A High-Throughput Study in Melanoma Identifies Epithelial Mesenchymal Transition as a Major...

2007;67:3450-3460. Cancer Res Soledad R. Alonso, Lorraine Tracey, Pablo Ortiz, et al. Metastasis

ofEpithelial-Mesenchymal Transition as a Major Determinant A High-Throughput Study in Melanoma Identifies

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A High-Throughput Study in Melanoma Identifies Epithelial-

Mesenchymal Transition as a Major Determinant of Metastasis

Soledad R. Alonso,1Lorraine Tracey,

1Pablo Ortiz,

4Beatriz Perez-Gomez,

5Jose Palacios,

1

Marina Pollan,5Juan Linares,

6Salvio Serrano,

7Ana I. Saez-Castillo,

6Lydia Sanchez,

2

Raquel Pajares,2Abel Sanchez-Aguilera,

1Maria J. Artiga,

1Miguel A. Piris,

1

and Jose L. Rodrıguez-Peralto3

1Molecular Pathology Programme and 2Histology and Immunohistochemistry Unit, Centro Nacional de Investigaciones Oncologicas;Departments of 3Pathology and 4Dermatology, Hospital Universitario 12 de Octubre; 5Centro Nacional de Epidemiologıa, Instituto de SaludCarlos III, Madrid, Spain; and Departments of 6Pathology and 7Dermatology, Hospital Universitario San Cecilio, Granada, Spain

Abstract

Metastatic disease is the primary cause of death in cutaneousmalignant melanoma (CMM) patients. To understand themechanisms of CMM metastasis and identify potentialpredictive markers, we analyzed gene-expression profiles of34 vertical growth phase melanoma cases using cDNA micro-arrays. All patients had a minimum follow-up of 36 months.Twenty-one cases developed nodal metastatic disease and 13did not. Comparison of gene expression profiling of metastaticand nonmetastatic melanoma cases identified 243 genes witha >2-fold differential expression ratio and a false discoveryrate of <0.2 (206 up-regulated and 37 down-regulated). Thisset of genes included molecules involved in cell cycle andapoptosis regulation, epithelial-mesenchymal transition(EMT), signal transduction, nucleic acid binding and tran-scription, protein synthesis and degradation, metabolism, anda specific group of melanoma- and neural-related proteins.Validation of these expression data in an independent seriesof melanomas using tissue microarrays confirmed that theexpression of a set of proteins included in the EMT group(N-cadherin, osteopontin, and SPARC/osteonectin) were sig-nificantly associated with metastasis development. Our resultssuggest that EMT-related genes contribute to the promotionof the metastatic phenotype in primary CMM by supportingspecific adhesive, invasive, and migratory properties. Thesedata give a better understanding of the biology of thisaggressive tumor and may provide new prognostic and patientstratification markers in addition to potential therapeutictargets. [Cancer Res 2007;67(7):3450–60]

Introduction

In primary cutaneous malignant melanoma (CMM) patients, itis essential to determine the molecular changes associated withmetastasis and to apply this knowledge to the fields of outcomeprediction and targeted treatment. This information will lead to abetter understanding of the biology of this tumor, and will probablyprovide prognostic information for defining subgroups of patients

with a less favorable prognosis as potential candidates for adjuvantor novel therapies.Currently, the prognosis of primary CMM is mainly based

on histopathologic criteria. The most important of these is theBreslow index, although it is merely a measure of tumor depth.New molecular markers that correlate with melanoma genesisand/or progression are continuously being identified but, to date,most of them have been obtained in experimental models andhave not yet been confirmed in series of human samples.The development of high-throughput screening techniques in

genomics and proteomics has enabled the analysis of theexpression of multiple genes and proteins in large seriesof tumor samples (1) and may contribute to the resolution ofsome specific issues of clinical relevance, such as identifying thesteps for melanoma progression and metastasis. Until now, moststudies characterizing CMM have been done using cell lines (2, 3),mouse models (4), or metastatic tumor samples (2, 5). However,data obtained from primary CMM samples are difficult to obtain,due in part to the lack of retrospective collections of frozenprimary melanomas samples or adequate follow-up (5–7).Molecular changes associated with the acquisition of metastatic

capacity in vertical growth phase melanoma are still to be fullydescribed. We might expect a repertoire of differentially expressedgenes defining the metastatic phenotype for primary CMM cases.The present study aimed to investigate the metastasis signaturein primary CMM. To this end, we compared the gene expressionprofile in a series of vertical growth phase primary CMMs that haddeveloped metastatic disease with vertical growth phase CMMwithout metastatic disease by the end of follow-up. Our resultsconfirm the multifactorial genesis of melanoma metastasis andidentify the epithelial-mesenchymal transition (EMT) and therelation with the extracellular matrix as key steps in melanomaprogression.

Materials and Methods

Patients and tissue samples. This study featured a series of 34 primaryCMMs provided by the Hospital San Cecilio (Granada, Spain) and the

Hospital 12 de Octubre (Madrid, Spain). Patients were recruited between

1990 and 2002 to ensure a minimum follow-up of 36 months. Samples were

collected and frozen according to standard protocols, and their histologywas reviewed by two pathologists (S.R.A. and J.L.R-P.) to confirm that they

contained at least 50% tumor cells. All selected cases corresponded to

consecutive melanoma tumors with potential metastatic capacity (i.e.,vertical growth phase cases with a Breslow index >1 mm). The cutoff was

selected taking into account the criteria of the American Joint Committee

on Cancer by which thin melanomas (V1 mm) have an excellent prognosis

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

Requests for reprints: Miguel A. Piris, Programa de Patologıa Molecular, CentroNacional de Investigaciones Oncologicas, C/Melchor Fernandez Almagro 3, Madrid28029, Spain. Phone: 34-91-224-69-00; Fax: 34-91-224-69-23; E-mail: [email protected].

I2007 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-06-3481

Cancer Res 2007; 67: (7). April 1, 2007 3450 www.aacrjournals.org

Research Article




(>90% survival at 5 years) compared with melanomas with a penetration

depth of >1 mm (8). Histologic review of the cases was done on paraffin-

embedded tissue, whereas frozen sections were examined for assessing the

proportion of the tumoral cells and the character of the neoplasticinfiltrate.

For tissue microarray (TMA) analysis, we used a retrospective cohort

of patients representing 127 primary vertical growth phase melanoma

cases ( formalin-fixed and paraffin-embedded tissue) collected from 1980

to 2000. These cases were obtained from the Hospital 12 de Octubre

(Madrid) and were included in six separate TMAs (1.5-mm core

diameter), with two representative duplicate cores for each case (9),

and constructed with a manual tissue arrayer (Beecher Instruments, Sun

Prairie, WI) using a standard method (10). Patient medical records were

reviewed to gather information on age, gender, localization, tumor

thickness, distant invasion (lymph node or skin), and follow-up of at

least 36 months. Patients were not treated before the development of

metastasis. The work was conducted in accordance with the Declaration

of Helsinki Principles and under the supervision of the Hospital 12 de

Octubre Ethics Committee.

RNA isolation and amplification. Total RNA was extracted from

frozen samples of primary CMM using TRIzol reagent (Invitrogen,

Carlsbad, CA) followed by purification with the RNeasy Mini kit (Qiagen

Inc., Valencia, CA) and digestion with RNase free DNase I according to

the manufacturer’s instructions. RNA quality and integrity were verified

using the Bioanalyzer system (Agilent Technologies, Palo Alto, CA).

Double-stranded cDNA was synthesized from 5 Ag of total RNA using

the Superscript Choice System for cDNA synthesis (Invitrogen) with an

oligo-dT primer containing a T7 RNA polymerase promoter. In vitro

transcription was carried out using the T7 Megascript in vitro trans-

cription kit (Ambion, Austin, TX) as previously described (11, 12). The

quality of the amplified RNA produced was checked by electrophoresis

and its concentration was measured.

Microarray procedures: preparation/synthesis of fluorescent cDNAand hybridization. For each melanoma sample, 5 Ag of amplified RNA

(aRNA) were directly labeled with cyanine 3-conjugated dUTP (Cy3),

whereas 5 Ag of aRNA from the Universal Human Reference RNA

(Stratagene, La Jolla, CA) were labeled with cyanine 5–conjugated dUTP

(Cy5). The Centro Nacional de Investigaciones Oncologicas (CNIO)

OncoChip platform (v.1.4) was used in all cases to perform the cDNA

microarray procedure. Basically, the CNIO OncoChip is a cDNA microarray

especially designed for the analysis of genes involved in cancer. It includes

2,489 cancer-relevant genes in addition to genes involved in drug response,

tissue-specific genes, and control genes. The platform used has a total

of 6,386 genes represented by 7,237 human clones purchased from

Research Genetics (Huntsville, AL). A list of these genes can be found

online.8 Sample hybridizations were done as described elsewhere (12).

After washing, the two fluorescent signals on the slides were scanned with

a standard two-color microarray scanner (Scanarray 5000XL, GSI

Lumonics, Kanata, Ontario, Canada). Images were analyzed with the

GenePix 4.1 software (Axon Instruments, Inc., Union City, CA). The clone

sequences of all the genes included in the OncoChip and the repro-

ducibility of the expression data of multiple genes have been previously

verified (12).

Data extraction and analysis. Data from each hybridization were

maintained in a database for analysis. Fluorescence intensity measurements

were subjected to automatic background subtraction. The Cy3/Cy5 ratioswere normalized to the value of the median ratio of all spots in the array.

The sum of the median background for each channel was calculated,

and spots with total intensities less than the calculated sum of median

backgrounds were discarded. Additionally, spots with background-subtracted signal intensities <500 fluorescence units (sum of the two

channels) and bad spots were excluded from the analysis. All ratio values

were log-transformed (base 2), and duplicated spots in the array wereaveraged. Inconsistent duplicates were discarded and all consistent

duplicate spots and genes were averaged. In addition, genes for which

fewer than 70% of the potential data were available were excluded from

further analysis. The median expression of each gene was calculated foreach patient group (metastatic and nonmetastatic primary CMM). Differ-

ences in expression between the two groups were analyzed by the Wilcoxon

test. To account for the effect of multiple hypothesis testing on the iden-

tification of significance, adjusted P values were also computed using themethod proposed by Hochberg and Benjamini (13) for controlling the false-

discovery rate (FDR). A single value for each gene was obtained. For our

purposes, a gene was deemed to be up-regulated or down-regulated if there

was an at least 2-fold difference in expression and if the FDR value was <0.2.The regulated genes were functionally classified manually on the basis of

exhaustive searches in PubMed, the Genecards database, and Gene

Ontology.TMA immunohistochemistry. We undertook immunohistochemical

analysis to identify at the protein level a selection of the statistically and

biologically significant genes obtained in the cDNA microarray study. This

was done in a TMA series of 127 vertical growth phase melanoma caseswith known follow-up. The proteins selected for validation featured the

group that proved to be biologically relevant in melanoma metastasis

development, including glypican 3 (polyclonal, Santa Cruz Biotechnology,

Santa Cruz, CA), N-cadherin (3B9, Zymed, San Francisco, CA), osteonectin/

Table 1. Antibodies used in the study indicating clone, source, dilution, visualization method, scoring, threshold, and positivecontrols

Protein Clone Source Dilution Visualizationsystem

Scoring Threshold Positive control

Glypican 3 Polyclonal Santa CruzBiotechnology

1:25 LSAB/DAB Pos/neg >10% positive cells Hepatocarcinoma

N-cadherin 3B9 Zymed 1:10 LSAB/DAB Pos/neg z5% positive cells,

membranous expression

Ovarian carcinoma,

cardiac muscleSPARC/

osteonectin

15G12 Novocastra 1:25 LSAB/DAB Pos/neg >10% positive cells, cytoplasm Endothelial cells in

malignant tumors

Osteopontin Polyclonal Abcam 1:1,500 LSAB/DAB Pos/neg >10% positive cells, cytoplasm Stromal cells in normal skin

PKCa H7 Santa CruzBiotechnology

1:25 LSAB/DAB High/low z50% positive cells, cytoplasm Small lymphocytes, nevus

Abbreviations: Pos/neg, positive/negative; LSAB, peroxidase-labeled streptavidin biotin; DAB, diaminobenzidine.

8 http://bioinfo.cnio.es/data/oncochip

Metastasis Signature in Melanoma

www.aacrjournals.org 3451 Cancer Res 2007; 67: (7). April 1, 2007




SPARC (15G12, Novocastra, Newcastle, United Kingdom), osteopontin(polyclonal, Abcam, Novus Biologicals, Littleton, CO), and protein kinase

Ca (PKCa; H7, Santa Cruz Biotechnology, Santa Cruz, CA). A heat-induced,

epitope-retrieval step was done in a solution of sodium citrate buffer

(pH 6.5) in the case of osteonectin/SPARC, osteopontin, and PKCa, and withEDTA buffer in the case of glypican 3 and N-cadherin. The slides were then

heated for 2 min in a conventional pressure cooker and rinsed in cool

running water for 5 min. They were then quickly washed in TBS (pH 7.4),

and incubated with the selected primary antibodies glypican 3 (1:25),N-cadherin (1:10), osteonectin/SPARC (1:25), osteopontin (1:1500), and

PKCa (1:25). Immunodetection was done with biotinylated secondary

antibodies, followed by peroxidase-labeled streptavidin biotin (Dakocyto-

mation, Glostrup, Denmark) visualization in the case of glypican,osteopontin, and PKCa and Envision (Dakocytomation) for N-cadherin

and SPARC. Diaminobenzidine chromogen was used as peroxidase

substrate. All immunostaining was done in a TechMate 500 automaticimmunostaining device (DAKO, Glostrup, Denmark). Incubations omitting

the specific antibody were used as a control of the technique. Scoring of the

results and selection of the thresholds, internal controls for the antibodyreactivity, and tissue controls for the series were done according previously

published methods (9).

Scoring systems. Immunostaining results were evaluated by two

different pathologists (S.R.A. and J.L.R-P.) and scored using clear cutoffcriteria to facilitate the reproducibility of the method. Discrepancies were

resolved by simultaneous reevaluation. Briefly, the result was recorded as

positive or negative and high versus low expression, taking into account

the expression in tumoral cells and the specific cutoff for each marker(see Table 1 for description of thresholds). As a general criterion, the cutoffs

were selected to facilitate reproducibility and, when possible, to translate

biological events. Scoring for the sample replicates was highly reproducible

in this series (96%).Statistical analysis. To validate protein expression in the series analyzed

by TMA, the relationship between marker expression in the patient samples

and disease-free survival (DFS) curves were derived by the Kaplan-Meier method. Statistical significance of associations between individual

variables and DFS was determined using the log-rank test. Cox univariate

Figure 1. Representative diagram of thestudy profile. Metastatic disease wasconsidered as end point for DFS. VGP,vertical growth phase; CMM, cutaneousmalignant melanoma; EMT, epithelialmesenchymal transition; FDR, falsediscovery rate; DFS, disease-free survival;TMA, tissue microarray. K-M,Kaplan-Meier.

Cancer Research





proportional hazard analysis was also done independently for each variable.Significance was concluded for values of P < 0.05. All statistical tests were

two-sided. A multivariate model including the Breslow index as a

continuous variable was also developed by backward elimination. All

statistical analyses were carried out using the Stata statistical program(StataCorp 2001, release 9.0).

Results

Clinical features. The study included two sets of patients.The training set was composed of 34 patients (14 males and 20females) with primary vertical growth phase CMM and >1 mm(Breslow index). After a median follow-up of 67.3 months (range16–166 months), 21 developed metastasis (median 9.4 months,range 0.73–139).The validation set of patients, analyzed by TMAs, included 127

patients (57 males and 70 females) with primary vertical growthphase CMM and a median age at diagnosis of 60 years (range 21–91years). The median follow-up was 116.8 months (range 1–276.5months), during which 73 patients developed metastatic diseaseand 54 did not (median 52.7 months, range 1–222). Figure 1 showsan outline of the study.Global changes in gene expression between metastatic and

nonmetastatic primary vertical growth phase CMM cases. Toidentify genes that might be potential markers for melanomametastases, cDNA microarray studies were done to compareprimary invasive CMM cases with and without metastatic disease.

From the 5,253 clones, corresponding to 2,945 known genesand 1,984 expressed-sequence tags that were suitable for analysisafter filtering steps, 243 genes were differentially expressed(>2-fold, FDR<0.2) in primary vertical growth phase melanomaswith metastases compared with primary nonmetastatic verticalgrowth phase melanomas. Of these, 206 genes were up-regulatedand 37 genes were down-regulated (see Supplementary Table S1for details). These genes were categorized according to theirmain biological function and/or mechanism of action. As seen inTable 2, six biological process classes (cell cycle and apoptosis,EMT, immune modulation, metabolism, nucleic acid binding andtranscription, protein synthesis and degradation, and signaltransduction) were identified as relevant in our metastatic genesignature. A group of genes with unknown function was alsoidentified (see Supplementary Data for details). Figure 2 shows theexpression profile of the significant genes in the training seriesclassified by these gene-functional categories.Interestingly, one of the most important functional clusters

recognized here as markers of metastatic melanoma includedgenes involved in EMT, a category that includes genes associatedwith cell adhesion, cell motility, migration, and extracellular matrixinteraction and organization (Table 3). This cDNA microarrayscreening identified several genes involved in the EMT phenom-enon (N-cadherin, SPARC , and WNT receptor frizzled) and thephysical link between the actin cytoskeleton, the extracellularmatrix (mainly mediated by receptors of the integrin family, suchas integrin aV), and supporting vessels (endothelin receptor B).

Table 2. Functional categories of differentially expressed genes

Functional categories Gene name

Cell cycle and apoptosis MAPRE1, SKP1A, FAIM, PMP22, SET, CDKN1A, CDC5L, WEE1,

CASP5, DAD1, APEX1, VDAC1, BCCIP, CKS1B, CYC1, HBXIP,TP53I3, KIF11, BCL2L10, TOB2

EMT LUM, SDCBP, MFAP1, CTSB, PRKCA, RAB1A, H2-ALPHA, RRAGA,

TUBB3, CLIC4, SPARC, ANLN, RAN, ENC1, EMP1, DSG2, SEPP1,

ITGAV, TUBA2, HMMR, MASA, TUBA3, CX3CR1, CDH2, EDNRB,CSPG2, CD63, SMARCA1, KIT, KRT8, ANXA5, FGG, PFN1, SPA17,

FZD1, APLP2, SERPINA3, SEMA3B, MMP2, TUBA1, EDN2, PCDH9,

CDH10, WNT2, PAPPA

Immune modulation CXCL12, MAGEA6, IFITM2, CCBP2Metabolism PRDX5,VKORC1, ACADM, ALDH1A2, GLO1, HIBADH, PMM1, NDUFAB1,

NDUFB11, ENO1, MDH1, ACSL3, ATP6V0B, MGC4172, OSBP, PFKM,

SLC39A1, ATP6V0D1, YWHAQ, LRPAP1, ADK, SLC30A6, GGH, GSTA4,DCK, ATP5L2, LIPA, COX8A, CA9, AKR7A2, SCD, AK3L1, TXNRD1,

SLC25A17, NPC2, NDUFB3, PAICS, COX6B1, ADSL, CYP7B1, CYP19A1, IDS

Nucleic acid binding and transcription factors NCOA4, HIST1H4C, RBM4, MAP4K3, NOLA2, BZW1, ARID5B, CBFB,

POLR3K, TRIP3, RDBP, ASH2L, CDYL, XRCC5, HIST1H4B, KLF10,TCEB1, HIST1H3D, GTF2H2, FOXO1A, REST, OLIG2

Protein synthesis and degradation HSPA8, MRPL15, CCT5, EIF5, UBE2D2, MRPL37, MRPS10, RPL10A,

VBP1, RPS22, UBL5, CCT6A, GC20, EIF3S3, RPL35A, SECISBP2

Signal transduction CAV1, NME2, SRI, PPP1R3C, S100A10, WBP5, GABARAP, IGFBP1, PPP1R8,C11orf15, PRKAR1A, PGR1_GPR153, SEC5L1, CGI-141, MYB, SPRY2,

PPP2CB, STK24, GNG12, GRB2, MAL, SHB, DUSP12, TGFBR2, PC4,

IDE, CGA, IL1RL1, PPP2R5C, TIMM10, PDE5A, RABAC1, GIMAP6,

TXK, EPS15, NTF5, AKAP13

NOTE: Categories are based on Gene Cards, National Center for Biotechnology Information, HUGO gene nomenclature committee, and PubMed. EMT is

a category that includes genes associated with cell adhesion, cell motility, migration, and extracellular matrix organization.






Figure 2. Results of global expressionprofiling between primary CMM withmetastasis at follow-up (red squares ) andprimary CMM without metastasis (yellowsquares ). Expression data for each genein each profiled sample are presented inthe form of a heat map (green to red scale )of log2-transformed ratios. Names on theright represent the main functionalcategory for each gene. Up-regulatedgenes are at the top and down-regulatedgenes are at the bottom.

Cancer Research





Interestingly, the increase in the expression of N-cadherin isassociated with the loss of the type II cadherin 10 (CDH10).A substantial number of the genes in the metastatic signature

could be included in a generic category named melanoma- andneural-related genes, which was created consistently with theneural origin of the melanocytes (Table 4).This series included two primary cases with metastatic disease

samples whose expression profiles somewhat resembled those

identified in the nonmetastatic group, in spite of not exhibitingdifferences in the Clark level or presence of ulceration, whencompared with the rest of nonmetastatic cases. These two caseshad in common the expression of the transforming growth factorh receptor II (TGFBR2), a signal transduction protein whosemutations have been associated with tumor progression in somesolid tumors (14). Moreover, case mel-08 had altered cell cycle andapoptosis regulator genes with a demonstrated role in melanoma

Table 3. EMT-related genes

Gene name Gene description n-fold change Log2 change

LUM Lumican 12.278 3.618

SDCBP Syndecan binding protein (syntenin, melanoma differentiation-associated

protein 9-MDA; pro-TGF-a cytoplasmic domain-interacting protein18; scaffold protein Pbp1)

6.753 2.756

MFAP1 Microfibrillar-associated protein 1 4.147 2.052

CTSB Cathepsin B 3.487 1.802PRKCA Protein kinase Ca 3.207 1.681

RAB1A RAB1A member RAS oncogene family 2.986 1.578

H2-ALPHA a-Tubulin isotype H2-a 2.934 1.553

RRAGA Ras-related GTP binding A 2.917 1.545TUBB3 Tubulin h3 (other designation: tubulin h4) 2.874 1.523

CLIC4 Chloride intracellular channel 4 2.870 1.521

SPARC Secreted protein, acidic, cysteine-rich (osteonectin) 2.840 1.506

ANLN Anillin, actin binding protein 2.764 1.467RAN RAN, member RAS oncogene family 2.745 1.457

ENC1 Ectodermal-neural cortex (with BTB-like domain). Other designations:

nuclear restricted protein, BTB domain-like (brain); tumor protein

p53 inducible protein 10

2.660 1.412

EMP1 Epithelial membrane protein 1 2.645 1.403

DSG2 Desmoglein 2 2.629 1.395

SEPP1 Selenoprotein P, plasma, 1 2.628 1.394ITGAV Integrin aV (vitronectin receptor, a polypeptide, antigen CD51) antigen

identified by monoclonal antibody L230

2.563 1.358

TUBA2 Tubulin a2 2.457 1.297

HMMR Hyaluronan-mediated motility receptor (RHAMM) intracellular hyaluronicacid binding protein

2.439 1.286

MASA L1 neuronal cell adhesion molecule, MASA (mental retardation, aphasia,

shuffling gait and adducted thumbs)

2.425 1.278

TUBA3 Tubulin a3 2.400 1.263CX3CR1 Chemokine (C-X3-C motif) receptor 1 2.380 1.251

CDH2 Cadherin 2, type 1, N-cadherin [neuronal N-cadherin 1; cadherin 2, N-cadherin

(neuronal); cadherin 2, type 1; calcium-dependent adhesion protein,neural cadherin]

2.375 1.248

EDNRB Endothelin receptor type B 2.315 1.211

CSPG2 Versican (chondroitin sulfate proteoglycan 2) 2.261 1.177

CD63 Melanoma 1 antigen 2.205 1.141SMARCA1 SWI/SNF-related matrix-associated, actin-dependent regulator of chromatin,

subfamily a, member 1

2.199 1.137

FGG Fibrinogen g chain 2.149 1.104

PFN1 Profilin 1 2.142 1.099SPA17 Sperm autoantigenic protein 17 2.129 1.090

FZD1 Frizzled homologue 1 (Drosophila) 2.121 1.085

APLP2 Amyloid h (A4) precursor-like protein 2 2.098 1.069SERPINA3 Serine (or cysteine) proteinase inhibitor, clade A (a-1 antiproteinase, antitrypsin), member 3 2.076 1.054

MMP2 Matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase) 2.046 1.033

TUBA1 Tubulin a 1 (testis-specific) 2.018 1.013

EDN2 Endothelin 2 �2.279 �1.189PCDH9 Protocadherin 9 �2.345 �1.230CDH10 Cadherin 10 type 2 (T2-cadherin) �2.397 �1.262WNT2 Wingless-type MMTV integration site family member 2 �3.274 �1.711PAPPA Pregnancy-associated plasma protein A, pappalysin 1 �3.323 �1.733






progression, such as overexpression of p21 (CDKN1A; ref. 9) ordown-regulation of BCL2L10. The mel-09 case showed up-regulation of ENC1 (ectodermal neural cortex 1), a gene associatedrelated with the Wingless (WNT) pathway and whose expressionhas been linked with colorectal carcinogenesis (15), and meningi-oma progression (16).Validation of gene expression results by immunohistochem-

istry on TMAs. To corroborate the gene expression data and toidentify in situ the cells expressing the various markers,immunohistochemical studies of an independent series of primaryvertical growth phase CMMs were done using TMAs. The relevanceand intensity in the changes of expression in genes linked withEMT in the training series led us to concentrate on validating somesignificantly up-regulated proteins (>2-fold change and FDR<0.2) inthis category (N-cadherin, osteonectin/SPARC , and PKCa). Promp-ted by a literature search and the nearly significant results reportedhere, we also studied glypican 3 , which has recently been describedas a novel tumor marker for melanoma (17), and osteopontin , aprotein belonging to this EMT group that has a crucial role inmelanoma (18, 19) and seems to be diagnostically useful in thedetection of mesothelioma in serum obtained from peripheralblood (20).Figure 3 shows the results of the univariate analyses of selected

proteins and representative immunohistochemistry for N-cadherin,osteonectin/SPARC, and osteopontin, which were the statisticallysignificant markers. These results are closely consistent with thecDNA microarray data.

Immunostaining for these proteins confirmed that they wereexpressed by tumoral melanoma cells. The adhesion molecule N-cadherin was found with a membrane and cytoplasmic pattern.Osteonectin (SPARC) was recognized in tumoral melanocytes andin endothelial vessels close to the tumor, as described in otherneoplasms (21, 22). Osteopontin is an acidic matrix protein (bonesyaloprotein) commonly localized within normal elastic fibers ofthe skin and secreted by many transformed cells. Immunolocaliza-tion of this protein in our melanoma cases was found in tumoralcells and also in the stroma (see Fig. 3B). As shown in the table inFig. 3A , the expression of these three proteins was significantlyassociated with an increased incidence of melanoma metastases[P = 0.013 for N-cadherin; P = 0.006 for osteonectin (SPARC);P = 0.05 for osteopontin], thereby confirming its potentialprognostic value in CMM.PKCa, a protein involved in melanoma progression (23), was also

expressed in the cytoplasm of tumoral cells, as previously described(24), but the univariate study failed to show any significantassociation with tumor progression in this series. However, themultivariate analysis including N-cadherin, osteopontin, osteonec-tin, PKCa, and Breslow index showed that only PKCa wasindependent of the Breslow index (Table 5).

Discussion

The findings reported here confirm microarray data previouslyobtained in cell lines (e.g., lumican; ref. 3) or in other experimental

Table 4. Melanoma and neural-related genes

Gene name Gene description n-fold change Log2 change

SDCBP Syndecan binding protein (syntenin, melanoma differentiation-associated

protein 9-MDA; pro-TGF-a cytoplasmic domain-interacting protein

18; scaffold protein Pbp1)

2.7555 6.753

CHN1 Chimerin 1 (GTPase-activating protein, U, 2); n-chimerin 1.523 2.874

PMP22 Peripheral Myelin Protein 22 (growth arrest-specific 3) 1.505 2.838

S100A10 S100 calcium binding protein A10 [Annexin II ligand, calpactin I, lightpolypeptide (p11)]

1.491 2.811

ENC1 Ectodermal-neural cortex (with BTB-like domain). Other designations:

nuclear restricted protein, BTB domain-like (brain); tumor protein

p53 inducible protein 10

1.4115 2.66

EMP1 Epithelial membrane protein 1 1.403 2.645

SEPP1 Selenoprotein P, plasma 1 1.394 2.628

MASA L1 neuronal cell adhesion molecule, MASA (mental retardation, aphasia,

shuffling gait and adducted thumbs)

1.278 2.425

PTPRZ1 Protein tyrosine phosphatase, receptor-type, Z polypeptide 1 1.255 2.387

MITF Microphthalmia-associated transcription factor 1.2505 2.379

MAGEA6 Melanoma antigen family A, 6 1.2075 2.309AKT1S1 AKT1 substrate 1 (proline-rich) 1.1705 2.251

CD63 Melanoma 1 antigen 1.141 2.205

MAL Mal, T-cell differentiation protein 1.138 2.201

KIT V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homologue,C-Kit, CD117, PBT, SCFR

1.123 2.178

APLP2 Amyloid h (A4) precursor-like protein 2 1.069 2.098

PDGFRA Platelet-derived growth factor receptor, a polypeptide 1.055 2.078

ALS4 Amyotrophic lateral sclerosis 4 1.053 2.075PCDH9 Protocadherin 9 �1.2295 �2.345CDH10 Cadherin 10 type 2 (T2-cadherin) �1.2615 �2.397NTF5 Neurotrophin 5 (neurotrophin 4/5) �1.791 �3.461OLIG2 Oligodendrocyte lineage transcription factor 2 �1.9555 �3.879

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assays (e.g., osteopontin; ref. 19), ENDRB (25), PKCa (26), and seemto imply that the metastatic melanoma signature in humansamples is recognized by the increased expression of clusters ofgenes involved in the control of EMT, cell cycle and apoptosis,immune response, metabolism, transcription regulation, proteinsynthesis and degradation, and signal transduction.In accordance with the results obtained, we have focused our

attention on the validation step in the EMT because a large andhighly significant group of up-regulated genes were members of

this functional cluster. EMT is the process by which an epithelialcell suffers transitory changes in cell structure and becomes a moremotile mesenchymal cell with migratory and invasive properties(27). As a result, cell-cell junctions are altered; cells lose polarity,express mesenchymal markers, and the actin cytoskeleton becomesreorganized. Consequently, tumoral cells lose contact withneighboring cells, become motile, and interact with extracellularmatrix, invading surrounding territories and acquiring capacity formetastasis. The relevance of the EMT signature to the origin of

Figure 3. A, Kaplan-Meier analysis of DFS among the validation set of 127 vertical growth phase melanoma cases included in the TMA. The genes evaluated wereN-cadherin, osteonectin (SPARC ), osteopontin, glypican 3 , and PKCa . Kaplan-Meier plots show the curves for significant proteins. 95% CI, confidence interval.B, representative immunohistochemical staining of significantly up-regulated EMT candidate genes identified from the microarray data. Brown staining denotes apositive signal. Magnification, �4 (inset �20).






metastasis is not restricted to melanoma, and has been identifiedas a determinant of local invasion and metastasis in other tumortypes, such as breast (28), gastrointestinal, or prostate carcinomas(29), among others.The present results suggest that migration and invasion in

melanoma are the result of a specific interaction between tumoraland stromal cells (30, 31) associated with the expression of a setof molecules involved in EMT (31). Thus, the study revealsN-cadherin, lumican, glypican, osteonectin (SPARC), osteopontin,metalloproteinases, and integrins, among others, as being involvedin the melanoma metastatic process. Although it is difficult toverify EMT experimentally in vivo due to the reversible nature ofthe process, expression profiling identifies a molecular signaturelinked with this phenomenon. Moreover, the immunohistochemicalstudies of N-cadherin, osteopontin, glypican 3, osteonectin(SPARC), and PKCa validate the data presented and confirm thatat least these components of the EMT signature are expressed bythe melanoma cells themselves.One of most remarkable findings of this study is the presence

of what has been called the switch of the cadherin class: loss ofepithelial cadherins (E-cadherin) with gain of neural cadherins(N-cadherin). Cadherins are calcium-dependent, cell-adhesionmolecules that are critical for the development and maintenanceof epithelial architecture. As described in carcinoma models,N-cadherin is a crucial molecule in the EMT event, acting as anoncogene in many tumors by promoting tumor invasiveness andprogression (32). In our study, increased N-cadherin was matchedby loss of cadherin 10 expression, a type II cadherin with near-equivalent binding strength to that of E-cadherin (33) as describedpreviously in cell line models (34, 35) and in desmoplasticmelanoma (36). Although melanoma cells are not epithelial innature, the EMT for this tumor is well recognized and the relevanceof the cadherin switch has been previously supported byexperimental approaches, demonstrating that melanoma cell lines

transfected with N-cadherin are morphologically transformed froman epithelial-like shape to a fibroblast-like shape; by contrast,adenoviral reexpression of E-cadherin in melanoma cells down-regulates endogenous N-cadherin and reduces their malignantpotential (37). The present study not only confirms the role ofN-cadherin in melanoma progression but also shows for the firsttime how gain of N-cadherin and loss of class II type cadherin canfacilitate metastatic dissemination in human primary CMMsamples.Osteonectin (SPARC or BM40) is a secreted extracellular matrix

glycoprotein involved in tumor cell migration, invasion, andangiogenesis (38). Data presented here are consistent with previousreports of SPARC overexpression as a marker of aggressiveness orlocal invasion in CMM (39, 40).Osteopontin is a secreted phosphoprotein with a crucial role

in tumor progression and metastasis in many kinds of tumors,including melanoma (19). It promotes antiapoptotic signalingand angiogenesis and induces matrix metalloproteinases (MMP;ref. 18). Osteopontin expression may be acquired in the early stagesof melanoma invasion, with moderate to high levels in primaryinvasive and metastatic melanoma samples and null or low levelsof expression in benign nevi, dysplastic nevi, or in situ melanoma(19). Here, cytoplasmic osteopontin expression in invasive mela-nomas was associated with metastatic disease, mimicking previousexperimental models in which osteopontin allowed murinemelanocytes to adhere, spread, and survive in three-dimensionalcollagen gels (41).The serine/threonine kinase PKCa is a protein with a well-

recognized role in regulating cell growth and progression (42).Melanocytes and melanoma cells express several PKC isoforms,with proposed roles in melanoma genesis, invasion, and metastasisthrough the WNT signaling pathway or by regulating integrinmolecule expression of integrin. The specific role of PKCa in CMMhas been a controversial issue, with some results suggesting that

Table 5. Kaplan-Meier, log-rank test, and Cox regression univariate and multivariate analyses of DFS

Univariate and Breslow-adjusted analyses

Marker No. cases Cases with metastatic disease Hazard ratio (95% CI) P Adjusted by Breslow

Hazard ratio (95% CI) P

N-cadherin 108 60 1.95 (1.15–3.31) 0.013 1.49 (0.86–2.58) 0.155

Osteonectin 112 66 1.99 (1.21–3.25) 0.006 1.48 (0.89–2.46) 0.126

Osteopontin 102 61 1.88 (1.00–3.55) 0.05 1.72 (0.91–3.23) 0.095Glypican 3 107 64 1.51 (0.80–2.83) 0.199 1.19 (0.62–2.26) 0.603

PKCa 119 69 1.21 (0.75–1.95) 0.429 2.03 (1.19–3.44) 0.009Multivariate analysis

Marker Hazard ratio SE z P > z 95% CI

PKCa 2.313 0.7188 2.89 0.004 1.3092–4.0870

Breslow 1.415 0.0655 7.50 0.000 1.2925–1.5497

NOTE: Data were measured from the time of primary vertical growth phase CMM to clinical or histopathologic metastatic disease. The P values areshown for the univariate analysis of the validation TMA set before and after introducing the Breslow index (bivariate Cox model). Significant markers

(P < 0.05) are shown in bold.

Abbreviation: 95% CI, 95% confidence interval.

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PKCa and PKCy isoforms suppress cell growth, whereas othersimply its involvement in cell motility (42, 43) or in metastasispromotion (44). Expression profiling data in this series identifiesPKCa as one of the genes up-regulated in melanoma withmetastasis, and the multivariate analysis showed that it is Breslowindependent, implying that there is a relationship between tumorgrowth and proliferation. This awaits further investigation.

Lumican , the most highly up-regulated gene in melanoma caseswith metastasis, is a proteoglycan of the extracellular matrixinvolved in collagen-fiber organization, epithelial-cell migration,and tissue repair. Lumican is an established EMT marker (45)whose protein expression level is associated with tumorigenesisand progression in a variety of tumors (46, 47). Data presented hereare also consistent with those obtained from the comparison ofnormal melanocytes with melanoma cell strains from advancedlesions (3).Cell-matrix interactions are closely related to EMT category. The

integrins are a family of cell surface adhesion molecules thatcoordinate cell-cell and cell-matrix interactions. This studyidentified up-regulation of integrin aV (ITGAV ) as a marker ofmelanoma metastases, confirming a previous report (48). Closelyrelated are the MMP family of proteins, which are involved in thebreakdown of the extracellular matrix in normal physiologicprocesses and in cancer invasion and metastasis. Here, MMP2(which degrades type IV collagen) and ADAM9 (disintegrin andmetalloproteinase domain 9) are associated with melanomaprogression, confirming previous findings regarding invasion (49).Two of the metastatic cases analyzed here (mel-08 and mel-09)

displayed a nonmetastatic phenotype, with the exception of theincreased expression of TGFBR2, p21 (CDKN1A), or ENC1. It isknown that melanoma and endothelial cells express type 2 TGF-hreceptor (50), which can enhance adhesion of melanoma cells toendothelium and favor invasion through activation of the TGF-hpathway. In the same way, p21 overexpression has been proposedas a marker of melanoma progression (9). The specific relevance of

each functional cluster of genes, as identified here, requires furtherstudy, both in human samples and experimental models.Interestingly, the multivariate study showed that three of the

protein markers analyzed in TMA were associated with localinvasion and metastasis, thereby linking the two phenomena.Overall, our results lead us to propose that metastasis in melanomais determined by the interaction of sets of molecules involved inthe regulation of EMT immune response, cell metabolism, nucleicacid binding and transcription, protein synthesis and degradation,and signal transduction. Moreover, validation of protein expressionin TMA confirms how some of the up-regulated genes arepredictors of metastasis development. Taking all our results intoconsideration, it seems that the control of the EMT plays a pivotalrole in the metastatic process in melanoma. This confirms andbuilds on findings linking EMT genes with melanoma progressionobtained in experimental models and human samples studies, suchas cadherins, MMPs, integrins, lumican, osteopontin, EDNRB, Snail,and others (6, 49).

Acknowledgments

Received 9/19/2006; revised 1/17/2007; accepted 1/31/2007.Grant support: FIS grant PI 040641, MMA 2005-085, and European Commission

(Translational and Functional Onco-Genomics) grant LSHC-CT-2004-503438. S.R.Alonso was supported in part by FIS grant CM03/00034 and by the CNIO (Madrid,Spain). L. Tracey received support though grants from the CNIO and the HigherEducation Authority of Ireland through the Department of Haematology/Institute ofMolecular Medicine, St. James Hospital, Dublin, Ireland.

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

We thank the Tumour Bank of the Hospital San Cecilio; the Spanish NationalTumour Bank Network, CNIO; Laura Cereceda (CNIO) and Alicia Maroto (CNIO,Hospital 12 de Octubre) for their valuable help in providing specific tissue samples forthis study; Pilar Sandoval (CNIO, Hospital Gregorio Maranon) for her time withgridding; Dr. David Hardisson and Raquel Marcos (CNIO, Hospital Universitario LaPaz) for helping with the SPARC immunostaining; Ramon Diaz for helping with theWilcoxon test; Amancio Carnero for their valuable guidance with gene classification;and Phil Mason for his help with the English edition of the final version of themanuscript.

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