C5a receptor (CD88) promotes motility and invasiveness of gastric cancer by activating RhoA

Abstract

Purpose: Anaphylatoxin C5a is a strong chemoattractant of the complement system that binds the C5a receptor (C5aR). The expression of C5aR is associated with poor prognosis in several cancers. However, the role of C5aR in gastric cancer (GC) is unknown. The aim of this study was to examine the role of C5aR on GC cell motility and invasion.

Experimental design: The mechanism of invasion via C5aR was assessed by analyzing cytoskeletal rearrangement and RhoA activity after C5a treatment. Moreover, we investigated the relationship between C5aR expression and the prognosis of GC patients.

Results: Two human GC cell lines (MKN1 and MKN7) had high C5aR expression. An invasion assay revealed that C5a stimulation promoted the invasive ability of MKN1 and MKN7 cells and that this was suppressed by knockdown of C5aR using siRNA or a C5aR-antagonist. Moreover, overexpression of C5aR in GC cells enhanced the conversion of RhoA-guanosine diphosphate (RhoA-GDP) to RhoA-guanosine triphosphate (RhoA-GTP) after C5a stimulation and caused morphological changes, including increased expression of stress fibers and filopodia. Examination of tumor specimens from 100 patients with GC revealed that high C5aR expression (35 of 100 samples, 35.0%) was associated with increased invasion depth, vascular invasion and advanced stage. The 5-year overall survival of patients with high or low C5aR expression was 58.2% and 68.5%, respectively (p=0.008).

Conclusions: This study is the first to demonstrate that C5aR promotes GC cell invasion by activating RhoA and is associated with a poor prognosis in GC patients. Therefore, this study provides a biomarker for GC patients who require an advanced therapeutic strategy.

Keywords: C5a receptor; CD88; RhoA; gastric cancer.

Figure 1. C5aR-expression in gastric cancer cell lines

A., Western blots demonstrating the level of C5aR-expression in gastric cancer cell lines. B and C., growth assays using Cell Counting Kit-8 showing the growth of C5aR positive and negative gastric cancer cells following stimulating with rC5a. C5aR: C5a receptor, PBMC: peripheral blood mononuclear cell, rC5a: recombinant C5a, GC: gastric cancer.

Figure 2. C5aR-stimulation with rC5a promotes invasion of gastric cancer cells

A., lower surfaces of an invasion membrane when MKN1 cells were assayed for invasion. B and C., rC5a significantly enhanced the invasive ability of MKN1 and MKN7 cells. D., rC5a did not enhance the invasive ability of AGS cells. E–H., suppression of C5aR-expression using two kinds of siRNA significantly decreased the invasive ability of MKN1 and MKN7 cells. I and J., W-54011, a C5aR-antagonist, significantly suppressed the invasive ability of MKN1 and MKN7 cells. C5aR, C5a receptor; rC5a, recombinant C5a; DMSO, Dimethyl sulfoxide; W-54011, C5aR-antagonist; *p<0.05; **p<0.01. NS, not significant.

Figure 3. C5aR stimulation enhanced the invasive ability and motility of NUGC3 cell with C5aR-overexpression

A., Flow cytometry showed that C5aR proteins were overexpressed in the cellular membrane of NUGC3/C5aR cells. B., rC5a significantly promotes the invasive ability of NUGC3/C5aR cells, but did not significantly promote the invasive ability of NUGC3/mock cells. C., W-54011 significantly decreased the invasive ability of NUGC3/C5aR cells. D and E., rC5a significantly promotes the mobility and the total distance of cell migration of NUGC3/C5aR cells but did not significantly promote the mobility of NUGC3/mock cells. F and G., W-54011 significantly decreased the mobility of NUGC3/C5aR cells. C5aR, C5a receptor; rC5a, recombinant C5a; DMSO, Dimethyl sulfoxide; W-54011, C5aR-antagonist; **p<0.01. NS, not significant.

Figure 4. C5a-C5aR signaling enhances the production of RhoA-GTP and changes the cellular morphology of C5aR-expressing NUGC3 cells

A., NUGC3/C5aR and NUGC3/mock cells were incubated with rC5a (10 nM) and fixed at the indicated times. F-actin was visualized by immunofluorescence staining with Alexa 488–conjugated phalloidin. Scale bars, 10 μm. Orange and yellow arrows and arrowheads indicate filopodia, stress fibers and membrane ruffling, respectively. B., analysis of the activation of RhoA using a G-LISA on the lysates of NUGC3/C5aR and NUGC3/mock cells were extracted at the indicated times after rC5a treatment. C., diagram of C5a-C5aR signaling via the RhoA pathway in gastric cancer cells. C5aR, C5a receptor; rC5a, recombinant C5a; GDP, guanosine diphosphate; GTP, guanosine triphosphate; *p<0.05; **p<0.01. NS, not significant.

Figure 5. Relationship between C5aR expression and the prognosis of patients with gastric cancer

A., gastric cancer tissues were immunohistochemically stained with anti-C5aR antibody. One-hundred cases were scored from 0 to 3 according to the extent of C5aR staining in the cancer area. Scale bar: 100μm. B., Relapse-free survival curves for 86 patients who underwent a gastrectomy for gastric cancer (excluding stage IV patients), which were stratified by low- and high-expression of C5aR. C., Overall survival curves for 100 patients who underwent gastrectomy for gastric cancer, which were stratified by low- and high-expression of C5aR. C5aR, C5a receptor; RFS, relapse-free survival; OS, overall survival.