Epigenetic basis of lung cancer—DNA methylation and tumor suppressor gene inactivation

Lung cancers turn out to have at least as many
epigenetic alterations as genetic changes. Epige-
netic phenomena are heritable characteristics (phe-
notypes) that cannot be explained by differences
in the primary structure of DNA. In normal cells,
genomic DNA is packaged into chromatin. Chro-
matin regulates the spatial arrangement and acces-
sibility of DNA to transcription factors in the nu-
cleus. DNAmethylation is an important component
of epigenetic gene regulation in normal cells and its
dysregulation is crucial to cellular transformation
on at least two levels: genome-wide hypomethyla-
tion and gene-specific promoter hypermethylation.
Genome-wide hypomethylation affects heterochro-
matic regions of the genome, which do not ordinar-
ily code for protein. These regions were believed to
be transcriptionally inert, or “junk” DNA, but recent
evidence suggests that the transcriptional capacity
genome has been underestimated, and thus could
encode sequences important for cancer [20].
Genome-wide hypomethylation has several im-
plications in preneoplastic cells, affecting both
transcription and genetic integrity. Transcriptional
effects include loss of imprinting, re-expression
of genes involved in fetal development, and
transcriptional activation of repetitive elements
[19,30,31]. The genetic effects are indirect andinvolve larger-scale processes such as overall chro-
matin architecture, aneuploidy, and DNA replica-
tion [20,32,33].
There is overwhelming evidence that tumor-
acquired promoter hypermethylation, leading to
loss of expression of the associated gene, is a com-
mon event during themultistep pathogenesis of hu-
man lung cancer [26,34–37]. Over the past decade,
nearly 150 genes have been identified that show
tumor-specific methylation in primary tumor sam-
ples, includingmany in lung cancer (Table 4.2) [38].
Certain loci are preferentially methylated in certain
cancer types [39,40].Gene-specific promoter hyper-
methylation is an early event in tumorigenesis and
occurs in conjunction with transcriptional silencing
of the associated gene. In addition, aberrant pro-
moter hypermethylation often coincides with loss
of heterozygosity resulting in complete loss of ex-
pression and thus function of the affected locus
[16,37]. However, the molecular mechanisms that
drive tumor-acquired promoter hypermethylation
in cancer progression are not yet known [41].
DNA methylation-dependent transcriptional si-
lencing frequently affects genes that are involved
in transcriptional regulation, DNA repair, negative
regulation of the cell cycle, as well as growth reg-
ulatory signaling pathways (Table 4.2). Similar to
genetic changes, promoter hypermethylation in-
creases during tumor progression. However, in-
creasing promoter hypermethylation also occurs
with increasing age and with carcinogen exposure-
related cancers such as that of the colon and lung
[42]. In the lung, a continuumof increasingmethy-
lation fromhyperplasia through invasive carcinoma
is evident [27,28,34,43,44]. Aberrant promoter hy-
permethylation has been found in a variety of pre-
neoplastic lesions, which supports the hypothesis
that this epigenetic alteration is an early event in
carcinogenesis. This observation has resulted in sub-
stantial interest from the medical community in
that detection of methylation in sputum, blood, or
bronchial washingsmay have utility in the early de-
tection of cancer.
Some genes, such as the important TSG p53, are
never inactivated by promoter hypermethylation
because they do not have a promoter region CpG
islands. Other genes, such as the tumor suppressorgene RASSF1A, which has a prominent CpG island
are nearly always inactivated by LOH and promoter
hypermethylation in both SCLC andNSCLC. Thus, a
curious feature of aberrant promoter hypermethy-
lation is that it does not appear to affect all genes
with equal probability. An even more conspicuous
example of this phenomenon is evidenced by the
difference between p16 and RB; the protein prod-
ucts of these two genes interact directly and inac-
tivation of one or the genes (and thus this regu-
latory pathway) is nearly universal in tumors. In-
terestingly in SCLC, RB (13q14) is nearly univer-
sally inactivated, whereas in NSCLC, it is usually
p16 (9p21) that is lost. Both genes have large CpG
islands in their promoter regions, but only p16 is
methylated with significant frequency, whereas in-
activation of RB almost always occurs through DNA
mutations. This suggests tumor-acquired promoter
hypermethylation is nonrandom, and that there is
something about certain loci that makes them par-
ticularly susceptible to aberrant methylation or to
mutation [37,45].