Epigenetic inactivation of checkpoint kinase 2 gene in non-small cell lung cancer and its relationship with clinicopathological features

doi:10.1016/j.lungcan.2009.03.011

Lung Cancer

Volume 65, Issue 2, August 2009, Pages 247-250

https://doi.org/10.1016/j.lungcan.2009.03.011 Get rights and content

Abstract

Lung cancer is the leading cause of cancer deaths worldwide and is usually associated with late diagnosis and poor prognosis. Tumor-acquired methylation of the promoter CpG islands (CGIs) is an important mechanism for silencing tumor suppressor genes. The checkpoint kinase 2 (CHK2) is a tumor suppressor that plays a crucial role in regulating cell-cycle checkpoints and apoptosis following DNA damage. The methylation statuses of two CGIs, distal and proximal, of human CHK2 gene were determined in non-small cell lung cancers (NSCLCs) using a nested methylation-specific PCR and bisulfite sequencing. The methylation of distal CHK2 CGI was found in 39 (28.1%) of the 139 NSCLCs. Its frequency was significantly more frequent in squamous cell carcinomas than in adenocarcinomas (40.0% vs 19.0%, p = 0.006) and was also higher in ever-smokers than in never-smokers with a borderline significance (31.7% vs 17.1%, p = 0.071). RT-PCR analysis showed that the distal CGI methylation correlated with CHK2 mRNA expression. However, the methylation of the proximal CHK2 CGI is not specific to tumors and not related to gene expression. These results suggest that the down-regulation of CHK2 gene via distal CGI methylation may play a role in the pathogenesis of NSCLC, particularly squamous cell carcinoma. However, further studies with large numbers of patients are needed to confirm our findings.

Introduction

Lung cancer is a major public health problem in the world and remains a leading cause of cancer deaths [1]. The late diagnosis and the lack of effective treatments for lung cancer are related to unsatisfactory prognosis and high mortality. Importantly, lung cancer is a heterogeneous group of diseases made up of entities characterized by distinctive clinical, pathological, morphologic, and genetic features [2]. In addition, the epidemiologic characteristics of lung cancer in East Asian countries are also remarkably different from those of Western countries [3]. However, the molecular basis of these variations in behavior and epidemiology has not been well known. Therefore, more detailed understanding of the biological features of lung carcinogenesis is needed to improve its diagnosis and prognosis.

Epigenetic-mediated gene silencing constitutes an alternative or complementary mechanism to mutational events in tumorigenesis [4], [5]. An emerging picture of genetic and epigenetic changes and their relationship is helping to unravel the biological network responsible for human cancer. Several genes are commonly the targets of promoter hypermethylation in lung cancer [6], [7], [8]. The predisposition of smokers in acquiring multiple epigenetic alterations in key cellular regulatory genes within the respiratory tract [9], suggests that DNA methylation could serve as a biomarker for the early stages of lung carcinogenesis.

Checkpoint kinase 2 (CHK2), the mammalian homolog of the yeast Rad53 and Cds1, is a versatile and multifunctional kinase that regulates the cell's response to DNA damage and maintains the integrity of the genome [10], [11]. Both germline and somatic loss-of-function CHK2 mutations occur in a range of human tumors, indicating that CHK2 is a major rate-limiting factor for cancer suppression in many tissues [12], [13], [14]. Interestingly, down-regulation of CHK2 expression via promoter methylation has occurred in a proportion of sporadic cancers such as lung, vulva, breast, colon, ovarian cancers as well as lymphoid malignancies [15], [16], [17], [18], [19], [20]. Most studies have focused on the distal CpG islands (CGIs) located upstream of the transcription start site (TSS) of the CHK2 gene [15], [16], [17], [18], [19], whereas one report evaluating CHK2 methylation status in 10 lung cancer samples investigated the proximal CGIs located within intron 1 [20]. However, there are shortcomings of study on the distal CGI, such as its location within intron 1 rather than upstream of transcriptional start site, it is does not even meet the most modest definition of a CGI (just 10 CpGs in 200 bp), its methylation in normal tissue, and absence of association with gene expression. Moreover, the biological role of CHK2 methylation in lung cancer is still poorly understood. In this study, therefore, the methylation status of two CGI clusters of CHK2 gene in non-small cell lung cancers (NSCLCs) was analyzed and the results correlated with the clinicopathological characteristics.

Section snippets

Patients and tissue samples

Tumor and corresponding nonmalignant lung tissue specimens were obtained from 139 Korean NSCLC patients who underwent curative resection at the Kyungpook National University Hospital (Daegu, Korea) from January 2002 to July 2006. None of these patients received chemotherapy and radiotherapy before the surgery. Informed consent was obtained from each patient before the surgery. This study was approved by the institutional review board of the Kyungpook National University Hospital. The

Methylation status and expression of CHK2 in tissue samples of NSCLCs

The methylation frequency of the distal CGI of CHK2 gene was determined in 139 resected NSCLCs and their corresponding nonmalignant lung tissues using a nested MSP. Representative examples of the MSP analysis are illustrated in Fig. 1A. Unmethylated (U) bands were detected in all the nonmalignant and malignant tissues, thus confirming the integrity of the DNA in these samples. Because the tumor specimens represented macroscopically isolated samples that contained both tumor and nonmalignant

Discussion

The present study has shown that methylation of the distal CGI of CHK2 gene was detected in 39 (28.1%) of the 139 NSCLCs and its methylation was related to loss of CHK2 gene expression. These results represent the first example of aberrant methylation-associated CHK2 down-regulation in NSCLCs. Furthermore, we have found methylation of the proximal CGI in all primary tumor samples analyzed, being consistent with the previous reports [19], [20] that the proximal CHK2 CGI was methylated in all

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by a grant from the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (grant no. 0720550-2) and by the Brain Korea 21 Project in 2006.

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Short communicationEpigenetic inactivation of checkpoint kinase 2 gene in non-small cell lung cancer and its relationship with clinicopathological features

Abstract

Introduction

Section snippets

Patients and tissue samples

Methylation status and expression of CHK2 in tissue samples of NSCLCs

Discussion

Conflict of interest

Acknowledgements

Cancer Cell

Trends Genet

Lung Cancer

DNA Rep

Cancer Cell

Blood

Cancer statistics 2007

CA Cancer J Clin

Worldwide trends in lung cancer pathology

Respirology

The fundamental role of epigenetic events in cancer

Nat Rev Genet

Gene-promoter hypermethylation as a biomarker in lung cancer

Nat Rev Cancer