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Cancer Genes and Genomics Mutational and Functional Analysis Reveals ADAMTS18 Metalloproteinase as a Novel Driver in Melanoma Xiaomu Wei 1 , Todd D. Prickett 1 , Cristina G. Viloria 4 , Alfredo Molinolo 2 , Jimmy C. Lin 5 , Isabel Cardenas-Navia 1 , Pedro Cruz 1 , NISC Comparative Sequencing Program 1 , Steven A. Rosenberg 3 , Michael A. Davies 6,7 , Jeffrey E. Gershenwald 8,9 , Carlos López-Otín 4 , and Yardena Samuels 1 Abstract The disintegrin-metalloproteinases with thrombospondin domains (ADAMTS) genes have been suggested to function as tumor suppressors as several have been found to be epigenetically silenced in various cancers. We performed a mutational analysis of the ADAMTS gene family in human melanoma and identified a large fraction of melanomas to harbor somatic mutations. To evaluate the functional consequences of the most commonly mutated gene, ADAMTS18, six of its mutations were biologically examined. ADAMTS18 mutations had little effect on melanoma cell growth under standard conditions, but reduced cell dependence on growth factors. ADAMTS18 mutations also reduced adhesion to laminin and increased migration in vitro and metastasis in vivo. Melanoma cells expressing mutant ADAMTS18 had reduced cell migration after short hairpin RNAmediated knockdown of ADAMTS18, suggesting that ADAMTS18 mutations promote growth, migration, and metastasis in melanoma. Mol Cancer Res; 8(11); 151325. ©2010 AACR. Introduction It is widely accepted that genetics plays a major role in cancer development (1). The progression of melanoma, which is one of the most aggressive forms of skin cancer (2), is accompanied by a series of genetic changes that affect at least several oncogenes and tumor-suppressor genes (3). Further identification of such genes in melanoma is crucial to promote our understanding of the disease and to develop successful molecular targeted therapies. In this study, we systematically evaluated the disintegrin metalloproteinases with thrombospondin domains (ADAMTS) genes through their comprehensive mutational analysis in melanoma. The ADAMTS gene family is part of a superfamily of zinc-based proteinases, the metzincins (4). The matrix metalloproteinase enzymes, which also belong to the metzincins superfamily, have recently been shown to be highly mutated in melanoma (5). All ADAMTS proteins have proteolytic potential, but have not yet been studied in detail in cancer (6). There is, however, an emerging concept that a number of ADAMTSs may have tumor- suppressor activities (6-8). In particular, Viloria et al. recently showed that ADAMTS15 is somatically mutated in colorectal cancer, and functional evaluation of its muta- tions revealed it to be a tumor-suppressor gene (7). As ADAMTS genes encode extracellular proteins, their acces- sibility to systematically delivered drugs makes them excel- lent therapeutic targets. In the current study, we examined melanoma samples for somatic mutations in 19 human genes that encode ADAMTS proteins. Remarkably, we found that one ADAMTS gene, ADAMTS18, which was highly mutated in melanoma, was previously found to be a candidate can- cer gene (CAN gene) in large-scale whole exome sequenc- ing of colorectal cancer (9, 10). In addition to the ability of the mutated versions of this gene to cause increased prolif- eration of melanoma cells, we found that they increased cell migration and metastasis. These results suggest that genetic alteration of ADAMTS18 plays a major role in melanoma tumorigenesis. Materials and Methods Tumor tissues Tissue and melanoma cell lines used for the discovery and first validation in this study were described previously (5). For the melanoma second validation set, optimum cut- ting temperatureembedded frozen clinical specimens were Authors' Affiliations: 1 NIH Intramural Sequencing Center, National Human Genome Research Institute, NIH; 2 Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH; 3 National Cancer Institute, NIH, Bethesda, Maryland; 4 Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain; 5 Ludwig Center for Cancer Genetics and Therapeutics, and Howard Hughes Medical Institute at the Johns Hopkins Kimmel Cancer Center, Baltimore, Maryland; and Departments of 6 Melanoma Medical Oncology, 7 Systems Biology, 8 Surgical Oncology, and 9 Cancer Biology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). X. Wei and T.D. Prickett contributed equally to this work. Corresponding Author: Yardena Samuels, National Human Genome Research Institute, Room 5140, Building 50, 50 South Drive, MSC 8000, Bethesda, MD 20892-8000. Phone: 301-451-2628; Fax: 301-480- 2592. E-mail: [email protected] doi: 10.1158/1541-7786.MCR-10-0262 ©2010 American Association for Cancer Research. Molecular Cancer Research www.aacrjournals.org 1513 on June 4, 2021. © 2010 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Published OnlineFirst October 13, 2010; DOI: 10.1158/1541-7786.MCR-10-0262

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  • Published OnlineFirst October 13, 2010; DOI: 10.1158/1541-7786.MCR-10-0262

    Cancer Genes and Genomics Molecular

    Cancer

    Research

    Mutational and Functional Analysis Reveals ADAMTS18Metalloproteinase as a Novel Driver in Melanoma

    Xiaomu Wei1, Todd D. Prickett1, Cristina G. Viloria4, Alfredo Molinolo2, Jimmy C. Lin5, Isabel Cardenas-Navia1,Pedro Cruz1, NISC Comparative Sequencing Program1, Steven A. Rosenberg3, Michael A. Davies6,7,Jeffrey E. Gershenwald8,9, Carlos López-Otín4, and Yardena Samuels1

    Abstract

    Authors' AGenome RNational InCancer Insty Biología MOviedo, OTherapeuticKimmel C6Melanoma9Cancer BiHouston, T

    Note: SupCancer Res

    X. Wei and

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    The disintegrin-metalloproteinases with thrombospondin domains (ADAMTS) genes have been suggested tofunction as tumor suppressors as several have been found to be epigenetically silenced in various cancers. Weperformed a mutational analysis of the ADAMTS gene family in human melanoma and identified a large fractionof melanomas to harbor somatic mutations. To evaluate the functional consequences of the most commonlymutated gene, ADAMTS18, six of its mutations were biologically examined. ADAMTS18 mutations had littleeffect on melanoma cell growth under standard conditions, but reduced cell dependence on growth factors.ADAMTS18 mutations also reduced adhesion to laminin and increased migration in vitro and metastasis in vivo.Melanoma cells expressing mutant ADAMTS18 had reduced cell migration after short hairpin RNA–mediatedknockdown of ADAMTS18, suggesting that ADAMTS18 mutations promote growth, migration, and metastasisin melanoma. Mol Cancer Res; 8(11); 1513–25. ©2010 AACR.

    Introduction

    It is widely accepted that genetics plays a major role incancer development (1). The progression of melanoma,which is one of the most aggressive forms of skin cancer(2), is accompanied by a series of genetic changes that affectat least several oncogenes and tumor-suppressor genes (3).Further identification of such genes in melanoma is crucialto promote our understanding of the disease and to developsuccessful molecular targeted therapies. In this study, wesystematically evaluated the disintegrin metalloproteinaseswith thrombospondin domains (ADAMTS) genes throughtheir comprehensive mutational analysis in melanoma.The ADAMTS gene family is part of a superfamily of

    zinc-based proteinases, the metzincins (4). The matrix

    ffiliations: 1NIH Intramural Sequencing Center, National Humanesearch Institute, NIH; 2Oral and Pharyngeal Cancer Branch,stitute of Dental and Craniofacial Research, NIH; 3Nationalitute, NIH, Bethesda, Maryland; 4Departamento de Bioquímicaolecular, Instituto Universitario de Oncología, Universidad de

    viedo, Spain; 5Ludwig Center for Cancer Genetics ands, and Howard Hughes Medical Institute at the Johns Hopkinsancer Center, Baltimore, Maryland; and Departments ofMedical Oncology, 7Systems Biology, 8Surgical Oncology, andology, The University of Texas M.D. Anderson Cancer Center,exas

    plementary data for this article are available at Molecularearch Online (http://mcr.aacrjournals.org/).

    T.D. Prickett contributed equally to this work.

    ding Author: Yardena Samuels, National Human GenomeInstitute, Room 5140, Building 50, 50 South Drive, MSCesda, MD 20892-8000. Phone: 301-451-2628; Fax: 301-480-il: [email protected]

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    metalloproteinase enzymes, which also belong to themetzincins superfamily, have recently been shown to behighly mutated in melanoma (5). All ADAMTS proteinshave proteolytic potential, but have not yet been studiedin detail in cancer (6). There is, however, an emergingconcept that a number of ADAMTSs may have tumor-suppressor activities (6-8). In particular, Viloria et al.recently showed that ADAMTS15 is somatically mutatedin colorectal cancer, and functional evaluation of its muta-tions revealed it to be a tumor-suppressor gene (7). AsADAMTS genes encode extracellular proteins, their acces-sibility to systematically delivered drugs makes them excel-lent therapeutic targets.In the current study, we examined melanoma samples

    for somatic mutations in 19 human genes that encodeADAMTS proteins. Remarkably, we found that oneADAMTS gene, ADAMTS18, which was highly mutatedin melanoma, was previously found to be a candidate can-cer gene (CAN gene) in large-scale whole exome sequenc-ing of colorectal cancer (9, 10). In addition to the ability ofthe mutated versions of this gene to cause increased prolif-eration of melanoma cells, we found that they increasedcell migration and metastasis. These results suggest thatgenetic alteration of ADAMTS18 plays a major role inmelanoma tumorigenesis.

    Materials and Methods

    Tumor tissuesTissue and melanoma cell lines used for the discovery and

    first validation in this study were described previously (5).For the melanoma second validation set, optimum cut-

    ting temperature–embedded frozen clinical specimens were

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    obtained from Melanoma Informatics, Tissue Resource,and Pathology Core, and the Central Nervous SystemTissue Bank at The University of Texas M.D. AndersonCancer Center under institutional review board–approvedprotocols. Hematoxylin and eosin (H&E)–guided dissec-tion and isolation of DNA from the tumor-enrichedisolates has been described previously (11).

    PCR, sequencing, and mutational analysis ofmelanoma samplesPCR and sequencing was done as previously described

    (5, 7, 12). The primary-phase mutation screen was analyzedusing Consed (13). Variants were called using Polyphred6.11 (14) and DIPDetector, an indel detector for improvedsensitivity in finding insertions and deletions. Sequencetraces of the secondary screen were analyzed using theMutation Surveyor software package (SoftGenetics).To increase our confidence that themutations in theM.D.

    Anderson set, for which no matched normal DNA samplewas available, did not represent germline polymorphisms,we searched the corresponding exons of ADAMTS18 in atotal of 145 DNA samples and detected no abnormalities.

    Construction of wild-type and mutant ADAMTS18expression vectorHuman ADAMTS18 (NM_199355.2) was cloned by

    PCR as previously described (5) using a clone purchasedfrom Open Biosystems with primers in SupplementaryTable S5. The PCR product was cloned into themammalianexpression vector pCDF-MCS2-EF1-Puro (Systems Bio-sciences, Inc.) or pCDNA3.1 (−) (Invitrogen) via the XbaIand NotI restriction sites. The G312E, P452S, C638S,Q904X, Q1002X, and P1035S point mutants were madeusing Phusion PCR for site-directed mutagenesis usingthe primers listed in Supplementary Table S5.

    Cell culture and transient expressionMetastatic melanoma tumor lines were maintained as pre-

    viously described (15). HEK 293T cells were purchased fromAmerican Type Culture Collection and maintained in com-plete DMEM supplemented with 10% fetal bovine serum,1× nonessential amino acids, 2 mmol/L L-glutamine, and0.75% sodium bicarbonate. A375 cells were purchased fromNational Cancer Institute, Division of Cancer Treatment,Developmental Therapeutics Program, Frederick, MD, andmaintained in RPMI 1640 and supplemented with 10%fetal bovine serum. Mel-STR cells were described previously(5). HEK 293T cells were transfected with Lipfoctamine2000 reagent (Invitrogen) at a 6:1 ratio with DNA (μL:μg)using 3 to 5 μg of plasmid DNA per T75 flask.

    Immunoprecipitation and Western blottingTransfected cells were gently washed three times in PBS

    and then lysed using 0.5 to 1.0 mL of 1%NP40 lysis buffer[1% NP40, 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/LNaCl, Complete Protease Inhibitor tablet, EDTA-freeRoche], 1 μmol/L sodium orthovanadate, 1 mmol/L sodiumfluoride, and 0.1% β-mercaptoethanol per T-75 flask for

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    20 minutes on ice. Lysed cells were scraped and transferredinto a 1.5-mL microcentrifuge tube. Extracts were centri-fuged for 10 minutes at 14,000 rpm at 4°C. The supernatant(500 μL) was immunoprecipitated overnight by using 20 μLof anti-FLAG (M2) beads (Sigma-Aldrich). Conditionedmedium was immunoprecipitated as previously described(5). The immunoprecipitates were washed and subjected toSDS-PAGE and Western blotting as previously described(16). The primary antibodies used in our analysis were anti-FLAG horseradish peroxidase conjugated (Sigma-Aldrich)and anti–α-tubulin (Calbiochem-EMD Biosciences).

    Pooled stable expressionTo make lentivirus, pCDF-MCS2-EF1-Puro ADAMTS18

    constructs were cotransfected into HEK 293T cells seeded at1.5 × 106 per T75 flask with pVSV-G and pFIV-34N (kindgifts from Todd Waldman, Georgetown University) helperplasmids using Lipofectamine 2000 as described by the manu-facturer.Virus-containingmediumwas harvested 48 to 60hoursafter transfection, filtered, aliquoted, and stored at −80°C.A375 cells were seeded at 1.5 × 106 per T75 flask 24 hours

    before infection. Lentivirus for ADAMTS18 [wild-type(WT), G312E, P452S, C638S, Q904X, Q1002X, andP1035S] or empty vector control were used to infect A375cells as previously described (17). Stable expression ofADAMTS18 proteins (WT and mutants) was determinedby SDS-PAGE analysis followed by immunoprecipitationand immunoblotting with anti-FLAG and anti–α-tubulinto show equivalent expression among pooled clones.Mel-STR cells were seeded at 1.5 × 106 per T75 flask

    24 hours before transfection with ADAMTS18 (WT,G312E, P452S, C638S, Q904X, Q1002X, and P1035S)or empty vector control in pcDNA3.1(−) using Fugene6(Roche 11814443001) as per the manufacturer's protocol.Transfected cells were selected using normal completegrowth medium supplemented with 300 μg/mL G418and pooled for future experiments. Stable expression ofADAMTS18 proteins (WT and mutants) was determinedby SDS-PAGE analysis followed by immunoprecipitationand immunoblotting with anti-FLAG and anti–α-tubulinto show equivalent expression among pools.

    Lentiviral short hairpin RNAConstructs for stable depletion of ADAMTS18 were

    obtained from Open Biosystems, and two were confirmedto efficiently knock down ADAMTS18 at the messageand protein levels. Lentiviral stocks were prepared as pre-viously described (5). Melanoma cell lines (5T, 12T, 85T,and A375) were infected with short hairpin RNA (shRNA)lentiviruses for each condition (vector and scrambled controlsand three independent ADAMTS18-specific shRNAs whosesequences are presented in Supplementary Table S6). Selec-tion and growthwere done as described above. Stably infectedpooled clones were tested in functional assays.

    Reverse transcription-PCRTotal RNA was extracted from pooled clones of melano-

    ma cells A375 stably knocked down for endogenous

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  • Mutation and Function of ADAMTS18 in Melanoma

    Published OnlineFirst October 13, 2010; DOI: 10.1158/1541-7786.MCR-10-0262

    ADAMTS18 following the manufacturer's protocol forRNeasy Mini Kit (Qiagen). Total RNA was eluted in30 μL DEPC-treated dH2O. A total of 1 μg of totalRNA was used for single-strand cDNA synthesis using aSuperScript III First Strand kit (Invitrogen). cDNA wasamplified using the oligo dT20 primer supplied in thekit. To test for loss of ADAMTS18 message, we used1 μL of cDNA in the PCR with either ADAMTS18primers (forward primer 5′-accctggtctcagtgttcca-3′ andreverse primer 5′-tgcaggtctcttccaagtcc-3′), (forward primer5′-ccgtttgtggcttgagtatg-3′ and reverse primer 5′-cggcta-gaacctggacagaa-3′), or GAPDH primers (forward primer5′-tggaaggactcatgaccaca-3′ and reverse primer: 5′-tgctgtagccaaattcgttg-3′). The product was then analyzedon a 1% agarose gel.

    Proliferation assaysTo examine growth potential, pooled A375 and Mel-STR

    ADAMTS18 clones were seeded into 96-well plates at 250cells per well in either 1%, 2.5%, or 10% serum-containingmedium and incubated for 13 to 17 days. Cells were countedevery 48 hours by lysing cells in 50 μL of 0.2% SDS perwell and incubating for 2 hours at 37°C before addition of150 μL/well of SYBRGreen I solution [1:750 SYBRGreen I(Invitrogen-Molecular Probes) diluted in dH2O]. Plateswere analyzed using a BMG Labtech FLOUstar Optima.

    Foci formation assaysA375 andMel-STR pooled clones were seeded at 500 cells

    per T25 flask in normal complete serum–containingmediumand incubated for 8 to 10 days before staining with Hema3 Stat Pack (Protocol) to visualize foci for counting.

    Migration assaysA375 or melanoma cells with stable knockdown of

    ADAMTS18were seeded into preconditionedmigrationwells(8.0 μm; BD Biocoat, BD Biosciences) at 10,000 to 30,000cells per well in serum-free medium in the top chamber andincubated for 16 to 18 hours with complete serum-containingmedium in the bottom chamber before harvesting. Insertswere fixed and stained using Hema 3 Stat Pack (Protocol).Inserts were analyzed and counted for cells that migrated perfield view and quantitated using ImageJ (NIH software).

    Adhesion assayNinety-six-well plates were coated with 5 μg/mL

    laminin-I or 5 μg/mL fibronectin in 200 μL of 1× PBSand incubated overnight at 4°C. Before plating cells, thecoated wells were washed once in PBS and then blockedwith 0.1 mg/mL heat-inactivated bovine serum albumin(BSA; dissolved in PBS) for 1 hour at room temperature.Cells were seeded into the plates at 30,000 per well andincubated at 37°C for 2 hours with the lid off. Cells wereshaken off plate vigorously then washed three times withPBS. The remaining cells were fixed using 4% paraformal-dehyde in PBS overnight at 4°C. Plates were then washedthree times using ddH2O. Attached cells were stainedwith 0.1% crystal violet (w/v; 20% methanol in PBS)

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    for 30 minutes at room temperature followed by threewashes with ddH2O. Dye was solubilized with 0.1 NHCl for 10 minutes at room temperature. Absorbancewas measured at 610 nm on Molecular Devices Spectra-Max and quantitated using Microsoft Excel.

    Immunoflourescence analysis of pooled clonesA375 pooled ADAMTS18 clones expressing either WT,

    G312E, C638S, and P1035S or empty vector were seededon eight-well chamber slides at a cell density of 50,000 perwell. Cells were grown for 24 hours before fixing and stain-ing. Chamber slides were washed once with 1× PBS followedby fixation with 4% paraformaldehyde (PBS) for 15 minutesat room temperature. Cells were subsequently washed threetimes with ice-cold 1× PBS for 5 minutes per wash. Slideswere blocked with 1% BSA (PBS-T) for 30 to 60 minutesat room temperature followed bywashingwith 1× PBS twice.Chamber slides were immunostained with anti-FLAG(rabbit; Sigma) in 1% BSA (PBS-T) at a dilution of 1:50for 18 hours at 4°C. Chamber slides were washed three timeswith PBS at 5 minutes per wash followed by incubationwitha anti-rabbit (Alexa Fluor 568; Invitrogen) diluted at1:200 in 1% BSA (PBS-T) for 2 hours at room temperature.Slides were washed three times in PBS followed by fixation/mountingwith 4′,6-diamidino-2-phenylindole and analyzedon a Zeiss AX10 (Scope.A1) at 40× using SPOT imagingsoftware for image acquisition. Further analysis was doneusing Adobe Photoshop and ImageJ/NIH software.

    Xenograft studies in miceNonobese diabetic (NOD)/severe combined immuno-

    deficient (SCID) mice were purchased from The JacksonLaboratory. All mice were housed in a pathogen-free facilityand were given autoclaved food and water. Mel-STR pooledclones expressing empty vector or with WT ADAMTS18 ormutant ADAMTS18 were grown in T-75 flasks to 70% to80% confluency. Cells (1 × 106) were resuspended in 100 μLof sterile 1× PBS and injected s.c. into 11-week-old maleNOD/SCID mice. Mice were monitored biweekly, andfinal tumor weights were measured after the tumor wasexcised from euthanizedmice at day 22 postinjection. Lungsfrom each mouse were harvested in 4% paraformaldehydeand embedded in paraffin for H&E staining followed byhistologic evaluation.

    Statistical analysisStatistical analyses were done using the R statistical environ-

    ment and Microsoft Excel (two-tailed t test, binomial test).

    Results and Discussion

    Comprehensive mutational analysis of the ADAMTSgene family in human melanomaThe human ADAMTS family consists of 19 genes (Supple-

    mentary Table S1). To evaluate whether these are geneticallyaltered in melanoma, we analyzed the coding exons of thisgene superfamily in 31melanomapatients. A total of 408 exonsfrom the ADAMTS genes were extracted from genomic

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  • Table 1. Mutations identified in ADAMTSs

    Gene RefSeqaccession*

    CCDSaccession*

    No. of mutations(% tumors affected)†

    Tumor Exon Nucleotide‡ Amino acid‡ Functional domain

    ADAMTS2 NM_014244.1 CCDS4444.1 5 (5.1%) 71T 3 C655T P219S None104T 12 G1891A D631N None104T 18 C2713T R905C Thrombospondin type 1 domain85T 19 C2858T S953F Thrombospondin type 1 domain55T 22 C3430T L1144F None

    ADAMTS3 NM_014243 CCDS3553.1 1 (1.3%) 32T 22 C3277T H1093Y NoneADAMTS6 NM_197941 CCDS3983.2 3 (2.5%) 85T 3 C629T S210L None

    21T 10 G1511A G504E None21T 11 G1513A E505K None

    ADAMTS7 NM_014272 CCDS32303.1 4 (5.1%) 83T 2 C148T R50X Propeptide63T 2 C377T P126L Propeptide59T 7 G1090A G364S/LOH Reprolysin4T 16 G2197T A733S ADAM-TS spacer 1

    ADAMTS8 NM_007037 CCDS41732.1 3 (3.8%) 7T 5 C1289A A430D None16T 6 G1580A G527E/LOH ADAM cysteine-rich domain48T 7 G1814A G605E None

    ADAMTS10 NM_030957 CCDS12206.1 4 (3.8%) 43T 15 G1936A E646K None55T 21 C2750T A917V Thrombospondin type 1 domain7T 22 C2791T P931S/LOH Thrombospondin type 1 domain7T 23 G3193A G1065S/LOH None

    ADAMTS13 NM_139027 CCDS6972.1 2 (2.5%) 33T 14 A1652G D551G None4T 20 C2495T S832L None

    ADAMTS15 NM_139055 CCDS8488.1 3 (3.8%) 24T 3 G1237A D413N Reprolysin32T 8 C2548T P850S Thrombospondin type 1 domain48T 8 C2642T A881V Thrombospondin type 1 domain

    ADAMTS18 NM_199355 CCDS10926.1 14 (17.7%) 13T 3 G319A E107K Propeptide44T 5 G851A R284K None63T 5 G860A G287E None23T 5 G935A G312E Reprolysin12T 9 C1354T P452S Reprolysin17T 13 T1912A C638S/LOH Thrombospondin type 1 domain10T 13 G1969A E657K None36T 14 G2061T K687N None55T 14 T2081A F694Y None98T 18 C2710T Q904X Thrombospondin type 1 domain

    (Continued on the following page)

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  • Table 1. Mutations identified in ADAMTSs (Cont'd)

    Gene RefSeqaccession*

    CCDSaccession*

    No. of mutations(% tumors affected)†

    Tumor Exon Nucleotide‡ Amino acid‡ Functional domain

    85T 19 C3004T Q1002X Thrombospondin type 1 domain104T 20 C3103T P1035S Thrombospondin type 1 domain71T 21 G3281A R1094Q Thrombospondin type 1 domain7T 23 C3556T P1186S None

    ADAMTS19 NM_133638.3 CCDS4146.1 3 (3.8%) 63T 9 G1568A R523Q Reprolysin16T 14 G2233A G745R None91T 19 G2906A R969Q/LOH Thrombospondin type 1 domain

    ADAMTS20 NM_025003 CCDS31778.1 12 (11.4%) 48T 2 C203T S68F Propeptide32T 4 G684A M228I None39T 8 C1124T S375L Reprolysin83T 10 C1442T S481L ADAM cysteine-rich domain22T 11 G1610A G537E ADAM cysteine-rich domain74T 13 G1771A G591R Thrombospondin type 1 domain17T 14 C1957T R653C None

    106T 22 C3133T R1044W Thrombospondin type 1 domain39T 30 G4489A D1497N Thrombospondin type 1 domain55T 36 A5350G R1784G GON domain55T 38 G5608A G1870R GON domain55T 38 G5609A G1870E GON domain

    NOTE: “X” refers to stop codon. “LOH” refers to cases wherein the WT allele was lost and only the mutant allele remained. “None” refers to mutations outside anyidentifiable domain.Abbreviation: CCDS, Consensus Coding Sequence project.*Accession numbers for mutated ADAMTSs in Santa Cruz and Genbank.†Number of nonsynonymous mutations observed and percentage of tumors affected for each of the 11 genes in the panel of 79 melanoma cancers.‡Nucleotide and amino acid change resulting from mutation.

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    databases. These exons were amplified by using PCR fromtumor genomic DNA samples, using the primers listed inSupplementary Table S2, and directly sequenced with dyeterminator chemistry. We then determined whether a muta-tion was somatic (i.e., tumor specific) by examining thesequence of the gene in genomic DNA from normal tissueof the relevant patient. From the ∼8Mb of sequence informa-tion obtained, we identified 11 genes containing somaticmutations (Table 1). Genes found to have one nonsynon-ymous mutation or more were then further analyzed formutations in an additional 48 melanomas. Through thisapproach, we identified 54mutations in 11 genes, thus affect-ing 37% of the melanoma tumors analyzed (Table 1 andexamples in Fig. 1A). The number of C>Tmutations in themelanoma tumors was significantly greater than other nucle-otide substitutions, resulting in a high prevalence of C:G>T:Atransitions (P < 0.0001; Supplementary Fig. S1), confirmingpreviously reported melanoma signatures (18).

    ADAMTS18 is highly mutated in melanomaTo evaluate the most highly mutated gene, ADAMTS18

    (affecting ∼18% of cases analyzed), we further extended oursequencing analysis to an additional human melanomatumor panel consisting of 65 melanoma specimens (11).In this screen, we detected 10 nonsilent somatic mutationsand 1 silent somatic mutation, affecting ∼14% of the casesanalyzed (Supplementary Table S3 and examples in Sup-plementary Fig. S2A), thus reaching a similar frequencyof mutations observed in the first melanoma set. The mu-tations that arise during tumorigenesis may give a selectiveadvantage to the tumor cell (driver mutations) or have nofunctional effect on tumor growth (passenger mutations).The combined genetic analysis of ADAMTS18 in bothmelanoma panels identified 24 nonsynonymous and 4synonymous mutations, yielding a ratio of nonsy nonymousto synonymous changes (N/S ratio) of 6:1; this is significant-ly higher than the N/S ratio of 2:1 predicted for nonselectedpassenger mutations (P < 0.004; ref. 9). These data are con-sistent with the hypothesis that mutations in ADAMTS18are positively selected for during tumorigenesis.ADAMTS18 has been previously reported to be mutat-

    ed in kidney and colorectal cancer (refs. 9, 10; http://www.sanger.ac.uk/genetics/CGP/Census/). Importantly,two of the previous mutations reported in colorectalcancer lie near our reported mutations (K455T and2085_2086insT). We therefore expanded our study toencompass 50 colorectal cancer samples. These studiesrevealed mutations in ADAMTS18 in 4 of 50 colorectalcancer samples, all of which were somatic (SupplementaryFig. S2B). Interestingly, two of these mutations occurredexactly at the same location and cause the deletion of thesame region as did the mutations in melanoma samples98T and 85T (Table 1). A schematic representation ofADAMTS18 protein and the location of all the mutationsidentified is presented in Fig. 1B. The clinical informationassociated with the melanoma and colorectal tumorscontaining somatic ADAMTS mutations is provided inSupplementary Table S4.

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    ADAMTS18 mutations promote growth factor–independent cell proliferationTo evaluate the effect of some of these mutations on

    ADAMTS function, we decided to focus on ADAMTS18,which was the most highly mutated gene, harboring 14 so-matic mutations in our initial screen (Table 1). The largenumber of mutations identified in ADAMTS18 and the factthat the affected residues in ADAMTS18 are highly con-served evolutionarily, retaining identity in rat and mouse,suggest that these mutations may be functionally importantin melanoma.To assess the effects of ADAMT18 mutations on tumor-

    igenic phenotypes, we created stable clones expressingWT or six tumor-derived ADAMTS18 mutants (G312E,P452S, C638S, Q904X, Q1002X, and P1035S) in twodifferent human melanoma cell lines (A375 and Mel-STR; ref. 19), both of which were confirmed to harborWTADAMTS18. We chose to focus on these six particularmutations as these were found in important functionaldomains and showed high species conservation. Westernblot analysis showed a similar expression level ofADAMT18 in A375 cells in all clones except mutationsQ904X and P1035S, which had lower expression levels(Supplementary Fig. S3). These clones were used for suc-ceeding studies.We first assessed the transformation abilities of the

    ADAMTS18 mutants. As seen in Fig. 2Ai-ii, expression ofall the ADAMTS18 mutants in either A375 or Mel-STRcells (except mutant Q1002X in Mel-STR cells) elicited asignificantly higher cell transformation ability comparedwith clones expressing vector or WT ADAMTS18 (P <0.05, t test). When the same set of clones was evaluatedfor growth, it was apparent that expression ofWTor mutantADAMTS18 genes did not affect the growth rate of A375and Mel-STR cells in tissue culture in the presence ofmedium with 10% serum (Fig. 2Bi-ii). However, if theserum concentration was reduced to 2.5% (for A375 cells)or 1% (for Mel-STR cells), WT clones grew at a slower ratethan mutant clones on plastic (Fig. 2Biii-iv).

    Mutant ADAMTS18 increases cell migration throughmodulation of cell adhesionADAMTS18 is a secretory protein and similarly to virtu-

    ally all ADAMTS family members, it is strongly associatedwith the extracellular matrix at the pericellular space. Thislocation likely facilitates the interaction of ADAMTS18withintegrins and other extracellular matrix components as wellas with growth factors such as vascular endothelial growthfactor and hepatocyte growth factor (20). To test whethermutant forms of ADAMTS18 have alterations in theseinteractions, we performed an adhesion assay by using eitherfibronectin or laminin-I as substrates. Analysis of celladhesion to these extracellular matrix components revealedthat WT ADAMTS18–expressing cells had an increasedadhesion to laminin-I when compared with cells expressingADAMTS18 mutations (Fig. 3Ai). In contrast, adhesionto fibronectin was similar between WT and mutantADAMTS18-expressing cells (Fig. 3Aii).

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    Alterations in adhesion have previously been shown to leadto changes in signaling properties. Based on the above results,mutant forms of ADAMTS18 will likely have profoundeffects on signaling proteins that have the ability to inducechanges in gene expression programs. These effects on geneexpression will ultimately result in further differences in celladhesion and migration between ADAMTS18-mutant cellsandWT cells. This situation is not unprecedented and it hasbeen previously described in detail for other extracelullarmetalloproteinases, including members of the ADAMTSfamily such as ADAMTS12 (21-23).

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    It has previously been shown that mutations inADAMTS genes can affect their cell localization (7).To test whether the identified mutations affect theirlocalization, we immunoprecipitated concentrated condi-tioned medium or cell lysates derived from the A375pooled clones using FLAG-M2 beads followed byWestern blot analysis. Equal levels of FLAG-taggedADAMTS18 bands were seen in immunoprecipitatedcell lysates expressing WT and mutant ADAMTS18immunoblotted for FLAG (Fig. 3Bi). However, only WTADAMTS18 was observed in immunoprecipitates from

    FIGURE 1. Somatic mutationsidentified in ADAMTS18 in differentcancer types. A, representativesequence chromatograms ofmutations identified in ADAMTS18.In each case, the lower sequencechromatogram was from amelanoma tumor. The topsequence chromatograms werefrom normal tissue of the relevantpatient. Arrows indicate thelocation of missense mutations.The nucleotide and amino acidalterations are shown at thebottom of each chromatogram.B, protein schematic ofADAMTS18 is presented withconserved functional domainsindicated as colored blocks.Black arrowheads indicatepositions of nonsynonymoussomatic mutations identified in79 melanoma specimens.Mutations found in the validationmelanoma or colorectal specimensare labeled as red or blue arrows,respectively. The mutationsanalyzed in this study areindicated with an asterisk. Propep,reprolysin family propeptidedomain; Repro, reprolysin familyzinc metalloprotease domain;ACR, ADAM cysteine-rich domain;TS, thrombospondin type 1domain; Spacer, ADAMTS spacer1 domain; PLAC, protease andlacunin domain.

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    conditioned medium using the same clones (Fig. 3Bi).Similar results were observed when the same clones wereanalyzed by immunofluorescence staining. As seen inFig. 3Bii, more total protein is retained and attachedto the cell surface for mutant ADAMTS18-expressing

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    cells, explaining its absence in the conditioned medium.In contrast, immunostaining of WT ADAMTS18-expressingcells revealed diminished cell surface localization ofFLAG-tagged WT protein (Fig. 3Bii). The effect ofADAMTS18 mutations on its localization could be the

    FIGURE 2. Mutant ADAMTS18 causes reduced cell dependence on growth factors. A, focus formation assay of A375 (i) or Mel-STR (ii) pooled clonesexpressing the indicated constructs was done. The graph indicates the number of colonies observed after 2 weeks of growth. B, cellular proliferation ofA375 (i and iii) or Mel-STR (ii and iv) pooled clones transduced with an empty vector, WT ADAMTS18, or the indicated ADAMTS18 mutants was assessed inplastic culture plates in the presence of 10% serum (i and ii) or reduced serum (iii and iv) for 12 days. The average cell number at each time point wasmeasured by determining DNA content in eight replicate wells using SYBR Green I.

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  • Mutation and Function of ADAMTS18 in Melanoma

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    underlying mechanism for the differential adhesiondescribed above.As previous studies reported that reduced adhesion

    facilitates cell migration (24, 25), our finding that cellsexpressing mutant ADAMTS18 have reduced laminin-Iadhesion prompted us to investigate whether these cellsalso have increased migration ability. Boyden chamberassays showed that A375 mutant ADAMTS18-expressingclones had an increased ability to migrate through porousmembrane (Fig. 3Ci-ii; P < 0.05, t test). Based on theseresults, we can postulate that ADAMTS18 modulates celladhesion, and this might be a candidate mechanism toexplain how mutated forms of this protease stimulate cellmigration.

    Mutant ADAMTS18 is required for migration inmelanoma cellsTo assess whether melanoma cells harboring endogenous

    ADAMTS18 mutations are dependent on ADAMTS18 formigration,we used shRNA to stably knock downADAMTS18in melanoma lines harboring eitherWT (5T, A375) or mutantADAMTS18 (85T, Q1002X; 12T, P452S). We confirmedspecific targeting of ADAMTS18 by shRNAs in transfectedHEK 293T cells and in one of the melanoma cell lines byreverse transcription-PCR (Supplementary Fig. S4A and B).Two unique shRNA constructs targeting ADAMTS18 hadminimal effect on the migration of cells expressing WTprotease but substantially reduced the migration of melanomalines carrying mutant ADAMTS18 (Fig. 3Di-vi). Thus, mu-tant ADAMTS18 is essential for the migration of melanomacells harboring these mutations.

    Mutant ADAMTS18 causes increased metastases in vivoTo determine whether ADAMTS18 mutations affect

    growth in vivo, Mel-STR clones expressing empty vector,WT, or mutant ADAMTS18 were s.c. injected intoNOD/SCID mice. Twenty-two days after injection, themice were evaluated for skin ulceration and metastasisformation by examining H&E-stained sections of paraffin-embedded lungs. As seen in Fig. 4A, most of thetumors expressing mutant ADAMTS18 presented withulcerations, whereas few of the mice with cells expressingWT ADAMTS18 had ulcerating lesions. In addition,most mice injected with mutant ADAMTS18-expressingcells had micrometastases. In contrast, no lung metas-tases were found in the mice injected with WT clones(Fig. 4B and C). This suggests that, in some cases,ADAMTS18 may have an assay-specific suppressive ef-fect. This scenario has precedent and has been describedfor Mdm2 (26). Although the underlying mechanism forthe lack of a metastatic phenotype seen in the WT cellsis unclear, it is consistent with ADAMTS proteins beinginhibited by proteins such as tissue inhibitor of metallo-proteinases in vivo (27). It must be noted, however, thatthe number of identified endogenous protease inhibitorsis significantly lower than that of proteases (28). It istherefore conceivable that another, as yet unknown,ADAMTS18 inhibitor is being expressed in vivo, specif-

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    ically inhibiting the metastasis of cells expressing WTADAMTS18.Clearly, ADAMTS18 mutations are dispersed through-

    out its domains. This is reminiscent of the driver muta-tions reported in ERBB4 (12), CARD11 (29), and FLT3(30). In addition to the catalytic domain, ADAMTSproteins have noncatalytic ancillary domains that regulateinteraction of the protein with substrates or inhibitors suchas the tissue inhibitor of metalloproteinases, and thesedomains have been shown to mediate recognition andcleavage of numerous substrates (31-33). Furthermore,the ancillary domain of several ADAMTS proteins is mod-ified by COOH-terminal proteolysis (34-36), which mightalter substrate recognition and enzyme localization. Asseveral of the mutations identified in ADAMTS18 liewithin the COOH terminus and some cause its trunca-tion, they might affect this recognition. Thus, elucidationof the specific interactions of ADAMTS18 with particularsubstrate(s) will provide an important understanding of thebiochemical effects of the discovered mutations. Recently,a novel method of detecting protease substrates has beendeveloped by Kleifeld et al. (37). Upon radioisotopic label-ing of the NH2-termini amine groups of cellular proteinsand enzymatic reaction with known proteases, fragmentedpeptides are purified and run on a tandem mass spec-trometer, thus identifying new cleavage sites. Utilizationof such a detection method in determining the physiologicsubstrates for ADAMTS18 will prove beneficial.As mentioned above, several ADAMTSs have been

    suggested to harbor antitumorigenic properties (38, 39).These reports focused on the antiangiogenic effects ofADAMTS1 and ADAMTS8 (40, 41) as well as the modu-lation of the extracellular signal-regulated kinase pathwayby ADAMTS12 through extracellular matrix interactions(21). El Hour et al. (42) showed that lack of ADAMTS12in mice resulted in increased angiogenesis and tumorprogression. In addition, ADAMTS15 was shown to havea protective role in breast cancer as increased expressionalong with decreased expression of ADAMTS8 resulted inprolonged relapse-free progression in these patients (43).Genetic inactivation of ADAMTS15 through somatic mu-tation lead to decreased ability to suppress colony formationor invasion of colorectal cancer cells compared with expres-sion of the WT gene (7). Furthermore, epigenetic silencingof ADAMTS genes such as ADAMTS9 or ADAMTS18 hasbeen observed in different types of carcinomas implyingthem to be tumor suppressors (44, 45).Conversely, genetic silencing of ADAMTS20 in mice re-

    sulted in increased melanoblast apoptosis, decreased solubleKit ligand stimulation of the prosurvival pathway, and de-creased processing of the extracellular matrix protein versican(46). These results suggest that ADAMTS20 is a prosurvivalmolecule that might act as an oncogene in melanoma. This issupported by the observation that ADAMTS20 is over-expressed in brain, colon, andbreast cancers (47). Importantly,ADAMTS20was the secondmost highlymutated gene in ourstudy, harboring 12 somatic mutations (11.4%). Futurefunctional evaluation of the identified mutations in

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    FIGURE 3. Mutant ADAMTS18 is essential for melanoma cell migration possibly through modulating cell adhesion. A, adhesion assay of A375 clonesexpressing the indicated constructs was done. Laminin-I (i)– or fibronectin (ii)–coated plates were assessed for adhesion after 1-hour incubation by crystalviolet staining. Plates were analyzed by reading the absorbance at 610 nm. B, localization of ADAMTS18 proteins was assessed using immunoprecipitationand immunofluorescence staining. (i) Cellular conditioned medium and lysates were immunoprecipitated using anti-FLAG (M2) beads and analyzed byWestern blot analysis. Cell lysates were blotted with anti–α-tubulin as a loading control. (ii) A375 pooled ADAMTS-18 clones (WT, G312E, C638S, P1035S,or empty vector) were plated and fixed on slides for immunofluorescence staining with anti-FLAG and 4′,6-diamidino-2-phenylindole (DAPI) for nuclearlocalization. C, A375 clones expressing the indicated constructs were grown in Boyden chambers and assessed for their ability to migrate. (i) The graphindicates the number of cells that migrated 18 hours after seeding. (ii) Representative pictures of migrated cells. D, melanoma cell lines expressing WTADAMTS18 (i and ii) or mutant ADAMTS18 (iii and iv) were infected with either control shRNAs or two different shRNA constructs targeted againstADAMTS18 (#4 and #5). The migration ability of the cells was assessed and plotted. Representative images of migrated cells are shown in v and vi.

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    ADAMTS20 will further verify whether ADAMTS20 partici-pates in melanoma progression.Taken together, our results provide genetic, cellular, and

    in vivo evidence that ADAMTS18 has a role in promoting

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    melanoma tumorigenesis. We postulate that the geneticalteration of ADAMTS18 contributes to the aggressivebiological behavior of melanoma through modulationof proliferative, migratory, and metastatic mechanisms.

    FIGURE 4. Mutant ADAMTS18 causes increased lung metastasis in vivo. Mel-STR clones expressing empty vector, WT, or mutant ADAMTS18 (G312E,C638S, Q904X, P1035S) were s.c. injected into NOD/SCID mice. Twenty-two days after injection, the mice were evaluated for lung metastasis formationby examining H&E-stained sections of paraffin-embedded lungs. A, number of mice with ulcerations, where 0.5 was used to score minor tumor ulcerations(

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    Importantly, this is the first genetic identification of anADAMTS gene that is functionally proven to have anoncogenic role in human disease.

    Disclosure of Potential Conflicts of Interest

    No potential conflicts of interest were disclosed.

    Acknowledgments

    We thank Allison Burrell, Catherine Y. Cheng, and Dr. Kristin E. Yates fortechnical assistance; Dr. Robert Weinberg (Ludwig Center for MolecularOncology, MIT, Cambridge, MA) for the Mel-STR cell line; Dr. Gabriel Capellá(Institut Català d'Oncologia, Barcelona, Spain) for colorectal cancer samples;Drs. Lynn Matrisian, Suneel Apte, Daniel McCulloch, and Paul Meltzer for their

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    helpful comments; and the members of the NIH Intramural Sequencing CenterComparative Sequencing Program for providing leadership in the generation ofthe sequence data analyzed here.

    Grant Support

    National Human Genome Research Institute, NIH (Y. Samuels); Ministerio deCiencia e Innovación-Spain, Fundación “M. Botín”, European Union (FP7 Micro-EnviMet; C. López-Otín); and in part by the University of Texas M.D. AndersonCancer Center Melanoma Specialized Programs of Research Excellence and the Mel-anoma Informatics, Tissue Resource, and Pathology Core (grant P50 CA93459).

    The costs of publication of this article were defrayed in part 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 06/15/2010; revised 09/03/2010; accepted 09/07/2010; publishedOnlineFirst 10/13/2010.

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