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Serological Identification of Tektin5 As a Cancer/ Testis Antigen and Its Hanafusa et al. BMC Cancer 2012, 12:520 http://www.biomedcentral.com/1471-2407/12/520 RESEARCH ARTICLE Open Access Serological identification of Tektin5 as a cancer/ testis antigen and its immunogenicity Tadashi Hanafusa1, Ali Eldib Ali Mohamed2,5, Shohei Domae3, Eiichi Nakayama4 and Toshiro Ono1* Abstract Background: Identification of new cancer antigens is necessary for the efficient diagnosis and immunotherapy. A variety of tumor antigens have been identified by several methodologies. Among those antigens, cancer/testis (CT) antigens have became promising targets. Methods: The serological identification of antigens by the recombinant expression cloning (SEREX) methodology has been successfully used for the identification of cancer/testis (CT) antigens. We performed the SEREX analysis of colon cancer. Results: We isolated a total of 60 positive cDNA clones comprising 38 different genes. They included 2 genes with testis-specific expression profiles in the UniGene database, such as TEKT5 and a CT-like gene, A kinase anchoring protein 3 (AKAP3). Quantitative real-time RT-PCR analysis showed that the expression of TEKT5 was restricted to the testis in normal adult tissues. In malignant tissues, TEKT5 was aberrantly expressed in a variety of cancers, including colon cancer. A serological survey of 101 cancer patients with different cancers by ELISA revealed antibodies to TEKT5 in 13 patients, including colon cancer. None of the 16 healthy donor serum samples were reactive in the same test. Conclusion: We identified candidate new CT antigen of colon cancer, TEKT5. The findings indicate that TEKT5 is immunogenic in humans, and suggest its potential use as diagnostic as well as an immunotherapeutic reagent for cancer patients. Keywords: TEKT5, SEREX, CT antigen Background The serological identification of antigens by the recom- Colon cancer is one of the major human malignancies. binant expression cloning (SEREX) methodology has Over the past decade, the overall incidence and resulting been successfully used for the identification of CT anti- deaths have been declining due to early diagnosis [1]. gens. Using cDNA libraries of cancer and normal testis However, a majority of cases still have a poor prognosis tissues, SSX2 [4], SYCP-1 [5], NY-ESO-1 [6], XAGE-1 even with the advances in chemotherapy and molecular [7], and CCDC62-2 [8] have been identified. targeted therapy. Recent progress in tumor immunology In this study, we performed SEREX analysis of colon based on the molecular identification of tumor antigens cancer, and isolated a novel CT antigen, Tektin5 (TEKT5), may allow immunotherapy to become another promising in addition to a previously defined CT-like antigen, A kin- treatment to improve outcomes. ase anchoring protein 3 (AKAP3). Tektins (TEKTs) are A variety of tumor antigens have been identified by composed of a family of filament-forming proteins in the several methodologies. Among antigens, cancer/testis male germ cell-lineage in centrioles and basal bodies and (CT) antigens have became promising targets for diag- within ciliary and flagellar doublet microtubules [9]. In nosis and immunotherapy for patients with various mammals, five Tektin proteins (TEKT1 – 5) have been tumors because of their unique expression pattern [2,3]. identified [10]. We isolated human TEKT5,andshowed that it has the characteristics of a CT antigen and that it * Correspondence: [email protected] elicits a strong immune response in a subset of patients 1Department of Radiation Research, Advanced Science Research Center, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan with cancer, including colon cancer. Full list of author information is available at the end of the article © 2012 Hanafusa et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hanafusa et al. BMC Cancer 2012, 12:520 Page 2 of 8 http://www.biomedcentral.com/1471-2407/12/520 Methods Sequence analysis of reacted clones Ethics statement The clones that reacted positively were subcloned to Patient samples were collected in accordance with the monoclonality, purified, and excised in vivo to pBK- Declaration of Helsinki and approved by the Ethics Com- CMV plasmid forms (Stratagene). The plasmid DNA mittee of Okayama University Graduate School of Medi- was prepared using the Quantum Prep Plasmid Mini- cine, Dentistry, and Pharmaceutical Sciences (No. 842). prep Kit (Bio-Rad, Hercules, CA, USA). The nucleotide sequences of the cDNA inserts were determined using Tissues and sera an ABI 3130x1 Genetic Analyzer (Applied Biosystems, Tumor specimens and sera were obtained from patients at Foster City, CA, USA), and sequence alignment was per- Okayama University Hospital. Written informed consent formed with BLAST software and sequences in the Gen- was obtained from all patients and healthy donors involved Bank database. in our study in accordance with the university guidelines. Total RNA isolation and cDNA synthesis Total RNA was isolated from the tumor tissues using an Preparation of the cDNA library RNeasy Mini Kit (Qiagen, Hilden, Germany). Total RNA mRNA was purified from the colon cancer tissue C164 from normal testis tissues was obtained commercially using a Quick Prep mRNA Purification Kit (Amersham (BD Bioscience Clontech, Palo Alto, CA, USA). The Pharmacia, Piscataway, NJ, USA). mRNA was also purified RNA (2 μg) was reverse-transcribed into single-strand from normal testicular total RNA. Then, a cDNA expres- cDNA using Moloney murine leukemia virus reverse γ sion library was prepared in a ZAP Express vector using transcriptase (Ready-To-Go You-Prime First-Strand a cDNA library kit (Stratagene, La Jolla, CA, USA). Beads, GE Healthcare, Buckinghamshire, UK) and oligo (dT)15 as a primer. The cDNA samples were tested for Immunoscreening of cDNA libraries integrity by the amplification of G3PDH in a 30-cycle A cDNA expression library of C164 colon cancer tissues reaction. was screened with autologous patient sera. A testicular cDNA expression library was screened with 2 different Quantitative real-time RT-PCR colon cancer patient sera. The screening procedure was Two-step real-time RT-PCR was performed using a Ste- described previously [11]. In brief, serum samples that pOne Real-Time PCR System (Applied Biosystems). had been diluted 1:10 were preabsorbed with lysate from cDNA was synthesized using a High-capacity cDNA Re- Eschericia coli Y1090/Y1089 and coupled to Sepharose verse Transcription kit (Applied Biosystems). TaqMan 4B (BioDynamics Lab Inc., Tokyo, Japan). Recombinant Gene Expression Assays (Applied Biosystems) were used phages (approximately 4,000 pfu) on agar in a plastic to measure the mRNA levels of TEKT5 (Assay ID: dish (140-mm diameter) were amplified for 8 hr and Hs01025979_m1). mRNA levels were expressed as n-fold then transferred to 135-mm diameter nitrocellulose differences relative to G3PDH (internal standard) and the membranes (Schleicher & Schuell, Dassel, Germany) for levels in normal testis (calibrator). PCR was performed 15 hr at 37°C. The membranes were then blocked with using TaqMan PCR Master Mix (Applied Biosystems), 5% non-fat milk and pre-screened by incubation with and the thermal cycling conditions comprised an initial peroxidase-conjugated Fc fragment-specific goat anti- denaturation at 95°C for 10 min, then 40 cycles at 95°C human IgG (Jackson ImmunoResearch, West Grove, PA, for 15 sec, and 60°C for 1 min. The parameter Ct was USA) (1:2,000 dilution) for 1 hr at room temperature. defined as the threshold cycle number at which the fluor- Color was developed using 3, 3'-diaminnobenzidine escence generated by cleavage of the probe passed above (Sigma, St. Louis, MO, USA), and IgG-encoding clones the baseline. The TEKT5 target message was quantified were marked so that they could be excluded from subse- by measuring the Ct value, and transcripts of G3PDH quent examinations. The membranes were then incu- were quantified as an endogenous RNA control using bated overnight at room temperature with the TaqMan human G3PDH control regents (Applied preabsorbed serum diluted to 1:200. The membranes Biosystems). were incubated with peroxidase-conjugated Fc fragment- specific goat anti-human IgG (Jackson ImmunoRe- Gene expression microarrays search) (1:2,000 dilution) for 1 hr at room temperature, Gene expression was examined using Agilent Human and then the color was developed. Positive clones were 1A oligomicroarrays containing 60-mer DNA probes in collected and subcloned to monoclonality by 2nd and a 22K format (Agilent Technologies). Of 19,061 spots, 3rd screenings using 82- and 47-mm diameter mem- 18,086 are non-controls, and there are 17,086 unique branes, respectively. A randomly chosen negative clone transcript sequences from 15,989 unique human genes. was included in each assay as a negative control. Five hundred ng of total RNA from 3 colon cancer Hanafusa et al. BMC Cancer 2012, 12:520 Page 3 of 8 http://www.biomedcentral.com/1471-2407/12/520 0 tissues for the test samples and normal colon tissue for KT-as2 (reverse: 5 -ATTGTCGACGGTGTGGCCCAC 0 the reference samples were used to synthesize labeled CAGGCGCGG-3 ) were designed to encompass the entire cRNA (Low RNA Input Linear Amp Kit, Agilent Tech- coding sequence of the gene corresponding to amino acid nologies) in the presence of cyanine 3-dCTP and cyanine positions 1–486. The isolated GST fusion protein was 5-dCTP (Perkin-Elmer Life Sciences, Boston, MA), re- purified on a gel filtration column (Hiload 16/60 Superdex spectively. Differentially labeled test and reference sam- 200 pg, GE Healthcare). ples were mixed with Agilent control targets before being hybridized onto the oligomicroarrays for 17 hr at ELISA 37°C in a rotating oven. The fluorescence intensities of Recombinant TEKT5 protein (1 μg/ml) in 0.05 M car- the targets were detected using a laser confocal scanner bonate buffer (pH 9.6) was absorbed onto 96-well plates (Agilent Technologies), and the resulting images were (Nunc) at 4°C overnight.
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