Tumor antigens eliciting autoantibody response in cancer of gingivo-buccal complex

RESEARCH ARTICLE

Tumor antigens eliciting autoantibody response in

cancer of gingivo-buccal complex

Sanjeev Shukla1, Rukmini B. Govekar1, Ravi Sirdeshmukh2, Curam S. Sundaram2,Anil K. D’Cruz3, K. Alok Pathak3, Shubhada V. Kane3 and Surekha M. Zingde1

1 Advanced Centre for Treatment, Research and Education in Cancer (ACTREC),Tata Memorial Centre, Navi Mumbai, India

2 Centre for Cellular and Molecular Biology, Hyderabad, India3 Tata Memorial Hospital, Tata Memorial Centre, Mumbai, India

Cancer of the gingivo-buccal complex (GBC) is a major cancer in Indian men. This study reportsthe identification of tumor antigens, which elicit an antibody response in cancer of GBC usingimmunoproteomics. Proteins from KB cells separated by 2-D PAGE, were immunoblotted withIgG from sera of 28 cancer patients, 12 patients with leukoplakia, and 28 healthy individuals.Antigens detected by the IgGs from the patient’s sera were different among different individualswith presence of any single antigen ranging from 7 to 79%. Several of these antigens have beenidentified by MS and confirmed by immunostaining. They are three forms of a-enolase, peroxi-redoxin-VI, annexin-II, HSP70, pyruvate kinase, a-tubulin, b-tubulin, ATP-synthase, phos-phoglycerate mutase (PGM), aldose reductase, triosephosphate isomerase, and cyclophilin-A.Except, HSP70, these antigens are being reported in cancer of GBC for the first time. Pyruvatekinase and aldose reductase have not been reported to elicit autoantibody response in any othercancer earlier. Initial results show that autoantibody response against a-enolase, HSP70, annexin-II, peroxiredoxin-VI, and aldose reductase are also seen in patients with leukoplakia of GBC, whichsuggest early occurrence of these autoantibodies during the process of oral carcinogenesis. Theseantigens can be further validated for their use in cancer management by immune intervention.

Received: February 26, 2007Revised: June 2, 2007

Accepted: June 26, 2007

Keywords:

Autoantibody / Cancer of gingivo-buccal complex / Immunoproteomics / Tumor anti-gens

1592 Proteomics Clin. Appl. 2007, 1, 1592–1604

1 Introduction

Oral cancer is the sixth common malignancy and is a majorcause of cancer morbidity and mortality worldwide. Globallyabout 500 000 new oral and pharyngeal cancers are diag-

nosed annually, and three quarters of these are from thedeveloping world [1, 2]. In India, at the Tata Memorial Hos-pital [3], cancers of the head and neck comprise of ,25% ofcancers presenting in males with cancers of the oral cavityconstituting 12% of the total cancer load and 52% of the headand neck cases. Of these, cancers of the gingivo-buccal com-plex (GBC) are ,59% of the oral cavity. Most of the GBCcancers present at stage III and IV.

There is increasing evidence for an immune response tocancer in humans, as demonstrated in part by the identifica-tion of autoantibodies against a number of intracellular andsurface antigens detectable in sera from patients with differ-ent cancer types [4–7]. Early studies involving the immunesystem investigated the circulating immune complexes toidentify antigen–antibody complexes in circulation [8]. This

Correspondence: Dr. Surekha M. Zingde, Advanced Centre forTreatment, Research and Education in Cancer (ACTREC), TataMemorial Centre, Kharghar, Navi, Mumbai 410210, IndiaE-mail: [email protected]: 191-22-27405095

Abbreviations: CyP-A, Cyclophilin A; GBC, gingivo-buccal com-plex; PGM, phosphoglycerate mutase; PMF, peptide mass fingerprint; Prx, peroxiredoxin; TPI, triose phosphate isomerase

DOI 10.1002/prca.200700206

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clinical.proteomics-journal.com

Proteomics Clin. Appl. 2007, 1, 1592–1604 1593

was followed by SEREX analysis wherein a cDNA expressionlibrary from tumor tissue is screened with autologous/het-erologous sera [9, 10]. This approach is limited by the neces-sity to construct expression libraries and the analysis isusually restricted to one or a few patients. The analysis doesnot allow the characterization of antibodies directed againstPTMs. Another approach involves the screening of a randompeptide library with patient’s sera [11].

Several studies have identified autoimmunity againstsingle different proteins such as p53, HSP70, c-erbB-2/HER2/neu, and mucin-related antigens in breast cancer[12–15]. These studies have shown the presence of auto-antibodies in variable amounts ranging from 10 to 20%suggesting that several different factors may contribute tohumoral response in individuals. A related study which hasaddressed the presence of p53 antigen and its antibody indifferent cancers [16] and also in head and neck tumors [16,17] shows, that p53 antibodies are associated with high-grade tumors and poor survival. Several recent investiga-tions have used the 2-D proteomics approaches coupledwith immunostaining with auto/heterologous sera to iden-tify tumor antigens eliciting an immune response. Some ofthe antigens are b-tubulin, SM 22-a/CAI, annexin I and II,PGP9.5, RS/DJ I, MUC I, and CK8 in different cancers [4,18–24].

This study reports the identification of proteins, whichelicit an antibody response in cancers of GBC, using 2-Dproteomics approaches coupled with immunostaining withIgGs from sera of patients with cancer of GBC, sera frompatients with leukoplakia of GBC, sera from healthy individ-uals, and from other cancers. The proteins are a-enolase,annexin II, HSP70, peroxiredoxin VI, ATP synthase, a-tubu-lin, b-tubulin, pyruvate kinase, triose phosphate isomerase(TPI), phosphoglycerate mutase (PGM), aldose reductase,and cyclophilin A (CyP-A), which elicit an autoantibody re-sponse in cancer of GBC. Autoantibody response to some ofthese antigens is also observed in sera of patients withleukoplakia of GBC. Except HSP70, none of these proteinshave been reported earlier to elicit an autoantibody responsein cancers of GBC and may therefore be considered as newmarkers for cancer of GBC. These tumor antigens couldhave clinical utility in improved early detection, and cancermanagement.

2 Materials and methods

2.1 Sera

This study was approved by the ACTREC scientific reviewcommittee and appropriate ethical clearance was obtainedfrom the Hospital Ethics Committee of the Tata MemorialHospital. Blood was collected from 28 patients with cancer ofGBC after obtaining informed consent and the isolatedserum was stored at 2807C till further use. Blood was alsocollected from 28 healthy individuals, ten patients each with

cancers of breast and esophageous, seven patients with lungcancer, and 12 patients with leukoplakia of GBC, sera iso-lated, stored at 2807C and used as control.

2.2 Cells and their maintenance

The human oral cancer cell line, KB was used in this study.KB cells were grown in DMEM (Gibco) supplemented with10% FBS (JRH Biosciences), streptomycin (1 g/L), gentami-cin (80 mg/L), amphotericin B (2.5 mg/L).

2.3 Sample preparation

Cells were harvested and washed with chilled PBS, and celllysates were prepared for 2-D analysis in buffer consistingof 8 M Urea, 2 M Thiourea, 2% CHAPS, 1% DTT, and thesame centrifuged at 55 000 rpm for 1 h at 47C in theBeckman TLD ultracentrifuge. The supernatant wasremoved and stored in aliquots at 2807C. Protein wasestimated in one of the aliquots by the modified Lowrymethod [25].

2.4 Purification of IgG from serum

IgGs were purified from serum with Protein A Sepharosebeads from Amersham Biosciences (17-0780-01) or MelonGel IgG Spin Purification Kit (45206) from Pierce Bio-technology and the protein estimated.

2.5 Electrophoresis and Western blotting

2-D SDS-PAGE of the proteins was done essentially accord-ing to Laemmli [26]. Proteins on the gel were visualized bysilver staining/CBB R-250 (Sigma). For 2-D PAGE, 7 cm(pH 3–10) IPG dry strips (BioRad) were rehydrated withproteins dissolved in 125 mL rehydration buffer (8 M Urea,2 M thiourea, 1% DTT, and 2% CHAPS) and focused in theProtean IEF Cell (BioRad). Separation in the second dimen-sion was carried out in the PROTEAN 3 Dodeca Cell(BioRad) on a 12% SDS polyacrylamide gel. After electro-phoresis, proteins in the gels were transferred to PVDFmembrane or visualized by silver staining. The membraneswere incubated for 1 h in blocking buffer containing 5%milk powder in 20 mM Tris-Cl (pH 7.5), 0.15 M NaCl, 0.1%Tween 20 (TBST). The membranes were incubated overnightat 47C with IgG obtained from serum of patients or healthyindividuals as a source of primary antibody at a concentra-tion of 5 mg IgG/mL TBST. After three washes with washingbuffer (TBS containing 0.05% Tween 20), the membraneswere incubated with HRP-conjugated sheep antihumanIgGs (Amersham Biosciences, NJ) at a dilution of 1:10 000for 1 h at RT. Immunodetection was done using the ECL pluskit from Amersham Biosciences and the signals captured onX-ray film (Kodak). After immunodetection, the membraneswere stripped with destainer (40% methanol and 10% aceticacid) washed with washing buffer and stained with Colloidal


1594 S. Shukla et al. Proteomics Clin. Appl. 2007, 1, 1592–1604

Gold (CG) (BioRad) to obtain the pattern of the separatedproteins. Autograph patterns on the X-ray film were over-layed on the CG-stained blots to identify proteins, whichreact with the IgGs from cancer sera or control IgGs andtheir patterns were then matched to the silver-stained gel runsimultaneously. This was further confirmed with the assis-tance of Microsoft PhotoDraw V2 software. A similar exercisewas done when blots were probed with commercial anti-bodies against the antigens identified.

2.6 MS analysis

Protein spots were identified by MALDI-TOF-TOF analysis.Silver-stained gel plugs were destained with 100 mL ofdestaining solution (30 mM potassium ferricyanide/100 mM sodium thiosulfate mixed 1:1 v/v). After thoroughrinsing with water, the gel was dehydrated in 100% ACNwhich was removed by drying in a speed-vac. The proteinsin the plugs were then trypsinized overnight with 100 ng of10 ng/mL trypsin (Sigma) in 25 mM ammonium bicarbo-nate in water and the peptides were recovered by extractionwith 50% ACN/5% TFA. Tryptic protein digests werereconstituted in 50% ACN with 0.1% TFA solvent beforesubjecting them to mass analysis. The dried digests weredissolved in about 5 mL of the solvent and about 1 mL of thereconstituted digest was premixed with equal volume ofCHCA matrix, vortexed well before spotting on 384-wellMALDI plate. Peptide mass fingerprint (PMF) data wasacquired on 4800 MALDI-TOF-TOF system (ABI, Framing-ham, USA) in reflector mode. Mass calibration was carriedout using peptide mixture of five known peptides spanningmass range of 800–4000 m/z and was set to 10 ppm. Accel-erating voltage of 20 kV was applied to the first TOF tube.The MS data was acquired in an automated manner using asolid state YAG laser at 337 nm. The resulting PMF datawas processed and further analyzed using Global Proteom-ics solutions (GPS) software (ABI). The data was searchedagainst NCBI database with Homosapiens species usingMASCOT search engine with a peptide mass tolerance of100 ppm and S/N threshold of 10 in the mass range of 800–4000 m/z. Only those proteins identified by MASCOTsearch criteria with the top score were considered for fur-ther validation. The protein IDs were examined forsequence coverage, number of peptides matched, agree-ment between theoretical and experimental gel MW and pIvalues and matching of major peaks of PMF with the pep-tides identified in the protein. The identification of proteinwas further confirmed by MS-MS experiment. From the listof peptides identified in PMF, major intense peaks werechosen for fragmentation in the second TOF tube and MS-MS spectra were generated using high laser power. The MS-MS ions of a given peptide were searched against NCBI database using MASCOT search engine for protein ID withprecursor tolerance of 100 ppm and MS-MS fragment tol-erance of 0.2 Da. Protein is considered as identified if theMS-MS ion score of individual peptide was above the

threshold set by the search engine. From the peptides sub-mitted for searches, those with a high MS-MS ion scorewere chosen. In addition, combined analysis using bothPMF and MS-MS data were carried out using the same cri-teria.

2.7 Confirmation of identities of the tumor antigens

with corresponding antibodies

Identities of a-enolase, annexin II, a and b-tubulin, ATPsynthase, peroxiredoxin VI, HSP70, and TPI, obtained byMS-MS analysis were confirmed by immunostaining 2-Dblots of KB cell lysate with corresponding antibodies. Anti-body against TPI (IMG-3793) was from Imgenex. Anti-annexin II antibody (03–4400) was obtained from Zymedlaboratories. Antibodies against HSP70 (ab31010), peroxi-redoxin VI (ab16946), and ATP synthase (ab14730) wereprocured from Abcam. Anti-a enolase antibody (sc-15343)was obtained from SantaCruz Biotech. Antibodies against a-(T6199) and b-tubulin (T5293) were from Sigma. Secondaryantibodies for antimouse IgG HRP (NA 931) and antirabbitIgG HRP (NA934) were obtained from Amersham Bio-sciences. Antigoat IgG HRP (sc-2020) was a product ofSantaCruz Biotech.

2.8 Preparation of GST tagged Æ-enolase and HSP70

pCMV-SPORT6 a-enolase construct was obtained from Dr.Peter J. Hornsby (University of Texas, USA) and pDS-HSP70 construct was obtained from Dr. Marja Jaattela(Danish Cancer Institute, Denmark) through Dr. SantoshKumar (RGCB, India) as a kind gift. a-Enolase and HSP70were subcloned into pGEX4T1 vector between the Bam H1and Sal1 and Eco R1 and Sal1 sites, respectively. Therecombinant plasmids and the empty vector were trans-formed in BL21 (DE3) Escherichia coli. Recombinant GST-a-enolase, GST-HSP70, and GST proteins were then expres-sed following induction with 0.1 mM isopropyl-b-D-thioga-lactoside. These proteins were purified by affinity chroma-tography using glutathione Sepharose 4B (GE Healthcare)and eluted with buffer containing 10 mM reduced glutathi-one and 50 mM Tris (pH 8).

2.9 Detection of autoantibody against Æ-enolase and

HSP70 in patient’s sera

Recombinant GST-a enolase, GST-HSP70, and GST werespotted on PVDF membrane. The ability of the IgG from seraof patients and of healthy individuals to detect the proteinswas assessed by immunoblotting as described in Section 2.5above.



3 Results

3.1 Detection of autoantibodies to protein antigens

in sera from patients with cancer of GBC

The presence of autoantibodies in sera from patients withcancer of GBC was investigated by using KB cell line pro-teins as the antigen source. KB cell line proteins wereseparated by 2-D PAGE and transferred onto PVDF mem-branes. For every experiment, sets of three or more gels to amaximum of nine gels were run simultaneously. One gelwas used for visualization by silver staining, proteins in therest of the gels were transferred to PVDF membrane andimmunostained with IgGs purified from sera of patientswith cancer of GBC, patients with other cancers and ageand sex matched healthy individuals. For ten samples, theexperiment was repeated twice, the autoantibody responsewas reproducible.

Table 1 shows the clinical status of the patients with can-cer of GBC. Figure 1 shows the pattern of separation of theKB cell lysate proteins and the numbers given for the same.

Table 1. Clinical information on patients with cancer of GBC

S. No. Samplenumber

Age Sex TNMstaging

Differentiationstatus

1 82 68 F NA MD2 660 37 M T1N0M0 MD3 493 29 F T2N0M0 MD4 697 56 M T2N1M0 WD5 190 55 F T2N1M0 SqC6 666 38 M T2N2CM0 MD7 825 40 F T2N2M0 MD8 623 51 M T3N0M0 WD9 495 43 M T3N0M0 SSC10 417 35 M T3N0M0 PD11 902 60 F T3/4N0M0 WD12 420 46 M T3/4N0M0 MD13 451 35 M T3/4N1M0 WD14 365 50 F T3/4N2aM0 PD15 883 65 F T4N0M0 MD16 756 60 F T4N0M0 MD17 766 50 F T4N0M0 MD18 136 49 F T4N0 SqC19 358 65 M T4N0M0 WD20 460 52 M T4N0M0 PD21 356 43 M T4N1M0 PD22 792 40 M T4N1M0 WD23 828 60 F T4N1M0 PD24 867 56 M T4N1M0 PD25 423 58 F T4N1M0 MD26 922 55 M T4N1M0 PD27 925 55 F T4N1M0 PD28 670 80 F T4N2M0 MD

NA, not available; SqC, squamous carcinoma; SSC, SarcomatoidSq carcinoma; MD, moderately differentiated; PD, poorly differ-entiated; WD, well differentiated

Figure 2 shows the pattern of the signals obtained on theautographs for two patients with cancer of GBC and twohealthy individuals. Sera IgGs from 28 patients with cancerof GBC, were screened for the presence of autoantibodies toKB cell line proteins. Table 2 shows the autoantibody re-sponse to each of the proteins numbered in the KB cell lysateprotein profile in Figure 1. Most of the sera recognized spots59a, 59b, 59c, 52, 19, and 16. Antigens, which were detectedconsistently in the patient’s sera, were targeted for furtheridentification by MALDI-TOF-TOF MS.

To ensure specificity of autoantibodies, which weredetected in sera of patients with cancer of GBC, 2-D blots ofKB cell lysate proteins were also immunostained with IgGspurified from sex matched healthy individuals in the sameage group.

To determine specificity of autoantibodies detected incancers of GBC, sera from different cancers were also used.2-D blots of KB cell lysate proteins were immunostained withIgGs purified from sera of ten patients each with cancer ofbreast and esophagus and seven patients with lung cancer.Figures 3 and 4 show representative profiles of the autographsignals obtained. Some of the spots, which were detected byIgGs purified from sera of patients with GBC, were alsodetected by IgGs purified from sera of patients with lung,esophageal, and breast cancer, but in lower percentage of thesamples analyzed. Table 2 shows the percentage occurrenceof each of the spots in each of the cancers.

Leukoplakia is a lesion of the oral cavity which is reportedto turn into cancer in 8–10% of cases [27]. To assess whetherany of the identified antigens elicit an antibody response,IgGs from sera of patients with leukoplakia of GBC wereused to screen 2-D blots of KB cell lysate. The antibody re-sponse is given in Table 2 and representative autographs areshown in Fig. 4. These patients had not presented with anyother cancer earlier.

3.2 Identification of tumor antigens

3.2.1 MS

To identify the proteins, which elicited an antibody response,KB cell line protein lysate was resolved by 2-DE and the gelswere stained with silver/coomassie/Sypro ruby, as describedin Section 2. The 12 proteins of interest were excised fromthe gel and analyzed by MALDI-TOF-TOF MS. The resultingspectra were used to identify the proteins, using the MAS-COT search engine. Figure 1 shows the position of the 12protein spots in the 2-D profiles of the KB cell lysate proteins,and Table 2 shows the 12 identified proteins and the per-centage of autoantibody response and Table 3 shows detailsof the MS analysis. The proteins belong to diverse groupsconsisting of metabolic enzymes (a-enolase, ATP synthase,pyruvate kinase, TPI, aldose reductase), chaperones(HSP70), microtubular proteins (a- and b-tubulin), celladhesion proteins (annexin II), antioxidants (peroxiredoxinVI), and signaling proteins (CyP-A).



Figure 1. Silver-stained 2-D profile of KB cell lysate, showing position of identified tumor antigens, which elicit an antibody response. ThepH gradient of first dimension electrophoresis is shown on top of the gel and migration of molecular weight markers on SDS-PAGE insecond dimension is shown on the right side.

Table 2. Autoantibody response in healthy individuals, patients with leukoplakia and cancer of GBC, lung, esophagus and breast

Spot Protein identity % Occurrence

Healthy individuala)

(n = 28)Leukoplakia*(n = 12)

GBC cancera)

(n = 28)Lung cancera)

(n = 7)Esophagus cancera)

(n = 10)Breast cancera)

(n = 10)

59b a-Enolase 11 17 79 43 50 4059c a-Enolase 21 75 75 57 60 5059a a-Enolase 7 25 50 43 10 1052 Peroxiredoxin VI – 8 54 – 10 –16 Annexin II – 25 50 14 20 1019 HSP70 3 17 43 –26 Pyruvate kinase 3 25 32 10 –15 a-Tubulin – 8 29 14 20 –10 b-Tubulin – 17 29 14 1065 ATP synthase – 8 25 – – 1069a PGM – 8 25 – 10 –41 Aldose reductase 3 17 18 – 1069b TPI – 8 14 – –70b CyP-A – – 7 – – –

a) Detailed information about the occurrence of autoantibody response to tumor antigens is given in Supporting Information S1–S6.

3.2.2 Immunostaining

2-D blots of KB cell lysate proteins were immunostained withcorresponding commercial antibodies against a-enolase,

annexin II, a- and b-tubulin, ATP synthase, peroxiredoxinVI, HSP70, and TPI. The signals obtained for each antibodycorresponded to the gel spot, which was subjected to MS,thereby confirming the identity of these proteins (Figure 5).



Figure 2. (A and B) Autographs of Western blots of KB cell lysate separated by 2-DE and probed with serum IgG from two patients withcancer of GBC. Protein spots, which elicit an antibody response in several patients, are indicated by arrows. (C and D) Autographs ofWestern blots of KB cell lysate separated by 2-DE and probed with serum IgG from two healthy individuals. Arrows show absence ofimmunoreactivity against protein spots, which are identified to elicit an antibody response in patients with cancer of GBC.

3.3 Auto antibody response to identified antigens

Among the proteins identified, autoantibodies to three forms(spot 59a, 59b, 59c) of a-enolase were seen in 50, 79, and75%, respectively of patients with cancer of GBC. Other pro-teins identified by the tumor sera were peroxiredoxin VI(54%), annexin II (50%), HSP70 (43%), pyruvate kinase(32%), a-tubulin (29%), b-tubulin (29%), ATP synthase(25%), PGM (25%), aldose reductase (18%), TPI (14%), andCyP-A (7%) (Table 2).

Sera of healthy individuals had autoantibodies against a-enolase, 59a, 59b, and 59c in 7, 11, and 21%, respectively.Autoantibodies against aldose reductase, pyruvate kinaseand HSP70 were seen only in 1 out of 28 healthy indi-viduals.

Sera from patients with leukoplakia had autoantibodiesagainst a-enolase, 59a, 59b, and 59c in 25, 17, and 75%,respectively. Other proteins identified were peroxiredoxin VI(8%), annexin II (25%), HSP70 (17%), pyruvate kinase(25%), a-tubulin (8%), b-tubulin (17%), ATP synthase (8%),PGM (8%), aldose reductase (17%), and TPI (8%) (Table 2).

Autoantibodies to three forms (spot 59a, 59b, 59c) of a-enolase were seen in only 10–60% in sera of patients withcancer of lung, breast, and esophageous. Some of the otherproteins elicited a lower antibody response ranging from 10to 20%.

3.4 Confirmation of autoantibody response

The autoantibody response detected above was confirmed byimmunoblot analysis using GST-a enolase and GST-HSP70.Figure 6 shows the signals obtained with IgGs from sera ofpatients with cancer of GBC and from healthy individuals.Optimal reactivity was obtained with 500 ng of GST-a eno-lase and GST-HSP70. Majority of the sera of patients withcancer of GBC, which detected a-enolase and HSP70 on 2-Dblots of KB cell lysate proteins also detected recombinant a-enolase and HSP70 on the dot blot (Fig. 6). Majority of IgGsfrom sera of healthy individuals did not react or showed verylow signal with these recombinant proteins except for CS-4,which showed strong signals for both proteins and CS-27



Figure 3. (A–D) Autographs of Western blots of KB cell lysate separated by 2-DE and probed with serum IgGs from two patients with eso-phageal cancer (A and B) and two patients with lung cancer (C and D).

which showed a strong signal for a-enolase only. GST alonedid not show any reactivity with IgGs from sera of patients aswell as healthy individuals (data not shown).

4 Discussion

Immunoproteomics has emerged as an important approachto identify tumor-associated proteins which elicit an antibodyresponse. Several approaches have been used by differentinvestigators. In one approach, resolved proteins from thetumor tissue are screened by Western blotting to identify thepresence of autoantibodies. Our initial attempts using thisapproach did not clearly identify antigens, as they are prob-ably present in variable and low amounts in the hetero-genous tumor tissue. Another approach to identify antigenswhich elicit an antibody response has been to enrich theantigens by immunoprecipitation with sera IgGs followed byWestern blotting. This approach has problems with the pre-cipitating IgGs interfering with the detection of the antigensprecipitated. In the third approach, which has been used in

this study, proteins from a cancer cell line provide the con-centrated source of human epithelial cell proteins, to identifyantigens, which elicit an antibody response. To ensuredetection of specific antibody reactivity, purified IgG at 5 mg/mL has been used which corresponds to 1:2000 dilution ofserum considering average IgG concentration of 10 mg/mL inserum.

Using 2-DE, immunoblot analysis, and MS, a set of pro-teins that elicit humoral immune response in patients withcancer of GBC have been identified (Table 2). The identifiedantigens are a-enolase, peroxiredoxin VI, annexin II, HSP70,pyruvate kinase, a- and b-tubulin, ATP synthase, PGM,aldose reductase, TPI, and CyP-A.

Analysis of Table 2 shows that in general, most of the seraIgG from patients with cancer of GBC recognized multiplespots on the 2-D pattern of the KB cell lysate. Antigensdetected by the sera from patients with cancer of GBC weredifferent among different individuals with presence of anysingle antigen ranging from 7 to 79% (detailed analysis isgiven in the Table in Supporting Information). Some of the12 tumor antigens identified in this study have been reported



Figure 4. (A–D) Autographs of Western blots of KB cell lysate separated by 2-DE and probed with serum IgG from two patients with breastcancer (A and B) and two patients with leukoplakia (C and D).

to elicit an antibody response in various conditions. Of these,a-enolase, annexin II, a-tubulin, peroxiredoxin VI, pyruvatekinase, TPI, and aldose reductase have not been reported toelicit an antibody response in cancer of GBC, while pyruvatekinase and aldose reductase have not been reported to elicitantibody response in any cancer.

a-Enolase has been characterized as a highly conservedcytoplasmic glycolytic enzyme that catalyzes the formation ofphosphoenolpyruvate from 2-phosphoglycerate, the second ofthe two high-energy intermediates that generate ATP in glyco-lysis [28]. a-Enolase has been considered to play an importantrole in tumorogenesis. Tumor cells have higher metabolic rateand a-enolase, is an important protein in cell metabolism.There is evidence to suggest that, a-enolase translocates to thecell membrane and in this position it acts as a receptor forplasminogen [29]. It may therefore be involved in cell invasionand metastasis. Autoantibodies to a-enolase have been re-ported earlier in lung cancer [30–33], and in CML [34].

Tubulins are integral component of microtubules andmostly occur as heterodimer of a- and b-tubulin. Auto-antibodies for b-tubulin have been reported earlier in headand neck cancer [23], CML [34], neuroblastoma [18], and innasopharyngeal carcinoma [35].

Heat shock proteins function as molecular chaperones,help in transport, assembly, and degradation of intracellularpolypeptides. Heat shock protein synthesis is acceleratedunder the influence of nonphysiological conditions or anystress to aid cell survival. Thus, over-production of heat shockproteins protects malignantly transformed cells from apop-totic cell death and provides resistance to chemotherapeuticagents and irradiation [36]. Autoantibodies to HSP70 havealso been reported in lesions of the oral cavity [37, 38], headand neck cancer [23], leukemia [34], and in prostate cancer[39].

Annexin II has been implicated in cell–cell adhesion andin plasminogen activation and may function as a cell surfacereceptor [40]. Annexin II tetramers have been shown tointeract with procathepsin B on the surface of tumor cellsand may be involved in extracellular proteolysis, facilitatingtumor invasion, and metastasis. Autoantibodies to annexinII have been reported in lung cancer [20].

Peroxiredoxins (Prx) are antioxidant enzymes that havean important role in cell differentiation, proliferation, andapoptosis [41]. Peroxiredoxin VI plays a role in protectingcancer cells from oxidative stress thereby increasing cell sur-vival. Prx are located in various subcellular locations includ-



Table 3. Mass spectroscopic analysis of identified antigens

MS analysis MS/MS analysis

Spot ID Accessionnumber

TheoreticalMr/pI

ObservedMr/pI

Score Sequencecoverage

Peptidematched

Peptides Score

59a a-Enolase P06733 47.3/6.99 50/6.8 198 54 17/33

59b a-Enolase P06733 47.3/6.99 50/7.1 175 58 19/50 YNQLLRIEEELGSKAAVPSGASTGIYEALELRLAMQEFMILPVGAANFR

233

59c a-Enolase P06733 47.3/6.99 50/7.4 274 65 21/35 NFRNPLAKIGAEVYHNLKGNPTVEVDLFTSKYISPDQLADLYKLAQANGWGVMVSHRVVIGMDVAASEFFRVNQIGSVTESLQACKYNQLLRIEEELGSKAAVPSGASTGIYEALELRLAMQEFMILPVGAANFRFTASAGIQVVGDDLTVTNPKEIFDSRGNPTVEVDLFTSKAGYTDKVVIGMDVAASEFFRFTASAGIQVVGDDLTVTNPKRSGETEDTFIADLVVGLCTGQIK

602

52 Peroxiredoxin VI P30041 25/6 25/7 150 56 11/32 DFTPVCTTELGRPGGLLLGDVAPNFEANTTVGRPGGLLLGDVAPNFEANTTVGRIR

305

19 HSP70 P08107 70.2/7 70/6.5 257 48 20/50 ARFEELCSDLFRINEPTAAAIAYGLDRQTQIFTTYSDNQPGVLIQVYEGER

320

16 Annexin II P07355 38.6/7.5 37/8 247 65 22/50 GVDEVTIVNILTNRSNAQRQDIAFAYQRSLYYYIQQDTKGDYQK

249

69a Phosphoglycratemutase 1

P18669 28.7/6.75 26/7.3 263 65 24/50 ALPFWNEEIVPQIKDAGYEFDICFTSVQKRSYDVPPPPMEPDHPFYSNISK

211

69b TPI P60174 26.8/6.51 25/7.3 249 77 18/47 FFVGGNWKVVLAYEPVWAIGTGKVPADTEVVCAPPTAYIDFAR

325

10 b-Tubulin P07437 50/4.78 50/5.3 290 56 29/37 LHFFMPGFAPLTSRISVYYNEATGGKYVPRSGPFGQIFRPDNFVFGQSGAGNNWAK

182

15 a-Tubulin P68363 50.8/4.94 50/5.5 281 59 21/35 YMACCLLYRLDHKFDLMYAKQLFHPEQLITGKAVFVDLEPTVIDEVRNLDIERPTYTNLNRIHFPLATYAPVISAEKVGINYQPPTVVPGGDLAKAVCMLSNTTAIAEAWARTIGGGDDSFNTFFSETGAGKFDGALNVDLTEFQTNLVPYPRTIGGGDDSFNTFFSETGAGKHVPR

716

65 ATP synthase P06576 56.5/5.26 55/5.5 119 21 8/8 AHGGYSVFAGVGER 130

26a Pyruvate kinasem1/m2

P14618 58.3/7.95 55/8 182 49 23/50 FGVEQDVDMVFASFIREAEAAIYHLQLFEELRR

161



Table 3. Continued

MS analysis MS/MS analysis

Spot ID Accessionnumber

TheoreticalMr/pI

ObservedMr/pI

Score Sequencecoverage

Peptidematched

Peptides Score

26b Pyruvate kinase m1/m2 P14618 58.3/7.95 55/8.2 345 58 25/35 GDYPLEAVRLDIDSPPITARNTGIICTIGPASRIISKIENHEGVRIYVDDGLISLQVKGADFLVTEVENGGSLGSKRFDEILEASDGIMVARGDLGIEIPAEKVFLAQKLNFSHGTHEYHAETIK

312

41 Aldose reductase P15121 36/6.56 35/7 159 47 15/35 LIQYCQSKMPILGLGTWKLWCTYHEKYKPAVNQIECHPYLTQEK

110

70b Cyclophilin A P62937 18.1/7.82 14/8 109 75 10/30 IIPGFMCQGGDFTRSIYGEKFEDENFILKVNPTVEFDIAVDGEPLGR

273

Figure 5. Western blots showing reactivity of a-enolase, a- and b-tubulin, HSP70, annexin II, TPI, peroxiredoxin VI, and ATP synthase withcorresponding commercial antibodies. The position of the corresponding protein spot is shown for each on the close up of the silver-stained 2-D gel image of KB cell proteins.



Figure 6. Western blot showing reactivity of (A) individual sera IgGs from 20 patients with cancer of GBC with GST-a enolase (B) individualsera IgGs from 20 healthy individuals with GST-a enolase (C) individual sera IgGs from 20 patients with cancer of GBC with GST-HSP70 (D)individual sera IgGs from 20 healthy individuals with GST-HSP70. CG-stained image of dot blot is given below each Western blot imageand 6 signs below each Western blot indicate whether the signal was seen (1) or not seen (2) on the 2-D blots of KB cell lysate afterimmunostaining with corresponding sera IgGs.

ing peroxisomes and mitochondria where oxidative stress ismost evident. Overexpression of Prx have been reported inbladder cancer [41] and breast cancer [42]. Autoantibodies toPrx VI have been reported in adenocarcinoma of lung [32].

ATP synthase is a remarkably conserved enzyme. ATPsynthase is located in the mitochondrial membrane and cat-alyses the synthesis of ATP. Autoantibodies to ATP synthasehave been reported earlier in hepatocellular carcinoma [24]and head and neck cancer [23].

Cyclophilin A (CyP-A) is a member of the immunophilinfamily of proteins, typically studied for their binding of var-ious immunosuppressive drugs, most notably cyclosporin A,and their role in cellular signaling pathways [43]. CyP-A hasalso been shown to possess peptidyl prolyl cis–trans isomer-ase activity and thus may have a role in protein folding [43].Cyp-A has been identified as overexpressed protein in lungcancer [43] and also in cancer of GBC [44]. Although CyP-Ahas numerous known activities, its role in cellular growthand differentiation, transcriptional control, cell signaling,

and immunosuppression suggests that it could be involvedin an important aspect of oncogenesis [43]. Autoantibodies toCyp-A have been reported in head and neck cancer [23].

Pyruvate kinase is a key glycolytic enzyme, which isresponsible for ATP production. Proliferating cells andtumor cells in particular express the pyruvate kinase iso-enzyme type M2 (M2-PK) [45]. There are no other reports ofan autoantibody response in cancer.

TPI is a glycolytic enzyme, which catalyzes conversion ofdihydroxyacetone phosphate to glyceraldehyde-3-phosphate.Autoantibodies to TPI have been reported in adenocarci-noma of lung [32].

PGM is an enzyme of the glycolytic and gluconeogenicpathways that catalyzes the interconversion of 3-phospho-D-glycerate (3-PGA) and 2-phospho-D-glycerate (2-PGA) in theEmbden–Meyerhoff pathway. Autoantibodies to PGM havebeen reported in lung carcinoma [33].

Aldose reductase is NADPH dependent oxidoreductasewhich detoxifies a variety of reductive aldehydes species and



metabolizes certain steroids and neurotransmitter metabo-lites and glucouronate [46]. Autoantibodies to aldose reduc-tase have not been reported earlier in any cancer.

In a very recent study, Li et al. [47] have shown presenceof autoantibodies to a-enolase (72%), PGM (5%), TPI (5%),annexin II (16%), and several other proteins in healthy Chi-nese individuals from Beijing. In their study, 2-D blots ofproteins from three different cell types were probed with1:300 diluted sera, this corresponds to 33 mg IgG/mL, con-sidering average IgG concentration of 10 mg/mL in serum. Inthe present investigation, more stringent conditions wereused. The blots were probed with IgG (5 mg/mL TBST) puri-fied from the sera. This corresponds to a 1:2000 dilution ofsera. The autoantibody response seen for patients with can-cer and leukoplakia are therefore specific. Under these con-ditions, the autoantibody response to a-enolase is highest at21% for spot 59c and 7 and 11% for spots 59a and b, respec-tively in healthy individuals.

Crosstabulation analysis of occurrence of autoantibodyresponse to particular antigen vis-a-vis differentiation status ofthe tumor has been done using the SPSS software. Resultsshow that autoantibody response against a-enolase (59c and59a), peroxiredoxin VI, and a-tubulin correlates with differ-entiation status (PD.MD.WD) (Table 4). Most of thepatients were T3 or T4 with no distal metastasis and no corre-lation was evident vis-a-vis the nodal status (data not shown).

The immunoproteomics approach in this study has yiel-ded a battery of autoantibodies, which characterize humoralimmune response in cancers of GBC. It is becoming appar-ent that immune system responds to self-antigens, which areincreased in expression, mutated, modified, or present in analtered cellular location [7]. In a parallel inquiry in our labo-ratory, we have seen increased expression of a-enolase(manuscript submitted for consideration) in cancers of GBC,which is probably one of the reasons for the antibody re-sponse observed. Increased expression of a-enolase, pyruvatekinase, HSP70, TPI, annexin II, and CyP-A has also beenreported in cancer of buccal mucosa by Chen et al. [44].Overexpression of HSP70 and PGM, has been reported inoral cancer [48, 49]. ATP synthase is reported to be over-expressed in tongue squamous cell carcinoma [50] whichcould be a reason for the antibody response. The exactmechanism of humoral immune response against tumorantigens is, however, not yet clear.

As evident from the discussion above, several of theidentified tumor antigens are reported to elicit humoralimmune response in other cancers, and have also beenshown in this study. Autoantibodies against a-enolase,HSP70, pyruvate kinase, a- and b-tubulin, ATP synthase, andaldose reductase were found in patients with esophageal,lung, and breast cancer, which indicates that some of theautoantibodies might be universal markers of cancer. Thisneeds to be confirmed with increased sample size.

The observation that there are autoantibodies against a-enolase, annexin II, HSP70, pyruvate kinase, and aldosereductase in sera of patients with leukoplakia of GBC, indi-

Table 4. Crosstabulation of the autoantibody response with dif-ferentiation status

Spot Protein PD (%)(n = 9)

MD (%)(n = 11)

WD (%)(n = 5)

59b a-Enolase 7(77) 11(100) 3(60)59c a-Enolase 8(88) 9(81) 3(60)19 HSP70 3(33) 6(54) 1(20)59a a-Enolase 6(66) 6(54) 1(20)16 Annexin II 4(44) 7(63) 2(40)52 Peroxiredoxin VI 7(77) 6(54) 2(40)26 Pyruvate kinase 3(33) 4(36) 1(20)15 a-Tubulin 4(44) 3(27) 1(20)10 b-Tubulin 3(33) 3(27) 2(40)65 ATP synthase 2(22) 2(18) 2(40)70b Cyclophilin A 0 2(18) 041 Aldose reductase 2(22) 3(27) 069a Phosphoglycerate

Mutase1(11) 5(45) 1(20)

69b TPI 1(11) 3(27) 0

The number of patients in each grade is indicated (n). PD, poorlydifferentiated; MD, moderately differentiated; WD, well differ-entiated. (% in parenthesis).

cates that they could be used, preferably in combination, asearly markers for onset of cancer. This is supported by theobservation that beside a-enolase (spot 59c), the other anti-gens do not elicit as high (21%) antibody response in healthyindividuals.

A multiplex array of these protein antigens or the corre-sponding antibodies could be used for early detection andmanagement of cancer of GBC to overcome problems aris-ing from variable expression of any particular antigen attri-butable to tumor heterogeneity. Studies have been initiatedtowards this end.

This work was supported in part by research grants fromCouncil of Scientific and Industrial Research. S. S. was supportedwith the Fellowship from ACTREC. We thank Atul Pranay for hishelp in making recombinant GST proteins and immunostainingof dot blots. Sera from patients with lung, esophageous, and breastcancer were obtained with support from Dr. R. A. Mistry and Dr.R. A. Badwe of Tata Memorial Hospital. Mass spectrometricanalysis was carried out at Centre for Cellular and Molecular Bi-ology (Hyderabad, India). We thank Dr. Bharat Joshi (FDA,USA) for his generous gift of antibodies against annexin II, per-oxiredoxin VI, ATP synthase, and triose phosphate isomerase.

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Tumor antigens eliciting autoantibody response in cancer of gingivo-buccal complex