Identification of Tumor-associated Autoantigens for the Diagnosis of Colorectal Cancer in Serum Using High Density Protein Microarrays
2009; Elsevier BV; Volume: 8; Issue: 10 Linguagem: Inglês
10.1074/mcp.m800596-mcp200
ISSN1535-9484
AutoresIngrid Babel, Rodrigo Barderas, Ramón Díaz‐Uriarte, Jorge L. Martı́nez-Torrecuadrada, Marta Sänchez‐Carbayo, J. Ignacio Casal,
Tópico(s)vaccines and immunoinformatics approaches
ResumoThere is a mounting evidence of the existence of autoantibodies associated to cancer progression. Antibodies are the target of choice for serum screening because of their stability and suitability for sensitive immunoassays. By using commercial protein microarrays containing 8000 human proteins, we examined 20 sera from colorectal cancer (CRC) patients and healthy subjects to identify autoantibody patterns and associated antigens. Forty-three proteins were differentially recognized by tumoral and reference sera (p value <0.04) in the protein microarrays. Five immunoreactive antigens, PIM1, MAPKAPK3, STK4, SRC, and FGFR4, showed the highest prevalence in cancer samples, whereas ACVR2B was more abundant in normal sera. Three of them, PIM1, MAPKAPK3, and ACVR2B, were used for further validation. A significant increase in the expression level of these antigens on CRC cell lines and colonic mucosa was confirmed by immunoblotting and immunohistochemistry on tissue microarrays. A diagnostic ELISA based on the combination of MAPKAPK3 and ACVR2B proteins yielded specificity and sensitivity values of 73.9 and 83.3% (area under the curve, 0.85), respectively, for CRC discrimination after using an independent sample set containing 94 sera representative of different stages of progression and control subjects. In summary, these studies confirmed the presence of specific autoantibodies for CRC and revealed new individual markers of disease (PIM1, MAPKAPK3, and ACVR2B) with the potential to diagnose CRC with higher specificity and sensitivity than previously reported serum biomarkers. There is a mounting evidence of the existence of autoantibodies associated to cancer progression. Antibodies are the target of choice for serum screening because of their stability and suitability for sensitive immunoassays. By using commercial protein microarrays containing 8000 human proteins, we examined 20 sera from colorectal cancer (CRC) patients and healthy subjects to identify autoantibody patterns and associated antigens. Forty-three proteins were differentially recognized by tumoral and reference sera (p value <0.04) in the protein microarrays. Five immunoreactive antigens, PIM1, MAPKAPK3, STK4, SRC, and FGFR4, showed the highest prevalence in cancer samples, whereas ACVR2B was more abundant in normal sera. Three of them, PIM1, MAPKAPK3, and ACVR2B, were used for further validation. A significant increase in the expression level of these antigens on CRC cell lines and colonic mucosa was confirmed by immunoblotting and immunohistochemistry on tissue microarrays. A diagnostic ELISA based on the combination of MAPKAPK3 and ACVR2B proteins yielded specificity and sensitivity values of 73.9 and 83.3% (area under the curve, 0.85), respectively, for CRC discrimination after using an independent sample set containing 94 sera representative of different stages of progression and control subjects. In summary, these studies confirmed the presence of specific autoantibodies for CRC and revealed new individual markers of disease (PIM1, MAPKAPK3, and ACVR2B) with the potential to diagnose CRC with higher specificity and sensitivity than previously reported serum biomarkers. Colorectal cancer (CRC) 1The abbreviations used are:CRCcolorectal cancerCEAcarcinoembryonic antigenTAAtumor-associated autoantigenTMAtissue microarrayCIconfidence intervalROCreceiver operating characteristicAUCarea under the curveMAPKAPKmitogen-activated protein kinase-activated protein kinaseERKextracellular signal-regulated kinase.1The abbreviations used are:CRCcolorectal cancerCEAcarcinoembryonic antigenTAAtumor-associated autoantigenTMAtissue microarrayCIconfidence intervalROCreceiver operating characteristicAUCarea under the curveMAPKAPKmitogen-activated protein kinase-activated protein kinaseERKextracellular signal-regulated kinase. is the second most prevalent cancer in the western world. The development of this disease takes decades and involves multiple genetic events. CRC remains a major cause of mortality in developed countries because most of the patients are diagnosed at advanced stages because of the reluctance to use highly invasive diagnostic tools like colonoscopy. Actually only a few proteins have been described as biomarkers in CRC (carcinoembryonic antigen (CEA), CA19.9, and CA125 (1Crawford N.P. Colliver D.W. Galandiuk S. Tumor markers and colorectal cancer: utility in management.J. Surg. Oncol. 2003; 84: 239-248Crossref PubMed Scopus (54) Google Scholar, 2Sidransky D. Emerging molecular markers of cancer.Nat. Rev. Cancer. 2002; 2: 210-219Crossref PubMed Scopus (560) Google Scholar, 3Duffy M.J. van Dalen A. Haglund C. Hansson L. Holinski-Feder E. Klapdor R. Lamerz R. Peltomaki P. Sturgeon C. Topolcan O. Tumour markers in colorectal cancer: European Group on Tumour Markers (EGTM) guidelines for clinical use.Eur. J. Cancer. 2007; 43: 1348-1360Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar)), although none of them is recommended for clinical screening (4Locker G.Y. Hamilton S. Harris J. Jessup J.M. Kemeny N. Macdonald J.S. Somerfield M.R. Hayes D.F. Bast Jr., R.C. 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Moreover, cDNA libraries might not be representative of the protein expression levels in tumors as there is a poor correspondence between mRNA and protein levels. Protein arrays provide a novel platform for the identification of both autoantibodies and their respective TAAs for diagnostic purposes in cancer serum patients. They present some advantages. Proteins printed on the microarray are known "a priori," avoiding the need for later identifications and the discovery of mimotopes. There is no bias in protein selection as the proteins are printed at a similar concentration. This should result in a higher sensitivity for biomarker identification (13Hudson M.E. Pozdnyakova I. Haines K. Mor G. Snyder M. Identification of differentially expressed proteins in ovarian cancer using high-density protein microarrays.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 17494-17499Crossref PubMed Scopus (230) Google Scholar, 35Ramachandran N. Raphael J.V. Hainsworth E. Demirkan G. Fuentes M.G. Rolfs A. Hu Y. LaBaer J. Next-generation high-density self-assembling functional protein arrays.Nat. Methods. 2008; 5: 535-538Crossref PubMed Scopus (264) Google Scholar, 36Robinson W.H. DiGennaro C. Hueber W. Haab B.B. Kamachi M. Dean E.J. Fournel S. Fong D. Genovese M.C. de Vegvar H.E. Skriner K. Hirschberg D.L. Morris R.I. Muller S. Pruijn G.J. van Venrooij W.J. Smolen J.S. Brown P.O. Steinman L. Utz P.J. Autoantigen microarrays for multiplex characterization of autoantibody responses.Nat. Med. 2002; 8: 295-301Crossref PubMed Scopus (641) Google Scholar). In this study, we used commercially available high density protein microarrays for the identification of autoantibody signatures and tumor-associated antigens in colorectal cancer. We screened 20 CRC patient and control sera with protein microarrays containing 8000 human proteins to identify the CRC-associated autoantibody repertoire and the corresponding TAAs. Autoantibody profiles that discriminated the different types of CRC metastasis were identified. Moreover a set of TAAs showing increased or decreased expression in cancer cell lines and paired tumoral tissues was found. Finally an ELISA was set up to test the ability of the most immunoreactive proteins to detect colorectal adenocarcinoma. On the basis of the antibody response, combinations of three antigens, PIM1, MAPKAPK3, and ACVR2B, showed a great potential for diagnosis. For microarray screening, serum samples from 12 individuals were collected after CRC diagnosis (Hospital Universitario de Salamanca). Those samples were selected for having CRC in advanced stages as well as for developing metastasis to liver (seven patients), liver and lung (four patients), or liver and bone marrow (one patient). The median age for the CRC patients was 64.5 years (range, 41–84 years). Eight control serum samples were obtained from healthy subjects and were selected to match both the median age of the CRC population and the same proportion of male and female subjects. Clinical data from the patients are provided in Table I. For ELISA validations, another set of 52 serum samples from CRC patients representative of the different Dukes stages (A–D) and 42 control serum samples from healthy subjects was used for the validation screening (supplemental Table S1).Table IClinical information of the CRC patients tested in the high density human protein microarraysSerumAgeaIn years.GenderbM, male; F, female.OutcomecOutcome of the CRC patients after collecting the serum.Survival timedSurvival time in months after collecting the serum samples. —, survival time was longer than five years.MetastasisVH184FAlive—LiverMH160FDead15LiverMHP165MDead64Liver-lungMHP241MDead62Liver-lungMH255MDead14LiverMHP362MDead51Liver-lungVP171FAlive—Lung-boneVH275MAlive—LiverMH376MDead31LiverMH464MDead28LiverVHP151MAlive—Liver-lungVH374MAlive—Livera In years.b M, male; F, female.c Outcome of the CRC patients after collecting the serum.d Survival time in months after collecting the serum samples. —, survival time was longer than five years. Open table in a new tab All sera were processed using identical procedures. Blood samples were left at room temperature for a minimum of 30 min (and a maximum of 60 min) to allow clot formation and then centrifuged at 3000 × g for 10 min at 4 °C. The serum was frozen and stored at −80 °C until use. Twenty serum samples (12 from the CRC tumor group and eight from the control group; Table I) were probed in the Human ProtoArrayTM v4.0 (Invitrogen). These microarrays contained 8000 human GST-tagged proteins expressed in Sf9 insect cells and spotted in duplicate. ProtoArrays were used according to the recommendations of the manufacturer. Briefly the slides were equilibrated at 4 °C for 15 min and then incubated with blocking buffer (1% BSA in 0.1% Tween 20, PBS) for 1 h at 4 °C with gentle agitation. Then 150 µl of human serum (diluted 1:50 in blocking buffer) was overlaid on the arrays, covered with cover glass (Corning), and incubated for 90 min at 4 °C. The slides were washed three times for 10 min with probe buffer (1% BSA, 0.5 mm DTT, 5% glycerol, 0.05% Triton X-100 in PBS). Human bound antibodies were detected after incubation with Alexa Fluor 647-labeled goat anti-human IgG (Invitrogen; diluted 1:2000 in probe buffer) for 90 min at 4 °C. The arrays were washed and dried by centrifugation at 1000 rpm for 1 min at room temperature. As a first control, ProtoArrays v4.0 were probed with goat anti-GST antibody to check the uniformity of the proteins spotted in the array followed by incubation with Alexa Fluor 555-labeled anti-goat IgG. The other control array was only incubated with the secondary antibody Alexa Fluor 647-labeled anti-human IgG for background determination. Finally the slides were scanned on a ScanArrayTM 5000 (Packard BioChip Technologies) to produce red (Alexa Fluor 647) or green images (Alexa Fluor 555). The Genepix Pro 5.1 (Axon Laboratories) image analysis software was used for the quantification. A cDNA encoding the full-length human PIM1 was introduced into the pET28a expression vector (Novagen). The His6-PIM1 fusion protein was then expressed in Escherichia coli strain BL21(DE3) (Invitrogen) and purified by affinity chromatography on a HiTrap chelating column (GE Healthcare) followed by gel filtration on a Superdex 200 column (GE Healthcare). Human MAPKAPK3 protein was purchased from GenWay (San Diego, CA). Human ACVR2B cDNA was cloned into the pDEST527 (a gift from Dr. J. L. Hartley, National Institutes of Health), expressed, and purified as mentioned above. Human Annexin IV cDNA was cloned into pTT3 expression vector (kindly provided by Dr. Y. Durochet, Biotechnology Research Institute, Montreal, Canada) and expressed in HEK293-EBNA cells. The recombinant protein was produced by the transiently transfected cells and purified by affinity chromatography on a nickel-chelating resin (GE Healthcare). CEA and human seroalbumin were purchased from Sigma. Antibodies against MAPKAPK3 and PIM1 used in ELISA were purchased from Abnova. Antibodies against MAPKAPK3, PIM1, and ACVR2B used for immunoblotting and tissue microarray were purchased from Abcam. CRC cell lines (RKO, HCT116, HCT15, SW48, SW480, and Colo205) and control BxPc3 (pancreatic adenocarcinoma) and Molt4 (lymphoblastoid) cells were grown according to established protocols. Neutrophiles (Neut) and lymphocytes (lymph) were isolated from peripheral blood cells from a healthy individual. Murine embryo fibroblasts were immortalized by infecting a primary culture with the Epstein-Barr virus and grown according to established protocols. Protein extracts from paired tissues from CRC patients were prepared as described previously (6Alfonso P. Núñez A. Madoz-Gurpide J. Lombardia L. Sánchez L. Casal J.I. Proteomic expression analysis of colorectal cancer by two-dimensional differential gel electrophoresis.Proteomics. 2005; 5: 2602-2611Crossref PubMed Scopus (175) Google Scholar). Briefly protein extracts were obtained after lysis with 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate in 150 mm NaCl, 5 mm EDTA, 10 mm Tris-HCl (pH 7.2) containing protease inhibitor mixture (Roche Applied Science). After clarifying by centrifugation at 12,000 × g for 15 min, protein concentrations were determined with the 2-D Quant kit (GE Healthcare). For Western blot, 50 µg of protein extracts were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Hybond-C Extra) according to standard procedures (37Barderas R. Desmet J. Timmerman P. Meloen R. Casal J.I. Affinity maturation of antibodies assisted by in silico modeling.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 9029-9034Crossref PubMed Scopus (111) Google Scholar). After blocking, membranes were incubated overnight at 4 °C with PIM1 (dilution, 1:100), MAPKAPK3 (dilution, 1: 500), and ACVR2B (dilution, 1:200) antibodies. Immunodetection on the membranes was achieved by using either peroxidase-labeled, anti-goat IgG (Dako Cytomation) at a dilution of 1:5000 for ACVR2B and 1:20,000 for PIM1 or peroxidase-labeled anti-chicken IgY (Jackson ImmunoResearch Laboratories) at 1:20,000 for MAPKAPK3. The signal was developed by ECL (GE Healthcare). Tissue microarrays (TMAs) specific for colorectal cancer with 45 different paired samples (tumoral and normal) were prepared as described previously (7Madoz-Gúrpide J. Cañamero M. Sanchez L. Solano J. Alfonso P. Casal J.I. A proteomics analysis of cell signaling alterations in colorectal cancer.Mol. Cell. Proteomics. 2007; 6: 2150-2164Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Sections were cut at a thickness of 3 µm and dried for 16 h at 56 °C before being dewaxed in xylene and rehydrated through graded ethanol series to water. A heat-induced epitope retrieval step was performed in 0.01 m trisodium citrate solution with heating for 2 min in a conventional pressure cooker. After heating, slides were rinsed in cool running water for 5 min and quickly washed in TBS (pH 7.4). TMAs were incubated with mouse monoclonal anti-PIM1 (dilution, 1:50) and goat polyclonal anti-ACVR2B (dilution, 1:10). Specific binding was followed by anti-IgG conjugated with biotin. Visualization of specific interaction was monitored by using the EnVision FLEX system (Dako Cytomation) or Bond (Vision BioSystems). Diaminobenzidine (DAB+) was used as substrate chromogen after the sections were counterstained with hematoxylin. A positive control was included within each staining experiment to ensure consistency between consecutive runs. The evaluation of the TMA was performed by two independent pathologists according to the following scale: 0, no staining; 1, weak staining; 2, normal staining; and 3, strong staining of the tissue cylinder. An ELISA was developed to test the ability of target proteins to screen for CRC status in sera. Briefly, microtiter plates (Maxisorp, Nunc) were coated overnight with 0.3 µg of the purified recombinant proteins, including human seroalbumin as negative control in 50 µl of PBS. After washing three times with PBS, plates were blocked with 3% skimmed milk in PBS (MPBS) for 2 h at room temperature. Then serum samples (dilution, 1:50 in 3% MPBS) were incubated for 2 h at room temperature. After washing, peroxidase-labeled anti-human IgG (Dako) (dilution, 1:3000 in 3% MPBS) was added for 2 h at room temperature. Then the signal was developed with 3,3′,5,5′-tetramethylbenzidine substrate for 10 min (Sigma). The reaction was stopped with 1 m H2SO4, and absorption was measured at 450 nm. Microarrays were analyzed with the ProtoArray Prospector Analyzer 4.0 (Invitrogen), which relies on Chebyshev's inequality principle (13Hudson M.E. Pozdnyakova I. Haines K. Mor G. Snyder M. Identification of differentially expressed proteins in ovarian cancer using high-density protein microarrays.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 17494-174
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