Methylation status of ANAPC 1 , CDKN 2 A and TP 53 promoter genes in individuals with gastric cancer

1Colegiado de Biomedicina, Universidade Federal do Piauí, Parnaíba, PI, Brasil 2Departamento de Morfologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brasil 3Laboratório de Citogenética Humana, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, Brasil 4Serviço de Cirurgia, Hospital João de Barros Barreto, Belém, PA, Brasil 5Instituto de Investigaciones Biomedicas, Madri, Espanha 6Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil


Introduction
Gastric cancer is the forth most frequent malignancy and the second most common cause of cancer death worldwide (1).In Northern Brazil, gastric cancer is the second most frequent neoplasia in males (11/100,000) and the third most common in females (6/100,000) (2).Diet may be associated with the high incidence of this neoplasia in the State of Pará, especially because of the high consumption of salt-preserved food, low use of refrigerators and low consumption of fresh fruit and vegetables (3).
DNA methylation is the most studied epigenetic alteration, occurring by the addition of a methyl radical to the cytosine base adjacent to guanine (4).In cancer, DNA methylation of the promoter region of a normal tumorsuppressor gene leads to the aberrant silencing of its functions.
Three genes were chosen for evaluation considering their function and/or their potential role in gastric carcinogenesis: ANAPC1, CDKN2A and TP53.
Our group previously described a line of adenocarcinoma cells in which polyploidization due to endoreduplication was detected (5).Atkin (6) suggested that polyploidization could be a characteristic of carcinogenesis because the aggressiveness of a cancer is related to the degree of genomic instability associated with chromosome selection.This genomic instability is a frequent finding in gastric cancer (7).Accurate segregation of sister chromatids during mitosis is necessary to prevent the aneuploidy found in many cancers.The spindle checkpoint has been shown to be defective in cancers with chromosomal instability.
This checkpoint regulates the anaphase-promoting complex or cyclosome.The product of ANAPC1 is the largest subunit of the anaphase-promoting complex (8).Thus, ANAPC1 may be a possible candidate for causing the chromosomal instability seen in gastric cancer.However, the methylation pattern of ANAPC1 had never been described.The product of CDKN2A is a protein that regulates phosphorylation of the retinoblastoma protein and negatively regulates the G1-S transition in the cell cycle (9).CDKN2A hypermethylation may play a key role in the progress of gastric cancer (10,11).TP53 is one of the most studied tumor-suppressor genes and acts especially in cell cycle arrest and induction of apoptosis (12).Inactivation of the TP53 pathway is a common feature of neoplasia.Dysregulation of the TP53 pathway has been shown to involve mutations of TP53, increased expression of the TP53 inhibitor HDM-2, or epigenetic silencing of the TP53 promoter (13).However, its methylation status has never been studied in gastric carcinogenesis.
In the present study, we evaluated the methylation status of ANAPC1, CDKN2A and TP53 promoters in gastric adenocarcinoma samples and their possible associations with clinical and pathological characteristics, such as gender, age, histopathology, tumor extension, and presence of lymph node or distant metastasis.

Samples
The study included 66 samples of gastric tissue.Of 66 patients, 47 were male and 19 were female, and mean age was 59 ± 9.8775 years (range 27-76).Among gastric cancer samples, 20 were non-neoplastic gastric mucosa of gastric cancer patients (distant location of primary tumor) and 46 sporadic gastric adenocarcinoma samples.Gastric samples were surgically obtained at the João de Barros Barreto University Hospital (HUJBB), State of Pará, Brazil, and were stored at -80°C before DNA extraction.Patients had never been submitted to chemotherapy or radiotherapy prior to surgery, nor did they have any other diagnosed cancer.All patients signed an informed consent with the approval of the HUJBB and Ribeirão Preto Medical School Clinical Hospital Institutional Review Board.All 46 gastric cancer samples were classified according to Laurén (14): 20 were diffuse-type and 26 were intestinaltype.Tumors were staged using standard criteria by tumor, node, metastasis (TNM) staging (15).

Methylation-specific PCR
Genomic DNA (2 µg) was modified by bisulfite treatment, converting unmethylated cytosines to uracils and leaving methylated cytosines unchanged.Methylation-specific PCR (MSP) was performed on treated DNA as previously described (16).Specific primers for MSP (Table 1), located within CpG island in the gene promoter region, were designed with the assistance of the Methprimer software (17).
The PCR product was carried out in a volume of 50 µL with 200 µM dNTPs, 200 µM MgCl 2 , 50 ng DNA, 200 pM primers, and 1 unit AmpliTaq GOLD (Applied Biosystems, Foster City, CA, USA).After initial denaturing for 2 min at 94°C, 35 cycles at 94°C for 40 s, 1 min at different temperatures with the primers (Table 1) and 72°C for 40 s were followed by a final extension for 5 min at 72°C.PCR products were separated and visualized by electrophoresis on 8% polyacrylamide gel and stained with 10% silver nitrate (Figure 1).Water was used as negative control.MSP results were scored when there was a clearly visible band on electrophoresis gel with the methylated and unmethylated primers (16).

Statistical analysis
Statistical analyses were performed using the Fisher exact test to assess associations between methylation status and frequency, and clinical and pathological characteristics, such as gender, age, histopathology, tumor extension, and presence of lymph node or distant metastasis.P values less than 0.05 were considered to be significant.

Results
According to Laurén's classification (14), 20 were diffuse-type and 26 were intestinal-type.All gastric cancer samples were in advanced stage: 0% was at pT1, 26.1% were at pT2, 63% were at pT3, and 10.9% were at pT4.It was observed that 34.8% were at N0 stage, 58.7% at N1 Figure 1. Figure 1. Figure 1. Figure 1. Figure 1.Gel electrophoresis using ANAPC1, CDKN2A and TP53 methylation-specific polymerase chain reaction primers.L: size marker; M: methylated; U: unmethylated.Table 1.Table 1.Table 1.Table 1  None of the non-neoplastic samples showed methylation of any gene promoter.Furthermore, none of the samples showed methylated sequences for the ANAPC1 and TP53 promoters.The methylation frequency of CDKN2A promoter was 30.43% in gastric cancer samples.CDKN2A methylation was associated with gastric cancer samples compared to non-neoplastic samples (P = 0.0009).CDKN2A methylation was associated with both diffusetype and intestinal-type compared to control samples using the Fisher exact test (P = 0.0024 and P = 0.0137, respectively; Table 2).
We analyzed whether CDKN2A methylation was associated with clinical and pathological characteristics, and detected a tendency for this gene methylation in intestinaltype gastric cancer with smaller tumor extension (T2) compared to T3 and T4 stages (P = 0.0572).

Discussion
DNA methylation is a stable but reversible epigenetic signal that silences gene expression.Somatic alterations E.M. Lima et al.
www.bjournal.com.br in genomic methylation patterns contribute to the etiology of human cancers and aging.It is tightly interwoven with the modification of histone tails and other epigenetic signals (18).
Lima et al. ( 5) developed and characterized cytogenetically a cell line called ACP01 from a gastric adenocarcinoma.In ACP01, polyploidization due to endoreduplication was observed.The spindle checkpoint has been shown to be defective in cancers with chromosomal instability.This checkpoint regulates the anaphase-promoting complex or cyclosome.ANAPC1 product is the largest subunit of the anaphase-promoting complex (8).To our knowledge, the methylation status of ANAPC1 has never been evaluated.In the present study, we did not detect ANAPC1 methylation.Our findings suggest that the ANAPC1 methylation is probably not important for the control of the chromosomal instability in our samples.
Methylation of the CDKN2A promoter was detected in 30.43% of gastric cancer samples, which was not significantly different from frequencies previously described (range 21 to 43%), and was also associated with carcinogenesis process (19)(20)(21)(22).Lee et al. (23) proposed that CDKN2A methylation may contribute to the malignant transformation of gastric precursor lesions.Tahara et al. (24) also suggested that the evaluation of CDKN2A status can help predict gastric cancer risk in non-neoplastic gastric epithelium, because methylation levels of CDKN2A seem to accumulate in the progression of gastric mucosa atrophy and intestinal metaplasia, and thus may be associated with the presence of gastric cancer especially for intestinal-type histopathology.In our sample, diet habits may play a role in CDKN2A methylation especially in the beginning of intestinal-type gastric cancer.
TP53 is one of the most studied tumor-suppressor genes and acts especially in cell cycle arrest and induction of apoptosis.TP53 is a gene related to the majority of human cancers.Methylation of TP53 was reported as a mechanism for its inactivation in some neoplasias, such as acute lymphoblastic leukemia, multiple myeloma, malignant glioma cells, and brain metastases of solid tumors (13,(25)(26)(27).To our knowledge, the methylation status of TP53 has never been evaluated in gastric samples.In the present study, we did not detect TP53 methylation.Thus, methylation of TP53 is not an important event for gastric carcinogenesis in the population studied.
The identification of epigenetic modifications in tumor suppressor genes and the determination of the frequency of these alterations may be useful in the development of a more specific cancer therapy.This is the first study that evaluated the methylation status of ANAPC1, CDKN2A and TP53 promoters in gastric samples in a population from Northern Brazil.In this study, the methylation pattern of ANAPC1 and TP53 promoter was not altered by the gastric carcinogenesis process.However, more studies are necessary to evaluate if the absence of methylation of these genes is a common finding in gastric cancer or if this is a regional characteristic.
CDKN2A promoter methylation was associated with gastric carcinogenesis.However, only about 30% of gastric cancer samples were methylated.CDKN2A methylation can be specific to a subset of gastric cancer and probably plays a role in the beginning of intestinal-type gastric cancer.

Table 2 .Table 2 .Table 2 .Table 2 .Table 2 .
Methylation number and frequency of CDKN2A in gastric samples for comparison of gastric cancer with non-neoplastic samples.Data are reported as number of individuals with percent within parentheses.M = methylated; U = unmethylated.Fisher exact test was used for statistical analysis.