p16 promoter hypermethylation is associated with increased risk of nasopharyngeal carcinoma

Abstract

The present study aimed to investigate the effects of p16 hypermethylation on the risk of nasopharyngeal carcinoma (NPC) quantitatively, through a meta-analysis of available case-control studies including malignant and normal NPC tissue samples. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were extracted and pooled to assess the strength of the association between p16 hypermethylation and NPC risk. A total of 9 studies, including 406 NPC and 376 control cases, were identified for meta-analysis. Statistically significant ORs of p16 hypermethylation were obtained from the NPC and control groups (OR=19.53; 95% CI: 9.54–39.97; P=0.685). The meta-analysis indicated that p16 hypermethylation significantly increases NPC risk.

Introduction

Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic malignant tumor that originates from the epithelial cells lining the nasopharynx (1). Several histological entities exist, but undifferentiated NPC is the most frequent histological type. NPC differs significantly from other head and neck cancers due to its specific multifactorial etiology and geographical distribution. NPC is relatively rare in most parts of the world, particularly in Europe and North America; however, it has a high incidence in Southern China. Outside of these areas, intermediate incidences are observed in the Eskimo population of Arctic regions and in Arabians of North Africa, including Tunisia, Algeria and Morocco (2–4).

Recently, a number of epigenetic changes, such as methylation and histone deacetylation, have been found to be involved in the tumorigenesis of NPC. In addition, growing evidence demonstrates that epigenetic changes contribute to this process by altering the functions of multiple genes playing critical roles in cell cycle regulation, apoptosis, signal transduction, adhesion and differentiation of the nasopharyngeal epithelial cells (5). The most prevalent epigenetic mechanism implicated in this type of cancer is the hypermethylation of CpG islands in the promoter regions of the genes.

p16, one of the most commonly inactivated tumor suppressor genes in human cancer, is a cyclin-dependent kinase (CDK) inhibitor that regulates tumor cell progression through the G1 phase of the cell cycle (6). The downregulation of p16 expression due to promoter hypermethylation frequently occurs in NPC. Aberrant DNA hypermethylation has been recognized as a frequent molecular alteration in cancer (7,8). This epigenetic modification occurs at the cytosines of CpG dinucleotide-rich regions (9), which are mostly unmethylated in normal tissues. The hypermethylation of CpG islands in the gene promoter regions of numerous tumor suppressor and DNA repair genes is associated with chromatin condensation, replication delay, inhibition of the initiation of transcription and gene silencing (10). Similar to a number of other genes, p16 is commonly inactivated by the hypermethylation of its CpG-rich promoter region (11). The association between p16 promoter methylation and NPC susceptibility has been extensively investigated; however, the results have been inconsistent. Thus, a comprehensive meta-analysis of the most recent and relevant studies was performed to identify statistical evidence of the association between p16 gene hypermethylation and NPC risk. Therefore, a systematic review and meta-analysis was conducted to evaluate this correlation quantitatively.

Materials and methods

Meta-analysis

This meta-analysis was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (12) and the recommendations of the Cochrane Collaboration (13). The study was approved by the Institutional Review Board of the Kunming University of Science and Technology.

Identification and eligibility of relevant studies

To identify eligible studies, the terms ‘p16’, ‘methylation’, ‘nasopharyngeal cancer’, ‘nasopharyngeal tumor’ and ‘nasopharyngeal carcinoma’ were searched for in PubMed, Web of Science and EBSCO with an English language restriction. The search results were updated until February 24, 2014. Only published full-text articles were included in the analysis. The following criteria were used in selecting eligible articles: i) independent case-control, malignant and benign nasopharyngeal tissue studies with reliable methods; ii) studies investigating p16 methylation and NPC risk; iii) sufficient data for estimating odds ratios (ORs) (14) with 95% confidence intervals (CIs).

Data extraction

Shao and Tang independently conducted reviews by extracting data via a standardized approach. The publication information (year of publication and name of first author) and p16 hypermethylation rates were collected using standard data extraction forms. Any discrepancies were resolved through discussion.

Statistical analysis

The ORs and 95% CIs were calculated to estimate the strength of the association between p16 hypermethylation and NPC risk. A Chi-square-based Q-test was performed to assess between-study heterogeneity. If P>0.10, the studies were considered to lack heterogeneity and the pooled ORs were calculated using the fixed-effects model according to the Mantel-Haenszel method (15). Otherwise, the random-effect model was used according to the DerSimonian-Laird method (16). All the statistical analyses in the present study were performed using Review Manager 5 (http://tech.cochrane.org/revman) and Stata software ( version 8.2;StataCorp LP, College Station, TX, USA).

Results

Study characteristics

A total of 9 studies met the inclusion criteria and were included in the present meta-analysis. The studies involved 406 NPC and 376 control cases. When the same investigators reported results obtained from the same cohort of patients in several publications, only the largest series was included in the analysis. A cohort of patients was excluded due to duplicate reports. The main characteristics of the included studies are summarized in Table I.

Table I.

Data from 9 studies on p16 methylation and nasopharyngeal cancer (NPC) risk included in this study.

		NPC cases (n=406)		Controls (n=376)
First author	Year	Methylated	Unmethylated	Methylated	Unmethylated	(Refs.)
Tian et al	2013	9	31	1	40	(28)
Challouf et al	2012	12	24	4	158	(29)
Hutajulu et al	2011	35	18	0	25	(30)
Tan et al	2013	6	13	0	3	(31)
Xiang and Zhang	2005	42	48	0	30	(32)
Wong et al	2004	17	24	1	42	(33)
Chang et al	2003	10	23	0	37	(34)
Tong et al	2002	13	15	0	26	(35)
Kwong et al	2002	17	16	0	6	(36)
Lo et al	1996	6	27	0	3	(37)

p16 hypermethylation in NPC and control groups

Data regarding the comparison of p16 hypermethylation between the NPC and control groups were collected from 9 studies. The NPC and control groups included 167 (41.1%) and 6 (1.6%) p16 hypermethylated cases, respectively. The pooled analysis demonstrated that the ORs of the NPC group during p16 hypermethylation significantly increased compared to those of the controls (OR=19.53; 95% CI: 9.54–39.97; P=0.685) (Fig. 1).

Figure 1.

Forest plot of odds ratios (ORs) of nasopharyngeal cancer risk according to p16 promoter hypermethylation when compared to the control groups. The squares and horizontal lines correspond to each study-specific OR and 95% confidence interval (CI). The area of the squares reflects the study-specific weight. The diamonds represent the pooled ORs and 95% CIs.

Discussion

The precise etiology of NPC (17) remains to be clearly elucidated; however, this disease has been found to be highly associated with specific geographical distributions, environmental factors and genetic alterations (18). CpG islands are located in the promoter or first exon region of genes and are normally unmethylated. Over the last few years, promoter hypermethylation has been recognised as a common mechanism underlying the inactivation of tumor suppressor genes in human cancer (19–21). However, in cancer cells, hypermethylation of these regions is associated with transcriptional silencing or a decrease in expression. The promoter hypermethylation of tumor suppressor genes, including p16, hMLH1 and VHL, has been established as a common mechanism for tumor suppressor gene inactivation in human cancer and has become a promising new molecular target for its detection. Aberrant DNA methylation is being increasingly recognized as a frequent molecular alteration in NPC. The product of the INK4A locus, p16, encodes a CDK (9) inhibitor that functions as a negative regulator of cyclin/CDK complexes. p16 binds preferentially to CDK4/6 and prevents the association of CDK4/6 with D-type cyclins, thus inhibiting pRB phosphorylation and progression through the cell cycle (22,23). p16 also plays a significant role in the maintenance of normal cellular properties and prevention of centrosome dysfunction and genomic instability (24). p16 inactivation occurs during the early stage of carcinogenesis (25) and the loss of p16/RB activity occurs through various mechanisms, including the deletion, mutation and hypermethylation of p16 (23). Aberrant hypermethylation has been suggested to be a useful biomarker, with implications for NPC etiology, diagnosis and management. Gene-specific promoter alterations are common epigenetic aberrations in human NPC. However, the epigenetic changes in p16 gene hypermethylation specific to NPC etiology remain elusive. The present pooled analysis comprehensively assessed the correlation between p16 gene hypermethylation and the incidence of NPC based on 9 studies, which included 406 NPC cases and 376 controls. Using the pooled crude ORs from the studies, we demonstrated that p16 gene hypermethylation is associated with a 20.88-fold increased risk of NPC compared to the control group.

The present study had several potential limitations. First, the possibility of information and selection bias and unidentified confounders cannot be completely excluded, as all the studies were observational. Second, the majority of the studies included in this meta-analysis were conducted in various ethnic groups.

In conclusion, despite the discrepancy in p16 hypermethylation in nasopharyngeal cancer and control groups, we observed that p16 hypermethylation is associated with an increased risk of NPC. p16 hypermethylation, which mediates the inactivation of the p16 gene, plays a significant role in nasopharyngeal carcinogenesis. Therefore, it may have useful clinical applications in the detection of NPC at the early stages. Currently available cancer therapies use DNA methylation inhibitors, including 5-aza-2′-deoxycytidine (26), to reactivate the repressed p16 gene. The reactivation of the p16 gene in cells with methylated genes restores normal cell growth control (27). According to the present study, the p16 promoter methylation status may be used for NPC diagnostic and therapeutic purposes.