Aberrant methylation of the FHIT gene in chronic smokers with early stage squamous cell carcinoma of the lung
Jin Seuk Kim1,
Hojoong Kim2,
Young Mog Shim3,
Joungho Han4,
Joobae Park1,5 and
Duk-Hwan Kim1,6
1 Center for Genome Research, Samsung Biomedical Research Institute, Seoul, Korea 135-710, 2 Division of Pulmonary and Critical Care Medicine, Samsung Medical Center, Sungkyunkwan University, School of Medicine, Seoul, Korea 135-710, 3 Department of Thoracic Surgery, Samsung Medical Center, Sungkyunkwan University, School of Medicine, Seoul, Korea 135-710, 4 Department of Pathology, Samsung Medical Center, Sungkyunkwan University, School of Medicine, Seoul, Korea 135-710 and 5 Department of Molecular Cell Biology, Sungkyunkwan University, School of Medicine, Suwon, Korea 440-746
6 To whom correspondence should be addressed Email: dukhwan.kim{at}samsung.com
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Abstract
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Fragile histidine triad (FHIT) gene plays an important role in the pathogenesis of lung cancer. However, the clinicopathological significance of CpG island hypermethylation of FHIT gene in non-small cell lung cancer (NSCLC) remains to be elucidated. We studied FHIT methylation in 254 NSCLCs in order to further understand the clinicopathological and prognostic significance of FHIT methylation in NSCLC. Methylation status of the FHIT gene was examined using Methylation-Specific PCR. All statistical analyses were two-sided, with a 5% type I error rate. Hypermethylation of the FHIT gene occurred more frequently in squamous cell carcinoma than adenocarcinoma. For 93 adenocarcinomas there was no statistically significant association between FHIT methylation and age, gender, smoking history, pathologic stage and p16 methylation. However, FHIT methylation in 125 squamous cell carcinomas was associated with exposure to tobacco smoke and p16 methylation, but not with age, gender and pathologic stage. Hypermethylation of FHIT in squamous cell carcinomas occurred more frequently in current smokers (45%) than in never-smokers (13%). FHIT methylation was significantly associated with p16 methylation in current- and ex-smokers (P = 0.02 and P = 0.01, respectively) with squamous cell carcinoma and in patients with pathologic stage I squamous cell carcinoma (P = 0.001). Patients with p16 methylation were 3.74 times [95% confidence interval (CI) = 1.62 7.95; P = 0.001] more likely to have FHIT methylation in squamous cell carcinoma. FHIT methylation in squamous cell carcinoma occurred at a 4.62 times (95% CI = 1.26 34.97; P = 0.02) higher prevalence in current smokers than in never-smokers. No prognostic effect of FHIT methylation was observed in stage I and stage II NSCLCs. In conclusion, hypermethylation of the FHIT gene did not have a prognostic significance in early stage NSCLCs. The FHIT methylation is associated with the p16 methylation and smoking in squamous cell carcinoma, suggesting that FHIT may cooperate with p16 for the development of squamous cell carcinoma of lung in individuals exposed to tobacco smoke.
Abbreviations: CI, confidence interval; FHIT, Fragile histidine triad; NSCLC, non-small cell lung cancer
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Introduction
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Fragile histidine triad (FHIT) is a tumor suppressor gene that spans the FRA3B common fragile site at chromosome 3p14.2, a site that frequently harbors chromosomal aberrations in a variety of cancers, including lung cancer (13). Since FHIT was first cloned by exon trapping and positional cloning in 1996 (4), there was considerable controversy about whether the FHIT gene is a classic tumor suppressor gene. However, recently many have suggested its role as a tumor suppressor (513). The transfer of FHIT gene into FHIT+/ knock mice prevented tumor development (9). FHIT-deficient mice exhibited tumors of forestomach and squamocolumnar junction (11). FHIT re-expression in FHIT negative cells lacking FHIT protein expression was found to suppress tumor formation by induction of apoptosis and control of cell cycle progression in vivo and in vitro (7,10,1215). FHIT gene transfer has been shown to induce apoptosis of and to reduce the tumor growth of epithelial cancer cells from lung (7,12), pancreatic cancer (10), cervical cancer (12), breast cancers (13) and esophageal (16). FHIT protein over-expression also alters the cell cycle profile of malignant cells (7,12,14,16).
Aberrant FHIT transcripts were detected in 80100% of small cell lung cancer (SCLC) and in 4080% of non-small cell lung cancer (NSCLC) specimens (17). In addition, FHIT expression is lost or reduced in NSCLC (1820). Although point mutation within the FHIT gene has not been reported in lung cancer, homozygous deletion and loss of heterozygosity (LOH) at the FHIT locus have been frequently observed in NSCLC (5,21,22). An alternative mechanism to intragenic mutational inactivation of tumor suppressor gene is CpG island hypermethylation at the promoter region. Aberrant methylation of normally unmethylated CpG islands at promoter regions of tumor suppressor genes has an important regulatory effect on gene expression, resulting in transcriptional silencing of genes (23). Abnormal hypermethylation of CpG islands has been found in a variety of human cancers including lung cancer (24).
Recently, aberrant methylation of the FHIT gene was reported in primary NSCLCs, and the methylation was associated with loss of FHIT mRNA and protein expression (25). However, the clinicopathological significance of FHIT hypermethylation in NSCLC remains to be elucidated clearly. In this study, we aimed at examining clinical significance of FHIT hypermethylation in NSCLC. We also investigated the relationship between FHIT and p16 hypermethylation as an approach for further understanding the role of FHIT in the carcinogenesis of NSCLC, since the biological and clinical significance of p16 hypermethylation in NSCLC is known well.
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Materials and methods
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Study population
254 patients who underwent curative surgical resection for NSCLC during the period April 1994 to November 2001 in the Department of Thoracic Surgery at the Samsung Medical Center in Seoul, Korea, participated in this study. Written informed consent for the use of paraffin-embedded tissues was obtained from all patients before the operation. Information regarding smoking habits, demographics and lifestyle factors, were obtained using an interviewer-administered questionnaire. There were 155 males and 99 females, with an age range of 2983 years (mean age 66 ± 10 years at diagnosis). 159 patients had stage I, 68 stage II, 23 stage III and four had stage IV disease. Histological subtypes included 125 squamous cell carcinomas, 93 adenocarcinomas and 36 other cell types, which included large-cell carcinomas and typical carcinoids.
DNA extraction from paraffin block
Formalin-fixed, paraffin wax-embedded tissues were cut into 10 µm thick sections. Before DNA extraction, the sections were placed on slides and stained with hematoxylineosin to evaluate the admixture of non-tumorous tissues. Areas corresponding to tumor or surrounding normal lung tissue were microdissected separately. Microdissected tissues were collected in 15 ml centrifuge tubes, and deparaffinized overnight at 63°C in xylene. After centrifugation at full speed for 5 min, the supernatant was removed. Ethanol was added to the pellet to remove residual xylene, and then removed by centrifugation. After ethanol evaporation, the tissue pellet was re-suspended in lysis buffer ATL (DNeasy Tissue kit, Qiagen) and the genomic DNA was isolated using DNeasy Tissue Kit according to the manufacturer's instruction.
Methylation-Specific PCR
The methylation status of the promoter regions of the FHIT and p16 was determined by Methylation-Specific PCR (MSP), as described by Herman et al. (26) (Figure 1). Two sets of primers were designed, one specific for DNA methylated at the promoter region of each gene and the other specific for unmethylated DNA. The primers and annealing temperatures used for MSP have been described previously (25,26). Briefly, 1 µg of genomic DNA was denatured by incubation with 0.2 M NaOH for 10 min at 37°C. Aliquots of 3 M sodium bisulfite (pH 5.0) (Sigma Chemical, St Louis, MO) and 10 mM hydroquinone (Sigma Chemical) were then added, and the solution was incubated at 50°C for 16 h. The modified DNA was then purified using the Wizard DNA purification System (Promega, Madison, WI), followed by ethanol precipitation. The PCR mixture contained 1 x PCR buffer (50 mM KCl, 67 mM Tris, pH 8.7, 1.5 mM MgCl2), deoxynucleotide triphosphates (each 1.25 mM), primers (300 ng each per reaction), 2.5 U of Taq polymerase and bisulfite-modified DNA (50 ng). Reactions were hot started at 94°C before adding 2.5 U of Taq polymerase. Amplification was carried out over 35 cycles (1 min at 94°C, 1 min at the annealing temperature, 1 min at 72°C), followed by 4 min at 72°C. Fifteen microliters of the PCR reaction were loaded on 2% agarose gel, stained with ethidium bromide (Life Technologies), and visualized under UV illumination. DNA from peripheral blood lymphocytes of a healthy individual was treated with SssI methyltransferase (New England Biolabs, Beverly, MA), subjected to bisulfite modification, and used as a positive control for methylated alleles. DNA from normal lymphocytes served as a positive control for unmethylated alleles and as a negative control for methylated alleles. Negative control samples without DNA were also included in each PCR set.

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Fig. 1. Methylation analysis. MSP for the FHIT and p16 genes was performed using unmethylation-specific (U) and methylation-specific (M) primer sets. Twenty microliters of PCR product was run on 2% metaphore agarose gel, stained with ethidium bromide and visualized under UV illumination. The numbers shown are sample identification numbers.
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Statistical analysis
The MantelHaenszel
2 test was first used to examine whether the association between FHIT methylation and the clinicopathological features is homogenous across histologic subtypes. The Wilcoxon rank sum test and Fisher's exact test (or the
2 test) were used for continuous and categorical variables in univariate analysis, respectively. Multivariate logistic regression was conducted to estimate the relationship between hypermethylation of FHIT gene and the covariates found to be statistically significant in univariate analysis, and to calculate Odds Ratio (OR). The effect of FHIT methylation on time to death was estimated using the KaplanMeier method, and differences between two groups were compared using the log-rank test. Cox proportional hazard regression analysis was used to estimate the hazard ratios of independent factors for survival, after controlling for potential confounding factors such as age, sex, pathologic stage, smoking status. All statistical analyses were two-sided, with a 5% type I error rate.
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Results
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Clinicopathological characteristics and FHIT methylation
The association between FHIT methylation and the clinicopathological features of patients are listed in Tables I and II. FHIT methylation was detected in 68 (27%) of 254 cases with NSCLC. Many groups have reported that the loss of Fhit is more prevalent in squamous cell carcinomas than in adenocarcinomas, suggesting that the effect of Fhit loss on tumorigenesis of lung differs according to histology. Thus, histology itself may be an effect modifier in the pathogenesis of lung cancer. Therefore, we first examined whether the association between FHIT methylation and the clinicopathological features is homogenous across histologic subtypes, using a MantelHaenszel
2 test. The effect of some clinicopathological variables including smoking status and p16 methylation on FHIT methylation was not constant across histology (data not shown), thus we stratified data by histology and analyzed stratum-specific estimates. The mean age of patients was 66 years and was similar in patients with and without FHIT methylation. FHIT methylation occurred in 44 of 155 men (28%) and in 24 of 99 women (24%). Hypermethylation of the FHIT gene was associated with smoking variables (Table I), but their relationship was significantly different according to histologic subtypes (Table II). Hypermethylation of the FHIT gene was not associated with packyears in adenocarcinomas (P = 0.12) but was significantly associated with packyears in squamous cell carcinoma (P = 0.008; Table II). The prevalence of FHIT methylation in adenocarcinomas was similar in current- and never-smokers (30 versus 33%, respectively; Table II), but its prevalence in squamous cell carcinomas was significantly different between current- and never-smokers (45 versus 13%, respectively; P = 0.03; Table II). We did not find a significant relationship between FHIT methylation and pathologic staging. FHIT methylation occurred more frequently in squamous cell carcinomas (32%) than in adenocarcinomas (26%) and other cell types (11%), and this difference was statistically significant (P = 0.04).
Relationship between FHIT and p16 methylation
The relationship between FHIT and p16 methylation was investigated as an approach for further understanding the role of FHIT in the tumorigenesis of lung, since the biological and clinical significance of p16 in NSCLCs has been studied intensively and is known well. The frequency of FHIT methylation in NSCLCs was higher in tumors with p16 methylation than in tumors without p16 methylation (44 versus 20%, respectively; P = 0.001; Table I). However, their relationship showed a significant difference according to histologic subtypes (Table III). In adenocarcinomas, the prevalence of FHIT methylation in patients with p16 methylation was higher than that in patients without p16 methylation (36 versus 23%, respectively), but this difference was not statistically significant (P = 0.20). In contrast, FHIT methylation in squamous cell carcinomas occurred more frequently in patients with p16 methylation than in those without p16 methylation (53 versus 22%, respectively; P = 0.001).
The association between FHIT and p16 methylation in squamous cell carcinomas was further investigated according to smoking status and pathologic stage (Tables IV and V), since abnormalities of FHIT and p16 genes are known to occur in the early stage of NSCLCs and to be associated with exposure to tobacco smoke. A strikingly significant difference was evident between the prevalence of FHIT methylation in patients with and without p16 methylation according to the smoking status in squamous cell carcinomas (Table IV). For current smokers with squamous cell carcinoma FHIT methylation occurred in 65% (13 of 20) of patients with p16 methylation compared with 32% (10 of 31) of those without p16 methylation (P = 0.02). Ex-smokers with squamous cell carcinoma also showed a significant association between FHIT and p16 methylation (P = 0.01). No relationship was found between FHIT and p16 methylation in never-smokers with squamous cell carcinomas (P = 1.00).
The relationship between FHIT and p16 methylation also showed a significant difference according to pathologic stage in squamous cell carcinomas. For those with stage I squamous cell carcinoma FHIT methylation occurred in 58% (15 of 26) of patients with p16 methylation, whereas 18% (9 of 49) of patients without p16 methylation had FHIT methylation. This difference was statistically significant (P = 0.001) (Table V). With a progression of squamous cell carcinomas, no relationship was found between FHIT and p16 methylation.
Multivariate logistic regression analysis
Multivariate logistic regression was performed to control for the potential confounding effects of variables, such as age, sex and pathologic stage, and to calculate OR. This analysis was conducted according to histology, separately. In adenocarcinomas, p16 methylation and smoking status were not associated with FHIT methylation (data not shown). However, in squamous cell carcinomas, patients with p16 methylation had an increased risk of FHIT methylation compared with those without p16 methylation [OR = 3.74, 95% confidence interval (CI) = 1.627.95; P = 0.001; Table VI]. FHIT methylation occurred at 4.62 times higher prevalence in current smokers with squamous cell carcinoma than in never-smokers with squamous cell carcinoma (95% CI = 1.2634.97; P = 0.02).
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Table VI. Multivariate logistic regression analysis of the association between FHIT methylation and clinicopathological features in 124 squamous cell carcinomas
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FHIT methylation and patient prognosis
Data were stratified by disease stage and histology for survival analysis, because stage is an independent risk factor in NSCLCs, and because the effect of FHIT methylation on lung tumorigenesis is considered to be histology dependent. KaplanMeier survival curves demonstrated that the overall survival times in stage I and stage II adenocarcinomas, and in stage I and stage II squamous cell carcinomas were not significantly different (Figure 2). Stratified Cox proportional hazards regression analysis was performed to determine whether FHIT methylation is an independent prognostic factor, after controlling for potential confounding factors, including age, sex, smoking status, p16 methylation and pathologic stage (Table VII). Patients with FHIT methylation were found to have slightly poorer prognosis than those without FHIT methylation in squamous cell carcinoma (HR = 1.27; 95% CI = 0.845.91), but this difference was not statistically significant (P = 0.41).

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Fig. 2. KaplanMeier survival curve. There was no significant difference in survival curves between those patients with and those without FHIT methylation in (a) stage I adenocarcinomas, (b) stage I squamous cell carcinomas, (c) stage II adenocarcinomas and (d) stage II squamous cell carcinomas. The dotted and solid lines indicate groups with and without FHIT methylation, respectively.
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Discussion
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We analyzed the methylation status at the 5' CpG island of FHIT gene in 254 NSCLCs to investigate the clinical significance of FHIT methylation in NSCLCs. FHIT methylation in the present study was found to occur more frequently in squamous cell carcinomas than in adenocarcinomas, supporting previous findings that squamous cell carcinoma is significantly more likely to be Fhit-negative than adenocarcinoma (18,19,27) and that LOH at the FHIT locus is more prevalent in squamous cell carcinoma than in adenocarcinoma (5,21,22). At present, the molecular mechanisms responsible for differential susceptibility to FHIT hypermethylation according to cell type remain unclear, but are probably related to several factors; namely, difference in basal gene expression level according to cell type, differential selective advantage for gene-specific aberrant methylation, differential exposure to environmental carcinogens, differential expression levels of transcription factors responsible for recruiting a repressor complex containing DNA methyltransferases to a specific target, and varying intrinsic susceptibility to epigenetic inactivation.
The relationship between FHIT methylation and the clinicopathological features was analyzed according to histologic subtype. A number of studies have reported that the FHIT gene is specifically targeted by carcinogens in cigarette smoke and the frequency of FHIT abnormalities at the DNA or protein level increase in smokers (18,20,22,27,2832). The present study found that hypermethylation of FHIT was significantly associated with pack-years smoked (P = 0.008) in squamous cell carcinomas. Those with hypermethylation of FHIT in squamous cell carcinomas were found to be more likely to be current smokers than those without FHIT promoter methylation: FHIT methylation in adenocarcinoma occurred at a similar frequency in never-smokers and current smokers (33 versus 30%, respectively; P = 0.45), whereas the prevalence of FHIT methylation in squamous cell carcinoma was higher in current smokers (45%) than in never-smokers (13%) (P = 0.03). However, the small sample size (especially eight never-smokers with squamous cell carcinoma) used in this analysis leads to the broad confidence intervals observed in the regression model. Accordingly, further work is necessary to evaluate the relationship between FHIT methylation and exposure to tobacco in a large sample.
There are also many reports that hypermethylation of p16 is associated with exposure to tobacco smoke. Lung cancers induced by the inhalation of plutonium and cigarette smoke in F344/N rats showed a high frequency of de novo methylation at p16 promoter (33). Hypermethylation of the p16 promoter was detected in 94% of rat adenocarcinomas induced by tobacco specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (34). In addition, hypermethylation of the p16 gene was associated with the duration of smoking in primary NSCLC (35), and was detected in the bronchial epithelium and in the sputum of current and former smokers (36). In our data, the relationship between p16 and FHIT methylation also depended on smoking status. In adenocarcinoma, there was no relationship between p16 and FHIT methylation in smokers and never-smokers. However, we found a statistically significant association between p16 and FHIT methylation in current- and ex-smokers with squamous cell carcinoma, suggesting that Fhit and p16 may cooperate for the development of squamous cell carcinoma in smokers.
Squamous cell carcinoma usually presents at central masses with endobronchial growth, while adenocarcinoma tends to present as peripheral nodules or masses with pleural involvement. Thus, the more centrally located cells of the airways giving rise to squamous cell carcinoma are exposed to a greater concentration of tobacco carcinogens. The effect on carcinogen exposure depends on carcinogen deposition that is influenced by lipid solubility and diffusion between the air interface and the capillary bed. Thus, the profile of carcinogens and perhaps concentration would differ between the central and peripheral lung. Therefore, it is possible that FHIT and p16 in the centrally located cells of airways are more easily methylated by more deposition of carcinogens in smokers than in never-smokers and that this contributes to the development of squamous cell carcinoma in individuals exposed to tobacco smoke.
The loss of FHIT is a very early event in lung cancer (18,19,30), suggesting that FHIT is involved in the initiation of lung cancer tumorigenesis rather than in the progression of lung cancer. No association was found between FHIT methylation and pathologic stage in the present study, indicating that FHIT methylation plays a role in the early pathogenesis of lung cancer rather than in its later metastatic stage. Besides, p16 methylation is known to occur early in the pathogenesis of lung cancer (34). We also found no association between p16 methylation and pathological stage in NSCLC, suggesting that p16 methylation occurs in the early stage of NSCLC. Moreover, we found a statistically strong association between p16 and FHIT methylation in only stage I squamous cell carcinomas. These observations suggest that p16 and FHIT cooperate to inhibit the initiation of squamous cell carcinoma development.
In our cases, the methylation status of p16 and FHIT was the same in 70% (177 of 254) of NSCLCs, whereas 30% of NSCLCs were methylated at either FHIT or p16. In addition, FHIT methylation was found to be significantly associated with p16 methylation in squamous cell carcinomas, but not in adenocarcinomas. However, it is not clear that the two genes cooperate by participating in the same or different biochemical pathway. Simultaneous inactivation of Rb and the p16 locus in the same signaling pathway was rarely found in glioblastomas or in lung cancers (37), whereas epigenetic silencing of genes in the Caspase and GATA signaling pathways was reported (38,39). Hopkins-Donaldson et al. (38) demonstrated aberrant methylation of CpG islands of the death receptor and caspase-8 genes in small cell lung carcinoma cell lines and tumors. Akiyama et al. (39) showed the epigenetic silencing of genes in the GATA signaling pathway in colorectal cancers and gastric cancers. Accordingly, it needs further work to understand the interaction of FHIT and p16 genes at the molecular level.
Finally, we studied the prognostic effects of FHIT methylation in NSCLCs. Several groups have reported the effect of FHIT loss on patient survival, but results are conflicting (18,19,21,27,30,31,40,41). In the present study, we found no prognostic effect of aberrant methylation of FHIT gene in stage I and stage II adenocarcinomas and squamous cell carcinomas, after controlling for potential confounding factors.
In conclusion, CpG island hypermethylation of FHIT did not have a prognostic significance in early stage NSCLCs. The FHIT methylation is associated with the p16 methylation and smoking in squamous cell carcinomas, suggesting that FHIT may cooperate with p16 for the development of squamous cell carcinoma of lung in individuals exposed to tobacco smoke.
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Acknowledgments
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The authors thank Jin-Hyuk Kim for his critical reading of the manuscript. We also thank Eunkyung Kim for assistance with data collection and management. This work was supported by grants from the Samsung Biomedical Research Institute and the Samsung Advanced Institute of Technology.
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Received March 23, 2004;
revised May 31, 2004;
accepted June 10, 2004.