Background
Lung cancer is the number one cancer killer in China and soon will reach epidemic levels worldwide [
1]. Epidemiological evidence has documented that approximately 41.8 men and 19.3 women per 100,000 Chinese individuals died of lung cancer in 2005 [
2]. This disease is largely associated with smoking. While in developed countries smoking rates are decreasing, the use of tobacco products is increasing in the developing countries. In combination with a spike in the number of lung cancer cases in never smokers, this ensures that lung cancer will remain a major health problem [
3]. Lung cancer is clinically divided into two subtypes, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The latter is the most common type, accounting for 85-90% of the total cases [
3]. Although recent studies have shown that adjuvant chemotherapy improves survival in completely resected NSCLC [
4,
5], only 5-15% of treated individuals ultimately benefit [
6]. Despite the fact that the cause of most lung cancer is well know, the disease has proven difficult to diagnosis early and treat successfully, reflecting limited advances in our understanding of the molecular mechanisms underlying lung carcinogenesis.
Currently, mutations in the
K-ras oncogene,
p53 tumor suppressor gene, and epidermal growth factor receptor (
EGFR) gene have been found frequently in lung tumors and implicated in lung carcinogenesis [
7‐
9]. In addition, genomic abnormalities represent another major signature of neoplastic transformation and tumor progression [
10]. Chromosome copy number abnormalities in lung cancer have been frequently identified using comparative genomic hybridization (CGH) assay [
11,
12], including genomic amplification of
PIK3CA which codes the phosphatidylinositol-3-kinase (PI3K) catalytic subunit α [
13]. PIK3CA is generally activated by a series of cell surface tyrosine kinase receptors [
14]. Upon activation, PIK3CA binds to its heterodimer, p85, and promotes the phosphorylation of Akt. Activated Akt phosphorylates down-stream protein effectors and amplifies the signaling cascade, enhancing cell proliferation and survival [
15]. The previous studies have shown that
PIK3CA amplification was more frequently observed in squamous cell carcinomas (SCC) than adenocarcinomas (ADC), which was closely associated with increased Akt activity in SCC [
12,
13], suggesting that
PIK3CA amplification, in addition to
K-ras and
EGFR mutations, may be another major cause of overactivation of PI3K/Akt pathway that promotes lung tumorigenesis.
In addition to genetic factors, promoter methylation is an alternative mechanism underlying inactivation of tumor-associated genes in lung carcinogenesis [
16,
17]. Currently, methylated gene profiles have been widely studied in lung cancer [
18,
19]. Of note, although it has been suggested that epigenetic alterations of genes can occur as a consequence of, or coexist with, aberrant signaling of certain oncogenic pathways activated by genetic alterations, such as
PIK3CA amplification,
EGFR mutation and
K-ras mutation [
20,
21], the interaction between genetic and epigenetic alterations in lung cancer still remains poorly understood.
In the present study, we used quantitative methylation-specific PCR (Q-MSP) to evaluate methylation levels of a panel of cancer-related genes in a cohort of clinically well-characterized NSCLC samples, including CALCA, CDH1, DAPK1, EVX2, PAX6, and RASSF1A, and further explore the association of promoter methylation of these genes with PIK3CA amplification.
Discussion
In the present study, we investigated the promoter methylation of the 6 genes in a large cohort of well-characterized NSCLC samples using Q-MSP technique and their relationships to
PIK3CA amplification. Promoter methylation of these genes, as a mechanism for their silencing, has been frequently observed in NSCLC, particularly
CALCA, CDH1, DAPK1, and
RASSF1A [
26‐
28]. In addition, this study also involved two new methylation markers,
EVX2 and
PAX6, which were highly specific for tumor-associated methylation, and little or no methylation was observed in tumor-adjacent normal lung tissue [
29]. Our findings showed that the overall methylation level from 5 of 6 genes in tumor tissues was significant higher than in non-cancerous lung tissues, including
CALCA, CDH1, EVX2, PAX6, and
RASSF1A. With 100% diagnostic specificity, excellent sensitivity, ranging from 45.8 to 84.1%, was obtained for each of the 6 genes. However, the analysis of hypermethylation still had a limitation in the present study, which the values of hypermethylation were not established a priori, but were calculated to maximize sensitivity given 100% specificity, the resulting sensitivities might be biased overestimates. To obtain non-biased sensitivity (and specificity) values, cut-offs need to be validated in a separate independent sample. In the present study, we also observed that promoter methylation of certain genes was associated with histologic type. Methylation of
CALCA, CDH1, DAPK1, and
EVX2 was common in SCC compared to ADC, particularly in
CDH1 (OR = 2.63, 95% CI = 1.05-6.60) and
DAPK1 (OR = 6.64, 95% CI = 1.85-23.8). Conversely, there was a trend toward a higher frequency of
RASSF1A methylation in ADC than SCC, which is consistent with a recent study [
26]. Of note, there was a trend toward an association between methylation of
CALCA and
DAPK1 and invasion or adhesion and lymph node metastasis, suggesting that aberrant methylation of these genes is associated with oncologic outcomes of NSCLC patients. Similar to a previous study [
30],
RASSF1A methylation was negatively associated with smoking history, lymph node metastasis, and invasion or adhesion, respectively. In addition,
RASSF1A was more frequently methylated in early tumor stage (data not shown), suggesting that
RASSF1A methylation may be an early event in lung tumorigenesis.
Although tobacco smoking plays a dominant role in the development of lung cancer, we did not observe significant association between promoter methylation and smoking history in the present study, in agreement with most studies [
31‐
34]. However, several studies have reported aberrant methylation of tumor-related genes was associated with tobacco smoking [
32,
35,
36]. It is possible that smoking-associated lung cancer is complex disease which involved many unique genetic and epigenetic events. Thus, better understanding of the molecular mechanisms underlying this disease would undoubtedly improve the outcomes of such patients.
It has recently become clear that PI3K/Akt pathway is frequently activated in human cancers [
15,
37], and plays an important role in the regulation of cell growth, proliferation, and survival and is involved in human tumorigenesis [
15]. In recent years, many oncologists have mainly focused on PIK3CA. In addition to
PIK3CA mutations [
38,
39],
PIK3CA amplification was frequently found in lung cancer, and promoted lung tumorigenesis through overactivation of PI3K/Akt signaling pathway [
12,
13,
25,
39]. Similar to the previous studies, in the present study, we found high frequency of
PIK3CA amplification in Chinese NSCLC population. Moreover,
PIK3CA amplification was significantly associated with smoking history and histologic type, which was more frequent in smokers compared to never-smokers, and in SCC compared to in ADC. Of note, although no statistical difference was noted,
PIK3CA amplification was negatively associated with pleural indentation in NSCLC. However, pleural indentation is a well-known imaging sign on chest computed tomography (CT) that suggests a possible pleural invasion by peripheral NSCLC, particularly ADC [
40,
41]. Importantly, pleural involvement was significantly correlated with a poor prognosis in NSCLC, suggesting that pleural involvement may be one of most important factors to affect on the prognosis of NSCLC [
42]. One possibility to explain this contradiction is that,
PIK3CA gene was more frequently amplified in SCC, not ADC, whereas pleural indentation is more common in ADC.
It has been suggested the epigenetic alterations might addict cells to certain oncogenic pathways, predisposing cells to the accumulations of genetic mutations, which drives tumor progression [
43]. On the other hand, overactivation of certain oncogenic pathways can affect the activity of methytransferase, and potentially the methylation activity and regulation of gene transcription, such as RAS/RAF/MEK/ERK pathway (MAPK pathway) [
44‐
47]. Recently, a number of tumor-related genes were found to be aberrantly methylated in association with the MAPK pathway overactivated by
BRAF mutation in human cancers, such as
hMLH1 in colon cancer [
48], and
SLC5A8 in thyroid cancer [
49]. Of note, our previous study showed that
PTEN gene was aberrantly methylated in association with activating genetic alterations in PI3K/Akt pathway, including
PIK3CA amplification [
20]. Moreover, another study showed that the differences in the evolvement of epigenetic alterations between the
EGFR and
K-ras mutation-mediated tumorigenesis and suggested that the specific interation of genetic and epigenetic events in lung tumorigenesis [
21]. In the present study, we found that promoter methylation of
CALCA, EVX2, and
PAX6 was significantly associated with
PIK3CA amplification in NSCLC, however, such association was not seen with other gene methylation, suggesting that epigenetic alterations of these three genes may specifically occur as a consequence of overactivation of PI3K/Akt pathway in NSCLC.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MJ and PH conceived and designed the experiments. MJ, HG and CG performed the experiments. MJ and PH collected the samples and analyzed the data. BS and PH contributed reagents/materials/analysis tools. MJ and PH Wrote the paper. All authors are in agreement with the content of the manuscript and this submission.