Epigenetic Changes in Lung Cancer: Pathobiological and Clinical Aspects

Within a decade after the publication of the first human genome
sequence, 1 and even before a full understanding of all
its implications has been attained, a new frontier has emerged:
epigenetics. 2 The study of factors superimposed on the genes,
or “epi”genetics, focuses on mitotically heritable modifications
of DNA and histones, and the associated chromatin components
that affect gene expression without altering gene sequence.
3 Epigenetics is one of the most exciting new frontiers
in genome analysis. Two of the most widely studied epigenetic
modifications are DNA methylation and histone modification.
Many of the interacting proteins that bind directly or indirectly
to methylated DNA or modified histones catalyze the
formation or removal of other alterations, forming a complex
regulatory network that is only beginning to be deciphered. 3–6
It has become clear that epigenetic deregulation contributes
very importantly to cancer development and progression. 7–11
Profound epigenetic changes are seen in all cancer types, including
lung cancer. 12–16 Epigenetic alterations in lung cancer
show potential as molecular markers that could be applied to
early detection, tumor classification, risk assessment, prognostication,
and monitoring of cancer recurrence. 17–20 In addition,
understanding the consequences of epigenetic changes
can help dissect the molecular basis of lung cancer, providing
new focal points for targeted therapies.
One of the most exciting aspects of epigenetic changes
is their inherent reversibility. This has encouraged the development
of novel drugs for cancer treatment, such as histone
deacetylase inhibitors (HDACI) and DNA methylation
inhibitors. 21,22 A number of these drugs are in clinical trials
for numerous cancers, including those of the lung. With the
advent of ever more powerful tools for genome-wide assessment,
23 our understanding of the lung cancer epigenome and
its application to diagnosis and treatment promises to increase
dramatically in the years to come. 7 Here, the basic concepts of
epigenetics will be reviewed, and our current knowledge concerning
epigenetic alterations in lung cancer will be discussed,
including the type of changes identified and their pathological
and clinical implications. Given the very large number of
epigenetic alterations analyzed to date and the dramatic acceleration
in acquired data, it is impossible to be comprehensive
in one short chapter. Therefore, the important advances made
in lung cancer research will be illustrated based on a limited
number of key examples, and reviews will be cited throughout
as a source of more detailed information.
GENETIC AND EPIGENETIC INTERACTIONS
Initial research into the molecular basis of lung cancer focused
on genetic alterations, such as mutations, loss of heterozygosity,
deletions, and gene amplification. 24,25 Examples of genetic
alterations in lung cancer include mutations in KRAS and the
epidermal growth factor receptor (EGFR), loss of heterozygosity
at chromosome 3p, and MYC gene amplification. However, it
has become abundantly clear that epigenetic alterations contribute
equally importantly to lung cancer development and progression.
12–14 Epigenetic alterations seen in lung cancer consist of
DNA methylation changes (both loss and gain of methylation),
changes in histone modifications, and alterations in chromatin
structure and chromatin- associated proteins. Interactions between
genetic and epigenetic hits in cancer cells can result in further alterations,
2,11,26,27 as outlined in Figure 7.1. For example, genetic
alterations in the genes encoding components of the epigenetic
machinery (such as DNA methyltransferases and HDACs) can
affect the activity of these enzymes and thereby the transcriptional
activity of many additional genes. In numerous cancers,
including lung cancer, somatic changes in parts of the epigenetic
machinery are seen. 27 This potential for genetic alterations to affect
epigenetics is underscored by the reported link between lung
cancer risk and genetic polymorphisms in several genes encoding
epigenetic enzymes. 27 Conversely, epigenetic alterations can lead
to further genetic damage. For example, hypermethylation of
DNA repair genes or genes encoding detoxification enzymes can
affect the cell’s susceptibility to mutagenesis and could result in
the genetic (in)activation of additional genes. 26 DNA methylation
of 6-O-methylguanine DNA methyltransferase (MGMT),
an enzyme involved in repair of alkylated guanine, is commonly
seen in lung cancer. 15 Inactivation of MGMT has been linked
to an increase in RAS gene mutation frequency. 28 In support of
their potential to affect cancer development, polymorphisms in
MGMT and other DNA repair genes have been linked to lung
cancer risk in various populations. 29–31 These examples illustrate
that genetic and epigenetic changes should not be seen as independent
but as components of a complex interactive network
that is responsible for the development and progression of lung
cancer (Fig. 7.1). Combined analysis of both types of molecular
changes will accelerate the elucidation of the molecular pathways
affected in lung cancer, and could be especially helpful in characterizing
particular types of lung cancer (e.g., histological subtypes
or lung cancer from smokers vs. nonsmokers). This holistic view
of (epi)genetic alterations is also highly relevant to the clinic, as
the use of certain cytotoxic drugs may potentiate or inhibit the
efficacy of epigenetic drugs and vice versa. 21,22
DNA METHYLATION
In mammals, DNA methylation occurs at the 5-position of cytosine,
in the context of a mini palindrome: a cytosine-phosphateguanine
(CpG) dinucleotide. The palindromic nature of methylation
allows the propagation of this modification following
DNA replication. In the normal mammalian genome, some areas
are heavily methylated, such as sections of the inactive X chromosome
in women, pericentromeric regions, and parentally imprinted
genes. Indeed, DNA methylation is essential for proper
development and viability. 3 Methylation in mammals is carried
out by at least three enzymes, the maintenance DNA methyltransferase
DNMT1 (which methylates daughter strands following
DNA replication) and de novo DNA methyltransferases
DNMT3A and 3B. 32 All three genes are essential, as illustrated
by mouse knockout experiments. 32 A large number of splice
isoforms exists, a number of which appear to target particular
genes or areas of the genome and some of which are implicated
in cancer. 33,34 In lung cancer, overexpression of the deltaDNMT3B4
variant correlates strongly with RASSF1A methylation,
and knockdown of this methyltransferase resulted in a rapid
demethylation of the RASSF1A CpG island. 35 This effect was
gene-specific, as no changes in methylation of CDKN2A were
observed.
CpG dinucleotides exist in two general environments in normal
cells: sparsely distributed and clustered. On the one hand,
CpGs are sprinkled throughout the genome, and these CpGs
are usually methylated. Spontaneous deamination of methyl-C
results in thymine, which is less efficiently repaired than the uracil
resulting from deamination of unmethylated cytosine. This has
resulted in depletion over time of CpGs in areas that are usually
methylated. 36 Thus, the remaining dense clusters of CpGs, called
CpG islands, 37 are presumed to be normally unmethylated. It is
estimated that 40% of human genes contain such CpG islands in
their promoter regions. 1
In cancer, a profound disruption of DNA methylation is
seen (see Fig. 7.2, top). 7–11 Global hypomethylation occurs,
which has been proposed to occur very early during cancer development
and results in a net loss of methyl-C. This is thought
to contribute to carcinogenesis in two possible ways: the transcriptional
activation of previously methylated sequences and the
loss of chromosome stability. In contrast, the local hypermethylation
at promoter CpG islands contributes to carcinogenesis
through gene inactivation, silencing a wide variety of growth
control and tumor suppressor genes, such as genes involved in
growth, adhesion, apoptosis, cell cycle, differentiation, signaling,
and transcription.
DNA methylation is by far the best-studied epigenetic
change in many cancers, including lung cancer. This is, on the
one hand, because promoter CpG island hypermethylation
is linked to gene silencing, and such silencing is thought to
play a key role in the development and progression of cancer.
On the other hand, DNA methylation has been extensively
analyzed because it promises to provide powerful molecular
markers for lung cancer. 14,15,38 Importantly, straightforward
techniques exist to assess this modification. 39 Initially, analysis
strategies were based on a target gene approach, utilizing DNA
methylation-sensitive restriction enzymes and polymerase chain
reaction (PCR)-based methods that rely on bisulfite conversion.
Bisulfite conversion, a chemical treatment that converts
unmethylated cytosine to uracil while methylated cytosine is
protected, 40 allows methylation information to be incorporated
into the DNA sequence (otherwise it would be lost during PCR).
Bisulfite-converted DNA can be analyzed by many methods,
depending on the design and location of the PCR primers.
The most common methods are bisulfite genomic sequencing,
methylation-specific PCR (MSP), and its real-time version,
MethyLight or its variation quantitative MSP (QMSP).
Bisulfite genomic sequencing consists of amplification followed
by cloning and sequencing and provides information
on the methylation status of all the Cs on the same DNA
strand in the amplified area. 41 MSP utilizes primers that cover
a number of methylation sites, 42 allowing interrogation of one
or more CpGs in a small area. MethyLight incorporates the
inclusion of a fluorescent probe between the primers, enabling
real-time PCR detection, and includes control reactions on
fully methylated DNA (treated with Sss I enzyme), allowing
quantitative measurement of methylation. 43,44 More recently,
higher throughput and more genome-wide approaches have
been developed, such as restriction landmark genomic scanning
45 and specific amplification/purification of methylated
versus unmethylated DNA (using restriction enzymes 46 or
binding enrichment) 47,48 followed by probing of microarrays.
Expression profiling of cancer cell lines treated with demethylating
drugs has also been used to identify DNA methylationsilenced
genes (e.g., in non–small cell lung cancer [NSCLC]). 49
In the future, direct sequencing with high-throughput methods
23 using either methylation-enriched or bisulfite-treated
DNA will be applied. Although reports based on these latter
methods are beginning to be published, 50 these techniques are
in their infancy and many technical hurdles remain. In addition,
they are extremely costly. At this time, high-throughput
bead-based PCR methods combine the best of both worlds in
richness of data and affordability, allowing the reproducible
and rapid interrogation of thousands of targeted loci (Illumina
Inc., GoldenGate platform,). 51 This approach was successfully
applied to lung adenocarcinoma (Table 7.1) 51 and has recently
been further developed to provide close to genome-wide representation
of CpG islands (Illumina Inc., Infinium platform).
All of these methods have yielded a great amount of information
on DNA methylation changes in many cancers including
lung cancer. This knowledge, and further epigenomic profiling,
promise to change the way in which lung cancer is detected
and treated.
Effects of Hypomethylation in Lung Cancer Because
an overall hypomethylation is observed in cancer cells, it had
originally been assumed that the cancer-causing effect of methylation
changes was based on loss of promoter CpG island
methylation resulting in proto-oncogene activation. 52 Indeed,
loss of methylation can lead to gene activation in lung cancer,
although the activated genes are not necessarily considered canonical
proto-oncogenes. One category of such genes is the
parentally imprinted genes—genes for which either the maternally
or paternally inherited allele is normally methylated.
Hypomethylation can result in loss of imprinting, thereby
contributing to cancer development; biallelic expression of the
normally imprinted insulin-like growth factor 2, mesodermspecific
transcript and H19 genes has been seen in lung cancer
and is thought to contribute to the carcinogenic phenotype. 53,54
Another type of gene that can be activated by hypomethylation
is the family of testis-specific antigens—these genes are usually
methylated and silent in all somatic tissues but the testes. 55
Expression of testis-specific antigens has been noted in many
tumor types including lung cancer, and these antigens are seen
as potential immunotherapy targets. 55–57 Loss of methylation
of transposable elements and repeats is also observed in lung
cancer 58 and can lead to mobility of such elements, causing
further genetic damage. 11 In addition, read-through from such
demethylated elements may result in the aberrant activation of
neighboring genes. Hypomethylation might also play a role in
the activation of microRNAs (miRNAs), many of which are
deregulated in cancer. 59,60 In the lung, the normally methylated
let-7a-3 miRNA was found to be hypomethylated in two
out of eight lung adenocarcinomas and forced overexpression
of this miRNA increased the oncogenic properties of lung cancer
cell line A549. 61
Besides contributing to carcinogenesis through gene activation,
a second consequence of hypomethylation is thought to
be genomic instability. Mice genetically engineered to underexpress
DNA methyltransferases show an increased frequency of
loss of heterozygosity and an elevated incidence of hematopoietic
malignancies. 62 Inactivation of DNMT1 and DNMT3b
in a human colorectal cancer cell line led to aneuploidy. 63
However, there appears to be little evidence that hypomethylation
is severely deleterious in this way in lung cancer. A recent
analysis of methylation of five human squamous cell lung
carcinomas and normal matched tissue showed prominent
hypomethylation of repetitive elements but little methylation
loss in single-copy sequences. 58 This supports the notion that
the effect of hypomethylation in lung cancer might be limited
and that clinical benefits might be achieved with methylationblocking
therapies. Importantly, the leukemia-prone DNMT
hypomorphic mice mentioned previously show a lower incidence
of intestinal cancer, pointing to a protective effect of
hypomethylation in certain tumor types. 64 Indeed, treatment
of a mouse xenograft model for human lung cancer with DNA
methylation and histone deacetylation inhibitors suppressed
tumor growth without apparent toxicity. 65 A similar treatment
of a murine lung cancer model cut lung tumor development
in half, emphasizing the potential of epigenetic drugs for lung
cancer treatment.