Our results demonstrated that MAGE-A1, -A3 and -B2 antigens are expressed in the airway epithelial cells of a proportion of chronic smokers without lung cancer, supporting our hypothesis that these antigens are activated even prior to the malignant transformation of bronchial cells. This novel finding contradicts the current perception that these antigens are expressed only in cancer cells, and has important clinical implications. Since these antigens are not expressed in normal lung tissue, their activation and expression in heavy smokers might represent an early carcinogenic state. Thus, for the first time, this finding draws attention to the novel significance of MAGE antigens as chemopreventive targets.
To date, type 1 MAGE
antigens have been considered tumor-specific (5
), since they are not expressed in adult normal tissues apart from the testis, (14
), but are expressed in a wide variety of histologically distinct tumor types (17
), including NSCLC (7
). These observations, coupled with the fact that these antigens are immunogenic and can be recognized by autologous cytotoxic T cells (18
), have aroused a great deal of interest and suggest these antigens are ideal cancer immunotherapy targets. One clinical trial investigating patients with malignant melanoma demonstrated anti-tumor activity by vaccination with a MAGE-A3
). In a recently completed randomized phase II clinical trial for patients with surgically resected early stage NSCLC, long-term administration of a MAGE-A3
peptide resulted in an improvement in cancer-free survival (Vansteenkiste J, et al, J Clin Oncol 25: abs. 7554, 2007). This approach is associated with minimal side effects since the testis, the only adult normal tissue expressing MAGE
, does not express MHC I/II molecules and therefore would be immune-exempt, as germ cells cannot present MAGE
proteins. Additionally, the testis has a blood-testis barrier, and is therefore considered an immunologically privileged site (13
). Therefore, immunization of a subject against these antigens, if successful, would be expected to be cell-specific with no direct effects on normal tissues. Thus, immunotherapeutic approaches against these antigens have emerged as a promising and feasible anticancer approach.
However, the present study expands the potential of MAGE
antigens from a therapeutic to a prevention setting. We analyzed 123 bronchial brush samples from 123 unrelated individual former chronic smokers, which allowed for the determination of the frequencies of MAGE
antigen activation in this type of cohort, as well as the relationship with smoking history and cessation. Our observations firmly establish that type 1 MAGE
antigens are expressed in non-malignant adult epithelial cells chronically exposed to tobacco carcinogens. Our findings differ from other reports, which failed to find MAGE
expression in normal tissues (20
). This discrepancy is most likely attributable to the use of different primer sets and a smaller amplicon size in our study, which could have affected the PCR amplification efficiency. It is also possible that the normal lung tissues used in other studies may have contained a limited amount of lung epithelial cells, whereas the bronchial brush samples used in our study consisted mainly of lung epithelial cells.
Considering the malignant potential of these antigens, there is a possibility that the smokers who express these antigens are at a higher risk of lung cancer development as compared to other subjects, since these antigens are known to function as oncoproteins. MAGE-A1
acts as a potent transcriptional repressor by binding to Ski interacting protein and recruiting histone deacetylase 1 (HDAC) (22
). Transfection of a human MAGE-A3
vector into murine myoblast cells was found to enhance resistance of the cells to apoptosis induced by prolonged ER stress, possibly through its binding and inhibiting murine caspase-12 (23
). This suggests that MAGE-A3
may protect malignant or premalignant cells from apoptosis and, therefore, may provide them with a survival advantage. In fact, MAGE A3
expression has been found to be associated with lung tumor progression (11
Expression of MAGE 1
genes could be indicative of a carcinogenic process, since they have been demonstrated to be linked to overall DNA demethylation (24
). Global hypomethylation is a hallmark of most cancer genomes, and is associated with tumor progression (25
). Thus, it is conceivable that MAGE-A
expression is linked to progressive disease. This aberrant activation could confer a growth advantage to these cells as compared to normally methylated cells. The acquisition of DNA demethylation by susceptible cells enables them to grow rapidly and to then progressively overwhelm other cells. Targeting these precancerous cells by specific CTL is attractive, since the remaining not-so-transformed cells would probably show less aggressiveness. Thus, a cancer vaccine preventing the emergence of demethylated MAGE
-expressing precancerous cells could halt the formation and development of cancer.
Although the mechanisms of tobacco-induced expression of MAGE
antigens are yet to be fully elucidated, it is possible that tobacco carcinogens deregulate the methylation pattern of these genes. Alternatively, they may cause aberrant histone modifications and result in expression of MAGE
antigens. Bollati et al
) observed that low-dose exposure to airborne benzene was associated with a significant reduction in LINE-1 (−2.33% for a 10-fold increase in airborne benzene levels; P=0.009), AluI methylation (−1.00%; P=0.027) and hypomethylation in MAGE 1
(−0.49%; P=0.049). In this regard, it is important to note that we recently reported a new subfamily of DNA methyltransferase 3B termed ΔDNMT3B (28
). We observed expression of at least seven variants resulting from alternative splicing in bronchial brush samples from cancer-free chronic smokers (data not shown), suggesting that ΔDNMT3B variants potentially regulate DNA methylation in early carcinogenesis. Some of these variants, particularly ΔDNMT3B5, ΔDNMT3B6 and ΔDNMT3B7, which lack the 3′ C terminal catalytic domain, may be in competition with other ΔDNMT3B variants, resulting in DNA hypomethylation on pericentromeric satellite regions, thus leading to transcriptional activation of MAGE
antigens. However, this hypothesis remains to be confirmed. As chronic smokers often present with chronic pulmonary inflammation, such as chronic bronchitis, it is possible that the MAGE
antigens are activated through the reactive oxygen species and oxidative DNA damage produced by cigarette smoke, which reduces the binding affinity of the methyl-CpG binding protein 2, thereby resulting in epigenetic alterations (29
). Although it is possible that certain hematologic stem cells in the inflammation field might express MAGE
antigens, we previously demonstrated the expression of MAGE
antigens in non-cancerous bronchial epithelium. Since most of the bronchial brush samples we analyzed contained a high purity of epithelial cells, the expression observed in this study was likely derived from bronchial epithelial cells.
We further assessed the effect of smoking cessation on the expression of these antigens. Notably, the frequencies of the expression were not significantly reduced in those who had quit smoking for more than 10 years compared with those who had quit smoking for less than 5 years, suggesting that the activation of these tumor antigens persists even after long-term smoking cessation. This finding is not surprising, and supplements previous observations that molecular alterations observed in the bronchial epithelium of chronic smokers persist for a long time after the cessation of smoking (30
). This might be one of the reasons for the fact that former smokers remain at high risk for developing lung cancer (32
). Future studies with a quantitative RT-PCR approach will further elucidate the true effect of smoking continuance or cessation on MAGE 1
Nevertheless, our findings do suggest that MAGE 1 antigens are ideal immunotherapeutic targets for lung cancer chemoprevention studies, as they are not expressed in normal lung tissue, but are present in a certain subset of heavy smokers, possibly putting those smokers at a higher risk of lung carcinogenesis than others who lack expression but have a comparable smoking history. Although our study was not designed to determine whether individuals with MAGE antigen expression carry a high risk for lung cancer development, the oncogenic properties of these antigens raise the possibility that the expression of MAGE antigens may be a biomarker for lung cancer risk. The peptides derived from these antigens may be utilized for immunotherapeutic approaches to lung cancer prevention, and the identification of individual MAGE genes would allow for an individualized chemoprevention approach using specific tumor antigens for each subject. However, our results also indicate that the presence of markers is heterogeneous, and a subject expressing one marker may or may not express another. In approximately one-third of the cases, more than one of the subtypes was expressed. The high frequency of coexpression of MAGE antigens calls for a polyvalent vaccination approach. Use of multiple immunogens is important, as it increases the probability of inducing a specific immune response and reduces the risk of tumor escape from the immune system by selection of antigen-loss variants.
In conclusion, we demonstrated that MAGE antigens are frequently expressed in lungs with chronic tobacco exposure, even prior to overt malignant transformation. Since these antigens have oncogenic properties, smokers who express these antigens might be at higher risk of lung carcinogenesis, and must be considered for a chemoprevention program. The expression of MAGE antigens in noncancerous cells indicates that the monitoring of these antigens may be of potential interest for determining new immunotherapeutic agents, and warrants development of widely applicable, polyvalent vaccines. Considering the promising clinical results and the safety profile of MAGE antigen vaccines, our findings suggest a possibility of applying the vaccination strategy in a chemoprevention setting for heavy smokers with positive MAGE antigen expression.