Neddylation Blockade Diminishes Hepatic Metastasis by ... · Translational Cancer Mechanisms and Therapy Neddylation Blockade Diminishes Hepatic Metastasis by Dampening Cancer Stem-Like

Translational Cancer Mechanisms and Therapy

Neddylation Blockade Diminishes HepaticMetastasis by Dampening Cancer Stem-LikeCells and Angiogenesis in Uveal MelanomaYanli Jin, Ping Zhang, Yun Wang, Bei Jin, Jingfeng Zhou, Jing Zhang,and Jingxuan Pan

Abstract

Purpose: Liver metastasis is the major and direct cause ofdeath in patients with uveal melanoma (UM). There is noeffective therapy for patients with metastatic UM. Improvedtreatments of hepaticmetastatic patientswithUMwere urgent-ly needed. Inspired by readily detectable key components inthe neddylation pathway in UM cells, we aimed at exploringwhether neddylation pathwaywas a therapeutic target for livermetastatic UM.

Experimental Design: Expression of key proteins in theneddylation pathway inUMwas detected byWestern blotting,real-time quantitative RT-PCR (qRT-PCR), and immunohis-tochemical staining. Cellular proliferation, apoptosis, cellcycle, migration, and cancer stem-like cells (CSCs) propertieswere examined upon treatment with MLN4924, a potent andselective NAE inhibitor. Antitumor activity and frequency ofCSCswere determined byusing aNOD-SCIDmouse xenograftmodel. 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 MLN4924repressed the CSCs properties in UM (capacities of tumor-sphere formation and serially replating, aldehyde dehydro-genase-positive cells, and frequency of CSC) through Slugprotein degradation. MLN4924 treatment disturbed theparacrine secretion of NF-kB-mediated VEGF-C and itsdependent angiogenesis. The inhibitory effect of neddyla-tion blockade on proliferation, which was confirmed byxenografted UM tumor in NOD-SCID mice, was involved inactivation of ATM-Chk1-Cdc25C DNA damage response,and G2–M phase arrest. Neddylation inhibition profoundlyinhibited hepatic metastasis in UM.

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

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

IntroductionMetastasis is themajor anddirect cause of death inpatientswith

cancer at the terminal stage. Unfortunately, there are no effectivetherapies for metastatic patients. The complicated metastasisprocess is principally composed of intravasation, circulation,extravasation, and colonization in the target organ (1). Intravasa-tion is referred to as the process that the cancer cells in the primarytumor sites break out of physical barriers in the surroundingtissues, and enter the blood or lymphatic stream to becomecirculating tumor cells (CTCs; ref. 2). Only a small portion ofCTCs that acquire stemness features can successfully escape anoi-kis, immune response, and shear stress (2). The CTCs undergoextravasation in which they leave from the bloodstream fordistant 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 even-tually form clinically overt metastatic foci. CTCs and MICs sharemany features (e.g., self-renewal, quiescence, and asymmetricdivision) with cancer stem-like cells (CSC; ref. 3).

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

UM biologically distinct from cutaneous melanoma mayprovide a naturally clinical phenotype and typical example oforgan-specifically liver metastasis (5). UM, the most commonocular malignancy in adults, usually originates from melano-cytes of the choroid, ciliary body, and iris (6). Althoughsuccessful treatment of the primary tumor with enucleation orradiotherapy can be achieved, half of the UM patients developmetastasis. Eighty-five percent of the patients with metastaticUM exhibit single liver metastasis (7). Little is known about theunderlying mechanism of such liver-specific metastasis; there isno effective therapy for patients with metastatic UM, with amedian survival of less than 12 months (8). Whole-genome

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, SunYat-sen University, Guangzhou, China.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Jingxuan Pan, Sun Yat-sen University ZhongshanOphthalmic Center, 54 South Xianlie Road, Guangzhou 510060, People'sRepublic of China, Phone: 86-20-87334279; Fax: 86-20-87334279; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-17-1703

�2017 American Association for Cancer Research.

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sequencing has demonstrated that mutually exclusive gain-of-function mutations in GNAQ or GNA11 are found in 80% ofUM, which may lead to activation of the mitogen-activatedprotein kinase (MAPK) pathway and transcriptional factor YAPpathway 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 metas-tasis of UM.

Most of stemness-related proteins are of short-life withstability sensitive to posttranslational modifications (11). Inaddition, exocytosis, intracellular trafficking, and secretion ofinflammatory cytokines are regulated by protein maturationand posttranslational modifications (12). Neural precursor cellexpressed, developmentally downregulated 8 (NEDD8)-conju-gation, also called neddylation, is first activated by an E1enzyme [NEDD8-activating enzyme (NAE); a heterodimer con-sisting of NAE E1 subunit 1 (NAE1) and ubiquitin-like modifieractivating enzyme 3 (UBA3)], transferred to an E2 enzyme(UBC12), and then conjugated to target substrates throughcullin-RING ligases (CRL; ref. 13). Neddylation controls pro-tein turnover of a number of CRL targets with essential roles inregulation of oncogenic transformation and pathogenesis.MLN4924 (pevonedistat), a potent and selective first-in-classNAE1 inhibitor (14), is currently in phase I clinical trials insome solid tumors and hematologic malignancies (15, 16).

We hypothesized that blocking the neddylation pathwaymight disturb the protein homeostasis between synthesis anddegradation, which might in turn impact the stemness-relatedproteins to diminish CSCs, and decrease secretion of themicroenvironment cytokine(s) in UM, both of which are fun-damental for metastasis (12). The hypothesis was supported bythe recent observation in a zebrafish xenograft model thatMLN4924 inhibits migration and proliferation of UM cells(17) with the mechanism yet unknown. We tested the hypoth-esis 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 agentfor the treatment of UM patients with hepatic metastasis.

Materials and MethodsCell culture

Genetic status of the known altered genes in human primary(92.1, Mel270) andmetastatic (Omm1, Omm2.3) UM cells usedin this study is summarized in Supplementary Table S1. 92.1 cellswere established in Leiden University Medical Center, Leiden, theNetherlands (18). Mel270 and Omm2.3 cells originally derivedfrom patients at the Bascom Palmer Eye Institute, University ofMiami School of Medicine, Florida (19), and Omm1 cells wereestablished by Luyten G at Rotterdam University Hospital, theNetherlands (20). TheseUMcell lineswere cultured inRPMI1640medium (Invitrogen) supplementedwith 10% fetal bovine serum(Hyclone) and 2 mmol/L L-glutamine (7, 21) and authenticatedinHAKEGeneticsCo., Ltd. byusing short tandemrepeatmatchinganalysis at August 8, 2017. The cells were kept at 37�C in ahumidified incubator with 5% CO2. No mycoplasma (Thermo-Fisher Scientific) contamination was detected.

Tumor samples of patients with UMHuman tissues from primary tumors were collected from

enrolled patients (n¼ 45) with UM in The Sun Yat-sen UniversityZhongshan Ophthalmic Center during 2012–2015, afterinformed consent according to the institutional guidelines andthe Declaration of Helsinki principles. The seventh edition of theAmerican Joint Committee on Cancer tumor, node, metastasis(TNM7) classification for eye cancer was used (22, 23). Detailedinformation of UM patients is shown in Supplementary Table S2.The studies were approved by Institutional Review Board, TheSun Yat-sen University Zhongshan Ophthalmic Center.

Immunohistochemical (IHC) staining and evaluation of NAE1IHC staining with anti-NAE1 antibody and quantification of

NAE1 expression were performed as described (17, 21, 24), withdetails provided in the Supplementary information. The associ-ation between NAE1 expression and clinicopathologic features inUM patients is summarized in Supplementary Table S3.

Cell viability, colony-formation, apoptosis, cell cycle, Westernblotting, real-time quantitative RT-PCR, luciferase activity,enzyme-linked immunosorbent analysis, retrovirus, andlentivirus infection

All above methods were performed as described previouslyreported (21, 25), with details provided in the Supplementaryinformation.

Tumor xenograft experimentsMale NOD-SCID mice (4–6-week-old) purchased from Vital

River Laboratory Animal Technology Co. and bred at the animalfacility of Sun Yat-sen University were used for evaluation the invivo antineoplastic efficacy of MLN4924 against UM as previouslyreported (21). After 4 weeks, the mice were randomly separatedinto two groups (n ¼ 8 per group) and received treatment withvehicle (10% 2-hydroxypropyl-b-cyclodextrin) or MLN4924(60 mg/kg/day, i.p.) for 14 days. The mice were euthanized, andtumor xenografts were immediately removed, recorded, fixed,and stored at �80�C.

Translational Relevance

The clinical single site-specific organ metastasis pattern incertain types of cancer (e.g., hepatic metastasis in uveal mel-anoma, UM) may provide a study model to realize andcircumvent metastasis. Most of stemness-related proteins areof short-life and sensitive to posttranslationalmodifications ofproteins. We hypothesized that blocking the neddylationpathway may disturb homeostasis of proteins, which areessential for cancer stem–like cells (CSCs), and the microen-vironment cytokine(s) in UM cells. We tested this hypothesisand found thatMLN4924, apotentNEDD8-activating enzyme(NAE) inhibitor, repressed the CSC properties in UM throughSlug protein degradation. MLN4924 treatment disturbed theparacrine secretion of nuclear factor-kB (NF-kB)-mediatedvascular endothelial growth factor-C (VEGF-C) and its depen-dent angiogenesis. Notably, xenografted tumor experiments inmice revealed that MLN4924 abrogated growth and hepaticmetastasis in UM. Our findings validate the neddylationpathway as a promising therapeutic target for the treatmentof patients with hepatic metastasis of UM.

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

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

Liver metastasis mouse modelTwenty-four hours after Omm2.3-luciferase cells (1 � 106 in

50 mL PBS per mouse) were intrasplenically injected into theNOD-scid-IL2Rg�/� (NSI) mice (25, 27), the mice were randomlyseparated into two groups (n ¼ 6 per group) and receivedtreatment with vehicle or MLN4924 (90 mg/kg, bid, i.p.) 5 dayson/2 days off for four cycles. Liver metastasis was detected by exvivo bioluminescence imaging using the IVIS Lumina II (Perki-nElmer) or counted the nodules on liver paraffin sections afterHematoxylin and eosin (H&E) staining. All animal studies wereconducted with the approval of the Sun Yat-sen University Insti-tutional Animal Care and Use Committee.

Statistical analysisAll experiments were performed three times, and results are

reported as mean � standard deviation (SD), unless otherwisestated. GraphPad Prism 5.0 was used for statistical analysis.Comparisons between two groups were analyzed by two-tailedStudent t test and comparisons of multiple groups by one-wayANOVAwith post hoc intergroup comparisonwith Tukey test. AP<0.05 was considered statistically significant.

ResultsNAE1 expression is readily detectable in UM

We first examined certain key proteins involved in globalNEDD8 conjunction in human UM cells. Western blotting resultsindicated that the levels of NAE1, UBA3, and UBC12 were higherin 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 mRNAlevels of NAE1, UBA3, UBC12, and NEDD8 displayed 3.9- to6.0-, 2.9- to 4.1-, 5.0- to 7.9-, and 4.7- to 7.0-fold increase in theUM cells compared with ARPE-19 cells, respectively (P < 0.01,Fig. 1B–E). These results suggest that the readily detectable statusof these key proteins may occur at the transcriptional level.

After verifying the specificity of antibody against NAE1 (Sup-plementary Fig. S1),we conducted IHCanalysis ofUMspecimens.The results showed that no staining of NAE1 in the adjacentnormal tissueswas detected. In contrast, readily detectable expres-sion ofNAE1 scored from low to highwas observed in 77.8% (35/45) patients with UM (Fig. 1F–H). Of importance, NAE1 expres-sion was positively correlated to the largest basal diameter (P ¼0.0373) and thickness (P ¼ 0.0461) of the primary tumors weanalyzed, two important independent predictors of UM meta-static death (28, 29) and the TNM stage (P ¼ 0.0374; Supple-mentary Table S3).

MLN4924 specifically inhibits the neddylation pathwayTo explore whether the neddylation pathway was a thera-

peutic target for UM, we first examined the specificity ofMLN4924 against the neddylation pathway in UM cells. The

results indicated that MLN4924, but not proteasome inhibitorsMG132 and bortezomib, suppressed the global NEDD8 con-junction or cullin1 neddylation (Fig. 2A). Consequently, thesubstrates of CRLs (e.g., p21 and p27) were accumulated overall treatments; however, cyclin B1, a non-CRL substrate, wasaccumulated upon MG132 and bortezomib treatment butnot upon MLN4924 treatment (Fig. 2A). Similarly, MLN4924treatment increased the protein levels of the CRL substrates(e.g., NRF2, phospho-IkBa) in a concentration-dependentmanner (Fig. 2B). MLN4924 treatment also decreased cullin1neddylation in a time-dependent manner (Fig. 2C). Consistentwith previous report (30), MLN4924 treatment inhibited 70%to 97% and 90% to 95% (P < 0.001) of the NF-kB-dependentreporter gene activity in Mel270 and Omm2.3 cells, respectively(Fig. 2D). These data suggest that MLN4924 specificallyinhibits the neddylation pathway, leading to accumulation ofCRL substrates.

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

MLN4924 induces apoptosis in UM cellsWe next ascertained the ability of MLN4924 to induce apo-

ptosis in UM cells. MLN4924 increased the dead cell populationin a concentration- (Fig. 3A) and time-dependent manner (Sup-plementary Fig. S2A) in UM cells. Western blotting analysisshowed a concentration- (Fig. 3B) and time-dependent (Supple-mentary Fig. S2B) PARP cleavage and caspase-3 activation. Addi-tionally, treatmentwithMLN4924 increased cytochrome c releasefrom the mitochondria into the cytosol detected by immuno-blotting (Fig. 3C) as well as the cell population with loss ofmitochondrial potential (Dcm) detected by flow cytometry (Sup-plementary Fig. S2C). These data together suggest that MLN4924induces mitochondrial damage and triggers intrinsic apoptosispathway.

MLN4924 disturbs balance of Bcl-2 family members inUM cells

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

Because Bcl-XL (a known NF-kB target gene) was overex-pressed in 10% of patients with UM extrapolated from TheCancer Genome Atlas (TCGA) database (http://www.cbioportal.org), which apparently correlated with poor overall survival(P ¼ 0.0174; Fig. 3E). The mRNA levels of BCL2L1 gene werelowered by up to 63.9% and 56.9% in the MLN4924-treatedMel270 and Omm2.3 cells, respectively (SupplementaryFig. S2D). We next examined the role of Bcl-XL in MLN4924-induced apoptotic cell death in UM cells. Forced overexpressionof Bcl-XL attenuated MLN4924-induced apoptosis in UM cellsas reflected by caspase-3 activation (Fig. 3F) and trypan bluestaining cells (19.3% decrease in Mel270, P < 0.01; 10.2%

Neddylation Inhibition Suppresses Hepatic Metastasis in UM

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Figure 1.

NAE1 expression is readily detectable in cells and specimens of human UM. A, Protein levels of NAE1, UBA3, UBC12, cullin1 neddylation, and globalNEDD8 conjugation in human UM cells (e.g., Mel270, 92.1, Omm1, and Omm2.3) and human adult retinal pigmented epithelium (ARPE-19) cells weredetermined 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 proteinexpression of NAE1 was classified into four levels (negative, low, medium, and high). Brown: choroid pigment, Red: NAE1 staining. Scale bar: 200 mm(100�), 100 mm (200�). G, NAE1 expression was increased in UM specimens (n ¼ 45) compared with adjacent normal tissues (n ¼ 14). H, The percentageof 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.

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Figure 2.

MLN4924, a potent and selective inhibitor of NAE, counteracts proliferation of UM cells. A, MLN4924 specifically inhibited the neddylation pathway. HumanUM cells were treated with MLN4924 (1.0 mmol/L), MG132 (20.0 mmol/L), or Bort (bortezomib; 1.0 mmol/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 theexpression of CRL substrates. Human UM cells were treated various concentrations of MLN4924 for 48 hours, cullin1 neddylation and protein levels of CRLsubstrates were determined by Western blotting analysis. C, MLN4924 time-dependently inhibited cullin1 neddylation. D, MLN4924 dramaticallyinhibited the activity of NF-kB-dependent reporter gene in UM cells. Mel270 and Omm2.3 cells were transfected with NF-kB-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 cellsand ARPE-19 cells were treated with escalating concentrations of MLN4924 for 72 hours; cell viability was measured by MTS assay. Dose-response curves from3 independent experiments are shown. F, MLN4924 concentration-dependently suppressed clonogenicity of UM cells in drug-free soft agar culture.






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 flowcytometry after dual staining with Annexin V-FITC/propidium iodide (PI). Representative histograms (top) for 92.1 cells and quantitative analysis (bottom) from threeindependent experiments are shown.Data representmean� SEM. �� ,P<0.01; �� ,P<0.0001, one-wayANOVA,post hoc intergroup comparisons, Tukey test.B,MLN4924induced apoptosis-specific cleavage of PARP and caspase-3 activation in a concentration-dependent manner. UM cells were treated with increasing concentrations ofMLN4924 for 48 hours,Western blotting analysiswas performedwith the specific antibodies, respectively.C,MLN4924 treatment induced release of cytochrome c into thecytosol. Cells were treated with MLN4924 (1.0 mmol/L) for the indicated durations, and the cytosolic fractions extracted with digitonin buffer were subjected toimmunoblotting analysis for cytochrome c. D, Expression of apoptosis-related proteins was analyzed byWestern 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 overallsurvival (bottom).FandG,EctopicBcl-XLexpressionabrogated theMLN4924-inducedapoptosis. Twenty-fourhoursafter transfectedwithemptyvector(pCMV6)orBcl-XL

(pCMV6-BCL2L1) constructs, Mel270 (left) and Omm2.3 (right) cells were exposed to the indicated concentrations of MLN4924 for 48 hours, followed by Westernblotting 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.

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decrease in Omm2.3, P < 0.05; at 1.0 mmol/L treatment;Fig. 3G). Conversely, silencing Bcl-XL by siRNA duplexes poten-tiated the lethal effect of MLN4924 in UM cells (Supple-mentary Fig. S2E and F; increased dead cells by 25.6% and27.9% in Mel270; 23.8% and 24.2% in Omm2.3, P < 0.01; at1.0 mmol/L treatment).

Next, we evaluated the synergistic effect between MLN4924and the conventional chemotherapeutic agent vinblastine inUM cells (17). MTS results showed that combinational treat-ment with MLN4924 and vinblastine synergistically (CIvalues <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 andWestern blotting results suggest that combinational treatmentwith MLN4924 and vinblastine induced enhanced cell apo-ptosis as reflected by the increase of Annexin Vþ cells, PARPcleavage, and caspase-3 activation, respectively (Supplemen-tary Fig. S3B and C).

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

Previous studies have shown that MLN4924 can induceDDR and cell cycle perturbation to inhibit cell growth inseveral types of cancer (32, 33). We first assessed the keycomponents involved in DDR. Western blotting analysisshowed that MLN4924-treated UM cells displayed an appre-ciably increased g-H2AX, an indicative hallmark of DNA dou-ble strand breaks (DSB; Supplementary Fig. S4A). g-H2AX notonly indicated the existence of MLN4924-mediated DSBs but

also reflected the activation of DDR. Indeed, the levels of p53and phospho-p53 (S15) were increased by MLN4924 (Sup-plementary Fig. S4A).

Ataxia telangiectasia-mutated (ATM) kinase and ATM-Rad3-related (ATR) kinase function at the level of sensors and transdu-cers of DNA damage signaling pathway in response to DSBs andsingle strand breaks (SSB), respectively (34). Our results showedthat the phosphorylation of ATMwas concentration-dependentlyincreased in the MLN4924-treated UM cells (Supplementary Fig.S4A), suggesting a steady activation of ATM after MLN4924treatment. A slight and nonconcentration-dependent increase inATR phosphorylation was observed in the cells treated withcertain lower concentrations of MLN4924 (Supplementary Fig.S4A). Thesefindings togetherwith the g-H2AX change further hintthat DSBs may be the major DNA damage form in response toneddylation inhibition.

We also detected DDR checkpoint kinase 1 (Chk1) and Chk2.The results revealed that the phosphorylation of both Chk1 andChk2 was steadily increased by MLN4924 (Supplementary Fig.S4A). These data collectively indicate that neddylation inhibitioninduces DSBs and elicits DDR predominantly by ATM, Chk1, andChk2.

Increased reactive oxygen species (ROS) generation isinvolved in MLN4924 action mechanism and may lead to DNAdamage (35). To explore the reason that MLN4924 inducedDDR in UM, we detected the intracellular ROS levels. ROSgeneration was found to be increased by 1.4- to 2.7-fold inMLN4924-treated Mel270 cells (Supplementary Fig. S4B). Pre-treated with N-acetylcysteine, the ROS scavenger, significantly

Figure 4.

MLN4924 inhibits outgrowth ofxenograft UM cells in NOD-SCID mice.A, Representative tumors removedfrom the mice of each group are shown.B, The growth curves of subcutaneousxenografts of Omm1 cells were plotted.� , P < 0.05; �� , P < 0.01; �� , P < 0.0001,Student t test. C,MLN4924 significantlylowered tumor weights on day 15postinoculation. �� , P < 0.01, Student ttest. D, H&E staining and IHC analysiswith anti-Ki67 and -p27 of tumor tissuesfrom mice. Scale bar: 20 mm. E,MLN4924 administration led to inhibitthe neddylation pathway, accumulationin CRL substrates such as p21 and p27,as well as decreased expression of Slug,Bcl-XL, and survivin in xenograftedtumor tissues fromNOD-SCIDmice. Celllysates prepared from four xenograftsof each group were detected byWestern blotting with the indicatedantibodies.






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prevented MLN4924-mediated DNA damage, as reflected byg-H2AX in UM cells (Supplementary Fig. S4C). These datasuggest that ROS generation may mediate DNA damage in UMcells upon MLN4924 exposure.

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

Flow cytometry analysis showed that MLN4924 treatmentled to increase in G2–M phase population in a concentration-and time-dependent manner in UM cells, respectively (Supple-mentary Fig. S4D and E). Correspondingly, overt decrease ofphospho-histone H3 (S10), a hallmark of M phase, was notedin the MLN4924-treated UM cells, suggesting a stalled G2 phase(Supplementary Fig. S4F). Further analysis of G2–M checkpointmechanism revealed that a remarkable increase of Wee1 (awell-defined CRL substrate and an inhibitor of G2–M phasetransition), phospho-Cdc25C (S216), and phospho-Cdc2(Y15) was observed in the MLN4924-treated UM cells (Sup-plementary Fig. S4F).

Inhibiting Chk1 and Wee1 synergistically potentiates thelethality of MLN4924 in UM cells

We next examined whether MLN4924 was synergistic withMK-8776 (Chk1 inhibitor) and MK-1775 (Wee1 inhibitor). Theresults indicated that the combination between MLN4924 andMK-8776 or MLN4924 and MK-1775 synergistically retardedthe cell growth as measured by MTS assay (SupplementaryFig. S5A) and induced enhanced apoptosis, reflected by enhancedspecific cleavage of PARP as detected by Western blotting analy-sis (Supplementary Fig. S5B) in UM cells.

MLN4924 inhibits outgrowth of xenografted UM cells inNOD-SCID mice

We evaluated the in vivo antineoplastic efficacy of MLN4924against 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 randomlydivided into two groups (n ¼ 8) and received treatmentwith vehicle or MLN4924 for 14 days. The tumor size was muchsmaller in MLN4924-treated group than vehicle-treated mice(Fig. 4A). MLN4924 administration abrogated the tumor out-growth (Fig. 4B). Consistently, tumor weight was decreased by

58%(P<0.01) in theMLN4924-treatedmice (Fig. 4C).MLN4924administration inhibited tumor proliferation as reflected by theexpression of Ki67. Moreover, the expression of p27, a well-known CRL substrate, was increased in tumor tissues from theMLN4924-treated mice (Fig. 4D). Western blotting results of celllysates showed inhibited neddylation pathway with the proteinlevels of CRL substrates p21 and p27 notably increased. Theexpression of Slug, Bcl-XL, and survivin was, however, decreasedin MLN4924-treated group (Fig. 4E), which were consistent withthe in vitro findings. These data demonstrate the in vivo antineo-plastic efficacy of MLN4924.

MLN4924 inhibits CSCs traits through Slug degradation inUM 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 conferredelimination of CSCs in UM. We first employed melanosphereformation and serially replating assay. As expected, MLN4924treatment inhibited the self-renewal of spherogenic UM cells(P < 0.05 for all comparisons) in UM cells (Fig. 5A). Aldehydedehydrogenase activity (ALDH) was a widely accepted biomarkerfor CSCs in solid tumors (37). We observed that MLN4924treatment reduced ALDHþ cells by 28.6%, 43%, 2.1%, and 8%inMel270, 92.1, Omm1, andOmm2.3 cells, respectively (Fig. 5B;Supplementary Fig. S6). Of importance, in vivo limitingdilution assay showed that neddylation inhibition reducedUM CSCs frequency by 98% (Control: 2.31 � 10�6; MLN4924:3.84 � 10�8; Fig. 5C; Supplementary Table S4). To explore thereason that MLN4924 inhibited CSC properties in UM cells, wedetermined the levels of epithelial-to-mesenchymal transition(EMT)- and stemness-related transcriptional factors (38). Theresults demonstrated that the levels of Slug were decreased in theMLN4924-treated UM cells (Fig. 5D).

In order to elucidate the mechanism by which MLN4924regulates Slug, we examined whether neddylation inhibitionaffected protein stability of Slug. Time-chase experimental resultsshowed thatMLN4924 treatment led to 1.9- and 1.6-fold increasein turnover rates of Slug protein in 92.1 and Omm2.3 cells,respectively (Fig. 5E). The MLN4924-mediated decrease in Slugprotein was effectively rescued by the proteasome inhibitorMG132 treatment (Fig. 5F). Because FBXO11 was the bona fideE3 ligase of Slug (11), we examined FBXO11 expression.

Figure 5.MLN4924 impairs properties of CSCs through lowering Slug in UM cells. A, MLN4924 suppressed the formation and serially replating capacity ofmelanospheres 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 ALDHþ cells in UM. Mel270 and 92.1 cells were treated with MLN4924 for 48 hours, detection of ALDHþ cells wasperformed. 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 isshown (right). D, MLN4924 treatment decreased the levels of Slug. The protein levels of stemness-related proteins in control or MLN4924-treated cellswere detected by Western blotting. E, MLN4924 treatment accelerated the turnover rate of Slug protein. UM cells (92.1 and Omm2.3) were treatedwith MLN4924 (1.0 mmol/L) for 48 hours, then exposed to 50 mg/mL of cycloheximide for the indicated durations, Slug expression was detected byWestern blotting analysis (upper). The Western blots were quantified by densitometry (bottom). Levels of Slug were normalized to the levels of relevantb-actin, and then normalized relative controls incubated with DMSO containing medium. The graphs (bottom) were one representative result fromthree independent experiments. F,MG132 treatment rescued MLN4924-mediated decrease of Slug protein level. G,MLN4924 treatment increased the levels ofE3 ligase FBXO11. 92.1 and Omm2.3 cells were treated with concentrations of MLN4924 for 48 hours, FBXO11 expression was detected by Westernblotting analysis. H, Enforced expression of FBXO11 inhibited protein levels of Slug. Omm2.3 cells were transfected with FBXO11-pcDNA3.1 and FBXO11-DFbox-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 forresults 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.






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Consistent with the previous study (35), MLN4924 treatmentincreased the levels of FBXO11 (Fig. 5G). Overexpression ofFBXO11 but not the F-box deletion mutant of FBXO11(FBXO11-DF) decreased Slug protein level (Fig. 5H), suggestingthat the amino-terminal F-box domain of FBXO11may be essen-tial for Slug protein degradation. Taken together, the data suggestthat MLN4924 decreases Slug through protein degradation inUM cells.

Slug is fundamental for MLN4924-mediated eliminationof CSCs

To functionally characterize the role of Slug in MLN4924-mediated elimination of CSCs in UM cells, we first analyzed theimpact of ectopic expression of Slug on stemness traits. Theresults showed that Slug overexpression increased the ability ofmelanosphere formation and serially replating in CSC culturemedium compared with the cells transfected with empty vector(P < 0.001; Supplementary Fig. S7A-C). Similarly, ectopicexpression of Slug in UM cells increased 1.6- and 4.5-fold thesubpopulation of ALDHþ cells (Supplementary Fig. S7D andE). Conversely, Slug knockdown decreased the ability of mel-anosphere formation and serially replating (P < 0.001) as wellas percentage of ALDHþ cells by 87% and 70% in 92.1 andOmm2.3 cells, respectively (Supplementary Fig. S7F-J). TheMLN4924-mediated decrease in melanosphere formation wasrescued by forced expression of Slug in UM cells (Supplemen-tary Fig. S7K). Collectively, these results indicate that Slugpositively regulates CSC properties, and that decreasing Slugby MLN4924 confers eradication of CSCs in UM cells.

MLN4924 diminishes migration and invasion of humanUM cells

It was previously reported that MLN4924 blocked prolifer-ation and migration of UM cells in a zebrafish xenograft model(17). By using wound-healing scratch assay, we similarly foundthe inhibitory effect of MLN4924 on the scratch healing abilityof 92.1 and Omm2.3 cells (Supplementary Fig. S8A). TranswellBoyden chamber assay further revealed that the migration andinvasion capacities were suppressed in the MLN4924-treatedUM cells, respectively (P < 0.001; Supplementary Fig. S8B andC). Western blotting analysis showed that the levels of metas-tasis-associated MMP-9 and MMP-2 were decreased afterMLN4924 treatment (Supplementary Fig. S8D). Taken togeth-

er, our data suggest that MLN4924 diminishes migration andinvasion of human UM cells.

Neddylation inhibition by MLN4924 suppresses hepaticmetastasis in UM

Using a liver metastasis mouse model by intrasplenicinjection of Omm2.3-Luc cells in NSI mice, we found thatMLN4924 treatment reduced bioluminescence signal by 76%in liver (P < 0.01; Fig. 6A). H&E staining of liver sectionsindicated 55% decrease in the number of metastatic nodulesin livers of the MLN4924-treated mice (P < 0.01; Fig. 6B).These results suggest that MLN4924 treatment reduces hepaticmetastasis in UM. Of note, we observed that the microvasculardensity was decreased by 74% (P < 0.05) upon MLN4924treatment (Fig. 6C), indicating impaired angiogenesis. Becausethe conditioned medium (CM) derived from cancer cells canpromote 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 migra-tion (decreased by 44%–74% and 51%–69%) and tube for-mation (decreased by 73%–86% and 74%–88%) of HUVECswhen exposed to the CM derived from MLN4924-treatedOmm1 and Omm2.3 cells, respectively (Fig. 6D and E). Withan independent approach of CAM assay, we noted that theCM derived from MLN4924-treated UM cells caused 55% to70% reduction of angiogenesis when compared with the CMderived from DMSO-treated UM cells (Fig. 6F). Taken togeth-er, these results suggest that the neddylation pathway block-ade impairs angiogenesis.

Given the critical role of VEGF in both angiogenesis andlymphangiogenesis (39) and the existence of VEGF in the CM ofUM cells (40), we examined the impact of MLN4924 on thesecreting ability of VEGF in UM cells. Among the all tested fiveangiogenic factors (VEGF-A, VEGF-B, VEGF-C, VEGF-D, andb-FGF) by qRT-PCR analysis, only the mRNA level of VEGF-Cwas inhibited in MLN4924-treated Omm1 and Omm2.3 cells(Fig. 6G; Supplementary Fig. S9A). The mRNA level of VEGF-Awas undetectable (data not shown). We therefore chose VEGF-Cin the subsequent experiments. ELISA assay showed that theprotein levels of VEGF-C in the CM derived from MLN4924-treated UM cells decreased by up to 70% (Fig. 6H). Ectopicoverexpression of VEGF-C cDNA in UM cells rescued, whiletreatment with neutralizing anti-VEGF-C antibody or silencingVEGF-C augmented, the inhibitory effect of CM derived from

Figure 6.MLN4924 inhibits hepatic metastasis of UM cells by attenuating VEGF-C secretion. A, Representative images and quantitative analysis of liver metastasismeasured by luciferase-based bioluminescence imaging in NSI mice in which Omm2.3-luc cells were inoculated intrasplenic injection. B, H&E staining ofliver section and quantification of liver metastatic nodules. Scale bar: 500 mm (left), 200 mm (right). C, MLN4924 inhibited angiogenesis in vivo asindicated by the microvascular density after IHC staining of liver section with anti-CD31 antibody. Scale bar: 50 mm. D, Representative images and quantitativeanalysis of migrated HUVEC cells. Twenty-four hours after cultured with addition of the conditioned medium (CM) derived from UM cells treated withMLN4924, the HUVEC cells underwent transwell migration assay. Scale bar: 200 mm. E, Representative images and quantitative analysis of HUVECs tubeformation cultured on matrigel-coated plates with CM derived from UM cells after treatment with MLN4924. Scale bar: 200 mm. F, Representative images andquantitative analysis of blood vessels of chick embryo CAM with CM derived from Omm2.3 cells after treatment with MLN4924. G, MLN4924 inhibitedtranscription 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 weredetected by ELISA. I and J, Overexpression of VEGF-C attenuated MLN4924 treatment-mediated HUVEC migration and tube formation. K and L, Treatmentwith neutralizing anti-VEGF-C antibody enhanced MLN4924 treatment-mediated HUVEC migration and tube formation. M, A proposed working model ofneddylation blockade induces apoptosis and diminishes organ-specifically hepatic metastasis in UM. NAE1 inhibition by MLN4924 disturbed proteinhomeostasis, lowers Bcl-XL to induce intrinsic apoptosis, and elicits DNA damage due to ROS generation to induce cell cycle arrest in UM cells. Neddylationinhibition diminishes hepatic metastasis by dampening Slug-dependent features of CSCs and disrupting niche secretion of NF-kB-mediated VEGF-Cand its dependent angiogenesis in UM.






the MLN4924-treated UM cells on migration and matrigel tubeformation of HUVECs (Fig. 6I–L; Supplementary Fig. S9B–G).These results indicated that MLN4924 suppresses angiogenesisby impairing the VEGF-C secretion in UM.

Given that NF-kB can bind the promoter of VEGF-C gene toinitiate its transcription (39), and that treatment with MLN4924decreases NF-kB transcriptional activity (Fig. 2E; ref. 30), weexamined whether NF-kB was a mediator of MLN4924-mediatedsuppression in VEGF-C. Luciferase reporter assay showed thatMLN4924 reducedVEGF-C genepromoter activity by49% to72%and 39% to 52% in Omm1 and Omm2.3 cells, respectively(Supplementary Fig. S9H). Overexpression of p65 increased2.8- and 3.3-fold VEGF-C mRNA levels, and rescued VEGF-Cdecrease in MLN4924-treated UM cells (Supplementary Fig.S9I). The results suggest that NF-kB is responsible forMLN4924-mediated suppression in VEGF-C.

DiscussionIn the present study, we found that NAE1 expression was

readily detectable in UM cells, and that inhibition of the neddyla-tion pathway byMLN4924 suppressed the proliferation, survival,migration and invasion, and hepatic metastasis. The inhibitoryeffect of neddylation blockade on proliferation, which was con-firmed by xenografted UM tumor in NOD-SCID mice, wasinvolved in activation of ATM-Chk1-Cdc25C DDR, and G2–Mphase arrest. Moreover, MLN4924 treatment eliminated CSCswith stemness-related transcriptional factor Slug suppressed, anddisturbed the paracrine secretion of VEGF-C and its dependentangiogenesis. With an NSI mouse model, we identified thatMLN4924 inhibited hepatic metastasis in UM.

Neddylation inhibition impairs maintenance of CSCsWe demonstrated that MLN4924 suppressed CSC properties

in UM. To our knowledge, this is the first study to provide invitro and in vivo evidence to demonstrate that inhibition of theneddylation pathway eliminates CSCs in solid tumors. Con-sistent with our findings, Knorr and colleagues showed that invitro treatment of MLN4924 exerts a cytotoxic effect on mye-lodysplastic syndrome and acute myelogenous leukemia stemand progenitor cell populations (41). In contrast, Zhou andcolleagues reported that MLN4924 at low nanomolar concen-trations (0–100 nmol/L) exerted a stimulating effect on tumor-sphere formation of non-small cell lung cancer cells whencultured in serum-free medium (42). The discrepancy may beattributed to their serum-starved culture condition, suggestingthat blockage of neddylation modification to regulate CSCself-renewal may be serum-, drug concentration-, and cancertype-dependent. At this view point, we carefully and system-ically evaluated the functional parameters of CSCs (e.g.,ALDHþ cells, melanosphere and serially replating capacity,and the frequency of CSCs) in UM. The results unequivocallyrevealed that the CSC features were diminished after MLN4924treatment.

Neddylation inhibition diminishes SlugPrevious studies have demonstrated that Slug alone or in

collaboration with transcriptional factor SOX9 is required todetermine the characteristics of CSCs in breast and ovariancancer (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 thatthe change of Slug is most impressive in the MLN4924-treatedUM cells. We therefore determined the role of Slug in theMLN4924-mediated elimination of CSCs in UM. Our resultsincluding Slug cDNA rescue experiments indicated that Slug isessential for sustaining the CSC properties in UM. Mechanis-tically, MLN4924 treatment leads to the degradation of Slugthrough the E3 ubiquitin ligase FBXO11, which was consistentwith the previous report (35).

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

In the complex colonization process, establishment of tumors'own angiogenesis may be a turning point. Regarding UM, wefound that VEGF-C is a mediator of the angiogenesis in liver. Thisis supported by several lines of evidence. First, the CM derivedfromUMcells can promote angiogenesis, indicating that UM cellsmay secrete certain cytokine(s). Second, MLN4924 treatmentsignificantly attenuated the UM cells secreting such cytokine(s).Third, ectopic overexpression of VEGF-C decreased the suppres-sion effect of MLN4924. Fourth, treatment with neutralizingantibody against VEGF-C suppressed the angiogenesis-promotingeffect of UM CM and potentiated the effect MLN4924. Finally,MLN4924 significantly decreased the hepatic metastasis andangiogenesis in mice.

Mechanistically, we found that NF-kB p65 was critical in theMLN4924-mediated inhibition of VEGF-C gene transcription inUM cells. MLN4924 treatment led to accumulation of IkBa,which elicited p65 degradation in UM cells, which was consistentwith the precious report in lymphoma cells (39). In the canonicalNF-kB pathway, IkBa phosphorylation at S32 and S36 and theresultant polyubiquitination and degradation of IkBa proteinare critical steps for nuclear localization of p65. Polyubiquitina-tion of IkBa is regulated by SCFbTRCP, which is controlled byNAE (44). Consistent with this notion, a dose-dependent IkBaphosphorylation at S32 and S36 was noted in the MLN4924-treated UM cells.

Blockade of neddylation lowers Bcl-XL to induce intrinsicapoptosis

Proliferation and evading apoptosis are also critical forforming overt metastases as well as growth of primary tumor.We found that MLN4924 at nanomolar concentrations exertbroad tumoricidal activity across a panel of UM cells. Phase Istudies of pevonedistat (MLN4924) showed that maximalplasma concentration (Cmax) reached to 2.7 to 8.1 mmol/L atmaximum tolerated dose (45), which is sufficient to kill UMcells, as extrapolated from our data.

MLN4924 treatment induces apoptosis with mitochondrialdamage in UM cells. Bcl-XL exhibited a marked decrease in theMLN4924-treated UM cells. In line with our findings, Nimati andcolleagues reported that Bcl-XL is an attractive therapeutic target inUM patient-derived xenografts (46). Targeting Bcl-XL may have aparticular implication because BCL2L1 overexpression is a poorprognostic factor based on the analysis of UM patients obtainedfrom the TCGA database.

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

Our results highlighted that prolonged (up to 48 hours) expo-sure of UM cells to MLN4924 changed cell cycle distribution

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featured G2–Mphase also observed in pancreatic cancer (47), andgastric cancer cells (48), but not S phase accumulation. Theincreased g-H2AX together with increased phospho-ATM andphospho-Chk1 unequivocally indicated that MLN4924 inducedDSBs, because ATM-Chk1-Cdc25C axis is predominantly respon-sible for DSBs. However, MLN4924 treatment also caused phos-phorylation in ATR and Chk2, suggesting occurrence of SSBs.Consistent with previous report (35), MLN4924-induced DNAdamage due to ROS generation in UM cells. Taken together,MLN4924 causes accumulation of CRL substrates, which areresponsible for G2–M phase arrest and DDR.

BRCA1 associateprotein-1 (BAP1) mutations are reportedlypresent in the majority of metastatic UM (49). BAP1, a de-ubiquitinating enzyme, is a member of the polycomb-groupproteins (PcG) of highly conserved transcriptional repressorsrequired for long-term silencing of genes that regulate cell fatedetermination and stemness features. It is possible that mutationof BAP1 is involved in the efficacy of neddylation inhibition inmetastatic UM.However, silencing BAP1 or forced overexpressingBAP1 in Mel270 and Omm2.3 cells did not alter the sensitivity toMLN4924 (data not shown).

In conclusion, neddylation inhibition dampens Slug toeliminate CSCs, disturbs paracrine secretion of VEGF-C andits dependent angiogenesis. Blockade of neddylation lowersBcl-XL to induce intrinsic apoptosis, and elicits DNA damage toinduce cell cycle arrest in UM cells (proposed model, Fig. 6M).From a therapeutic standpoint, our work presents novelinsights into molecular strategies to effectively treat organ-specifically hepatic metastasis in UM. These findings warrant

a clinical trial of pevonedistat in hepatic metastatic patientswith UM.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Jin, J. PanDevelopment of methodology: Y. Jin, Y. Wang, B. Jin, J. Zhou, J. Zhang, J. PanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Jin, P. Zhang, Y. Wang, B. Jin, J. Zhou, J. Zhang,J. PanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Jin, Y. Wang, B. Jin, J. PanWriting, review, and/or revision of the manuscript: Y. Jin, J. PanStudy supervision: J. Pan

AcknowledgmentsThe authors thank Jager MJ (Leiden University Medical Center, Leiden, the

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

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 14, 2017; revised September 3, 2017; accepted December 5,2017; published first December 12, 2017.

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2018;24:3741-3754. Published OnlineFirst December 12, 2017.Clin Cancer Res Yanli Jin, Ping Zhang, Yun Wang, et al. MelanomaDampening Cancer Stem-Like Cells and Angiogenesis in Uveal Neddylation Blockade Diminishes Hepatic Metastasis by

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