The
Oncologist
®
Aberrant DNA Methylation in Lung Cancer:
Biological and Clinical Implications
SABINE ZÖCHBAUER-MÜLLER,a JOHN D. MINNA,b ADI F. GAZDARb
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
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
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;7:451-457
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
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: 43-1-40400-4429; Fax: 43-1-40400-4451; e-mail:
sabine.zoechbauer@akh-wien.ac.at Received July 19, 2002; accepted for publication August 15, 2002. ©AlphaMed Press
1083-7159/2002/$5.00/0
The Oncologist 2002;7:451-457 www.TheOncologist.com
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A BSTRACT
Methylation in Lung Cancer
452
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.
RARβ
Patient
Patient
Patient
Patient
Controls
NL TU
NL TU NL TU
NL TU POS NEG
U M U M U M U M U M U M U M U M U M UM
p16
Patient
Patient
Patient
Patient Controls
NL TU
NL TU
NL TU POS NEG
NL TU
U M U M U M U M UM U M UM U M 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.
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
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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].
Zöchbauer-Müller, Minna, Gazdar
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
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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 transcriptionPCR (RT-PCR).
The p16INK4a (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 O6-methylguanineDNA-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, p14ARF [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.
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Methylation in 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. Patients 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 5AZA-CdR in patients with stage IV NSCLC. Patients were
administered an 8-hour i.v. infusion of 5-AZA-CdR for one
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22% of bronchial lavage samples, and in 16% of bronchial
brushings from patients with lung cancer.
Zöchbauer-Müller, Minna, Gazdar
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. However, in recent years, aberrant methylation as an epigenetic abnormality has come into focus, and
it has been shown that it may be the most common mechanism for silencing TSGs. Both genetic as well as epigenetic
abnormalities can be detected in preneoplastic lesions from
clinically cancer-free smokers, which could be used in lung
cancer risk assessment and monitoring the effect of chemopreventive drugs. The fact that methylation can be reversed
in vitro and the synergistic effect of the demethylating agent
5-AZA-CdR with the histone deacetylase inhibitor TSA in
vitro raise hope for new treatment strategies for lung cancer
patients. If this concept works in cancer patients, then an
essential step toward prevention and improved treatment of
lung cancer patients could be taken.
ACKNOWLEDGMENT
This work was supported by a National Cancer Institute
Lung Cancer SPORE grant (P50 CA70907).
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