Role of the PEBP4 protein in the development and metastasis of gastric cancer

Phosphatidylethanolamine-binding protein 4 (PEBP4) has previously been reported to be upregulated in various cancers. However, the physiological functions of PEBP4 in gastric cancer are still unknown. Aiming to clarify the properties and role of PEBP4 in the development and invasion of gastric cancer, we performed several biological assays and a knockdown assay. The expression level of PEBP4 was shown to be significantly upregulated in gastric cancer tissue samples, and knockdown of the expression of PEBP4 induced significant inhibitory effects on cell proliferation, migration and invasiveness. In addition, it was demonstrated that PEBP4 was associated with the development and invasion of gastric cancer cells through activation of the PI3K/Akt signaling pathway. Our findings supported the hypothesis that PEBP4 might be a novel potential drug target for the treatment of gastric cancer.


INTRODUCTION
Gastric cancer is one of the main contributors to cancer-related mortality worldwide [1]. Despite recent developments in early diagnosis and other novel therapeutic strategies, the median survival time for advanced gastric cancer patients has still remained at approximately 7 months [2,3]. Thus, investigations into the molecular mechanisms of invasion and metastasis of gastric cancer seek to contribute to the development of a novel and effective drug target.
Phosphatidylethanolamine-binding protein 4 (PEBP4) is a member of the PEBP family, which shares a similar domain consisting of a C-terminal large β-sheet flanked by smaller β-sheets and 2 α-helices [4]. It has been reported that PEBP4 plays important roles in multiple biological processes, including the synthesis of the cell membrane, myoblast differentiation, neural development and tumorigenesis [5][6][7][8]. Recently, the upregulation of PEBP4 was reported to be associated with the proliferation, invasion and drug resistance of tumor cells [9,10]. However, direct evidence for the role of PEBP4 in cancer development and invasion is still lacking. In this study, we silenced the expression of PEBP4 by specific siRNA in gastric cancer cell lines and investigated the effects of PEBP4 on cell proliferation, migration and invasion. Our results show that the effects of PEBP4 on the tumorigenesis and metastasis of gastric cancer depend on the dysregulation of the PI3K/Akt signaling pathway.

PEBP4 is highly expressed in gastric cancer tissue and cell lines
To explore the potential role of PEBP4 in the tumorigenesis of gastric cancer, the mRNA levels of PEBP4 were studied in gastric cancer tissue samples and the corresponding adjacent normal tissue samples from 11 patients using RT-qPCR. The mRNA levels of PEBP4 in human gastric cancer tissue were significantly higher than those in the adjacent normal gastric tissue, as shown in Figure 1A. The western blot results showed that the expression level of the PEBP4 protein was markedly www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 11), pp: 18177-18184 Research Paper higher in human gastric cancer tissue samples than in normal gastric tissue samples ( Figure 1B). Similarly, the mRNA and protein expression levels of PEBP4 were also increased in gastric cancer cell lines ( Figure 1C and 1D).

Knockdown of PEBP4 inhibited the proliferation, migration and invasion of gastric cancer cells
To clarify the role of PEBP4 on cell proliferation, migration and invasion, the TSGH and AGS cells were transfected with siRNA-PEBP4 or siRNA-control. The western blot results demonstrated a remarkable downregulation of PEBP4 expression in TSGH and AGS cells (Figure 2A). The results of the cell proliferation assay indicated that knockdown of PEBP4 greatly suppressed the proliferation of both types of cells in a time-dependent manner ( Figure 2B). Additionally, knockdown of PEBP4 resulted in a remarkable reduction in tumor cell migration and invasion ( Figure 3).
Interestingly, knockdown of PEBP4 was found to retard the epithelial-mesenchymal transition (EMT) phenotype in gastric cancer cells. The RT-qPCR results indicated that knockdown of PEBP4 resulted in a significant increase in the mRNA levels of E-cadherin and a decrease in N-cadherin in TSGH cells ( Figure 4A) and AGS cells ( Figure 4B). Similarly, the western blot results also suggested an inhibitory effect on the EMT in gastric tumor cells upon knockdown of PEBP4 ( Figure 4C).
It was observed that knockdown of PEBP4 greatly inhibited the phosphorylation of PI3K and Akt, suggesting that PEBP4 might exert its physiological functions via modulation of the PI3K/Akt signaling pathway ( Figure 5).

DISCUSSION
PEBP4 has been reported to be involved in the tumor development and metastasis of various cancers [11]. For example, PEBP4 has been reported to be upregulated in pancreatic ductal adenocarcinoma (PDAC), and knockdown of PEBP4 has been shown to result in the attenuation of cancer cell growth, migration, and metastasis of PDAC cancer cells [10]. In lung cancer, PEBP4 is capable of enhancing the cell proliferation and invasion ability [12]. Liu et al. reported that PEBP4 in colorectal cancer tissues was markedly increased and the downregulation of PEBP4 significantly reduced the migration of colorectal cancer cells in a Transwell chamber [13].
In the present study, PEBP4 was first demonstrated to be upregulated in gastric cancer tissue samples and cell lines. Knockdown of PEBP4 inhibited cell proliferation, migration and invasiveness. EMT is an important process during tumor development, characterized by reduced intercellular adhesion and enhanced motility of epithelial cells [14]. As one of the markers of EMT, the measurement of the levels of E-cadherin and N-cadherin was a crucial approach to investigate the EMT phenotype and then evaluate tumor invasion [15]. In the present study, knockdown of PEBP4 was found to remarkably increase the expression of E-cadherin and decrease the expression of N-cadherin in TSGH and AGS cells. The results suggested that knockdown of PEBP4 retarded the EMT process in gastric cancer cells, which might have contributed to the inhibitory effect of the siRNA-PEBP4 on tumor migration and invasion.
The PI3K/Akt pathway was determined to be associated with proliferation, metastasis, apoptosis and EMT in gastric cancer [16][17][18][19][20][21]. Treatment of gastric cancer cells with a PI3K/Akt kinase inhibitor significantly   PI3K, total PI3K, phosphorylated Akt, and total Akt were analyzed using western blot analysis. TSGH cells were transfected with siRNA against PEBP4 and scrambled siRNA for 30 min. www.impactjournals.com/oncotarget retarded the EMT phenotype by inhibiting bone morphogenetic protein (BMP)-2 [22,23]. PEBP4 was previously found to be crucial for the phosphorylation of Akt at serine 473 [10]. Additionally, the silencing of hPEBP4 in TRAIL-resistant human prostate cancer cells inhibited Akt activation [24]. In this study, it was also found that knockdown of PEBP4 markedly inhibited the phosphorylation of PI3K and Akt in gastric cancer cells.
Together, the results in this study supported the hypothesis that knockdown of PEBP4 inhibits cell invasion and EMT through suppressing the PI3K/Akt signaling pathway. These results suggest that PEBP4 might be potential drug target for the treatment of gastric cancer.

Tissue sample collection
Gastric cancer tissue samples were obtained from 11 patients undergoing surgical treatment at the Department of Oncology, Tianjin Medical University General Hospital (China), during the period from 2014 to 2015 (average age=59.6). All of the patients received primary treatments by surgery, chemotherapy or hormone therapy. Healthy tissue samples were from non-pathologic areas distant from the tumors in surgical specimens. The fresh tissue specimens were immediately snap-frozen and stored in liquid nitrogen until use. The protocol for the use of patient samples was approved by the Biomedical Ethics Committee of the Tianjin Medical University General Hospital.

Cell proliferation assay
Cells were transfected with siRNA-PEBP4 and siRNA control. After 24 h of serum starvation, the cells were incubated with BrdU solution at 37°C for 24 or 48 h using the cell proliferation ELISA BrdU kit (Takara Biotechnology, Dalian, China). The amount of BrdU was measured at 450 nm by a microplate reader (Bio-Rad, Hercules, CA, USA).

Cell migration and invasion assays
The invasion and migration of gastric cancer cells upon downregulation of PEBP4 were analyzed by a Transwell chamber (Corning Costar Corp., Cambridge, MA, USA) assay. The TSGH and AGS cells treated with siRNA-PEBP4 or siRNA-control (1×10 5 cells/ml) were added to the upper chamber. The lower chamber of the Transwell was filled with 500 μl DMEM containing 10% FBS as a chemo-attractant. After 24 h incubation, cells on the surface of the upper chamber were removed by scraping with a cotton swab. The migrated cells on the lower surface of the chamber were stained with 0.1% crystal violet (Sigma, MO) and counted. These experiments were repeated three times.
For the invasion assay, cells were seeded onto a Matrigel-coated membrane matrix (BD Biosciences, Bedford, MA, USA) in a 24-well culture plate for 24 h after transfection. The cells that did not invade through the pores were carefully wiped with cotton wool. Then, cells located on the lower surface of the chamber were stained with 0.1% crystal violet (Sigma, MO) and counted. These experiments were repeated three times.