Identification of tumor-associated antigens in human thyroid papillar carcinoma

In this study two cDNA expressing libraries generated from thyroid papillar carcinomas were screened using SEREX approach. Thirty positive cDNA clones representing seventeen different genes were identified from both libraries. It is important to note, that three of them were isolated previously by other laboratories in SEREX screens of various types of human cancer. These include transcription factor NZF, a-catenin and ВАС RPU—a protein with unknown function. Moreover, we identified a whole panel of novel potential tumor-associated antigens, which would be further investigated. We are particularly interested in more detailed analysis of cathepsin H and transducer of ErbB2 (TOB2), which are differentially expressed in various types of human cancer. We will analyse the frequency of autoantibodies against identified antigens in sera of patients with various malignancies and healthy donors by heterologous screening. It is expected that among the clones isolated in this study, there might be novel cancer-associated markers.

Introduction.Thyroid cancer, although the most fre quent malignancy of the endocrine system, is in general a rare disease.It accounts for about 1 % of all human cancers, with a higher prevalence in women (5-9 of 100000) as compared with men (2-4 of 100000) [1].Normally, thyroid cancer is a disease with good prognosis, but about 30 % of tumors dedifferentiate and may finally develop into highly malignant anaplastic thyroid carcinomas with the mean survival time of less than 8 months.The thyroid gland is highly sensitive to radiation-induced on cogenesis.This is verified by numerous reports from survivors after Hiroshima and Nagasaki, the Nevada, Novaya Zemlya and Marshal Island atomic bomb tests [2].Investigations provided after Chernobyl nuclear plant accident have shown the increase of thyroid cancer two fold in adults and three fold in children.
The success of cancer therapy depends on the stage of the disease detection.Today, the specific markers for early detection of thyroid cancer are unknown and the search for them is a problem of outstanding value.
At present, several methods for the search of tumor-associated antigens exist, such as DNA Microarrays, SAGE, CNAPS, SEREX (Serological analysis of recombinant cDNA expressing libraries) with their advantages and deficiencies [3].SEREX method is based on the study of cancer patient's immune response.Samples of cancer tissue are used for the creation of expressing cDNA libraries further scree ned by autologous sera.This method is suitable for the identification of the proteins (or their immuno genic epitopes) that cause immune responses in can cer patients.
SEREX method has been successfully applied for the detection of tumor-associated antigens from vari ous types of human cancer, including breast, colo rectal, renal etc. [4][5][6].
These studies led to the identification of several novel antigens, which are currently used as markers of malignant transformation and for the production of anti-cancer vaccines [7 ].We have previously reported the identification of 15 immunoreactive clones isolated by SEREX screening of thyroid cancer cDNA exp ressing libraries [8 ].We have extended the search for novel antigens by generating and screening of two novel libraries from thyroid papillar cancer.Here, we report the identification of further 30 immunoreactive clones, representing 17 genes.It is important to note that three from identified genes have been previously found by SEREX approach from other types of human cancer.Heterologous screens of isolated clones with a panel of sera from healthy donors and patients with various types of cancer is currently in progress.
Materials and Methods.The samples of human thyroid papillar carcinomas and autologous sera were kindly provided by Dr. V. Usenko and Dr. V. Lysogubov (BIONTEC, Ukraine).
Purification of total and messenger RNA.Total RNA was purified from freshly frozen in liquid nitrogen tumor samples by the modified guanidineisothiocyanate method [9 J. mRNA was isolated by affinity chromatography using oligo(dT) Dynabeds matrix («Dynal», UK).The quality and quantity of the preparations obtained were estimated by spectrophotometrical methods and by the electrophoresis in 1 % formaldehyde-agarose gels.The mRNA prepa rations were stored under ethanol at -80 °С until further use.
Generation of cDNA expressing libraries.Five micrograms of purified mRNA preparations were applied for cDNA synthesis with the use of cDNA synthesizing kit («Stratagene», USA).The oligonuc leotide primer containing Xhol site and an oligo(dT) tail was used for the initiation of reverse transcription.The effectiveness of the synthesis was estimated with the use of the test RNA preparation from Stratagene kit followed by electrophoresis in 1 % agarose gels.
cDNA fragments accounting 0.5-3 kb were extracted from the gels using DNA extraction kit («Qiagen», USA).The fragments obtained were clo ned into bacteriophage Я DNA (UniZap vector) by Xhol and EcoRI restriction sites.The phagemide obtained was packed into phage particles with the use of Gigapack Gold III kit («Stratagene», USA).For the estimation of primary libraries titers Escherichia coli XL-1 Blue MRF' cells were infected by recombinant phages in appropriate dilutions.The percentage of non-recombinant phages was detected by blue/white plaque selection in the presence of IPTG and X-Gal («Sigma», USA).
Affinity purification of the sera.Sera samples from patients with thyroid cancer were diluted 1:1 with glycerol and stored at -20 °С until further use.For the affinity purification, prepared sera samples were diluted 1:10 by TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl) and incubated with affinity matrixes containing covalently crosslinked E. coli and Я phage proteins (Y1090 and BNN97 matrixes respectively).This technique allowed the elimination of serum immunoglobulins directed against bacterial and phage proteins.The depleted sera were further diluted by TBS 1:100 and stored at 4 °С with the addition of 0.02 % NaN 3 .
Immunoscreening of cDNA expression libraries.For the primary screening 600 /лі of E. coli XL-1 Blue MRF' cells were infected with 6* 10 s phage particles and plated onto 150 mm Petri dishes.The expression of recombinant proteins was induced by the addition of 1 mM IPTG for 5-7 hours.The transfer of the proteins to the nitrocellulose membrane Hybond-C was carried out overnight by standard method.
The identification of IgG expressing clones was performed by immunoblotting of filters with antihuman horse radish peroxidase conjugate (dilution 1:2000, «Sigma», USA).Detection of primary posi tives was carried out by probing filters with the autologous sera and anti-human alkaline phosphatase conjugate staining.Positive clones were extracted from the agar and stored in 0.5 ml of SM buffer with the addition of 20 ці of chloroform.
The secondary screening of isolated clones was performed on 90 mm Petri dishes by the same method.Plasmid DNA from positive clones was obtained by in vivo recombination in E. coli strain XL-1 Blue MRF'.
Restriction analysis and sequencing of isolated clones.The size of cDNA insertions was detected by restriction analysis with EcoRI and Xhol endonucleases followed by 1 % agarose gel electrophoresis.Sequencing of the inserts was performed by a stan dard protocol using the automatic sequencer ABI 373 (Applied Biosystem).The identification of positive clones and their analysis was performed with a help of EMBO, GenBank, dBest and SEREX databases.
Results and Discussion.Taking into conside ration the increase of thyroid cancer frequency and the absence of specific markers for early diagnostic purposes we have extended our search for thyroid cancer associated markers.In this study, we have used SEREX methodology aimed on the identification of tumor-associated proteins which induce immune response in cancer patients.We have successfully applied SEREX technique in previous studies, which led us to the identification of 25 immunoreactive clones from thyroid cancer and melanoma [8,10].
In order to extend the search for novel antigens, we have created two additional cDNA expressing libraries from thyroid papillar carcinomas with the titer of 1.05 and 1.4 • 10 6 respectively.The percentage of non-recombinant phages was less than 1 % for both libraries.These tests indicated that both li braries are suitable for immunoscreening by SEREX methodology.
The primary immunoscreening with autologous sera allowed us to isolate 170 primary positives clones, which showed various degree of immunoreactivity.It is important to note that we picked up clones which exhibited even a very weak immunoreactivity (a borderline with non-specific signal).There fore, a large number of primary clones were not confirmed by secondary screenings, leaving only 30 clones as true-positives.cDNA plasmids, correspon ding to positive clones, were rescued by in vitro recombination approach.The size of inserts in isola ted plasmids were determined by restriction analysis (data not shown).The identification of isolated clones was performed by sequence analysis followed by searching of various DNA and protein databases.The search revealed that 30 clones encode 17 genes (Table 1).Furthermore, 12 of them encode known proteins, while 5 represent so far genes with unknown func tions.The sequence of isolated clones and other relevant information were submitted to the SEREX database http://www.licr.org/SEREX.html.
The analysis of SEREX database revealed that three of these genes were identified earlier by other SEREX laboratories in screens of different types of human cancer (Table 2).These include a potential transcription factor NZF, a-catenin and a protein with unknown function ВАС RP11.Noteworthy, NZF protein was isolated from libraries generated from glioma, renal cell carcinoma, ovarian and colon can cer, teratoma and normal testis.On the other hand, a-catenin was cloned from melanoma, renal, breast, colorectal cancer, small cell carcinoma and testis.Further analysis of other antigens identified in pre sent study showed their relevance to malignant trans formation.
For example, a-and /?-catenins are involved in cadherin-mediated cell-cell adhesion.Disregulation of cellular functions of both a-and /ї-catenins is asso ciated with invasive potential of malignant cells [11][12][13].The overexpression of ErbB-2 antigen is ob served in 30 % of breast cancer and is associated with poor prognosis.Moreover, ErbB2 has been also found overexpressed in thyroid and other types of human cancer [14,15].In this study we have isolated the transducer of ErbB2 (clone Thy28).So far, very little is known about the function of the transducer of ErbB2.However, there is no doubt that the function of this gene in normal and transformed cells needs further investigation.
Phosphotyrosine independent ligand p62 for the Lck SH2 domain (Thy36) is a major component of intracytoplasmic hyaline bodies in hepatocellular car cinoma cells, while in non-neoplastic liver cells it was not observed [16].p62 protein binds ubiquitin and may act as an adapter linking ubiquitinated proteins to multienzyme proteosomal complexes to other pro teins.These features suggest a role for p62 in signal transduction and possibly also in carcinogenesis.
Elevated expression of proteases is observed in a variety of tumors.Clone Thy40 encodes a proteasecathepsin H, whose expression is increased in highgrade prostatic intraepithelial neoplasia and carci noma of the prostate [17 ].Two forms of cathepsin are known: a full length and a truncated version.Both of them are enzymatically active, but truncated form has a reduced lysosomal association when compared with a full-length cathepsin H.It was suggested that increased expression of cathepsin H may affect cellu lar functions especially those which are associated with tumor progression and metastasis.Short-chain collagen type VIII (Thy43) was observed throughout the development of hemangioma in the study of Tan et al. [18].It was also detected within mast cells during early proliferative phase.In several SEREX studies different ribosomal proteins have been iden tified as tumor-associated antigens [19,20].We have identified ribosomal protein S24 (Thy32 and 35) which has not been previously detected in serological screenings by SEREX or other approaches.Several clones corresponding to non-sarcomeric myosin light chain (Thy37, 38 and 42) have been also isolated in our screen.In our previous article [8] we have reported SEREX-based detection of CDC-42 binding protein kinase.Phosphorylation of myosin light chain by CDC-42 binding protein kinase leads to the activation of actin-myosin contractility [21 ].Dis regulation in actin-myosin contractility, induced by aberrant signaling may play a role in malignant invasiveness and metastatic growth [22 ].
The clone KY-Thy29 encodes for the solid tumor associated protein.Two STAG1/PMEPA1 mRNA transcripts of approximately 2.7 and 5 kb, with  identical coding regions but variant 3' untranslated regions, were predominantly expressed in normal prostate tissue and at lower levels in the ovary.The expression of this gene was upregulated in 87 % of RCC samples and also was upregulated in stomach and rectal adenocarcinomas.
In contrast, STAG1/PMEPA1 expression was barely detectable in leukemia and lymphoma samples.Analysis of expressed sequence tag databases showed that STAG1/PMEPA1 also was expressed in pancrea tic, endometrial, and prostatic adenocarcinomas.The STAG1/PMEPA1 cDNA encodes a 287-amino-acid protein containing a putative transmembrane domain and motifs that suggest that it may bind src homology 3-and tryptophan domain-containing proteins.This protein shows 67 % identity to the protein encoded by the chromosome 18 open reading frame 1 gene.Translation of STAG1/PMEPA1 mRNA in vitro sho wed two products of 36 and 39 kDa, respectively, suggesting that translation may initiate at more than one site.The upregulation of this gene in several solid tumors indicated that it may play an important role in tumorigenesis [23].The link between identified antigens and various aspects of malignant trans formation, based on the literature search, is summa rized in Table 3.Our further investigations will be

Table 3 A possible link between SEREX-derived antigens from human thyroid carcinoma and malignant transformation
VIII, alpha 2 The levels of catenin expression in tumor tissue differs from the normal ones a-Catenin meets the criteria of an invasion suppressor gene The expression in thyroid carcinomas correlates with the degree of aggressiveness and differentiationMajor component of intracytoplasmic hyaline bodies in hepatocellular carcinomaInvolved in invasion and metastasis of pancreatic cancerIncreased expression in high-grade prostatic intra epithelial neoplasia and carcinoma of the prostateInvolved in down-regulation of signalling pathways, inducing growth and proliferationShort-chain collagen is localized extracellulary throughout the development of hemangioma

В. Роднін, I. О. Тихонкова, P. Г. Кіямова, О. M. Гарифулін, І. Т. Гут, В. В. Філоненко Ідентифікація пухлиноасоційованих антигенів папілярної карциноми щитовидної залози людини Резюме З двох зразків тканини папіломи щитовидної залози людини
focused on the elucidation of the role of identified antigens in cancerogenesis.Heterologous screenings with a panel of sera from healthy donors and patients with various types of cancer would allow us to select those which have the properties of tumor specific markers or the potential for the development of anti-cancer vaccines.Acknowledgements.This study was supported in part by grants from the National Academy of Sciences of Ukraine, The Royal Society and INTAS.Authors are grateful to Dr. Usenko and Dr. Lyzogubov (BIO-NTEC, Ukraine) for providing the samples of thyroid cancer and autologous sera. M.