Peptide Mimic Isolated by Autoantibody Reveals Human Arrest Defective 1 Overexpression Is Associated with Poor Prognosis for Colon Cancer Patients
2010; Elsevier BV; Volume: 177; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2010.091178
ISSN1525-2191
AutoresBeihai Jiang, Tingting Ren, Bin Dong, Like Qu, Genglin Jin, Jianning Li, Hong Qu, Lin Meng, Caiyun Liu, Jian Wu, Chengchao Shou,
Tópico(s)Gastric Cancer Management and Outcomes
ResumoTumor-associated antigens, which induce the generation of autoantibodies, are useful as cancer biomarkers in early detection and prognostic prediction of cancer. To isolate a novel cancer marker, we used serum antibodies from colon cancer patients to screen a phage display peptide library. A positive peptide 249C (VPLYSNTLRYGF) that could specifically react with serum from colon cancer patients was isolated, and the corresponding antigen–human arrest defective 1 (ARD1A), which shares an identical LYSNTL motif with 249C, was identified. Both immunological assays and three-dimensional structure analysis showed that the LYSNTL region is an epitope of ARD1A. Using ELISA and immunohistochemistry, we found anti-ARD1A antibody levels in serum from patients with colon cancer were significantly higher than those in healthy volunteers (P < 0.001), and ARD1A expression was detected in 84.1% (227/270) of colon cancer tissues compared with 22.7% (55/242) of matched noncancerous tissues (P < 0.001) and 4.8% (2/42) of benign lesions (P < 0.001). Furthermore, multivariate analysis with Cox proportional hazards regression models revealed that ARD1A-positive patients had significantly shortened overall survival (OS) (HR, 1.91, P = 0.039) and borderline significantly shortened disease-free survival (DFS) (HR, 1.70; P = 0.068). Kaplan–Meier survival curves also showed that ARD1A expression was associated significantly with shortened DFS (P = 0.037) and OS (P = 0.019). These results indicate that ARD1A is a novel tumor-associated antigen and a potential prognostic factor for colon cancer. Tumor-associated antigens, which induce the generation of autoantibodies, are useful as cancer biomarkers in early detection and prognostic prediction of cancer. To isolate a novel cancer marker, we used serum antibodies from colon cancer patients to screen a phage display peptide library. A positive peptide 249C (VPLYSNTLRYGF) that could specifically react with serum from colon cancer patients was isolated, and the corresponding antigen–human arrest defective 1 (ARD1A), which shares an identical LYSNTL motif with 249C, was identified. Both immunological assays and three-dimensional structure analysis showed that the LYSNTL region is an epitope of ARD1A. Using ELISA and immunohistochemistry, we found anti-ARD1A antibody levels in serum from patients with colon cancer were significantly higher than those in healthy volunteers (P < 0.001), and ARD1A expression was detected in 84.1% (227/270) of colon cancer tissues compared with 22.7% (55/242) of matched noncancerous tissues (P < 0.001) and 4.8% (2/42) of benign lesions (P < 0.001). Furthermore, multivariate analysis with Cox proportional hazards regression models revealed that ARD1A-positive patients had significantly shortened overall survival (OS) (HR, 1.91, P = 0.039) and borderline significantly shortened disease-free survival (DFS) (HR, 1.70; P = 0.068). Kaplan–Meier survival curves also showed that ARD1A expression was associated significantly with shortened DFS (P = 0.037) and OS (P = 0.019). These results indicate that ARD1A is a novel tumor-associated antigen and a potential prognostic factor for colon cancer. Colon cancer is one of the most common cancers worldwide and is the second leading cause of cancer death in developed countries.1Levin B Lieberman DA McFarland B Andrews KS Brooks D Bond J Dash C Giardiello FM Glick S Johnson D Johnson CD Levin TR Pickhardt PJ Rex DK Smith RA Thorson A Winawer SJ Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps, 2008: a joint guideline from the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology.Gastroenterology. 2008; 134: 1570-1595Abstract Full Text Full Text PDF PubMed Scopus (1701) Google Scholar The 5-year survival rate of patients with colon cancer has improved during the past decade because of advances in screening and systemic treatment, but the survival rate remains relatively poor in patients with stage III and IV cancer.2Wolpin BM Mayer RJ Systemic treatment of colorectal cancer.Gastroenterology. 2008; 134: 1296-1310Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar Colon cancer is treated mainly by surgery, and it would be beneficial if the prognosis after initial surgery could be predicted. 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Considerable evidence has shown that an immune response in the form of autoantibodies to various tumor antigens was developed in patients with cancer.3Wang X Yu J Sreekumar A Varambally S Shen R Giacherio D Mehra R Montie JE Pienta KJ Sanda MG Kantoff PW Rubin MA Wei JT Ghosh D Chinnaiyan AM Autoantibody signatures in prostate cancer.N Engl J Med. 2005; 353: 1224-1235Crossref PubMed Scopus (528) Google Scholar, 4Mintz PJ Kim J Do KA Wang X Zinner RG Cristofanilli M Arap MA Hong WK Troncoso P Logothetis CJ Pasqualini R Arap W Fingerprinting the circulating repertoire of antibodies from cancer patients.Nature Biotechnol. 2003; 21: 57-63Crossref Scopus (290) Google Scholar, 5Soussi T p53 Antibodies in the sera of patients with various types of cancer: a review.Cancer Res. 2000; 60: 1777-1788PubMed Google Scholar, 6Disis ML Calenoff E McLaughlin G Murphy AE Chen W Groner B Jeschke M Lydon N McGlynn E Livingston RB Moe R Cheever MA Existent T-cell and antibody immunity to HER-2/neu protein in patients with breast cancer.Cancer Res. 1994; 54: 16-20PubMed Google Scholar, 7Disis ML Pupa SM Gralow JR Dittadi R Menard S Cheever MA High-titer HER-2/neu protein-specific antibody can be detected in patients with early-stage breast cancer.J Clin Oncol. 1997; 15: 3363-3367Crossref PubMed Scopus (302) Google Scholar Thus, autoantibodies in the serum of patients with colon cancer could be used to isolate specific tumor-associated antigens. More sensitive and specific molecular markers have the potential to improve the preclinical diagnosis of primary and recurrent colon cancer, as well as hold the promise of prognostic value. Phage display libraries have proved to be a useful tool for identifying autoantigens or epitopes recognized by antibodies.8Chen YT Scanlan MJ Sahin U Türeci O Gure AO Tsang S Williamson B Stockert E Pfreundschuh M Old LJ A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening.Proc Natl Acad Sci USA. 1997; 94: 1914-1918Crossref PubMed Scopus (1093) Google Scholar, 9Naora H Montz FJ Chai CY Roden RB Aberrant expression of homeobox gene HOXA7 is associated with müllerian-like differentiation of epithelial ovarian tumors and the generation of a specific autologous antibody response.Proc Natl Acad Sci USA. 2001; 98: 15209-15214Crossref PubMed Scopus (76) Google Scholar, 10Boon T van der Bruggen P Human tumor antigens recognized by T lymphocytes.J Exp Med. 1996; 183: 725-729Crossref PubMed Scopus (782) Google Scholar, 11Stone B Schummer M Paley PJ Thompson L Stewart J Ford M Crawford M Urban N O'Briant K Nelson BH Serologic analysis of ovarian tumor antigens reveals a bias toward antigens encoded on 17q.Int J Cancer. 2003; 104: 73-84Crossref PubMed Scopus (59) Google Scholar, 12Cekaite L Haug O Myklebost O Aldrin M Østenstad B Holden M Frigessi A Hovig E Sioud M Analysis of the humoral immune response to immunoselected phage-displayed peptides by a microarray-based method.Proteomics. 2004; 4: 2572-2582Crossref PubMed Scopus (25) Google Scholar Selection of peptide libraries with mAbs and mixed serum from patients with disease has led to the isolation of immunoreactive peptide epitopes.4Mintz PJ Kim J Do KA Wang X Zinner RG Cristofanilli M Arap MA Hong WK Troncoso P Logothetis CJ Pasqualini R Arap W Fingerprinting the circulating repertoire of antibodies from cancer patients.Nature Biotechnol. 2003; 21: 57-63Crossref Scopus (290) Google Scholar, 12Cekaite L Haug O Myklebost O Aldrin M Østenstad B Holden M Frigessi A Hovig E Sioud M Analysis of the humoral immune response to immunoselected phage-displayed peptides by a microarray-based method.Proteomics. 2004; 4: 2572-2582Crossref PubMed Scopus (25) Google Scholar, 13Luo W Chen Y Wang M Chen Y Zheng Z Song H Chen H Guan Y Ng MH Zhang J Xia N Peptide mimics of a conserved H5N1 avian influenza virus neutralization site.Biochem J. 2009; 419: 133-139Crossref PubMed Scopus (6) Google Scholar, 14Jiang B Liu W Qu H Meng L Song S Ouyang T Shou C A novel peptide isolated from a phage display peptide library with trastuzumab can mimic antigen epitope of HER-2.J Biol Chem. 2005; 280: 4656-4662Crossref PubMed Scopus (55) Google Scholar The strategy has also led to the identification of some tumor-associated antigens from phage display libraries using patient serum.4Mintz PJ Kim J Do KA Wang X Zinner RG Cristofanilli M Arap MA Hong WK Troncoso P Logothetis CJ Pasqualini R Arap W Fingerprinting the circulating repertoire of antibodies from cancer patients.Nature Biotechnol. 2003; 21: 57-63Crossref Scopus (290) Google Scholar, 12Cekaite L Haug O Myklebost O Aldrin M Østenstad B Holden M Frigessi A Hovig E Sioud M Analysis of the humoral immune response to immunoselected phage-displayed peptides by a microarray-based method.Proteomics. 2004; 4: 2572-2582Crossref PubMed Scopus (25) Google Scholar In the present study, by screening a phage display peptide library with serum antibodies from patients with colon cancer, together with bioinformatic analysis and a series of immune experiments, we first revealed that human arrest defective 1 (ARD1A) may serve as an indicator of unfavorable prognosis in colon cancer. The LoVo colon adenocarcinoma cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). The LoVo cells were grown in DMEM (Gibco) supplement with 10% fetal bovine serum (FBS, PAA Laboratories, Pasching, Austria) and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin) at 37°C in a humidified incubator with 5% CO2. Sera from five patients with colon cancer before any therapeutic intervention were obtained from Tissue Bank of Peking University School of Oncology. The clinicopathological features and TNM staging are summarized in Supplemental Table S1 at http://ajp.amjpathol.org. Control serum samples were obtained from five healthy age- and sex-matched blood-donor volunteers. Informed consent was obtained from each person, and the study was approved by the Hospital Research Ethics Committee. The biopanning process consisted of four rounds to select the phages binding to IgGs in serum from the five patients with colon cancer. To enhance the selection of peptides binding to specifically IgGs associated with colon cancer, during each round, 1.5 × 1011 plaque-forming units (pfu) of the Ph.D.−12 phage display peptide library (New England Biolabs, Beverly, MA) were preincubated with IgGs purified from 50 μl of normal serum pooled from five healthy volunteers and immobilized on Protein G Sepharose 4 Fast Flow (GE Health care, Uppsala, Sweden) to remove nonspecific binding clones. After the preclearing step, the phage library was selected onto the pool of IgGs purified from patient serum. Phage clones bound to the patient-derived IgGs on beads were amplified by direct infection with host Escherichia coli strain ER2738 grown to the log phase. Amplified phages were used in the next cycle. After four rounds of selection, 1000 plaques were picked up randomly and analyzed for reactivity with serum from colon cancer patients using phage ELISA. Amplified phages from a single clone were added to the wells of a microtiter plate precoated with a 1:200 dilution of serum from colon cancer patients or healthy volunteers in 0.1 M of bicarbonate buffer (pH 9.5) and then blocked with 5% skim milk in PBS. The plate was incubated at room temperature for 1 hour and washed five times with 0.05% Tween-20/PBS. Bound phages were detected by incubation with HRP-conjugated anti-M13 antibody (Amersham Biosciences, Piscataway, NJ) for 1 hour, followed by washing and addition of a peroxidase substrate (o-phenylenediamine, 0.4 mg/ml) in citrate-phosphate buffer (pH 5.0) containing 0.02% (v/v) H2O2. The reaction was stopped with 50 μl of 12.5% H2SO4. Absorbance at 490 nm was determined using a microplate reader (Bio-Rad model 550, Bio-Rad, Hercules, CA). Single-stranded phage DNA was prepared from 10 immunopositive clones using standard techniques as described in the phage display peptide library kit and sequenced by Sangon Company (Shanghai, China). To make the glutathione S-transferase −249C (GST-249C) fusion protein, the sense (5′-GATCCGTTCCTCTGTATAGTAATACGCTTCGTTATGGGTTTTGATATCC-3′) and the antisense (5′-TCGAGGATATCAAAACCCATAACGAAGCGTATTACTATACAGAGGAACG-3′) oligonucleotide fragments encoding the positive clone 249C, with stop codon, EcoRV site, and sticky ends of BamHI/XhoI shown in italics, were annealed and inserted into the pGEX-4T-1 vector (Amersham Biosciences). To prepare His-ARD1A protein, ARD1A cDNA fragment containing the full-length coding region was amplified using the polymerase chain reaction (PCR) method with plasmid pGEX-5X-3-ARD1A15Ren T Jiang B Jin G Li J Dong B Zhang J Meng L Wu J Shou C Generation of novel monoclonal antibodies and their application for detecting ARD1 expression in colorectal cancer.Cancer Lett. 2008; 264: 83-92Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar as a template and two primers (5′-ATTGGATCCATGAACATCCGCAATGCGAG-3′ and 5′- AATGTCGACTAGGAGGCTGAGTCGGAG-3′). The PCR products were purified and inserted into a prokaryotic expression system pET-28a (+) (Novagen, Gibbstown, NJ). DNA of recombinants was verified by digestion with restriction endonucleases and then confirmed by DNA sequence analysis. For the preparation of GST-249C and His-ARD1A proteins, transformed bacteria were cultured in 200 ml of LB medium with ampicillin and kanamycin, respectively, to an optical density of 0.8 at 600 nm. Next, 0.5 mmol/L isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added, and the cultures were further incubated for 5 hours at 30°C. Cells were collected by centrifugation and resuspended in 20 ml PBS with 1 mmol/L phenylmethanesulfonyl fluoride and 1 mg/ml lysozyme. Cell suspensions were sonicated, followed by centrifugation at 12,000 rpm for 10 minutes at 4°C. The GST-249C fusion protein was purified with glutathione beads according to the manufacturer's instructions (Amersham Biosciences), and the His-ARD1A protein was purified with Ni-NTA Agarose (Qiagen, Valencia, CA). To detect whether peptide 249C is an epitope of ARD1A, GST-249C and His-ARD1A proteins were prepared. Six- to eight-week-old female BALB/c mice (obtained from the Animal Center of the Chinese Medical Academy) were immunized subcutaneously with 40 μg of GST-249C or GST emulsified in complete Freund's adjuvant (Sigma, St. Louis, MO). Mice received three boosters of the antigen every three weeks. Serum was collected on days 0 (preimmune) and 7 after the fourth immunization. The reactivity of antipeptide 249C serum with ARD1A was tested by Western blotting or immunoprecipitation (IP) analysis using His-ARD1A or lysates from LoVo cells expressing endogenous ARD1A.15Ren T Jiang B Jin G Li J Dong B Zhang J Meng L Wu J Shou C Generation of novel monoclonal antibodies and their application for detecting ARD1 expression in colorectal cancer.Cancer Lett. 2008; 264: 83-92Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar For Western blotting, 100 ng of His-ARD1A protein or 100 μg cell lysates (prepared in lysis buffer containing 50 mmol/L Tris-Cl [pH 7.4], 50 mmol/L NaCl, 1% Triton X-100, 1 mmol/L EDTA, 1 mmol/L phenylmethanesulfonyl fluoride, 1 μg/ml aprotinin, and 1 μg/ml leupeptin) were subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk in PBS and probed with either a 1:50 dilution of serum from mice immunized with GST-249C or with monoclonal antibody (14D4) against ARD1A15Ren T Jiang B Jin G Li J Dong B Zhang J Meng L Wu J Shou C Generation of novel monoclonal antibodies and their application for detecting ARD1 expression in colorectal cancer.Cancer Lett. 2008; 264: 83-92Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar for 1 hour at room temperature. Preimmunized mouse serum (1:50 dilution), and serum from mice immunized with GST (1:50 dilution) were used as negative controls. After washing with PBST, the antigen-antibody complex on the membrane was detected by incubating with HRP-conjugated secondary antibody and then developed with an enhanced chemiluminescence system (Amersham Biosciences). For IP, 200 μg lysates from LoVo cells were incubated with anti-ARD1A mAb, 10C12,15Ren T Jiang B Jin G Li J Dong B Zhang J Meng L Wu J Shou C Generation of novel monoclonal antibodies and their application for detecting ARD1 expression in colorectal cancer.Cancer Lett. 2008; 264: 83-92Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar or 10 μl of serum from mice immunized with GST-249C for 2 hours at 4°C followed by the mixing with protein G beads. Preimmunized mouse serum and serum from mice immunized with GST were used as negative controls. Immunocomplexes were eluted in the sample buffer, boiled, and subjected to Western blotting. The sequences of the peptides were compared with protein sequence databases by basic local alignment search tool (BLAST) homology search. To analyze the location of the sequence shared by 249C and ARD1A, the amino acid residue sequence of ARD1A was compared with the primary sequences in the Protein Data Bank through WU-Blast2 in http://www.ebi.ac.uk. The best match, 2OB0, a complex of human Mak3 homolog with acetyl-coenzyme A, was used as a structural model. The three-dimensional structure of ARD1A was modeled and refined by the Homology program and Discover program in Insight II (2000) software package (Accelrys Inc., San Diego, CA). All specimens were obtained from Tissue Bank of Peking University School of Oncology. The samples from patients who had undergone preoperative chemotherapy or radiotherapy were excluded from the present study. Informed consent was obtained from each patient, and the study was approved by the Hospital Research Ethics Committee. Serum samples from 198 patients (range, 19–80 years) with colon cancer before surgery and 200 healthy blood donors (range, 24–76 years) were collected between 2003 and 2008. Clinicopathological features and TNM staging of the patients were summarized in Supplemental Table S2 at http://ajp.amjpathol.org. Complete follow-up data were available for 149 patients for a median of 34 months (range, 1–60 months). Specimens from 270 patients with colon cancer were collected during primary tumor surgical resection over the period 1996–2002. The median age at diagnosis was 63 years (range, 23–85 years). Formalin-fixed paraffin-embedded specimens from these patients and from 242 matched adjacent noncancerous tissues were collected for immunohistochemical analysis. Primary histological examination was performed by two senior pathologists to confirm the diagnosis of colon cancer, establish the pathological stage, and select areas that contained at least 75% tumor cells. The clinicopathological characteristics of patients are detailed in Supplemental Table S3 at http://ajp.amjpathol.org. Follow-up data were available for all patients, with a median follow-up of 48 months. In addition, 27 colitis and 15 colonic adenoma specimens were examined. Another 40 cancer tissue specimens, as well as the matched noncancerous colon tissue both adjacent to (at a distance of 2 cm) and distant from (at a distance of 5 cm or surgical margin) were obtained from patients immediately after surgery during the period 2007–2008 and stored at −80°C until processed for Western blotting. Serum from each patient was also collected for detecting the antibody against ARD1A. The clinical features of the 40 patients in the study are shown in Supplemental Table S4 at http://ajp.amjpathol.org. The reactivity of human serum antibody to ARD1A protein was tested by ELISA. The microplates were coated with 5 μg/ml of His-ARD1A in 0.1 M of bicarbonate buffer (pH 9.5) then blocked with 5% skim milk in PBS. A 1:200 dilution of patient serum was added to each well. Bound antibodies were detected using HRP-conjugated anti-human IgG antibody (Zhong Shan Co., Beijing, China) with a peroxidase substrate, as described earlier. The cutoff used was the mean absorbance of the healthy volunteers plus 2 SDs. To detect ARD1A expression in cancerous, adjacent noncancerous, and distant noncancerous colon tissues from the same patient, specimens of approximately the same size (∼100 mm3) were washed several times with PBS then crushed in 200 μl of lysis buffer. Protein concentrations were measured using the Protein BCA Assay (Pierce Rockford, IL). Forty micrograms of total protein were subjected to Western blotting. Nitrocellulose membranes were incubated with ARD1A antibody 14D4. Band intensity was quantified using National Institutes of Health Scion Image software and normalized to GAPDH. The specificity of the mouse mAb 14D4 against ARD1A was characterized in our previous study.15Ren T Jiang B Jin G Li J Dong B Zhang J Meng L Wu J Shou C Generation of novel monoclonal antibodies and their application for detecting ARD1 expression in colorectal cancer.Cancer Lett. 2008; 264: 83-92Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar For immunohistochemical staining, all specimens were fixed in freshly prepared 10% neutral buffered formalin, embedded in paraffin, and cut into 5-μm sections. After baking at 60°C overnight, sections were dewaxed and rehydrated. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 10 minutes at room temperature. After blocking with 5% skim milk, sections were incubated with the 14D4 antibody at 4°C overnight followed by incubation with second antibody from the EnVisionTM kit (Dako Cytomation, Carpinteria, CA) for 30 minutes at room temperature. The reaction product was visualized with diaminobenzidine (Sigma) for 5 minutes at room temperature. Sections were counterstained with hematoxylin. Purified IgG from normal mouse serum was used as a negative control. ARD1A immunoreactivity was evaluated independently by two experienced pathologists without any knowledge of the clinical data. Tissue samples were estimated in a consecutive analysis to ensure maximal internal consistency. We assessed both the percentage of positive cells and the intensity of cytoplasmic staining in 10 randomly chosen microscopic fields. Assays were scored as negative if <10% of tumor cells were stained and positive if ≥10% of tumor cells were stained. Staining intensity was classified as follows: 0, no staining or staining in 50% of tumor cells.16Xing X Peng L Qu L Ren T Dong B Su X Shou C Prognostic value of PRL-3 overexpression in early stages of colonic cancer.Histopathology. 2009; 54: 309-318Crossref PubMed Scopus (35) Google Scholar The Fisher's exact test was performed to evaluate the possible differences in serum anti-ARD1A antibody levels between patients with colon cancer and healthy volunteers. In immunohistochemical analysis, colon cancer specimens were categorized as either ARD1A-positive or ARD1A-negative. The association between ARD1A expression and clinicopathological variables was analyzed using the Fisher's exact test. The impact of ARD1A expression on DFS and OS was assessed with the hazard ratio (HR; relative risk of relapse or death in the ARD1A-positive group) calculated by Cox multivariate proportional hazards regression model. The multivariate model was adjusted for ARD1A expression in tumors, age, sex, clinical stage, histological type. Survival curves were estimated using the Kaplan–Meier method and compared with the log-rank test. Statistical analyses were performed using SPSS software 13.0 (SPSS, Inc., Chicago, IL). All P values are two-sided, and P < 0.05 was considered statistically significant. We used a random 12-mer phage display peptide library composed of 1.5 × 1011 independent phage clones to select peptides specifically binding to IgGs associated with colon cancer. The total number of phages bound to serum from patients with colon cancer increased from 1.8 × 106 pfu in the first round to 4.0 × 107 pfu in the fourth round. After four rounds of selection, roughly 1% (10/1000) of phage clones exhibited binding activity to serum from patients with colon cancer (data not shown). DNA from 10 positive clones that specifically reacted with serum from patients was sequenced, and their amino acid sequences were shown in Table 1. Among 10 phage clones examined, five types of peptides were obtained. 164B and 249C had a TLRYG motif, whereas no obvious consensus motif was found in other clones.Table 1Peptide Sequences Displayed by Phage Binding to Purified IgGs of Patients with Colon CancerPeptidesSequence249CVPLYSNTLRYGF*249C and 164B had a ‘TLRYG’ motif shown in bold type.164BSYLTTLRYGNMS238BTQQSVFSTTLMY93AIPLPPPSRPFFK211BQPVDMPYFRTHP* 249C and 164B had a ‘TLRYG’ motif shown in bold type. Open table in a new tab The peptide 249C showed significant homology to the residues 121–126 (LYSNTL) of ARD1A by BLAST homology search (Figure 1A). The epitope recognized by the anti-249C antibody in serum of colon cancer patients appeared to be a linear fragment of the protein ARD1A. To confirm that peptide 249C is an epitope of ARD1A, the anti-249C antibody was obtained by immunization of BALB/c mice with GST-249C fusion protein and used in Western blotting. As shown in Figure 1B lane 4, the antibody against peptide 249C specifically reacted with recombinant His-ARD1A protein from bacteria and endogenous ARD1A from colon cancer LoVo cells. Furthermore, the molecular mimicry between the selected peptide 249C and ARD1A was shown by reciprocal co-IP assays (Figure 1C). These data clearly demonstrated that anti-249C and anti-ARD1A antibodies recognized the same antigen, ARD1A. By sequence alignment, 23% sequence homology was found between ARD1A and human Mak3 homolog in complex with acetyl-coenzyme A (PDB ID: 2OB0). Using 2OB0 as the model, a three-dimensional structure of ARD1A was predicted. As shown in Figure 1D, the LYSNTL region was on the surface of ARD1A and might contribute to an important external epitope of ARD1A. Although peptides 164B, 283B, 93A, and 211B, which specifically reacted with serum from patients with colon cancer, were also obtained, we did not identify the corresponding target antigens for these peptide mimics (data not shown). We speculated that the peptide 249C was a distinct epitope on ARD1A. Thus, we determined the presence of antibodies to recombinant ARD1A in serum from 198 colon cancer patients and 200 healthy volunteers with ELISA. When the cutoff was set at OD = 0.415, serum from patients with colon cancer was more likely to have detectable antibodies specific for ARD1A than samples from healthy volunteers, indicating that humoral immunity against ARD1A increased significantly (P < 0.001) from 2.0% (4/200) in healthy volunteers to 14.7% (29/198) in colon cancer patients (Figure 2A). Although we found no association between anti-ARD1A antibody immunity and clinicopathological variables (see Supplemental Table S2 at http://ajp.amjpathol.org), the observations suggested that humoral immunity to ARD1A readily distinguished patients with colon cancer from control subjects. Because the presence of circulating autoantibodies against ARD1A was associated with colon cancer, we further investigated the expression of ARD1A in matched cancerous, adjacent noncancerous, and distant noncancerous colon tissues from 40 patients by Western blotting. Compared with adjacent and distant noncancerous tissue, ARD1A expression was up-regulated in 28 (70.0%) and down-regulated in 12 (30.0%) of 40 patients in the cancer tissues by Western blotting (Figure 2B, see Supplemental Figure S1, and Supplemental Table S4 at http://ajp.amjpathol.org). These data suggested that ARD1A expression was higher in cancerous tissues than in noncancerous tissues. However, more colon samples are required to further confirm this results. Next, we sought to examine the expression level of ARD1A in large cohort of tissue sample by immunohistochemical analysis. The frequency of ARD1A expression is shown in Supplemental Table S5 at http://ajp.amjpathol.org. When the frequency of expression was stratified into two groups—negative (0) and positive (1+, 2+, and 3+)—84.1% (227/270) of cancer tissues stained for ARD1A in ≥10% of tumor cells. As shown in Figure 2C, ARD1A staining exhibited strong peri-nuclear pattern. In contrast, ARD1A expression was detected in 22.7% (55/242) of matched adjacent noncancerous tissues. Only 4.8% (2/42) of benign lesions showed ARD1A expression, indicating that ARD1A expression was significantly more frequent in colon cancer tissues than in matched adjacent noncancerous tissues (P < 0.001) and benign lesions (P < 0.001) (see Supplemental Table S5 at http://ajp.amjpathol.org). We further investigated the clinical relevance of differential ARD1A expression in tumor tissues. Clinicopathological variables of ARD1A-positive and ARD1A-negative patients were summarized in Supplemental Table
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