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Keywords:

  • lung adenocarcinoma;
  • SEREX;
  • XAGE-1;
  • cancer/testis antigen

Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

By serologic identification of antigens by recombinant expression cloning (SEREX) analysis using an autologous lung adenocarcinoma cell line, OU-LU-6, as a cDNA library source, we demonstrated that XAGE-1 was the dominant antigen recognized by serum from a patient. By immunoscreening, we obtained 38 positive cDNA clones consisting of 16 genes designated as OY-LC-1 to -OY-LC-16. OY-LC-1, represented by 18 clones, was identical to XAGE-1. OY-LC-2 to -16, represented by either a single or 2 clones, were identical to known genes shown to be ubiquitously expressed in various normal tissues. RT-PCR analysis showed that of 4 XAGE-1 transcripts—XAGE-1a, b, c and dXAGE-1b was expressed in OU-LU-6 dominantly. Furthermore, XAGE-1b mRNA was expressed in 4 of 10 lung cancer tissues, whereas no expression was observed in normal tissues. Of 4 XAGE-1b mRNA positive cancer tissues, 3 were adenocarcinoma and one was poorly differentiated squamous cell carcinoma. Of 32 sera from lung cancer patients, 8 sera were reactive with the XAGE-1b product. Those 8 sera were from patients with adenocarcinoma. These findings indicated strong immunogenicity of XAGE-1b in lung adenocarcinoma and suggested its potential use as a target for vaccine-based immunotherapies. © 2003 Wiley-Liss, Inc.

Lung cancer is one of the leading causes of cancer death in the world.1 The predominant lung cancer is non small cell lung cancer (NSCLC) consisting of squamous cell carcinoma, large cell carcinoma and adenocarcinoma, and the 5-year survival is poor even in the early stage.2 Recent progress in tumor immunology based on the molecular identification of tumor antigens may allow immunotherapy to be a promising treatment for lung cancer.

Serologic identification of antigens by recombinant expression cloning (SEREX) analysis of a wide range of tumors has identified more than 2,000 immunogenic tumor products,3, 4 including cancer/testis (CT) antigens,5, 6 mutational antigens,7 over-expressed antigens,8, 9 differentiation antigens,5, 10 splice-variant antigens11 and viral antigens.12 Among them, CT antigens have received particular attention because of their unique expression pattern and their potential as targets for cancer vaccines.13 The defining characteristics of CT antigens are high levels of expression in male germ cells, lack of expression in other normal tissues and aberrant expression in a wide range of tumor types.14 To date, more than a dozen genes or gene families coding for CT antigens, such as MAGE,15 BAGE,16 GAGE,17 SSX,5, 18 NY-ESO-1/LAGE,6, 19 SCP-1,20 CT7/MAGE C1,21 CTp11,22 OY-TES-1,23 etc., have been characterized by T-cell epitope cloning, SEREX, representational difference analysis and database mining.

Using the SEREX approach, several lung cancer antigens have been identified.24, 25, 26 A screening of a testicular cDNA expression library with pooled lung cancer patient sera has led to the identification of a novel CT antigen, CAGE-1.27 In our study, we showed that XAGE-1 was the dominant antigen recognized by sera from a lung adenocarcinoma patient using a cultured autologous tumor cell line, OU-LU-6, as a cDNA library source in SEREX analysis. We also showed that XAGE-1 was expressed frequently in lung adenocarcinoma and highly immunogenic for antibody production in patients.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Tissues, cell lines and sera

Tumor specimens were surgically obtained from patients at Okayama University Hospital. Lung cancer and adjacent normal lung tissues were obtained in the same samples from 10 specimens. OU-LU-6 is a lung cancer cell line established from pleural effusion of a 38-year-old male with adenocarcinoma. Sera were obtained from 27 healthy donors and 32 lung cancer patients in accordance with the university guidelines after receiving written informed consent.

Preparation of a cDNA library from a lung cancer cell line

mRNA was purified from a lung adenocarcinoma cell line, OU-LU-6, using a Quick Prep mRNA Purification Kit (Amersham Pharmacia, Piscataway, NJ). A cDNA expression library was prepared in a λZAP Express vector using a cDNA library kit (Stratagene, La Jolla, CA).

Immunoscreening of the cDNA library and characterization of selected immunoreactive clones

cDNA expression library of the lung cancer cell line OU-LU-6 was screened with autologous patient serum. The screening procedure was described previously.28 In brief, serum samples diluted 1:10 were preabsorbed with lysate from Escherichia coli Y1090/Y1089 and bacteriophage-infected Y1090 coupled to sepharose 4B (BioDynamics Lab Inc., Tokyo, Japan). Nitrocellulose membranes containing the phage plaques at a density of about 4,000 pfu/140 mm plate were incubated overnight at room temperature with the preabsorbed autologous serum diluted 1:200. Reacted clones were detected by peroxidase-conjugated goat anti-human immunoglobulin (Ig)G (Jackson Immuno Research, West Grove, PA) and visualized with 3-3′-diaminobenzidine (Sigma, St. Louis, MO). XAGE-1b, XAGE-1c and selected immunoreactive clones were tested for reactivity against serially diluted sera from lung adenocarcinoma patients using the same plaque assay. A negative clone randomly chosen was included in each assay as a negative control.

Sequence analysis

The positive clones were subcloned to monoclonality, purified and excised in vivo to pBK-CMV plasmid forms (Stratagene). Plasmid DNA was prepared using a Quantum Prep Plasmid Miniprep Kit (Bio-Rad, Hercules, CA). The nucleotide sequence of cDNA inserts was determined by an ABI PRISM R310 Genetic Analyzer (PerkinElmer, Foster City, CA), and sequence alignments were performed with BLAST software and compared to sequences in the Genbank and EST databases.

RT-PCR

To amplify cDNA segments from tumor and normal tissues, primers for the respective XAGE-1 transcripts were designed (Table I). Total RNA was isolated from different tumor tissues, cell lines and normal lung tissues using an RNeasy Mini Kit (Qiagen, Hilden, Germany). The isolated RNA was reverse-transcribed into a single-strand cDNA using Moloney murine leukemia virus reverse transcriptase (Ready-To-Go You-Prime First-Strand Beads, Amersham Pharmacia) and oligo(DT)15 as a primer. A normal tissue panel obtained commercially (Clontech, Palo Alto, CA) was also used for PCR reaction. RT-PCR was performed using 30 and 35 cycles at an annealing temperature of 60°C, and the products were analyzed by agarose gel electrophoresis. cDNA was tested for integrity by amplification of G3PDH and β-actin transcripts in a 30-cycle reaction.

Table I. XAGE-1 PCR and 5′ Race Primers Used in This Study
PrimerSequenceComments
X-15′-TTTCTCCGCTACTGAGACAC-3′XAGE-1 common (sense)
X-25′-CAGCTTGCGTTGTTTCAGCT-3′XAGE-1 common (anti-sense)
X-35′-TGCAGATCACCTTCCATGTC-3′XAGE-1 common (anti-sense)
X-45′-CTGGGAGTTGAAGTGTGAGT-3′XAGE-1c specific (sense)
X-55′-TCTGGGGAGTCCAGAATCTT-3′XAGE-1c specific (anti-sense)
X-65′-CAGGTGCTGGGAAGGGAAAT-3′XAGE-1d specific (sense)
X-75′-ACCTCAGTGCGCATGTTCAC-3′XAGE-1a specific (sense)
X-GSP15′-GCTCTTGCAGATCACCTTCCATGTC-3′For 5′ RACE
X-GSP25′-TCTGTCTGCTGCCCAGGAGTAGGAT-3′For 5′ RACE

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Identification of OU-LU-6 genes using autologous serum by SEREX

A cDNA expression library of 1.5 × 106 clones was prepared from the lung adenocarcinoma cell line OU-LU-6. Immunoscreening of 2.4 × 105 clones with autologous patient serum yielded a total of 38 positive clones. As shown in Table II, the nucleotide sequences of the cDNA inserts identified 16 different genes, designated as OY-LC-1 through OY-LC-16. Three genes, OY-LC-3, 5 and 9, were in the SEREX database (http://www.licr.org/SEREX.html). OY-LC-1, represented by 18 clones, was identical to Homo sapiens G antigen, family D2 (GAGED2) or XAGE-1, which was recently identified as a CT-associated gene.29, 30 Four XAGE-1 transcripts—XAGE-1a, b, c and d—were identified (Fig. 1 and NCBI database, http://www.ncbi.nih.gov/entrez/query). Of the 18 clones, 16 were homologous to XAGE-1b. The size of the cDNA ranged from 378 bp to 468 bp (Table III). The longest insert (468 bp) contained a 13-nucleotide addition at the 5′ end of the XAGE-1b transcript (455 bp) in the database (NCBI database). Two of the 18 clones had a cDNA insert of 511 bp in length and contained an intronic sequence from intron 1, being identical to the XAGE-1c transcript. EST database search and UniGene cluster analysis revealed that OY-LC-2 to OY-LC-16 were expressed in normal tissues ubiquitously.

Table II. Genes Identified from the Lung Adenocarcinoma Cell Line OU-LU-6 by Autologous Serum Screening
GeneNo. of clonesIdentity/similaritiesSEREX database IDUniGene clusterExpression
OY-LC-118Homo sapiens G antigen, family D 2 (GAGED2) (XAGE-1)Hs. 112208Cancer/testis
OY-LC-21Activating transcription factor 4 (Tax-responsive enhancer element B67)Hs. 181243Ubiquitous
OY-LC-31LIM and senescent cell antigen-like domain 13987Hs.112378Ubiquitous
OY-LC-41Anaplastic large cell lymphoma kinase (ALK)Hs.9614Ubiquitous
OY-LC-52Lactate dehydrogenase A1157Hs.2795Ubiquitous
OY-LC-61Coatomer protein comlex, subunite epsilon (COPE)Hs.10326Ubiquitous
OY-LC-72Cold shock domain protein A, clone MGC:12695Hs.198726Ubiquitous
OY-LC-81Carbonyl reductase 1 (CBR1)Hs.88778Ubiquitous
OY-LC-92Zink finger protein 36, C3H type-like 2 (ZFP36L2)1175Hs.78909Ubiquitous
OY-LC-101P4HB procollagen-proline, 2-oxoglutarate 4-dioxygenaseHs.410578Ubiquitous
OY-LC-111Methylene tetrahydrofolate dehydrogenase (NAD+ dependent), methenyltetrahydrofolate cyclohydrolase (MTHFD2)Hs.154672Ubiquitous
OY-LC-121Transcription elongation regulator 1 (CA150) (TCERG1)Hs.13063Ubiquitous
OY-LC-132Ras and Rab interactor 3 (RIN 3)Hs.180040Ubiquitous
OY-LC-141Neuronal protein 17.3Hs.433328Ubiquitous
OY-LC-151RNA helicase (RIG-I)Hs.145612Ubiquitous
OY-LC-162Similar to integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA receptor)Hs.265829Ubiquitous
thumbnail image

Figure 1. Schematic representation of the XAGE-1 gene and transcripts. Exons are shown as shaded boxes and introns as open boxes. Open reading frames are shown in hatched boxes. Arrows indicate primers used in our study.

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Table III. Summary of 18 XAGE-1 Clones
No. of clonescDNA insert size, bpComments
5468XAGE-1b
2464XAGE-1b
2456XAGE-1b
2447XAGE-1b
4381XAGE-1b
1378XAGE-1b
2511XAGE-1c

XAGE-1a, b, c and d mRNA expression in tumors and normal tissues

We investigated the expression of XAGE-1a, b, c and d transcripts in OU-LU-6 and normal testis using specific PCR primers (Table I and Fig. 1). RT-PCR was performed at 30 and 35 cycles. As shown in Figure 2a, no XAGE-1a was detected in either OU-LU-6 or normal testis. XAGE-1c was detected in normal testis but not OU-LU-6 after 35 cycles. XAGE-1d was detected in both OU-LU-6 and normal testis after 35 cycles. Using the common primer pair X-1 and X-2, a PCR product of 346 bp in length was observed in both OU-LU-6 and testis at 30 cycles. XAGE-1d contains a 16 bp intronic sequence between exons 2 and 3 in XAGE-1b. The sequence of the PCR products by X-1 and X-2 primers showed no 16-bp intronic sequence, indicating that they were derived from XAGE-1b (data not shown). The results indicated that XAGE-1b is the predominantly expressed XAGE-1 transcript in OU-LU-6 and normal testis. In adult normal tissues other than normal testis, no expression was observed (Fig. 2b).

thumbnail image

Figure 2. RT-PCR analysis. Primers used (see Table I and Fig. 1) are X-7 and X-2 for XAGE-1a, X-1 and X-2 for the common region of XAGE-1, X-4 and X-2 for XAGE-1c and X-6 and X-2 for XAGE-1d. In (a), PCR was performed at 30 and 35 cycles. OU-LU-6 is the lung adenocarcinoma cell line used as a source of cDNA library in the SEREX analysis. In (b), X-1 and X-2 primers were used. In (c) and (d), T and N denote lung cancer and the adjacent normal tissues, respectively. G3PDH and β-actin (not shown) were used for internal controls. XAGE-1b was confirmed by negative expression of XAGE-1a and nucleotide sequence.

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XAGE-1 mRNA expression was further examined with 10 lung cancers and the adjacent normal tissues by RT-PCR at 30 cycles. As shown in Figure 2c and 2d, XAGE-1b mRNA expression was observed in 4 of the 10 lung cancers, but not in the normal tissues. Three of 4 XAGE-1b positive lung cancers were adenocarcinoma and one was poorly differentiated squamous cell carcinoma. XAGE-1c mRNA expression was observed weakly in 1 (T5) of the 10 lung cancers. No expression of XAGE-1a or d was observed (data not shown). XAGE-1b mRNA expression was further investigated in other malignant tissues. The expression was observed in 2 of 4 breast cancers, 1 of 21 bladder cancers and 1 of 9 malignant melanomas. No expression was observed in liver, stomach, colon and renal cancers (Table IV). In cancer cell lines, the expression was observed in lung cancer and melanoma cell lines with high frequency. No expression was observed in colon cancer cell lines.

Table IV. XAGE-1b mRNA Expression in Tumors and Normal Tissues
TumormRNA positive/totalNormal tissuemRNA positive/total
  • 1

    Three Clontech normal tissue panels (MTC panel, CLONTECH) with different lot number were used.

  • 2

    Additional normal lung and testis were included.

Lung cancer4/10Brain0/31
Heart0/3
Breast cancer2/4Kidney0/3
Liver cancer0/4Liver0/3
Stomach cancer0/5PBL0/3
Lung0/132
Colon cancer0/14Pancreas0/3
Bladder cancer1/21Placenta0/3
Skeletal muscle0/3
Renal cancer0/10Colon0/3
Melanoma1/9Ovary0/3
  Prostate0/3
Lung cancer cell lines12/20Small intestine0/3
Colon cancer cell lines0/5Spleen0/3
Testis8/82
Melanoma cell lines4/6Thymus0/3

Reactivity of allogeneic sera with OY-LC-1 to OY-LC-16 antigens

Serum reactivity was investigated against the 16 SEREX-defined antigens by plaque assay. Serum samples were obtained from 27 healthy donors and 32 lung cancer patients including 18 adenocarcinomas, 9 small cell carcinomas and 5 squamous cell carcinomas. As shown in Table V, 8 of the 32 sera from the lung cancer patients were reactive with OY-LC-1. None of the healthy donor serum was reactive. All 8 positive sera were derived from patients with adenocarcinoma consisting of a stage IIIb and 7 stage IV. With the OY-LC-2 to -16 antigens, a reaction was observed with some sera from both healthy donors or lung cancer patients.

Table V. Reactivity of Sera from Healthy Donors and Lung Cancer Patients Against Serex-defined Antigens
AntigenSera
Healthy donorsLung cancer patients
OY-LC-10/278/32
OY-LC-23/276/32
OY-LC-312/279/32
OY-LC-44/274/32
OY-LC-57/276/32
OY-LC-63/275/32
OY-LC-72/274/32
OY-LC-83/274/32
OY-LC-93/275/32
OY-LC-102/273/32
OY-LC-115/273/32
OY-LC-123/274/32
OY-LC-139/276/32
OY-LC-145/274/32
OY-LC-153/273/32
OY-LC-163/273/32

Titration was performed with the 8 OY-LC-1 positive sera against XAGE-1b, XAGE-1c and 3 other antigens. As shown in Table VI, 7 of the 8 sera were cross-reactive with XAGE-1c. Patient 8's serum was reactive only with XAGE-1b.

Table VI. Reactivity of Serially Diluted Sera from Lung Adenocarcinoma Patients to XAGE-1b, XAGE-1c and Three Serex-Defined Antigens1
SeraAntigens
XAGE-1bXAGE-1cOY-LC-3OY-LC-9OY-LC-13
  • 1

    Diluted serum was tested by the plaque assay.

  • 2

    Autologous serum used in SEREX.

Patient 12     
 Stage IV     
  1/200+++++
  1/500+++++
  1/1000+++++
  1/5000+++
Patient 2     
 Stage IV     
  1/200+++
  1/500+
  1/1000
  1/5000
Patient 3     
 Stage IV     
  1/200++
  1/500++
  1/1000+
  1/5000
Patient 4     
 Stage IV     
  1/200+++++
  1/500++++
  1/1000+++
  1/5000+
Patient 5     
 Stage IV     
  1/200+++++
  1/500+++++
  1/1000++++
  1/5000++
Patient 6     
 Stage III B     
  1/200++++
  1/500+++
  1/1000+
  1/5000
Patient 7     
 Stage IV     
  1/200++++
  1/500+++
  1/1000++
  1/5000++
Patient 8     
 Stage IV     
  1/200+
  1/500+
  1/1000+
  1/5000+

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

In our study, we demonstrated that XAGE-1 was the dominant antigen recognized by serum from a lung adenocarcinoma patient using the autologous tumor cell line OU-LU-6 as a cDNA library source in SEREX analysis. By immunoscreening, we obtained 38 cDNA clones consisting of 16 genes designated OY-LC-1 to -16. OY-LC-1, represented by 18 clones, was identical to XAGE-1. OY-LC-2 to -16, represented by either a single or 2 clones, were identical to known genes shown to be ubiquitously expressed in various normal tissues.

XAGE-1 was originally identified by EST database mining29 and found to be highly expressed in normal testis and Ewing's sarcoma.30 The XAGE-1 gene is located on chromosome Xp11.21–Xp11.22, consists of 4 exons31, 32 and shows homology to GAGE/PAGE genes. It has been reported that there are 4 transcript variants, XAGE-1a, b, c and d.31, 32 Within those, XAGE-1b was shown to be the major transcript. A higher expression of XAGE-1b mRNA was observed in Ewing's sarcoma and metastatic lesions of melanoma compared to XAGE-1a.31

We showed that XAGE-1b mRNA was expressed only in the testis in normal adult tissues and in 4 of 10 lung cancer tissues. No expression was observed in the normal lung. Of 4 XAGE-1b mRNA-positive lung cancers, 3 were adenocarcinoma and one was poorly differentiated squamous cell carcinoma. A weak expression of XAGE-1c mRNA was observed in one XAGE-1b positive lung cancer. Egland et al.33 showed that XAGE-1 mRNA was expressed in a significant number of lung cancers. XAGE-1b seemed to be the dominant transcript and XAGE-1a showed a weak expression. Wang et al.34 reported that L552S, identical to the XAGE-1c transcript, was over-expressed in lung adenocarcinoma. The discrepancy between those studies, including ours, appeared to be caused by the use of a specific tumor specimen that happened to express XAGE-1c mRNA and subsequent cloning of the cDNA in the latter study.34 We also showed XAGE-1b mRNA expression in other tumor tissues such as breast cancer (2/4), malignant melanoma (1/9) and bladder cancer (1/12).

By a screening plaque assay, we showed that of 32 sera from lung cancer patients, 8 sera reacted with XAGE-1b. Those 8 sera were derived from patients with adenocarcinoma. XAGE-1c was also recognized by 7 of 8 XAGE-1b reactive sera probably by cross reaction because of a shared open reading frame. The findings indicated high immunogenicity of XAGE-1b and suggested its potential use as a target for immunotherapy in lung adenocarcinoma. The T cell immune response is now being studied using cultured CD8 and CD4 T lymphocyte cell lines reactive with OU-LU-6 obtained from a patient's pleural effusion.

The transcription start site of XAGE-1b is controversial.31, 33 Previously, Zendman et al.32 reported a 455-bp transcript as XAGE-1b by 5′ rapid amplification of cDNA ends (RACE) analysis. Recently, Egland et al.33 reported a 466-bp transcript as XAGE-1b by a primer extension analysis using total RNA isolated from Ewing's sarcoma cell line and normal testis. In 5′ RACE analysis using total RNA from OU-LU-6 as a template, we obtained a 468-bp cDNA that was longer at the 5′ end than those described above. Due to the GC-rich nucleotide sequence at the 5′ end, the 5′ RACE may not reveal the full-length extension of the cDNA. However, the 468-bp PCR product appeared not to be derived from XAGE-1a in OU-LU-6 because no expression of XAGE-1a mRNA was observed. The 468-bp size was the same as that of the longest SEREX-defined clones. Furthermore, we obtained a PCR product that was identical to the 468 bp. These findings suggested that the 468-bp transcript would be the XAGE-1b transcript.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Drs. L.J. Old and J. Skipper for continuous encouragement and advice during our study. We also thank Mr. Y. Isomoto (Central Research Laboratory, Okayama University Medical School) for DNA sequencing, Ms. M. Isobe and Ms. T. Akimoto for excellent technical assistance and Ms. J. Mizuuchi for preparation of the article.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES