Abstract
Background: Formyl peptide receptor 1 (FPR1) as a regulator of innate inflammatory response has been implicated in tumor progression of gliomas. The purpose of the present study was to evaluate the prognostic significance and the ligand-receptor interaction of FPR1 in gastric cancer (GC). Patients and Methods: FPR1 was immunohistochemically-analyzed in tissue sections originating from 116 GC patients. Reverse transcription-polymerase chain reaction (RT-PCR) was used for the assessment of interaction between FPR1 and the FPR1 ligand annexin A1 (AnxA1) in GC cells. Results: High FPR1 expression was significantly associated with stage IV disease, submucosal invasion, serosal invasion, and clinical outcome of GC. Multivariate analysis showed that high FPR1 expression was an independent risk factor of poor overall survival in GC patients. FPR1 expression increased significantly when AnxA1 overexpression was present in GC cells. A positive feedback regulation of FPR1 was involved in the AnxA1-FPR1 signal transduction. Conclusion: FPR1 expression may be used as a novel indicator to predict outcome in GC patients after gastrectomy.
Gastric cancer (GC) is the fourth most common cancer and the second leading cause of cancer-related death in the world (1). Though the incidence and mortality rates have steadily declined in many developed countries for decades, an upward trend in young patients in several countries around the globe is being observed (2). Adequate surgery is currently the cornerstone of GC treatment. But achievement of locoregional control for advanced GC is still difficult even if combination of chemotherapy and radiotherapy are employed (3). Although many targeted-agents have led to better outcomes regarding other malignancies, only trastuzumab has shown clinical benefits in response rates and survival on combination treatment with chemotherapy for advanced GC (4, 5). Since development of more targeted-therapy options is mandatory, researchers should put more effort in investigating novel targets based on molecular carcinogenetic mechanisms underlying GC.
The formyl peptide receptor 1 (FPR1) is a seven transmembrane, G protein-coupled receptor involving innate inflammatory response regulation. The human FPR sub-family so far consists of three proteins, FPR1, FPR2/FPRL1, and FPR3/FPRL2. FPRs can promote cell motility and microbicidal respiratory burst of leukocytes upon recruitment and activation (6). Epithelial FPR1/NOX1-dependent redox signaling pathway is involved in promoting intestinal mucosal wound repair (7). In addition to inflammation regulation, more evidence suggests that FPRs play important roles in tumor progression. FPR1 contributes to tumor progression of malignant human gliomas by mediating tumor cell chemotaxis, proliferation, and production of VEGF (8). In a previous study of our group, we found that expression of annexin A1 (AnxA1), an endogenous ligand of FPRs, was associated with GC invasiveness and overall survival. High AnxA1 expression was associated with more serosal invasion, more peritoneal metastasis, and poorer overall survival in GC patients (9). However, there was no direct evidence on the relationship between expression of FPRs and GC, except for one association study regarding FPR1 polymorphisms and GC susceptibility (10). In the present study, we evaluated FPR1 expression in GC tissues and analyzed its prognostic significance by clinicopathological correlation. Besides, we attempted to illustrate the mechanistic interaction between FPR1 and its ligand AnxA1.
Patients and Methods
Patients and specimens. Tissue specimens for the study were derived from a set of 116 consecutive histologically-verified GC patients treated with surgical resection at the National Taiwan University Hospital between November 1999 and February 2003. The histological diagnosis was performed according to the recommendations of the World Health Organization. Tumor node metastasis status was based on the 7th edition of the American Joint Commission on Cancer (AJCC 7th) Staging System. The corresponding clinical and pathological data were retrieved for analysis. All patients had been followed-up for at least 7 years. The surgical specimens were obtained from patients after written informed consent was obtained in the National Taiwan University Hospital.
Immunohistochemical staining. After rehydration, sections (5 μm) of paraffin-embedded tissue blocks were incubated in 3% hydrogen peroxide to block for endogenous peroxidase activity. Following citrate buffer antigen retrieval, the sections were blocked by incubation in 3% bovine serum albumin in phosphate-buffered saline. The slides were incubated with anti-FPR1 polyclonal antibody (GTX71283, GeneTex, Irvine, CA) and incubated at a 1:100 dilution overnight at 4°C. After washes in phosphate-buffered saline, the samples were treated with a SuperPicture Polymer Detection kit (Invitrogen, Carlsbad, CA). The slides were stained with diaminobenzidine, washed, counterstained with hematoxylin, dehydrated, treated with xylene, and mounted. FPR1 expression in lymphoplasma cells served as an internal positive control. Appropriate negative controls were prepared using non-immune rabbit immunoglobulin G. The results of immunohistochemical staining were classified using the extent of stained cells: level 0 (negative staining), level 1 (<10% of tumor cells stained), level 2 (10%-50% of tumor cells stained), and level 3 (>50% of tumor cells stained). The pathologists assessing immunostaining intensity were blinded to the patients' information. Institutional review board approval was obtained to procure and analyze the tissues used in this study.
Cell culture. The human gastric cancer cell lines AGS and N87 were purchased from the American Type Culture Collection (Manassas, VA, USA). MKN45 was purchased from the Health Science Research Resources Bank (Osaka, Japan). These cell lines were cultured in RPMI-1640 medium (GIBCO) containing 10% fetal bovine serum (Bioserum, Victoria, Australia) in a humidified atmosphere containing 5% CO2 at 37°C.
Reverse transcription–polymerase chain reaction. Messenger RNAs were isolated and amplified as previously described (11). For human FPR1, sense primer 5’-CCTCACATTGCCAGTTATCATTC-3’ and anti-sense primer 5’-CTTTGCCTGTAACTCCACCTCT-3’ generated a 586-bp product. For FPR2, sense primer 5’-GAGAA AAATGGCCTTTTGGCTG-3’ and anti-sense primer 5’-CATTGCCTGTAACTCAGTCTCTGC-3’ generated a 791-bp product. For FPR3, sense primer 5’-CTGGCCACACCGTTCTGT-3’ and anti-sense primer 5’-AACGTGTTCATTACCATGGCC-3’ generated a 536-bp product. Reverse transcription–polymerase chain reaction (RT-PCR) was performed with human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control.
Construction of AnxA1 expression vectors and transient transfection. ANXA1 cloned into pcDNA3.1 V5/His-TOPO plasmid (Invitrogen, Carlsbad, CA) was amplified using specific AnxA1 primer sets, in either the sense or anti-sense orientation. The cloned DNA fragments were verified by direct sequencing (ABI Prism 377 DNA sequencer; PE Biosystems, Foster City, CA). The AnxA1 expression vectors were transfected into test cells with Lipofectamine 2000 transfection reagents (Invitrogen).
Statistical analysis. For comparison of clinicopathological parameters, the Mann-Whitney test was employed for scale variables and the Pearson Chi-square test for nominal variables. To determine whether FPR1 expression is an independent prognostic factor for survival, hazard ratios were calculated using the Cox proportional hazards model. Data were analyzed with the SPSS program for Windows, version 11.0 (SPSS Inc., Chicago, Illinois, USA). ANOVA was used to evaluate the statistical significance of the mean values. All statistical tests included two-way analysis of variance. We used significance level of 0.05 for all our statistical analyses.
Results
Clinicopathological features. Among the 116 GC patients, there were 77 men and 39 women, and the average age was 61.2 ±15.1 years (median=64.5 years, range=26-88 years). Tumor distribution was as follows: 19 in the proximal third, 21 in the middle third, 64 in the distal third, and 12 involving more than two parts of the stomach. Among stage IV disease patients, 4 had resectable liver metastases and 17 had peritoneal metastases detected during operation. Among GC specimens, the majority (75.9%, 88/116) showed negative/low expression (level 0 and 1) whereas a subset (24.1%, 28/116) of GC showed high expression (level 2 and 3) of FPR1 (Figure 1A-C). We then compared the FPR1 expression among GC patients with different clinicopathological characteristics (Table I). High FPR1 expression was associated with older age (p=0.029), stage IV disease (p=0.019), submucosal invasion (p=0.039), serosal invasion (p=0.027), and clinical outcome (p=0.001). There was no statistically significant correlation of the level of FPR1 expression with gender, tumor location, histological type, differentiation, lymphovascular invasion, lymph node involvement, or peritoneal metastasis.
FPR1 expression associates with survival in GC patients. Kaplan-Meier analysis showed that overall survival rates for GC patients with high FPR1 expression were significantly lower than those with negative/low expression (median=12.0 vs. 49.0 months, p<0.001) (Figure 2A). When patients are stratified into TNM stage I/II and III/IV, the correlation between FPR1 status and overall survival rates persisted in both groups (Figure 2B, C). On Cox univariate proportional hazard analysis, we found that poor overall survival correlated significantly with older age (p=0.001), stage III or IV (p<0.001), submucosal involvement (p=0.008), serosal invasion (p<0.001), lymphovascular invasion (p<0.001), lymph node metastasis (p=0.002), peritoneal seeding (p<0.001), and high FPR1 expression (p<0.001). On multivariate analysis, only age (p=0.002), lymphovascular invasion (p=0.018), peritoneal seeding (p=0.040), and high FPR1 expression (p=0.002) were independent risk factors for poor overall survival in GC patients (Table II).
Immunohistochemical analysis of FPR1 expression in representative gastric cancer tissue. (A) Negative control with immunoglobulin G. (B) Negative/low FPR1 expression (<10% of tumor cells stained). (C) High FPR1 expression (>10% of tumor cells stained). Black arrows indicate the lymphoplasma cells with FPR1 immunoreactivity. Scale bars, 50 μm.
FPR1 expression response to changes of AnxA1 in GC cells. Some recent reports have shown micro-environmental AnxA1 to be important in promoting tumorigenesis through interaction with FPRs (12, 13). Whether FPR1 plays a role as a primary effector or a secondary responder in the GC tumorigenesis process is still unclear. Therefore, we examined the FPR1 expression profiles after altering AnxA1 levels in GC cells. As shown by RT-PCR analysis, FPR1 expression increased in both AGS and N87 cancer cells overexpressing AnxA1 (Figure 3A). The AGS cancer cells overexpressing AnxA1 showed significantly increased expression of FPR1 with a dosing effect in comparison to controls (Figure 3B). The MKN45 cancer cells with down-regulated AnxA1 did not show obvious change of FPR1 expression compared to control (Figure 3C). The expression patterns of FPR2 and FPR3 were similar to FPR1. These results suggested a positive feedback regulation of FPRs including FPR1 to be involved in the AnxA1-FPR1 signal transduction pathway. However, AnxA1 down-regulation alone could not alter FPR1 expression.
Discussion
The present study revealed high FPR1 expression to be associated with more submucosal invasion, more serosal invasion, more advanced tumor stage, and poorer outcome of GC patients. GC patients with high FPR1 expression in tumors had lower overall survival rates than those with negative/low FPR1 expression. To the best of our knowledge, this is the first reported study of FPR1 expression in GC. There are few studies of FPR1 expression in human malignancies. FPR1 expression has been correlated with clinicopathological parameters traditionally associated with poor prognosis, such as higher tumor grade in gliomas (8) and drug resistance in acute lymphoblastic leukemia (14). Considering personalized cancer treatment, more independent prognostic markers other than conventional ones may be of help with regard to clinical practice (15). In the present study, FPR1 expression may be used as a potential molecular marker to predict for outcome in GC patients treated with gastrectomy. Cox multivariate analysis confirmed that FPR1 expression was an independent prognostic factor for poorer overall survival in GC patients. Tumor levels of FPR1, regardless of T stage or nodal status may be used to determine for patient outcome.
The complexity of ligand-receptor relationship makes it difficult to predict the FPR1 effects based solely on our knowledge on AnxA1. The present study showed that forced expression of AnxA1, which possibly increases AnxA1 secretion, as shown in our previous study (9), up-regulated FPR1 expression with a dosing effect. This suggested a positive feedback pattern between the receptor FPR1 and its ligand AnxA1 in GC cells. However, AnxA1 down-regulation alone could hardly alter FPR1 expression. One of the possible explanations for this phenomenon is that there are several ligands to FPR1 other than AnxA1. In addition to microbe-derived ligands such as the prototypical formylated peptide fMLF from Escherichia coli and some HIV-derived peptides, mitochondrial formyl peptides and cathepsin G are known host-derived ligands for FPR1 (16-18). Cathepsin G, a serine protease involved in host immunity, is highly expressed and ubiquitinated in acute myeloid leukemia blasts (19). Furthermore, cathepsin G could induce cell aggregation in breast cancer cells (20). Future studies should clarify the roles of different FPR1 ligands in tumor progression.
Association of FPR1 expression and clinicopathological features in GC patients.
High FPR1 expression was associated with older age in our study. The risk of developing cancer increases dramatically with the process of aging, which negatively-impacts on the development and function ability of the host immune system (21). A decline in immunity eventually results in a cluster of mutations capable of growing in that weakened immune environment (22), and FPR1 is one of the targets “hijacked” by cancer cells for tumor progression (23). Besides, age-related changes have been reported in other G protein-coupled receptors. Previous studies have shown age-related hyper-inflammation effects with up-regulation of some G protein-coupled receptors, such as TNF receptor-1 and alpha-2A adrenergic receptor (24, 25). Future studies on analyzing these receptors concurrently may elucidate the effects of aging process in tumorigenesis.
Kaplan-Meier estimates and log-rank tests of GC patients with different FPR1 expression for overall survival (A), overall survival in stage I/II (B), and overall survival in stage III/IV (C).
In summary, results of the present study showed that high FPR1 expression was associated with more advanced tumor stage and poorer overall survival in GC patients. A positive feedback regulation of FPR1 in the AnxA1-FPR1 signal transduction contributed important information on the role of FPR1 in GC cells. These findings suggest that FPR1 expression may be used to predict patient outcome in GC after gastrectomy, and may serve as a potential target for attenuating tumor invasiveness in GC.
FPR1 expression responce to AnxA1 levels in gastric cancer cells. (A) RT-PCR assay shows AnxA1-associated expression levels of the FPRs in AGS and N87 cancer cells. (B) RT-PCR assay shows expression of FPRs in AGS cells over-expressing AnxA1. (C) RT-PCR assay shows expression of FPRs in MKN45 cells with down-regulated AnxA1. GAPDH, Human glyceraldehyde 3-phosphate dehydrogenase.
Cox proportional analysis for the predictors of mortality in GC patients.
- Received December 16, 2013.
- Revision received January 22, 2014.
- Accepted January 24, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
