Aberrant hypermethylation of Wnt antagonists has been observed in gastric cancer. A number of studies have focused on the hypermethylation of a single Wnt antagonist and its role in regulating the activation of signaling. However, how the Wnt antagonists interacted to regulate the signaling pathway has not been reported. In the present study, we systematically investigated the methylation of some Wnt antagonist genes (
Gastric cancer (GC) is the fifth most commonly diagnosed cancer and the second primary cause of death worldwide (Ferlay et al., 2010). However, the etiology and pathogenesis of GC remain unclear. Both genetic and epigenetic factors play key roles in the development and progression of GC (Yoda et al., 2015).
Wnt/β-catenin signaling is known to regulate cell differentiation, proliferation, migration, and organogenesis during embryonic development (Cadigan and Nusse, 1997). Recent studies have revealed that aberrant activation of Wnt signaling is also involved in gastric carcinogenesis and progression (Ooi et al., 2009). Wnt ligands bind to the Frizzled (FZD) family of receptors and the LRP5/LRP6 coreceptor, subsequently activating the canonical and non-canonical Wnt pathways (Gonzalez-Sancho et al., 2004; Liu et al., 2005; Oishi et al., 2003). In the canonical pathway, signal transduction activates the protein Dishevelled (Dsh), which inhibits the activity of GSK3β, resulting in the accumulation of β-catenin in the cytoplasm (Giles et al., 2003). β-catenin acts a transcriptional switch; when it enters the nucleus, it interacts with T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors to stimulate downstream target oncogenes such as
Aberrant activation of the Wnt signaling pathways may be caused by β-catenin-activating mutation and
Considering the complexity of the interaction of Wnt antagonists for regulating Wnt signaling, we propose that methylation of a single Wnt antagonist gene might play only a minor role in signaling activation and that the joint effect of the co-methylation of multiple antagonist genes might be more important for the activation of the signaling pathway. In the present study, we systematically and quantitatively investigated the methylation status and mRNA expression levels of six Wnt/β-catenin pathway inhibitor genes using pyrosequencing and real-time reverse-transcription polymerase chain reaction (PCR) in samples of GC tissue. We analyzed the correlation between the methylation of Wnt antagonist genes and clinical pathologic characteristics and evaluated whether quantitative methylation of Wnt antagonist genes can serve as a potential prognostic biomarker for GC. We also analyzed TCGA data to further validate the hypothesis that co-methylation of Wnt antagonist genes cooperatively drive the activation of signaling. Additionally, a demethylation drug was used to study the relationship between methylation and gene expression.
A total of 92 GC samples were collected from 72 male and 20 female surgical patients. These samples included 52 formalin-fixed paraffin-embedded (FFPE) samples and 40 samples of frozen GC tissue along with adjacent normal tissue. The mean age of the patients was 61.2 years (ranged 35–87). All the samples were classified by TNM (UICC 2009) staging, and 27 cases of stage I and II cancer and 65 cases of stage III and IV cancer were determined. Follow-up information about the 52 FFPE specimens was obtained from patients at the time of operation. All patients provided informed consent, and the study protocol was approved by the Ethics Committee of the Shaanxi Provincial People’s Hospital. In addition, information about 262 GC samples with the methylation and mRNA expression data of six genes, as well as β-catenin expression data of matched 255 samples, were downloaded from the results of a TCGA group work (
Genomic DNA from the samples of GC and normal adjacent tissue and FFPE samples were isolated using a Tissue DNA Kit and an FFPE DNA kit (Omega Bio-Tek, USA), respectively. Next, 1 μg DNA was bisulfite-modified using the EpiTect Fast DNA Bisulfite kit (Qiagen, Germany), according to the manufacturer’s protocol.
Total RNA was extracted from 40 frozen samples of GC and the paired adjacent non-cancerous tissues using TRIZOL reagent (Life Technologies, USA). The quantitative mRNA expression levels were determined by real-time PCR (Applied Biosystems, Life Technologies ViiA 7 DX). The glyceralde-hyde-3-phosphate dehydrogenase gene (
The CpG islands of the six genes were obtained using an online software, Methprimer (
BGC823 and MKN-45 cell lines were purchased from the Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences, Cell Resource Center. These two cell lines were incubated in an RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 95% air and 5% CO2. In order to analyze gene demethylation and mRNA restoration, cells were seeded at a density of 3 × 104 cells/cm2 in a 6-well plate and treated with 5-Aza-2′-deoxycytidine, also called decitabine (trade name Dacogen [DAC]), (2 μM and 10 μM) on days 1, 2, and 3. The drug and the medium were replaced every 24 h. Control cells were incubated without DAC.
A two-sided unpaired t test and a paired t test were performed to analyze the differences in methylation and expression levels of the genes, respectively, between GC and normal tissues. An unsupervised hierarchical clustering analysis was performed using the correlation uncentered and average linkage algorithm on Cluster 3.0, and the heatmap was constructed using TreeView. The scatter plot matrix was obtained using R software. The DAC treatment experiment was analyzed using ANOVA. Survival curves were plotted using the Kaplan-Meier method, and survival differences were determined using the log rank test. The multivariable Cox proportional hazard model was used to estimate the adjusted HR. All the statistical analyses were completed using SPSS PASW Statistics, and
In order to systematically investigate the regulatory role of epigenetic silencing of Wnt antagonists in GC, the methylation levels of
In order to understand the concurrent methylation status of each gene, a scatter plot matrix was prepared to compare the correlations between methylation of these Wnt antagonist genes. A significant positive correlation between
In addition, unsupervised clustering was performed for the Wnt antagonist genes in the 40 paired samples to test whether the methylation levels of these genes could be used to distinguish between cancerous and normal samples. As shown in Fig. 5A, 37 and 43 samples were classified as low- and high-methylation group, respectively, based on these six genes (with 38 CpG loci), and 23 (62.2%) normal and 26 (60.5%) GC samples were clustered into the low- and high-methylation groups, respectively. When unsupervised clustering was performed by combining
To determine the function of the methylation of Wnt antagonists, the mRNA expression levels of Wnt antagonist genes were assessed by quantitative PCR in 40 GC samples and matched normal controls. As expected, the mRNA expression levels of both
The data about methylation of Wnt antagonist genes and expression levels of β-catenin were downloaded from the TCGA database to confirm our hypothesis that concurrent hypermethylation of
We next investigated the association between the methylation status of Wnt antagonist genes and the clinicopathological features of GC. The clinicopathological data of the methylated genes are summarized in
To analyze the relationship between overall survival and the methylation status of Wnt antagonist genes, patients were divided into two groups according to the median methylation level of each gene. The Kaplan-Meier survival curves showed that methylation of
We next evaluated whether promoter methylation of Wnt antagonists was functionally associated with their mRNA expression levels in GC cell lines. To address this question, BGC823 and MKN-45 GC cell lines were treated with a methyltransferase inhibitor, Dacogen (DAC), for three days. As shown in Fig. 9, the DAC treatment resulted in a clear decrease in promoter methylation of
Constitutive Wnt/β-catenin signaling is a major contributor to gastric carcinogenesis. Ooi et al. demonstrated that Wnt/β-catenin pathways were activated in 46% of GC cases (ranged 43% to 48%) (Ooi et al., 2009). Interestingly, mutations in
Although several previous studies on the methylation of Wnt antagonist genes in GC have been reported, however, how these Wnt antagonists interacted to regulate the signaling pathway has not been reported. In the present study, we investigated the co-methylation of Wnt antagonist genes and its functions in Wnt signaling pathway activation in GC.
In our study, we found that
The mRNA expression data revealed that
β-catenin as a key mediator of the canonical Wnt signaling pathway (Giles et al., 2003), and its accumulation in the cytoplasm/nucleus is a critical mechanism for the activation of this pathway. In this study, we analyzed the relationship between the methylation status of Wnt antagonist genes and the expression levels of β-catenin using TCGA data. When the methylation status of each gene was separately analyzed, we found no association between them. As expected, patients who showed co-methylation of
Several studies have reported aberrant hypermethylation of Wnt inhibitor genes in GC, but have not established a correlation between methylation and clinicopathological characteristics. Hirata et al. (2009) indicated that the methylation frequency of
Most patients with GC are diagnosed at an advanced tumor stage, where metastasis to lymph nodes has already occurred. Consequently, most patients face a poor prognosis. Therefore, it is important to identify prognostic markers that can reliably predict patient outcome. Several previous studies have demonstrated that the activation of the Wnt/β-catenin pathway and its components could indicate the clinical prognosis in GC (Ooi et al., 2009; Yu et al., 2009). In the present study, we investigated the clinical significance and prognostic value of the methylation of Wnt antagonist genes in 52 patients. We found that a high methylation rate of
We confirmed that the expression levels of all six Wnt antagonist genes were restored after DAC treatment in at least one of the two cell lines. Four of six genes (
In summary, our results show that the methylation levels of several Wnt antagonist genes significantly increased in GC, while their mRNA expression levels decreased. Concurrent hypermethylation of