EPIGENETIC SILENCING AND GENE METHYLATION

In addition to the role of genetic alterations in the pathogenesis
of lung cancer, epigenetic modification, or DNA methylation,
also plays a key role in human carcinogenesis (see also
Chapter 7). Gene promoter regions have CpG islands that are
subject to aberrant hypermethylation by cytosine-DNA methyltransferases.
When this occurs, the composition of chromatin
around the island is modified, and access to the promoter
region by key regulatory proteins involved in transcription is
denied. Through the amplification of methylated alleles in the
promoter region of specific genes, the methylation-specific
PCR (MSP) assay allows for rapid detection of methylation
in genes of interest. As a result, several key genes have been
identified, which are altered by DNA methylation in the development
of lung cancer.
Genes involved in all aspects of cellular function—
regulation of cell cycle, DNA repair, RAS signaling and invasion,
apoptosis—are affected by epigenetic silencing in the development
of NSCLC. Methylation of the death-associated protein
kinase (DAPK) is found in up to 48% of adenocarcinomas
and approximately 25% to 33% of squamous cell carcinomas.
149 DAPK is a serine/threonine kinase involved in apoptosis
resulting from DNA damage, through TNF- , FAS-, or
- interferon–associated pathways, or by downstream activation
of p53. An association has been made between methylation
of DAPK and increasing pathologic stage in NSCLC 150 ; for
those patients with resected stage I tumors, methylated DAPK
has been associated with poorer disease-specific and overall
survival. 151
The family of cadherins includes cell-surface glycoproteins,
which are responsible for adhesion and cell recognition.
Two of these, E-cadherin and H-cadherin , are methylated in
NSCLC. Methylation of E-cadherin ranges from 16% to 45%,
whereas that of H-cadherin was found in approximately 43% of
NSCLCs. 152,153 Impaired or absent expression of E-cadherin
has been linked to poor differentiation, lymph node metastasis
and poorer prognosis and survival in patients with NSCLC. 154
Metastatic potential is also regulated by the tissue inhibitor
of metalloproteinases (TIMPs), which inhibit the proteolytic
activity of MMPs. Methylation of TIMPs has been observed in
19% to 26% of NSCLCs. 153
One of the best studied genes in DNA methylation and
NSCLC is p16 CDKN2A . It is affected in up to 67% of adenocarcinomas
and 70% of squamous cell carcinomas. 155 In experimental
models of cancer development and progression,
methylation of p16 CDKN2A is an early event in lung carcinogenesis,
and its prevalence increases with disease progression. 156
In addition, methylation of p16 CDKN2A is associated with increased
tobacco exposure. 157 In a large study of NSCLCs in
which methylation status of five genes was examined, Toyooka
et al. 158 determined that only methylation of p16 CDKN2A was
associated with poor survival.
The RASSF1 gene is a member of a family of genes that
encode for ras -binding proteins; several different transcripts
are produced by alternative promoter selection and splicing.
mRNA expression of RASSF1A is often lost in NSCLCs; this
led to the observation that RASSF1 is methylated in 30% to
40% of primary NSCLCs. 159,160 However, the association between
methylation of RASSF1A and patient survival is controversial.
While Burbee et al. 159 determined that methylation of
RASSF1A in lung cancer was associated with shorter overall
survival, Toyooka et al. 158 were unable to confirm these findings
in a larger study.
In 2003, Harden et al. 161 examined promoter methylation
in 90 primary stage I lung cancers and their associated lymph
nodes. Methylation at p16 was demonstrated in 15/90 tumors
(17%); 14/90 (16%) at O 6 -methylguanine-DNA-methyltransferase
(MGMT); 7/90 (8%) at glutathione S-transferase P1
(GSTP1), 15/90 (17%) at the DAPK1 gene, and 65/90 (72%)
at the adenomatous polyposis coli (APC) gene. Methylation of
both APC and GSTP1 were more often associated with squamous
morphology and a worse clinical outcome. Interestingly,
data from gene expression profiles have demonstrated significantly
diminished levels for APC in adenocarcinomas, and that
tumors with lower levels of expression showed a trend toward
worse outcome. 102
Gu et al. 162 examined the methylation status in a cohort
of 155 patients with stages I to III NSCLC. Nine genes
were studied: p16, CDH1, TIMP3, RASSF1A, HFIT, APC,
DAPK, MGMT, and GSTP1. The investigators calculated a
methylation index (MI), defined as the ratio of the number
of methylated genes to the number studied, nine in this case.
They determined that the MI was significantly higher in adenocarcinomas
relative to SCCs and in patients with 50 packyear
smoking history relative to 50 pack-years. In addition,
the MI was higher in older patients ( 66 years) relative to the
MI of patients younger than 66 years of age. However, there
was no difference between early stage tumors and late-stage
tumors. Survival analysis revealed that patients with methylation
of CDH1 had a significantly longer survival than those
without. In contrast, patients with p16 methylation had worse
survival than those without p16 methylation. As the number
of unfavorable methylation events increased, the median survival
time was significantly diminished.
Recently, Brock et al. 163 demonstrated a relationship between
gene methylation, specific combinations of gene methylation
and tumor recurrence for patients with resected stage I
NSCLC. From a cohort of 71 patients, seven genes were studied
from tumor and associated mediastinal lymph nodes sampled at
the time of surgery. The four genes with the largest differences
in the frequency of methylation between tumors and controls
were p16, the H-cadherin gene 13 ( CDH13 ), RASSF1A , and
APC . A higher number of methylated genes in each sample was
associated with poorer survival. Specifically, patients with two or
more methylated genes of interest in either the primary tumor
or mediastinal lymph nodes had a 5-year recurrence-free survival
rate of 27.3%, compared to 65.3% for patients with fewer
than two methylated genes of interest. There was an additive
effect to patients whose samples demonstrated methylation of
both p16 and CDH13 . Methylation of both genes in either the
primary tumor, regional or mediastinal lymph nodes, was associated
with significantly shorter recurrence-free survival. Although
these results suggest a possible role for using methylation status
as a means of detecting occult micrometastasis, large-scale, prospective,
multi-institution studies have yet to be conducted.