4924 induced DDR in UM, we detected the intracellular ROS levels. ROS generation was found to be increased by 1.4- to 2.7-fold in MLN4924-treated Mel270 cells (Supplementary Fig. S4B). Pretreated with N-acetylcysteine, the ROS scavenger, significantly prevented MLN4924-mediated DNA damage, as reflected by γ-H2AX in UM cells (Supplementary Fig. S4C). These data suggest that ROS generation may mediate DNA damage in UM cells upon MLN4924 exposure.

MLN4924 induces G2–M phase arrest with increased phosphorylation in Chk1 and Wee1

Flow cytometry analysis showed that MLN4924 treatment led to increase in G2–M phase population in a concentration- and time-dependent manner in UM cells, respectively (Supplementary Fig. S4D and E). Correspondingly, overt decrease of phospho-histone H3 (S10), a hallmark of M phase, was noted in the MLN4924-treated UM cells, suggesting a stalled G2 phase (Supplementary Fig. S4F). Further analysis of G2–M checkpoint mechanism revealed that a remarkable increase of Wee1 (a well-defined CRL substrate and an inhibitor of G2–M phase transition), phospho-Cdc25C (S216), and phospho-Cdc2 (Y15) was observed in the MLN4924-treated UM cells (Supplementary Fig. S4F).

Inhibiting Chk1 and Wee1 synergistically potentiates the lethality of MLN4924 in UM cells

We next examined whether MLN4924 was synergistic with MK-8776 (Chk1 inhibitor) and MK-1775 (Wee1 inhibitor). The results indicated that the combination between MLN4924 and MK-8776 or MLN4924 and MK-1775 synergistically retarded the cell growth as measured by MTS assay (Supplementary Fig. S5A) and induced enhanced apoptosis, reflected by enhanced specific cleavage of PARP as detected by Western blotting analysis (Supplementary Fig. S5B) in UM cells.

MLN4924 inhibits outgrowth of xenografted UM cells in NOD-SCID mice

We evaluated the in vivo antineoplastic efficacy of MLN4924 against UM using NOD-SCID mouse xenograft model. The mice (4–6-week-old) were subcutaneously injected with Omm1 cells. When the tumor reached ∼50 mm3, the mice were randomly divided into two groups (n = 8) and received treatment with vehicle or MLN4924 for 14 days. The tumor size was much smaller in MLN4924-treated group than vehicle-treated mice (Fig. 4A). MLN4924 administration abrogated the tumor outgrowth (Fig. 4B). Consistently, tumor weight was decreased by 58% (P < 0.01) in the MLN4924-treated mice (Fig. 4C). MLN4924 administration inhibited tumor proliferation as reflected by the expression of Ki67. Moreover, the expression of p27, a well-known CRL substrate, was increased in tumor tissues from the MLN4924-treated mice (Fig. 4D). Western blotting results of cell lysates showed inhibited neddylation pathway with the protein levels of CRL substrates p21 and p27 notably increased. The expression of Slug, Bcl-XL, and survivin was, however, decreased in MLN4924-treated group (Fig. 4E), which were consistent with the in vitro findings. These data demonstrate the in vivo antineoplastic efficacy of MLN4924.

Figure 4.

MLN4924 inhibits outgrowth of xenograft UM cells in NOD-SCID mice. A, Representative tumors removed from the mice of each group are shown. B, The growth curves of subcutaneous xenografts of Omm1 cells were plotted. *, P < 0.05; **, P < 0.01; ***, P < 0.0001, Student t test. C, MLN4924 significantly lowered tumor weights on day 15 postinoculation. **, P < 0.01, Student t test. D, H&E staining and IHC analysis with anti-Ki67 and -p27 of tumor tissues from mice. Scale bar: 20 μm. E, MLN4924 administration led to inhibit the neddylation pathway, accumulation in CRL substrates such as p21 and p27, as well as decreased expression of Slug, Bcl-XL, and survivin in xenografted tumor tissues from NOD-SCID mice. Cell lysates prepared from four xenografts of each group were detected by Western blotting with the indicated antibodies.

Figure 4.

MLN4924 inhibits outgrowth of xenograft UM cells in NOD-SCID mice. A, Representative tumors removed from the mice of each group are shown. B, The growth curves of subcutaneous xenografts of Omm1 cells were plotted. *, P < 0.05; **, P < 0.01; ***, P < 0.0001, Student t test. C, MLN4924 significantly lowered tumor weights on day 15 postinoculation. **, P < 0.01, Student t test. D, H&E staining and IHC analysis with anti-Ki67 and -p27 of tumor tissues from mice. Scale bar: 20 μm. E, MLN4924 administration led to inhibit the neddylation pathway, accumulation in CRL substrates such as p21 and p27, as well as decreased expression of Slug, Bcl-XL, and survivin in xenografted tumor tissues from NOD-SCID mice. Cell lysates prepared from four xenografts of each group were detected by Western blotting with the indicated antibodies.

Close modal

MLN4924 inhibits CSCs traits through Slug degradation in UM cells

Because proteasome-ubiquitin cascade is involved in self-renewal of CSCs, and CSCs were reported to exist in UM cells (36), we determined whether MLN4924 treatment conferred elimination of CSCs in UM. We first employed melanosphere formation and serially replating assay. As expected, MLN4924 treatment inhibited the self-renewal of spherogenic UM cells (P < 0.05 for all comparisons) in UM cells (Fig. 5A). Aldehyde dehydrogenase activity (ALDH) was a widely accepted biomarker for CSCs in solid tumors (37). We observed that MLN4924 treatment reduced ALDH+ cells by 28.6%, 43%, 2.1%, and 8% in Mel270, 92.1, Omm1, and Omm2.3 cells, respectively (Fig. 5B; Supplementary Fig. S6). Of importance, in vivo limiting dilution assay showed that neddylation inhibition reduced UM CSCs frequency by 98% (Control: 2.31 × 10−6; MLN4924: 3.84 × 10−8; Fig. 5C; Supplementary Table S4). To explore the reason that MLN4924 inhibited CSC properties in UM cells, we determined the levels of epithelial-to-mesenchymal transition (EMT)- and stemness-related transcriptional factors (38). The results demonstrated that the levels of Slug were decreased in the MLN4924-treated UM cells (Fig. 5D).

Figure 5.

MLN4924 impairs properties of CSCs through lowering Slug in UM cells. A, MLN4924 suppressed the formation and serially replating capacity of melanospheres in human UM cells. Twenty-four hours after treated with different concentrations of MLN4924, UM cells (92.1, Mel270, Omm1, and Omm2.3) were harvested and plated in stem cell culture medium in ultra-low attachment 24-well plate (3, 000 cells/well). Melanospheres were counted on day 7. The cells were then harvested and replated for the second and third rounds, respectively; melanospheres were counted on day 7 after each round of culture. B, MLN4924 reduced the percentage of ALDH+ cells in UM. Mel270 and 92.1 cells were treated with MLN4924 for 48 hours, detection of ALDH+ cells was performed. C, Administration of MLN4924 in mice suppressed the frequency of CSCs in Omm1 performed by limiting dilution assay in NOD-SCID mice. Representative image of tumors removed from the mice of each group are shown (left). The frequency of CSCs in UM after MLN4924 treatment is shown (right). D, MLN4924 treatment decreased the levels of Slug. The protein levels of stemness-related proteins in control or MLN4924-treated cells were detected by Western blotting. E, MLN4924 treatment accelerated the turnover rate of Slug protein. UM cells (92.1 and Omm2.3) were treated with MLN4924 (1.0 μmol/L) for 48 hours, then exposed to 50 μg/mL of cycloheximide for the indicated durations, Slug expression was detected by Western blotting analysis (upper). The Western blots were quantified by densitometry (bottom). Levels of Slug were normalized to the levels of relevant β-actin, and then normalized relative controls incubated with DMSO containing medium. The graphs (bottom) were one representative result from three independent experiments. F, MG132 treatment rescued MLN4924-mediated decrease of Slug protein level. G, MLN4924 treatment increased the levels of E3 ligase FBXO11. 92.1 and Omm2.3 cells were treated with concentrations of MLN4924 for 48 hours, FBXO11 expression was detected by Western blotting analysis. H, Enforced expression of FBXO11 inhibited protein levels of Slug. Omm2.3 cells were transfected with FBXO11-pcDNA3.1 and FBXO11-ΔFbox-pcDNA3.1 constructs for 48 hours, Slug protein level was then examined by Western blotting analysis. *, P < 0.05; *** P < 0.001, Student t test for results in B and E. *, P < 0.05; **, P < 0.01; ***, P < 0.0001, one-way ANOVA, post hoc intergroup comparisons, Tukey test for results in A.

Figure 5.

MLN4924 impairs properties of CSCs through lowering Slug in UM cells. A, MLN4924 suppressed the formation and serially replating capacity of melanospheres in human UM cells. Twenty-four hours after treated with different concentrations of MLN4924, UM cells (92.1, Mel270, Omm1, and Omm2.3) were harvested and plated in stem cell culture medium in ultra-low attachment 24-well plate (3, 000 cells/well). Melanospheres were counted on day 7. The cells were then harvested and replated for the second and third rounds, respectively; melanospheres were counted on day 7 after each round of culture. B, MLN4924 reduced the percentage of ALDH+ cells in UM. Mel270 and 92.1 cells were treated with MLN4924 for 48 hours, detection of ALDH+ cells was performed. C, Administration of MLN4924 in mice suppressed the frequency of CSCs in Omm1 performed by limiting dilution assay in NOD-SCID mice. Representative image of tumors removed from the mice of each group are shown (left). The frequency of CSCs in UM after MLN4924 treatment is shown (right). D, MLN4924 treatment decreased the levels of Slug. The protein levels of stemness-related proteins in control or MLN4924-treated cells were detected by Western blotting. E, MLN4924 treatment accelerated the turnover rate of Slug protein. UM cells (92.1 and Omm2.3) were treated with MLN4924 (1.0 μmol/L) for 48 hours, then exposed to 50 μg/mL of cycloheximide for the indicated durations, Slug expression was detected by Western blotting analysis (upper). The Western blots were quantified by densitometry (bottom). Levels of Slug were normalized to the levels of relevant β-actin, and then normalized relative controls incubated with DMSO containing medium. The graphs (bottom) were one representative result from three independent experiments. F, MG132 treatment rescued MLN4924-mediated decrease of Slug protein level. G, MLN4924 treatment increased the levels of E3 ligase FBXO11. 92.1 and Omm2.3 cells were treated with concentrations of MLN4924 for 48 hours, FBXO11 expression was detected by Western blotting analysis. H, Enforced expression of FBXO11 inhibited protein levels of Slug. Omm2.3 cells were transfected with FBXO11-pcDNA3.1 and FBXO11-ΔFbox-pcDNA3.1 constructs for 48 hours, Slug protein level was then examined by Western blotting analysis. *, P < 0.05; *** P < 0.001, Student t test for results in B and E. *, P < 0.05; **, P < 0.01; ***, P < 0.0001, one-way ANOVA, post hoc intergroup comparisons, Tukey test for results in A.

Close modal

In order to elucidate the mechanism by which MLN4924 regulates Slug, we examined whether neddylation inhibition affected protein stability of Slug. Time-chase experimental results showed that MLN4924 treatment led to 1.9- and 1.6-fold increase in turnover rates of Slug protein in 92.1 and Omm2.3 cells, respectively (Fig. 5E). The MLN4924-mediated decrease in Slug protein was effectively rescued by the proteasome inhibitor MG132 treatment (Fig. 5F). Because FBXO11 was the bona fide E3 ligase of Slug (11), we examined FBXO11 expression. Consistent with the previous study (35), MLN4924 treatment increased the levels of FBXO11 (Fig. 5G). Overexpression of FBXO11 but not the F-box deletion mutant of FBXO11 (FBXO11-ΔF) decreased Slug protein level (Fig. 5H), suggesting that the amino-terminal F-box domain of FBXO11 may be essential for Slug protein degradation. Taken together, the data suggest that MLN4924 decreases Slug through protein degradation in UM cells.

Slug is fundamental for MLN4924-mediated elimination of CSCs

To functionally characterize the role of Slug in MLN4924-mediated elimination of CSCs in UM cells, we first analyzed the impact of ectopic expression of Slug on stemness traits. The results showed that Slug overexpression increased the ability of melanosphere formation and serially replating in CSC culture medium compared with the cells transfected with empty vector (P < 0.001; Supplementary Fig. S7A-C). Similarly, ectopic expression of Slug in UM cells increased 1.6- and 4.5-fold the subpopulation of ALDH+ cells (Supplementary Fig. S7D and E). Conversely, Slug knockdown decreased the ability of melanosphere formation and serially replating (P < 0.001) as well as percentage of ALDH+ cells by 87% and 70% in 92.1 and Omm2.3 cells, respectively (Supplementary Fig. S7F-J). The MLN4924-mediated decrease in melanosphere formation was rescued by forced expression of Slug in UM cells (Supplementary Fig. S7K). Collectively, these results indicate that Slug positively regulates CSC properties, and that decreasing Slug by MLN4924 confers eradication of CSCs in UM cells.

MLN4924 diminishes migration and invasion of human UM cells

It was previously reported that MLN4924 blocked proliferation and migration of UM cells in a zebrafish xenograft model (17). By using wound-healing scratch assay, we similarly found the inhibitory effect of MLN4924 on the scratch healing ability of 92.1 and Omm2.3 cells (Supplementary Fig. S8A). Transwell Boyden chamber assay further revealed that the migration and invasion capacities were suppressed in the MLN4924-treated UM cells, respectively (P < 0.001; Supplementary Fig. S8B and C). Western blotting analysis showed that the levels of metastasis-associated MMP-9 and MMP-2 were decreased after MLN4924 treatment (Supplementary Fig. S8D). Taken together, our data suggest that MLN4924 diminishes migration and invasion of human UM cells.

Neddylation inhibition by MLN4924 suppresses hepatic metastasis in UM

Using a liver metastasis mouse model by intrasplenic injection of Omm2.3-Luc cells in NSI mice, we found that MLN4924 treatment reduced bioluminescence signal by 76% in liver (P < 0.01; Fig. 6A). H&E staining of liver sections indicated 55% decrease in the number of metastatic nodules in livers of the MLN4924-treated mice (P < 0.01; Fig. 6B). These results suggest that MLN4924 treatment reduces hepatic metastasis in UM. Of note, we observed that the microvascular density was decreased by 74% (P < 0.05) upon MLN4924 treatment (Fig. 6C), indicating impaired angiogenesis. Because the conditioned medium (CM) derived from cancer cells can promote the migration and tube formation of HUVECs (24), we examined the effect of the CM derived from MLN4924-treated UM cells. The results showed that the ability of migration (decreased by 44%–74% and 51%–69%) and tube formation (decreased by 73%–86% and 74%–88%) of HUVECs when exposed to the CM derived from MLN4924-treated Omm1 and Omm2.3 cells, respectively (Fig. 6D and E). With an independent approach of CAM assay, we noted that the CM derived from MLN4924-treated UM cells caused 55% to 70% reduction of angiogenesis when compared with the CM derived from DMSO-treated UM cells (Fig. 6F). Taken together, these results suggest that the neddylation pathway blockade impairs angiogenesis.

Figure 6.

MLN4924 inhibits hepatic metastasis of UM cells by attenuating VEGF-C secretion. A, Representative images and quantitative analysis of liver metastasis measured by luciferase-based bioluminescence imaging in NSI mice in which Omm2.3-luc cells were inoculated intrasplenic injection. B, H&E staining of liver section and quantification of liver metastatic nodules. Scale bar: 500 μm (left), 200 μm (right). C, MLN4924 inhibited angiogenesis in vivo as indicated by the microvascular density after IHC staining of liver section with anti-CD31 antibody. Scale bar: 50 μm. D, Representative images and quantitative analysis of migrated HUVEC cells. Twenty-four hours after cultured with addition of the conditioned medium (CM) derived from UM cells treated with MLN4924, the HUVEC cells underwent transwell migration assay. Scale bar: 200 μm. E, Representative images and quantitative analysis of HUVECs tube formation cultured on matrigel-coated plates with CM derived from UM cells after treatment with MLN4924. Scale bar: 200 μm. F, Representative images and quantitative analysis of blood vessels of chick embryo CAM with CM derived from Omm2.3 cells after treatment with MLN4924. G, MLN4924 inhibited transcription of VEGF-C gene in UM cells as measured by qRT-PCR assay. H, The levels of secretory VEGF-C protein in the CM collected from UM cells were detected by ELISA. I and J, Overexpression of VEGF-C attenuated MLN4924 treatment-mediated HUVEC migration and tube formation. K and L, Treatment with neutralizing anti-VEGF-C antibody enhanced MLN4924 treatment-mediated HUVEC migration and tube formation. M, A proposed working model of neddylation blockade induces apoptosis and diminishes organ-specifically hepatic metastasis in UM. NAE1 inhibition by MLN4924 disturbed protein homeostasis, lowers Bcl-XL to induce intrinsic apoptosis, and elicits DNA damage due to ROS generation to induce cell cycle arrest in UM cells. Neddylation inhibition diminishes hepatic metastasis by dampening Slug-dependent features of CSCs and disrupting niche secretion of NF-κB-mediated VEGF-C and its dependent angiogenesis in UM.

Figure 6.

MLN4924 inhibits hepatic metastasis of UM cells by attenuating VEGF-C secretion. A, Representative images and quantitative analysis of liver metastasis measured by luciferase-based bioluminescence imaging in NSI mice in which Omm2.3-luc cells were inoculated intrasplenic injection. B, H&E staining of liver section and quantification of liver metastatic nodules. Scale bar: 500 μm (left), 200 μm (right). C, MLN4924 inhibited angiogenesis in vivo as indicated by the microvascular density after IHC staining of liver section with anti-CD31 antibody. Scale bar: 50 μm. D, Representative images and quantitative analysis of migrated HUVEC cells. Twenty-four hours after cultured with addition of the conditioned medium (CM) derived from UM cells treated with MLN4924, the HUVEC cells underwent transwell migration assay. Scale bar: 200 μm. E, Representative images and quantitative analysis of HUVECs tube formation cultured on matrigel-coated plates with CM derived from UM cells after treatment with MLN4924. Scale bar: 200 μm. F, Representative images and quantitative analysis of blood vessels of chick embryo CAM with CM derived from Omm2.3 cells after treatment with MLN4924. G, MLN4924 inhibited transcription of VEGF-C gene in UM cells as measured by qRT-PCR assay. H, The levels of secretory VEGF-C protein in the CM collected from UM cells were detected by ELISA. I and J, Overexpression of VEGF-C attenuated MLN4924 treatment-mediated HUVEC migration and tube formation. K and L, Treatment with neutralizing anti-VEGF-C antibody enhanced MLN4924 treatment-mediated HUVEC migration and tube formation. M, A proposed working model of neddylation blockade induces apoptosis and diminishes organ-specifically hepatic metastasis in UM. NAE1 inhibition by MLN4924 disturbed protein homeostasis, lowers Bcl-XL to induce intrinsic apoptosis, and elicits DNA damage due to ROS generation to induce cell cycle arrest in UM cells. Neddylation inhibition diminishes hepatic metastasis by dampening Slug-dependent features of CSCs and disrupting niche secretion of NF-κB-mediated VEGF-C and its dependent angiogenesis in UM.

Close modal

Given the critical role of VEGF in both angiogenesis and lymphangiogenesis (39) and the existence of VEGF in the CM of UM cells (40), we examined the impact of MLN4924 on the secreting ability of VEGF in UM cells. Among the all tested five angiogenic factors (VEGF-A, VEGF-B, VEGF-C, VEGF-D, and b-FGF) by qRT-PCR analysis, only the mRNA level of VEGF-C was inhibited in MLN4924-treated Omm1 and Omm2.3 cells (Fig. 6G; Supplementary Fig. S9A). The mRNA level of VEGF-A was undetectable (data not shown). We therefore chose VEGF-C in the subsequent experiments. ELISA assay showed that the protein levels of VEGF-C in the CM derived from MLN4924-treated UM cells decreased by up to 70% (Fig. 6H). Ectopic overexpression of VEGF-C cDNA in UM cells rescued, while treatment with neutralizing anti-VEGF-C antibody or silencing VEGF-C augmented, the inhibitory effect of CM derived from the MLN4924-treated UM cells on migration and matrigel tube formation of HUVECs (Fig. 6I–L; Supplementary Fig. S9B–G). These results indicated that MLN4924 suppresses angiogenesis by impairing the VEGF-C secretion in UM.

Given that NF-κB can bind the promoter of VEGF-C gene to initiate its transcription (39), and that treatment with MLN4924 decreases NF-κB transcriptional activity (Fig. 2E; ref. 30), we examined whether NF-κB was a mediator of MLN4924-mediated suppression in VEGF-C. Luciferase reporter assay showed that MLN4924 reduced VEGF-C gene promoter activity by 49% to 72% and 39% to 52% in Omm1 and Omm2.3 cells, respectively (Supplementary Fig. S9H). Overexpression of p65 increased 2.8- and 3.3-fold VEGF-C mRNA levels, and rescued VEGF-C decrease in MLN4924-treated UM cells (Supplementary Fig. S9I). The results suggest that NF-κB is responsible for MLN4924-mediated suppression in VEGF-C.

In the present study, we found that NAE1 expression was readily detectable in UM cells, and that inhibition of the neddylation pathway by MLN4924 suppressed the proliferation, survival, migration and invasion, and hepatic metastasis. The inhibitory effect of neddylation blockade on proliferation, which was confirmed by xenografted UM tumor in NOD-SCID mice, was involved in activation of ATM-Chk1-Cdc25C DDR, and G2–M phase arrest. Moreover, MLN4924 treatment eliminated CSCs with stemness-related transcriptional factor Slug suppressed, and disturbed the paracrine secretion of VEGF-C and its dependent angiogenesis. With an NSI mouse model, we identified that MLN4924 inhibited hepatic metastasis in UM.

Neddylation inhibition impairs maintenance of CSCs

We demonstrated that MLN4924 suppressed CSC properties in UM. To our knowledge, this is the first study to provide in vitro and in vivo evidence to demonstrate that inhibition of the neddylation pathway eliminates CSCs in solid tumors. Consistent with our findings, Knorr and colleagues showed that in vitro treatment of MLN4924 exerts a cytotoxic effect on myelodysplastic syndrome and acute myelogenous leukemia stem and progenitor cell populations (41). In contrast, Zhou and colleagues reported that MLN4924 at low nanomolar concentrations (0–100 nmol/L) exerted a stimulating effect on tumorsphere formation of non-small cell lung cancer cells when cultured in serum-free medium (42). The discrepancy may be attributed to their serum-starved culture condition, suggesting that blockage of neddylation modification to regulate CSC self-renewal may be serum-, drug concentration-, and cancer type-dependent. At this view point, we carefully and systemically evaluated the functional parameters of CSCs (e.g., ALDH+ cells, melanosphere and serially replating capacity, and the frequency of CSCs) in UM. The results unequivocally revealed that the CSC features were diminished after MLN4924 treatment.

Neddylation inhibition diminishes Slug

Previous studies have demonstrated that Slug alone or in collaboration with transcriptional factor SOX9 is required to determine the characteristics of CSCs in breast and ovarian cancer (43). However, the role of Slug in maintaining the self-renewal of UM CSCs has not been reported. By screening the EMT- and stemness-related proteins in UM cells, we found that the change of Slug is most impressive in the MLN4924-treated UM cells. We therefore determined the role of Slug in the MLN4924-mediated elimination of CSCs in UM. Our results including Slug cDNA rescue experiments indicated that Slug is essential for sustaining the CSC properties in UM. Mechanistically, MLN4924 treatment leads to the degradation of Slug through the E3 ubiquitin ligase FBXO11, which was consistent with the previous report (35).

Blockade of neddylation disturbs paracrine secretion of VEGF-C and its dependent angiogenesis

In the complex colonization process, establishment of tumors' own angiogenesis may be a turning point. Regarding UM, we found that VEGF-C is a mediator of the angiogenesis in liver. This is supported by several lines of evidence. First, the CM derived from UM cells can promote angiogenesis, indicating that UM cells may secrete certain cytokine(s). Second, MLN4924 treatment significantly attenuated the UM cells secreting such cytokine(s). Third, ectopic overexpression of VEGF-C decreased the suppression effect of MLN4924. Fourth, treatment with neutralizing antibody against VEGF-C suppressed the angiogenesis-promoting effect of UM CM and potentiated the effect MLN4924. Finally, MLN4924 significantly decreased the hepatic metastasis and angiogenesis in mice.

Mechanistically, we found that NF-κB p65 was critical in the MLN4924-mediated inhibition of VEGF-C gene transcription in UM cells. MLN4924 treatment led to accumulation of IκBα, which elicited p65 degradation in UM cells, which was consistent with the precious report in lymphoma cells (39). In the canonical NF-κB pathway, IκBα phosphorylation at S32 and S36 and the resultant polyubiquitination and degradation of IκBα protein are critical steps for nuclear localization of p65. Polyubiquitination of IκBα is regulated by SCFβTRCP, which is controlled by NAE (44). Consistent with this notion, a dose-dependent IκBα phosphorylation at S32 and S36 was noted in the MLN4924-treated UM cells.

Blockade of neddylation lowers Bcl-XL to induce intrinsic apoptosis

Proliferation and evading apoptosis are also critical for forming overt metastases as well as growth of primary tumor. We found that MLN4924 at nanomolar concentrations exert broad tumoricidal activity across a panel of UM cells. Phase I studies of pevonedistat (MLN4924) showed that maximal plasma concentration (Cmax) reached to 2.7 to 8.1 μmol/L at maximum tolerated dose (45), which is sufficient to kill UM cells, as extrapolated from our data.

MLN4924 treatment induces apoptosis with mitochondrial damage in UM cells. Bcl-XL exhibited a marked decrease in the MLN4924-treated UM cells. In line with our findings, Nimati and colleagues reported that Bcl-XL is an attractive therapeutic target in UM patient-derived xenografts (46). Targeting Bcl-XL may have a particular implication because BCL2L1 overexpression is a poor prognostic factor based on the analysis of UM patients obtained from the TCGA database.

Neddylation blockade elicits DSBs and induces G2–M phase arrest in UM cells

Our results highlighted that prolonged (up to 48 hours) exposure of UM cells to MLN4924 changed cell cycle distribution featured G2–M phase also observed in pancreatic cancer (47), and gastric cancer cells (48), but not S phase accumulation. The increased γ-H2AX together with increased phospho-ATM and phospho-Chk1 unequivocally indicated that MLN4924 induced DSBs, because ATM-Chk1-Cdc25C axis is predominantly responsible for DSBs. However, MLN4924 treatment also caused phosphorylation in ATR and Chk2, suggesting occurrence of SSBs. Consistent with previous report (35), MLN4924-induced DNA damage due to ROS generation in UM cells. Taken together, MLN4924 causes accumulation of CRL substrates, which are responsible for G2–M phase arrest and DDR.

BRCA1 associateprotein-1 (BAP1) mutations are reportedly present in the majority of metastatic UM (49). BAP1, a de-ubiquitinating enzyme, is a member of the polycomb-group proteins (PcG) of highly conserved transcriptional repressors required for long-term silencing of genes that regulate cell fate determination and stemness features. It is possible that mutation of BAP1 is involved in the efficacy of neddylation inhibition in metastatic UM. However, silencing BAP1 or forced overexpressing BAP1 in Mel270 and Omm2.3 cells did not alter the sensitivity to MLN4924 (data not shown).

In conclusion, neddylation inhibition dampens Slug to eliminate CSCs, disturbs paracrine secretion of VEGF-C and its dependent angiogenesis. Blockade of neddylation lowers Bcl-XL to induce intrinsic apoptosis, and elicits DNA damage to induce cell cycle arrest in UM cells (proposed model, Fig. 6M). From a therapeutic standpoint, our work presents novel insights into molecular strategies to effectively treat organ-specifically hepatic metastasis in UM. These findings warrant a clinical trial of pevonedistat in hepatic metastatic patients with UM.

No potential conflicts of interest were disclosed.

Conception and design: Y. Jin, J. Pan

Development of methodology: Y. Jin, Y. Wang, B. Jin, J. Zhou, J. Zhang, J. Pan

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Jin, P. Zhang, Y. Wang, B. Jin, J. Zhou, J. Zhang, J. Pan

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Jin, Y. Wang, B. Jin, J. Pan

Writing, review, and/or revision of the manuscript: Y. Jin, J. Pan

Study supervision: J. Pan

The authors thank Jager MJ (Leiden University Medical Center, Leiden, the Netherlands) for generously providing 92.1, Mel270, Omm1, and Omm2.3 cells. This study was supported by grants from National Natural Science Funds (No. U1301226 and No. 81025021 to J. Pan); the Research Foundation of Education Bureau of Guangdong Province, China (Grant cxzd1103 to J. Pan). Present address of Yanli Jin: Institute of Tumor Pharmacology, College of Pharmacy, Jinan University, Guangzhou, China.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Chaffer
CL
,
Weinberg
RA
. 
A perspective on cancer cell metastasis
.
Science
2011
;
331
:
1559
64
.
2.
Massague
J
,
Obenauf
AC
. 
Metastatic colonization by circulating tumour cells
.
Nature
2016
;
529
:
298
306
.
3.
Oskarsson
T
,
Batlle
E
,
Massague
J
. 
Metastatic stem cells: sources, niches, and vital pathways
.
Cell Stem Cell
2014
;
14
:
306
21
.
4.
Obenauf
AC
,
Massague
J
. 
Surviving at a distance: organ specific metastasis
.
Trends Cancer
2015
;
1
:
76
91
.
5.
Amaro
A
,
Gangemi
R
,
Piaggio
F
,
Angelini
G
,
Barisione
G
,
Ferrini
S
, et al
The biology of uveal melanoma
.
Cancer Metastasis Rev
2017
;
36
:
109
40
.
6.
Singh
AD
,
Turell
ME
,
Topham
AK
. 
Uveal melanoma: trends in incidence, treatment, and survival
.
Ophthalmology
2011
;
118
:
1881
5
.
7.
Carvajal
RD
,
Schwartz
GK
,
Tezel
T
,
Marr
B
,
Francis
JH
,
Nathan
PD
. 
Metastatic disease from uveal melanoma: treatment options and future prospects
.
Br J Ophthalmol
2017
;
101
:
38
44
.
8.
Augsburger
JJ
,
Correa
ZM
,
Shaikh
AH
. 
Effectiveness of treatments for metastatic uveal melanoma
.
Am J Ophthalmol
2009
;
148
:
119
27
.
9.
Royer-Bertrand
B
,
Torsello
M
,
Rimoldi
D
,
El Zaoui
I
,
Cisarova
K
,
Pescini-Gobert
R
, et al
Comprehensive genetic landscape of uveal melanoma by whole-genome sequencing
.
Am J Hum Genet
2016
;
99
:
1190
8
.
10.
Yu
FX
,
Luo
J
,
Mo
JS
,
Liu
G
,
Kim
YC
,
Meng
Z
, et al
Mutant Gq/11 promote uveal melanoma tumorigenesis by activating YAP
.
Cancer Cell
2014
;
25
:
822
30
.
11.
Zheng
H
,
Shen
M
,
Zha
YL
,
Li
W
,
Wei
Y
,
Blanco
MA
, et al
PKD1 phosphorylation-dependent degradation of SNAIL by SCF-FBXO11 regulates epithelial-mesenchymal transition and metastasis
.
Cancer Cell
2014
;
26
:
358
73
.
12.
Stow
JL
,
Murray
RZ
. 
Intracellular trafficking and secretion of inflammatory cytokines
.
Cytokine Growth Factor Rev
2013
;
24
:
227
39
.
13.
Watson
IR
,
Irwin
MS
,
Ohh
M
. 
NEDD8 pathways in cancer, Sine Quibus Non
.
Cancer Cell
2011
;
19
:
168
76
.
14.
Soucy
TA
,
Smith
PG
,
Milhollen
MA
,
Berger
AJ
,
Gavin
JM
,
Adhikari
S
, et al
An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer
.
Nature
2009
;
458
:
732
6
.
15.
Sarantopoulos
J
,
Shapiro
GI
,
Cohen
RB
,
Clark
JW
,
Kauh
JS
,
Weiss
GJ
, et al
Phase I study of the investigational NEDD8-activating enzyme inhibitor Pevonedistat (TAK-924/MLN4924) in patients with advanced solid tumors
.
Clin Cancer Res
2016
;
22
:
847
57
.
16.
Shah
JJ
,
Jakubowiak
AJ
,
O'Connor
OA
,
Orlowski
RZ
,
Harvey
RD
,
Smith
MR
, et al
Phase I study of the novel investigational NEDD8-activating enzyme inhibitor Pevonedistat (MLN4924) in patients with relapsed/refractory multiple myeloma or lymphoma
.
Clin Cancer Res
2016
;
22
:
34
43
.
17.
Brustmann
H.
Expression of cellular apoptosis susceptibility protein in serous ovarian carcinoma: a clinicopathologic and immunohistochemical study
.
Gynecol Oncol
2004
;
92
:
268
76
.
18.
De Waard-Siebinga
I
,
Blom
DJ
,
Griffioen
M
,
Schrier
PI
,
Hoogendoorn
E
,
Beverstock
G
, et al
Establishment and characterization of an uveal-melanoma cell line
.
Int J Cancer
1995
;
62
:
155
61
.
19.
Verbik
DJ
,
Murray
TG
,
Tran
JM
,
Ksander
BR
. 
Melanomas that develop within the eye inhibit lymphocyte proliferation
.
Int J Cancer
1997
;
73
:
470
8
.
20.
Luyten
GP
,
Naus
NC
,
Mooy
CM
,
Hagemeijer
A
,
Kan-Mitchell
J
,
Van Drunen
E
, et al
Establishment and characterization of primary and metastatic uveal melanoma cell lines
.
Int J Cancer
1996
;
66
:
380
7
.
21.
Zhou
J
,
Jin
B
,
Jin
Y
,
Liu
Y
,
Pan
J
. 
The antihelminthic drug niclosamide effectively inhibits the malignant phenotypes of uveal melanoma in vitro and in vivo
.
Theranostics
2017
;
7
:
1447
62
.
22.
Kivela
T
,
Kujala
E
. 
Prognostication in eye cancer: the latest tumor, node, metastasis classification and beyond
.
Eye (Lond)
2013
;
27
:
243
52
.
23.
Kujala
E
,
Damato
B
,
Coupland
SE
,
Desjardins
L
,
Bechrakis
NE
,
Grange
JD
, et al
Staging of ciliary body and choroidal melanomas based on anatomic extent
.
J Clin Oncol
2013
;
31
:
2825
31
.
24.
Liu
L
,
Lin
C
,
Liang
W
,
Wu
S
,
Liu
A
,
Wu
J
, et al
TBL1XR1 promotes lymphangiogenesis and lymphatic metastasis in esophageal squamous cell carcinoma
.
Gut
2015
;
64
:
26
36
.
25.
Jin
Y
,
Zhou
J
,
Xu
F
,
Jin
B
,
Cui
L
,
Wang
Y
, et al
Targeting methyltransferase PRMT5 eliminates leukemia stem cells in chronic myelogenous leukemia
.
J Clin Invest
2016
;
126
:
3961
80
.
26.
Jiang
L
,
Song
L
,
Wu
J
,
Yang
Y
,
Zhu
X
,
Hu
B
, et al
Bmi-1 promotes glioma angiogenesis by activating NF-κB signaling
.
PLoS One
2013
;
8
:
e55527
.
27.
Costa-Silva
B
,
Aiello
NM
,
Ocean
AJ
,
Singh
S
,
Zhang
H
,
Thakur
BK
, et al
Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver
.
Nat Cell Biol
2015
;
17
:
816
26
.
28.
Damato
B
,
Duke
C
,
Coupland
SE
,
Hiscott
P
,
Smith
PA
,
Campbell
I
, et al
Cytogenetics of uveal melanoma: a 7-year clinical experience
.
Ophthalmology
2007
;
114
:
1925
31
.
29.
Shields
CL
,
Furuta
M
,
Thangappan
A
,
Nagori
S
,
Mashayekhi
A
,
Lally
DR
, et al
Metastasis of uveal melanoma millimeter-by-millimeter in 8033 consecutive eyes
.
Arch Ophthalmol
2009
;
127
:
989
98
.
30.
Milhollen
MA
,
Traore
T
,
Adams-Duffy
J
,
Thomas
MP
,
Berger
AJ
,
Dang
L
, et al
MLN4924, a NEDD8-activating enzyme inhibitor, is active in diffuse large B-cell lymphoma models: rationale for treatment of NF-κB-dependent lymphoma
.
Blood
2010
;
116
:
1515
23
.
31.
Chou
TC
,
Talalay
P
. 
Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors
.
Adv Enzyme Regul
1984
;
22
:
27
55
.
32.
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
.
33.
Zhou
L
,
Chen
S
,
Zhang
Y
,
Kmieciak
M
,
Leng
Y
,
Li
L
, et al
The NAE inhibitor pevonedistat interacts with the HDAC inhibitor belinostat to target AML cells by disrupting the DDR
.
Blood
2016
;
127
:
2219
30
.
34.
Zhou
BB
,
Elledge
SJ
. 
The DNA damage response: putting checkpoints in perspective
.
Nature
2000
;
408
:
433
9
.
35.
Nawrocki
ST
,
Kelly
KR
,
Smith
PG
,
Espitia
CM
,
Possemato
A
,
Beausoleil
SA
, et al
Disrupting protein NEDDylation with MLN4924 is a novel strategy to target cisplatin resistance in ovarian cancer
.
Clin Cancer Res
2013
;
19
:
3577
90
.
36.
Kalirai
H
,
Damato
BE
,
Coupland
SE
. 
Uveal melanoma cell lines contain stem-like cells that self-renew, produce differentiated progeny, and survive chemotherapy
.
Invest Ophthalmol Vis Sci
2011
;
52
:
8458
66
.
37.
Giraud
J
,
Failla
LM
,
Pascussi
JM
,
Lagerqvist
EL
,
Ollier
J
,
Finetti
P
, et al
Autocrine secretion of progastrin promotes the survival and self-renewal of colon cancer stem-like cells
.
Cancer Res
2016
;
76
:
3618
28
.
38.
Asnaghi
L
,
Gezgin
G
,
Tripathy
A
,
Handa
JT
,
Merbs
SL
,
van der Velden
PA
, et al
EMT-associated factors promote invasive properties of uveal melanoma cells
.
Mol Vis
2015
;
21
:
919
29
.
39.
Lin
C
,
Song
L
,
Liu
A
,
Gong
H
,
Lin
X
,
Wu
J
, et al
Overexpression of AKIP1 promotes angiogenesis and lymphangiogenesis in human esophageal squamous cell carcinoma
.
Oncogene
2015
;
34
:
384
93
.
40.
Notting
IC
,
Missotten
GS
,
Sijmons
B
,
Boonman
ZF
,
Keunen
JE
,
van der Pluijm
G
. 
Angiogenic profile of uveal melanoma
.
Curr Eye Res
2006
;
31
:
775
85
.
41.
Knorr
KL
,
Finn
LE
,
Smith
BD
,
Hess
AD
,
Foran
JM
,
Karp
JE
, et al
Assessment of drug sensitivity in hematopoietic stem and progenitor cells from acute myelogenous leukemia and myelodysplastic syndrome ex vivo
.
Stem Cells Transl Med
2017
;
6
:
840
50
.
42.
Zhou
X
,
Tan
M
,
Nyati
MK
,
Zhao
Y
,
Wang
G
,
Sun
Y
. 
Blockage of neddylation modification stimulates tumor sphere formation in vitro and stem cell differentiation and wound healing in vivo
.
Proc Natl Acad Sci U S A
2016
;
113
:
E2935
44
.
43.
Guo
W
,
Keckesova
Z
,
Donaher
JL
,
Shibue
T
,
Tischler
V
,
Reinhardt
F
, et al
Slug and Sox9 cooperatively determine the mammary stem cell state
.
Cell
2012
;
148
:
1015
28
.
44.
Read
MA
,
Brownell
JE
,
Gladysheva
TB
,
Hottelet
M
,
Parent
LA
,
Coggins
MB
, et al
Nedd8 modification of cul-1 activates SCF(β(TrCP))-dependent ubiquitination of IκBα
.
Mol Cell Biol
2000
;
20
:
2326
33
.
45.
Bhatia
S
,
Pavlick
AC
,
Boasberg
P
,
Thompson
JA
,
Mulligan
G
,
Pickard
MD
, et al
A phase I study of the investigational NEDD8-activating enzyme inhibitor pevonedistat (TAK-924/MLN4924) in patients with metastatic melanoma
.
Invest New Drugs
2016
;
34
:
439
49
.
46.
Nemati
F
,
de Montrion
C
,
Lang
G
,
Kraus-Berthier
L
,
Carita
G
,
Sastre-Garau
X
, et al
Targeting Bcl-2/Bcl-XL induces antitumor activity in uveal melanoma patient-derived xenografts
.
PLoS One
2014
;
9
:
e80836
.
47.
Wei
D
,
Li
H
,
Yu
J
,
Sebolt
JT
,
Zhao
L
,
Lawrence
TS
, et al
Radiosensitization of human pancreatic cancer cells by MLN4924, an investigational NEDD8-activating enzyme inhibitor
.
Cancer Res
2012
;
72
:
282
93
.
48.
Lan
H
,
Tang
Z
,
Jin
H
,
Sun
Y
. 
Neddylation inhibitor MLN4924 suppresses growth and migration of human gastric cancer cells
.
Sci Rep
2016
;
6
:
24218
.
49.
Harbour
JW
,
Onken
MD
,
Roberson
ED
,
Duan
S
,
Cao
L
,
Worley
LA
, et al
Frequent mutation of BAP1 in metastasizing uveal melanomas
.
Science
2010
;
330
:
1410
3
.