Genetic and epigenetic alterations of the LKB1 gene and their associations with mutations in TP53 and EGFR pathway genes in Korean non-small cell lung cancers
Introduction
The epidermal growth factor receptor (EGFR) signaling pathway plays an important role in lung carcinogenesis, including cell proliferation, survival, invasion and metastasis. These effects are mediated by activation of downstream signaling pathways, including RAS-RAF-mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K)-AKT-mammalian target for rapamycin (mTOR) and signal transduction and activator of transcription pathways [1], [2].
Liver kinase 1 (LKB1), also known as serine threonine kinase 11, regulates multiple biological processes by activation at least 14 downstream kinases, including cellular energy metabolism, cell polarity, the epithelial-mesenchymal transition, cell cycle arrest and proliferation, apoptosis, and angiogenesis [3], [4], [5], [6]. Key among these functions is activation of AMP-activated protein kinase (AMPK), which, in turn, activates tuberous sclerosis complex protein 2 (TSC2), resulting in inhibition of mTOR [7], [8]. This LKB1-AMPK-TSC-mTOR pathway plays a central role in the regulation of cellular metabolism and cell growth by integrating information regarding intracellular energy and oxygen status, the presence of growth factors, and nutrient availability [3], [4], [5], [6], [7], [8].
Germline inactivating mutations in LKB1 predispose Peutz-Jeghers syndrome, an autosomal dominant disorder characterized by gastrointestinal hamartomatous polyps, mucocutaneous pigmentation, and an increased risk of a variety of cancers, including lung cancer [9], [10]. In addition, cumulative evidence suggests that somatic inactivation of LKB1 is involved in the pathogenesis of sporadic lung cancer [3], [11], [12], [13], [14]. Genetic alterations of LKB1 in sporadic lung cancer occur mainly as a results of non-sense mutations, small insertions and deletions, or large intragenic deletions at one allele plus large chromosomal deletions at the other, leading to complete absence of the LKB1 protein [11], [15], [16], [17]. Several studies have reported that inactivation of LKB1 by a mutation or homozygous deletion occurs in approximately one-third of non-small cell lung cancers (NSCLCs) from Caucasian populations [11], [16], [17]. In contrast to the frequent occurrence of genetic alterations of LKB1, epigenetic inactivation by aberrant promoter methylation is infrequent in Caucasian NSCLCs [11], [18], [19].
LKB1 inactivation by genetic alterations in NSCLCs occurs preferentially in adenocarcinomas (ACs) of male smokers and frequently coexist with mutations of other important tumor suppressor genes such as KRAS and TP53 but rarely with those of EGFR [11], [15], [16], [17], [20]. Notably, several studies have shown that the prevalence of genetic alterations of LKB1 vary remarkably depending on the patient ethnicity; LKB1 mutations are less frequent in Asian patients than in Caucasian patients, and homozygous deletion of the LKB1 locus is less frequent in African-Americans patients than in Caucasians patients [17], [20], [21], [22].
Although LKB1 genetic alterations have been widely studied in lung cancer, the majority of studies were performed on Caucasian populations. Additionally, few studies have investigated the prevalence of epigenetic inactivation of LKB1 in NSCLCs. The epidemiological characteristics of lung cancer in Korea are remarkably different from those of Western countries. In Korea, lung cancer occurs predominantly in male smokers, and the ratio of AC to squamous cell carcinoma (SCC) is relatively low, which may be due to the very high smoking rate (87.3%) among males and a low smoking rate (20.3%) among females [23]. Considering the differences in genetic and environmental factors related to lung cancer, it is possible that the prevalence of LKB1 inactivation in Korean patients with NSCLC may be different from patients in Western countries. Therefore, we searched for genetic and epigenetic alterations of LKB1 in Korean NSCLCs and correlated the results with clinicopathological features. We also investigated the relationship between genetic and epigenetic alterations of LKB1 and mutations in the TP53 gene and EGFR signaling pathway genes.
Section snippets
Patients and genomic DNA isolation
Tumor and corresponding non-malignant lung tissue specimens (n = 159) were provided by the National Biobank of Korea – Kyungpook National University Hospital (KNUH), which is supported by the Ministry of Health, Welfare, and Family Affairs. All materials derived from the National Biobank of Korea – KNUH were obtained under institutional review board approved protocols. None of the patients had any clinical manifestations of Peutz-Jeghers syndrome. This cohort of patients was investigated
Genetic and epigenetic alterations of the LKB1 gene
We tested LOH of the LKB1 locus in primary tumors using D19S886 and D19S878 microsatellite markers. LOH was detected in 31 (19.5%) of 159 tumors specimens; eight tumors showed LOH at both markers, and 23 tumors showed LOH at one marker (Table 1). In the mutational analysis of the entire coding region (exons 1–9) and exon-intron boundaries of the LKB1 gene, one (male smoker AC) of the 159 tumors examined had a somatic frameshift mutation (842delC, stop at codon 286).
The methylation status of CGI
Discussion
In the present study, we examined the prevalence of genetic and epigenetic alterations of the LKB1 gene in Korean NSCLCs and explored the relationship between LKB1 alterations and clinicopathological features including the presence of mutations in TP53 and EGFR signaling pathway genes. LOH or promoter methylation of the LKB1 gene were found in 48 (30.2%) of 159 NSCLCs and were associated with down-regulation of gene expression. The LKB1 mutation was extremely rare (one [0.6%] of 159 tumors) and
Conflict of interest statement
None declared.
Acknowledgments
This study was supported by the National R&D Program for Cancer Control Ministry of Health & Welfare (0720550-2) and the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (2010-0010000).
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These two authors contributed equally to this paper.