Comparative Analysis of the Protein Profiles from Primary Gastric

Jun 4, 2007 - Tumors and Their Adjacent Regions: MAWBP Could Be a New ... University School of Oncology, Beijing Cancer Hospital/Institute, 52 Fucheng...
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Comparative Analysis of the Protein Profiles from Primary Gastric Tumors and Their Adjacent Regions: MAWBP Could Be a New Protein Candidate Involved in Gastric Cancer Jun Zhang,†,§ Bin Kang,†,§ Xiaohui Tan,‡ Zhigang Bai,| Yumei Liang,O Rui Xing,†,§ Jianmin Shao,†,§ Ningzhi Xu,†,§ Rong Wang,*,†,§,# Siqi Liu,*,†,§ and Youyong Lu*,†,‡,§ Beijing Genomics Institute, Chinese Academy of Sciences, Beijing Airport Industrial Zone B-6, Shunyi, Beijing, China 101318, Peking University School of Oncology, Beijing Cancer Hospital/Institute, 52 Fucheng Road, Haidian District, Beijing, China 100036, Beijing Proteomics Institute, Beijing Airport Industrial Zone B-6, Shunyi, Beijing, China 101318, Peking University People’s Hospital, Xi-Wai Street, Western District, Beijing, China 100034, Department of Pathology of General Hospital of PLA, 28 Fuxing Road, Beijing, China 100853, and Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York 10029 Received June 4, 2007

The study of tumor biomarkers is generally facilitated by the adoption of proteomic strategies. With limitations of techniques and individual varieties of biological samples, the biomarkers for gastric cancer (GC) have not reached a common agreement derived from the proteomic investigations. Herein, we reported a new set of data for screening the biomarkers from the gastric tissues, on the basis of the proteomic strategy developed in our laboratory. Ten pairs of the clinic samples were collected and treated with protein extraction. The gastric proteins were well-resolved by 2-DE, and the GC-associated proteins were identified by MALDI-TOF/TOF MS following image analysis, including 12 up-regulated and 13 down-regulated unique ones. MAWBP was found to be one of the new GC proteins which appeared with lower expression in the GC tissues. We expanded a systematical examination to deeply pursue the relevance between MAWBP and GC. Quantitatively, we measured the expression of MAWBP with Western blot and Real-Time PCR. Extendedly, we estimated the existence of MAWBP with immunohistochemical staining in a large number of the GC cases. Specifically, we inquired whether MAWD, a protein with high affinity to MAWBP, could coexpress and interact with MAWBP in vivo. On the basis of all the results, we concluded that MAWBP could be a new GC-related protein even though its physiological roles remain unexplored. Keywords: Gastric cancer • MAWBP • MAWD • proteomics • tissue microarray

Introduction Gastric cancer (GC) remains one of the most commonly diagnosed cancers and is the second leading cause of cancerrelated death worldwide, killing more than 700 000 people every year.1 The GC incidence in Asian countries, particularly in East Asia, is significantly higher than that in other parts of the world.1 This cancer creates a serious public health problem in this region. In the past decades, the mortality of GC patients has decreased somewhat; however, this improvement did not mainly result from revolutionary therapies, but from the early diagnosis of GC. Development of diagnosis techniques, thus, * To whom correspondence should be addressed. E-mails: (Y. L.) [email protected]; (S.L.) [email protected]; (R. W.) [email protected]. Phone: +86-10-80485325. Fax: +86-10-80485324. † Beijing Genomics Institute, Chinese Academy of Sciences. ‡ Peking University School of Oncology. § Beijing Proteomics Institute. | Peking University People’s Hospital. O Department of Pathology of General Hospital of PLA. # Mount Sinai School of Medicine. 10.1021/pr0703425 CCC: $37.00

 2007 American Chemical Society

is urgently required for GC prevention as well as therapeutics. While endoscopic evaluation is considered as a gold standard in diagnosis of GC, this technique has evident disadvantages, such as invasiveness and expense. The endoscopic mucosal resection also has difficulty in detecting the lesions located at the gastric cardia or lesser curvature. Therefore, it is necessary to develop molecular biological techniques which are less invasive and more sensitive to improve the early diagnostics of GC. During the past decade, a great effort has been made to better define biological profiles of GC. Kang et al. analyzed 28 cases of primary gastric adenocarcinoma and found some correlations between novel amplified or deleted regions of chromosomes and clinical status. For instance, the losses at position 4q or 14q could be evaluated for the metastatic status of GC.2 Using serial analysis of gene expression (SAGE), Aung et al. defined 54 GC-specific candidate genes and further confirmed the results with quantitative RT-PCR, immunohistochemistry (IHC), and enzyme-linked immunosorbent assay Journal of Proteome Research 2007, 6, 4423-4432

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research articles (ELISA).3 Although the clinical relevance of these potential biomarkers has not been determined yet, they could still contribute to expand our understanding of the GC biology. The field of proteomics has evolved considerably recently, and proteomic screening has been widely employed to explore cancer biomarkers.4-9 With the use different proteomic approaches, several laboratories have achieved significant progress to validate new putative diagnostic protein markers in GC specimens.10-14 A breakthrough for GC early diagnosis, however, has not come yet, and no protein biomarker with sufficient sensitivity and specificity has been generally adopted at the bedside. At present, the efforts to investigate cancer biomarkers are generally divided into two phases, which aim to (i) discover differential proteins as potential biomarker candidates and (ii) validate the proteomic discovery. How to integrate the techniques and extract the valuable information from the two phases are critically important in this field. At the discovery phase, we must pay attention to the quality of proteomic survey generated from different techniques; at the validation phase, we should carefully examine in vivo the status of these candidate biomarkers distributed along clinical samples. According to our knowledge, there is a lack of a systematic investigation of the protein biomarkers of GC. On the basis of these considerations, we have initiated this project to identify the GC-associated biomarkers from the clinical tissue samples. In the present communication, we reported new proteomic data acquired from screening the protein profiles of GC with the strategy developed in our laboratory. At the first phase, 10 pairs of clinical samples consisting of the tissue portions of GC and their adjacent regions were carefully estimated for the differential proteomes using 2-DE as well as mass spectrometry. Upon identification of these differential 2-DE spots, immunoblot and quantitative RT-PCR were further employed to confirm the proteomic conclusions. To validate these potential GCrelated proteins, large numbers of tissue samples were examined with the specific antibodies using tissue microarray (TMA). Furthermore, we found that MAWD binding protein (MAWBP) was a new type of biomarker which had an attenuated protein expression in the GC tissues. Corresponding to the declined MAWBP expression, MAWD, a protein having an affinity to MAWBP, was also detected with low expression in the GC tissues. These data suggested that MAWBP, MAWD, and their interactions would be significant in studying carcinogenesis of GC even though the relevant molecular mechanisms remain to be explored.

Materials and Methods Gastric Tissue Specimens and Cell Line. For the proteomic study, 10 pairs of the gastric tissues of patients consisting of the GC portions and their corresponding noncancerous gastric mucosa were obtained from Beijing Cancer Hospital/Institute. To ensure the purity of the GC tissues, the specimens were excised from the cancerous cores. The tissue portions 5 cm apart from the border of gastric tumors, at least, were defined as the controls from each GC patient. The samples were washed with physiological saline to remove contaminants and subsequently frozen in liquid nitrogen. The diagnosis for GC was routinely conducted by the senior pathologists based upon the HE staining and Lauren’s classification. The patients’ information is listed in Supporting Information Table 1. For the TMA study, 88 pairs of tissues were collected from The People’s Hospital of Peking University and Beijing Cancer 4424

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Hospital & Institute. All the samples were fixed with formalin and examined following the same procedure described above. For co-immunoprecipitation study, BGC823, a gastric cancer cell line originated from gastric adenocarcinoma, was cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37 °C and 5% CO2. Sample Preparation and Protein Separation by 2-DE. Protein extraction and 2-DE analysis were performed as previously described with minor modifications.15 Briefly, 50 mg of tissue/sample was crushed by metal mortar merged in liquid nitrogen, and then precipitated with 10% TCA/acetone for 2 h. The precipitate was washed with precooled acetone. After removing acetone with vacuum evaporation, the pellet was dissolved in the lysis buffer containing 20 mM Tris-HCl, pH 7.5, 8 M urea, 4% 3-[(3-cholamidopropyl) dimethylammonio]1-propanesulfonate (CHAPS), 0.5% Pharmalyte (pH 3-10L), 10 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 2 mM ethylenediamine tetraacetic acid (EDTA). The lysate was sonicated with a probe sonicator for 5 min followed by centrifugation at 40 000g for 30 min. After quantitative measurements of protein concentrations by Bradford method, the supernatant was stored at -80 °C until use. Approximately 200 µg of protein was loaded onto an IPG strip (pH 3-10L, 18 cm, Amersham Biosciences, Sweden) and subjected to isoelectric focusing in IPGphor (Amersham Biosciences, Sweden). Briefly, the strips were rehydrated for 4 h at 0 V and for 8 h at 50 V. The isoelectric focusing was carried out with a program of gradient voltage at 500, 1000, and 8000 V, each for 1 h and finally remained at 8000 V until the value of Vh reached 56 000. The focused strip were reduced with 1% DTT and alkylated with 2.5% iodoacetamide (IAM) in the buffer containing 6 M urea, 50 mM Tris-HCl, pH 8.8, 30% glycerol, 2% SDS, and trace bromophenol blue. The treated strips were subjected to 12% SDS-PAGE (260 mm × 200 mm) in Ettan DALT II System (Amersham Biosciences, Sweden) for the secondary electrophoresis. The proteins on the 2-DE gels were visualized using silver staining. The Image Analysis of Differential 2-DE Spots. All the 2-DE gels were scanned by Imagescanner (Amersham Biosciences, Sweden) with 300 dpi. Relative spot volumes were estimated with ImageMaster 2-D Platinum, version3.0 (Amersham Biosciences, Sweden). To minimize the individual differences among the patients’ samples, the “mixed pools” were prepared and set as the references, in which equal amounts of protein from each sample, either the GC or the adjacent tissue, were evenly pooled and run on 2-DE gels. On the 2-DE gels, those spots which appeared on parallel gels of the “mixed pools” were selected for image analysis. First, the 2-DE gels obtained from the samples of “mixed pools”, the GC tissue and the adjacent tissue were compared to identify the spots with significant changes in relative spot volume (> 3-fold); second, the differential spots found in the “mixed pools” were further rechecked in 10 pairs of 2-DE images from the individual samples to identify the ones with high incidence rate (>50%), respectively. Protein Identification by Mass Spectrometry. After image analysis, the differential spots were manually excised from gels and transferred into 96-well plates. The gel particles were reduced with 10 mM DTT, alkylated with 55 mM IAM, and subjected to in-gel digestion with 0.01 µg of trypsin (Sigma) at 37 °C overnight. The digestive reaction was stopped using 0.1% triflouroacetic acid (TFA). The peptides generated from tryptic digestion were spotted onto Anchorchip (Bruker, Germany) and

MAWBP, a New Candidate of Gastric Cancer Biomarker

cocrystallized with cyano-4-hydroxycinnamic acid (CHCA) (4 mg/mL). The mass spectra of peptides were acquired by an Ultraflex MALDI-TOF/TOF MS (Bruker, Germany). A commercial peptide mixture was used to calibrate the accuracy and resolution of mass spectrometry. All peptide mass fringerprintings (PMFs) were analyzed with the protein search engine MASCOT (Matrix Science, U.K.) against NCBI’s nonredundant human genome database (NCBInr). The protein identification criteria for this work were based on probability-based MOWSE scoring algorithm with 95% confident level in MASCOT.16 The PMF peaks with high mass intensities were selected for MS/ MS analysis. On the basis of b- and y-ions, the corresponding sequences of peptides could be attained as the advanced evidence for protein identification. Generation of Antibodies against MAWBP and MAWD. The full lengths of MAWBP and MAWD cDNA were obtained by PCR amplification using a human liver cDNA library. The amplified fragments were inserted into an Escherichia coli expression vector, pET28a. After the confirmation of nucleotide sequences, these vectors were transformed into E. coli BL-21(DE3) strain for protein expression. These bacteria were lysed in the buffer containing 10 mM Tris-Cl, pH 8.0, 150 mM NaCl, and 5 mM imidazole, and the recombinant proteins were purified by Ni2+-NAT affinity chromatography (Qiagen). To generate monoclonal antibodies against MAWBP and MAWD, the recombinant proteins of MAWBP-His and MAWDHis were used to immunize animals. Three Balb/c mice (female, 4-6 weeks old) were immunized subcutaneously 4 times in 10-14 days intervals. The spleens of the mice were removed 3 days after the final immunization, and the spleen cells from the mice were isolated and fused with Sp2/0 myeloma cells at a ratio of 1-2 Sp2/0 cell/10 spleen cells with polyethylene glycol 1500. The fused cells were cultured in semisolid IMDM medium containing 25% new born calf serum, 2% methyl cellulose, and 2% hypoxanthine-aminopterin-thymidine (HAT). Single hybridoma colonies were collected individually after 7 days incubation. Appropriate amounts of the supernatants generated from hybridoma cell culture were used for screening for the immuneaffinity ELISA. The positive hybridomas were selected for further culture and titer determination. To generate polyclonal antibodies against MAWBP and MAWD, two New Zealand rabbits were immunized by approximately 500 µg of recombinant protein/rabbit in complete Freund’s adjuvant (1:1), followed by several boosts with the same amount of proteins in incomplete Freund’s adjuvant (1: 1). The rabbit sera were collected and purified through protein A affinity chromatography (Bio-Rad). Western Blot. The proteins used in the 2-DE analysis were further examined with Western blot. The electric-blotted PVDF membranes (Millipore) were soaked in 5% skimmed milk and 0.1% Tween 20 overnight and incubated with the primary antibodies, such as cathepsin D (1:2000, Santa Cruz Biotechnology), tropomyosin (1:2000, Sigma), Annexin I (1:2000, Santa Cruz Biotechnology), MAWBP (1:1000, our lab), and MAWD (1: 2000, our lab) at room temperature for 2 h, respectively. The anti-GAPDH antibody (1:2000, Santa Cruz Biotechnology) was used as a reference to normalize the intensities of immunoreactions with different antibodies. The antibody against rabbitIgG or mouse-IgG conjugated with horseradish peroxidase (Zhongshan Golden Bridge Biotechnology, China) was used as the secondary antibody. The immuno-recognition signals were visualized with enhanced chemiluminescence system (Amer-

research articles sham Biosciences, Sweden) according to the manufacturer’s instructions. Real-Time PCR. The mRNA levels of MAWBP and MAWD were quantitatively estimated through real-time PCR using an ABI PRISM 7300 system (Foster City, CA). The cDNA libraries were generated from the total RNA preparations of the gastric tissues. The primers were designed as follows: MAWBP, 5′GGG TCT GCA CAC GCT GTT C-3′ (forward) and 5′-TAA TGT CAA CCC TTC CGT CT-3′ (reverse); MAWD, 5′-GGG ACA GGA TAA ACT GTT ACG C-3′ (forward) and 5′-AGC ATG ATC CCA AAG TCG AAC-3′ (reverse); GAPDH, 5′-GAA GGT GAA GGT CGG AGT-3′ (forward) and 5′-GAA GAT GGT GAT GGG ATT TC-3′ (reverse). The Ct values of GAPDH were adopted to normalize the expression of MAWBP or MAWD in the tissues. The comparison of mRNA levels between the samples was achieved on the basis of the relative values of gene expression calculated by 2∆∆Ct method. TMA Analysis with Immunohistochemistry. The immunohistochemical staining was conducted in TMA analysis for the gastric tissues, either for MAWBP or MAWD. Tissue cylinders with a diameter of 0.6 mm and a height of 1 mm mounted in a puncher (Beecher Instruments, Silver Springs, MD) were employed to punch the selected areas of the waxed tissues, and the punched samples were transferred into a paraffin block. The 4-µm thick slices were generated from the block, placed onto glass slides, and baked overnight at 60 °C. Prior to immunohistochemical staining, the slides were deparaffined with xylene and rehydrated in a graded alcohol series. Antigen retrieval was carried out with a microwave oven in 1 mM EDTA solution, pH 8.0 (Zhongshan Golden Bridge Biotechnology, China). The enzyme activity of endogenous peroxidase was blocked by incubation in 3% H2O2 at room temperature for 10 min. After incubation of 3% milk to prevent nonspecific binding, the slides were incubated with the primary antibody, polyclonal anti-MAWBP (1:100, our lab) or polyclonal anti-MAWD (1:400, Santa Cruz), at 4 °C overnight in a moist chamber. Following the thorough washes with phosphate buffered saline (PBS), the slides were incubated with the secondary antibody conjugated with peroxidase (Zhongshan Golden Bridge Biotechnology, China). Immunohistochemical staining was developed to yield the brown signals using the commercial DAB kit (Zhongshan Golden Bridge Biotechnology, China). To distinguish the cellular portions of nucleus and cytoplasm, the slides were counterstained with hematoxylin. The TMA data were evaluated statistically using SPSS version 11.5 (SPSS Company). The χ2 and leave-one-out cross validation (LOOCV) tests were used to define the significant differences among the pathological samples, and P-values less than 0.05 were set as a significant threshold. Co-immunoprecipitation of MAWBP and MAWD. BGC823 cells were harvested and suspended in the lysis buffer containing 50 mM Tris-HCl, pH7.5, 150 mM NaCl, 1% NP40, 1 mM EDTA, and 2% protease inhibitor cocktail. The lysates were precleaned by incubation of the protein A resins bound with the preserum originated from the preimmunized rabbits for 2 h. After centrifugation at 1000g for 5 min, the supernatants were transferred to the slurry containing monoclonal anti-MAWBP antibody or polyclonal anti-MAWD antibody (the two antibodies were generated in our lab) and protein A resins for overnight incubation. The preserum was used as a control. The beads of protein A were washed thoroughly with the lysis buffer, and the immunoprecipitated Journal of Proteome Research • Vol. 6, No. 11, 2007 4425

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Figure 1. The typical 2-DE images for the GC and adjacent noncancerous tissues. MN and MT indicate the samples of “mixed pool” from the GC and adjacent noncancerous tissues, respectively. The differential spots were labeled with arrowheads. The experimental conditions were described in Materials and Methods.

Figure 2. The mass spectra of MALDI-TOF/TOF MS for identification of MAWBP protein. (A) The close-up 2-DE images of spot no. 15 from the 10 paired samples. The sample numbers represent the individual ones in this study. (B) The mass spectrum of PMF generated from the digested peptides of the 2-DE spot no. 15. Inset, the MS/MS spectrum derived from the parent ion at 1659.82, in which the amino acid sequence, PGGQTQAFDFYSR, was deduced based upon these b-ions and y-ions.

complexes bound to the protein A resins were eluted and dissolved in 5% SDS solution. The immunoprecipitated proteins were resolved by 12% SDS-PAGE followed by electric-blot to PVDF membrane to further examine the protein components in the complexes. The antibody, polyclonal anti-MAWD or monoclonal anti-MAWBP, was used for the immuno-recognitions. 4426

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Results Comparison of 2-DE Images in the Paired GC Samples. To elucidate the proteins differentially expressed in the gastric tissues, we processed 2-DE gels and analyzed 10 pairs of samples consisting of the GC tissues and their corresponding adjacent sections. In Figure 1, we observed that the staining

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MAWBP, a New Candidate of Gastric Cancer Biomarker Table 1. The Up-Regulated Proteins Identified in the GC Tissues spot number

accession number

description

MW (kDa) the./exp.

pI the./exp.

Mascot score

sequence coverage

fold change

incidence rate (%)

1 2 3 4 5 6 7 81 9 10 11 12

gi|3123283 gi|27597085 gi30582659 gi|51479152 gi|662841 gi|500638 gi|57108093 gi|23274163 gi|21361120 gi|40225338 gi|105247 gi|4505371

SM22 R-Tropomysin Cathepsin D ATP synthase HSP27 HnRNP A2 Actin alpha2 SEPT2 protein Calponin 1 ACTG1 protein Aldehyde dehydrogenase NADH dehydrogenase Fe-S protein 8

23/25 33/28 45/34 25/25 22/31 36/37 42/26 43/46 33/37 19/26 57/25 24/26

8.8/8.6 4.5/6.9 6.1/5.4 5.1/5.2 7.8/5.3 8.7/9.2 5.2/5.5 6.4/6.5 9.1/9.4 5.2/5.2 6.5/5.2 6.0/5.0

129 70 64 77 78 165 71 87 110 82 190 68

57% 42% 25% 46% 34% 44% 24% 40% 50% 47% 32% 38%

3.17 4.13 3.12 4.27 16.77 3.96 9.06 ∞ 5.82 3.42 ∞ ∞

80 70 60 50 50 50 50 50 50 50 50 50

fold change

incidence rate (%)

∞ 0.17 0.04 ∞ 0.32 0.08 0.13 ∞ ∞ 0.21 0.22 0.18 0.11

80 80 80 70 70 50 50 50 50 50 50 50 50

Table 2. The Down-Regulated Proteins Identified in the GC Tissues spot number

accession number

description

MW the./exp.

pI the./exp.

Mascot score

13 14 15 16 17 18 19 20 21 22 23 24 25

gi|19684166 gi|48145973 gi|16307296 gi|15679996 gi|62897391 gi|62896933 gi|442361 gi|31416989 gi|30582427 gi|556516 gi|54696548 gi|460771 gi|13279254

Acyl-CoA dehydrogenase NDUFV2 protein MAWBP Hypothetical protein Isocitrate dehydrogenase Aldehyde dehydrogenase 6A1 Annexin I Pyruvate kinase3, isoform1 Aldo-keto reductase family1, B10 Dihydrodiol dehydrogenase isoform DD1 Carbonyl reductase 1 HnRNP-E1 HSDL2 protein

45/42 27/28 32/34 23/21 51/47 58/58 35/40 58/63 36/39 35/39 31/35 38/43 37/48

8.1/6.5 8.2/6.7 6.0/6.5 8.0/8.0 8.7/8.4 8.7/8.1 7.6/7.8 7.9/8.0 7.1/7.5 8.1/8.0 8.5/7.7 6.7/7.1 5.8/8.2

103 95 139 72 68 103 93 71 76 103 124 88 64

spots were well-resolved by 2-DE with sharp focusing as well as wide distribution along pH 3-10. Approximately 1050 spots per gel were detected. We set two criteria to judge the gel quality to ensure that these images were sufficient for comparative analysis: (1) the overlapp rate for all the parallel gels derived from the same samples should be over 80% and (2) the correlation coefficients (CC) for all the gels derived from the different samples in the same tissue should be no less than 80%.17 The statistic data indicated that all the 2-DE images were qualified for comparative analysis by passing the criteria. The number of spots was automatically determined, and their normalized volume was quantified as percent volume (%v), where %v ) spot volume/Spot volumes of all spots resolved in the gel.18 These spots located at the similar 2-DE positions in different gels with 3-fold differences in relative spot volumes were defined as the differential spots in these 2-DE gels.19 With the two-step strategy described in Materials and Methods, we have found 40 differential 2-DE spots, 19 upregulated and 21 down-regulated, in the tumor tissues, which appeared in 50% samples at least. One example is the spot no. 15 on 2-DE with the apparent values of MW of 34 kDa and pI of 6.5, whose abundance was significantly attenuated (8.72fold) in the GC tissues in most cases (8 out of 10). The comparison of local 2-DE images for this spot is present in Figure 2A. Protein Identification by MALDI-TOF/TOF MS and Western Blot. We employed MALDI-TOF/TOF MS to identify the 40 differential 2-DE spots. To ensure the accuracy of protein identification, we adopted three stringent criteria: (a) the total coverage of identified peptides to the full length of protein to be >20%, (b) the identified proteins to rank at the top with five matched peptides at least, and (c) one PMF signal to be confirmed by MS/MS at least. As shown in Figure 2B, the spot

sequence coverage

27% 42% 45% 29% 20% 24% 29 24% 33% 28% 42% 33% 20%

no. 15 was identified as MAWBP, 15 unique peptides matching to this protein (45% coverage) and the PMF at m/z ) 1659.82 further giving an amino acid sequence of PGGQTQAFDFYSR. Of 40 differential 2-DE spots, 36 were identified as proteins by MALDI-TOF/TOF MS, corresponding to 25 unique proteins, 12 up-regulated and 13 down-regulated in the GC tissues. These identifications were listed in Tables 1 and 2. Furthermore, we confirmed the identification results with Western blot. We selected several identified proteins which were previously reported to be tumor-related. Figure 3 revealed the intensity changes of immunostaining for the three proteins in the paired tissues. After semiquantitative estimation, cathepsin D and tropomysin were up-regulated 17.2- and 3.3-fold, respectively, and annexin I was down-regulated 14.1-fold in the GC tissues as compared with the adjacent tissues. The data coincided with the proteomic conclusions drawn from 2-DE analysis. The Decreased Expression of MAWBP and MAWD Gene in the GC Tissues. To confirm the differential expression of MAWBP protein, antibodies were generated in our lab. Using the monoclonal antibody against MAWBP, we confirmed all the GC tissues containing low protein expression of MAWBP with Western blot (Figure 4 gives three typical pairs of the samples). Although the biochemical properties of MAWBP are poorly understood, it is generally accepted that MAWBP is a protein with high affinity to MAWD, a protein containing WD40 repeats that contributes to protein/protein interactions leading to inhibition of transcriptional activation mediated by TGF-β. Is the expression of MAWBP correlated with MAWD protein expression in the GC tissues? We applied a polyclonal antibody against MAWD to screen the gastric tissues and found that the levels of MAWD protein were significantly lower in the GC tissues than those of the adjacent portions (Figure 4). Journal of Proteome Research • Vol. 6, No. 11, 2007 4427

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Figure 3. Analysis of Western blot to confirm the proteomic observations for the differential proteins in the GC tissues. The results of Western blot upon three cases from the 10 paired samples were shown here, in which three antibodies, cathepsin D, tropomysin, and annexin I, were used as the potential markers for GC and GAPDH was adopted as a reference protein. The experimental details were described in Materials and Methods.

Figure 4. Comparison of MAWBP or MAWD expression at protein or mRNA level in the paired samples. (A) The results of Western blot upon three cases from the 10 paired samples using the antibodies against MAWBP or MAWD. (B) Comparison of the relative abundances of MAWBP and MAWD proteins in 10 paired samples with Western blot. (C) Comparison of the relative Ct values of MAWBP and MAWD mRNAs in six paired samples with quantitative PCR. In all the experiments, GAPDH was used as a reference for normalization in quantitative estimations.

Furthermore, we adopted Real-Time PCR to measure the quantitative data of MAWBP and MAWD at the mRNA level. After normalization by GAPDH expression, the results demonstrated that the expression of MAWBP and MAWD in the GC tissues were significantly lower than that of their adjacent regions (p < 0.001, Figure 4). The Coexpression of MAWBP and MAWD and Their Clinicopathologic Relevance in the Gastric Tissues. After careful evaluation for these potentially tumor-related proteins, we expanded the clinical cases to further validate these protein markers, particularly for MAWBP and MAWD. The TMA slide with 88 GC paired samples was used in this study. Figure 5 exhibited the typical images of IHC-TMA. With the use of anti4428

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MAWBP or anti-MAWD antibody, the immunohistochemical staining for the normal gastric tissues was shown in Figure 5A,D, in which the relatively small pleomorphic brown bodies appeared in the cytoplasm but not in nucleus, suggesting that MAWBP and MAWD were localized in the cytoplasm of the epithelial cells. In addition, we observed that the heavy staining occurred in the epithelial cells of the GC-adjacent regions with normal-appearing mucosa, but the faint or negative staining occurred in the GC tissues (Figure 5). Statistically, the strongly positive staining of MAWBP or MAWD was detected in 70.5% (62 of 88) or 47.7% (42 of 88) of the GC-adjacent regions, respectively. In the corresponding GC tissues, the positive staining rate for MAWBP or MAWD was approximately 40.1%

MAWBP, a New Candidate of Gastric Cancer Biomarker

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Figure 5. The images of immunohistochemical staining with the antibodies against MAWBP or MAWD in normal and GC tissues. (A-C) The IHC staining with MAWBP antibody; (D-F) the IHC staining with MAWD antibody. (A and D) The normal gastric tissues; (B and E) the GC tissues well-differentiated; (C and F) the GC tissues poorly-differentiated.

(36 of 88) or 27.3% (24 of 88), respectively. These data demonstrated the significant differences of MAWBP or MAWD protein expression between the GC tissues and their adjacent portions (p < 0.001 for MAWBP, p < 0.05 for MAWD). Furthermore, we examined the coexpression rates of MAWBP and MAWD in the GC tissues. The positive staining for the coexpression in these samples was 15.9% (14 of 88), and the negative staining for that was 47.7% (42 of 88). Taking all the rates together, the χ2 test revealed that the expression status of MAWBP and MAWD was tightly correlated with the tumorigenesis in gastric tissues (p < 0.05). We performed a leaveone-out cross validation (LOOCV) test to examine the accuracy of the χ2 test for TMA. Randomly dropping one sample out from the pool with 88 paired samples, the LOOCV evaluations revealed that the rates of GC prediction using MAWBP, MAWD, or both were all qualified as the significant biomarker of GC with total accuracy of 65%. On the basis of the image analysis of immunohistochemical staining for TMA, we then tried to seek how the expression status of MAWBP and MAWD were associated with the differentiation status of GC. In Figure 5B,E, the faint immunohistochemistry staining of either MAWBP or MAWD was detected in the GC tissues well-differentiated. In Figure 5C,F, the negatively immunohistochemical staining for both proteins

appeared in the GC tissues poorly differentiated. We applied χ2 test to evaluate the correlations of the immunohistochemical data and the differentiated status of GC and concluded that MAWBP and MAWD could be the indicators for the GC differentiations (p < 0.05) (Supporting Information Table 2). We also statistically examined the correlations between MAWBP or MAWD expression and the other clinicopathological parameters. As shown in Supporting Information Table 2, the expression of MAWBP or MAWD was independent from ages, genders, locations of tumors, histological types, tumor stages, depth of wall invasion, lymph node metastasis and distant metastasis (Supporting Information Table 2). Interactions of MAWBP and MAWD in BGC823 Cells. All the evidence above revealed MAWBP and MAWD have the same trends of their coexpression in the gastric tissues. Since these data were derived from tissues, we still inquired whether the two proteins in the gastric cells were truly interacting with each other. Since there was a lack of the normal and cultured gastric cells, we used BGC823 cells in this study, which was widely accepted in the GC investigations. As depicted in Figure 6, the two proteins in BGC823 exhibited the interactions with each other. When the MAWBP antibody was applied to catch the MAWBP proteins in the cells, a coprecipitated protein band was recognized by the MAWD antibody (Figure 6, top panel). Journal of Proteome Research • Vol. 6, No. 11, 2007 4429

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Figure 6. Confirmation of the interactions between MAWBP and MAWD in BGC823 cells with immunoprecipitation approach. Top panel showed that the MAWBP proteins in the cell lysates were immunoprecipitated by monoclonal anti-MAWBP, and the coprecipitated protein was detected by polyclonal anti-MAWD antibody; bottom panel showed that the MAWD proteins in the cell lysates were immunoprecipitated by polyclonal anti-MAWD, and the coprecipitated protein was detected by monoclonal antiMAWBP antibody.

Conversely, when the MAWD antibody was used for immunoprecipitation of the MAWD proteins, the MAWBP band appeared in the coprecipitated products (Figure 6, bottom panel). We have drawn a conclusion, therefore, that the coexpression of the two proteins in the gastric tissues could represent their close interactions.

Discussion The epithelial cells on the gastric mucosa are believed to be a primary place of gastric malignancy.20 The layers of the gastric mucous membrane are quite thin, and many cellular events, such as angiogenesis and invasion of myofibroblast, occur along with tumorigensis.21 These factors make the accurate dissection of gastric tumors difficult in surgery, even though laser capture has been employed. To the present, 5 groups have reported proteomic studies on the clinically paired specimens from the primary gastric adenocarcinoma and noncancerous mucosa using 2-DE coupled with MALDI-TOF MS22,23,25,26 or LC-MS/MS24 and obtained quite diverse results. He et al. from Hong Kong collected 10 paired specimens and identified 21 protein candidates, 12 up-regulated and 9 down-regulated;22 Ebert et al. from Germany gathered 10 paired samples and found 18 unique proteins related with GC, 5 up-regulated and 13 down-regulated;23 Yoshihara et al. from Japan established the study of 5 cases and finally confirmed 9 proteins associated with GC, 2 up-regulated and 7 down-regulated;24 Nishigaki et al. from Japan collected 14 patient samples and identified 22 GC-associated proteins, 9 up-regulated and 13 down-regulated;25 Ryu et al. from Korea had 11 GC specimens and verified 14 proteins related with GC, 7 up-regulated and 7 downregulated.26 Of a total of 84 GC-associated candidates identified by those groups, none was identified by all the groups. There were only two proteins co-identified by several laboratories. For instance, CA11 was reported in four reports, and apolipoprotien A1 was documented by three papers.22-24,26 In this study, our data demonstrated again that the proteomic data from the GC tissues was poorly overlapped with other reports. As shown in Tables 1 and 2, only three proteins, HSP27, carbonate anhydrase I, and carbonate anhydrase II, were detected by other two laboratories.25,26 Which lessons do we learn from the published data? First, technique is a fundamental issue (how we can acquire the accurate data from proteomic measurements and statistical comparisons). Second, we have 4430

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Zhang et al.

to realize that the proteomic measurement is able to provide an average level of protein abundances in a tissue, but unable to distinguish the protein expression varied at different tissue regions as well as cells. Third, the sizes of clinical samples are critically required to validate the GC candidates. The maximum size in the previous reports with 2-DE proteomics for GC was 14 collected specimens. Considering the experimental errors and the individual differences, we decided to screen the potential biomarkers in a large number of GC samples (88 pairs). According to these estimations to study the GC biomarkers, we have developed the corresponding strategy to reduce the misinterpretations in this study. We strictly selected the clinical samples, dissecting the tissues with careful surgery and examining the GC samples with senior pathologists. We have semiquantitatively analyzed the 2-DE images, through “mixed pools” image comparison and individual image validation. We have employed the tandem mass spectrometry to acquire accurate mass signals and precise identification of proteins, on the basis of PMF and MS/MS information. We have used the different analytic means to validate the potential candidates for GC, quantitatively estimating the differential expression of genes by real-time PCR and Western blot, and specifically localizing the GC-associated proteins in tissues by IHC. We have expanded the sample sizes to further confirm the proteomic conclusions based upon the TMA analysis. Because of all these efforts which we have taken, we reasoned we have achieved convincing data for the GC biomarkers. Of these protein candidates related with GC, we were intensely attracted by the protein MAWBP. To our knowledge, this is the first report regarding MAWBP involvement in GC. We have employed multiple approaches, such as proteomic, real-time PCR, and IHC, detected the MAWBP expression in tissues or cells, and finally provided the convincing evidence to validate the conclusion that this protein was down-regulated in the GC tissues. It was first reported by Iriyama et al., who claimed the MAWBP gene was widely expressed in many human tissues, such as brain, heart, lung, liver, pancreas, kidney, and placenta.27 By now, however, there is lack of the systematical investigation focusing on its biological functions. The limited reports were mostly achieved from the screening experiments of DNA array and proteomic survey.28-31 In a mouse model with folate deficiency, two differential 2-DE spots of MAWBP, one up-regulated and the other down-regulated, were identified, but the modified forms and the corresponding significance were not further clarified.30 Recently Solomon et al. observed that the expression of MAWBP protein in H411E liver cells could be induced by TNF-R; nevertheless, the authors did not pay attention seeking its physiological roles.31 Apparently, it is necessitated to strengthen the functional investigations of MAWBP protein. At the initiation stage, a cell system with overexpression or knockdown of the MAWBP expression has been established in our laboratory. During the effort of pursuing the functions of MAWBP, MAWD may be an elementary factor involved in the formation of the functional complex with MAWBP. Using yeast two-hybrid screening with MAWD as a bait gene, Iriyama et al. had confirmed that MAWBP and MAWD possessed high affinity to each other and could form a functional complex.27 MAWD, also called Strap, is ubiquitously expressed and localized in both cytoplasm and nucleus. It contains the WD-motif repeats which facilitate the assembling process of macromolecular complexes.32 Once MAWD interacts with transforming growth factor

MAWBP, a New Candidate of Gastric Cancer Biomarker

beta (TGF-β) receptor and Smad 7, these interactions can inhibit TGF-β signaling which may promote cell growth leading to tumorigenicity.33 In this study, we first identified MAWBP to be a GC-associated protein, and further proved the expression of MAWBP and MAWD exhibiting the co-attenuated patterns in the GC tissues. Finally, we found that the two proteins indeed interact together and formed the complexes in BGC823 cells. In the light of the model of TGF-β signaling and our observations, we hypothesize that the interactions of MAWBP and MAWD may act in a key role in the tumorigenesis of GC. Thus, an accurate estimation for these interactions could lead to a new clue for the carcinogenic mechanisms of GC.

Acknowledgment. This work was supported by grants of science and technology project in Beijing (D0905001040331), National Key Basic Research Program Project (2004CB518708), National Bio-Tech 863 program (2002-BA711A11, 2006AA02Z492) of China and the CAS International Partnership Program for Creative Research Teams. We also thank Tissue Bank of Beijing Cancer Hospital/Institute for gastric tissue specimens. Supporting Information Available: Figure of the comparison of MAWBP or MAWD expression at the protein level in the paired gastric tissue samples; tables listing the clinicopathologic features of the samples and the statistic estimations to the relationships of MAWBP or MAWD expression and clinicopathologic features. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Max Parkin, D.; Bray, F.; Ferlay, J.; Pisani, P. Global Cancer Statistics, 2002. CA Cancer J. Clin. 2005, 55, 74-108. (2) Kang, J. U.; Kang, J. J.; Kwon, K. C.; Park, J. W.; Jeong, T. E.; Noh, S. M.; Koo, S. H. Genetic alterations in primary gastric carcinomas correlated with clinicopathological variables by array comparative genomic hybridization. J. Korean Med. Sci. 2006, 21, 656-65. (3) Aung, P. P.; Oue, N.; Mitani, Y.; Nakayama, H.; Yoshida, K.; Noguchi, T.; Bosserhoff, A. K.; Yasui, W. Systematic search for gastric cancer-specific genes based on SAGE data: melanoma inhibitory activity and matrix metalloproteinase-10 are novel prognostic factors in patients with gastric cancer. Oncogene 2006, 25, 2546-57. (4) Deng, B.; Ye, N.; Luo, G.; Chen, X.; Wang, Y. Proteomics analysis of stage-specific proteins expressed in human squamous cell lung carcinoma tissues. Cancer Biomarkers 2005, 1, 279-86. (5) Hsu, P. I.; Chen, C. H.; Hsieh, C. S.; Chang, W. C.; Lai, K. H.; Lo, G. H.; Hsu, P. N.; Tsay, F. W.; Chen, Y. S.; Hsiao, M.; Chen, H. C.; Lu, P. J. Alpha1-antitrypsin precursor in gastric juice is a novel biomarker for gastric cancer and ulcer. Clin. Cancer Res. 2007, 13, 876-83. (6) Canelle, L.; Bousquet, J.; Pionneau, C.; Hardouin, J.; ChoquetKastylevsky, G.; Joubert-Caron, R.; Caron, M. A proteomic approach to investigate potential biomarkers directed against membrane-associated breast cancer proteins. Electrophoresis 2006, 27, 1609-16. (7) Hwang, S. I.; Thumar, J.; Lundgren, D. H.; Rezaul, K.; Mayya, V.; Wu, L.; Eng, J.; Wright, M. E.; Han, D. K. Direct cancer tissue proteomics: a method to identify candidate cancer biomarkers from formalin-fixed paraffin-embedded archival tissues. Oncogene 2007, 26, 65-76. (8) Fujita, Y.; Nakanishi, T.; Hiramatsu, M.; Mabuchi, H.; Miyamoto, Y.; Miyamoto, A.; Shimizu, A.; Tanigawa, N. Proteomics-based approach identifying autoantibody against peroxiredoxin VI as a novel serum marker in esophageal squamous cell carcinoma. Clin. Cancer Res. 2006, 12, 6415-20. (9) Zhu, Y.; Wu, R.; Sangha, N.; Yoo, C.; Cho, K. R.; Shedden, K. A.; Katabuchi, H.; Lubman, D. M. Classifications of ovarian cancer tissues by proteomic patterns. Proteomics 2006, 6, 5846-56.

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