Identification of Tumor Antigens in Human Lung Squamous

Dec 29, 2006 - Synopsis. Proteomic approach was applied to identify proteins that commonly elicit humoral response in lung squamous carcinoma (LSC)...
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Identification of Tumor Antigens in Human Lung Squamous Carcinoma by Serological Proteome Analysis Fang Yang,†,‡,# Zhi-qiang Xiao,†,# Xiu-zhi Zhang,‡ Cui Li,† Peng-fei Zhang,† Mao-yu Li,† Ying Chen,‡ Ge-qin Zhu,‡ Yi Sun,‡ Ying-fu Liu,‡ and Zhu-chu Chen†,‡,* Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha, Hunan, China, and Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan, China Received May 12, 2006

Autoantibodies against tumor antigens are promising means for cancer diagnosis and prognosis. In this study, we applied a proteomic approach to identify proteins that commonly elicit humoral response in lung squamous carcinoma (LSC). Sera from 20 newly diagnosed patients with LSC and 20 matched healthy individuals were analyzed for antibody-based reactivity against LSC proteins separated by twodimensional electrophoresis. Autoantibodies against triosephosphate isomerase (Tim) and superoxide dismutase [Mn] (MnSOD) were detected in sera from over 20% patients with LSC but none from the normal controls. Furthermore, the occurrence of autoantibodies against Tim and MnSOD was evaluated by ELISA in an additional 40 LSC patients, 30 other types of cancer (OTC) patients, and 50 noncancer controls (NC). Results showed that frequency of autoantibody against Tim (27.5%) in LSC patients was significantly higher than that in OTC patients (6.7%, p ) 0.027) and in NC (6%, p ) 0.005). Likewise, frequency of autoantibody against MnSOD in LSC (20%) patients was significantly higher than that in NC (4%, p ) 0.016), however, there was no significant difference when comparing to that in OTC patients (6.7%, p ) 0.115). We also observed significantly increased expression and secretion of Tim and MnSOD in LSC, which possibly account for their autoantibody development. Our results indicate that autoantibody and antigen of Tim and MnSOD may be useful for screening and diagnosis of the lung squamous carcinoma. Keywords: human lung squamous carcinoma • tumor antigen • autoantibody • serological proteome analysis • Tim • MnSOD

Introduction Lung cancer is a worldwide leading cause of cancer death, with gradually increased incidence and mortality.1,2 Despite significant progress on the molecular pathology of lung cancers has been made recent years,3 diagnostic techniques and therapeutic strategies for lung cancer are little improved.4,5 Early detection of lung cancer remains one of the biggest challenges. To achieve this goal, extensive studies have been performed to identify biomarkers by analyzing gene overexpression in lung cancer6-8 or protein elevation in sera from lung cancer patients, which lead to the identification of several markers, including neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), cytokeratin 19 fragments (CYFRA 21-1), squamous cell carcinoma antigen (SCCA), cancer antigen CA 125, and tissue polypeptide antigen (TPA).9 Nevertheless, few of these markers are acceptable for routine clinical use, because of the conflicting * To whom correspondence should be addressed. Key Laboratory of Cancer Proteomics of Chinese ministry of Health, Xiangya Hospital, Central South University, 87# Xiangya Road, Changsha, 410078, Hunan, China. Tel: (86)731-4327239; Fax: (86)731-4327332; E-mail: [email protected]. † Xiangya Hospital. ‡ Xiangya School of Medicine. # These authors contributed equally to this work. 10.1021/pr0602287 CCC: $37.00

 2007 American Chemical Society

results from different studies or low sensitivity and specificity that is not reliable for clinical diagnosis.10 Therefore, it is essential to identify clinical reliable biomarkers for lung cancer to develop an effective approach for early diagnosis. The humoral immune response to cancer in humans has been well demonstrated by identification of autoantibodies to a number of different intracellular and surface antigens in patients with various types of tumors.11-15 At the early stage of cancer, the amount of tumor antigens in tumor cells or in the circulation is usually very low.16,17 The immune response to such antigens generates a remarkable biological amplification for weak signals of tumor antigens, and autoantibodies can detect the tumor antigens at a low concentration that might not be directly detected due to the subtle alterations in antigen proteins.18 Furthermore, the identified tumor antigens that induce humoral immune response are potential targets for cancer-specific therapy. Therefore, tumor antigens and the elicited autoantibodies are attractive biomarkers for cancer diagnosis and therapy. Anti-tumor antibodies have been discovered in serum by various approaches, such as screening tumor-derived cDNA expression libraries and phage display libraries by immunoassay of serum autoantibodies against a range of tumors.19-21 Journal of Proteome Research 2007, 6, 751-758

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research articles The proteomic approach is emerging as a powerful method to identify the potential cancer markers.22 Compared with the recombinant DNA methods, the proteomic approach has the important advantage of revealing intact proteins with potentially critical post-translational modifications that are recognized by autoantibodies in patient sera. A growing number of tumor autoantibodies discovered with the proteomic approach have been reported.17 In lung cancer, autoantibodies against a four-member group of 25 kDa proteins identified as PGP 9.5 were described, and circulating PGP 9.5 antigen was also detected in sera of lung cancer patients.23 Annexins I and II are additional two tumor antigens associated with lung cancer, which are identified by the proteomic approach. Sera from 60% of patients with lung adenocarcinoma and 33% of patients with lung squamous carcinoma exhibited IgG-based reactivity to annexin I and/or II. Further studies revealed that the immunoreactivity to annexin I in lung cancer patients was N-glycosylation dependent.24 In this study, we aim to identify tumor antigens that elicit humoral response in lung squamous carcinoma (LSC) using a proteomic approach. 2-DE was used to simultaneously separate thousands of individual cellular proteins from tumor tissues. Separated proteins were transferred onto PVDF membranes, and then sera from LSC patients and normal controls were individually screened for antibodies that reacted with separated tumor proteins by Western blot analysis. Proteins that were specifically recognized by sera from cancer patients were identified by mass spectrometry. We reported the identification of autoantibodies against Tim and MnSOD in 25 and 20% LSC patients, respectively, but in none of the normal controls. Moreover, circulating Tim was also detected in patient sera. The tumor antigens identified in this study may be utilized in lung squamous cancer screening and diagnosis. These tumor antigens have also a potential use in lung cancer immunotherapy.

Materials and Methods Tissue and Serum Specimens. Lung squamous carcinoma tissues and serum specimens were obtained from Xiangya Hospital of Central South University and Hunan Provincial Tumor Hospital, Hunan, China. All samples were collected at the time of diagnosis, before any therapy following informed consent, and were prepared as previously described.25 The tissue samples were diagnosed by histopathological analysis. Tumor tissues and autologous sera from 20 patients with LSC as well as sera from 20 healthy subjects were investigated for the autoantibodies against lung squamous carcinoma by proteomic approach. This patient population consisted of 18 males and 2 females with an age range of 30-73 years (median, 56 years). Of the 20 cases, 7 were of clinical stage I, 9 were of clinical stage II, and 4 were of clinical stage III. These samples included 6 well differentiated, 8 moderately differentiated, and 6 poorly differentiated squamous carcinomas. As normal control, 20 serum samples from age- and gender-matched healthy subjects without a prior history of cancer were obtained from a “healthy screening” program at the Xiangya Hospital of Central South University. 2-DE and Western Blot. The tissue specimens were either used immediately for total protein extraction or stored at -80 °C until use. To extract protein, tissues were ground to powder in liquid nitrogen and dissolved in lysis buffer (7 M urea, 2 M thiourea, 2% NP-40, 1% Triton X-100, 100 mM DTT, 4% CHAPS, 2% Pharmalyte, 0.5 mM EDTA, 40 mM Tris, 5 mM PMSF). 752

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Protein concentration was determined with 2D Quantification kit (Amersham Biosciences). Proteins were separated by 2-DE as described previously.25 Briefly, for each sample, three gels were run simultaneously. One gel loaded with 800 µg protein was used for visualization and the protein patterns were visualized by Coomassie blue G-250 staining. The other two gels loaded with 200 µg protein were used for Western blot analysis. The separated proteins were transferred onto a PVDF membrane (Immoblion P, Millipore) and transferring efficiency was checked by staining of the membranes with Ponceau S. For hybridization with serum, the membranes were blocked with 5% nonfat dry milk in TBS containing 0.1% Tween 20 (TBS-T) for 2 h at room temperature and then were washed with TBS-T 3 times for 15 min each time. The membranes were incubated with either patient autologous sera or control sera (1:100 dilution) overnight at room temperature. Following another three washes with TBS-T, the membranes were incubated with horseradish peroxidase-conjugated anti-human IgG antibody (1:2000 dilution, Amersham Biosciences) for 1 h at room temperature and then washed as mentioned above. Immunodetection was accomplished with an enhanced chemiluminescence (ECL) system (Amersham Biosciences) followed by autoradiography on hyperfilm MP (Amersham Biosciences). Protein Identification. The stained 2-DE gels, the Ponceau S-stained PVDF membranes, and the films exposed to the blots were scanned with LabScan software on an ImageScanner (Amersham Biosciences). Spot detection, quantification, and alignment were performed with ImageMaster 2D Elite 4.01 analysis software. The protein pattern on the membrane was the same as that of 2-DE gel visualized by Coomassie blue. The Ponceau S-stained spots on membrane were used as landmarks for the image of the film. By comparing the exposed films with the membranes, we localized the exposed spots on the PVDF membrane and matched spots on the membrane with the 2-DE map of the same sample. The protein spots selectively reacted with sera from the LSC patients were excised from the Coomassie blue-stained gels, destained, and in-gel digested as described previously.26 Briefly, the gel spots were destained with 100 mM NH4HCO3 in 50% acetonitrile, dried in a vacuum centrifuge, and incubated in the digestion solution (40 mM NH4HCO3, 9% acetonitrile, and 20 µg/mL proteomics grade trypsin) at 37 °C for 14-16 h. The resulted peptides were extracted with 50% acetonitrile/2.5% TFA, purified with ZipTip C18 column (Millipore) and mixed with CCA matrix solution followed by analysis with Voyager System DE-STR 4307 MALDITOF Mass Spectrometer (ABI). The standard peptide mixture was spotted at the same time to correct the machine. The parameters of MALDI-TOF were set up as follows: positive ionreflector mode, accelerating voltage 20 kV, grid voltage 64.5%, mirror voltage ratio 1.12, N2 laser wavelength 337 nm, pulse width 3 ns, the number of laser shots 50, acquisition mass range 500-3000 Da, delay 100 ns, and vacuum degree 4 × 10-7 Torr. The protein spots identified by MALDI-TOF MS were also subjected for analysis of ESI-Q-TOF-MS (Micromass). The samples were loaded on to a precolumn (320 µm × 50 mm, 5 µm C18 sillica beads, Waters) at 30 µL/min flow rates for concentrations and fast desalting through a Waters CapLC autosampler, then eluted to the reversed-phase column (75 µm × 150 mm, 5 µm, 100 Å, LC Packing) at a flow rate of 200 nL/min after flow splitting for separation. MS/MS spectra were performed in data-depended mode in which up to 4 precursor ions above an intensity threshold of 7 counts/second (cps) were selected for MS/MS analysis from each survey “scan”. The

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Identification of Tumor Antigens in Human LSC

nanospray parameters were 3000 V for capillary voltage, 45 V for cone voltage, 80 °C for source temperature, and 15 psi collision gas back pressure. Database Analysis. In PMF map database searching, Mascot Distiller was used to get the monoisotopic peak list from the raw mass spectrometry files. All mass spectra were used to search the Swiss-Prot database with the Mascot search engine (www.matrixscience.com). The searching parameter was set up as follows: the taxonomy was selected as homo sapiens; the mass tolerance was 200 ppm; the missed cleavage sites were allowed up to 1; the fixed modifications were selected as carbamidomethyl (cysteine); the variable modification was selected as oxidation (methionine) or none. In tandem mass spectrometry database query, the PKL format files that generated from MS/MS were searched SwissProt database using a Mascot MS/MS ion search. The searching parameter was set up as follows: the taxonomy was selected as homo sapiens; the peptide mass tolerance was 1.0 Da; the MS/MS tolerance was 0.5 Da; the missed cleavage sites were allowed up to 1; the fixed modifications were selected as carbamidomethyl (cysteine); the variable modification was selected as oxidation (methionine) or none; the data format was selected as Micromass PKL format; and the instrument was selected as ESI-Q-TOF. ELISA Analysis. To validate the presence of autoantibodies against Tim and MnSOD in sera from LSC patients, an additional 120 serum samples were applied for ELISA analysis, 40 from LSC patients with an age range of 28-73 years (median, 56 years), 30 from the patients with other types of cancer with an age range of 29-80 years (median, 51 years), including 9 with nasopharyngeal carcinoma, 8 with esophageal carcinoma, 6 with breast cancer, and 7 with gastrointestinal tumor, and 50 from noncancer controls including 40 healthy subjects without a prior history of cancer or autoimmune disease, which were gender- and age-matched with the LSC patients, and 10 other subjects with inflammatory lung disease. Of the 40 LSC patients, 16 were of clinical stage I, 16 were of clinical stage II, and 8 were of clinical stage III. We detected the autoantibodies against tumor antigens in sera using indirect ELISA as described previously.27,28 The antigen proteins used to coat the ELISA microplates (Costar) were GST-fusion proteins of Tim and MnSOD (Abnova). Briefly, each well of the microplate was coated with 100 ng of individual recombinant protein or GST (as a background) in a carbonate buffer (50 mM sodium carbonate, pH 9.6) overnight at 4 °C, followed by blocking with PBS containing 5% nonfat dry milk and 0.1% Tween 20. To adsorb the reactivity of the serum samples to GST, the serum samples were diluted (1:100) and incubated with 30 µg/mL of GST in PBS containing 5% nonfat dry milk and 0.1% Tween 20 for 2 h at room temperature, and then the adsorbed serum samples were added to the coated wells and incubated at 37 °C for 2 h. The microplates were washed and incubated with horseradish peroxidase-conjugated anti-human IgG antibody (1:2000 dilution, Amersham Biosciences) for 1 h at 37 °C. After washing, TMB one solution (Promega) was added to each well. The reaction was stopped by 2 M H2SO4 and optical density was measured at 450 nm. All samples were assayed in triplicate and the results were the mean values of the reading. For each serum sample, OD value of the fusion partner GST was subtracted from OD value of the fusion protein. The cutoff value of reactivity was defined as mean OD of sample plus 3 folds of standard deviations (SD) of normal sera.

Statistical Analysis. A comparison of the frequency of autoantibodies against Tim and MnSOD was performed among LSC patients, OTC patients, and NC. The statistic analysis was done with Statistical Package For Social Science software (SPSS for windows, version 10.01). The chi-square test was performed to determine the differences between groups. p < 0.05 was considered of statistically significance. Detection of Tim and MnSOD Expression in Lung Squamous Cancer. Western blot analysis was performed to detect the expression level of Tim and MnSOD in LSC tissues and paired normal bronchial epithelial tissues adjacent to tumors from additional 20 LSC patients, which consisted of 17 males and 3 females with an age range of 43-71 years (median, 55 years). Tissues were ground to powder in liquid nitrogen and dissolved in lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 1 mM PMSF, 25 µg/mL aprotinin, 25 µg/mL leupeptin). After incubation on ice for 30 min, lysates were centrifuged at 11 000 × g for 15 min at 4 °C to remove cell debris. Then, protein concentration was determined by using Bradford reagent (Bio-Rad). The protein samples were separated by 12% SDS-PAGE and subsequently transferred to PVDF membranes. After blocking with 5% nonfat dry milk in TBS-T for 2 h at room temperature, the membranes were incubated with goat anti-human Tim antibody (1:200, Santa Cruz) or rabbit anti-human MnSOD antibody (1:5000, Stressgen) for 2 h at room temperature, followed by incubation in a 1:2000 dilution of secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. Protein bands were detected using ECL detection system (Amersham Biosciences) followed by autoradiography on hyperfilm MP. The mouse anti-β-actin antibody (1:5000, Sigma) was hybridized simultaneously as a loading control. Analysis of Secretion Character of Tim and MnSOD. To determine whether Tim and MnSOD could be secreted, Western blot analysis was performed to detect the presence of Tim and MnSOD in the culture supernatant of human lung squamous carcinoma cell line HTB-182. Briefly, HTB-182 cells were cultured in RPMI 1640 medium with 10% FCS. When the cells grew to 80% confluent, the medium was removed, and the cells were rinsed five times with hank’s balanced salt solution, followed by incubating in serum-free medium for another 24 h. Then, the culture supernatant was recovered and concentrated using centrifugal filter units (Millipore), and cultured cells were simultaneously harvested by scraping and dissolved in lysis buffer. Both proteins from the whole-cell extracts and the concentrated culture supernatant were separated by 12% SDS-PAGE and transferred to PVDF membrane for Western blot analysis with anti-Tim, anti-MnSOD, or antiβ-actin antibodies. Likewise, Western blot analysis was performed to investigate for the presence of circulating Tim and MnSOD in 30 sera from LSC patients and 30 sera from normal controls. Serum proteins were separated by 12% SDS-PAGE and then transferred onto PVDF membranes, followed by hybridizing with anti-Tim and anti-MnSOD antibodies as described above.

Results Reactivity of Sera from LSC Patients with LSC Proteins. Sera from 20 newly diagnosed patients with LSC and 20 matched healthy subjects were investigated for the presence of autoantibodies against tumor tissue proteins separated by 2-DE. The results showed that 63 protein spots were recognized by patient sera (Figure 1). Forty-nine out of 63 protein spots Journal of Proteome Research • Vol. 6, No. 2, 2007 753

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Figure 1. Screening of autoantibodies against LSC proteins in sera from patients with LSC. (A) Coomassie blue staining of LSC proteins separated by 2-DE. (B) Ponceau S staining of proteins transferred onto PVDF membrane. (C) Representative result of Western blot analysis performed with sera from LSC patients. (D) Representative result of Western blot analysis performed with sera from healthy individuals. Table 1. List of 20 Selected Reactive Protein Spots Identified by MALDI-TOF MS spot

accession number

protein name

frequency (%)

MW (kD)

pI

coverage (%)

1,2,3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

P06733 Q6F137 P40121 P14550 P78417 P30041 P60174 P04179 P07355 P49411 Q06323 P08107 P47756 P06576 P52907 P10809 P18669

alpha enolase (NNE) IDH1 protein macrophage capping protein (CAPG) alcohol dehydrogenase (AKA1) glutathione-s-transferase omega 1(GSTO1) peroxiredoxin 6 (Prx VI) triosephosphate isomerase, splice isoform 1 (Tim) superoxide dismutase [Mn] (MnSOD) annexin A2 (ANXA2) Tu translation elongation factor (EF-Tu) proteasome activator complex subunit 1 (PSME1) heat shock 70kDa protein 1(HSP70-1) F-actin capping protein beta subunit (CapZ beta) mitochondrial ATP synthetase, beta subunit (ATPMB) F-actin capping protein alpha-1 subunit (CapZ alpha-1) heat shock protein 60 (HSP-60) phosphoglycerate mutase 1 (PGAM-B) not determined

60 15 25 25 15 30 25 20 30 20 20 30 30 20 15 15 20

47.35 46.94 38.78 36.76 27.83 25.00 26.52 24.88 38.78 50.19 28.88 70.28 30.82 56.53 33.07 61.19 28.77

6.99 6.53 5.88 6.34 6.23 6.02 6.51 8.35 7.57 7.26 5.78 5.48 5.69 5.26 5.45 5.70 6.75

36 31 38 57 45 35 33 38 59 50 34 47 50 65 64 49 35

were specifically recognized by sera from one or more independent patients, and 20 out of 63 protein spots were recognized by sera from at least 3 patients. On the other hand, 17 out of the 20 lung cancer patient serum samples showed reactivity to at least two of the 20 protein spots, and 15 out of the 20 patient serum samples reacted with at least 3 of the 20 protein spots. 754

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MS Identification of the Proteins Specifically Recognized by LSC Patient Sera. The 20 protein spots recognized by sera of at least 15% LSC patients were excised from the gels, digested with trypsin, and subsequently analyzed by MALDI-TOF MS. Nineteen of these protein spots were successfully identified (Table 1). These proteins consisted of members of proteins with diverse functions, including proteins related to cell structure

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Figure 2. Detection of tumor proteins reacted with LSC patient sera. Twenty reactive protein spots are indicated on the 2-DE gel stained with Coomassie blue (A). Protein spots 9 and 10 are shown by Commassie blue staining (B, top panel) and immunodetected by LSC patient serum (B, 2 middle panels) but not control serum (B, lower panel).

(annexin A2, F-actin capping protein), glycometabolism (Renolase, triosephosphate isomerase), antioxidative stress (peroxiredoxin 6, superoxide dismutase) and chaperones (HSP60 and HSP70). Figure 2A shows the location of the 20 reactive protein spots on 2-DE gel stained by Coomassie blue. We next focused on two proteins, which reacted with sera of more than 20% LSC patients but not with the normal controls. The protein spot 9, with a molecular weight of 26.52 kD and a pI value of 6.51, recognized by the sera of 25% (5/20) patients, was identified as triosephosphate isomerase (Tim). Protein spot 10, with a molecular weight of 24.88 kD and pI value of 8.35, detected in sera of 20% (4/20) patients, was identified as superoxide dismutase [Mn] (MnSOD) (Figure 2B). To confirm the protein identification, proteins were further analyzed by ESI-Q-TOF MS and the results were consistent with that obtained by MALDI-TOF MS analysis (Figure 3). Frequency of Autoantibodies Against Tim and MnSOD in Sera of the LSC Patients. We next used ELISA to determine the frequency of anti-Tim and anti-MnSOD autoantibodies in sera from patients with LSC as well as patients with other type of cancer (OTC) and noncancer controls (NC). As shown in Table 2, the frequency of anti-Tim antibody was about 27.5% (11/40) in LSC patients, 6.7% (2/30) in OTC patients, and 6% (3/50) in NC. The frequency of anti-Tim autoantibody was significantly greater in LSC patients than that in OTC patients (χ2 ) 4.920, p ) 0.027) and NC (χ2 ) 7.820, p ) 0.005). The frequency of anti-MnSOD autoantibody was 20% (8/40) in LSC patients, 6.7% (2/30) in OTC patients, and 4% (2/50) in NC. The frequency of anti-MnSOD antibody in LSC patients was significantly higher than that in NC (χ2 ) 5.760, p ) 0.016), but not statistic difference comparing to that in OTC patients (χ2 ) 2.489, p ) 0.115). Neither anti-Tim nor anti-MnSOD autoantibody had significant difference between OTC patients and NC. Expression of Tim and MnSOD in Lung Squamous Carcinoma. Given the high incidence of recognition by LSC patient serum, we further examined expression of Tim and MnSOD in tumor tissues. Twenty lung squamous cancer tissues and paired normal bronchial epithelial control tissues were analyzed using Western blot analysis. The expression levels of target proteins were normalized with the level of β-actin in the same blot, and each tumor tissue was compared with its paired normal

Figure 3. MALDI-TOF MS and ESI-Q-TOF MS analysis of spot 9. (A) MALDI- TOF MS mass spectrum of spot 9 was shown, which was identified as Tim according to the matched peaks. (B) Peptide with m/z 801.6376 was identified as VVLAYEPVWAIGTGK by ESIQ-TOF MS and matched with residues 160-174 of Tim. Protein sequence of Tim was shown, and matched MS/MS fragmentation was underlined. Table 2. Frequency of Tim and MnSOD Autoantibodies Detected by ELISA

subjectsa

number of subjects

LSC OTC NC

40 30 50

positive autoantibodies in subject sera Tim

MnSOD

11 (27.5%) 2 (6.7%) 3 (6%)

8 (20%) 2 (6.7%) 2 (4%)

a LSC ) lung squamous cancer; OTC) other types of cancer; NC ) noncancer controls.

bronchial epithelial tissue. The ratio of protein expression in tumor tissue vs paired normal control for Tim and MnSOD were 3.02 ( 2.36 and 1.65 ( 0.87 separately. Seventeen out of 20 (85%) tumor samples showed 1.5-12 folds increased in Tim compared to the paired normal bronchial epithelial tissues (Figure 4). In contrast, only 6 out of 20 (30%) tumor samples showed 1.5-4 folds increased in MnSOD compared to their matched controls. Secretion of Tim and MnSOD. To determine whether Tim and MnSOD were secreted, Western blot was performed using Journal of Proteome Research • Vol. 6, No. 2, 2007 755

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Figure 4. Representative result of Western blot analysis showing changes in the expression levels of Tim and MnSOD in lung squamous carcinoma (T) and paired normal bronchial epithelia (N). β-actin was used as a loading control.

Figure 5. Western blot analysis showing the presence of Tim and MnSOD in culture supernatant of HTB-182 cell. Proteins from either whole-cell extracts (WC) or culture supernatant (SP) were immunodetected for expression of Tim, MnSOD, and β-actin.

whole-cell extracts and culture supernatant of human lung squamous carcinoma cell line HTB-182. To exclude the possible proteins released from dead cells, cytoskeleton protein β-actin was used as an internal control. Tim and MnSOD were detected in both whole-cell extracts and culture supernatant of HTB182 cells, whereas β-actin was only detected in the whole-cell extracts (Figure 5). These results suggest that Tim and MnSOD could be secreted by cultured lung squamous carcinoma cell. Next, we sought to investigate the expression of Tim and MnSOD in patient sera. Thirty LSC patient sera and 30 normal control sera were immunoblotted with anti-Tim and antiMnSOD antibodies. Results showed that Tim was detected in sera of 6 patients (6/30, 20%), but no detectable Tim was found in any of the sera of the 30 controls (Figure 6). Consistent with the previous studies that showed that MnSOD was present in serum,29 in this study, we also observed MnSOD in sera from both LSC patients and the controls (data not shown).

Discussion We have implemented a proteomics-based approach to identify proteins that elicit a humoral immune response in LSC patients. This approach combines with western blot to screening the patient sera for autoantibodies that react with tumor cell proteins separated by 2-DE technique. In this study, we identified 20 protein spots that were specifically recognized by sera from 3 or more LSC patients, but not by sera from the matched normal controls. Our study showed that Tim and MnSOD reacted specifically with sera of more than 20% of LSC 756

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Figure 6. A representative result of Western blot analysis showing the presence of Tim in LSC patient sera. Sera from patients with lung squamous cancer (LSC) or from matched normal controls (NC) were immunodetected with anti-Tim antibody.

patients and were secreted into culture medium of a LSC cell line in vitro and circulating in patient sera in vivo. Tim is a ubiquitously expressed enzyme that catalyzes interconversion between dihydroxyacetone phosphate (DHPA) and glycerol dehyde-3-phosphate in the energy metabolism pathway. Tim is ubiquitously distributed in the cytoplasm of all tissues, and tissues with high glycolytic activity generally contain the high level of Tim. In clinic, Tim deficiency causes a rare autosomal recessive inherited disorder characterized by non-spherocytic hemeolytic anaemia, recurrent infections, cardiomyopathy, and severe and fatal neuromuscular dysfunctions.30,31 Consistent with previous report of overexpression of Tim in lung adenocarcinoma and squamous cell carcinoma of bladder,32,33 our results found up-regulation of Tim expression in LSC. Previous studies reported that IgM-type autoantibodies against Tim were present in patients who suffered acute virus infection with either infectious mononucleosis or malaria,34,35 and the generation of anti-Tim autoantibodies might play pathological role in hemolytic anemia of the infected patients.35,36 IgG-type autoantibodies against Tim were also detected in patients with neuropsychiatric lupus and osteoarthiritis.37,38 Our study, for the first time to our knowledge, reported the finding of anti-Tim autoantibody in cancer patients. These findings might help to elucidate tumorigenesis of lung squamous carcinoma. MnSOD is a mitochondrial anti-oxidative enzyme, which dismutates the superoxide free radicals to hydrogen peroxide

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Identification of Tumor Antigens in Human LSC

and molecular oxygen. Numerous studies have shown that MnSOD can protect against pro-oxidant insults resulting from cytokine treatment, ultraviolet light, irradiation, certain tumors, and ischemia/reperfusion.39 The enzyme activity and expression level of MnSOD are often found decreased during development of cancer and implicated as a candidate of tumor suppressor gene.39-42 In contrast, our data presented in this study show that increased expression of MnSOD and present of autoantibodies against MnSOD are detected in 30% and 20% of LSC patients, respectively. These results do not come out complete surprising. Previous study showed that in lung carcinoma tissue, the enzyme activity determined by enzymatic assay was in the same order of magnitude as in uninvolved tissues. However, the enzyme content determined by immunochemical assay was significantly higher in adenocarcinoma than in the uninvolved lung tissue.43 The apparent discrepancy between the amount of immunoreactive MnSOD and the enzymatic activity found in human lung cancer suggested that an immunoreactive but enzymatically inactive MnSOD protein existed. The precise molecular mechanisms involved with this loss in activity are not well understood. Recently studies showed that IgM autoantibodies against MnSOD detected in sera of patient with acute EBV infection were capable of blocking the function of MnSOD.44 To evaluate the specificity of anti-Tim and anti-MnSOD autoantibodies in patients with LSC, we investigated the frequency of autoantibodies against Tim and MnSOD by ELISA in an additional 40 patients with LSC, 30 patients with other types of cancer, and 50 noncancer controls. The frequency of anti-Tim and anti-MnSOD autoantibodies in LSC patients was 27.5 and 20%, respectively, and was significantly higher than that in noncancer controls, which corresponded well with the result of screening by a proteomic approach. As to the tumor specificity of anti-Tim and anti-MnSOD autoantibodies, although one patient with gastrointestinal carcinoma and one patient with esophageal carcinoma occurred anti-Tim autoantibody, the difference in frequency between LSC and OTC patients was of statistical significance; the frequency of antiMnSOD autoantibody in LSC patients was also higher than that in OTC patients, but there was no significant difference between LSC and OTC patients. Neither anti-Tim nor antiMnSOD autoantibodies frequency showed different between OTC patients and noncancer controls. These results indicate that Tim and MnSOD may be tumor antigens, and the immune response to Tim may mainly be in patients with LSC, whereas MnSOD may elicit immune response not only in LSC but also in other types of cancer. It was reported that autoantibodies against MnSOD were developed in acute viral illness, due to cross-reactive epitopes with EBV.45 Therefore, it is important to eliminate the possibility that the MnSOD antibody detected in our LSC patients is just a reflection of cancer-induced reactivation of EBV. In our study, none of the nine patients with nasopharyngeal carcinoma tested in the ELISA experiment was MnSOD autoantibody positive, which suggested that MnSOD antibody detected in our LSC patients may not be related to EBV infection. To further demonstrate this deduction, we collected another group of 20 sera from nasopharyngeal carcinoma patients who are EBVVCA IgA positive to detect MnSOD antibody by ELISA. As a result, only 1(5%) patient was MnSOD antibody positive. Furthermore, antibodies against MnSOD developed in acute EBV infection were IgM subclass, which regularly appear at the beginning of acute EBV infection and disappear with patient’s

recovery,44,46 but the antibodies detected in our study were IgG subclass and could exist in serum for some time. For these reasons, we presume that an increased frequency of MnSOD antibody (IgG) in LSC patients may not be caused by EBV infection. Autoimmune response is found in a wide range of tumors.11,17 However, mechanism for the development of autoantibodies against certain proteins in cancer remains largely unknown. Previous studies suggest that a C to T mutation resulting a Thr to Ile conversion in Tim creates a new epitope to increase immune response.47 This mutation may happen in a fraction for cancer cases. In this study, we had checked Tim cDNA by sequencing in several tumor samples from patients with autoimmunity to Tim, but no mutation was found (data not shown). Although it is not clear why some patients develop immunoreactivity to a particular antigen, most of tumorassociated antigens are not products of mutated genes but are overexpressed proteins or antigens related to cell differentiation.11,48 The immunogenicity depends on not only sufficient amount of antigen proteins, but also effective processing of APC cells, so the release or secretion of normal, mutated, cleaved, or otherwise modified proteins can also contribute to immunogenicity development. Our finding of overexpression and secretion of Tim and MnSOD into patient serum may account for at least one of the mechanism of developing autoantibodies in lung cancer. Proteomics technology is a powerful approach to discover tumor-specific or -associated proteins; moreover, its combination with immunological methods can identify the autoantibodies to tumor proteins and has a great potential impact on cancer biomarker discovery. Applying proteomics-based technologies, we found that Tim and MnSOD are novel potential antigens in human lung squamous carcinoma, and the development of their immunogenicity is possibly due to high expression and secretion into circulation. These results will likely help us to develop biomarkers for lung squamous carcinoma.

Acknowledgment. This work was supported by a grant from National 973 Project of China (2001CB510207) for Outstanding Scholars of New Era from Ministry of Education of China (2002-48), National Natural Science Foundation of China (30000028, 30240056, 30370642), key research program from Science and Technology Committee of Hunan, China (04XK1001-1, 05SK1004-1), and key research program from Public Health Bureau of Hunan Province, China (Z02-04). References (1) Jemal, A.; Tiwari, R. C.; Murray, T.; Ghafoor, A.; Samuels, A.; Ward, E.; Feuer, E. J.; Thun, M. J. CA Cancer J. Clin. 2004, 54, 8-29. (2) Hoffman, P. C.; Mauer, A. M.; Vokes, E. E. Lancet 2000, 355, 479485. (3) Granville, C. A.; Dennis, P. A. Am. J. Respir. Cell Mol. Biol. 2005, 32, 169-176. (4) Gazdar, A. F.; Minna, J. D. J. Natl. Cancer Inst. 1999, 91, 299301. (5) Bunn, P. A., Jr. Clin. Lung Cancer 2004, 6, 85-98. (6) Wang, T.; Hopkins, D.; Schmidt, C.; Silva, S.; Houghton, R.; Takita, H.; Repasky, E.; Reed, S. G. Oncogene 2000, 19, 1519-1528. (7) Wikman, H.; Kettunen, E.; Seppanen, J. K.; Karjalainen, A.; Hollmen, J.; Anttila, S.; Knuutila, S. Oncogene 2002, 21, 58045813. (8) Bangur, C. S.; Switzer, A.; Fan, L.; Marton, M. J.; Meyer, M. R.; Wang, T. Oncogene 2002, 21, 3814-3825. (9) Stieber, P.; Aronsson, A.C.; Bialk, P.; Kulpa, J.; Lamerz, R.; Molina, R.; Van Dalen, H. Anticancer Res. 1999, 19, 2817-2819. (10) Alaiya, A. A.; Franzen, B.; Auer, G.; Linder, S. Electrophoresis 2000, 21, 1210-1217.

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