Promoter methylation of APC and RAR-β genes as prognostic markers in non-small cell lung cancer (NSCLC)

Abstract

Aberrant promoter hypermethylations of tumor suppressor genes are promising markers for lung cancer diagnosis and prognosis. The purpose of this study was to determine methylation status at APC and RAR-β promoters in primary NSCLC, and whether they have any relationship with survival. APC and RAR-β promoter methylation status were determined in 41 NSCLC patients using methylation specific PCR. APC promoter methylation was detectable in 9 (22.0%) tumor samples and 6 (14.6%) corresponding non-tumor samples (P = 0.391). RAR-β promoter methylation was detectable in 13 (31.7%) tumor samples and 4 (9.8%) corresponding non-tumor samples (P = 0.049) in the NSCLC patients. APC promoter methylation was found to be associated with T stage (P = 0.046) and nodal status (P = 0.019) in non-tumor samples, and with smoking (P = 0.004) in tumor samples. RAR-β promoter methylation was found associated with age (P = 0.031) in non-tumor samples and with primary tumor site in tumor samples. Patients with APC promoter methylation in tumor samples showed significantly longer survival than patients without it (Log-rank P = 0.014). In a multivariate analysis of prognostic factors, APC methylation in tumor samples was an independent prognostic factor (P = 0.012), as were N1 positive lymph node number (P = 0.025) and N2 positive lymph node number (P = 0.06). Our study shows that RAR-β methylation detected in lung tissue may be used as a predictive marker for NSCLC diagnosis and that APC methylation in tumor sample may be a useful marker for superior survival in NSCLC patients.

Introduction

Lung cancer is the leading cause of cancer-related deaths in the world. Lung cancer survival tends to be poorer in developing countries as a result of increasing use of tobacco product compared with developed countries (Jemal et al., 2011, Molina et al., 2008). Non-small cell lung cancer (NSCLC) comprises the majority (85–90%) of lung cancer (Zhang et al., 2011b). Despite advances in the detection and treatment of NSCLC, the overall 5-year survival rate remains under 15% (Kim et al., 2006). The poor outcome of NSCLC patients may be attributed partially to late diagnosis and tumor recurrence (Henschke et al., 2006). Thus, developing new markers for early diagnosis and prognostic prediction may help to improve survival rate and life quality of patients with NSCLC.

DNA methylation is an epigenetic modification causing the silence of target genes, and can be induced by environmental factors such as exposure to smoking (Salskov et al., 2011). The silencing of tumor suppressor genes by promoter methylation is a common mechanism in the genesis of human cancers (Cheishvili et al., 2014). As promising biomarkers for early detection of NSCLC, methylation of multiple genes has been studied, including CADM1, CDH1, CDH13, p16, GSTP1, MGMT, and RASSF1A (Zhang et al., 2011b, Ji et al., 2011, Anglim et al., 2008). Moreover, methylation of some tumor suppressor genes has also been studied for prognosis in different cancers. For example, Henrique et al. (2007) reported that high promoter methylation levels of Adenomatous polyposis coli (APC) gene predict poor prognosis in prostate cancer patients, and Chen and Chiu (2009) showed that methylation in the APC promoter may serve as a predictor for the prognosis of Taiwanese colorectal cancer patients. In patients with stage I NSCLC, Kim et al. (2006) suggested that cohypermethylation of p16 and FHIT genes may be a valuable biomarker for predicting the recurrence-associated prognosis of the disease. Saito et al. (2010) reported that LINE-1 hypomethylation was an independent marker of poor prognosis in stage IA NSCLC.

APC and retinoic acid receptor-beta (RAR-β) are two important tumor suppressor genes (Houle et al., 1993, Virmani and Gazdar, 2003). However, Khuri et al. (2000) demonstrated that strong expression of RAR-β correlated with worse outcome of early-stage NSCLC. In addition, Brabender et al. (2001) reported that high APC promoter methylation is associated with inferior survival whereas Safar et al. (2005) showed that patients whose tumors were hypermethylated at APC enjoyed substantially longer 1- and 2-year survival using recursive partitioning. Additional studies are needed to elucidate the prognostic values of the methylation status of these two genes. Here we examined the methylation status of APC and RAR-β promoters in tumor tissue and corresponding non-tumor lung tissue in a cohort of 41 Chinese patients with NSCLC, and analyzed whether the methylation of APC or RAR-β, combined with or without other clinicopathological variables, could be used to predict the clinical outcomes of patients with primary NSCLC after surgical resection.