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Clinical Cancer Research Vol. 11, 2466-2470, April 2005
© 2005 American Association for Cancer Research

Human Cancer Biology

Differential DNA Hypermethylation of Critical Genes Mediates the Stage-Specific Tobacco Smoke-Induced Neoplastic Progression of Lung Cancer

Andrea L. Russo1, Arunthathi Thiagalingam1, Hongjie Pan1,2, Joseph Califano4, Kuang-hung Cheng1,2,3, Jose F. Ponte1,2, Dharmaraj Chinnappan1, Pratima Nemani1, David Sidransky4 and Sam Thiagalingam1,2,3

Authors' Affiliations: Departments of 1 Medicine (Genetics Program and Cancer Research Center), 2 Genetics and Genomics, and 3 Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts and 4 Department of Otolaryngology-Head and Neck Surgery, Head and Neck Cancer Research Division, Johns Hopkins Medical Institutions, Baltimore, Maryland

Requests for reprints: Sam Thiagalingam, Genetics Program, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118. Phone: 617-638-6013; Fax: 617-638-4275; E-mail: samthia{at}bu.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results and Discussion
 References
 
Promoter DNA methylation status of six genes in samples derived from 27 bronchial epithelial cells and matching blood samples from 22 former/current smokers and five nonsmokers as well as 49 primary non–small cell lung cancer samples with corresponding blood controls was determined using methylation-specific PCR (MSP). Lung tumor tissues showed a significantly higher frequency of promoter DNA methylation in p16, MGMT, and DAPK (P < 0.05; Fisher's exact test). p16 promoter DNA methylation in tumors was observed at consistently higher levels when compared with all the other samples analyzed (P = 0.001; Fisher's exact test). ECAD and DAPK exhibited statistically insignificant differences in their levels of DNA methylation among the tumors and bronchial epithelial cells from the smokers. Interestingly, similar levels of methylation were observed in bronchial epithelial cells and corresponding blood from smokers for all four genes (ECAD, p16, MGMT, and DAPK) that showed smoking/lung cancer–associated methylation changes. In summary, our data suggest that targeted DNA methylation silencing of ECAD and DAPK occurs in the early stages and that of p16 and MGMT in the later stages of lung cancer progression. We also provide preliminary evidence that peripheral lymphocytes could potentially be used as a surrogate for bronchial epithelial cells to detect altered DNA methylation in smokers.

Key Words: DNA Methylation • MSP • tobacco smoke • lung cancer • blood lymphocytes • diagnostic markers

Lung cancer is the leading cause of cancer deaths in the United States, accounting for 28% of all cancer deaths and ~157,200 deaths every year (1). Unfortunately, 36% of non–small cell lung cancer cases are detected at an advanced stage often after micrometastasis has developed, leading to an alarmingly low 5-year survival rate of only 14.9% (1). Because nearly 87% of lung cancer cases are due to smoking, yet only 10% of smokers develop lung cancer, it may be worthwhile to screen for susceptible individuals to administer effective preventive measures (1, 2). Thus, the establishment of a combination of early diagnostic markers that could be analyzed in clinical samples obtained using relatively noninvasive procedures could become an asset to efficient detection of changes in preneoplastic tissue before tumor formation and metastasis can occur.

Differential DNA methylation at CpG islands has been associated with regulation of gene expression and is essential for normal development, X-chromosome inactivation, imprinting, suppression of parasitic DNA sequences, and cancer (3–5). Aberrant differential methylation of CpG islands in the promoter region of genes that are implicated in different roles including carcinogen activation or detoxification (CYP1A1 and GSTP1), tumor suppression (p14, p15, p16, p73, APC, and BRCA1), DNA repair (hMLH1 and MGMT), and metastasis and invasion (CDH1, ECAD, TIMP1, and DAPK) occurs in several cancers, including lung cancer (3–10). Thus, the DNA methylation status of critical genes is not only ideal for use as diagnostic markers but also as therapeutic targets for lung cancer. In this study, we chose to analyze ECAD, p16, DAPK, MGMT, GSTP1, and SMAD8 based on their likely involvement in the genesis of lung cancer. The loss of expression of E-cadherin (ECAD), a Ca2+-dependent adhesion molecule responsible for mediating intercellular contacts in morphogenesis and tissue structure maintenance, has been implicated in a number of cancers, including lung cancer (11). Death-associated protein kinase (DAPK) is involved in DNA damage–induced apoptosis and has been shown to be inactivated via hypermethylation in a number of studies involving non–small cell lung cancer (12, 13). Glutathione S-transferase pi (GSTP1), a gene involved in the detoxification of xenobiotics and oxygen radicals, has been shown to be frequently hypermethylated in glandular cancers such as prostate, breast, and liver cancers but rarely in lung cancer (14).

One of the extensively studied examples for promoter methylation associated decrease in transcription is the tumor suppressor gene p16 (15–19). Alterations in p16 occur frequently in lung and in most common forms of human cancers including gastric, head and neck, and breast cancers and in leukemia. In general, point mutations in the p16 gene are rare and the loss of p16 gene function occurs frequently via transcriptional silencing associated with abnormal DNA methylation of the transcription start site region. O6-Methyl-guanine-DNA methyltransferase (MGMT) is a DNA repair gene that removes adducts from the O6 position of guanine, hence providing protection from alkylating agents. It has recently been shown to be hypermethylated in lung tumors of both smokers and nonsmokers with increased frequency in the later stages of tumorigenesis (20). Our final gene of interest is SMAD8, a receptor-regulated Smad involved in bone morphogenetic protein signaling that has been shown to be inactivated in many cancers but rarely in lung cancer in a limited study (21). Because it seemed to be a major target for inactivation in other cancers except for lung cancer, we decided to extend the analysis of SMAD8 to a larger set of tumors and preneoplastic lung cancer samples to verify and validate the observations made in the initial study.

In summary, we investigated the methylation status of a select group of genes to determine whether we could find unique combinations of methylated genes that could identify distinct stages of non–small cell lung cancer progression providing early diagnostic and therapeutic markers.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results and Discussion
 References
 
Subject enrollment and tissue collection. This study, approved by the Boston University Medical Center Institutional Review Board, recruited volunteers who were nonsmokers, former and current smokers, and those suspected of having lung cancer. Subjects (n = 27) provided information on smoking history and family cancer incidence. Subjects were asked to undergo a bronchoscopy in order to collect bronchial brushings in addition to providing 15 mL of blood.

Tumor specimens. Primary lung carcinoma tissues and pretreatment blood were collected from participants who consented and enrolled with institutional review board approval from the Johns Hopkins Hospital. Primary tumors were snap frozen immediately after resection until further analysis.

Genomic DNA Isolation. Blood samples were allowed to sit in the heparinized tube for 45 to 60 minutes at 37°C to allow for separation of serum and dark red blood. The upper layer containing serum and peripheral blood lymphocytes was combined with 1 mL PBS in a 15-mL tube and centrifuged at 1200 rpm for 25 minutes to pellet the lymphocytes. Genomic DNA isolated from the lymphocytes/microdissected tumor was routinely resuspended in genomic DNA sample buffer [100 mmol/L NaCl, 25 mmol/L EDTA (pH 8), 10 mmol/L Tris-Cl (pH 8), and 0.5% SDS] and digested with proteinase K in 0.1% SDS. Overnight incubation at 58°C was followed by standard phenol-chloroform extraction and sodium acetate and ethanol precipitation.

Bronchial DNA samples were isolated from the DNA fraction of the RNA isolation procedure using the Trizol (Invitrogen, Carlsbad, CA) method and further processed by three sodium citrate washes in 10% ethanol. After the final wash, DNA was suspended in 75% ethanol, centrifuged at 2000 x g for 5 minutes, air dried, and dissolved in 8 mmol/L NaOH.

Methylation-specific PCR. Genomic DNA was chemically modified with sodium bisulfite, which converts all unmethylated cytosines to uracil while all methylated cytosines remain unchanged. Briefly, 0.5 to 1.0 µg of genomic DNA was treated with 2 mol/L NaOH. After a 10-minute incubation at room temperature, samples were treated with 10 mmol/L hydroquinone and 3 mol/L sodium bisulfite and incubated for 16 to 20 hours at 50°C. DNA was purified using the Wizard DNA Purification System (Promega, Madison, WI) according to the manufacturer's protocol. Samples were then desulfonated with 3 mol/L NaOH, precipitated with ammonium acetate, ethanol, and glycogen, and resuspended in distilled H20.

Primer sequences, designed to amplify specifically methylated or unmethylated CpG islands in the promoter region, are listed in Table 1. PCR amplification was carried out using ~50 ng of treated DNA template, 300 ng forward and reverse primers, 0.4 µL of 25 mmol/L deoxynucleotide triphosphates, 2.5 µL 10x PCR buffer, 0.1 µL Platinum Taq polymerase (Invitrogen), and 1.5 µL DMSO. PCR conditions were as follows: 94°C for 2 minutes, 30 to 35 cycles (gene dependent) at 94°C for 30 seconds, 60°C to 66°C (see Table 1) for 40 seconds, 70°C for 40 seconds followed by a final extension at 70°C for 10 minutes. The methylation-specific PCR (MSP) products were routinely analyzed by gel electrophoresis in a 6% mini-acrylamide gel, stained with ethidium bromide, and visualized under UV light.


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  		Table 1. Primers used for MSP

 

   Results and Discussion
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 Abstract
 Materials and Methods
 Results and Discussion
References
 
Methylation-specific PCR was used to evaluate the promoter DNA methylation status of six genes in samples derived from 27 bronchial epithelial cells and matching blood samples from 22 former/current smokers and five nonsmokers as well as 49 primary non–small cell lung cancer samples with matching normal controls.

We screened likely preneoplastic (smoke-exposed bronchial epithelial cells) and neoplastic (primary lung tumors) lesions with their matching blood samples to evaluate the methylation status of six selected genes (ECAD, p16, DAPK, MGMT, GSTP1, and SMAD8) of interest using MSP analyses. Figure 1A illustrates representative examples of the MSP assays that were used in our studies. We wished to identify methylation patterns in smoke-exposed epithelium before any detectable presence of lung cancer by comparing it to primary lung tumors. In examining these differences, we hoped to find specific genes as targets for inactivation through DNA methylation that could eventually be used as early diagnostic markers and therapeutic targets. In addition, by combining our methylation data with that of genetic alterations, it is our hope to identify a minimal set of markers to provide a highly accurate determination of genetic predisposition to and/or the extent of the progression/spread of lung cancer at the time of diagnosis.



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  		Fig. 1. Methylation-specific PCR analysis of selected genes for lung cancer. A, representative examples of MSP analysis of selected genes. MSP was done using primers specific for methylated and unmethylated DNA for the indicated genes. Each reaction included a water control (dH2O), a negative control for methylation using placental DNA (pDNA), and a positive control for methylation using in vitro methylated DNA (IVM). PCR reactions were analyzed on a 6% acrylamide gel and the DNA bands were visualized after staining with ethidium bromide under UV light. BL and BR, blood and bronchial epithelial cells; T and N, tumors and their corresponding blood as the normal control, respectively. B, a comparison of the percentage of promoter hypermethylation of primary tumors and smokers' bronchial epithelial cells with their corresponding blood samples. The percentage of promoter hypermethylations were determined by MSP analysis for ECAD, p16, DAPK, and MGMT. Columns, genomic DNA from primary lung tumors (diagonal stripes) versus corresponding blood samples (filled) and bronchial epithelial cells of smokers (vertical stripes) versus corresponding blood samples from these smokers (open). MGMT was most frequently methylated in tumors followed by DAPK, p16, and ECAD, respectively. GSTP1 and SMAD8 showed no methylation (data not shown).

 
Methylation in preneoplastic bronchial epithelium and blood. We analyzed the bronchial epithelium and blood of the 27 subjects representing the preneoplastic stage, composed of both smokers and nonsmokers for altered promoter DNA methylation patterns (Table 2). Aberrant methylation of ECAD was detected in 27% of smokers' bronchial epithelium (n = 22) and in 41% of smokers' blood. Interestingly, no methylation was found for ECAD in either the blood or bronchial epithelium of nonsmokers (n = 5; data not shown). Similarly, 35% of smokers' bronchial epithelium and 41% of smoker' blood exhibited hypermethylation for DAPK, whereas the remaining samples from nonsmokers were unaffected. Surprisingly, only one sample of the smokers' bronchial epithelial cells and one from the smokers' blood was methylated for p16, whereas one bronchial epithelial cell sample and two blood samples exhibited methylation for MGMT. Methylation of nonsmoker samples remained consistent in that no methylation was observed. GSTP1 and SMAD8 were rarely or never methylated in both smokers and nonsmokers (data not shown).


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  		Table 2. The frequency of promoter DNA hypermethylation in primary lung tumors, bronchial brushings, and blood

 
In summary, analysis of the four frequently methylated sites (DAPK, ECAD, p16, and MGMT) in this study revealed that smoker blood is methylated in at least one site in 64% of samples, whereas bronchial epithelium is methylated 41% of the time. Alternatively, nonsmoker samples showed methylation in zero of four sites, 100% of the time (n = 5). Although a recent report suggest that one can detect a low level of DAPK methylation in normal lymphocytes using quantitative MSP, in the current study, using standard MSP, we clearly detected methylation only in the blood and bronchial epithelial cell samples from the smokers and not in the blood and bronchial epithelial cell samples of nonsmokers (data not shown), which has also been confirmed in other previous reports (13, 22, 23). Although our data strongly support that high levels of DAPK hypermethylation is diagnostic of lung cancer predisposition, the low levels of DAPK hypermethylation detected using a more sensitive quantitative MSP method is also interesting and requires further study as it could have physiologic implications (22, 24).

Methylation in primary lung tumors and corresponding pretreatment blood. We analyzed 49 resected tumors and corresponding blood samples to determine the aberrant methylation of p16, ECAD, DAPK, MGMT, GSTP1 and SMAD8 by MSP assays (Fig. 1A and B). Overall, our results indicated that four of six genes (ECAD, p16, MGMT, and DAPK) are frequently methylated (more than 35%), whereas two genes (GSTP1 and SMAD8) are seldom or never methylated in tumor tissues (Table 2 and data not shown). The corresponding blood samples showed significantly lower levels of methylation: ECAD (22%), p16 (8%), MGMT (18%), DAPK (4%), GSTP1 (0%), and SMAD8 (0%). GSTP1 and SMAD8 were not methylated in any of the samples analyzed. MGMT was the most frequently methylated gene (55%) in tumor tissues, which was followed by DAPK (45%), p16 (44%) and ECAD (37%) in a decreasing order of incidence.

Overall, there was a significantly higher level of methylation observed for the p16 gene in tumors compared with all the other samples analyzed (P = 0.001/0.003; Fisher's exact test; Fig. 1B; Table 2). Interestingly, the levels of methylation observed in pretreatment blood matching the tumors were observed at relatively lower frequencies. When we focused on the four genes most frequently differentially methylated (p16, MGMT, DAPK, and ECAD), methylation occurrence was zero of the four sites in 35% of corresponding blood samples, whereas only 16% of the tumor samples exhibited lack of methylation. This observation may be derived from the natural turnover of blood cells and corresponding decrease in smoking frequency after diagnosis of lung cancer in these patients. In contrast, methylation was observed in four of four sites in 10% of malignant tissue, whereas it was 0% in all the other samples tested (i.e., blood, bronchial epithelial cells). Furthermore, DNA methylation in two or more sites analyzed was observed at significantly higher levels in the tumors compared with either smokers' bronchial epithelial cells (55% versus 19%, P = 0.02) or the smokers' blood (55% versus 27%, P = 0.04) samples.

Differential DNA methylation patterns could be used to distinguish preneoplastic cells from tumors. ECAD and DAPK exhibited statistically insignificant differences in their levels of methylation among the tumor and bronchial epithelial cell and blood samples from smokers (Table 2). On the contrary, promoter DNA methylation was observed at a relatively higher frequency for the MGMT and p16 genes in tumors when compared with the other test samples (Table 2). Interestingly, similar levels of methylation were observed in bronchial epithelial cells and blood from the smokers for all four genes (ECAD, p16, MGMT, and DAPK), whereas no methylation was detectable for the same genes in nonsmokers' bronchial epithelial cells and blood (Table 2, Fig. 1B, data not shown). In addition, neither GSTP1 nor SMAD8 exhibited lung cancer- or smoking-related increases in promoter DNA methylation (data not shown).

In summary, our data suggest that ECAD and DAPK are targeted for methylation in the earliest stages of lung cancer, whereas DNA methylation silencing of p16 and hMGMT are likely alterations that occur in the later stages of cancer progression and are often diagnostic of advanced lung cancer (Fig. 2). Interestingly, a recent study reporting the analysis of tobacco smoke–induced murine lung tumors also suggested that promoter DNA methylation of DAPK is an early event in adenocarcinoma development (25). Our data also suggest that when the four genes (ECAD, p16, MGMT, and DAPK) that exhibited differential methylation upon exposure to tobacco smoke or in lung cancer are evaluated, the frequency of methylation in two or more sites could be used as diagnostic of cancer provided that the altered/tumor cells are present in the clinical samples.



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  		Fig. 2. Tumor stage–specific targeting for promoter DNA methylations in lung cancer. A unique group of genes is targeted for gene silencing during distinct stages of lung cancer progression. ECAD and DAPK methylation are early and p16 and hMGMT methylation likely late events during lung cancer progression.

 
Conclusions
We found that DNA methylation changes that affect critical gene expression patterns occur at increments from the initial stages of lung cancer to late stages of lung cancer (Fig. 2). Our studies identify that promoter DNA methylation at DAPK and ECAD are early events, whereas DNA methylation at p16 and MGMT are late events (Fig. 2). Thus, these and other alterations identified from comprehensive studies could aid in predicting the extent of cancer progression or predisposition to subsequent cancer development. Furthermore, our studies also showed that peripheral lymphocytes from the smokers could potentially substitute for test samples such as bronchial epithelial cells that are acquired by using more invasive methods to detect DNA methylation changes as diagnostic/prognostic markers. Thus, in terms of determining susceptibility to lung cancer at the preneoplastic stage, our findings provide strong preliminary data, which upon confirmation in a larger population study could lead to the use of blood from the smokers as a reliable surrogate for detecting similar changes that could be found in bronchial epithelial cells. Therefore, our findings could have implications for the use of alternate clinical sample sources for diagnostic testing for lung cancer risk and susceptibility and could enable one to secure clinical samples with minimum discomfort to the patient in an economically feasible manner and which are within reach of all those who are likely to be affected.


   Acknowledgments
 
We thank our colleagues Avrum Spira, Gang Liu, and Jerome Brody (Pulmonary Center, Boston University School of Medicine) for generously helping to collect clinical samples and Jerome Brody for critical reading of the manuscript.


   Footnotes
 
Grant support: National Institute of Environmental Health Sciences grant ES 10377 (S. Thiagalingam) and NIH training grant T32HL07035 (J.F. Ponte). S. Thiagalingam is a Dolphin Trust investigator supported by the Medical Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: A. Thiagalingam is presently at Bayer Corporation, 333 Coney Street, East Walpole, MA 02032.

Received 9/23/04; revised 11/16/04; accepted 12/30/04.


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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
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