The characterization of the invasion phenotype of uveal melanoma tumour cells

shows the presence of MUC18 and HMG-1 metastasis markers and leads

to the identification of DJ-1 as a potential serum biomarker

Mar�ıa Pardo1*, �Angel Garc�ıa1, Benjamin Thomas2, Antonio Pi~neiro3, Alexandre Akoulitchev2,Raymond A. Dwek

1and Nicole Zitzmann

1

1Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, United Kingdom2Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom3Instituto Galego de Oftalmolox�ıa (INGO), R�ua Ram�on Baltar s/n, 15706, Santiago de Compostela, Spain

Uveal malignant melanoma (UM) is the most frequent primary in-traocular tumour in adult humans. Because the survival rate ofpatients with UM has changed little in the past few decades, a bet-ter understanding of the molecular events governing UM develop-ment and the identification of markers indicating the potential formetastasis at the time of diagnosis are necessary to designimproved and more specific treatments. In this study, we investi-gated UM tumour development by comparing two recently estab-lished UM cultures with different invasion potential by two-dimensional gel electrophoresis. Protein features expressed differ-entially were identified by mass spectrometric analysis. Potentialmarkers were assayed in both cultures and in long-term estab-lished UM cell lines (UW-1, OCM-1, SP6.5 and 92.1) by Westernblotting and their role in invasion analysed using Matrigel mem-branes. Comparative analysis revealed that UM cultures with low-and high-grade invasion potential differ in their cellular metabo-lism and, more interestingly, in several cancer-associated proteins,including those implicated in cell adhesion and migration, prolif-eration and various oncogenes. Our data indicate a correlationbetween MUC18 and HMG-1 expression and the invasiveness ofUM cells. We also demonstrate the expression and secretion ofDJ-1 oncoprotein by UM cells. We suggest a possible role forMUC18 and HMG-1 proteins in UM cell invasion. The secretionof DJ-1 by UM cells, and the ability to detect this protein in UMpatients’ sera implicate it as a potential noninvasive biomarkerfor this malignancy.' 2006 Wiley-Liss, Inc.

Key words: uveal melanoma; invasion; proteomics; MUC-18; DJ-1

Uveal malignant melanoma (UM) is the most frequent intraoculartumour in adult humans.1 Unlike cutaneous melanoma, uveal mela-noma disseminates mainly through the blood stream and preferen-tially establishes metastases in the liver. Metastatic liver disease isthe leading cause of death in uveal melanoma and can develop aftera long disease-free interval, which suggests the presence of occultmicrometastatic disease at the time the primary eye tumour is diag-nosed and treated.2 Unfortunately, advances in eye cancer treatmenthave not paralleled those made in the management of other types ofcancer, and the survival rate of patients with uveal melanoma haschanged little in the past few decades.3 A better understanding of themolecular events governing uveal melanoma development and theidentification of markers indicating the potential for metastasis at thetime of diagnosis are necessary to design improved and more spe-cific treatments. Various clinical and molecular prognostic factorshave been suggested in uveal melanoma, but none has proved to besufficiently useful or viable for routine clinical use.4,5

Currently, there are many challenging technologies and app-roaches to identify tumour markers for prognostic and therapeuticpurposes.6 Transcriptional studies alone are not sufficient, with sev-eral investigators reporting a poor correlation between mRNA andprotein abundance.7 Furthermore, a single gene can encode for morethan one mRNA species through differential splicing, and proteinscan undergo as many as 200 posttranslational modifications.8 Forthis reason, proteomics has become a complementary technique togenomic analysis. Studies of global protein expression by proteo-mics technology in human tumours have yielded information about

tumour heterogeneity and have led to the identification of variouspolypeptide markers which are potentially useful as diagnostictools.9 We recently applied this approach to obtain the first proteomeanalysis of a previously well-characterized uveal melanoma primarycell culture named UM-A.10,11 This analysis represented the firststep towards the utilization of proteomics in the study of uveal mela-noma tumours and a novel approach to search for potential markers.The UM-A primary culture, characterized by the presence of hypo-phosphorylated and unexpectedly inactive antitumoural protein Rb,changed into a phenotypically different cell line after 7 passages.10

On the basis of extracellular matrix (ECM) invasion assays thatshowed a low and high degree of metastatic potential in the UM pri-mary cell culture (UM-A < 7) and cell line (UM-A > 7), respec-tively, we present here the differential proteome analysis of both cul-tures. The identification of differentially regulated features revealsan increase in the cellular metabolism of the UM-A cell line and,interestingly, a different expression profile of cancer-related pro-teins. Proteins involved in cell adhesion and migration, proliferation,various oncogenes and others, many described for the first time inUM, are listed in this article. A selection of novel proteins is vali-dated by other techniques, showing a possible correlation betweenMUC18 and HMG-1 expression and UM invasiveness. We alsoshow the overexpression and secretion of DJ-1 oncoprotein in UMcells and the ability to detect it in certain UM patient sera, indicatingDJ-1 as a potential biomarker.

Material and methods

Cell culture

The UM-A primary cell culture, the resulting cell line and nor-mal melanocytes (NM) were obtained as described previously.10

UM-A < 7, UM-A > 7, NM and UM cell lines (UW-1, OCM-1,SP5.6 and 92.1) were cultured in RPMI medium containing 5%inactivated fetal calf serum (FCS), 2 mM glutamine and standardantibiotics at 37�C in 5% CO2.

Immunoblotting

Whole cell lysates were prepared by direct lysis of subconfluentcells in cold RIPA buffer, as described previously.10 Equalamounts of protein (30 lg/lane) were separated on sodium dodecylsulphate–polyacrylamide gels (SDS–PAGE) and electroblottedonto nitrocellulose membranes. The membranes were probed suc-cessively with primary antibodies and horseradish peroxidase-labelled secondary antibodies (ECL, GE Healthcare, Uppsala,Sweden). Anti-DJ-1 and MUC18 antibodies were purchased from

This article contains supplementary material available via the Internet athttp://www.interscience.wiley.com/jpages/0020-7136/suppmatGrant sponsor: Oxford Glycobiology Institute Endowment.*Correspondence to: Glycobiology Institute, Department of Biochem-

istry, University of Oxford, South Parks Road, OX1 3QU Oxford, UK.Fax:144-1865-275216. E-mail: maruxapardo@hotmail.comReceived 24 November 2005; Accepted 7 February 2005DOI 10.1002/ijc.21942Published online 28 March 2006 inWiley InterScience (www.interscience.

wiley.com).

Int. J. Cancer: 119, 1014–1022 (2006)' 2006 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

Upstate (Lake Placid, NY) and anti-actin and HMG-1 from StaCruz Biotechnology (Santa Cruz, CA). To prepare cell culturesecreted proteins for Western blot, the cell culture medium wasconcentrated in a Centricon YM-10 (Millipore, MA) to a final vol-ume of 100 ll, according to the manufacturer’s instructions. AllWestern blot images shown in the figures are representative of atleast 3 independent experiments.

Extracellular matrix invasion assays

The invasion potential of UM cells was assessed using a BDBioCoatTM MatrigelTM Invasion Chamber (Becton Dickinson,MA), following the manufacturer’s indications. Briefly, 15 3 104

cells were seeded over an 8-lm pore size PET membrane withECM or without ECM (control) inserts in 0.1% FCS-RPMI me-dium for 48–72 hr. FCS-RPMI (5%) was added to the lower cham-ber as a chemoattractant. Noninvading cells were removed fromthe upper chamber by gently wiping the upper surface of the mem-brane with a cotton swab. Membranes were fixed in 100% metha-nol and stained in 1% toluidine blue. Levels of invasion wereassessed by counting the number of cells present in 5 differentfields in triplicate on the lower surface of the membrane under thelight microscope (403 magnification). Data are expressed as thepercent of invasion through the Matrigel matrix and relative to themigration through the control membrane.

Two-dimensional gel electrophoresis

UM-A < 7 and UM-A > 7 protein samples were obtained asdescribed previously.11 Briefly, cells were washed twice in phos-phate balance solution and disrupted by gentle sonication in sam-ple buffer (5 M urea, 2 M thiourea, 2 mM tributylphosphine,65 mM DTT, 65 mM CHAPS, 0.15 M NDSB-256, 1 mM sodiumvanadate, 0.1 mM sodium fluoride, 1 mM benzamidine). The su-pernatant was removed and 600 lg of protein was taken up in atotal volume of 375 ll sample buffer. 3–10NL immobilized pHgradient (IPG) strips were hydrated in the sample, and isoelectricfocusing was carried out for 70 kV hr at 17�C, according to themethod of Sanchez et al.12 Following focusing, the IPG strips

were immediately equilibrated for 10 min in 4 M urea, 2 mMthiourea, 12 mM DTT, 50 mM Tris (pH 6.8), 2% (w/v) SDS, 30%(w/v) glycerol and placed on top of the second dimension gels em-bedded in 0.5% melted agarose. Proteins were separated in thesecond dimension on SDS-PAGE gradient gels (9–16% T, 2.67%C) under running conditions of 10�C, 20 mA/gel for 1 hr, followedby 40 mA/gel for 4 hr. Following electrophoresis, the gels werefixed and stained with the fluorescent dye OGT MP17 (OxfordGlycosciences, Abingdon, UK), on the basis of Ref. 13. 16-Bitmonochrome fluorescence images were obtained at 200-lm reso-lution by scanning gels with an Apollo II linear fluorescent scan-ner (Oxford Glycosciences).

Differential image analysis

Scanned images were processed with a custom version ofMELANIE II (Bio-Rad Laboratories Ltd, Hemel Hempstead,UK). Four pI 3–10 gels were prepared in independent experimentsfor both UM-A < 7 and UM-A > 7 samples. Internal calibrationof the 2DE (two dimensional gel electrophoresis) gel images withregard to pI and molecular weight was carried out, as describedpreviously.14 For differential image analysis, a synthetic gel imagewas generated by means of accurate spot matching. This syntheticimage contained all protein features detected in UM-A < 7 andUM-A > 7 samples. Only the features present in at least 3 of 4individual gels belonging to either the UM-A < 7 or UM-A > 7samples were considered for differential analysis. Intensity (opti-cal density) was measured by summing pixels within each spotboundary (spot volume) and recorded as a percentage of the totalspot intensity of the gel: %V5 spot volume/R volumes of all spotsresolved in the gel. Variations in protein expression were calcu-lated as the ratio of average volumes (%V) and carefully validatedby repeated image analysis by human operators. Differentialexpression of a protein present in both UM-A < 7 and UM-A > 7gels was considered significant when the fold change was at least2 and p was no more than 0.05 after rank-sum test applied on %Vvalues.

In-gel digestion and peptide extraction

Protein features assigned to mass spectrometric analysis wereexcised from the gel by a software-driven robotic cutter. Therecovered gel pieces were dried in a speed-vac, and in-gel diges-tion was carried out by the automated DigestPro workstation(Abimed, Langenfeld, Germany), according to the protocol ofShevchenko et al.15

Mass spectrometric analysis

Mass spectrometric analysis was carried out using a Q-TOFMicro instrument (Micromass, Manchester, UK) coupled toCapLC (Waters, Milford, MA). The tryptic peptides were loadedand desalted on a 300 lm id/5 mm length C18 PepMap column(LC packings, San Francisco, CA). The peptides were resolved ona 75 lm id/150 mm length C18 Atlantis NanoEase column(Waters). The peptide mixture was eluted with 98% acetonitrilecontaining 0.1% formic acid over 60 min at a flow rate of 250 nl/min. The gradient was as follows: 0–3 min, constant 5% acetoni-trile; 3–30 min, acetonitrile increased to 40%; 30–35 min, acetoni-trile increased to 90%; 35–40 min, acetonitrile constant at 90%;40–45 min, acetonitrile decreased to 5%; 45–60 min, acetonitrileconstant at 5%. Mass spectrometry data were acquired and ana-lysed by Masslynx software version 4 (Micromass) using auto-matic switching between MS and MS/MS modes. The survey scan(1 sec) was obtained over the mass range m/z 300–1,600 in thepositive-ion mode with a cone voltage of 35 V and a capillaryvoltage of 3,500 V. When the signal reached a user-defined thresh-old (10 counts/sec), peptide precursor ions could be selected forMS/MS (8 sec total scan time) over the mass range m/z 50–2,000.Fragmentation was performed using argon as the collision gas andwith a collision energy profile (20–40 eV) optimized for variousmass ranges of precursor ions. The selected precursor ions were

FIGURE 1 – Different in vitro behaviour of the human uveal mela-noma primary cell culture UM-A < 7 and the resulting cell line UM-A > 7. (a) Phenotype of the UM cultures UM-A < 7 and UM-A > 7.(b) Matrigel invasion assay. (c) Rb phosphorylation status on UM-A< 7 and UM-A > 7.

1015PRESENCE OF MUC18 AND HMG-1 METASTASIS MARKERS IN UM TUMOURS AND IDENTIFICATION OF DJ-1 AS A BIOMARKER

automatically included in the exclusion list. The database searchwas performed with the MASCOT search tool (Matrix science,London, UK) screening Swiss-Prot (release 45.2 of November 23,2004) and TrEMBL (release 28.2 of November 23, 2004), andrestricting the database search to human taxonomy. Positive iden-tification was accepted only when the data satisfied the followingcriteria: (i) MS data were obtained for a full-length y-ion series ofa peptide comprising at least 8 amino acids and no missed cleav-age; (ii) MS data with 1 or 2 missing y-ions were obtained for 2 ormore different peptides comprising at least 8 amino acids and nomissed cleavage.

Results

Differential analysis of human uveal melanoma primary cellculture (UM-A\ 7) and the resulting cell line (UM-A[ 7)

We observed previously that cells from the UM-A primary cul-ture became phenotypically different from the cells of origin after

passage 7 (UM-A > 7).10 These cells were less firmly attached,exhibiting more rounded, refractile cellular bodies and an acceler-ated growth rate (Fig. 1a). In this study, we demonstrate that UM-A > 7 cells show a higher invasion potential in Matrigel assays(Fig. 1b), and contrary to the primary cell culture of origin (UM-A< 7), these cells are characterized by a highly phosphorylatedform of the antitumoural protein Rb (Fig. 1c).

On the basis of a recently performed proteome study of theUM-A primary cell culture, which revealed several candidates aspotential cancer markers,11 we present here the proteome differen-ces between UM-A < 7 and UM-A > 7 samples that may give rel-evant clues to further understand disease processes in these tumourcells. Comparing 2D gels of UM-A < 7 and UM-A > 7 celllysates (Fig. 2a), we found that most of the protein features identi-fied in the UM primary cell culture proteome11 were also presentin the derived cell line (UM-A > 7). These include spots contain-ing proteins related to the oncogenesis process, such as oncogeneDJ-1, oncoprotein 18/Stathmin and tumour-rejection Ag, among

FIGURE 2 – Differential proteomeanalysis of human uveal melanomaprimary cell culture (UM-A < 7)and the resulting cell line (UM-A >7). (a) Representative images of thepI 3–10 proteome coverage 2DEmap of UM-A < 7 and UM-A > 7.(b) Synthetic 2DE image represent-ing all protein features present in theanalysis. The location of some rele-vant cancer-related proteins on theUM 2D-proteome map is shown.Proteins present only in UM-A < 7are shown in bold characters andproteins present only in UM-A > 7are shown in italic; all other proteinsare present in both cultures.

1016 PARDO ET AL.

others (Fig. 2b). However, we were specifically interested in a dif-ferential analysis between both sets of gels, focusing on the identi-fication of disappearing and appearing spots, and at least a 2-foldup- or downregulation of spot intensities (with p < 0.05). Approx-imately 85% of protein features were present in at least 3 of the 4gels per group (UM-A < 7 or UM-A > 7), a requirement for beingconsidered for the analysis. These strict criteria for the inclusionof proteins into the analysis were used to avoid misidentificationsdue to gel-to-gel variations. Applying these criteria, 290 differen-tially regulated protein features from �1,100 spots were detectedin the pI 3–10 analytical range. Ninety percent of the differentiallyexpressed proteins were successfully identified by liquid chroma-tography and mass spectrometry (see Supplementary Table). Fromthose, 41% were present only in the cell line (UM-A > 7), 31%were present only in the primary culture (UM-A < 7), 12% wereupregulated in UM-A > 7 and 10% upregulated in UM-A < 7.

The differential analysis showed that UM-A < 7 containedincreased levels of actin-related and actin-binding proteins com-pared to that UM-A > 7 cells contained. However, cytokeratines 8and 18, together with vimentin, a combination characteristic ofuveal melanoma tumours, were present in both cell cultures (seeSupplementary Table). Interestingly, several features correspond-ing to proteins implicated in cell adhesion present in the primaryculture disappeared in the cell line, e.g., vinculin and ezrin. TheUM-A > 7 culture was characterized by the presence of proteinsrelated to the disassembly of actin filaments (e.g., macrophagecapping protein, WD-repeat protein) and also by various forms oftubulins. Furthermore, cellular metabolism seems to be increasedin UM-A > 7 cells, as reflected in the appearance of new spotsrelated to glycolysis enzymes (e.g., alpha enolase, fructose-biphosphate aldolase A-C, gamma-enolase, phosphoglyceratekinase, glyceraldehide 3 phosphate dehydrogenase) and energymetabolism (e.g. ATP synthases). Other spots containing nuclearproteins decreased their expression or disappeared in UM-A > 7culture, such as CENP-F, Oncogene FUS and nuclear protein Hcc,whereas new spots were detected in UM-A > 7, such as BRCA-1,HMG-1, nuclear protein SkiP, protein associated with myc andnew isoforms of nucleophosmin (Table I, Fig. 2b). In addition,various annexins were present in the differential analysis, sincethe corresponding spots were either absent or less intense in thecell line compared to that in the primary cell culture (e.g., annexinA1, A2, A5, A6). Similarly, LIM and SH3 domain protein 1, RhoGDP dissociating inhibitor, tumour protein D54 and S100A6 andA6 were identified only in spots from early passages of the culture(Table I, Fig. 2b and Supplementary Table). Interestingly, othernew proteins like HS1-binding protein, related to the promotion ofcell survival, CTCL tumour antigen se33-1, pelota homolog, newisoforms of GTP-binding nuclear protein RAN, HSP60b and thecell adhesion protein MUC18 were identified as exclusive featurespresent in the UM cell line (Table I, Fig. 2b and SupplementaryTable). The location of some relevant cancer-related proteins onthe UM 2D-proteome map is shown in Figure 2b.

Validation of potential markers for uveal melanoma: Role ofHMG-1 and MUC18 in in vitro cell invasion assays

We analysed further the potential biological relevance of someinteresting proteins identified in the differential proteome analysis.Because of our interest in studying the dissemination of cancerouscells in UM patients, we focused on the expression of 2 metasta-sis-associated proteins, the gene expression-regulating proteinHMG-1 and the cell adhesion protein MUC18 (Fig. 3a), by vali-dating their presence in whole cell extracts by Western blot (Fig.3b). To eliminate the possibility of cell line-specific expression,we included standard cell lines established by other groups usedfor the study of uveal melanoma, namely UW-1, SP6.5, OCM-1and 92.116 and a primary cell culture of NM established previ-ously by our group.10 HMG-1 protein was observed at higher lev-els in the UM-A cell line than in the primary cell culture, andoverall, was overexpressed in all the UM cell lines assayed (Fig.3b). In the case of MUC18, immunodetection showed its presence

at low levels in UM-A < 7, UW-1 and 92.1; it was overexpressedin UM-A > 7, OCM-1 and SP6.5 and was not present in NM (Fig.3b). Matrigel invasion assays showed a correlation between theinvasion potential and MUC18 expression in the UM cultures(Fig. 3c). This experiment showed a large difference in the inva-sion potential between the UM primary culture (20%) and the cellline (70%), paralleling the expression levels of MUC18 in thesecells (Figs. 3b and 3c). The same correlation was found with UW-1, OCM-1 and SP6.5, where expression levels of MUC18 was cor-related with the invasion potential; however, the UM cell line 92.1did not show this pattern even though the invasion potential wassimilar to OCM-1 (Figs. 3b and 3c). Although the levels of HMG-1 were high in all the UM cultures when compared to the normalcells, a clear correlation with the invasion potential assayed inMatrigel on these cells was not found (Figs. 3b and 3c).

Validating the presence of DJ-1 oncogene in uveal melanoma:DJ-1 as a potential biomarker in uveal melanoma patients’ sera

DJ-1 oncogene was not differentially regulated between UM-A< 7 and UM-A > 7 (Fig. 4a). However, we found that DJ-1 wasoverexpressed in all the UM cell lines, including the UM primarycell culture, when compared to NM (Fig. 4b). It has beendescribed that this protein can be secreted from malignant cells;17

therefore, we studied whether this was the case in our UM model.We analysed the presence of DJ-1 in the culture medium of UM-Aand other UM cell lines by Western blot. All these cultures exceptthe NM secreted DJ-1 into the cell culture media (Fig. 4b). Thisresult indicated that DJ-1 may be secreted by tumour cells into theblood stream; and indeed, preliminary trials revealed its presencein certain UM patients’ sera (data not shown).

Discussion

Differential proteome analysis of human uveal melanoma primarycell culture (UM-A\ 7) and the resulting cell line (UM-A[ 7)

To unravel alterations related to tumour progression, proteo-mics has recently attracted great attention, because it allows theidentification of qualitative and quantitative changes in proteincomposition, including posttranslational modifications, when com-paring healthy and cancerous cells. Recently, Hayashi et al.proved the value of this approach, comparing regressive and pro-gressive cancer cell lines, for the identification of molecularabnormalities in tumour progression.18 Molecular determinantsfor UM have been studied at the genomic level from 2 UM celllines with different invasion potentials.19 However, to our knowl-edge, the comparison of UM cell lines by proteomics has not beenperformed to date.

We have successfully established a UM primary cell culture(UM-A) that proved to be a valuable tool for the study of this neo-plasia in vitro.10,11 Seike et al. studied different cancer cell linesand found that most tissue-cultured cells retain characteristics thatreflect their in vivo origin and differentiation phenotype afterlong-term culture.20 This is also consistent with previous reportsthat tissue-cultured cells retain the gene expression signature oftheir original tissues.21,22 Taking into account the low incidence ofUM (7–10 per million per year) and that only about 5% of patientswith newly diagnosed disease undergo enucleation, the acquire-ment of significant numbers of fresh tissue samples for the estab-lishment of new primary cultures has been traditionally problem-atic in the study of UM.23 Mainly for this reason, the molecularsignature of UM is still very poorly known, and may explainwhy the survival rate of patients with UM has not paralleledthat of other cancer types, with no improvement made in the lastdecades.24

Here we continue a previous study whose aim was to bettercharacterize UM-A, by studying the molecular progression fromthe primary cell culture to cell line after 7 passages in vitro. Theoccurrence of differences between primary cell cultures and celllines is generally known, but very few reports have studied these

1017PRESENCE OF MUC18 AND HMG-1 METASTASIS MARKERS IN UM TUMOURS AND IDENTIFICATION OF DJ-1 AS A BIOMARKER

TABLE I – RELEVANT CANCER-RELATED PROTEINS IDENTIFIED IN THE UM-A < 7 VERSUS UM-A > 7 DIFFERENTIAL ANALYSIS

Protein name Protein ID MW pI Fold Biological function

Proteins present or over-expressed in UM-A<7Annexin A1 P04083 32,153 6.03 em Regulation of membrane trafficking

and cellular adhesion.31,501 5.75 emAnnexin A2 P07355 36,061 6.27 22.05 Plays a role in the regulation of

cellular growth and in signaltransduction pathways.

35,864 6.47 em25,592 5.41 em28,512 6.97 22.08

Annexin A5 P08758 33,406 5.27 22.16 Potential role in cellular signaltransduction, inflammation,growth and differentiation.

Annexin A6 P08133 79,521 5.76 22.09 Implicated in membrane-relatedevents along exocytotic andendocytotic pathways.

Calcyclin (S-100A6) P06703 10,542 5.12 em Involved in calcium signalling.Loss of expression inadvanced stage cancers.

10,477 5.19 em

Cathepsin B precursor P07858 25,784 5.34 em Participates in intracellular degradationand turnover of proteins.Related to tumour invasionand metastasis.

33,406 5.27 22.16

CENP-F kinetochore protein P49454 79,521 5.76 22.09 Chromosome segregation duringmitosis. Interacts withretinoblastoma protein (Rb),CENP-E and BUBR1.

Ezrin P15311 81,161 6.07 23 Cell adhesion, mobility and cellsurvival might participate intumour progression.

LIM and SH3 domain 1 Q14847 36,061 6.27 22.05 Focal adhesion protein necessaryfor cell migration and survival.35,864 6.47 em

Multidrug resistanceassociated protein MGr1-Ag

P08865 34,708 4.66 em Belongs to the S2P family ofribosomal proteins.

Nuclear protein Hcc-1 P82979 32,153 6.03 em Translational control of cell growth,metabolism and carcinogenesis.

Oncogene FUS P35637 62,874 5.98 em May play a role in maintenanceof genomic integrity.

Rho GDP-dissociation inhibitor 1 P52565 23,207 5.03 23.02 Belongs to the Rho GDI family.Rho GDP-dissociation inhibitor 2 P52566 26,367 5.15 22.05 Belongs to the Rho GDI family.S100 calcium-binding protein A16 Q96FQ6 9,096 6.37 em Belongs to the S-100 family.

Up-regulated in tumours.Tumour protein D54 O43399 28,669 5.24 em May play roles in calcium-mediated

signal transduction and cellproliferation.

30,113 5.11 em30,113 5.07 em

UV excision repair proteinRAD23 homolog B

P54727 55,431 4.86 22.36 Involved in DNA excision repair.

Vinculin P18206 149,390 5.92 em Involved in cell adhesion. May allowcancer cells to move awayfrom tumours.

135,273 5.94 em131,413 6.09 22.09134,715 6.21 em

Proteins present or over-expressed in UM-A>7Breast cancer type 1 susceptibilityprotein

P38398 47,523 6.15 lm Plays a central role in DNA repairby facilitating cellular responseto DNA repair.

CTCL tumour antigen se33-1 Q9H2G1 32,808 7.04 lm Tumour-associated antigen found inseveral cutaneous T-cell lymphomas(CTCL).

DNA excision repair proteinERCC-6

Q03468 17,746 6.32 lm Is involved in the preferentialrepair of active genes.

GTP-binding nuclear protein RAN P62826 25,418 6.91 lm Required for the import of proteins intothe nucleus and also for RNA export.Involved in chromatin condensationand control of cell cycle.

Heat shock protein HSP 90-b P08238 35,920 5.31 lm Molecular chaperone. Related tovarious cancers.108,646 5.18 em

Heterogeneous nuclearribonucleoproteins A2/B1

P22626 31,021 7.11 2.04 Pre-mRNA processing.

High mobility groupprotein 1 HMG-1

P09429 28,258 6.33 lm Has a role in the transcription of manygenes involved at different steps inthe metastatic cascade and has beenlinked with cancer in human andanimal models.

HS1-binding protein O00165 32,546 4.86 lm May function in promoting cellsurvival.

Continued

1018 PARDO ET AL.

changes in more depth.25,26 Interestingly, invasion assays withboth UM-A cultures showed a significant increase in the percent-age of cells able to degrade the ECM in the UM cell line (UM-A> 7) when compared to the primary culture (UM-A < 7). Takinginto consideration that more UM tumour samples and culturesneed to be analysed, and the difficulty in linking the enormousamount of information yielded by proteomics, we believe that thisanalysis may give relevant clues about disease stages, includingdissemination and metastasis.

Differentially expressed proteins between the UM-A primarycell culture and the resulting cell line correlate well with differen-ces observed when culturing these cells in vitro. The increasedamount of mitochondrial, protein processing and general metabo-lism proteins observed in the cell line explains their higher prolif-eration rate and consequently more active cell metabolism com-pared to that of the cells of origin. The fact that the Rb antitumou-ral protein is inactivated by phosphorylation in the UM-A > 7also reflects the increment on cell division of these cells. In agree-ment with the differences found in the invasion potential of UM-Acultures, we found that some proteins related to cell adhesion wereshown to be present only in the UM primary cell culture, whilevarious actin-disassembling features were present exclusively inthe cell line. In addition, several of the identified protein featurescorrelated well with proteins described previously at differentstages of cancer. The presence of spots containing Annexin A1,A5 and A6 was significantly decreased in the UM-A cell line, asdescribed previously in various oncogenesis processes, includingskin melanoma.27–29 The differentially expressed proteins RhoGDPI 1 and 2, mostly present in the UM primary cell culture, alsocorrelated well with previous reports involving the downregula-tion of those proteins in late stages of cancer.30,31

On the other hand, we found proteins previously linked to tumourdevelopment or related to cell migration and invasion that wereupregulated or exclusively present in the primary cell culture. Inter-estingly, new proteins were differentially present in the UM cellline like HMG-1, nuclear protein SkiP, and hnRNPA2/B1. Thisphenomenon may be related to new chromosome aberrations and tothe cell selection and dedifferentiation processes inherent in theestablishment of cell lines in vitro. However, both UM-A primarycells and the resulting cell line showed the presence of cytokeratins8 and 18 together with vimentin, constituting the interconvertedphenotype characteristic of uveal melanoma tumours.32

HMG-1 and MUC18 as potential invasion markers inuveal melanoma

The presence of a new isoform of the non-histone chromosome-binding protein HMG-1 in the UM cell line is interesting. Wedescribed previously the presence of HMG-1 in the UM-A pri-

mary cell culture, which coimmunoprecipitated with the hypo-phosphorylated form of the antitumoural protein Rb;10 however,the role of HMG-1 in UM is still unknown. We validated the pres-ence of HMG-1 and found high levels of this protein in UM cul-ture extracts. Most normal, differentiated mammalian cells expressextremely low levels of HMG-1. Only cells experimentally trans-formed and many neoplasms are characterized by high levels ofthis protein.33,34 In addition, HMG-1 is involved in the transcrip-tional regulation of a number of genes reported to play key rolesin different biological processes of neoplasm progression and me-tastasis.35 According to our results, HMG-1 may play a role inUM, even though we could not show a clear relationship betweenHMG-1 levels in UM cultures and its invasion potential in Matri-gel membranes. This result agrees with those reports that do notclarify whether HMG-1 has a metastasis-associated or metastasis-inducing role.34

MUC18 (also named MelCAM/CD146/S-Endo 1-associatedantigen), a cell adhesion molecule (CAM) belonging to the immu-noglobulin superfamily, is constitutively expressed in the wholehuman endothelium.36–38 MUC18 is upregulated during skin mel-anoma development in a stepwise fashion and coincides with theseparation of nevus cells from keratinocytes.39 Despite originatingfrom common precursor cells, UM and cutaneous melanomas ex-hibit several notable differences in tumour biology and behaviour,including their molecular signatures.40 However, similar to skinmelanoma, we found spots containing MUC18 in the UM-A cul-tures and also the expression of this protein in these and other UMcell lines. A positive association between the loss of the adhesionprotein ICAM-1 expression and increased risk of metastasis hasbeen suggested in UM;41 but to our knowledge, no relationshiphas been described to date between UM and MUC18. The expres-sion of MUC18 is limited not only to melanoma neoplasias, but ithas also been implicated with an increase of metastasis potentialin human prostate and bladder cancer cells.42,43 In agreement withthese data, we also show a correlation between MUC18 expressionin UM-A cultures and their invasion potential in vitro. This corre-lation was also found in all the UM cell lines except 92.1, whichmight suggest that more than one molecular event govern UMinvasion. More research is necessary to determine if the role ofMUC18 in UM agrees with previous data suggesting that MUC18may play a major role in metastasis by mediating not only mela-noma cell–cell interactions, but also melanoma–endothelial celladhesion.44 This process can be reverted in animal models by theuse of fully human antibodies against MUC18.45 Thus, the possi-bility of using this strategy in UM may be considered in the future.To follow up this further and to correlate it with clinical parame-ters, we are in the process of immunodetecting MUC18 in UMtumour sections (not shown).

TABLE I – RELEVANT CANCER-RELATED PROTEINS IDENTIFIED IN THE UM-A < 7 VERSUS UM-A > 7 DIFFERENTIAL ANALYSIS (CONTINUED)

Protein name Protein ID MW pI Fold Biological function

Macrophage capping protein P40121 40,499 6.03 lm Helps to modify actin structures inresponse to external signals, whichpermit rapid changes in shape duringdevelopment.

42,039 5.48 2.3146,521 6.89 lm39,015 5.60 lm14,262 6.42 lm

Melanoma-associatedantigen MUC18

P43121 36,597 6.31 lm May allow melanoma cells to interactwith cellular elements of the vascularsystem thereby enhancing hematogeneoustumour spread.

Nuclear protein SkiP Q13573 72,610 5.74 lm Interacts with the ski oncogene.Nucleophosmin P06748 35,825 5.11 lm May function in the assembly or transport

of ribosomes.34,882 4.73 lmPelota homolog Q9GZS6 44,341 5.98 lm Possibly participates in cell cycle regulation.Protein associated with Myc O75592 34,237 6.31 lm Interacts directly with the transcriptional

activating domain of the oncogene–Myc.Uveal autoantigen Q9BZF9 45,660 5.41 lm Myosin heavy chain with Ankyrin repeat.

Note: Protein ID, Swiss-Prot accession number. MW and pI, molecular weight and isoelectric point for identified protein features. Fold repre-sents fold change (UM-A < 7 versus UM-A > 7); a negative value indicates that the feature is present in a higher extend in the UM-A < 7; em,spot only present in UM-A < 7 and lm, spot only present in UM-A > 7. Biological function based on Swiss-Prot and PudMed databases.

1019PRESENCE OF MUC18 AND HMG-1 METASTASIS MARKERS IN UM TUMOURS AND IDENTIFICATION OF DJ-1 AS A BIOMARKER

DJ-1 oncogene as a potential serum marker in uveal melanoma

Finally, we show the expression of DJ-1 protein in both UM-Acultures and in different UM cell lines as well as its secretion tothe cell culture media. Preliminary results show that DJ-1 is alsodetectable in the serum of certain UM patients (data not shown).DJ-1 was originally cloned as a putative oncogene capable oftransforming NIH-3T3 cells in cooperation with H-ras.46 It hasalso been implicated in fertilization, the regulation of androgen re-ceptor signalling and oxidative stress.47–49 Further, mutations ofthe DJ-1 gene are associated with autosomal early-onset Parkin-

son’s disease.50 Several lines of evidence suggest that DJ-1 playsa role in human tumourigenesis, including breast cancer, nonsmallcell lung carcinoma and prostate cancer.17,51,52 Very recently, DJ-1 was identified as a negative regulator of the tumour suppressorPTEN, promoting cell survival in primary breast and lung cancerpatients.53 The role of a soluble form of DJ-1 is still unknown, butit has been described as a circulating tumour antigen in serumfrom 37% of newly diagnosed patients with breast cancer.17 Ourpreliminary data detecting DJ-1 in UM patients’ sera is now beinganalysed further by including a greater number of patients and cor-relating the presence of DJ-1 with the clinical data. The presenceof biomarkers in serum samples, indicating the potential for me-tastasis at the time of diagnosis, would be very useful in UM, par-ticularly since occult micrometastases are thought to be presentwhen the primary tumour manifests.2 There is a need to distin-guish 2 well-defined tumour groups previously described in UM,presenting high and low risk of metastasis.5,54 Crucial questionsnow are how large a tumour mass has to be in order to be detecta-ble by serum screening, and how does the sensitivity of thisapproach compare with imaging methods.

In summary, we present here the first comparative analysis of 2UM cell cultures with different invasion potentials. This approach is avaluable tool for the study of the molecular events governing UM de-velopment and for the identification of potential markers. As a resultof this study, we suggest the involvement of the cell adhesion proteinMUC18, and to a lesser extent HMG-1 protein, in the invasion poten-tial of UM cells. We also describe for the first time the overexpressionof the oncogene DJ-1 in UM and initial results showing its implicationas a potential serum biomarker for this malignancy.

Acknowledgements

The authors thank Prof. Capeans-Tom�e (Unidad de Oncolox�ıaOcular, Servicio de Oftalmolox�ıa, Complexo Hospitalario Univer-sitario de Santiago de Compostela, Spain) for her support inobtaining the UM samples. They further thank Dr. Blanco forkindly selecting the UM patients and providing the blood samples,and Dr. de la Fuente for obtaining the serum. The UM cell lines(UW-1, SP6.5, OCM-1 and 92.1) were kindly donated by Prof.Burnier (The Henry C. Witelson Ocular Pathology Laboratory,McGill University, Montreal, Canada). N.Z. is a Senior ResearchFellow at Linacre College, Oxford.

FIGURE 3 – Validation of potential invasion markers for uveal mela-noma. (a) Zoom images from 2DE gels showing the differentiallyregulated proteins HMG-1 and MUC18 in UM-A cultures. (b) Repre-sentative Western blot images showing the levels of expression ofHMG-1 and MUC18 in NM, UM-A < 7, UM-A > 7 and other UMcell lines (UW-1, OCM-1, SP6.5, 92.1). Equal protein loading wasconfirmed by measuring the amount of actin in the different cellextracts. (c) Graph showing the percentage of UM cells able tomigrate through Matrigel matrix and relative to the migration througha control membrane without ECM.

FIGURE 4 – Validation of the presence of DJ-1 oncogene as a poten-tial biomarker in uveal melanoma. (a) Zoom images from 2DE gelsshowing spots containing DJ-1 protein. (b) Representative Western blotimages showing the levels of expression and the secretion into the cellculture media of DJ-1 for NM, UM-A < 7, UM-A > 7 and other UMcell lines (UW-1, OCM-1, SP6.5, 92.1). Equal protein loading was con-firmed by measuring the amount of actin in the different cell extracts.

1020 PARDO ET AL.

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