FPR2 promotes invasion and metastasis of gastric cancer cells and predicts the prognosis of patients

Formyl peptide receptor 2 (FPR2), a classical chemoattractant receptor of G-protein-coupled receptors, is reported to be involved in invasion and metastasis of some cancers, but the role of FPR2 in gastric cancer (GC) has not yet been elucidated. In this study, we found that the levels of FPR2 expression in GC were positively correlated with invasion depth, lymph node metastasis and negatively correlated with the patients’ overall survival. Multivariate analysis indicated that FPR2 expression was an independent prognostic marker for GC patients. FPR2-knockdown significantly abrogated the migration and invasion stimulated by Hp(2–20) and Ac(2–26), two well-characterized ligands for FPR2 in GC cells. FPR2 deletion also reduced the tumorigenic and metastatic capabilities of GC cells in vivo. Mechanistically, stimulation with FPR2 ligands resulted in down-regulation of E-cadherin and up-regulation of vimentin, which were reversed by FPR2 knock-down, implying the involvement of epithelial–mesenchymal transition (EMT). Moreover, the activation of FPR2 was accompanied with ERK1/2 phosphorylation, which could be attenuated by FPR2 silencing or treatment with MEK inhibitor, PD98059. Altogether, our results demonstrate that FPR2 is functionally involved in invasion and metastasis, and potentially acts as a novel prognostic marker as well as a potential therapeutic target in human GC.

In this study, we evaluated the relevance between the expression of FPR2 and the clinical characteristics in GC. The results indicated that FPR2 was overexpressed in GC tissues and was an independent prognostic factor for the patients. Mechanistically, we demonstrated that FPR2 could enhance capabilities of invasion and metastasis of GC cells by activating MAPK/ERK pathway to induce EMT.

Results
FPR2 expression is associated with clinicopathological characteristics and outcome of GC patients. To elucidate the clinical relevance of FPR2 in the human GC, immunohistochemistry (IHC) was performed to detect the levels of FPR2 expression in tumor tissues and their adjacent normal tissues from 169 GC patients. The FPR2 protein was mainly localized in the cytomembrane and cytoplasm of the cancer cells. The expression of FPR2 was very low or absent in normal gastric mucosa (Fig. 1Aa). The highly expressed FPR2 was observed in cancer tissues as well as in metastatic lymph nodes (Fig. 1Ab-e). As shown in Fig. 1Ab-d, the staining intensity of FPR2 increased with invasion depth. Among GC specimens, 122 (72.2%) showed positive expression (FPR2 + ) and 47 (27.8%) showed negative expression of FPR2 (FPR2 − ), while in corresponding adjacent normal tissues, 131 (77.5%) were FPR2 − and 38 (22.5%) appeared FPR2 + (p < 0.001, Table S1). In a separate set of samples, quantitative analysis of FPR2 mRNA in 6 fresh surgical specimens indicated that 5 out of 6 tumor tissues had high level of FPR2 expression as compared with their adjacent normal tissues (Fig. 1B). The expression profiling by array from 2 reported datasets (GSE65801 and GSE27342) 23,24 showed that the mRNA expression of FPR2 was significantly higher in gastric cancer tissues than in the paired normal adjacent tissues (Fig. 1C). The correlation analysis between FPR2 expression in cancerous tissues and clinicopathological features showed that FPR2+ was positively related to TNM (Tumor, Node, Metastasis) stage (P = 0.002), serosal invasion (P = 0.015) and lymph node metastasis (P = 0.043), but not with histological grade (P = 0.812, Table 1).
Kaplan-Meier analysis was performed to analyze the association of FPR2 expression with the overall survival rates of GC patients. The patients with FPR2 + had shorter lifespan compared to those with FPR2 − (p = 0.0023, Fig. 1D). Univariate and multivariate analyses showed that the expression of FPR2 was an independent prognostic indicator for the overall survival of GC patients (p = 0.002 and p = 0.026, respectively) ( Table 2). To further reveal the prognostic significance of FPR2 expression in GC patients, Kaplan-Meier estimates were also performed in patients with different depth of invasion and with or without lymph node metastasis. In the serosal invasion group, patients with FPR2 + had worse overall survival compared to those with FPR2 − (P = 0.003, Fig. S1A), while similar lifespans of patients with FPR + or FPR − were observed in non-serosal invasion patients group (P = 0.9702, Fig. S1B). In the group of lymph node metastasis, the overall survival of FPR2 + patients was worse than that of FPR2 − patients (P = 0.0145, Fig. S1C), but the overall survival of patients was not associated with FPR2 expression in the group of non-lymph node metastasis (P = 0.1909, Fig. S1D). These results suggest that FPR2 may exert important roles in carcinogenesis and progress of GC and serve as a prognostic biomarker for the patients.

FPR2 promotes tumorigenesis and metastasis of GC cells in vivo.
Since FPR2 served as an inducer for migration and invasion of GC cells in vitro, we next examined the roles of FPR2 in tumorigenesis and metastasis of GC cells in vivo. Subcutaneous xenograft model was used to investigate the effect of FPR2 on tumorigenic ability in nude mice. Although both FPR2-knockdown shFPR2 SGC7901 or shFPR2 XN0422 cells and their mock cells at 1 × 10 5 cells/mouse had capability to form xenograft tumors in all nude mice, the size and weight of tumors derived from FPR2-knockdown cells were markedly smaller and lighter than that of tumors formed by paired mock cells (Fig. 3A,B). When the implantation was performed at 1 × 10 4 cells/mouse, not only the smaller and lighter xenograft tumors but also the reduced tumor-forming ratio (4/5 vs 5/5) were observed in FPR2-knockdown GC cells as compared to mock cells (Fig. 3A,B). An intraperitoneal metastasis model was employed to assess the role of FPR2 in metastasis of GC cells. shFPR2 SGC7901 and shFPR2 XN0422 and their mock cells were injected into the peritoneal cavity of nude mice at 2 × 10 4 cells/mouse for 4 weeks, respectively (n = 5). Metastatic nodules were found in all the four groups, but SGC7901 and XN0422 mock cells generated more metastatic nodules compared with shFPR2 SGC7901 and shFPR2 XN0422 cells (11.50 ± 2.89 vs 5.25 ± 2.22 *p < 0.05, **p < 0.01 and ***p < 0.001. and 14.50 ± 4.20 vs 6.75 ± 3.40, respectively. p < 0.05) (Fig. 3C,D). These results strongly suggest that FPR2 plays important roles in tumorigenesis and metastasis of GC.

The activation of FPR2 induces Epithelial-mesenchymal-transition in GC cells. Epithelial-
mesenchymal-transition (EMT), which converts tumor cells into an elongated, motile and invasive phenotype, has been well recognized as pivotal incident for tumor cells to invasion and metastasis. To clarify whether EMT is involved in the FPR2-promoting invasion and metastasis, the expression of EMT-related molecules E-cadherin and vimentin was examined in GC cells with or without FPR2 knockdown under presence or absence of FPR2 ligands. As shown in Fig. 4A, treatment with FPR2 ligands Hp (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and Ac(2-26) decreased mRNA expression of E-cadherin and enhanced mRNA expression of vimentin, while FPR2 knockdown reversed the expression patterns of these two molecules in SGC7901 cells (left panel) and XN0422 cells (right panel). Western blot assay confirmed these results at protein level (Fig. 4B). However, FPR2 knockdown could not abolish the effect of FPR2 ligands on the expression of EMT-related molecules, possibly due to the presence of FPR1 and FPR2 in GC cells and the non-specificity of the ligands. These results demonstrate that the activation of FPR2 can induce EMT of GC cells, which may be an important mechanism for FPR2-promoting invasion and metastasis of GC cells.

Discussion
All the members of FPR family, including FPR1, FPR2, and FPR3, are expressed in human GC cells 18 . The roles of FPR1 in GC have been contradictorily reported. Prevete et al. 27 found that FPR1 is a tumor suppressor by inhibiting angiogenesis in GC xenograft experiments. Otani et al. 28 reported that a specific FPR1 polymorphism, which reduced FPR1 activity, is positively associated with the risk of GC. While Cheng et al. 29 reported that FPR1 was highly expressed in GC tissues and significantly associated with stage IV disease, invasion depth, and clinical outcome of the patients. So far, there is a few data to correlate the expression of FPR2 and FPR3 with GC. In this study, we first examined the expression patterns of FPRs in 6 GC cells and 6 fresh GC specimens and found that all GC cells and specimens expressed all the members of FPRs, but 5 of 6 GC cells and 5 of 6 GC specimens had much higher expression levels of FPR2 than FPR1 and FPR3. These prompted us to further investigate the role of FPR2 in GC. We found that FPR2 was expressed more frequently in GC cancerous tissues than in adjacent tissues and increased expression levels in cancerous tissues were correlated with the invasion depth and lymph node metastasis as well as the poor survival of the patients. To our knowledge, this is the first clinicopathological study to link FPR2 to the clinicopathological features of GC and the outcome of GC patients. Invasion and metastasis are considered to be the main factors affecting the prognosis of patients with GC. Our clinical data suggested that FPR2 was a potential factor involved in facilitating invasion and metastasis of GC. It was confirmed by in vitro and in vivo experiments with GC cell line SGC7901 and primary GC cell XN0422. Silencing FPR2 expression significantly impaired the migratory and invasive potentials induced by Hp (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and Ac(2-26) as well as the capability of peritoneal metastasis in the GC cells. Our in vitro experiments were in line with the report of Prevete et al. 27 , in which FPR2 promoting EMT and migration in GC cells, and another report, in which FPR2-promoting migration and invasion in human pancreatic carcinoma cells 30 . In our experiments, we also found that FPR2 deletion markedly decreased the tumor formation in nude mice. However, the findings of Prevete et al. 27 showed that knockdown of FPR2 did not significantly affected GC cell tumor formation despite a significant decrease in cell growth in vitro.
EMT is a highly conserved and fundamental process that is critical for embryogenesis and tumor progression. More and more evidence suggests that the achievement of an EMT phenotype is associated with increased capability of invasion and metastasis in GC cells 31 . The up-regulation of mesenchymal markers and the down-regulation of epithelial markers are the major molecular features of EMT. Our and other's 27 works proved that FPR2 knockdown up-regulated mesenchymal marker vimentin and down-regulated epithelial maker E-cadherin in GC cells, suggesting that EMT is an important mechanism in FPR2-promoting GC invasion and metastasis.
In conclusion, we demonstrated that, for the first time, high expression of FPR2 in gastric cancer tissues is correlated with poor prognosis of GC patients. We also elucidated that FPR2 can enhance the invasion and metastasis of gastric cancer. A possible mechanism regarding these effects was that FPR2 promotes GC cell EMT by activating MAPK/ERK pathway. Thus, FPR2 could be potentially used as not only a prognostic biomarker but also a therapeutic target for GC patients. However, it is worth mentioning that the high FPR2 expression in gastric cancer might be a symptom of an underlying mechanism, which should be a target of therapeutic approaches besides FPR2 itself and its signaling pathway and needs to be further investigated.

Material and Methods
Patients and specimens. A total of 169 formalin-fixed and paraffin-embedded surgical carcinous and the corresponding adjacent normal tissues were collected from GC patients who were enrolled in the Southwest Hospital from January 2006 to December 2007. All patients had not received radiotherapy, chemotherapy or immunotherapy before surgery. Follow-up information was available for all patients for a period of minimum 80 months. All the specimens were routinely processed for pathological diagnosis according to the WHO classification. The study was approved by the Southwest Hospital Research Ethics Committees, and all patients were enrolled by written informed consent.

Cells and culture. Human gastric cancer cell line SGC7901 was purchased from Cell Bank of Shanghai
Institute of Cell Biology, Chinese Academy of Sciences and primary gastric cancer cell XN0422 was initiated by our laboratory. Both the cell line and primary cells were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium (Gibco, Grand island, USA) supplemented with 10% fetal bovine serum (BD Pharmingen, USA) in the condition of a humidified atmosphere containing 5% CO 2 at 37 °C. Cells in exponential growth phase (approximately 80% confluency) were used in all experiments.
Immunohistochemistry. After fixation in 4% formalin, cancerous and corresponding adjacent normal tissues from the 169 GC patients were dehydrated through an ascending series of graded ethanol, embedded in paraffin wax, and cut into 4-μm sections. After dewaxing and hydrating, antigen retrival, bloking of endogenous peroxidase activity, the sections were incubated with primary FPR2 antibody (1:100, Santa Cruz, USA) at 4 °C overnight. Following incubation with secondary antibody (Beijing Zhongshan Golden Bridge Biotechnology, China) at 37 °C for 30 minutes, the sections were visualized using diaminobenzidine solution (DAKO) and lightly counterstained with haematoxylin. The tumors were interpreted as FPR2-positive and FPR2-negative according to the cancer cells with or without staining of FPR2.

RNA extraction and quantitative real-time PCR (qRT-PCR). Total RNA was isolated using RNAiso
TRIzol reagent (TAKARA, Kyoto, Japan) according to the manufacturer's instructions. Reverse-transcription of RNA was performed in a final reaction volume of 20 μL containing 1000 ng of total RNA by using PrimeScript RT Master Mix (TAKARA, Kyoto, Japan). FPR2 mRNAs were detected by qRT-PCR with the SYBR Premix Ex TaqII (TAKARA, Kyoto, Japan). The sequences of all primers for RT-qPCR were presented in Table S2.
The protein gel images were acquired under automatic exposure settings on ChemiDoc ™ MP System (Bio-Rad, Hercules, California, USA) with Image Lab (Version 5.2 build 14) software. Tubulin was used as a loading control.
Lentivirus Production and Infection. Three shRNA sequences targeted against FPR2 and a non-targeting scrambled gene sequence were listed in Table S3. Lentivirus particles containing shFPR2 and control shRNA were obtained from Life Technologies Co. Ltd (Shanghai, China) and used to infect SGC7901 and XN0422 cells with 2 μg/mL of polybrene. The stable FPR2-knockdown cells were selected using 3 μg/mL puromycin. The efficacy of FPR2 knockdown at mRNA and protein levels were examined by RT-PCR and Western blot analysis, respectively (Fig. S2).
Wound-healing assay. A wound-healing assay was performed to examine the capability of cancer cell migration, as previously described 40 . Briefly, GC cells were grown in 24-well plates with RPMI-1640 medium supplemented with 10% FBS up to 90% confluence. A single scratch wound was generated with a 10 μL pipette tip. After removing the suspension cells by washing with PBS, fresh RPMI-1640 medium without FBS was added. With a Live Cell Imaging System (ZEISS, Germany), moving and growing of cells across the scratched lines were monitored every hour for 24 h. The migratory ability of the cells was presented as the gap distance recovered compared with the original gap.
Invasion assay in vitro and Chemotaxis assay. Invasion assay was performed as previously described 40 .
Briefly, GC cells were seeded in the upper chamber (8 μm, 24-well format) coated with 10 μL of matrigel (BD, USA)/RPMI-1640 (1:1, v/v) at 2 × 104 cells/well in 200 μL RPMI-1640 medium without FBS. The lower chambers were filled with 600 μL RPMI-1640 medium containing 10% FBS. After 24 h of incubation, the membranes were fixed with 4% paraformaldehyde for 20 min. Then the non-invaded cells (upper surface of the membrane) were removed with a cotton swab, and the cells on the lower surface of the membrane were stained with crystal violet solution (Beyotime, China). The number of invaded cells was counted in five randomly selected high powered fields under a microscope. Chemotaxis assay was performed as previously described 41 . Briefly, transwell chambers (8 μm pore size, Millipore) without matrigel coating were used. The upper wells of the chamber were added with 5 × 10 4 cells suspended in 200 μL serum-free RPMI-1640 medium. Lower wells of the chamber were added with 600 μL serum-free medium containing different concentrations of Hp (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) or Ac , which were synthesized by GL Biotech Ltd (Shanghai, China) according to their sequences (Table S3) and their biological activity was tested by chemotaxis (Fig. S5). After an incubation period of 6 h at 37 °C, migrated cells on the lower surface of membrane were counted in five randomly chosen fields.

Subcutaneous xenograft tumorigenicity and intraperitoneal metastasis assays.
To assess the effect of FPR2 on in vivo tumorigenecity, XN0422 and SGC7901 FPR2-knockdown and mock cells were injected subcutaneously into axilla of 6-week-old female nude mice (Laboratory Animal Center, Third Military Medical University) at 1 × 10 4 cells and 1 × 10 5 cells suspended in 0.2 mL Matrigel (1:1, v/v) per mouse, respectively (n = 5). The mice were euthanized at the end of 5 weeks after implantations. The xenografts were removed and measured. The tumorigenic capability was assessed by tumor weight. To examine the effect of FPR2 on in vivo metastasis, the GC cells with different treatments were injected intraperitoneally (1 × 10 4 cells per mouse). After 4 weeks, the mice were euthanized. The numbers of intraperitoneal nodules were counted. All animal procedures were approved by the Third Military Medical University Animal Committee.
Statistics. All data are expressed as mean ± SD of three independent experiments and analyzed using SPSS 18.0 statistical software. Kaplan-Meier analysis was used to evaluate the survival rates and chi-square test was used to detect the associations between FPR2 expression and clinicopathologic characteristics of GC patients. To determine whether FPR2 is an independent prognosis factor for survival, the Cox proportional hazards model was used to calculate the hazard ratios. The statistical significance of the mean values was evaluated using the unpaired Student's t test. Tests were assumed significant when the P < 0.05.
All the methods were carried out in accordance with relevant approved guidelines and regulations.