Abstract
Background: This study analysed the humoral immune response in asbestos exposed lung cancer patients to identify new surrogate markers of the carcinogenic risk in populations exposed to asbestos. Methods and Results: A serological analysis identified five distinct antigens reactive with IgG derived from a lung cancer patient with high asbestos exposure. In one of the isolated antigens, quantitative RT-PCR indicated that annexin A2 (AnxA2) was overexpressed in lung cancer tissues and normal lung from patients with high asbestos exposure. Antibody against AnxA2 was detected in 9/15 (60%) of lung cancer patients with high asbestos exposure; however, in only 1/12 (8%) of lung cancer patients with low asbestos exposure. AnxA2 was also overexpressed in malignant mesothelioma cells, and the antibody was also positive in 8/15 (53%) of patients with malignant mesothelioma. Conclusion: The antibody titer against AnxA2 may be a potentially useful new diagnostic surrogate marker for asbestos-related lung cancer and malignant mesothelioma.
Asbestos has been recognised as a potential health hazard since the 1940s. The workers in many manufacturing sectors such as building construction, automobile manufacturing and boiler building are at the greatest risk of exposure to asbestos, and therefore asbestos-related diseases have developed in many workers in such areas. Environmental exposure to asbestos can also occur for local residents close to asbestos producing or handling factories. The relationship between asbestos exposure and lung cancer was first demonstrated in the 1950s (1), and an aetiological relationship is now widely accepted. While some studies insisted on a close relationship between lung cancer and asbestosis (parenchymal fibrosis), other studies noted that the presence of asbestos fibres may cause lung cancer without asbestosis (2-5). A consensus report by the Helsinki Criteria for Diagnosis and Attribution on Asbestos, Asbestosis, and Cancer clearly stated that it is not necessary to demonstrate asbestosis on chest X-rays or in biopsied tissues in order to determine a causal relationship between asbestos and lung cancer (6). Therefore, the inhalation of asbestos fibres itself may increase the risk of lung cancer before causing any changes in the pleura or lung parenchyma. Various aetiological studies have so far demonstrated that the risk of lung cancer due to exposure to asbestos ranges from 6 to 23% (7-9), however, the precise mechanisms which render asbestos a lung carcinogen have not yet been fully studied.
Recent developments of molecular biological techniques have provided a powerful tool to detect the humoral immune response to cancer. SEREX (serological analysis of antigens by recombinant cDNA expression cloning) is a method that combines molecular cloning and immunological methods by using the patient's serum antibodies against tumour associated antigens (Ags) (10). Several SEREX studies of both non-small cell lung cancer (11-13) and small cell lung cancer (14) have been reported. There are humoral immune responses against tumour associated Ags, and antibodies against these tumour associated Ags may be applicable as tumour markers in the process of clinical follow-up (15-18).
The goal of this study was to identify Ags which potentially induce a humoral immune response in asbestos related lung cancer using the SEREX method.
Materials and Methods
The study protocol was approved by the Human Review Committees of the University of Occupational and Environmental Health, Japan and signed consent was obtained from each patient prior to tissue sampling in the present study.
Patient characteristics and the number of asbestos bodies. The present study was conducted by reviewing the hospital records of 573 lung cancer patients who were treated between January 2001 and December 2007. The subjects were 27 patients with a confirmed histopathological diagnosis of lung cancer and a history of asbestos exposure (Table I). Asbestos exposure was estimated from occupational history obtained by personal interviews, and also evaluated on the basis of the findings of pleural plaques, and diffuse pulmonary fibrosis on chest X-ray and thoracic computed tomography (CT). In addition, the number of asbestos bodies (ABs) in the resected lung was counted under light microscopy (19, 20). Briefly, the normal part of lung tissue resected by surgery was cut into small parts (2-3 mm thick) and dried. The dried samples were treated with hypochlorite digestion and membrane filtration. The number of ABs was counted under a light microscope. An AB count higher than 1,000 ABs/per gram dry weight (gdw) lung tissue indicated a probable occupational exposure (19, 20).
Cell lines and tissues. Surgically resected tumour and paired normal lung tissue specimens were frozen and kept at −80°C until analysis. The histological types of lung cancer cell lines used in this study, were: adenocarcinoma (A110L, A129L, C422L, D611L, F1121L), adenosquamous cell carcinoma (A529L), squamous cell carcinoma (B1203L, C1026L), large cell carcinoma (A904L), pleomorphic carcinoma (G603L), mesothelioma sarcomatoid type (K921MSO, L324MSO), mesothelioma biphasic type (N407MSO) and mesothelioma unknown type (ACC-MESO-1, ACC-MESO-4, 21, 22). A commercially available panel of 20 normal tissues including adrenal gland, bone marrow, brain, foetal brain, foetal liver, heart, kidney, liver, lung, placenta, prostate, salivary gland, skeletal muscle, testis, thymus, thyroid gland, trachea, uterus, colon and spinal cord (Clontech Laboratories, Palo Alto, CA, USA) were used as normal controls.
Construction of a cDNA library and the SEREX method. A cDNA expression library was constructed from K921MSO cells in a lambda ZAP Express vector using a cDNA library kit (Stratagene, La Jolla, CA, USA). A total of 6×106 phage plaques were immunoscreened with the serum from an asbestos exposed lung cancer patient (case no. 1) as described previously (15). The SEREX method can detect IgG Ab against specific antigen. Briefly, the serum samples obtained at surgery from the patient were pooled, frozen and kept at −20°C until used. The pooled serum was diluted 1:200 by Tris-buffered saline (TBS) containing 1% bovine serum albumin (BSA) and 0.02% NaN3, and was applied using the SEREX method. When the positive cDNA clones were obtained, each DNA base arrangement of all clones was sequenced. The sequencing reactions were performed using ABI PRISM™ 310 (PE Biosystems, Tokyo, Japan) automated sequencers (15-18).
Real-time quantitative RT-PCR. Total RNAs from the frozen tissue specimens and cell lines were obtained using the RNeasy kit (QIAGEN Science, MD, USA). RNA was converted to cDNA using a First Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Tokyo, Japan). Real-time quantitative PCR of the AnxA2 gene was performed using TaqMan Universal PCR Master Mix (Applied Biosystems, Foster, CA, USA) in the ABI PRISM 7000 System (Applied Biosystems). The PCR conditions were: initial denaturation at 95°C for 10 minutes, followed by 50 cycles of 9°C for 15 seconds, and 6°C for 1 minute. The primers for AnxA2 were: forward, ACT TCC GCA AGC TGA TGG TT (20 mer), reverse, TCC TCT TCA CTC CAG CGT CA (20 mer), probe, TGC TCT TCT ACC CTT TGC CAG GCC (24 mer), and the primers were synthesised commercially by Hokkaido System Science, Sapporo, Japan. The threshold of cycle number (CT) was defined as the fractional cycle number at which the amount of amplified target product reached a fixed threshold. ΔCT was obtained by comparing the CT of AnxA2 with CT of β-actin in the same amount of templates. Relative quantification was achieved by comparison with the ΔCT of normal lung tissue. The relative expression was calculated by the formula:
Analysis of the serum antibody titer against the identified antigens by phage plaque assay. A plaque assay was used for antibody titration against immunoreactive clones, as described previously (15-18). Briefly, the phages from positive clones were mixed with an irrelevant phage as an internal negative control at a ratio 1:2, and used to infect E. coli. Plaques were blotted onto nitrocellulose membranes and washed. Next, they were incubated with sera at 1:200, 1:1,000, 1:5,000 and 1:25,000 dilutions with Tris-buffered saline containing 0.02% NaN3, which had been pre-absorbed with E. coli. The spots were evaluated as being positive only if test clones were clearly distinguishable.
Analysis of the serum antibody titer against the identified antigens by ELISA. Three antigenic peptides (peptide1: 2-22aa, peptide2: 63-84aa. peptide3: 302-330aa) of AnxA2 were predicted with a computed algorithm system on the basis of their hydrophilicity, probability of surface exposure, flexibility and antigenic index from the base sequence of the open reading frame, and synthesised commercially to over 95% purity by BEX Co. (Tokyo, Japan). Each synthesised peptide of AnxA2 was diluted in PBS and transferred onto a flat-bottomed 96-well ELISA plate (Iwaki; Asahi Techno Glass, Chiba, Japan) at 4°C overnight. A serial dilution of the peptides (peptide2 and 3) revealed that the optimal peptide concentrations of AnxA2 were 1 μg/ml and 100 ng/ml, respectively. The plates were washed with PBS in Tween 80 and blocked with 1% BSA in PBS at room temperature for 1 h. The plates were washed three times and the patient sera (diluted 1/100, 1/500, 1/2,500 and 1/12,500 in PBS containing 1% BSA) were added and incubated at 4°C overnight. The plates were washed and incubated with the secondary antibody, horseradish peroxidase-conjugated goat antihuman IgG (H+L-chain; Medical and Biological Laboratories, Nagoya, Japan) at 1/4,000 dilution for 5 h at 4°C. The plates were washed and incubated with tetramethylbenzidine substrate solution (1, 2-phenylenediamine dihydrochloride; Thermo Fisher Scientific, Waltham, MA, USA) for 20 min at room temperature. The reaction was stopped by adding 0.18 M H2S04 (100 μl), and the absorbance was determined with an iMark Microplate Absorbance Reader (Bio-Rad Laboratories, Hercules, CA, USA) at 450 nm.
Measurement of IL-6 in serum. The serum levels of IL-6 in asbestos exposed lung cancer patients were measured using a commercially available ELISA kit (Endogen, Boston, MA, USA). IL-6 was measured according to the manufacturer's instructions and the sensitivity limit of the kit was 4 pg/ml.
Results
The number of asbestos bodies in the lung of asbestos related lung cancer patients. The ABs of resected lung tissues in 27 lung cancer patients were examined to evaluate asbestos exposure. All 27 patients were male, smokers and the range of ABs was 0 to 58,337/ gdw (Table I). Fifteen of 27 had a count higher than 1,000 ABs/gdw. In particular, 6 of 27 showed higher than 5,000 ABs/gdw. Antibodies derived from case no.1 were used in the following experiments, because case no. 1 had the highest number of ABs (58.337 ABs/gdw) in the resected lung tissues (Table I).
Identification of antigens recognised by IgG antibody derived from asbestos exposed lung cancer patients. The SEREX method using the serum of case no. 1 identified a total of five positive cDNA clones and the DNA base sequence of all clones were determined. A homology search using the BLAST (basic local alignment search tool) database revealed that all five genes corresponded to previously identified genes, which had no reported association with asbestos exposure (Table II). Clone 1 (annexin A2: AnxA2) encodes a member of the annexin family. Members of this calcium dependent, phospholipid binding protein family play a pivotal role in the regulation of cellular growth and in signal transduction pathways (23). Clone 2 is registered as transforming, acidic coiled-coil containing protein 3 (TACC3), and its function is not yet known. However, it may be involved in cell growth and differentiation, and the expression of this gene is ubiquitous and upregulated in some cancer cell lines (24). Clone 3 (coiled-coil domain containing 136: CCDC 136) is also known as NAG6. NAG6 gene maps at chromosome 7q31-32, the common loss of heterozygosity (LOH) site in various human malignancies (25). NAG6 is down-regulated in gastric cancer and thought to be a tumour suppressor gene (25). Clone 4 [TBC (tre-2 oncogene and the yeast cycle regulators BUB2 and cdc16) 1 domain family, member 2: TBC1D2] has been previously identified by SEREX in prostatic cancer (26). This gene may therefore play a role in the regulation of cell differentiation and growth in prostatic cancer. Clone 5 (protein kinase, cAMP-dependent, regulatory, type 1, beta: PRKAR1B) was cAMP-dependent protein kinase (27). The seroreactivity against the isolated Ags was measured in asbestos exposed lung cancer patients in order to examine the correlation between the detection of such Ags and asbestos exposure. Only clone 1 (AnxA2) showed seroreactivity in 10 of 27 patients by the phage plaque assay (Table II).
Expression levels of annexin A2 in asbestos-exposed lung cancer. The expression levels of AnxA2 were analysed by quantitative RT-PCR to examine the recognition of humoral immunity against AnxA2 in asbestos exposed lung cancer patients. Case no.1 had a greater than 4.9-fold higher AnxA2 mRNA level than the autologous lung cancer tissue, when the expression level of the normal lung tissues was considered to be 1.0. Interestingly, the expression in the autologous lung tissue of case no. 1 was 2.8-fold higher than the normal lung (Figure 1). AnxA2 expression was 1.2-fold higher in the heart and placenta among 20 normal tissues in comparison to normal lung tissues, and over 1.3-fold higher in 5 out of 10 lung cancer cells and 3 out of 5 malignant meso-thelioma cells (Figure 2). These results may indicate that overexpressed AnxA2 in tumour and lung tissues could be recognised by a humoral immune response in asbestos exposed lung cancer patients.
Construction of ELISA system for titration of annexin A2 antibody. Three antigenic peptides (peptide1: 2-22aa, peptide2: 63-84 aa, peptide3: 302-330 aa) of AnxA2 were prepared for the construction of the ELISA system. This study first evaluated the correlation of antibody titers against AnxA2 between the phage plaque assay and ELISA. The antibody titers against peptide2 (22 mer, 63-84 aa) plus peptide3 (29 mer, 302-330 aa) of AnxA2 were strongly correlated with the results of the phage plaque assay (Figure 3A). Therefore, the antibody titers against peptide2 and peptide3 of AnxA2 were measured in the ELISA system. The cutoff values of antibody titers were determined on the basis of the mean plus two times the standard deviation of a healthy person's titer. The estimated cutoff value was 0.421. The antibody against AnxA2 was positive in 10 out of 27 (37%) asbestos exposed lung cancer patients, in 4 out of 30 (13%) lung cancer patients and in 1 of 27 (4%) healthy donors (Figure 3B). The titers against AnxA2 were also positive in 8 out of 15 (53%) malignant mesothelioma patients (Figure 3B).
Correlation between the expression levels of annexin A2 and the antibody titer against it. The correlation between the expression levels of AnxA2 and the humoral response against AnxA2 was examined in asbestos exposed lung cancer patients. The 11 lung cancer patients with high asbestos exposure (more than 1,000 ABs) showed that the expression levels of AnxA2 both in tumour tissues (3.3) and lung tissues (3.0) were significantly higher than those in lung cancer patients with less asbestos exposure (less than 1,000 ABs; Figure 4A). Moreover, the antibody titer against AnxA2 was positive in 9 out of 15 (60%) of the high asbestos exposure lung cancer patients, whereas only one patient was positive among the 12 patients with less asbestos exposure (Figure 4B). These results suggest that a humoral immune response was elicited against overexpressed AnxA2 in high asbestos exposure lung cancer patients.
Serum interleukin-6 levels in asbestos-exposed lung cancer patients. Previously, Brichory et al. reported that the humoral immune response against AnxA2 in lung cancer patients is associated with high circulating levels of IL-6 (28). However, asbestos exposure also induces increased levels of serum IL-6 (29). The serum levels of IL-6 were measured in asbestos exposed lung cancer patients (Table I). The 15 lung cancer patients with high asbestos exposure (more than 1,000 ABs) showed higher serum levels of IL-6 than those in lung cancer patients with less asbestos exposure (less than 1,000 ABs; Figure 5A). Moreover, the serum levels of IL-6 were also higher in patients seropositive against AnxA2 among lung cancer patients with asbestos-exposure (Figure 5B). However, there was no significant correlation between them. These results suggest that IL-6 may also play a pivotal role in the humoral immunity of asbestos exposed lung cancer patients.
Discussion
Exposure to asbestos is a risk factor both for pleural mesothelioma and lung cancer. Although numerous studies have demonstrated the carcinogenicity of asbestos, the immunological effects by asbestos exposure have not been fully investigated. The major findings of the present study were: (i) Five distinct antigens have been identified by serum IgG derived from a lung cancer patient with high asbestos exposure; (ii) Among them, quantitative RT-PCR indicated that AnxA2 is overexpressed in lung cancer tissues and normal lung from patients with high asbestos exposure; (iii) Antibody against AnxA2 was detected in 9/15 (60%) of lung cancer patients with high asbestos exposure; however, it was detected in only 1/12 (8%) of lung cancer patients with low asbestos exposure; (iv) AnxA2 was also overexpressed in malignant mesothelioma cells, and the antibody against AnxA2 was also positive in 8/15 (53%) of patients with malignant mesothelioma.
The purpose of this study was the identification of antigens that commonly elicit a humoral immune response in asbestos related malignant disease. Over 1.000 Ags have been identified by SEREX in various malignancies, including lung cancer (10-14), however, some of these Ags are also recognised in an apparently non-cancer related manner and can be classified as naturally occurring autoantigens. IgG from highly asbestos exposed lung cancer patient sera was used to detect asbestos related humoral immune response of malignancies and cDNA from malignant mesothelioma cells. This study isolated five Ags which had not been reported to be associated with asbestos exposure. However, except for clone 5 (protein kinase, cAMP-dependent, regulatory, type 1, beta: PRKAR1B), four of the Ags have been reported to have an association with cancer. Clone 1 (annexin A2: AnxA2) is associated with cancer metastasis in various tumours (30-33). Clone 2 (transforming, acidic coiled-coil containing protein: TACC 3) is upregulated in some cancer cell lines (24). Clone 3 (coiled-coil domain containing 136: CCDC 136) is a tumour suppressor gene, called NAG6 (25). The expression of NAG6 is absent in 57.1% gastric cancer tissues. The downregulation of NAG6 in gastric cancer tissues occurs significantly more frequently than that in corresponding normal tissues. This suggests that NAG6 may be a putative tumour suppressor gene mapped at 7q31-32 loci and could be associated with the development of gastric carcinoma. Clone 4 (TBC1 domain family, member 2: TBC1D2) was identified by SEREX in prostatic cancer (26).
Asbestos exposure modulates the immune response and leads to significant changes of humoral and natural immunity. Hurbánková M et al. reported that asbestos exposure stimulates marked IgG and complement 4 production and IgA, IgM, complement 3 and transferrin production to a lesser extent (34). Alveolar macrophages exposed to asbestos produce TNF-alpha, and TGF-beta1, which may contribute not only to lung fibrosis but also to immune suppression (35). Ilavská et al. reported that occupational exposure to asbestos fibres caused significantly increased levels of serum IgE and such activation markers on eosinophils as CD66b, CD69 and interleukin-6 (29).
Annexins are a family of Ca2+-dependent membrane binding eukaryotic proteins that have been implicated in several functions including membrane trafficking, cell signaling, ion transport, inflammation, apoptosis and hemostasis (23). Human AnxA2 is abundantly expressed in various tissues such as the brain, spleen, kidney, lung, placenta, intestine and adrenal gland. Intracellular roles of AnxA2 may include membrane trafficking and cytoskeletal actin bundling, and extracellular AnxA2 may affect the fibrinolytic pathway (23). AnxA2 has been reported to be related to metastasis of various tumours, as a substrate for kinases and a receptor for the tissue-type plasminogen activator (30-33). Seroreactivity against AnxA2 was reported to be positive in sera from 18 of 54 (33%) patients with lung cancer (28). Interestingly, the IL-6 levels were significantly higher in the sera of antibody-positive lung cancer patients than in antibody negative patients and healthy donors (28). The serum levels of IL-6 were higher in AnxA2 seropositive lung cancer patients with asbestos exposure in the present study (Figure 5). These results indicate that the immune response in lung cancer is modulated by AnxA2 and it is also associated with high circulating levels of an inflammatory cytokine IL-6. The suppression of AnxA2 expression in prostate cancer cells by siRNA demonstrated reduction of IL-6 secretion and recovery of AnxA2 expression by transfection of the AnxA2 gene induced restoration of IL-6 secretion (36). Therefore, the present results suggest that AnxA2 and IL-6 are closely associated in malignancies. IL-6 regulates immune and inflammatory responses, but recent reports suggest that IL-6 expression is also implicated in the regulation of tumour growth and metastatic spread in various types of cancer, including lung cancer (37, 38).
In summary, humoral immunity against AnxA2 is up-regulated, not only in asbestos exposed lung cancer patients but also in malignant mesothelioma patients. AnxA2 could be used for early detection of lung cancer or malignant mesothelioma in the population with asbestos exposure. Future studies must therefore elucidate the function of AnxA2 in asbestos-related malignancies, which may allow for the development of new therapeutic approaches to control asbestos-related malignancies.
Acknowledgements
This work was supported in part by a Research Grant for Promotion of Occupational Health and High-Altitude Research Grant (both from the University of Occupational and Environmental Health, Japan), and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank Dr. Y Morimoto and Dr. A Ohgami (Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Japan) for helpful discussions, Dr. T Hamada (Department of Pathology, Kyushu Rosai Hospital, Japan) for the measurement of asbestos bodies count, and Ms Y Oshibuchi and Ms M Fukumoto for excellent technical assistance.
- Received May 12, 2010.
- Revision received June 6, 2010.
- Accepted May 11, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved