Decreased expression of prenyl diphosphate synthase subunit 2 correlates with reduced survival of patients with gastric cancer

This study conformed to the ethical guidelines of the World Medical Association Declaration of Helsinki–Ethical Principles for Medical Research Involving Human Subjects and has been approved by the Institutional Review Board of Nagoya University, Japan. Written informed consent for usage of clinical samples and data, as required by the institutional review board, was obtained from all patients [4].

Eleven GC cell lines (H111, KATOIII, MKN1, MKN28, MKN45, MKN74, NUGC2, NUGC3, NUGC4, SC-2-NU and SC-6-LCK) and CCL-241 (non-cancerous cell line derived from the small intestine) were obtained from the American Type Culture Collection (Manassas, VA, USA) or Tohoku University, Japan. The GC cell lines were cultured at 37°C in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum in an atmosphere containing 5% CO₂. For CCL-241, 30 ng/ml of the epidermal growth factor (Sigma-Aldrich) was added in the medium. Primary GC tissues and corresponding normal adjacent tissues were collected from 238 patients who underwent gastric resection for GC at Nagoya University Hospital between 2001 and 2012. Patients who received neoadjuvant therapy were excluded because it was difficult to obtain cancer cells from scarred tissues. Specimens were classified histologically using the 7th edition of the Union for International Cancer Control (UICC) classification [19]. Relevant clinicopathological parameters were acquired from medical records. To evaluate whether the expression level of PDSS2 correlated with tumor phenotype, patients were categorized into three groups according to the definition of GC subtypes according to the criteria of Shah et al. [9] as follows: proximal nondiffuse, diffuse, and distal nondiffuse type. Since 2006, adjuvant chemotherapy using S-1 (an oral fluorinated pyrimidine) is administered to all UICC stage II–III patients with GC unless contraindicated by the patient’s condition [20]. Patients were followed at least once every 3 months for 2 years after surgery and then every 6 months for 5 years or until death. Physical examination, laboratory tests, and enhanced computed tomography (chest and abdominal cavity) were performed at each visit. Chemotherapy for patients with distant metastasis or after recurrence was determined by physician’s discretion.

Tissue samples were immediately frozen in liquid nitrogen and stored at –80°C. Tumor samples without necrotic areas (approximately 5 mm²) were extracted by gross observation and only samples confirmed to comprise more than 80% tumor components by H&E staining were included in this study. Corresponding normal adjacent gastric mucosa samples >5°Cm from the edge of the tumors were obtained from the same patient [21].

Total RNAs (10 μg per sample) were isolated from 11 GC cell lines, CCL-241, 238 primary GC tissues and corresponding normal adjacent tissues were used to generate cDNAs, which were amplified using specific PCR primers (Additional file 1: Table S1). Real-time detection of SYBR® Green fluorescence intensity was conducted using an ABI StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The expression of GAPDH mRNA was quantified in each sample for standardization. The qRT-PCR reactions in each sample were performed in triplicate. The expression level of each sample is presented as the value of the PDSS2 amplicon divided by that of GAPDH[22]. PDSS2 mRNA expression was defined as decreased in GC tissues when its level was less than half that of the corresponding normal adjacent tissue.

The nucleotide sequence of the PDSS2 promoter region was analyzed to determine the presence or absence of CpG islands defined as follows: at least a 200-bp region of DNA with a high GC content (>50%) and an Observed CpG/Expected CpG ratio ≥0.6 [23]. We used CpG Island Searcher software (http://​cpgislands.​usc.​edu/​) to determine the locations of CpG islands [24].

PDSS2 possesses a CpG island near its promoter region, and we hypothesized that aberrant methylation is responsible for regulating the transcription of PDSS2 in GC. DNA samples from 11 GC cell lines treated with bisulfite were subjected to MSP and nucleotide sequence analysis [25]. The primer sequences used for MSP and bisulfite sequencing are listed in Additional file 1: Table S1.

To assess the relation of promoter hypermethylation to PDSS2 transcription, GC cells (1.5 × 10⁶) were treated with 5-aza-dC (Sigma-Aldrich) to inhibit DNA methylation and cultured for 6 days with medium changes on days 1, 3, and 5. RNA was extracted, and RT-PCR was performed as described [7].

IHC analysis of the localization of PDSS2 was performed using a mouse monoclonal antibody against PDSS2 (ab119768, Abcam, Cambridge, UK) diluted 1:150 in antibody diluent (Dako, Glostrup, Denmark) to probe 30 representative formalin-fixed and paraffin-embedded sections of well-preserved GC tissue described previously [3]. Staining patterns were compared between GCs and the corresponding normal adjacent tissues. To avoid subjectivity, the specimens were randomized and coded before analysis by two independent observers who were unaware of the status of the samples. Each observer evaluated all specimens at least twice to minimize intra-observer variation [7].

Correlations between the pattern of PDSS2 mRNA expression and clinicopathological parameters were evaluated according to the differences among the three GC subtypes. Subgroup analysis of survival according to GC subtype was performed to determine the influence of PDSS2 expression on patients’ outcomes.

Relative levels of mRNA expression (PDSS2/GAPDH) between GC and adjacent normal tissues were analyzed using the Mann–Whitney U test. The χ² test was used to analyze the significance of the association between the expression and methylation status of PDSS2 and clinicopathological parameters. Disease-specific and disease-free survival rates were calculated using the Kaplan–Meier method, and the difference in survival curves was analyzed using the log-rank test. We performed multivariate regression analysis to detect prognostic factors using the Cox proportional hazards model, and variables with P < 0.05 were entered into the final model. All statistical analyses were performed using JMP 10 software (SAS Institute Inc, Cary, NC, USA). A value of P < 0.05 was considered statistically significant.

A CpG island was identified in the PDSS2 promoter region using the CpG Island Searcher. The properties of the CpG island are as follows: 1655 bp, 55.9% GC, and 0.70 Observed CpG/Expected CpG ratio (Figure 1A). Therefore, we hypothesized that hypermethylation of the CpG islands regulates the expression of PDSS2 in GC.

Significant decreases in PDSS2 mRNA levels were detected in seven (73%) of 11 GC cell lines compared with the expression level of the CCL-241 cell (Data for GC tissues are described below). There was no apparent difference in expression levels between cell lines derived from differentiated and undifferentiated GCs (Figure 1B). Hypermethylation of the PDSS2 promoter was detected in MKN1, SC-2-NU, KATOIII, MKN45, and NUGC3 cells (Figure 1B). To determine whether hypermethylation of the PDSS2 promoter inhibited transcription, mRNA expression levels were compared before and after treating cells with the methylation inhibitor 5-aza-dC. PDSS2 mRNA levels were restored in cells with down-regulated PDSS2 expression accompanying hypermethylation after 5-aza-dC treatment (Figure 1B), indicating that promoter hypermethylation inhibited PDSS2 transcription in GC. Representative chromatograms of the sequence analysis of the PDSS2 promoter region in MKN28 (complete methylation) and NUGC4 (absence of methylation) cells are shown in Figure 1C.

The patient population included 179 males and 59 females aged from 20 to 84 years (65.3 × 11.7 years, mean × standard deviation). Pathologically, 139 and 99 patients were diagnosed with undifferentiated and differentiated GC, respectively. Patients were classified into the three GC phenotypes as follows: nondiffuse, 54; diffuse, 48; and distal nondiffuse, 136. According to the 7th edition of the UICC classification, 58, 40, 71 and 69 patients were in stages I, II, III and IV, respectively. One hundred sixty-four patients in stages I–III underwent R0 resection. Sixty of the 69 patients in UICC stage IV were diagnosed as stage IV due to positive peritoneal lavage cytology, localized peritoneal metastasis or distant lymph node metastasis. Eight of patients in stage IV had synchronous liver metastasis, one had lung metastasis, and they underwent gastrectomy aimed to control bleeding or obstruction to the passage of food.

The mean expression level of PDSS2 mRNA was lower in GC tissues compared with that of normal adjacent tissues (P <0.001); however, there was no significant difference in PDSS2 mRNA expression levels between patients with undifferentiated or differentiated GC (Figure 2A). The expression pattern of PDSS2 was evaluated using IHC. Representative cases with reduced PDSS2 staining of GC tissues are shown in Figure 2B. Overall, the staining patterns of PDSS2 were consistent with the qRT-PCR data.

The expression of PDSS2 mRNA in GC tissues was decreased in 76 (32%) of 238 patients (less than half the level of expression detected in the corresponding normal adjacent tissues). The disease-specific survival rate of patients with decreased levels of PDSS2 mRNA in GCs was significantly lower compared with those without (5-year survival rates, 36% and 64%, respectively, P <0.001, Figure 3A). Decreased levels of PDSS2 mRNA in GCs were significantly associated with carbohydrate antigen (CA) 19-9 > 37 IU/ml and lymph node metastasis (Table 1). Univariate analysis of disease-specific survival showed that GC subtype (proximal nondiffuse or diffuse), CA 19-9 > 37 IU/ml, tumor size (≥50 mm), pT4, undifferentiated tumor, lymphatic involvement, vessel invasion, invasive growth, lymph node metastasis, positive peritoneal lavage cytology, and decreased PDSS2 mRNA expression in GC tissues were significant prognostic factors of adverse outcomes. Multivariate analysis identified decreased PDSS2 mRNA expression as an independent prognostic factor (hazard ratio 1.95, 95% confidence interval 1.22–3.09, P = 0.005, Table 2). The proportional hazards assumption in the Cox model was assessed with models including time-by-covariate interactions and no significant violations were found in the model.

Of the 168 patients who underwent R0 resection, the disease-specific survival rate was significantly lower for those with decreased PDSS2 mRNA expression in GCs (n = 50) compared with those without (n = 118) (5-year survival rates, 50% and 77%, respectively, P = 0.006, Figure 3B). Patients with decreased PDSS2 mRNA expression in GCs experienced significantly earlier recurrences after surgery compared with those without (2-year recurrence-free survival rates, 64% and 84%, respectively, P = 0.022, Figure 3C). Initial recurrence sites of 43 relapsing patients with decreased PDSS2 mRNA expression in GCs were peritoneal in 21 (49%), liver in 6 (14%), lymph node in 13 (30%) and others (e.g. lung and bone) in 3 patients. On the other hand, those of 61 relapsing patients without decreased PDSS2 were peritoneal in 35 (57%), liver in 10 (16%), lymph node in 8 (13%) and others in 8 patients. Patients with decreased PDSS2 mRNA expression in GCs were likely to have a node relapse, though it did not reach the statistical significance.

Mean PDSS2 mRNA expression levels were equivalent in GC and normal adjacent tissues (Figure 4A). Similarly, the prognostic value of decreased PDSS2 mRNA expression in GCs was comparable among the three GC subtypes (Figure 4B).