Clinical Division of Oncology, Department of Medicine I, University Hospital, Vienna, Austria; b

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1 The Oncologist Aberrant DNA Methylation in Lung Cancer: Biological and Clinical Implications SABINE ZÖCHBAUER-MÜLLER, a JOHN D. MINNA, b ADI F. GAZDAR b a Clinical Division of Oncology, Department of Medicine I, University Hospital, Vienna, Austria; b Hamon Center for Therapeutic Oncology Research, The University of Texas, Southwestern Medical Center at Dallas, Dallas, Texas, USA ABSTRACT Key Words. Methylation Tumor suppressor gene Lung cancer Preneoplastic lesion Risk assessment Genetic abnormalities of proto-oncogenes and tumor suppressor genes are well-known changes that are frequently involved in lung cancer pathogenesis. However, another mechanism for inactivation of tumor suppressor genes is coming more and more into focus. Epigenetic inactivation of certain tumor suppressor genes by aberrant promoter methylation is frequently observed in lung carcinomas and seems to play an important role in the pathogenesis of this tumor type. While genetic abnormalities are associated with changes in DNA sequence, epigenetic events may lead to changes in gene expression that occur without changes in DNA sequence. Recent findings demonstrate that aberrant methylation can also be detected in the smoking-damaged bronchial epithelium INTRODUCTION Lung cancer is one of the most prevalent cancers and is the leading cause of cancer deaths in the world. So far, enormous progress has been made in understanding the molecular and cellular biology of lung cancer, however, a lot more work needs to be done to completely understand the pathogenesis of this disease. Tumor suppressor genes involved in cancer pathogenesis require inactivation of both alleles. One allele is frequently inactivated by allelic loss, while the other one is inactivated by multiple mechanisms, including point mutations and homozygous deletions, or by a process known as aberrant methylation, a process that is limited to certain cytosine from cancer-free heavy smokers, suggesting that aberrant methylation might be an ideal candidate biomarker for lung cancer risk assessment and monitoring of chemoprevention trials. Moreover, in vitro studies demonstrate that methylation can be reversed by demethylating agents resulting in gene re-expression. This concept is currently under investigation in clinical trials. In summary, recent studies demonstrate that aberrant methylation may be the most common mechanism of inactivating cancer-related genes in lung cancer, occurs already in smoking-damaged bronchial epithelium from cancer-free individuals, can be reversed in vitro by demethylating agents, and may be a useful biomarker for lung cancer risk assessment. The Oncologist 2002;7: nucleotides. Cytosine methylation is a postreplicative epigenetic modification of DNA that plays a crucial role in physiology and carcinogenesis [1]. In vertebrates, methylation is limited to the dinucleotide CpG. The distribution and methylation status of CpG sites are nonrandom. CpG sites occur relatively infrequently in much of the human genome except for discreet CpG-rich regions known as CpG islands. These islands are ~200-1,000 bp in length and often coincide with the 5 ends of genes. There are approximately 29,000 CpG islands in the human genome, although estimates vary widely, depending on the stringency of the definition [2]. It is becoming increasingly apparent that aberrant methylation (referred to as methylation) of the promoter regions of genes Correspondence: Sabine Zöchbauer-Müller, M.D., Clinical Division of Oncology, Department of Medicine I, University Hospital, Währinger Gürtel 18-20, 1090 Vienna, Austria. Telephone: ; Fax: ; sabine.zoechbauer@akh-wien.ac.at Received July 19, 2002; accepted for publication August 15, AlphaMed Press /2002/$5.00/0 The Oncologist 2002;7:

2 452 Methylation in Lung Cancer is the major mechanism of gene silencing in tumors [3]. Recently, DNA methylation and chromatin structure have been linked. Methylated DNA recruits methyl binding proteins, which attract a chromatin-remodeling complex along with proteins that modify histones by deacetylating them, thus closing down DNA to transcription [1]. Several methods can be used to detect aberrant promoter methylation, e.g., bisulfite genomic sequencing and methylation-specific polymerase (MSP) chain reaction (Fig. 1). The principle of the MSP assay was described by Herman et al. [4]. Treatment of genomic DNA with sodium bisulfite converts unmethylated, but not methylated, cytosines to uracil, which are then converted to thymidine during the subsequent polymerase chain reaction (PCR) step giving sequence differences between methylated and unmethylated DNA. PCR primers that distinguish between methylated and unmethylated DNA sequences were then used. The authors of that study reported that 0.1% of methylated DNA present in an otherwise unmethylated DNA sample could be detected consistently, a finding that was confirmed by other authors [5]. Thus, these results suggest that MSP is a very sensitive and fast method to detect methylation. Very recently, an approach to detect methylation by using fluorescence-based, real-time quantitative PCR has been described, and the authors of that study reported an even 10-fold higher sensitivity of that method compared with MSP [6-8]. FREQUENTLY METHYLATED TUMOR SUPPRESSOR GENES IN LUNG CARCINOMAS So far, several known and putative tumor suppressor genes (TSGs) have been identified that are involved in the pathogenesis of lung cancer and are frequently inactivated by methylation. The retinoic acid receptor β-2 (RARβ) gene located at 3p24 has been intensively studied in lung cancer and found to have defective function, thus making it a candidate TSG. RARβ functions as a key retinoid receptor, mediating growth control responses [9-12]. Frequent loss of RARβ mrna expression has been described in both primary nonsmall cell lung cancers (NSCLCs) and bronchial biopsy specimens from heavy smokers [12-14]. In addition, diminished or absent RARβ protein expression was seen in ~50% of resected NSCLCs [15]. Virmani et al. [16] identified methylation as the underlying mechanism for this frequent loss of RARβ expression. Twenty-one of 49 (43%) primary resected NSCLC samples showed RARβ methylation. In addition, the authors demonstrated that RARβ methylation was also important in the pathogenesis of small cell lung cancers (SCLCs), finding 62% of SCLCs methylated for RARβ. A close correlation between methylation of RARβ and loss of RARβ expression was found. UMUM UMUM RARβ U M U M p16 UM UM Another 3p TSG gene, located at 3p21.3, which is frequently deleted, is the RASSF1 gene. This gene has a predicted Ras association domain and homology to the Ras effector Nore 1 [17, 18]. The RASSF1 gene encodes several major transcripts that are produced by alternative promoter selection and alternative mrna splicing. mrna expression of one of these transcripts, RASSF1A, is frequently lost in lung cancer. While mutations are rare, Dammann et al. [17] identified methylation of the RASSF1A gene as the major mechanism for silencing this gene. Using sodium bisulfite sequencing, 40% of primary NSCLCs showed RASSF1A methylation. Burbee et al. [18] investigated the frequency of RASSF1A methylation in resected primary NSCLCs by MSP. Thirty-two of 107 (30%) NSCLC samples were RASSF1A methylated. However, 79% of SCLCs are RASSF1A methylated, suggesting a different pattern of methylation between NSCLCs and SCLCs [19]. The FHIT (fragile histidine triad) gene, a candidate TSG that spans the FRA3B common fragile site at 3p14.2, was found to be frequently abnormal in lung cancer [20, 21]. Aberrant FHIT transcripts were detected in 80% of SCLC and 40% of NSCLC specimens [20, 21], and absent Fhit protein expression was found in ~50% of all lung cancers [22, 23]. In a recent manuscript, we reported that FHIT was frequently methylated in primary NSCLCs [24]. Forty of 107 (37%) resected primary NSCLC samples were found to be FHIT methylated. In addition, there was a significant Controls POS NEG UM UM Controls POS NEG U M U M Figure 1. Examples for the methylation analysis of the genes RARβ and p16 in primary NSCLCs (TU) and their corresponding nonmalignant lung tissues (NL) from different patients by MSP [31]. Lane U = amplified product with primers recognizing unmethylated sequence; lane M = amplified product with primers recognizing methylated sequence.

3 Zöchbauer-Müller, Minna, Gazdar 453 correlation between FHIT methylation and loss of Fhit protein expression by immunostaining. The cadherins are a family of cell-surface glycoproteins responsible for homophilic cell recognition and adhesion [25]. Several family members, including CDH1 (E-cadherin) and CDH13 (H-cadherin), are located on the long arm of chromosome 16, a region of frequent allelic loss in lung cancers. CDH13 expression is frequently diminished in lung cancers. Toyooka et al. [26] reported that methylation is the major mechanism for inactivating CDH13. Fifteen of 30 (50%) NSCLC cell lines and 18 of 42 (43%) primary NSCLCs were found to be CDH13 methylated. Also CDH1 is frequently methylated in lung carcinomas [27]. The adenomatous polyposis coli (APC) gene is a tumor suppressor gene associated with both familial and sporadic colon cancer. Virmani et al. [28] reported that methylation of APC promoter 1A occurs frequently in lung cancers and correlates with loss of APC expression by reverse transcription- PCR (RT-PCR). The p16 INK4a (p16) gene was mapped to the critical region at chromosome 9p21, which frequently undergoes allele loss [29]. p16 functions in the pathway by binding to cyclin-dependent protein kinase 4 (CDK4) and inhibits the ability of CDK4 to interact with cyclin D1 [30]. Several authors reported that methylation of p16 occurs frequently in NSCLCs [31-33]. Belinsky et al. [34] even linked p16 methylation to an early stage in the pathogenesis of lung cancer. Other genes that are frequently silenced by promoter methylation are the DNA repair gene O 6 -methylguanine- DNA-methyltransferase (MGMT) and the apoptosis-associated gene death-associated protein kinase (DAPK) [31, 32]. In a recent study, we investigated the frequency of methylation of eight different genes (RARβ, tissue inhibitor of metalloproteinase-3 [TIMP-3], p16, MGMT, DAPK, CDH1, p14 ARF [p14], and glutathione S-transferase P1 [GSTP1]) in a large number of resected primary NSCLCs and also in the corresponding nonmalignant lung tissues [31]. While methylation of RARβ, TIMP-3, p16, MGMT, DAPK, and CDH1 occurred frequently in the tumors, it was not seen in the vast majority of corresponding nonmalignant lung tissues. A total of 82% of the NSCLCs showed methylation of at least one of these genes. Toyooka et al. [27] analyzed the pattern of methylation of seven different genes (APC, CDH13, GSTP1, MGMT, RARβ, CDH1, and RASSF1A) in 198 lung tumors consisting of SCLCs, NSCLCs, and bronchial carcinoids. The profiles of methylated genes in the neuroendocrine tumors (SCLCs and carcinoids) were very different from that of NSCLCs. While the overall pattern of methylation of carcinoids was similar to that of SCLCs, carcinoids had lower frequencies of methylation for some of the genes tested. Table 1. Frequencies of genes methylated in lung cancers and sputum samples from smokers Gene NSCLCs SCLCs Sputum (smokers) APC 46%-96% 15% ND CDH13 43%-45% 15% ND RARβ 40%-43% 45% 27% FHIT 37% 64% a 17% b RASSF1A 30%-40% 79%-85% ND TIMP-3 19%-26% ND ND p16 25%-41% 5% 16%-19% MGMT 16%-27% 16% 16% DAPK 16%-44% ND 17% CDH1 18%-33% 60% ND p14 6%-8% ND ND GSTP1 7%-12% 16% 6% a Data are from tumor cell lines b Data are from bronchial brushes ND = not done Data were extracted from [16, 18, 19, 24, 26-28, 31-33, 35-42]. These results demonstrate that methylation is a major mechanism for the inactivation of certain TSGs in lung cancers (Table 1). METHYLATION IN SERUM DNA AND SPUTUM FROM LUNG CANCER PATIENTS Using MSP, Esteller et al. [35] investigated whether methylation could be detected in serum DNA from lung cancer patients. The authors analyzed primary NSCLCs and serum from 22 patients for the methylation pattern of four TSGs (DAPK, GSTP1, p16, and MGMT). Methylation of at least one of these genes was detected in 68% of NSCLCs. Comparing primary tumors with methylation and matched serum samples, 73% of the matched serum samples were found to be methylated. In addition, none of the sera from patients with tumors not demonstrating methylation were positive. Usadel et al. [36] investigated the frequency of APC methylation in primary NSCLCs and paired preoperative serum or plasma samples of these patients by semiquantitative methylation-specific fluorogenic real-time PCR. Fortyseven percent of serum and/or plasma samples from patients with APC-methylated tumors carried detectable amounts of methylated APC promoter DNA. In contrast, no methylated APC promoter DNA was detected in serum samples from 50 healthy controls. Kersting et al. [37] analyzed the frequency of p16 methylation in exfoliative material from lung cancer patients. p16 methylation was detected in 35% of sputum samples, in

4 454 Methylation in Lung Cancer 22% of bronchial lavage samples, and in 16% of bronchial brushings from patients with lung cancer. assessment as well as for monitoring the response to chemopreventive agents (Table 1). METHYLATION IS AN EARLY EVENT IN THE PATHOGENESIS OF LUNG CANCER Belinsky et al. [34] were the first who determined the timing of p16 methylation in an animal model of lung carcinogenesis and in human squamous cell carcinomas. In the rat, p16 methylation was frequently detected in precursor lesions to lung tumors. In humans, p16 methylation was found in 75% of carcinoma in situ lesions adjacent to squamous cell carcinomas harboring this change. Interestingly, the frequency of p16 methylation increased during disease progression from basal cell hyperplasia to squamous metaplasia to carcinoma in situ. Moreover, the authors were able to detect p16 methylation in sputum samples from cancer-free individuals at high risk for lung cancer. p16 methylation in sputum samples from cancer-free chronic smokers has also been described by Kersting et al. [37]. Four of 25 (16%) sputum samples were p16 methylated. In addition, p16 methylation was found in 12% of bronchial lavage samples and in 8% of bronchial brushings. Palmisano et al. [38] investigated whether lung cancer could be predicted by detection of methylation in sputum samples. Using a highly sensitive PCR approach to detect methylated DNA sequences, the authors reported that methylation of p16 and/or MGMT could be detected in DNA from sputum in 100% of patients with squamous cell lung carcinomas up to 3 years before clinical diagnosis. In addition, the authors described that the prevalence of these markers in sputum from cancer-free, high-risk subjects approximated lifetime risk for lung cancer. We found the FHIT gene frequently methylated in primary lung carcinomas [24]. Moreover, we analyzed sputum samples from heavy smokers without clinical evidence for lung cancer for FHIT methylation and found 6 of 35 (17%) bronchial brushes FHIT methylated. These results suggest that FHIT methylation already occurs in preneoplastic lesions in the smoking-damaged bronchial epithelium of heavy smokers. Moreover, methylation of RARβ can be detected in sputum samples from heavy smokers [39]. Methylation of p16, DAPK, and GSTP1 was detected in bronchial brush samples from former cigarette smokers in 17%, 17%, and 6% of the samples, respectively [40]. However, no correlation was found between methylation in any of these genes and the smoking characteristics of the individuals analyzed. These results demonstrate that methylation of certain genes can be an early event in the pathogenesis of lung cancer and might be a useful biomarker for lung cancer risk DOES METHYLATION CORRELATE WITH CLINICOPATHOLOGICAL CHARACTERISTICS OF LUNG CANCER PATIENTS? The methylation pattern of several genes in primary lung tumors was compared with clinicopathological characteristics from these patients. Burbee et al. [18] reported that RASSF1A methylation may be of prognostic impact in NSCLC patients. s whose tumors were RASSF1A methylated had a shorter overall survival than patients whose tumors were not RASSF1A methylated. Stage I NSCLC patients whose tumors exhibited DAPK methylation had a statistically significantly poorer probability of overall survival at 5 years than those without DAPK methylation [41]. Moreover, the groups with and without DAPK methylation showed a striking difference in the probability of disease-specific survival [41]. A significantly longer survival for NSCLC patients with low APC methylation status than for patients with high APC methylation status was reported by Brabender et al. [8]. A higher prevalence of p16 methylation in squamous cell carcinomas compared with adenocarcinomas was described [42]. Moreover, methylation of p16 was significantly associated with pack-years smoked and duration of smoking, and negatively with the time since quitting smoking [42]. In our study, where we analyzed the methylation status of eight genes, we found lymph node involvement more frequently in samples with any gene methylated than in samples where no genes showed methylation [31]. Moreover, similar to the results of Kim et al. [42], p16 methylation was more frequent in squamous than adenocarcinomas and was only seen in smokers [31]. Although some genes that are frequently inactivated by methylation and are of prognostic impact for lung cancer patients have already been found, additional genes need to be identified. Thus, patients with a worse prognosis could be selected. These patients might benefit from a more aggressive treatment strategy. METHYLATION IS REVERSIBLE BY DEMETHYLATING AGENTS 5-aza-2 -deoxycytidine (5-AZA-CdR) is a potent inhibitor of DNA methylation and an inducer of cellular differentiation [43]. Numerous studies demonstrated that genes that have been silenced by methylation can be re-expressed after treatment with 5-AZA-CdR in vitro [16, 18, 24, 26]. Momparler et al. [44] performed a pilot clinical trial on 5- AZA-CdR in patients with stage IV NSCLC. s were administered an 8-hour i.v. infusion of 5-AZA-CdR for one

5 Zöchbauer-Müller, Minna, Gazdar 455 or more cycles at intervals of 5 to 7 weeks depending on recovery of their granulocyte count. The patients had not received any prior chemotherapy. Seven patients were included, and for all but one, the survival time increased with the number of cycles of 5-AZA-CdR administered. Interestingly, one patient survived more than 6 years. This patient had received five cycles of 5-AZA-CdR. In that study, the authors observed a delayed action of 5-AZA-CdR on tumor growth that began to occur after one or more cell divisions. The authors reported that it seemed that the antitumor activity of 5-AZA-CdR may require three or more cycles of treatment before it becomes evident. Recent studies suggest that there is a synergy between methylation and histone deacetylase (HDAC) activity [3]. This synergy can be mediated directly by HDAC interaction with DNA-methylating enzymes and by recruitment through complexes involving methyl-cytosine binding proteins. The combination of 5-AZA-CdR and the histone deacetylase inhibitor trichostatin A (TSA) has shown promising results on both cell kill and cancer-related gene reactivation in cancer cell lines [45, 46]. Clinical trials with HDAC inhibitors are currently under way [47, 48]. These agents potentially open a new area of lung cancer treatment. POTENTIAL CLINICAL APPLICATIONS OF DETECTING METHYLATION Methylation of certain genes is frequently observed in primary lung carcinomas and can also be detected in the bronchial epithelium of heavy smokers. These findings open a wide spectrum for potential clinical applications of detecting methylation. First of all, detecting methylation in primary lung carcinomas may influence treatment strategies for lung cancer patients. Finding genes of prognostic impact methylated in tumors could allow one to customize therapy for those lung cancer patients. Those patients potentially might benefit from a more aggressive treatment strategy or the use of demethylating agents. Secondly, detection of methylation in the bronchial epithelium of heavy smokers could be used in a population with greater risk for lung cancer to assess the individual risk. Determining the methylation status of several genes and performing a methylation profile could allow the identification of individuals with a very high risk. This approach is currently being tested in clinical trials. In addition, this subgroup of people could then undergo other, but more costly, procedures for early detection of lung cancer, such as spiral computerized tomography scans, and would also be ideal candidates for chemoprevention trials. For this approach, it is important that sputum samples can be obtained in a noninvasive, easy, fast, and inexpensive way. Moreover, the frequency of methylation in sputum samples seems to be comparable with the frequency in bronchial brushings, which can be obtained only by bronchoscopy. Thus, sputum samples are ideal for this kind of study. Finally, one of the most important findings is that methylation can be reversed in vitro. Several studies demonstrated that gene expression could be restored after treatment of cells with demethylating agents, such as 5-AZA-CdR. Although it is too early to make any conclusions about the effect and possible side effects of these drugs in lung cancer patients, this is a very promising concept and needs to be tested in clinical trials with monitoring of methylated markers. SUMMARY The occurrence of genetic abnormalities was thought to be the main mechanism for inactivating TSGs in the pathogenesis of lung cancer. 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