Biomarker Discovery from Uveal Melanoma Secretomes: Identification

Biomarker Discovery from Uveal Melanoma Secretomes: Identification of gp100 and Cathepsin D in Patient Serum Marıá Pardo,*,† AÄ ngel Garcıá,†,‡ Robin Antrobus,† Marıá Jose´ Blanco,§ Raymond A. Dwek,† and Nicole Zitzmann† Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom, and Servicio de Oftalmoloxıá, Complexo Hospitalario Universitario de Santiago de Compostela, Hospital de Conxo, Rua Ramoń Baltar s/n, 15706 Santiago de Compostela, Spain Received January 15, 2007

There is a necessity to better characterize uveal melanoma (UM) tumors according to their metastasis potential at an early stage. In this study we report the identification of potential biomarkers by a combination of proteomics-related approaches: the characterization of UM cell secretomes, the analysis of UM autoantibodies, and the differential depleted serum proteome analysis. We describe a possible role of cathepsin D, syntenin, and gp100 in UM as potential biomarkers. Keywords: uveal melanoma • proteomics • secretome • biomarker • cathepsin D • syntenin • gp100

Introduction Uveal malignant melanoma (UM) is the most frequent primary intraocular tumor in adult humans.1 Unlike cutaneous melanoma, uveal melanoma disseminates mainly through the blood stream and preferentially establishes metastases in the liver. Metastatic liver disease is the leading cause of death in uveal melanoma and can develop after a long disease-free interval, which suggests the presence of occult micrometastatic disease at the time the primary eye tumor is diagnosed and treated.2 For this reason there is an unmet need to be able to predict at an early stage the metastatic potential of UM to improve prognosis and increase the survival rate. Factors reported to be associated with an increased risk of metastatic disease from uveal melanoma include clinical features such as location, size, and configuration of the tumor, as well as histological factors such as cell type, microcirculation architecture, mitotic activity, tumor-infiltrating lymphocytes, and the presence of extrascleral extension.3 Other markers have been suggested, involving molecular factors such as cytogenetic changes (loss of chromosome 3),4 deregulation of cell cycle proteins,5,6 loss of cell adhesion proteins,7 or overexpression of apoptosis inhibitors.8 The discovery of tumor-specific biomarkers has been a challenge for cancer research for decades. Unfortunately, very few markers found are useful in a routine clinical setting, stressing the need for new clinically relevant sources such as * To whom correspondence should be addressed. Current address: Laboratorio de Endocrinologıá Molecular, Departamento de Medicina, Universidade de Santiago de Compostela, Rua San Francisco S/N, 15782 Santiago de Compostela (A Corun ˜ a), Spain. E-mail: [email protected]. Phone/ Fax: + 34 981572121. † University of Oxford. ‡ Current address: RIAIDT-Universidade de Santiago de Compostela, Edificio CACTUS-Campus Universitario Sur, 15782 Santiago de Compostela, Spain. § Hospital de Conxo.

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proteomics.9 Tumor cells alter and interact with their microenvironment by secreting a variety of proteins, which include growth factors, extracellular matrix-degrading proteinases implicated in tumor invasion, and cell motility factors that sustain cell migration and metastasis. Other factors such as immunoregulatory cytokines and molecules participating in cell to cell and cell to substrate interactions are involved in immunological escape, tumor invasion, and angiogenesis.10 Therefore, the secretome of cells and tissues may reflect a broad variety of pathological conditions and represents a rich source of biomarkers. The identity of secreted proteins, usually isolated from cell supernatants or body fluids, is hardly accessible by direct proteome analysis because these proteins are often masked by high amounts of proteins not secreted by the investigated cells. On the other hand, autoantibodies in cancer sera target important molecules involved in signal transduction, cell cycle regulation, cell proliferation, and apoptosis, all of which are key processes in carcinogenesis. Thus, autoantigens detected by antibody-based methods are candidates for oncogenic proteins.11-15 In recent years we have identified a large number of proteins by global and differential proteomics analysis in UM cells, which has yielded numerous insights into the biology of UM.15,16 We have now applied newly emerging proteomic approaches to the study of UM in an attempt to detect tumorspecific proteins liberated into the tumor surroundings, including those released into the serum of UM patients, capable of discriminating tumors according to their metastatic potential. We first characterized the secreted proteins or “secretome” of a short-term UM cell line established by us, UM-A,6 and other long-term UM cell lines established by other groups (SP6.5, UW-1, 92.1, OCM-1);17 we then proceeded to study UM autoantibodies present in patients’ sera, and finally we performed a differential analysis of healthy versus UM-depleted serum. This combined approach led to the identification, 10.1021/pr070021t CCC: $37.00

 2007 American Chemical Society

Biomarker Discovery from Uveal Melanoma Secretomes

among others, of cathepsin D, syntenin, and gp100 as potential biomarkers in UM.

Materials and Methods Cell Culture. The UM-A cell line and normal melanocytes (NMs) were obtained as described previously.6 All UM cell lines, including 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 standard antibiotics at 37 °C in 5% CO2. Secretomes. Cells were grown to 60-70% confluence before they were washed three times in PBS to eliminate any contaminants from serum present in the culture medium. They were then incubated in serum-free medium at 37 °C. After 36 h the media containing the proteins secreted from the cells during the incubation period were collected, centrifuged for 5 min at 1800 rpm, and filtered through a 0.45 µm syringe filter to eliminate any debris or floating cells. Secretomes were concentrated in a Centricon YM-10 (Millipore, Massachusetts) to a final volume of 1000 µL according to the manufacturer’s instructions. Secreted proteins were then TCA/acetone precipitated to be finally resuspended in 1× Laemmli sample buffer. An aliquot of control RPMI medium was processed in the same way as the secretomes to identify any proteins contained in the medium that could be possibly contaminating the sample. Proteins were separated in NuPAGE Bis-Tris 4-12% gels (Invitrogen, Carlsbad, CA), fixed, and stained using the fluorescent dye OGT MP17 (Oxford Glycosciences, Abingdon, U.K.).18 A total of 40 bands were excised and manually digested for MS analysis. Immunoblotting. Whole cell lysates were prepared by direct lysis of subconfluent cells in cold RIPA buffer as described previously.6 Equal amounts of protein lysates (30 µg/lane) or 50 µg of proteins from depleted serum was separated on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and electroblotted onto nitrocellulose membranes. The membranes were blocked for 2 h at room temperature in 0.2% casein and probed successively with primary antibodies and horseradish peroxidase-labeled secondary antibodies (ECL, GE Healthcare, Fairfield, CT). Anti-syntenin 1, cathepsin D, and gp100 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). For autoantibody studies, UM-A whole cell extracts separated by pI 4-7 2-D minigels were blotted onto PVDF membranes and incubated with nondepleted pooled healthy or UM serum as a source of primary antibodies, followed by anti-human IgG secondary antibody (ECL, GE Healthcare). Proteins that specifically reacted with sera from UM patients (not found in blots incubated with sera from healthy controls) were located on fluorescently stained gels that were superimposed with the blot, excised, and identified by LC-MS/MS. All Western blot images shown in the figures are representative of at least three independent experiments. Patients and Serum Depletion. After informed consent was given, sera were obtained from 11 patients attending periodical appointments at the Ocular Oncology Unit (Hospital Provincial de Conxo, Santiago de Compostela, Spain) and kept at -80 °C. The UM sample group included patients presenting small and medium tumors under observation or treated with brachytherapy (I125); none of them presented known metastasis at the time of serum collection. The samples were collected prior to enucleation if performed (Supporting Information (SI) Table 1). Sera from eight healthy individuals were used as the control. Pooled samples were depleted from the 12 most abundant

research articles proteins (albumin, IgG, transferrin, fibrinogen, IgA, R2-macroglobulin, IgM, R1-antitrypsin, haptoglobin, R1-acid glycoprotein, apolipoprotein A-I, apolipoprotein A-II) using the ProteomeLab TM IgY partitioning kit (Beckmann Coulter, Fullerton, CA). Two-Dimensional Gel Electrophoresis. A 120 µg sample of depleted serum was resuspended in 2-D sample buffer (5 M urea, 2 M thiourea, 2 mM tributylphosphine, 65 mM DTT, 65 mM CHAPS, 0.15 M NDSB-256, 1 mM sodium vanadate, 0.1 mM sodium fluoride, 1 mM benzamidine) in a total volume of 375 µL. IPG strips (18 cm, 3-10NL, GE Healthcare) were hydrated in the sample, and isoelectric focusing was carried out for 70 kV h at 17 °C, as previously described.19 Following focusing, the IPG strips were immediately equilibrated for 10 min in 4 M urea, 2 mM thiourea, 12 mM DTT, 50 mM Tris, pH 6.8, 2% (w/v) SDS, and 30% (w/v) glycerol and placed on top of the second dimension of gels embedded in 0.5% melted agarose. The proteins were separated in the second dimension on SDS-PAGE gradient gels (9-16% T, 2.67% C) under running conditions of 10 °C and 20 mA per gel for 1 h, followed by 40 mA per gel for 4 h. Following electrophoresis, the gels were fixed and stained with the fluorescent dye OGT MP17 (Oxford Glycosciences). Monochrome fluorescence images (16 bit) were obtained at 200 µm resolution by scanning gels with an Apollo II linear fluorescent scanner (Oxford Glycosciences). For autoantibody experiments, UM-A cell protein samples were obtained as described previously15 and taken to a final volume of 120 µL in 2-D sample buffer. The first dimension was carried out in 7 cm 4-7NL IPG strips (GE Healthcare), and the second dimension was performed on NuPAGE 4-12% BisTris ZOOM gel (Invitrogen). The gels were transferred for 2 h at 120 mA to PVDF membranes in a wet transfer system (Invitrogen). Differential Image Analysis. Scanned images were processed with a custom version of MELANIE II (Bio-Rad Laboratories Ltd., Hemel Hempstead, U.K.). Three pI 3-10 2-DE gels were prepared in independent experiments for both UM and control pooled serum samples. Internal calibration of the 2-DE gel images with regard to pI and molecular weight was carried out as described previously.15 For differential image analysis, a synthetic gel image was generated by means of accurate spot matching. This synthetic image contained all protein features detected in the UM and control samples. Only features present in at least two out of three individual gels belonging to either the UM or control samples were considered for differential analysis. The intensity (optical density) was measured by summing the pixels within each spot boundary (spot volume) and recorded as a percentage of the total spot intensity of the gel: V (%) ) spot volume/∑(volumes of all spots resolved in the gel). Variations in protein expression were calculated as the ratio of average volumes (V, %) and carefully validated by repeated image analysis by human operators. Differential expression of a protein present in both the UM and control gels was considered significant when the fold change was at least 2 and P was no more than 0.05 after the rank sum test applied on V (%) values. In-Gel Digestion and Peptide Extraction. Protein features assigned to mass spectrometric analysis were excised from the gel by a software-driven robotic cutter. The recovered gel pieces were dried in a speed-vac, and in-gel digestion was carried out by the automated DigestPro workstation (Abimed, Langenfeld, Germany) according to the protocol of Shevchenko et al.20 Journal of Proteome Research • Vol. 6, No. 7, 2007 2803

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Figure 1. Collection of UM secretomes. Serum-free conditioned medium was collected from UM cell cultures, concentrated, TCA/ acetone precipitated, and separated using NuPAGE Bis-Tris 4-12% gels. A total of 40 bands containing secreted proteins were excised and manually digested before MS analysis. Lanes: 1, RPMI control; 2, UM-A; 3, OCM-1; 4, UW-1; 5, 92.5; 6, SP6.5.

Excised bands containing secretome proteins were manually digested following the same protocol. Mass Spectrometric Analysis. Mass spectrometric analysis was carried out using a Q-TOF 1 spectrometer (Micromass, Manchester, U.K.) coupled to a CapLC system (Waters, Milford, MA). Tryptic peptides were concentrated and desalted on a 300 µm i.d./5 mm C18 precolumn and resolved on a 75 µm i.d./25 cm C18 PepMap analytical column (LC Packings, San Francisco, CA). Peptides were eluted to the mass spectrometer using a 45 min 5-95% acetonitrile gradient containing 0.1% formic acid at a flow rate of 200 nL/min. Spectra were acquired in positive mode with a cone voltage of 40 V and a capillary voltage of 3300 V. The MS to MS/MS switching was controlled in an automatic data-dependent fashion with a 1 s survey scan followed by three 1 s MS/MS scans of the most intense ions. Precursor ions selected for MS/MS were excluded from further fragmentation for 2 min. The spectra were processed using ProteinLynx Global server 2.1.5 and searched against the SWISS-PROT and MSDB databases using the MASCOT search engine version (Matrix Science, London, U.K.). Searches were restricted to the human taxonomy, allowing carbamidomethyl cysteine as a fixed modification and oxidized methionine as a potential variable modification. The data were searched, allowing a 0.5 Da error on all spectra and up to two missed tryptic cleavage sites to accommodate calibration drift and incomplete digestion; all data were checked for consistent error distribution. Positive identification was only accepted when the data satisfied the following criteria: (1) MS data were obtained for a full-length y-ion series of a peptide comprising at least eight amino acids and no missed cleavage; (2) MS data with 5070% y-ions were obtained for two or more different peptides comprising at least eight amino acids and no more than one missed cleavage.

Results Analysis of Uveal Melanoma Secretomes as a Source of Biomarkers. The secretome of the UM cell cultures was obtained as described in the Materials and Methods (Figure 1). We focused on the UM-A cell line previously established by our group but also analyzed other cell lines traditionally used for the study of uveal melanoma (OCM-1, SP6.5, UW-1, 92.1). The metastatic potential of all these cells has been recently 2804

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assayed by us.16 None of the cell lines were noticeably affected by the presence of serum-free medium, and all the cell lines proliferated during the 36 h incubation time (not shown). In the UM-A secretome we identified a total number of 133 proteins which were involved in cell growth and/or maintenance, protein metabolism, catabolism, signal transduction, cell adhesion, etc. Among those, 36 (27%) were extracellular or secreted proteins and 5.2% were membrane proteins (Table 1 and SI Table 2). Many of the identified proteins (66%) were new proteins not detected previously by global proteome profiling of UM-A cells in previous studies done in our laboratory. The variety and diversity of the proteins identified in other UM cell line secretomes were much lower than in the UM-A culture (see SI Tables 2-6). A total of 23 proteins were common in all the cultures such as MIF, melanocyte protein Pmel (gp100), MAC25, and MAC-2. The UM-A cell line shared 89 of the identified proteins with the SP6.5 cell line, 69 with OCM-1, 31 with 92.1, and 46 with UW-1. Although very few studies have been published to date analyzing cancer secretomes, 42 (32%) secreted proteins identified by us in the UM-A secretome have been found in the cell culture media of other cancer cell types. This is the case for agrin, GDF-15, galectin-3-binding protein (MAC-2 BP), transferring receptor, macrophage migration inhibitor factor (MIF), and others (Table 1 and SI Table 2).21-23 However, to the best of our knowledge, the remaining 91 (68%) UM-A-secreted proteins such as melanocyte protein Pmel 17 (gp100), MAC25, pigmented epithelium-derived factor (PEDF), and extracellular matrix protein 1, among others, have not been related to cancer secretomes to date. Interestingly, this approach allowed us to characterize cell membrane proteins previously linked to uveal melanoma such as hepatocyte growth factor receptor (c-met oncogene)24,25 and neural cell adhesion molecule L126,27 and new proteins such as lactadherin, semaphorin 5A, syntenin 1, and insulin-like growth factor II receptor (IGF-2R). Other interesting proteins identified were lysosomal proteases linked to invasive and metastatic behavior, such as cathepsins A, B, D, L, and Z. As shown in Table 1 and in SI Table 2, many of the UM-Asecreted proteins were also found in the other UM cell line secretomes. In addition to those proteins of interest found exclusively in the secretome of UM-A cells (see above), a selection of other interesting proteins such as DJ-1, Muc18, MIA, SPARC, basement-membrane-specific heparan sulfate proteoglycan core protein (Perlecan), galectin-3, and CD9 antigen, were found exclusively in the other UM cell lines (Table 2 and SI Tables 3-6). Study of Autoantibodies against Secreted Proteins in UM Patients’ Sera. To verify whether proteins found in UM secretomes were able to elicit humoral response in cancer patients, we studied UM cell proteins detected by potential autoantibodies present in patients’ sera as described in the Materials and Methods (Figure 2). From all 133 proteins identified in the UM-A secretome, 15 were found positive for specific antibodies present in UM patients’ sera such as R and β enolase, cyclophilin A, annexin A2, macrophage capping protein, elongation factor 2, etc. (Figure 2). Verification of Selected Secretome Proteins in Uveal Melanoma: Cathepsin D, gp100, and syntenin 1. We validated the secretome study by focusing on cathepsin D, gp100, and syntenin 1 on the basis of bibliographic research that indicated them as relevant in cancer cell spreading.28-32 Protease cathe-

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Biomarker Discovery from Uveal Melanoma Secretomes Table 1. UM-A Secretome Summary Tablea protein name 14-3-3 protein 14-3-3 protein ζ/δ adipocyte-derived leucine aminopeptidase precursor AGRIN precursor R enolase R-2-macroglobulin precursor annexin A2 annexin A5 apolipoprotein E precursor β enolase β-2-microglobulin precursor calsyntenin-1 precursor cathepsin B precursor cathepsin D precursor cathepsin L precursor cathepsin Z precursor cation-independent mannose-6-phosphate receptor precursor (insulin-like growth factor II receptor)25,65 cofilin, nonmuscle isoform extracellular matrix protein 1 precursor (secretory component p85) fibronectin precursor fructose-bisphosphate aldolase A (lung cancer antigen NY-LU-1) fructose-bisphosphate aldolase C γ enolase66 gelsolin precursor, plasma glia-derived nexin precursor glutathione S-transferase P glyceraldehyde 3-phosphate dehydrogenase, liver glyceraldehyde 3-phosphate dehydrogenase, muscle growth/differentiation factor 15 precursor (GDF-15) heat shock 70 kDa protein 4 heat shock protein HSP 90-R hepatocyte growth factor receptor precursor (c-met)24,25 insulin-like growth factor binding protein 7 precursor (MAC25 protein) lactadherin precursor lamin A/C laminin R-1 chain precursor67 laminin β-2 chain precursor67 laminin γ-1 chain precursor67 lysosomal protective protein precursor (cathepsin A) galectin-3-binding protein precursor (MAC-2 binding protein) macrophage capping protein macrophage migration inhibitory factor (MIF)50 melanocyte protein Pmel 17 precursor (95 kDa melanocyte-specific secreted glycoprotein)68 metalloproteinase inhibitor 1 precursor (TIMP-1) metalloproteinase inhibitor 2 precursor (TIMP-2) moesin neural cell adhesion molecule L1 precursor (N-CAM L1) nucleophosmin nucleoside diphosphate kinase A (tumor metastatic process-associated protein)69 nucleoside diphosphate kinase B (nm23-H2) peptidyl-prolyl cis-trans isomerase A (cyclophilin A) peptidyl-prolyl cis-trans isomerase B precursor (cyclophilin B) peroxiredoxin 2 pigment epithelium-derived factor precursor profilin I programmed cell death 6-interacting protein protein C19orf10 (IL-25) Ras-related protein Rap-1A semaphorin 5A precursor serum albumin precursor syntenin 1 (syndecan-binding protein 1) (melanomadifferentiation-associated protein-9) (Mda-9) tenascin precursor (glioma-associated extracellular matrix antigen) transferrin receptor protein 1 (CD71 antigen) transketolase tubulin R-ubiquitous chain tubulin, β polypeptide

function signaling signaling metallopeptidase basal lamina glycolysis plasma Ca-regulated membrane prot Ca-regulated membrane prot catabolism glycolysis MHC class I molecule cell membrane protease protease protease protease lysosomal membrane actin binding ECM ECM glycolysis glycolysis glycolysis actin binding protease inhib detoxication glycolysis

signal protein peptide ID

yes

yes yes

yes yes

yes yes

glycolysis TGF-B fam

yes

chaperone chaperone cell membrane IGF-I and -II binding adhesion/angiogenesis nuclear lamina extracellular/migration extracellular/migration extracellular/migration protease cell adhesion actin binding host defense melanoma Ag metalloproteinase inhib metalloproteinase inhib cytoskeleton cell adhesion

yes

yes yes yes

pI

P62258 P63104 Q9NZ08

29173 4.63 27745 4.73 105847 5.97

O00468 P06733 P01023 P07355 P08758 P02649 P13929 P01884 O94985 P07858 P07339 P07711 Q9UBR2 P11717

212883 47037 163277 38472 35805 36154 46855 13714 109792 37807 44552 37564 33867 274308

18502 8.22 60704 6.25

P02751 P04075

262606 5.45 39288 8.39

OCM-1 OCM-1

6.46 4.91 5.9 9.35 5.44 8.58

Ma11/WM/OHS UW-1/OCM-1 NPC/MesEx/Panc UW-1/OCM-1 NPC

P00354

35875 6.6

UW-1/OCM-1 MEx UW-1

Q99988

34168 9.79

P34932 P07900 P08581

94299 5.18 84542 4.94 155527 7.02

Q16270

29130 8.25

Q08431 P02545 P25391 P55268 P11047 P10619

yes

P01033 P16035 P26038 P32004

43122 74139 337157 196080 177606 54466

8.47 6.57 5.93 6.09 5.01 6.16

23170 24399 67688 140002

17298 8.52 17881 7.82

protein folding

P23284

22742 9.33

redox regulation angiogenesis inhib actin binding multivesicular body (MVB) IL-25 signaling membrane/axon guidance plasma adapter

P32119 21891 5.66 P36955 46342 5.97 P07737 14923 8.47 Q8WUM4 96023 6.13 Q969H8 18660 6.08 P62834 20987 6.39 Q13591 120571 6.9 P02768 69366 5.92 O00560 32444 7.06

yes

P02786 P29401 Q71U36 Q9BVA1

UW-1/OCM-1 NPC

UW-1/OCM-1

P22392 P62937

iron uptake metabolism microtubules microtubules

OCM-1 UW-1

UW-1/OCM-1 UW-1/OCM-1 UW-1/OCM-1 UW-1/OCM-1

nucleotide synthesis protein folding

P24821

UW-1/OCM-1

8.46 7.46 6.09 5.84

32575 4.64 17148 5.83

yes

Ma11/WM

MesEx UW-1/OCM-1 NPC/MesEx

UW-1/OCM-1 NPC/Ma11/WM/OHS/serum solid tumors 38517 5.88 13 UW-1 12345 8.24 UW-1/OCM-1 Ma11/OHS 70255 5.37 UW-1/OCM-1

P06748 P15531

cell adhesion

UW-1/ OCM-1 OCM-1 UW-1/OCM-1 5 UW-1/OCM-1 MEx/WM 7 UW-1/OCM-1 NPC/MesEx

65331 5.13

nuclear nucleotide synthesis

yes

others

39324 47137 85697 44002 23224 35922

P40121 P14174 P40967

yes

UM detection

P09972 P09104 P06396 P07093 P09211 P04406

Q08380

yes

aut Ab

5.97 UW-1/OCM-1 NPC 6.99 2 OCM-1 NPC/MEx/MesEx/Ma11/WM/OHS 6 UW-1/OCM-1 7.56 15 OCM-1 MEx/MesEx/Ma11/WM 4.94 UW-1/OCM-1 MEx/MesEx/Ma11/WM/OHS 5.65 UW-1/ OCM-1 Panc/Ma11/WM 7.73 3 UW-1/OCM-1 6.06 NPC/Ma11/WM/OHS 4.81 OCM-1 NPC 5.88 NCP/Panc 6.1 Panc/NPC/Ma11/WM 5.32 6.7 5.63

P23528 Q16610

yes

yes yes

MW

240866 4.79 84901 67877 50151 49953

Ma11/WM/OHS WM/OHS NPC/MEx/MesEx Panc

UW-1/OCM-1 8 UW-1/OCM-1 Panc/NPC OCM-1

NPC

UW-1

Ma11/OHS

UW-1/OCM-1 NPC OCM-1

OCM-1 OCM-1

MesEx MEx

UW-1/OCM-1

6.18 7.58 UW-1/OCM-1 Panc 4.94 14 Panc 4.78 14 MesEx

a Shown are the protein name and function, the presence of a signal peptide, the protein ID (Swiss-Prot accession number), molecular weight (MW), and isoelectric point (pI), the localization on Figure 2B if positive for autoantibodies, and the detection in other UM secretomes and in other cancer secretomes or exosomes. NCP ) nasopharyngeal carcinoma cell line secretome,21 Panc ) pancreatic cancer cell line secretome,22 MA11 ) breast cancer, WM 266-4 ) melanoma, and OHS ) aggressive osteosarcoma cell line secretomes,23 MesEx ) mesothelioma cell-secreted exosome,63 and Mex ) melanoma cell-derived exosome.64 Proteins that were described in UM tumors are indicated with a bibliography reference number.

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Table 2. UM Cell Lines (OCM-1, UW-1, SP6.5, and 92.1) Secretome Summary Tablea protein name

apolipoprotein D precursor basement-membrane-specific heparan sulfate proteoglycan core protein precursor (Perlecan) CD81 antigen CD9 antigen (motility-related protein) chondroitin sulfate proteoglycan 4 (precursor) c-myc promoter-binding protein (MPB-1) galectin-3 (MAC-2 antigen) legumain precursor (protease, cysteine 1) thrombospondin 1 precursor SPARC precursor (secreted protein acidic and rich in cysteine) (basement-membrane protein 40) protein DJ-1 (oncogene DJ1) cell surface glycoprotein MUC18 precursor (melanoma cell adhesion molecule) (CD146 antigen) 4F2 cell-surface antigen heavy chain (lymphocyte activation antigen 4F2 large subunit) (CD98 antigen) melanoma-derived growth regulatory protein precursor (melanoma inhibitory activity)

protein ID

signal peptide

P05090 P98160

MW

pI

yes yes

21275 468824

5.06 6.06

OCM-1 OCM-1/UW-1

yes

25809 25284 250451 37087 26057 49411 129412 34632

5.09 7.14 5.2 6.79 8.6 6.07 4.71 4.73

OCM-1/SP6.5/92.1 OCM-1/92.1 OCM-1/ UW-1 UW-1/SP6.5/92.1 OCM-1/UW-1/SP6.5 OCM-1/92.1/ NPC OCM-1 92.1

Q99497 P43121

19891 71607

6.33 5.58

92.1/SP6.5 SP6.5

P08195

57944

5.2

SP6.5

14508

9.04

SP6.5

P60033 P21926 Q92675 P06733 P17931 Q99538 P07996 P09486

Q16674

yes

detection

a Shown are the protein name, protein ID (Swiss-Prot accession number), presence of a signal peptide, molecular weight (MW), and isoelectric point (pI), as well as the UM cell line where it was detected.

Figure 2. Study of UM autoantibodies. (A) Representative images of Western blots using pooled serum from healthy controls and UM patients as the source for autoantibodies. UM-A cell extracts containing potential antigens were separated by 2-DE and transferred to a PVDF membrane for immunodetection. (B) Proteins that specifically reacted with sera from UM patients were located on fluorescently stained gels by superimposition with the blot. Numbers from 1 to 15 show the localization of these differences. (C) Table listing the proteins identified by LC-MS/MS. Asterisks indicate proteins whose autoantibodies have been found previously in other cancer types.

psin D, melanoma-specific antigen gp100, and adapter protein mda-9/syntenin 1 (melanoma differentiation-associated protein) were checked on NM and UM cell line secretomes by Western blotting (Figure 3). The three proteins were found to be positive in all the UM secretomes, although an accurate quantification of differences between secretomes was not possible due to variations in the efficiency of protein precipitation between experiments (Figure 3A). Interestingly, we observed that all the UM cells secrete the inactive precursor procathepsin D (52 kDa) and active mature forms of cathepsin D (31 kDa/16 kDa). UM-A, SP6.5, and 92.5 cell lines also contained intermediate active forms of this protein. We studied the expression of these proteins in UM cell extracts and found that an intermediate form of cathepsin D was overexpressed in all the UM cell lines compared to NM cells where none was detected (Figure 3B). Confirming this result, we observed that 2806


cathepsin D was less expressed in UM-A during early passages (primary culture extract, UM-A passage 7), which has a higher metastatic potential in vitro.16 In the case of the melanomaspecific marker gp100, we found that NM and UM-A cell cultures, established by us, were positive for that protein. We also show that OCM-1, UW-1, SP6.5, and 92.1 cell lines were positive for gp100 as previously described by others (Figure 3B).17 Unfortunately, we could not detect syntenin 1 in NM or UM cell extracts, although it was present in UM secretomes, as indicated above (not shown). Finally, we assayed the potential presence of these markers in UM patients’ and healthy volunteers’ sera. Equal amounts of sera were pooled from healthy controls and UM patients and submitted to depletion columns, removing the 12 most abundant proteins. Interestingly, we detected cathepsin D and

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Figure 3. Verification of selected proteins from UM secretomes. (A) Representative Western blot images showing the presence of syntenin, cathepsin D, and gp10 in UM secretomes. Lanes: 1, NMs; 2, UM-A; 3, OCM-1; 4, UW-1; 5, SP6.5; 6, 92.1. (B) Overexpression of cathepsin D and gp100 in UM cell extracts versus NMs and in UM patients’ pooled depleted sera versus healthy individuals’ sera. Lanes: 1, NMs; 2, UM-A early stages (passage 7); 4, OCM-1; 5, UW-1; 6, SP6.5; 7, 92.1; C, healthy individuals’ depleted sera; UM, UM patients’ depleted sera. Equal protein loading was confirmed by measuring the amount of actin in the different cell extracts.

Discussion

Figure 4. Proteomic differential analysis of depleted sera. (A) Representative 2-DE images corresponding to pooled and depleted sera from healthy controls and UM patients. The location of PEDF is shown. (B) Zoom images from 2-DE gels showing the decrease in PEDF levels in UM patients compared to healthy individuals.

gp100 in UM patients’ sera at higher levels compared to those of healthy individuals (Figure 3B). Syntenin-1 was not detected either in depleted sera from UM patients or in that of healthy volunteers (data not shown). Proteomic Differential Analysis of Depleted Sera. We investigated whether proteins identified by secretome analysis would also appear in a differential proteomics analysis comparing the depleted sera of healthy versus patient-derived samples (Figure 4). Previous analyses performed by our group using non-depleted serum failed to detect variations other than those of the most abundant proteins secreted by the liver. However, only small amounts of protein could be recovered after depletion, and the number of new proteins detected after analysis only improved modestly. We found 61 differences and were able to identify 38 proteins as shown in SI Table 7. None of the secreted proteins were found in this analysis. However, we detected the disappearance of one feature corresponding to the angiogenesis inhibitor pigment-epithelium-derived factor (PEDF) in UM patient’s sera.

UM tumor cells secrete proteins into the tumor environment which are subsequently spread through the surrounding vascular networks and virtually poured into the blood circulation. Therefore, proteins secreted by tumor cells which could be picked up by a simple blood test are potential biomarkers for disease diagnosis and/or prognosis. In this study we used three different approaches in an attempt to identify specific biomarkers shed by UM tumor cells: UM cell secretomes, UMspecific serum autoantibodies, and differential proteome analysis of depleted sera derived from healthy controls and UM patients. There are not that many studies for cancer secretomes published to date due to the relatively recent development of proteomics-based technology for biomarker studies; however, several publications have been launching this approach for the study of cancer in the past 2 years.21,22,33-35 We present here the first secretome analysis of UM tumors which shows the great potential of this method. We identified many new proteins including membrane proteins typically not easy to detect by 2-DE. We also describe a high percentage of nonsecreted proteins such as cytoplasmic actin and tubulin (SI Table 2). The elevated presence of these proteins may have masked the detection of less abundant secreted proteins. Although some cell autolysis may occur after incubation of cells with serumfree media for longer than 24 h, as described by Mbeunkui and co-workers,35 we hypothesize that UM cells also liberate exosomes (membrane vesicles from endosomal origin) to the extracellular medium. Indeed, some protein markers for these vesicles were found in UM secretomes, such as proteins associated with antigen presentation (heat shock proteins), microvesicular structure (cytoskeleton-associated and actinbinding proteins, annexins), and exosome targeting (lactadherin, CD81, CD9, MAC-2).36-38 Exosomes have been purified from the supernatant as well as sera and other body fluids of patients with cancer.39 It is also known that these vesicles contain cytosolic proteins and express molecular markers characteristic of the cell plasma membrane with an enhanced expression of tumor antigens in cancer cells.40 This may explain the identification of known and novel UM membrane antigens in our study. Unraveling the role of exosomes in UM might be very interesting since it has been suggested that they have Journal of Proteome Research • Vol. 6, No. 7, 2007 2807

research articles profound inhibitory effects on immune cells in vitro including the blocking of signaling and proliferation and the induction of cytotoxicity and apoptosis.41,42 They may be also useful for anticancer vaccine therapy.40 More than half of the identified proteins in UM-A cell secretomes were new proteins not detected previously by us in UM-A global proteome profiling. This fact is due to the different nature of samples and technical approaches, the simplicity of secretome samples compared to cell extracts, and the incapacity of 2-DE technology to properly resolve poorly soluble hydrophobic and very acidic and basic proteins.43 Interestingly, we detected a significant number of membrane proteins, such as receptors for liver-synthesized growth factors, such as hepatocyte growth factor receptor (HGFR/c-met protooncogen) and insulin growth factor receptor II (IGF-2R). c-met has been previously described in primary uveal melanomas and metastatic melanoma to the liver by other authors using immunohistochemistry;24,25 equally, IGF-1R was shown in UM,25 whereas the presence of IGF-2R had not been found in UM to the best of our knowledge. Finding of semaphorin 5A and the neural cell adhesion molecule L1 (N-CAM L1) in the secretome of UM-A cells is intriguing as semaphorins have been previously related to axon guidance, cell migration, morphogenesis, angiogenesis, and tumor progression,44 whereas Sema5A is a transmembrane protein regulated by heparan and chondroitin sulfate proteoglycans45 that can trigger the intracellular signaling of HGFR associated in a complex with plexin-B346 and may contribute to tumorigenesis or tumor progression.44 Indeed, both sulfate proteoglycans were found in the secretome of certain UM cells in this study (SI Tables 3-6). N-CAM L1 protein and its soluble form are tumor-associated proteins and potential markers for tumor staging as well as targets for therapeutic intervention. In cell culture, soluble L1 substrate was found to stimulate cellular motility and migration.47 The release of soluble L1 is not restricted to cells in culture, as soluble L1 was recently detected in serum samples from patients with ovarian and uterine tumors and is believed to lead to increased cell migration and metastatic spread in these malignancies.48 It has also been shown that shedding of L1 is a regulated process, triggered by the physiological factor HGF.49 Our UM secretome studies led to the identification of proteins such as MIF. It is known that UM tumors produce MIF to prevent lysis by natural killer (NK) cells.50 While NKcell-mediated lysis of uveal melanomas is inhibited in the eye, melanoma cells that disseminate from the eye are at risk for surveillance by these cells, especially those that metastasize to the liver, an organ with one of the highest levels of NK activity in the body. The variation of proteins found within different UM cell lines shows certain cell line specificity. The detection of melanoma inhibitory activity protein (MIA) and other UM potential biomarkers recently described by us in invasion phenotype studies such as MUC-18 and DJ-1 in UM secretomes is encouraging.16 Syntenin, Cathepsin D, and gp100. At least three cancerrelated proteins selected from UM-A secretomes deserve further discussion and study. Syntenin, also known as Mda-9, is a PDZdomain protein which was selected as it is overexpressed in many types of human cancers, where it is believed to have a role in tumor progression.28,29 It was shown previously that advanced metastatic cutaneous melanomas strongly express mda-9/syntenin compared to both melanocytic nevi and 2808


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primary melanomas;29 however, the identification of syntenin in UM tumors had not been published to date. Interestingly, we could not detect syntenin in whole UM cell extracts nor in patients’ sera, although this protein was immunodetected in the UM secretomes. Syntenin may be better represented in the secretomes since they are much simpler samples compared to whole cell extracts. The role of this protein in UM will be analyzed further. Melanocyte protein Pmel 17 (gp100/ME20M) was one of the proteins present in all UM cell secretomes. It is thought to represent an oncofetal self-antigen that is normally expressed at low levels in quiescent adult melanocytes but overexpressed by proliferating neonatal melanocytes and during tumor growth, as shown in our results using proliferating NM and UM cell lines (Figure 3B). It has been suggested that the release of the soluble form, ME20-S, could protect tumor cells from antibodymediated immunity.30 However, so far we could not determine whether UM cells secrete the soluble form of this protein because exosomes present in the cell supernatants may contain tumor cell membrane antigens. The lysosomal acid proteinase cathepsin D is believed to be associated with proteolytic processes leading to the invasion and seeding of tumor cells. An association between cathepsin D tissue concentration and aggressiveness of tumors has been detected in different cancer types, as well as in metastatic melanomas.31,32 This is the first time that cathepsin D secretion and its relation to UM invasion potential in vitro has been described in UM cells. However, the role of this proteinase in UM tumors should be further analyzed by immunohistochemistry in tumor sections, especially in invasion and metastasis phenotypes. Finally, the presence of both gp100 and cathepsin D in UM patients’ sera is a novel and an encouraging discovery which suggests their potential as biomarkers of clinical use. More studies including inmunohistochemical detection will be done in the near future to assess the clinical potential of these findings. Autoantibodies. The humoral immune response to cancer in humans has been evidenced by finding p53 and c-erbB-2/ HER2/neu autoantibodies in different cancer patients.51,52 We suggest that proteins secreted by UM cells may elicit an immune response once they have left the immunoprivilege site of the eye. In support of this hypothesis we detected autoantibodies for several proteins present in UM secretomes, some of them previously found in other types of cancer such as actin,53 R enolase,54,55 elongation factor 2,56 tubulin,57 and annexin A2.58 In future experiments we will use fresh UM tissue, instead of UM cell lines, correlated with the serum of the patient and healthy individuals to corroborate the efficiency and specificity of the approach. Differential Analysis of Pooled Sera. Finally, accordingly to previous work on cancer biomarker discovery,59-61 we have done a differential analysis using pooled serum from healthy controls and individuals suffering from UM to identify potential markers. Unfortunately, we could not detect any of the potential markers identified by the secretome approach apart from PEDF, the level of which was decreased in UM patients as shown previously for other cancer types.62 As shown in the Results, these experiments suffered from the presence of abundant plasma proteins which may have masked any relevant marker. Depletion columns used to address this problem did not circumvent the problem but, nevertheless,


improved the detection of less abundant serum proteins by Western blotting as shown for cathepsin D and gp100.

Conclusions In summary, we present here the identification of potential biomarkers from UM tumors by a combination of proteomicsrelated approaches that has never been applied to the study of this cancer before: the characterization of UM cell secretomes by one-dimensional gel electrophoresis and protein identification by MS and Western blotting, the analysis of UM autoantibodies in UM patients’ sera by Western blotting and MS, and the differential proteome analysis of healthy versus UM-depleted serum by 2-DE and MS. Considering the need to detect biomarkers capable of predicting the metastatic potential of UM cells, we demonstrated that the analysis of the secretomes is a promising tool in the hunt for markers. We also show the usefulness of depleting the serum of the most abundant proteins to detect by Western blotting potential markers present in UM patients’ sera. As a result of this, among others, we suggest a possible role of cathepsin D, syntenin, and gp100 in UM and encourage their further investigation as disease biomarkers. Indeed, we are currently undertaking a more clinical-oriented study based on the generation of a serum bank with a significant number of samples from ageand sex-matched individuals with UM classified as follows: healthy, benign nevi, small melanocytic choroidal lesions, and enucleated or brachytherapy-treated with or without metastasis.

Acknowledgment. We thank Prof. Capeans (Unidade de Oncoloxıá Ocular, Servicio de Oftalmoloxıá, Complexo Hospitalario Universitario de Santiago de Compostela, Spain), Dr. Pin ˜ eiro and Dr. de la Fuente (Instituto Galego de Oftalmoloxıá, Santiago de Compostela, Spain) for their support in obtaining the UM serum samples. 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). A.G. is a Parga Pondal Fellow (Xunta de Galicia, Spain). N.Z. is a Senior Research Fellow at Linacre College, Oxford. This research has been funded by the Oxford Glycobiology Institute Endowment. Supporting Information Available: Tables showing UM patients’ clinical data, proteins identified in the UM-A secretome, proteins identified in the OCM-1, UW-1, SP6.5, and 92.1 cell line secretomes, and proteome differential analysis of healthy versus UM patient pooled sera. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Singh, A. D.; Topham, A. Survival rates with uveal melanoma in the United States: 1973-1997. Ophthalmology 2003, 110, 962965. (2) Eskelin, S.; Pyrhonen, S.; Summanen, P.; Hahka-Kemppinen, M.; Kivela, T. Tumor doubling times in metastatic malignant melanoma of the uvea: tumor progression before and after treatment. Ophthalmology 2000, 107, 1443-1449. (3) Singh, A. D.; Shields, C. L.; Shields, J. A. Prognostic factors in uveal melanoma. Melanoma Res. 2001, 11, 255-263. (4) Sholes, A. G.; Damato, B. E.; Nunn, J.; Hiscott, P. Grierson, I.; Field, J. K. Monosomy 3 in uveal melanoma: correlation with clinical and histologic predictors of survival. Invest. Ophthalmol. Vis. Sci. 2003, 44, 1008-1011. (5) Mouriaux, F.; Maurage, C. A.; Labalette, P.; Sablonniere, B.; Malecaze, F.; Darbon, J. M. Cyclin-dependent kinase inhibitory protein expression in human choroidal melanoma tumors. Invest. Ophthalmol. Vis. Sci. 2000, 41, 2837-2843.

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research articles

Biomarker Discovery from Uveal Melanoma Secretomes (65) All-Ericsson, C.; Girnita, L.; Seregard, S.; Bartolazzi, A.; Jager, M. J.; Larsson, O. Insulin-like growth factor-1 receptor in uveal melanoma: a predictor for metastatic disease and a potential therapeutic target. Invest. Ophthalmol. Vis. Sci. 2002, 43, 1-8. (66) Royds, J. A.; Rennie, I. G.; Parsons, M. A.; Timperley, W. R.; Taylor, C. B. Enolase isoenzymes in uveal melanomas-a possible parameter of malignancy. Br. J. Ophthalmol. 1983, 67, 244-248. (67) Bajcsy, P.; Lee, S. C.; Lin, A.; Folberg, R. Three-dimensional volume reconstruction of extracellular matrix proteins in uveal melanoma from fluorescent confocal laser scanning microscope images. J. Microsc. 2006, 221, 30-45.

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Biomarker Discovery from Uveal Melanoma Secretomes: Identification

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