Large-Scale Proteomics Analysis of Human Ovarian Cancer for Biomarkers Sofia Bengtsson,† Morten Krogh,† Cristina Al-Khalili Szigyarto,‡ Mathias Uhlen,‡ Kjell Schedvins,§ Claes Silfverswa1 rd,| Stig Linder,| Gert Auer,| Ayodele Alaiya,|,⊥ and Peter James*,† Department of Protein Technology and Department of Computational Biology and Biological Systems, Lund University, BMC D13, 221 84 Lund, Sweden, The Human Proteome Resource, Department of Biotechnology, Royal Institute of Technology, Stockholm, Sweden, Department of Woman and Child Health, Division of Obstetrics and Gynaecology, and Department of Oncology and Pathology, Karolinska Hospital, Stockholm, Sweden, and Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia Received November 10, 2006
Ovarian cancer is usually found at a late stage when the prognosis is often bad. Relative survival rates decrease with tumor stage or grade, and the 5-year survival rate for women with carcinoma is only 38%. Thus, there is a great need to find biomarkers that can be used to carry out routine screening, especially in high-risk patient groups. Here, we present a large-scale study of 64 tissue samples taken from patients at all stages and show that we can identify statistically valid markers using nonsupervised methods that distinguish between normal, benign, borderline, and malignant tissue. We have identified 217 of the significantly changing protein spots. We are expressing and raising antibodies to 35 of these. Currently, we have validated 5 of these antibodies for use in immunohistochemical analysis using tissue microarrays of healthy and diseased ovarian, as well as other, human tissues. Keywords: Biomarkers • Ovarian cancer • DIGE • Proteomics • Statistical analysis
Introduction Epithelial ovarian carcinomas account for 85-90% of all cancers of the ovaries with the major subtypes being serous/ seropapillary and mucinous tumors. The tumors range from benign to aggressive malignant including an intermediate class referred to as borderline carcinomas.1 The prognosis of the disease is strongly dependent on tumor classification and early detection. Tumors are classified based on a subjective histopathological evaluation. Immunostaining can be used to determine the expression of various diagnostic markers and may increase reproducibility. Almost 70% of patients with ovarian cancer have an advanced stage (stage III-IV) at diagnosis. The 5-year survival rate for these patients is about 30%. In contrast, those patients diagnosed with stage I disease have a survival rate of over 90%, and in these cases, the disease may be cured by surgery alone.2 CA125 is the most thoroughly assessed biomarker for ovarian cancer. Elevated levels of CA125 are found in >90% of the * Author for correspondence; Peter James, Protein Technology, BMC D13, Lund University, SE-221 84 Lund, Sweden. E-mail,
[email protected]; fax, +46 46 222 1495. † Lund University. ‡ Royal Institute of Technology. § Department of Woman and Child Health, Division of Obstetrics, Karolinska Hospital. | Department of Oncology and Pathology, Karolinska Hospital. ⊥ King Faisal Specialist Hospital and Research Centre.
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Journal of Proteome Research 2007, 6, 1440-1450
Published on Web 02/22/2007
advanced-stage disease, but it lacks sensitivity for early stage diagnosis, as increased expression is only seen in approximately 50% of stage I disease.3 The concentration of CA125 is also found to be elevated in women with benign gynecologic conditions such as ovarian cysts, endometriosis, and uterine fibroids, as well as in other cancers (breast, bladder, pancreatic, liver, lung).4 The lack of sensitivity and specificity of CA125 has restricted its use in Sweden to monitoring for recurrence of the disease.2 The comparative analysis of ovarian cancer development may lead to new diagnostics, prognostics, and treatment approaches. Proteins are the logical targets, both for use as biomarkers for screening, as well as drug targets, and the best cancer markers known to date (e.g., prostate-specific antigen, R-fetoprotein, carcinoembryonic antigen) are tumorderived proteins. These are present in serum at very low concentrations (1-10 ng/mL). Searching for new markers in diseased tissue where the marker protein is present at a much higher concentration facilitates protein identification, and these highly expressed proteins are the ones most likely to be found being released into blood in amounts accessible for testing. Two-dimensional electrophoresis (2-DE) is the method that is most applicable to high-throughput analysis of highly expressed proteins in tissue, since samples can be run in parallel and multiplexed.5 It has been previously shown that cluster analysis of 2-DE protein expression data can be used for objective and accurate molecular classification of tumors.6 Differential in-Gel Electrophoresis, DIGE, greatly improves 10.1021/pr060593y CCC: $37.00
2007 American Chemical Society
Ovarian Cancer Biomarkers
reproducibility.7,8 We report the use of this technique to create protein expression maps for a set of sporadic epithelial ovarian tumors. The tumors were classified into three different groups based on histological evaluation: benign, borderline, and malignant tumors. In addition, prophylactic oophorectomies from healthy patients with a known BRCA1 mutation were included as a normal reference group after histological evaluation. Sixty-four samples were run in duplicate, and we can distinguish between benign and malignant tumors using unsupervised clustering. When supervised methods are used, these data sets can be separated into the four biological groups. We report proteins that are differentially expressed between benign, malignant, and borderline tumors as well as the histologically normal ovaries. These proteins can potentially be used as tumor markers for diagnosis. Most of the proteins identified in this study have not previously been reported to be involved in ovarian cancer, and we show that these are effective in immunohistochemical evaluation of tissue samples. We have produced antibodies against 5 of these biomarkers and show their specificity in distinguishing between malignant and benign tissue. A further 30 antibodies are in production and will be used to test their effectiveness in diagnosis using blood samples.
Materials and Methods Materials and Reagents. Cy2, Cy3, Cy5, immobilized pH gradient strips, and Pharmalytes were purchased from GE Healthcare (Uppsala, Sweden). Acrylamide, urea, Tris, magnesium acetate, DTT, iodoacetamide, and the Protein assay kit were bought from Sigma Aldrich (Buchs, Switzerland). Protein desalting spin columns were obtained from Pierce (Boule, Huddinge, Sweden). Extraction of Tumor Cells. Tissues were collected at the Karolinska Hospital and made anonymous after informed consent and approval by the Ethics committee. The resected sample was put on ice, and tumor cells were enriched as previously described.9 A pathologist first examined all samples to obtain representative, viable, and non-necrotic tumor tissue. One part of the tissue was used for sample preparation for 2-DE, and the adjacent tissue was formalin-fixed and paraffinembedded for histological characterization. A two-phase nylon mesh filter with 250 and 100 µm pore sizes was used to remove tissue fragments and connective tissues. Cell suspensions were underlaid with 2 mL of ice-cold Percoll/PBS solution (54.7% and density 1.07 g/mL) and then centrifuged at 1000g for 10 min at +4 °C using low acceleration and retardation in swingout buckets. The cells at the interface were collected and washed twice with PBS in the presence of protease inhibitors. The interphase layer was transferred into a preweighed Eppendorf tube, mixed with PBS and protease inhibitors, and then centrifuged at 800g for 3 min at +4 °C. The cell pellet was resuspended in PBS with protease inhibitors and centrifuged at 2700g at +4 °C for 5 min. The supernatant was discarded, and the remaining liquid on the wall of the tube was carefully removed. The wet weight (WW) was recorded, and the pellet was then stored at -80 °C until further processing. The histopathological characterization of all samples was done using hemeatoxylin-eosin-stained sections of formalin-fixed, paraffin-embedded specimens, and tumors were classified according to the WHO guidelines. Both histological and cytological smears of all the analyzed samples showed that the preparations usually contained more than 90% tumor cells. The nontumor tissue was treated in the same way and gave a purity
research articles of over 90% epithelial cells. The samples were kept on ice at all times, and no degradation was observed. Sample Preparation for 2D-PAGE Analysis. Each cell pellet was thawed on ice and resuspended in 1.89 µL of mQ water per mg WW (1.89 × WW µL) with PMSF and EDTA added (0.2 and 1.0 mM, respectively). The suspension was then frozen and thawed four times to break the cells. A volume of (0.089 × WW) µL of 10% SDS/33.3% mercaptoethanol was added, and the sample was incubated 5 min on ice after addition of 0.329 × WW µL of DNAse I (0.144 mg/mL 20 mM Tris-HCl with 2 mM CaCl2 × 2H2O, pH 8.8) and RNAse A (0.0718 mg/mL Tris). Samples were then frozen and lyophilised, and sample buffer including PMSF (0.2 mM), EDTA (1.0 mM), NP-40 (0.5%), and CHAPS (25 mM) was added (6 × WW, or 3 × WW if WW < 10 mg) carefully, mixed for 3 h, centrifuged for 15 min at 12 000 rpm to remove insoluble material, and finally stored at -80 °C. Duplicate or triplicate 1-µL samples were taken for microscale protein determination. Protein Desalting Spin Columns from Pierce were equilibrated with lysis buffer, containing 8 M urea, 30 mM Tris, 5 mM magnesium acetate, and 4% CHAPS, pH 8.5, according to the manufacturer’s buffer exchange procedure protocol. A total of 160 µg of protein lysate was added to the column. In case the volume was smaller than the required 30 µL, additional lysis buffer was added up to 30 µL. The proteins were eluted from the column, and the protein concentration was determined using Protein Assay Kit (Sigma) and stored at -80 °C. The procedure was repeated for all 64 samples. All 64 samples were thawed and labeled with Cy2 and Cy3, independently, according to the manufacturer’s protocol. A total of 600 pmol of dye per 50 µg of protein was used. Equal amounts of protein from each sample were mixed to form a pool. The pool was labeled with Cy5 as above. The samples were stored in the -80 °C freezer. The samples were thawed, and 30 µg of protein from each dye was combined and mixed with rehydration buffer (8M urea, 2% CHAPS, 0.002% bromophenol blue, 18.2 mM DTT, 0.5% Pharmalyte (pH 4-7)), left at room temperature for 30 min, and centrifuged for 10 min before it was applied to a 24 cm immobilized pH gradient strip (pH 4-7) for overnight rehydration. 2D DIGE and Image Analysis. First-dimension isoelectric focusing was carried out on an Amersham Biosciences IPGphor with a total focusing time of 67 kVh. Afterward, the strips were equilibrated in 15 mL equilibration solution (6 M urea, 75 mM Tris (pH 8.8), 30% (w/v) glycerol, 2% (w/v) SDS, and 0.002% bromophenol blue) and reduced with 65 mM DTT for 15 min, followed by 15 min equilibration in equilibration solution with 135 mM iodoacetamide added. The IPG-strips were then loaded and run on a 12.5% SDS-PAGE gel overnight (25 °C) at 1 W/gel until the bromophenol blue dye front had run off the base of the gel. The gels were fixed in 30% ethanol and 10% acetic acid for 30 min, and then kept in water. The gels were scanned with Amersham Biosciences Typhoon 9400 variable imager and the robotic equipment as described previously,10 using automated prescanning.11 Spot detection was carried out in DeCyder DIA for each gel. The estimated number of spots was set to 2500, and a small area filter (area B
3.20E-05 1.70E-04 6.70E-06 6.70E-05 1.70E-04
2.5 2.7 2.3 2.0 2.7
M>B M>B M>B M>B M>B
description Involved in the folding and assembly of proteins in the endoplasmic reticulum (ER)
454 461 466 1377 584
CRTC
P27797
0.89 0.86 0.92 0.88 0.86
826 1464
ERP29
P30040
0.88 0.84
6.40E-05 4.10E-04
2.5 2.1
M>B M>B
324 325 738
ENPL
P14625
PDIA1
P07237
0.90 0.91 0.85
1.50E-05 1.00E-05 3.10E-04
2.1 2.2 1.8
M>B M>B M>B
Catalyzes the rearrangement of -S-S- bonds in proteins
722
PDIA3
P30101
0.88
7.60E-05
2.2
M>B
Catalyzes the rearrangement of -S-S- bonds in proteins
1367 1202 1110
ACTB ACTG CAPG
P60709 P63261 P40121
0.93 0.79 0.86
Cytoskeletal Regulation 5.30E-04 3.3 M>B 4.60E-03 2.6 M>B 1.70E-04 2.4 M>B
Involved in cell motility, structure and integrity Involved in cell motility, structure and integrity Calcium sensitive, blocks the barbed ends of actin filaments
1366
TPM3
P06753
0.89
3.80E-05
2.4
M>B
Binds to actin filaments
1366
TPM4
P67936
1366
TPM2
P07951
1130 20
VIME CO6A1
P08670 P12109
0.87 0.88
5.50E-03 9.00E-04
2.7 3.0
M>B B>M
Class-III intermediate filaments Cell-binding protein
0.79
M>B
Catalyzes the overall conversion of 2-oxoglutarate to succinyl-CoA and CO(2)
21
Promoting folding, oligomeric assembly and quality control in the ER Processing of secretory proteins within the ER Functions in the processing and transport of secreted proteins
ODO2
P36957
0.88
8.80E-03 2.8 Metabolism 1.60E-04 2.1
1290
IPYR
Q15181
0.86
2.00E-04
3.0
M>B
Phosphate metabolism of cells
1225
ODPB
P11177
0.75
1.90E-02
2.7
M>B
Catalyzes the conversion of pyruvate to acetyl-CoA and CO(2)
1716
SODC
P00441
0.89
4.60E-05
1.8
M>B
Destroys radicals
1618
CBX5
P45973
0.90
Nuclear Function 3.00E-02 3.2 M>B
543
LAM1
P20700
0.77
5.80E-03
2.5
M>B
585
LAM2
Q03252
0.90
3.50E-04
1.9
M>B
14-3-3 protein sigma Cathepsin B
1435
1433S
P31947
0.91
Proliferation 5.90E-05 2.3
M>B
p53-regulated inhibitor of G2/M progression
1585
CATB
P07858
0.90
8.00E-05
2.4
M>B
Heterogeneous nuclear ribonucleoprotein K
1319
HNRPK
P61978
0.84
9.40E-03
2.5
M>B
Participate in degradation and turnover of proteins, has been implicated in tumor invasion and metastasis pre-mRNA binding protein, binds PKC, telomere maintenance
Nucleophosmin
1331 1730
NPM
P06748
0.83 0.76
9.80E-04 8.70E-03
3.2 2.6
M>B M>B
Peroxiredoxin 2 Prohibitin
1636 1421
PRDX2 PHB
P32119 P35232
0.91 0.87
1.20E-05 9.00E-05
2.6 2.0
M>B M>B
Receptor tyrosine-protein kinase erbB-3
1211
ERBB3
P21860
0.90
8.20E-05
2.2
M>B
FIBG
P02679
0.83
Signaling 7.10E-04 2.9
M>B
Dihydrolipoyllysineresidue succinyltransferase component of 2-oxoglutarate dehydrogenase Inorganic pyrophosphatase Pyruvate dehydrogenase E1 component beta subunit Superoxide dismutase [Cu-Zn] Chromobox protein homologue 5 Lamin B1 Lamin B2
Fibrinogen gamma chain
871
259 265 266 267
Splicing factor, arginine/serine-rich 5 Elongation factor 1-beta
0.86 0.90 0.91
B>M
1116
SFRS5
Q13243-1
1.00
1.80E-04 3.3 M>B 1.50E-05 3.8 M>B 1.20E-05 3.9 M>B Protein Synthesis 7.00E-03 2.5 M>B
1393
EF1B
P24534
0.91
8.20E-06
2.3
M>B
Component of heterochromatin. Mediates gene silencing Involved in nuclear stability, chromatin structure and gene expression Involved in nuclear stability, chromatin structure and gene expression
Bind single-stranded nucleic acids. May function in the assembly and/or transport of ribosome. Cytoplasmic antioxidant, may have a proliferative effect Negative regulator of cell proliferation, may be a tumor suppressor Involved in pathways which lead to cell proliferation or differentiation.
Forms fibrins and act as a cofactor in platelet aggregation.
Plays a role in constitutive splicing and can modulate the selection of alternative splice sites Translation elongation factor involved in the transfer of aminoacylated tRNAs to the ribosome
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Table 3. (Continued) protein name Hemoglobin beta subunit Lysosomal protective protein Serum albumin Transitional endoplasmic reticulum ATPase
spot no
short name
Swiss-Prot/ TrntEMBL
Fibrinogen gamma chain Transitional endoplasmic reticulum ATPase Serum albumin Heat shock 70 kDa protein 1 60 kDa heat shock protein Heterogeneous nuclear ribonucleoprotein K Keratin, type I cytoskeletal 9 Keratin, type II cytoskeletal 1 Protein disulfide-isomerase Fc fragment of IgG binding protein Endoplasmin Hypothetical protein DKFZp761K0511 Lamin A/C Protein disulfidesomerase A3 60 kDa heat shock protein Actin, cytoplasmic 1 Keratin, type II cytoskeletal 7 Keratin, type I cytoskeletal 18 Keratin, type I cytoskeletal 19 Fibrinogen beta chain Heterogeneous nuclear ribonucleoprotein A/B Actin, cytoplasmic 1 Heterogeneous nuclear ribonucleoprotein K ATP synthase beta chain Prohibitin Annexin A2 Rho GDPdissociation inhibitor 1 heat shock 70 kDa protein 1B Tumor protein D54 Heterogeneous nuclear ribonucleoprotein K Cathepsin B Prohibitin Ras-related protein Rab-7 Glutathione S-transferase P Peroxiredoxin 2 Adenylosuccinate lyase
ROC p-value
fold change trend
description
2145
HBB
P68871
Transport 0.92 1.10E-03
6.0
2167 1444 521
PPGB ALBU
P10619 P02768
0.90 4.70E-03 0.95 6.00E-06 0.86 2.00E-04
2.8 1.7 2.2
306
TERA
P55072
0.94 1.40E-03
2.8
M>B oxygen transport from the lung to the various peripheral tissues M>B M>B Glycoprotein which associates with lysosomal enzymes B>M Carrier protein for steroids, fatty acids, and thyroid hormones and stabilizes extracellular fluid volume M>B Vesicle transport and a role in mitosis
1.7
M>B
Q9BVV6 IPI00046437
0.89 3.80E-05 Unknown 0.84 1.90E-03 0.92 6.60E-06
2.6 2.7
B>M M>B
338 Protein KIAA0586 Similar to testis expressed sequence 13A SNRPF protein
ROC area
457 467
K0586
2115 260 260
FIBG TERA
Q6IBQ1; Q6P4I0 0.86 0.00017 2.5 M>B Spots with Multiple Proteins Identified P02679 0.85 1.50E-02 3.0 M>B P55072
525 525
ALBU HSP71
P02768 P08107
0.89 4.60E-05
2.5
B>M
688
CH60
P10809
0.95 2.10E-05
2.7
M>B
688
HNRPK P61978
688
K1C9
P35527
688
K2C1
P04264
694 694
PDIA1
0.88 3.00E-04
2.1
M>B
694 694
ENPL
902 902
LAMA PDIA3
P07237 O95784; Q9Y6R7 P14625 Q9H6X 9; Q9NTK6 P02545 P30101
0.92 7.50E-05
2.0
M>B
1005
CH60
P10809
0.88 5.40E-05
2.8
M>B
1005 1005
ACTB K2C7
P60709 P08729
1005
K1C18
P05783
1005
K1C19
P08727
1118
FIBB
P02675
0.88 5.40E-05
3.1
M>B
1118
ROAA
Q99729-2
1347 1347
ACTB HNRPK
P60709 P61978
0.82 6.80E-03
2.5
M>B
1347
ATPB
P06576
1453 1453 1508
PHB ANXA2 GDIR
P35232 P07355 P52565
0.87 1.10E-04
1.9
0.90 9.30E-05
2.1
M>B
1508
HSP71
P08107
1508 1508
TPD54 HNRPK
O43399 P61978
1591 1591 1616
CATB PHB RAB7
P07858 P35232 P51149
0.82 1.20E-03
2.7
M>B
0.90 1.50E-05
2.9
M>B
1616
GSTP1
P09211
1616 1616
PRDX2 PUR8
P32119 P30566
a The protein accession number for each spot is given together with the ROC and p-value indicating the degree of confidence that can be allocated to the spot when used to distinguish between benign and malignant. The fold change indicated the direction and magnitude of the change in expression level between the two states as well as a description of the function of the protein if known.
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Ovarian Cancer Biomarkers Table 4. Summary of Immunohistochemical Evaluation of Biomarkersa
ENSG00000171345 Keratin 19, type 1 ENSG00000065361 ErbB3 ENSG00000167085 Prohibitin ENSG00000100357 Adenylosuccinate lyase ENSG00000138448 Integrin alpha-5
follicle cells
stromal cells
cancer tissue analyzed as positive
+
-
11/12
1/12
-
-
4/12
8/12
-
Weak+
11/12
1/12
+
Weak+
11/12
1/12
-
+
12/12
0/12
normal tissue analyzed as positive
a Twelve pairs of tissues (normal and cancerous) not included in the group for 2D-PAGE analysis were used for IHC analysis. The location of the staining and the ability to distinguish between normal and cancer tissue is indicated.
A set of proteins that can be linked to cellular responses to stress and protein folding were identified. Six different chaperones were found up-regulated in the malignant tumors compared to the benign ones. Endoplasmin (GRP94) is the most abundant glycoprotein in the endoplasmic reticulum (ER) and shows elevated levels in other cancers, such as lung19 and colon cancer.20 Erp29 has recently been characterized as a novel 29 kDa endoplasmic reticulum protein and is thought to play a role in the processing of secretory proteins within the ER21 and to interact and bind to other ER chaperones such as GRP78.22 GRP78 is one of the major molecular chaperones involved in the unfolded protein response inside the endoplasmic reticulum. Elevated levels have been shown in several cancer studies including colon, mantle cell, and breast carcinoma.23-25 Protein disulphide-isomerases (PDI) and calreticulin are also known to be involved in protein folding. PDI has been found up-regulated in 2D gel based cancer studies,26,27 but no further confirmations has been made, while calreticulin has been reported to be up-regulated in several cancer forms, including bladder,28 neuroblastoma,29 and colon cancer.30 The up-regulation in malignant tumors of nucleophosmin, cathepsin B, 14-3-3 protein sigma, peroxiredoxin II, heterogeneous nuclear ribonucleoprotein K (hnRNP K), and prohibitin indicate alterations in apoptosis and in the p53 signalling pathway. p53 acts as a cellular negative regulator (tumor suppressor) to protect cells from stress-induced apoptosis. Nucleophosmin (NPM) is an oestrogen-regulated multifunctional protein that behaves like a molecular chaperone that is frequently overexpressed in proliferating cells including tumor and stem cells and interacts directly with p53 which might lead to deregulation of p53 in tumors.31,32 The presence of autoantibodies to NPM has been suggested as a predictor for recurrence in breast cancer patients,33 and overexpression of NPM has been shown in many blood malignancies. Cathepsin B is a lysosomal aspartic protease produced in a variety of tumors and plays a key role in metastatic spread by promoting the destruction of normal tissue architecture and stimulating tumor growth with a simultaneous increase of tumor angiogenesis. Cathepsin B overexpression has been investigated in different malignant diseases, such as colon34 and gastric cancer,35 where also the serum level of cathepsin B was increased in patients with gastric cancer compared to healthy controls, and overexpression has been shown to be associated
Figure 2. Immunohistochemical validation of marker proteins by immunohistochemistry. IHC of normal ovarian (right) and tumor tissues (left) are shown. The antibodies used were against adenylosuccinate lyase (a and b), integrin alpha-5 (c and d), keratin 19 (e and f), prohibitin (g and h) and ErbB3 (i and j). Journal of Proteome Research • Vol. 6, No. 4, 2007 1447
research articles with the stage of disease.18 Peroxiredoxin II was found downregulated in malignant skin tumors compared to benign ones,36 while it is indicated to be up-regulated in breast cancers.37 14-3-3 protein sigma was up-regulated in malignant tumors compared to benign, as well as in the borderline cases compared to the benign (Supporting Information). This protein is located downstream in the p53 pathway and is suggested to act as tumor suppressive factor.38 This protein is downregulated in some cancers such as prostate,39 while the opposite is seen in pancreatic cancer.40 Moreira et al.41 suggest that loss of expression of 14-3-3 sigma protein is not a frequent event in breast tumorogenesis. Our findings are confirmed by Kaneuchi et al.42 who showed positive staining of 14-3-3 sigma protein in the majority of serous, endometrioid, and mucinous ovarian adenocarcinoma tissues. Xiao et al.43 found 14-3-3 protein sigma was down-regulated in plasma from patients with early-stage lung cancer as is the case for patients with earlystage breast cancer.16 Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA binding protein that appears to influence the mRNA metabolism and transport, can bind directly to the promoter region of the human c-myc gene, and functions as a transcription factor.44 It is induced by DNA damage and serves as a cofactor for p53 and, thus, plays a key role in transcriptional responses to DNA damage.45 An earlier study showed a noticeable nuclear overexpression of hnRNP K in tumors when compared with normal lung.46 Prohibitin (PHB) has been shown to colocalize with pRb, p53, and E2F in various cell lines and is localized to many cellular compartments, having a distinct function in each, a chaperone protein, a tumor suppressor, an anti-proliferative protein, a regulator of cell-cycle progression, and a function in apoptosis.47 Prohibitin has been suggested as a potential biomarker in 2D-gel based cancer studies.48 The membrane protein ErbB3 is encoded by the HER-3 oncogene that is a member of the epidermal growth factor (EGF) receptor (EGFR) family of receptor tyrosine kinases. ErbB2/ErbB3 heterodimers have been demonstrated to be potent in mitogenic signalling .49 Amplification of the ErbB2 gene and overexpression of its protein have been reported in numerous cancers, including breast and ovarian tumors. This is the basis for the use of Trastuzumab (Herceptin, an antiErbB2 antibody), a successful immunotherapeutic agent for treatment of breast cancer. A recent study showed promising results when treating human breast cancer cell lines with antiHER3 antibodies,50 while Lee et al. only found 3 of 103 tumors positively stained for Her-3 in surface epithelial ovarian cancer .51 This is in line with our observations. The amount of ErbB2 increases in malignant tumors when analyzed by 2D-PAGE. However it is seen to be decreased when looking at the IHC images. Since membrane proteins are not commonly found on 2D-PAGE gels, it may be we are looking at an increase of a soluble form of the protein, which lacks the transmembrane portion and is being excreted into the local extracellular environment. Another area that has become increasingly interesting as target for anticancer therapies is anti-angiogenic therapy. Proliferation and migration of microvascular endothelial cells are essential for tumor growth and metastasis, and these processes have been likened to wound healing.52 In this study, fibrinogen gamma chain was found to be highly up-regulated in four different spots as well as in one of the spots with two proteins. Fibrin(ogen) plays an important role in tissue repair by providing an initial matrix that can stabilize wounds and 1448
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support local cell proliferation and migration and cytoskeletal rearrangements. We found upregulation in malignant tumors of cytoplasmic actin 1 and 2, as well as tropomyosin alpha 3 and alpha 4 chains, tropomyosin beta chain, and macrophage capping protein, implicating alterations of the cytoskeleton. These proteins were also up-regulated in borderline tumors compared to the benign ones. Collagen alpha I (IV) was found down-regulated in two spots in malignant tumors. Vimentin is another protein coupled with the cytoskeleton and belonging to the intermediate filaments. Expression of vimentin has been seen in several forms of cancers, but the trend is contradictory. Lang et al.53 showed presence of vimentin in tissue sections from prostate cancer, and it correlated positively to poorly differentiated cancers, while decreased expression of vimentin has been shown in endometrial hyperplasia and neoplasias with increasing malignancy.54
Conclusions Our data indicate that two-dimensional gel electrophoresis is useful to identify proteins that can be tested as potential blood markers. In this study, a majority of the identified proteins have not previously been reported in ovarian cancer. Some of the proteins are, however, involved also in other cancers, and finding such generic proteins is to be expected. The most important point, however, is the great variance between patients and hence the need to use large sample numbers to obtain statistically valid data. This confirms the need to develop multiparameter tests for diagnostic and prognostic use, since individual proteins are too variable to give high confidence results. The proteins must be chosen carefully to ensure that they are truly independent of one another and must be validated by immunohistochemistry before moving to blood test development. We have currently raised 5 antibodies against defined proteins, and another 35 are underway. The development of tissue microarray will allow us to verify the usefulness of the antibodies by screening against a large variety of normal and pathogenic tissue types. Abbreviations: DIGE, differential in-gel electrophoresis.
Acknowledgment. This work was supported by grants from Cancerfonden (to S.B. and P.J.), and the Swedish Science Foundation funding to CREATE Health (P.J.) and from the Knut and Alice Wallenberg Foundation (to M.K., M.U., and P.J.). Supporting Information Available: Figures and tables showing the statistical analysis of the differentiation between all the sample groups using a 2D Sammon plot as well a table summarizing the pairwise statistical analysis of the four sample types: normal, benign, borderline, and malignant; the ROC curves for each of the comparisons; and the details of the data used for the protein identification. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Feeley, K. M.; Wells, M. Precursor lesions of ovarian epithelial malignancy. Histopathology 2001, 38 (2), 87-95. (2) Jacobs, I. J.; Menon, U. Progress and challenges in screening for early detection of ovarian cancer. Mol. Cell. Proteomics 2004, 3 (4), 355-366. (3) Fritsche, H. A.; Bast, R. C. CA 125 in ovarian cancer: advances and controversy. Clin. Chem. 1998, 44 (7), 1379-1380. (4) Sjovall, K.; Nilsson, B.; Einhorn, N. The significance of serum CA 125 elevation in malignant and nonmalignant diseases. Gynecol. Oncol. 2002, 85 (1), 175-178.
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