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Ornithine aminotransferase and carbamoyl phosphate synthetase 1 involved in ammonia metabolism serve as novel targets for early stages of gastric cancer

Abstract Objective The sensitivity and specificity of current biomarkers for gastric cancer were insufficient. The aim of the present study was to screen novel biomarkers and determine the diagnostic values of ornithine aminotransferase (OAT) and carbamoyl phosphate synthetase 1 (CPS1) for detecting gastric cancer. Methods With stable isotope tags, we labelled an initial discovery group of four paired gastric cancer tissue samples and identified with LC‐ESI‐MS/MS. A validation group of 159 gastric cancer samples and 30 healthy controls were used to validate the candidate targets. GSEA was used to explore the pathways activated in gastric cancer. Results Four hundred and thirty one proteins were found differentially expressed in gastric cancer tissues. Of these proteins, OAT and CPS1 were found over‐expressed in gastric cancer patients, with sensitivity of 70.4% (95% CI: 63.3%–77.6%) and specificity of 80.5% (95% CI: 74.3%–86.7%) for ornithine aminotransferase, and with sensitivity of 68.6% (95% CI: 61.3%–75.8%) and specificity of 73% (95% CI: 66%–79.9%) for carbamoyl phosphate synthetase 1. The co‐expression of OAT and CPS1 in gastric cancer tissues has a sensitivity of 81% (95% CI: 73.2%–88.8%) and specificity of 89% (95% CI: 83%–95%). Furthermore, both OAT and CPS1 were overexpressed in patients with local invasion T3 and T4 stages than those in patients with T1 and T2 stages. The co‐expression of OAT and CPS1 was strongly correlated with histological grade I 68% (95% CI: 58.7%–77.3%) and TNM stage I/II 52% (95% CI: 42%–62%). The areas under ROC curves were up to 0.758 for the co‐expression of OAT and CPS1 in gastric cancer. GSEA results showed that two gene sets and 30 gene sets were activated in OAT high‐ and CPS1 high‐expression patients with gastric cancer, respectively. Conclusions The present findings indicated a tight correlation between the co‐expression of OAT and CPS1 and the histological grade, local invasion, and TNM stages of gastric cancer. Therefore, OAT and CPS1 might be predictors for gastric cancer invasion and potential targets for anticancer drug design for gastric cancer.


| INTRODUC TI ON
Gastric cancer (GC) is the fifth most common type of cancer and the third leading cause of cancer-related death worldwide. The 5-year relative survival rate was approximately 55% in patients with stage II or III gastric cancer. 1 The contributors to high incidence and mortality of gastric cancer include distant metastasis and poor prognosis in advanced stage of gastric cancer.
The past decades have witnessed great advances in the treatment of patients with gastric cancer, including preoperative endoscopy and postoperative chemotherapy. With the wide use of endoscopic diagnostic and therapeutic techniques, the proportion of cured gastric cancers has been continuously growing year by years. 2 However, there were still a large number of patients who presented with advanced gastric cancer stage due to the lack of early detection methods and biomarkers. The 5-year survival rate of early gastric cancer patients undergoing radical resection can reach about 90%, while that of advanced gastric cancer patients was less than 30%. The serum markers such as CA19-9, CEA, and CA72-4 were commonly used in clinical laboratory diagnosis for gastric cancer. However, the sensitivity and specificity of these markers were not ideal enough. For example, the sensitivity for CA19-9, CEA, and CA72-4 were 18.98%, 22.69%, and 22.69%, respectively. 3 Therefore, it was urgent for us to develop specific early diagnostic markers for gastric cancer.
In the present study, we aim to identify early screen biomarkers for gastric cancer. We used iTRAQ-labeled quantitative proteomics to determine the expression levels of proteins among gastric cancer and adjacent gastric tissues and benign gastritis as well. These results provided novel diagnostic and therapeutic targets for early detection and follow-up of gastric cancer.

| Sample collection
In this study, 159 cases of gastric cancer tissues and adjacent gas-

| Sample preparation and iTRAQ labeling
We used the discovery group including four pairs of gastric cancer tissue samples and surrounding gastric tissues for proteomics analysis. For the selection of four pairs of gastric cancer tissue samples, we set the criteria as follows: (1) ≥ 40 years; (2) adenocarcinoma; (3) no distant metastasis; (4) fresh gastrectomy specimens from patients with gastric cancer who did not receive preoperative radiotherapy or chemotherapy. All tissue samples were lysed in lysis buffer (7 M urea, 1 mg/ml DNase I, 1 mM Na 3 VO 4 , and 1 mM PMSF), centrifuged at 12,000 rpm, and the supernatant was collected for total protein concentration determination by using Bradford protein assay methods. Secondly, iTRAQ labeling was applied for each sample. We used 100 μg of sample protein for precipitation by adding six times volumes of cold acetone and then incubated at −20°C for 4 h.
After precipitation, the protein was dissolved in solution buffer and then denatured, and the cysteine was blocked according to the manufacturer's instructions (Applied Biosystems). Next, 20 μl of 0.25 μg/μl trypsin was used to digest each sample protein. The iTRAQ 8-plex reagent was dissolved in methanol. The tryptic peptide of each sample was added into iTRAQ dissolution buffer, then mixed with iTRAQ 8-plex reagent, and incubated for 1 h at room temperature. The labeled protein was pooled before further analysis.

| Strong cation exchange chromatography and MS/MS analysis
The iTRAQ-labeled samples were fractionated using a Shimadzu LC-20AB HPLC pump system fitted with a 5μm particle size, 4.6 × 250 mm Ultremex SCX column (Phenomenex). The column was eluted with a gradient of a buffer A (25 mM NaH 2 PO 4 in 25% ACN, pH 2.7) for 10 min, 5%-35% buffer B (25 mM NaH 2 PO 4 , 1 M KCl, 25% ACN, pH 2.7) for 11 min, and 35%-80% buffer B for 1 min. The fractions were collected at 1-min intervals. The eluted peptides were pooled into 20 fractions. The peptides dried by the Speed-Vac concentrator and desalted with C18 Sep-Pak. MS/MS analysis was conducted by using a LTQ Orbitrap Velos (Thermo) system connected to the HPLC. The SCX-fractionated peptides dissolved in 2% ACN, 0.1% FA. The sample volume was 10 μl per injection. A data-dependent procedure was applied to the MS scan for the most abundant precursor ions by using a threshold ion count of 5000. During the LC-MS mode, data were acquired in the m/z range of 350-2000 Da.

| Peptide identifications
The acquired data were searched against the International Protein Index (IPI) human database using the Mascot (Matrix Science version K E Y W O R D S amino metabolism, CPS1, gastric cancer, iTRAQ, OAT 2.2). The data were searched with a tolerance of 10 ppm on the precursors and 0.05 Dalton on the fragment ions. If there was least one such unique peptide match, a protein was considered identified. We set the threshold of a 1.2-fold increase or decrease as significant change in expression.

| Gene set enrichment analysis
Gene set enrichment analysis (GSEA) was used to elucidate pathways that were activated in gastric cancer. RNA-seq data of gastric cancer from the Gene Expression Omnibus database (GSE84437, https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE84437) was analyzed with GSEA software (V2.0; Broad Institute). Gene sets with NES >1, p value≤0.05 and FDR <0.25 were considered as significantly enriched.

| Statistical analysis
The χ 2 test was applied to calculate the correlation of protein expression with clinicopathological parameters in gastric cancer patients. GSEA V2.0 was used to perform gene set enrichment analysis. All statistical analyses were performed by SPSS 22.0 (SPSS) for Windows.

| Identification of OAT and CPS1, the potential biomarkers for gastric cancer
To identify the expression of OAT and CPS1, we used a discovery group of four pairs of technical replicates of gastric cancer tissues and adjacent gastric tissues to perform the iTRAQ quantitative proteomics. Our previous studies had showed that 224 proteins were significantly upregulated and 207 proteins were downregulated in gastric cancer relative to paired adjacent gastric tissues. 5 OAT was found to be upregulated with a 1.294 fold changes in gastric cancer tissues, and CPS1 was found to be upregulated with a 1.333 fold changes in gastric cancer tissues. Figure 1 showed the representative MS/MS peptides spectrum for OAT and CPS1 in gastric cancer, respectively.

| Comparison of pathological variables of OAT positive patients with OAT negative patients
To find out the correlation of OAT expression with the pathological variables of gastric cancer patients, we compared the pathological variables between OAT-positive and -negative patients. All gastric cancer tissues underwent immunohistochemistry of OAT expression evaluation. We selected histological type, T staging, and node metastasis for further analysis. We found that OAT was significantly overexpressed in patients with T3 and T4 stages compared with that of patients with T1 and T2 local invasion (p = 0.01) ( Table 2).

| Comparison of pathological variables between CPS1-positive patients and CPS1negative patients
The pathological variables of CPS1-positive expression patients and -negative expression patients are compared in Table 3. As can be seen from the Table 3 associated with the local invasion that CPS1 was highly expressed in patients with T3 and T4 stages than that of patients with T1 and T2 stages (p = 0.02) ( Table 3).

| The combined assay of OAT and CPS1 expression and its correlation with pathological variables of gastric cancer
To further analyze the association of combined OAT and CPS1 expression with gastric cancer, we divided the gastric cancer patients into several groups according to the pathological variables. As shown in Table 4, the coexpression of OAT and CPS1 was significantly associated with histological grade and TNM staging. There were 68% cases of gastric cancer patients with histological grade I who had a more positive rate of OAT and CPS1 coexpression than that of 32% in patients with histological grade II and III (p = 0.03). Moreover, the coexpression of OAT and CPS1 was significantly higher in gastric cancer patients with TNM III/IV stages than that of patients with TNM I/IIstages (p = 0.04) ( Table 4).

| Gene sets enrichment analysis to reveal genes activated in gastric cancer
We download the RNA-seq dataset of GSE84437 from the GEO database. We divided the subjects into OAT high group (top 50%) and OAT low group (bottom 50%) according to the expression of OAT in gastric cancer patients. We also divided the subjects into CPS1 high group (top 50%) and CPS1 low group (bottom 50%) based on CPS1 expression. We set the criteria of NES >1, p value ≤0.05 and FDR  Ornithine aminotransferase, a pyridoxal 5'phosphate-dependent mitochondrial enzyme, catalyzed the reversible interconversion of ornithine and α-ketoglutarate to l-glutamate semialdehyde, which cyclizes to Δ1-pyrroline-5-carboxylate (P5C) and l-glutamate. [6][7][8][9][10] Recently, studies showed that OAT was involved in proline metabolism, which was altered in cancer cells to sustain cell proliferation.
Moreover, cellular glutamate generated from OAT can be converted to glutamine by glutamine synthetase to synthesize de novo nucleotide.
Previous study found that OAT was significantly higher expressed in lung cancer specimens than that of normal lung tissues.
The over expression of OAT was significantly correlated with TNM III/IV staging. 11 Liu et al. 12   However, the OAT expression and potential mechanism in gastric cancer were still unknown. In the present study, we demonstrated that OAT was overexpressed in 70.4% cases of gastric cancer tissues, which was significantly higher than that of 19.5% cases of adjacent cancer tissues. Furthermore, OAT was significantly overexpressed in patients with T3 and T4 stages than that of patients with T1 and T2 stage (p = 0.01) ( Table 2). These data suggested that overexpression of OAT may affect gastric cancer cell local invasion and metastasis.
Previous studies suggested that OAT was a potential chemotherapy target for cancers. Silverman et al. 14   Contrary to previous studies, we found that CPS1 was highly expressed in 68.6% cases of gastric cancer tissues than that of 27% cases of adjacent gastric tissues and than that of 30% cases of benign gastritis (Table 1 and Figure 3A). On the other hand, another study found that CPS1 was significantly overexpressed in glioblastoma multiforme. 28 These discrepancies may be caused by the differences of sample sources, detection methods, and experimental conditions, which need to be further verified.
To screen the potential inhibitors for CPS1, Yao et al. 29 identified as a specific inhibitor H3B-120 for CPS1 through high-throughput screen. They found that H3B-120 binds to allosteric pocket to block ATP hydrolysis in the process of carbamoyl phosphate synthesis.
The analogues of H3B-120 can block CPS1 cellular activity in primary human hepatocytes. Furthermore, by using a high-throughput screening effort and structure based drug design, Alan et al. 30 found that piperazine H3B-616(25) as a potent allosteric inhibitor of CPS1.
Our GSEA analysis revealed that there were a number of genes ac- respectively. 3 Therefore, it was urgently required to find novel biomarkers to improve the diagnostic rate for early gastric cancer. In the present study, our validation study showed that the sensitivity and specificity of OAT were 70.4% and 80.5% for gastric cancer, respectively. The sensitivity and specificity of OAT in detecting gastric cancer were higher than that of CEA and CA-199. Furthermore, OAT was overexpressed in patients with T3 and T4 stages than that of patients with T1 and T2 stages, indicating OAT serves as a potential biomarker for gastric cancer.
Nevertheless, we were not content with the sensitivity and specificity obtained by OAT. We therefore combined analysis of OAT with CPS1 and obtained higher sensitivity and specificity for gastric cancer. For combined analysis of OAT and CPS1, we obtained 81% of sensitivity and 89% of specificity for gastric cancer ( Table 1 and Figure 3B). In addition, for combined analysis of OAT and CPS1, the AUC for gastric cancer was 0.758 ( Figure 4). Obviously, the combined analysis of OAT and CPS1 obtained a higher sensitivity, specificity, and AUC than those of traditional biomarkers, indicating that combined analyses of OAT and CPS1 were potential diagnostic biomarkers for screening and follow-up of gastric cancer. The roles of OAT and CPS1 in regulating ammonia metabolism in cancer cells are shown in Figure 6.
The limitations of the present study were as follows. (1), the sample size of the present study was relatively small. We will enlarge the sample size in future study to obtain more sufficient conclusions.
(2), because the OAT and CPS1 were expressed in a variety of tumors, we only tested the expression of OAT and CPS1 in gastric cancer and the adjacent normal gastric tissues in the present study. As long as conditions permit in the future, we will supplement the data from other tumor types to obtain more sufficient conclusions.

| CON CLUS ION
In conclusion, by using the iTRAQ based quantitative proteomics, the present study identified OAT and CPS1 that were significantly overexpressed in gastric cancer, which significantly correlated with clinical parameters, including histological grade, local invasion, and TNM staging, in patients with gastric cancer. Our study suggested that detecting of OAT and CPS1 can serve as novel diagnostic and therapeutic targets for early screen and follow-up for gastric cancer.

ACK N OWLED G M ENTS
We would like to thank the funding project of the National Natural

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The dataset (GSE84437) was available on the GEO database (https:// www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE84437). Other data are available from the corresponding author upon reasonable request.