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Comparative Proteomics of Ovarian Epithelial Tumors Hee Jung An,† Dong Su Kim,⊥,O Yong Kyu Park,| Sei Kwang Kim,| Yoon Pyo Choi,§ Suki Kang,‡ Boxiao Ding,§ and Nam Hoon Cho*,‡,§ Department of Pathology, Pocheon CHA University, Departments of Pathology and Gynecologic Oncology, Yonsei University College of Medicine, Brain Korea 21 Project for Medical Science, Yonsei University, and Division of Molecular and Life Sciences and Systems Bio-Dynamics Research Center, POSTECH, and Genomine Research Division, Genomine, Inc. Received December 14, 2005

We analyzed 12 ovarian epithelial tumors using 2D PAGE-based comparative proteomics to construct intra- and inter-tumoral distance map trees and to discover surrogate biomarkers indicative of an ovarian tumor. The analysis was performed after laser microdissection of 12 fresh-frozen tissue samples, including 4 serous, 5 mucinous, and 3 endometrioid tumors, with correlation with their histopathological characteristics. Ovarian epithelial tumors and normal tissues showed an apparent separation on the distance map tree. Mucinous carcinomas were closest to the normal group, whereas serous carcinomas were located furthest from the normal group. All mucinous tumors with aggressive histology were separated from the low malignant potential (LMP) group. The benign-looking cysts adjacent to the intraepithelial carcinoma (IEC) showed an expression pattern identical to that of the IEC area. The extent of change on the lineages leading to the mucinous and serous carcinoma was 1.98-fold different. The overall gene expression profiles of serous or endometrioid carcinomas appeared to be less affected by grade or stage than by histologic type. The potential candidate biomarkers screened in ovarian tumors and found to be significantly up-regulated in comparison to normal tissues were as follows: NM23, annexin-1, protein phosphatase-1, ferritin light chain, proteasome R-6, and NAGK (N-acetyl glucosamine kinase). In conclusion, ovarian mucinous tumors are distinct from other ovarian epithelial tumors. LMP mucinous tumors showing histologically aggressive features belong to mucinous carcinoma on the proteomic basis. Keywords: ovarian tumor • mucinous tumor • low malignant potential • proteomics • distance map tree

1. Introduction Carcinogenesis in ovarian epithelial cells is one of the least understood processes in cancer research. Most ovarian epithelial tumors are believed to be derived from the ovarian surface epithelium (OSE),1-3 and display a unique spectrum consisting of benign cystadenomas, low malignant potential (LMP) epithelial tumors, and frankly invasive carcinomas.4-5 LMP tumors show histopathological and biological features intermediate between those of clearly benign and clearly malignant tumors of the same cell type. However, the charac* Corresponding author. Nam Hoon Cho, Dept. of Pathology, Yonsei University College of Medicine, Sinchon-dong 134 Seodaemoon-ku, Seoul, South Korea. Tel.: 82-2-2228-1767. Fax: 82-2-362-0860. E-mail: [email protected]. † Pocheon CHA University. ‡ Department of Pathology, Yonsei University College of Medicine. § Brain Korea 21 Project for Medical Science, Yonsei University. | Departments of Gynecologic Oncology, Yonsei University College of Medicine. ⊥ Division of Molecular and Life Sciences and Systems Bio-Dynamics Research Center, POSTECH, and Genomine Research Division, Genomine, Inc. O Current address: Division of Molecular and Life Sciences and Systems Bio-Dynamics Research Center, Genomine Research Division, Genomine, Inc.

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terization of LMP tumors has been controversial for more than a century, and LMP tumors occasionally show bothersome clinical manifestations, such as peritoneal implants in serous tumors and pseudomyxoma peritonei in mucinous tumors.6 Although most ovarian carcinomas have been known to arise de novo from OSE, approximately 5-10% of carcinomas may arise in a stepwise manner from borderline tumors,5,7 and a continuous pathological spectrum based on multistep carcinogenesis has been accepted in some well-differentiated mucinous tumors, comprising benign mucinous cystadenomas, LMP mucinous tumors, intraepithelial carcinoma (IEC), mucinous tumors with stromal microinvasion, and widely invasive mucinous carcinomas.6,8 Many mucinous carcinomas often exhibit a spectrum of benign to malignant epithelia within the same tumor, and abrupt transition from benign to borderline or malignant epithelia are sometimes noted.8 According to most studies that have attempted to define the validity of the biological aspect of this spectrum, IEC with stromal microinvasionsdefined as more than one isolated focus of stromal invasion measuring less than 10 mm2shas been categorized as LMP, due to its favorable outcome. However, the molecular aspects of IEC with or without microinvasion and their similarities to LMP tumors have not yet been verified. More10.1021/pr050461p CCC: $33.50

 2006 American Chemical Society

Proteomics of Ovarian Epithelial Tumors

over, the duration of follow-up in previous studies have been too short to determine the clinical relevance of LMP tumors showing aggressive features by histology. Molecular studies on ovarian tumors are extremely rare, mainly due to their morphological heterogeneity, and it is difficult to completely rely on the literature regarding the molecular properties of these tumors as the extracted protein is often easily degenerated. Studies using proteomics or cDNA arrays of human tissues subsequently have been rarely performed,9,10 whereas molecular studies using patient sera have been focused.11-12 A proteomic analysis of protein expression in OSE-derived tumors may provide new insight into dysregulated cell signaling pathways, which may lead to the development of promising methods of treatment.10,13 With advances in the field of proteomics, molecular tools are becoming available to aid in diagnostic and therapeutic conundrums, especially in LMP tumors which can show either benign or malignant behavior. In the recent proteomics study, a distance tree based on intra- and inter-tumoral variations of gene expression patterns enabled us to determine similarities between several organs from different species of primates.14 Differences in apparent protein expression levels were used to calculate an overall map distance summarized over all genes.15 With advances in the field of proteomics, tools are becoming available to aid in diagnostic and therapeutic conundra, such as the issue of ovarian LMP tumors. In particular, quantitative changes in protein expression can detect between closely related tumors, and eventually reflect their phenotypic and biologic properties. In this study, we aimed to reclassify ovarian epithelial tumors with different histological types and grades and to determine the molecular similarities or differences between conventional LMP tumors and LMP tumors with histologically aggressive features, such as IEC or IEC with microinvasion, using a distance map tree.

2. Materials and Methods Tissue Preparation and Laser Capture Microdissection. All tumors and corresponding normal tissues with informed consent from donors were stored in sterile bottles in a deep freezer. The present study was approved by the ethical committee of the university. Prior to protein extraction, all cases were examined on cryosections and again matched with the paraffin-embedded tissues. All tumors were inspected by standard hematoxylin-eosin staining, then subjected to laser capture microdissection (LCM). A total of 12 ovarian epithelial tumors: four serous tumors including one LMP tumor and three serous carcinomas; five mucinous tumors, including two LMP tumors, two LMP tumors with aggressive features (one intraepithelial carcinoma and one stromal microinvasion), and one widely invasive mucinous carcinoma; and three endometrioid tumors, including one endometriotic cyst and two carcinomas, were analyzed using proteomics. Frozen slides were prepared from each case and microdissected by LCM with a Zeiss Axiovert 135 System (Zeiss, Oberkochen, Germany). Frozen tissue specimens were cut into a series of 6 µm-thick sections and mounted on slides coated with a thermoplastic membrane (PEN foil slides; Leica Microsystems, Wetzlar, Germany). Cystic tumor cells were selectively dissected by focal melting of the membrane with a UV-laser beam (337 nm) set to pulse at 80 mW with 10 000 shots, corresponding to 50 000-

research articles 70 000 cells, which typically contain roughly 50 µg protein. This procedure was performed within the set maximum time period of 30 min per slide. Microdissected fragments were dropped into cap-tubes under microscope inspection. To minimize degradation, slides were fixed in 70% ethanol for 1 min, washed in diethylpyrocarbonate (DEPC)-treated deionized water, and stained with a Histogene LCM kit (Arcturus, Mountain View, CA) to preserve the integrity of cellular nucleic acids. On the basis of careful review of the histologic sections, each microdissection was estimated to contain >98% of the desired cells. Areas with luminal secretion, blood, and necroinflammation were avoided. Protein Extraction and 2DE Gel Electrophoresis. Protein lysates were immediately prepared after microdissection by adding lysis buffer [40 mM Tris-HCl, 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropy) dimethyammonio]-1-propanesulfonate (CHAPS; Sigma-Aldrich, St. Louis, MO), and 100 mM 1,4dithioerythtitol (DTT; Sigma-Aldrich, St. Louis, MO) with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO)]. Subsequently, lysates were incubated at 4 °C for 40 min with vigorous shaking every 10 min, and then centrifuged at 4 °C for 30 min at 14 000 rpm. The protein-containing supernatant was transferred to a new tube, and then the protein concentration was determined using a Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Nonlinear gradient strips, pH 4-10, were equilibrated by applying 7 M urea containing 2% CHAPS, 1% DTT, 1% Pharmalyte, and 2 M thiourea for 12-16 h; 200 µg of sample was loaded onto each strip. Isoelectric focusing (IEF) was performed using a Multiphor II electrophoresis unit and an EPS 3500 XL power supply (Amersham Biosciences, Uppsala, Sweden) at 20 °C. For IEF, the voltage was linearly increased during 3 h from 150 to 3500 V for sample entry followed by constant 3500 V overnight. A total volt hour product of 98 kVh was used for the completion of focusing. Prior to the second dimension, the strips were incubated in equilibration buffer [6 M urea, 2% SDS, 50 mM Tris-HCl, pH 6.8, and 30% glycerol] for 10 min. At that time, 1% DTT was added the first time, and subsequently, 2.5% iodoacetamide (Sigma-Aldrich, St. Louis, MO) was added. The equilibrated strips were inserted into SDSPAGE gels (20 × 24 cm, 10-16% gradient gel format), and SDSPAGE was performed using a Hoefer DALT 2D system (Amersham Biosciences, Uppsala, Sweden). Protein spots on 2-DE gel were stained with the alkaline silver staining method as previously described.16 To secure the reliability of data and exclude the experimental variation, expression data were obtained from gel replicates of three samples from the same individual. Image Analysis. An image analysis was performed using a PDQuest software (version 7.0; Bio-Rad, Hercules, CA), and the quantity of protein in each spot was normalized by the total valid spot intensity according to the manufacturer’s instruction. Only the spots that apparently clearly showed a greater than 2-fold change in expression compared to controls were selected. Protein Profile Distance Comparison and Clustering Analysis. We applied a filtering method to the protein expression data to avoid including in the data analysis those proteins that did not vary or that were not highly expressed. We selected only proteins whose expressions showed over 2-fold spot quantity change in tumor samples compared with normal samples. The selected protein spots were filtered by the Student’s t-test (0.05 < P), and for these spots, we performed protein profile distance comparison and clustering analysis. Journal of Proteome Research • Vol. 5, No. 5, 2006 1083

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Table 1. Clinicopathologic Characteristic of Tested Samples Pathol. No.

1.

Age

1560 g/21 × 16 × 7 cm I

32

Microinvasive mucinous ca less than 6 mm2 LMP mucinous tumor, intestinal type

1230 g/25 × 15 × 7 cm I

28

540 g/8 × 6 × 4 cm

I

42

1550 g/8 × 6 × 2 cm

I

MT5: carcinoma

35

Endometriotic cyst

25 g/6 × 5 × 2 cm

NS

49

Endometrioid carcinoma, sertoliform, grade 3/3

533 g/12 × 10 × 8 cm

II

40

730 g/15 × 13 × 9 cm

I

37

Endometrioid carcinoma, conventional, grade 1/3 LMP serous tumor

45 g/7 × 4 × 2 cm

I

49

Papillary serous ca, grade 1/3

170 g/9 × 5 × 2.5 cm

I

50

Papillary serous ca, grade 3/3, 90 g/6 × 4 × 3 cm with node metastasis Papillary serous ca, grade 3/3, 75 g/6 × 4 × 4 cm with omental and parametrial invasion

ET6: endometriotic cyst, EN6: normal tissue ET7a: sertoliform adenoca, ET7b: conventional adenoca, EN7: corresponding normal ET8: carcinoma, EN8: corresponding normal ST9: borderline papillae, SN9: corresponding normal ST10: carcinoma, SN10: corresponding normal ST11: carcinoma, SN11: corresponding normal ST12: carcinoma, SN12: corresponding normal

01-6418 6. 01-1517 7. 01-15905 8. 01-18842 9. 00-16285 10. 01-17394 11. 00-22646 12. 00-24250

50

The overall distances between protein expression profiles were calculated using the formula below by summing up the absolute ratio of the proteins (n ) number of proteins calculated in the tissue). xj ia

n

∑|log i)1

2

xj ib

|

The normalized intensity of protein spots in each a and b sample tissue was used. The pairwise distance metric of protein expression profile was used to build distance trees. The values were entered into a MEGA software program (http://www.megasoftware.net), and the neighbor joining tree was constructed. The average of the pairwise distances measured between the triplicates for each tissue sample were used to estimate a tree depicting the overall differences in gene expression measured between individuals. A clustering analysis was performed for selected filtered spots. To calculate the protein expression ratios, the mean average spot volume of normal reference pools was used for individual normal and tumor samples. The Cluster and Tree View software were used (http://rana.Stanford.EDU/software/) to group proteins with similar expression patterns and to display the tree. We used average linkage clustering with a modified Pearson correlation as similarity metric, and protein and array was median-centered. MALDI-TOF MS. For each gel spot, a biopsy punch was prepared and transferred to a 1.5 mL siliconized Eppendorf tube (Ambion, Austin, TX). Subsequently, the transferred gelspots were destained in destaining solution [100 mM Na2S2O3 (Sigma-Aldrich, St. Louis, MO) and 30 mM K3Fe(CN)6 (SigmaAldrich, St. Louis, MO) (v/v, 1:1)]. The destained gel slices underwent prereduction using 100% acetonitrile (HPLC grade). Gel slices were dried in a Speed-Vac (GMI, Ramsey, MN). The dried gel slices were incubated at 37 °C for 12-16 h in ABC buffer [50mM ammonium bicarbonate (Sigma-Aldrich, St. Louis, MO), pH 8.0] containing 0.1 mg/mL sequencing grade 1084

MT1a: cystadenoma, MT1b: IEC, MN1: corresponding normal tissue MT2: invasive focus

LMP mucinous tumor, Mullerian type Mucinous adenocarcinoma

26 00-9254

5.

1150 g/25 × 20 × 8 cm I

Labels tested

MT3: intestinal-type LMP mucinous tumor, MN3: corresponding normal tissue MT4: borderline papillae

00-6856 4.

stage

LMP Mucinous tumor with IEC, noninvasive

00-6302 3.

Mass size

25 00-22172

2.

Tumor type

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III II

modified trypsin (Promega Biosciences, San Luis Obispo, CA). The peptide mixture, treated with trypsin, was concentrated using ZipTips (Millipore Corp., Billerica, MA). Peptide samples were mixed at a ratio of 0.5 µL of matrix (a-cyano-4-hydroxytranscinnamic acid; Sigma-Aldrich, St. Louis, MO) and 0.5 µL of sample, loaded into a 96 × 2 samples plate (P/N V700813), and crystallized. The crystallized samples were analyzed using an Applied Biosystems Voyager System 4307 MALDI-TOF Mass Spectrometer (Applied Biosystems, Foster City, CA). Parameters were set as follows: positive ion-reflector mode, accelerating voltage 20 kV, grid voltage 64.5%, mirror voltage ratio 1.12, N2 laser wavelength 337 nm, pulse width 3 ns, number of laser shots 300, acquisition mass range 800-3500 Da, delay 100 ns, and vacuum degree 4 × 10-7 Torr. In addition, des-Arg1Bradykinin, Glu1-Fibrinopeptide B, and ACTH (clip 18-39) were used as external standards for mass calibration. Database Analysis. The ProFound (http://129.85.19.192/ profound_bin/WebProFound.exe) was used to search a protein database for protein identification using peptide mass fingerprinting (PMF). The internal standard spectra were adjusted to a trypsin auto-digestion ion peak m/z (842.510, 2211.1046). Search parameters were assigned as follows: the databases used were Swiss-Prot (10.30.200) and NCBI (nr) (10.21.2003), the mass tolerance was (50 ppm, the missed cleavage site value was permitted up to 1, the species was Homo sapiens (human), monoisotope mass was used, and the test range was experimental pI ( 2 pH units, and Mr ( 20%. The analysis standard, Est’Z was applied, which is a method used to measure the identification confidence level by evaluating the z-score of various candidate groups (estimation of z). The z-score is a measurement of the standard deviation distance from the mean value and is obtained by the formula z ) (x - xj )/σ, where x is Gaussian random variable, xj is the mean value of x, and σ is the standard deviation of x. In this study, z-scores of 1.65, 2.326, and 3.090 represent confidence levels of 95%, 99%, and 99.9%, respectively.

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Figure 1. Selected areas for proteomics from fresh frozen tissues. (a) LMP mucinous tumor with intraepithelial carcinoma. MT1 is characteristic of carcinomatous transformation of a conventional LMP intestinal-type mucinous tumor restricted to the surface epithelium, without definite stromal invasion. MT1a was obtained from the benign-looking cysts (above to semicircle), MT1b (inside the semicircle), adjacent to MT1a, is magnified showing papillary and complex glandular proliferation of atypical mucinous cells (bottom left panel). The figure in the bottom right disclosed that severe cellular atypia approaching intraepithelial carcinoma is noted, obviously different from the bland-looking lining cells in other cysts (asterisk). (b) Stromal invasion in mucinous tumor. The criteria defining stromal invasion in LMP mucinous tumors is 10 mm2. Above figure is compatible with focal superficial microinvasion (MT2) measuring less than 6 mm2. The encircled areas were microdissected. Below figure is compatible with widely invasive mucinous carcinoma (MT5). On magnification of the close circle, infiltrative disorganized mucinous glands are shown along with desmoplasia. (c) Ovarian tumors of low malignant potential (LMP). There are two distinct subsets of mucinous LMP tumors: intestinal and Mullerian. LMP mucinous intestinal-type tumor (MT3) is similar to villous adenoma of the gastrointestinal tract, and LMP mucinous Mullerian-type tumor (MT4) shows the typical swollen papillae covered by endocervical-like epithelium. (d) Endometrioid tumors. The encircled area in the enodmetriotic cyst (ET6) composed of normal-looking proliferative endometrial glands and stroma was selected by microdissection. The cord-like or/and trabecular pattern in a sertoliform endometrioid carcinoma (ET7a) is shown along with a more typical endoemtrioid carcinoma (MT7b). A well-differentiated endometrioid carcinoma is demonstrated (ET8). (e) Serous tumors. Atypical proliferating serous tumor (ST9) is characterized by detached proliferating papillae with numerous cellular tufts within the lumen. All three cases of papillary serous carcinoma showed different histologic grades. The well-differentiated PSC (ST10) is an early stage tumor. ST11 and ST12 are poorly differentiated carcinomas, and ST11 showed numerous psammomatous calcifications within the carcinoma. All figures were visualized by standard hematoxylin-eosin staining.

3. Results 1. Clinicopathological Analysis. Clinical information on the ovarian tumor in this study is shown in Table 1. The mean ages of patients with mucinous, endometrioid, and serous tumors were 30.6, 41.3, and 46.5 years, respectively. Mucinous tumors were larger than the other types, and all were stage I except one endometrioid carcinoma (stage II) and two serous carcinomas (stage III and stage II). Case 1 was sampled from two different lesions, one from a benign cystadenoma (MT1a) and another from an intraepithelial carcinoma (MT1b). Cases 2, 4, and 5 had no corresponding normal tissues, probably due to the total replacement of the ovaries by the tumors. Figure 1shows the selected areas used for proteomics and the microscopic portraits of each tumor. MT1 exhibited characteristic stepwise carcinogenesis (intraepithelial carcinoma) directly arising from a conventional LMP mucinous tumor.

Adjacent to the IEC of MT1, a benign-looking cystadenoma (MT1a) was captured and labeled separately from that of IEC (MT1b) (Figure 1a). MT5 exhibited disorderly infiltrative nests with a desmoplastic stroma, whereas MT2 showed a small invasive focus of less than 10 mm2, which was compatible with LMP intestinal-type mucinous tumor with microinvasion (Figure 1b). LMP intestinal-type mucinous tumor (MT3) was microscopically similar to villous adenoma of the gastrointestinal tract, and LMP Mullerian-type mucinous tumor (MT4) showed typical swollen papillae covered by endocervical-like mucinous epithelium (Figure 1c). In contrast to the typical endometriotic cyst composed of normal-looking proliferative endometrial glands and stroma (ET6), the sertoliform endometrioid carcinoma (ET7a) was less differentiated than the conventional endometrioid carcinoma (ET7b), demonstrating cords and trabeculae (Figure 1d). For comparison with the Journal of Proteome Research • Vol. 5, No. 5, 2006 1085

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Figure 2. 2-DE gel images of ovarian cancers and their corresponding normal tissues and nomination of spots on 2-DE. (a) To compare the protein expression differences of the tumor and the normal samples, each histologically and biologically different tumor was categorized. (b) A total of 117 differential protein spots were selected. Of the 12 ovarian tumor samples analyzed, one is shown in this figure. A Gaussian synthetic image was used to designate the selected 117 spots. M, mucinous tumor with IEC/invasion; E, endometriotic carcinoma; S, serous carcinoma; N, normal; T, tumor.

poorly differentiated carcinoma, an extremely well-differentiated endometrioid carcinoma was included (ET8). The LMP serous tumor (ST9) was characterized by detached proliferating papillae with numerous cellular tufts toward the lumen. Three cases of papillary serous carcinomas showed different clinical stages. Whereas ST10 was a well-differentiated PSC of early stage, ST11 and ST12 were poorly differentiated advanced stage carcinomas. ST11 showed numerous psammomatous calcifications within the carcinoma (Figure 1e). 2. Proteomic Analysis. 2.1. 2-DE Analysis. A proteomic analysis was performed to assess the molecular similarities or differences between the specific types of ovarian epithelial tumors. For the comparison of LMP tumors, corresponding normal tissues and carcinomas were used as two reference groups (Figure 2a). In the analyzed samples including normal and tumor tissue, approximately 1400 protein spots were detected. To analyze similarities and differences in protein expression patterns among the three sample groups, quantitative data from 117 spots showing expression changes between normal and tumor groups (Figure 2b) were analyzed. 2.2. Image Analysis of 117 Spots and MALDI-TOF MS Analysis. In comparison with the control group, only spots that clearly showed 2-fold changes in quantitative protein expression were selected (Figure 3a) and represented for the histogram (Figure 3b). To individually confirm each spot, a MALDITOF analysis was performed. PMF maps obtained by MALDITOF MS were used for protein identification (Figure 3c). Six spots with possible relations to tumor biology were analyzed. The protein spots with a confidence level of higher than 90% and a z-score of over 1.65 were as follows: NM23-H1, annexin I, protein phosphatase-1, ferritin light chain, proteasome R-6, and NAGK (Table 2). 2.3. Distance Map Tree Construction. The tree was constructed as a neighbor joining tree by adding the calculated distance of 117 spots as pairwise comparisons and by generating a distance metric. The distance map constructed to assess similarities in the protein expression between samples is shown in Figure 4a. On clustering of distance map tree of ovarian 1086

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epithelial tumors constructed for estimation of similarities (Figure 4b), each tumor is categorized according to protein expression extents. Each number refers to the ratio between the changes common to ovarian epithelial tumors. Corresponding normal tissues from the remaining ovarian tissues spared by ovarian tumors aggregated closely to each other. The LMP Mullerian-type mucinous tumor (MT4) exhibited an expression pattern quite similar to the normal tissue. The LMP intestinal-type mucinous tumor (MT3), LMP serous tumor (ST9), and endometriotic cyst (ET6) were in the borderline range between the normal and carcinoma groups. The LMP tumors with ominous microscopic findings, such as IEC (MT1b) or microinvasion of less than 10 mm2 (MT2), were separated from the conventional LMP (MT3 and MT4) and normal groups. Even benign-looking cystadenoma areas (MT1a) showed an expression pattern similar to that of the adjacent IEC (MT1b). The invasive mucinous carcinoma (MT5) was located at a slightly greater distance from the LMP with IEC or microinvasion. The serous carcinomas (ST10, ST11, and ST12) were found in a cluster at the greatest distance from the normal group. Endometriotic carcinomas (ET7 and ET8) were located between the serous and mucinous carcinomas. The sertoliform endometrioid carcinoma (ET7a) showed a map tree similar to the conventional endometrioid carcinoma (ET7b). The overall protein expression profiles of serous and endometrioid carcinomas appeared to be less affected by grade or stage than by histologic type. The degree of similarity or difference between three distinct ovarian epithelial carcinomas was analyzed by the relative extent of expression ratio using the initially branched point (asterisk in Figure 4a) as a reference. When type-specific differences were compared, mucinous carcinoma was different from the PSC or endometrioid carcinoma by 1.98-fold. The ratio of endometrioid carcinoma to serous carcinomas was 1.1-fold, both of them being similar to each other (Figure 4c). 2.4. Clustering Analysis of Ovarian Tumors. In the previous distance analysis of protein expression profile, we showed that protein expression profiles discriminate the features of tumor

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Figure 3. Identification of differentially expressed proteins between the tumors and the paired normal samples. (a) According to the criteria of 2-fold changes, differentially expressed protein spots from the 2-DE gels were selected. (b) Histogram depicting the intensity of spots. (c) PMF of each spot selected in 2-DE image analysis. Peptide mass fingerprinting of six spots showing significant differences between human ovarian tumors and corresponding normal tissues in 2-DE map. Table 2. Protein Identification of Potential Endpoint Biomarkersa Spot no.

Protein name

%

pIb

MW (kDa)b

Prob

Estimated Z

Intensity ratec

2021 3110 4009 4207 4217 6128

NAGK Annexin I NM23-H1 Protein phosphatase 1 Ferritin light chain Proteasome alpha 6

25 49 32 35 72 44

5.8 5.6 5.4 5.8 5.6 6.3

37.70 36.48 19.86 37.97 16.43 27.84

1.0e + 000 1.0e + 000 1.0e + 000 1.0e + 000 1.0e + 000 1.0e + 000

2.30 2.38 2.16 2.29 2.46 2.40

+2.334806 +4.017177 +2.422712 +2.38945 +1.282543 +1.74117

a The peptide profiles of the protein spots treated with trypsin were analyzed by MALDI-TOF MS. The ProFound (http://129.85.19.192/profound_bin/ WebProFound.exe) was used to search protein database for protein identification using peptide mass fingerprinting (PMF). Database: Swiss-Prot (10.30.200) and NCBI (nr) (10.21.2003). b The mass and pI values specified are theoretically matched by database search. c Plus (+): the rate of increase in intensity (average tumor sample intensity/average normal sample intensity).

type classified by histologic observation. Although the profile distance analysis reflected relative distances of similarities or differences of expression profiles, these calculations neglected the orientation of the expression pattern. For example, expression patterns showing simultaneous 2-fold up-regulation or

down-regulation showed the same distance. For this reason, a hierarchical clustering analysis was performed to compare the similarity according to expression patterns. The same 117 spots applied to the previous profile distance analysis were adopted for clustering analysis. As shown in Figure 4d, 12 tumor samples Journal of Proteome Research • Vol. 5, No. 5, 2006 1087

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Figure 4. Map tree and clustergram of ovarian tumor expression profiles. (a) Distance map tree representing the relative extents of expression changes between ovarian tumors and corresponding normal tissues. (b) Clustering of distance map tree. Numbers refer to the ratio between the changes common to ovarian epithelial tumors. Each tumor is categorized according to protein expression extents. (c) Group average distance map tree of ovarian epithelial tumors. This represents the relative extent of expression difference between three different ovarian carcinomas with serous carcinoma group as a reference. (d) The left panel represents the expression patterns of 12 tumors. This clustergram was constructed from 117 differentially expressed spots as compared to normal groups. The right panel demonstrates the dendrogram showing the relationships between the 12 tumors. In scale bar, green represents down-regulation, and red represents up-regulation of protein expression.

were classified into three groups. The expression profiles of the serous carcinomas were isolated forming a subclass consisting of two endometrioid carcinomas in one group. Five mucinous tumors were clustered in one group. In the previous profile distance analysis, the MT4 mucinous tumor was separated from the rest of mucinous tumors showing expression profiles similar to normal tissues. In the clustergram demonstrating all normal and tumor samples, the pattern of MT4 was more similar to normal tissues than to the other mucinous tumors. The last group consisting of two borderline tumors was isolated from the two principal groups. In this analysis, three distinct groups were classified by protein expression profiles, although the distance relationship was not clarified.

4. Discussion

ovarian epithelial tumors, is the first application of distance map tree proteomics in human tumors. The distance map tree method was adopted from the neighbor-joining method for resconstructing phylogenetic trees.15 The principle of this method is to find pairs of operational taxonomic units (OTUs), called neighbors, which minimize the total branch length at each stage of OUT clustering starting with a starlike tree.15 A distance tree based on intra- and inter-tumoral variations in gene expression patterns was first applied to determine similarities between several organs from different species of primates.14 Comparisons between the species tested were made for each tissue, and the differences in apparent expression levels were used to calculate an overall distance summarized over all genes.14

The present study, which aimed to phylogenetically classify and determine similarities in protein phenotypes between

We focused on evaluating mucinous tumors, particularly conventional LMP and LMP with histologically ominous fea-

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research articles

Proteomics of Ovarian Epithelial Tumors

tures, such as IEC or microinvasion. This was feasible as there were quantitative differences in protein expression profiles between closely related epithelial tumors. There are numerous possible reasons for such expressional differences, including duplications and deletions of genes, promoter changes, changes in levels of transcriptional factors, and changes in cellular compositions of tissues.14 In general, mucinous tumors are more prevalent among younger patients and more frequently present as borderline or benign tumors than serous or endometrioid tumors.6,8 This may imply that mucinous tumors develop through stepwise genetic alterations from benign through malignant, in contrast to serous carcinomas, which are thought to arise de novo from the OSE and inclusion cysts.6,8,17 We recognized that ovarian mucinous tumors are apparently distinct from other OSEderived epithelial tumors in their gene expression profiles. Interestingly, we observed that morphologic heterogeneity in mucinous tumors does not affect proteomics pattern, as morphologically divergent areas within the same mucinous tumor showed similar proteomics profiles. The protein expression profiles of benign cystadenoma were closer to those of adjacent intraepithelial carcinoma than to those of the corresponding normal areas, and the phylogenetic tree of LMP mucinous tumors with microinvasion was very close to that of widely invasive mucinous carcinoma. All LMP tumors showing histologically ominous features, such as IEC or microinvasion, were distinctively different from LMP mucinous tumors, and the overall protein expression profiles were similar to those of mucinous carcinoma. Mucinous intraepithelial carcinomas have been defined in the same way as carcinoma in situ, and LMP tumors with foci of stromal invasion of less than 10 mm2 have been designated “LMP with microinvasion”, such cases demonstrating more favorable outcomes than mucinous carcinoma in previous studies.8,18-19 However, their biological behaviors remain unknown, not only because of a lack of differences in proteomics profiles between LMP and LMP with IEC or LMP with microinvasion, but also because there have been very few studies on these tumors. We speculate that the protein portraits of ovarian mucinous tumors may reflect areas with the worst histological features, based on the results of the present study demonstrating that the protein profiles of even benign or borderline areas are the same as those of the adjacent carcinoma. This is consistent with a previous study detecting the same genetic aberrance, that is, k-ras mutation, in separate areas exhibiting different histologic grades within the same mucinous tumor.17 However, the protein expression profiles of Mullerian LMP mucinous tumors are nearly identical to those of normal tissue, which may explain why LMP mucinous tumors are not fatal and show excellent clinical behavior.20-21 In the phylogenetic map tree, serous carcinomas were obviously separated from mucinous carcinomas. Serous carcinomas were located at the greatest distance from the normal and LMP groups, whereas mucinous carcinomas were closer to the normal group. This protein expression profile of serous carcinomas may explain their highly aggressive clinical behavior. As expected, advanced stage serous carcinomas (ST11 and ST12) were slightly separated from the stage 1 serous carcinoma (ST10). Endometrioid carcinomas were mapped between the serous and mucinous carcinomas. The sertoliform endometrioid carcinoma, a variant of poorly differentiated endometrioid carcinoma, demonstrated a lineage common with conventional endometrioid carcinoma at least in the overall protein expres-

sion profiles. When we compared type-specific differences, we found that the change of mucinous carcinomas was 1.98-fold that of serous carcinomas, and the ratio of endometrioid carcinoma to serous carcinomas was 1.1-fold. These results show that mucinous carcinomas are different from serous and endometrioid carcinomas, which may reflect the more favorable prognosis of mucinous carcinomas. Candidate gene approaches, as well as various subtractive and comparative methods,22-24 have made possible the identification of several genes differentially expressed in ovarian cancer. In the present study, gene expression profiles were screened using proteomics, and the following genes differed significantly in their expression levels: NM23-H1, annexin I, protein phosphatase-1, ferritin light chain, proteasome R-6, and NAGK. NM23-H1, a metastasis suppressor gene in chromosome 17q21.3, has been suggested to play a key role in cell metastasis suppression at different stages of ovarian cancer.25,26 NAGK, a key enzyme involved in the intracellular targeting of lysosomal enzymes, was documented to be elevated by 4-fold in ovarian tumors.27 Protein phosphatase-1 overexpression may play a role in ovarian cancer development, probably by altering the balance of intracellular tyrosine phosphorylation.28 Both ferritin and annexin are also valid biomarker candidates in ovarian tumors based on the fact that myc-activation, FasL, TNFa, and TRAIL disturbed cellular iron homeostasis in ovarian carcinoma.29 The transfer of iron ensures survival by reestablishing this homeostasis.29 In the present study, we identified that different histologic subtypes of ovarian malignant epithelial tumors showed distinctively different protein expression profiles. Serous carcinomas showed the greatest difference from the normal group, while the mucinous carcinoma was the least different. LMP with IEC and stromal microinvasion exhibited proteomics patterns closer to mucinous carcinomas rather than to LMP mucinous tumors, implying that LMP mucinous tumors showing histologically aggressive features may belong to mucinous carcinomas on the basis of protein phenotypes. Furthermore, the continuous morphologic spectrum within mucinous carcinoma showed the same protein expression profiles. The putative surrogate biomarkers, such as NM23-H1, annexin 1, protein phosphatase-1, ferritin light chain, proteasome R-6, and NAGK, demonstrated significantly different expression levels in OSE-derived tumors.

Acknowledgment. This study was supported by Women’s Cancer Biomarker Research Center, Yonsei University (N.H.C.) and Brain Korea 21 Project for Medical Science, Yonsei University (N.H.C.). Note Added after ASAP Publication. This manuscript was released ASAP on March 31, 2006, with incorrect author order and affiliation address footnote. The correct version was published on April 13, 2006.

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