Circulating Methylated XAF1 DNA Indicates Poor Prognosis for Gastric Cancer

Ethics Statement

De-identified human tissue samples and sera were obtained from Zhejiang Province Human Tissue Specimen Bank. The use of specimens was approved by the Institutional Review Board at Zhejiang Province Cancer Hospital. Written informed consent was obtained from each patient in accordance with the requirements of our institution’s board of ethics. 88 non-cancer volunteers provided written informed consent. Part of the specimens were from Zhejiang Province People’s Hospital and the First People’s Hospital of Chunan County. The Institutional Review Board on Medical Ethics of Zhejiang Province People’s Hospital and the First People’s Hospital of Chunan County approved the method of specimen collection including written informed consent from all patients, respectively.

Tissue Specimen

Paired tumor and para-cancerous histological normal tissues (PCHNT) specimens were collected at the time of surgery from 202 patients with primary gastric adenocarcinoma at Zhejiang Province Cancer Hospital, Zhejiang Province People’s Hospital and the First People’s Hospital of Chunan County from January 2008 to December 2009. The PCHNT was assessed microscopically for the presence of normal cells and absence of dysplastic cells. None of these cases had undergone any medical treatment before surgery. Demographic, clinical and histopathological parameters of these cases were shown in Table 1. The growth pattern of tumor cells was determined according to Ming’s classification [20]. All recruited patients had been followed-up periodically until the due date. Antral mucosa biopsy specimens from 88 non-cancer volunteers by gastroscopy were randomly collected as controls within the same period, including 54 men and 34 women, with an average age of 52.9 years old. Among these volunteers, 48 patients were diagnosed with chronic non-atrophic gastritis. Meanwhile, paired serum samples were collected before surgery or endoscopy.

Analysis of Helicobacter Pylori (H. pylori) Infection

Biopsies were obtained from all patients who had endoscopic examination. H. pylori status was determined by rapid Urease test and Giemsa staining methods [21], [22]. It was considered as H. pylori infection when both tests were positive, and the samples with single positive were excluded for statistical analysis [22].

Real-time RT-PCR

The mRNA expression of XAF1 was analyzed by real-time RT-PCR [23]. Total RNAs were extracted using the Trizol (Gibco). A total of 3 µg total RNAs was subjected to reverse transcription using M-MLV reverse transcriptase (Promega). The glyceraldehyde phosphate dehydrogenase (GAPDH) was selected as the internal reference. The sequences of XAF1 primers were as follows: (F) 5′-TGGGTGTAGGATTCTCCAGG-3′, (R) 5′- GGTTTGCCCAAG GACTACAA-3′. GAPDH primer sequences were as follows: (F) 5′-CATGA GAAGTATGACAACAGCCT-3′, (R) 5′-TAATTTTAGGTTAGAGGGTTATTGT- 3′. The 2^−ΔΔCt method was used to calculate relative changes in gene expression.

Immunohistochemical Staining

The expression of XAF1 protein was determined by immunohistochemical analysis with XAF1 monoclonal antibody (Santa Cruz Biotechnology). Immunohistochemical staining for XAF1 was carried out using representative paraffin-embedded specimens from the 202 GC patients. After deparaffinization, antigen retrieval in 0.01 M citrate buffer, and inactivation of endogenous peroxidase activity in 3% H₂O₂/methanol, we incubated the slides with antibody for XAF1 at 4°C overnight, and immunohistochemical staining, following a standard avidinbiotin-peroxidase complex technique, was carried out using 3,3′-diaminobenzidine (DAB) as the chromogen. Nuclei were counterstained with hematoxylin. The immunohistochemical staining score (ISS) is determined by three independent pathologists combining staining frequency and intensity as follows [24], [25]: no staining is scored as 0, 1∼10% of cells stained scored as 1, 11∼50% as 2, 51∼80% as 3, and 81∼100% as 4. Staining intensity is rated on a scale of 0 to 3, with 0, negative; 1, weak; 2, moderate; and 3, strong. When there is multifocal immunoreactivity and there are significant differences in staining intensities between foci, the average of the least intense and most intense staining was recorded. The raw data were converted to the ISS by multiplying the frequency score and the staining intensity score. Theoretically, the ISS could range from 0 to 12. An ISS of 9∼12 was considered strong immunoreactivity, 5∼8 was considered moderate, 1∼4 was considered weak, and 0 was scored as negative. Sections in which the staining could not be well characterized were considered equivocal.

Western Blot Analysis

Paired tumor and PCHNT specimens of 20 cases were randomly selected from 202 gastric cancer patients for western blot analysis. Total protein was extracted and then quantified using the Lowry method [26]. Western blot analysis was performed using anti-XAF1 monoclonal antibodies (Santa Cruz, CA) according to previous report [6]. β-tubulin was served as an internal control.

DNA Extraction, Bisulfite Modification and Real-time Methylation Specific-PCR (MSP)

Serial 5-mm sections that contained carcinoma and non-neoplastic tissues were mounted on non-coated glass slides and dried at 37°C overnight. After deparaffinization and staining with hematoxylin and eosin (H&E), we collected 5,000 nuclei from 5 to 10 serial sections using a 27G needle. The collected nuclei were treated with 40 ml of 200 mg/ml proteinase K (Sigma-Aldrich Co., St. Louis, MO) at 42°C, for 72 hr. The paramagnetic bead technology (AxyPrep Mag Blood gDNA kit, Axygen Scientific, Inc., Union City, CA) was utilized to isolate genomic DNA from fresh blood according to kit’s protocol. The protocol consists of the following step: lysis, binding, washing, and elution. Contaminants are removed during the binding and washing steps. The quality of DNA was assessed by the A260/280 ratio at NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE), DNA integrity was checked by denaturing agarose gel electrophoresis.

DNAs were modified by sodium bisulfite using the EpiTect Bisulfite kit (Qiagen Inc.) following manufactory’s instructions. Modified DNAs were analyzed by real-time MSP on the ABI7500 PCR (ABI Co.) using the SYBR Premix Taq ExTaq Kit (TaKaRa Co. Ltd). XAF1 methylation and unmethylation specific primers were designed as follows: XAF1 (MF) 5′-TTTGTAAGAAACG AAATTTAATCGA-3′, (MR) 5′- CCTACCCTTAAAACCCACGAT-3′; XAF1(UF) 5′-TTTGTAAGAAATGAAATTTAATTGA-3′, (UR) 5′-CTCCTACCCTTAAAACC CACAAT-3′ [23]. Human genomic DNA (NEB) treated by SssI methyltransferase in vitro was used as a positive control. A peripheral blood DNA from a healthy subject was used as a negative control. The percentage of methylated DNAs in the samples were calculated according to the references as previous described [27], [28]. Methylated DNAs index was scored according to the percentage of DNA methylation; 0, <20%; 1, 20%−40%; 2, 40%−60%; 3, 60%−80%; and 4, >80%. The index score of 0 is considered as DNA unmethylation and scores 1–4 were considered hypermethylated, respectively [7], [27]. The 20% cut off threshold for DNA hypermethylation was based on control normal samples and internal quality controls obtained in the real-time MSP analysis.

Cell Culture and Drugs Treatment

Human gastric cancer cell lines (AGS and KATO-III) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C and 5% CO_2, respectively. Cultured cells were treated with 5-aza-2′-deoxycytidine (5-Aza-CdR) (Sigma-Aldrich) at a final concentration of 1.0 µmol/L. Trichostatin A (TSA) (Sigma-Aldrich) at a final concentration of 20 ng/ml was administrated following 5-Aza treatment or alone for 24 h. Cells were collected and subjected to DNA, RNA and protein purification and subsequent analyses.

Statistical Analysis

SPSS 17.0 statistical software was adopted for data analysis. Counting data comparisons between groups were subjected to the χ ² test and Fisher’s exact test. Survival analysis was computered by means of the Kaplan-Meier method and significant levels were assessed by means of the log-rank test. A univariate analysis with the Cox regression model was used to determine identified prognostic factors, and multivariate analysis with the Cox regression model was used to explore combined effects. For all statistical analyses, p values <0.05 were considered to be statistical significance.

Down-regulation of XAF1 in Primary Gastric Tumors

To investigate XAF1 gene expression profile, we examined mRNA expression of XAF1 in 88 non-cancer volunteers, and 202 primary gastric cancer tissues and their corresponding PCHNTs. As shown in Figure 1A, the XAF1 expression was significantly reduced in gastric cancer samples as compared with that in normal controls and PCHNTs (p<0.001). However, there was no significant difference in XAF1 expression in PCHNTs as compared to non-cancer controls.

Furthermore, XAF1 expression was significantly lower in advanced tumors (stage III/IV) than that in early stage tumors (stage I/II) (p<0.0001, Figure S1A), and was significantly lower in poorly differentiated tumors than that in well/moderately differentiated tumors (p<0.0001, Figure S1B).

To check XAF1 protein level in gastric cancer tissues, we performed immunohistochemical analysis in the 202 gastric cancer tissues and their corresponding PCHNTs. Nuclear XAF1 expression was consistently present in the normal gastric epithelia showing high immunoreactive scores. The XAF1 protein expression was detected in high level in 97.5% (197/202) of PCHNTs (Figure 1B). However, high expression of XAF1 protein was only detected in 16.8% (34/202) of gastric cancer tissues; majority of gastric cancer tissues were absent or at a low level of XAF1 protein (Figure 1C). The immunohistochemical findings are summarized in Table 1. The immunohistochemical staining score in gastric cancer tissues was significantly lower than that in PCHNTs or in normal controls (p<0.0001, Figure 1D). The loss of XAF1 protein significantly correlated with H. pylori infection, tumor size, histological differentiation, lymphatic invasion, venous invasion, invasive depth, lymph node metastasis, distant metastasis and clinical stage (Table 1) (all p<0.05).

The immunohistochemical results of 20 gastric cancer tissues were further confirmed by means of western blot analysis. The representative western blotting results in two cases were shown in Figure 1E. The relative quantity of XAF1 protein expression was normalized to the β-tubulin of the same samples. The average XAF1 protein level in 20 gastric cancer tissues was significantly lower than that in PCHNTs (p<0.05) (Figure 1F).

Promoter Hypermethylation of XAF1 in Primary Gastric Tumors

To investigate the molecular mechanisms for the XAF1 silence in gastric cancers, we applied a real-time MSP technology to study the DNA methylation status of XAF1 promoter. The frequency of XAF1 hypermethylation in gastric cancer tissues, corresponding PCHNTs and non-cancer controls were 83.2% (168/202), 24.8% (50/202) and 5.7% (5/88), respectively. The hypermethylation frequency of XAF1 in cancers is significantly higher than that in PCHNTs and non-cancer controls (all p<0.0001, Figure 2).

Of note, the methylated status of XAF1 significantly correlated with some clinico-pathological parameters in gastric cancer, such as lymph node metastasis, T-stage, H. pylori infection, etc (all p<0.05, Table 2). No significant correlation between the hypermethylation of XAF1 and gender, age at diagnosis, tumor site and distant metastasis was evidenced (all p>0.05) (Table 2).

XAF1 Promoter Hypermethylation is Associated with its Transcriptional Silencing in Gastric Cancer Cells

To examine the relationships between XAF1 methylation and XAF1 expression, we compared the XAF1 methylation level with XAF1 mRNA level and protein levels determined either by immunohistochemical analysis (Figure 3A) or western blotting (Figure 3B) by the Spearman correlation analysis. As shown in Figure-3A, XAF1 protein expression (determined by immunohistochemical analysis) in 202 GC tissues was closely correlated with XAF1 mRNA level [lg(T/N)] (determined by RT-PCR) (γ = 0.841, p<0.0001). More important, XAF1 methylation level was significantly associated with XAF1 mRNA level (γ = - 0.846, p<0.0001) and XAF1 protein level (γ = −0.969, p<0.0001). Similar results were obtained when analyzing the correlation of XAF1 promoter methylation and the XAF1 protein expression determined by western blot analysis (Figure 3B). These results strongly indicted that XAF1 expression is regulated by XAF1 promoter methylation.

5-Aza-CdR Administration Restores the Expression of XAF1

To further confirm the epigenetic regulation of XAF1 expression, AGS and KATO-III cells were treated with 5-Aza-CdR and/or TSA. XAF1 mRNA expression was reactivated in both gastric cancer cell lines, accompanied by demethylation of XAF1 promoter (Figure 4), indicating that XAF1 is transcriptionally silenced in these cells by DNA hypermethylation. Interestingly, TSA treatment alone was effective in restoring XAF1 expression in AGS and KATO-III without significant change of XAF1 methylation level, suggesting that histone modifications may also be involved in regulating XAF1 expression. However, administration of TSA following 5-Aza-CdR had an additive effect in restoring gene expression with a further decrease in the methylation level of XAF1. These results are in agreement with recent studies that suggested that TSA can have a demethylation effect in a gene-specific manner [5]. The western blot analysis using AGS cells, as a model, confirmed the up-regulation of XAF1 proteins following the 5-Aza-CdR and 5-Aza-CdR/TSA treatments (Figure 4).

Detection of Circulating XAF1 Methylation

We detected high frequency of XAF1 methylation in primary gastric cancer (83.2%), suggesting that it may be a good biomarker if XAF1 methylation could be detectable in serum. Therefore, we examined XAF1 methylation in the matched serum DNAs from the 202 GC patients. XAF1 methylation was detected in 141 (69.8%) serum DNAs from the 202 gastric cancer patients (Figure 5). In contrast, XAF1 methylation was not detected in serum DNAs from the 88 non-cancer controls.

Next, we confirmed the consistency in XAF1 methylation between tumor tissues and corresponding serum. In 168 cases that showed XAF1 methylation in tumor tissues, 141 displayed XAF1 methylation in their paired serum, giving a consistency of 83.9% between them. And in the 34 gastric cancer patients without XAF1 methylation in their gastric cancer tissues, no methylation was found in all the serum DNAs (Figure S2).

XAF1 Methylation as a Biomarker in the Diagnosis of Gastric Cancer

To evaluate the value of XAF1 methylation as a biomarker in diagnosing GC, we plotted receiver operating characteristic (ROC) curves and calculated area under the curve (AUC) values using DNA methylation data from both GC tissues and sera. Both the ROC analyses of XAF1 methylation in tissues and sera revealed significant discriminative capacity (Figure 6); the AUC value of tissues XAF1 methylation was 0.849 (95% confidence interval, 0.806–0.892; p<0.0001), the AUC value of serum AXF1 methylation was 0.909 (95% confidence interval, 0.875–0.942; p<0.0001).

In addition, like XAF1 methylation status in gastric cancer tissues and the sera significantly correlated with lymph node metastasis, T-stage, clinical stage, and other clinico-pathological parameters (all p<0.05, Table 2).

XAF1 Methylation Correlated with Prognosis of Gastric Cancer Patients

The significant correlation of XAF1 methylation with many clinico-pathological parameters (Table 2) suggested that it may associate with the prognosis of gastric cancer patients. Until the due date of Follow-up, 113 of 168 patients with XAF1 hypermethylation in gastric cancer tissues went rapid disease progression or died. The Median disease free survival (DFS) was only 23.4 months. In contrast, in the 34 patients without XAF1 hypermethylation in their gastric cancer tissues, only 5 patients were deteriorating, and the Median DFS was 39.6 months. Kaplan-Meier analysis demonstrated that patients with XAF1 hypermethylation in their gastric cancer tissues exhibited an obvious worse survival than that without XAF1 hypermethylation (p<0.0001) (Figure 7A). Similarly, Kaplan-Meier analysis proved that patients with positive serum XAF1 methylation had significantly lower DFS than that in the patients without serum XAF1 methylation (p<0.0001) (Figure 7B), indicating that XAF1 promoter methylation in serum was an unfavorable predictor for the gastric cancer patients.

Cox regression analysis revealed that XAF1 methylation in sera is an independent factor on patients’ survival: patients with XAF1 methylation had worse prognosis (p<0.0001; Hazard ratio, 5.710; 95% CI, 3.474∼9.383). In addition, TNM stages and age at diagnosis could be considered as the influencing factor of prognosis in gastric cancer, only when the effect of XAF1 methylation was eliminated. Moreover, circulating methylated XAF1 in blood had a greater impact on the prognosis than that in TNM stages (Table S1).

Positive Transition of Circulating XAF1 Methylation Predicts Tumor Recurrence and Poor Prognosis

Among the 202 patients who were evaluated for preoperative circulating XAF1 methylation, 72 patients received follow-up examinations of circulating XAF1 methylation 2∼5 times at intervals of 1∼6 months after surgery. Among 12 recurrent patients, 10 patients displayed negative-to-positive transition in XAF1 methylation status in their sera, the other two showed always positive for XAF1 methylation, suggesting that monitoring of XAF1 methylation in serum may be a good marker for predicting tumor recurrence (Table 3).

Conclusions

Dysfunction of XAF1 is frequent and is regulated through XAF1 promoter hypermethylation in gastric cancer. Circulating methylated XAF1 DNA was associated with tumor burden and malignant progression, which may be a valuable biomarker for diagnosis of gastric cancer, predicting patients’ prognosis and monitoring tumor recurrence after surgery treatment.