Protease-activated receptor-2 activation in gastric cancer cells promotes epidermal growth factor receptor trans-activation and proliferation

Abstract

Dysregulated epidermal growth factor receptor (EGFR) signaling is involved in gastric cancer (GC) cell growth. However, the mechanism that sustains EGFR signaling in GC remains unknown. Since protease-activated receptor-2 (PAR-2), a G protein-coupled receptor, has been shown to trans-activate EGFR in several cell types, we examined the role of PAR-2 in GC. We show here that in vitro activation of PAR-2 enhances the growth of two GC cell lines, AGS and MKN28. In both these cell lines, PAR-2 trans-activated EGFR and inhibition of EGFR tyrosine kinase activity by AG1478 or specific EGFR siRNA completely prevented PAR-2-driven proliferation. Antibody blockade of EGF-like ligands to EGFR did not modify EGFR signaling or cell growth induced by PAR-2 activation. In contrast, PAR-2 promoted Src activation and interaction of this kinase with EGFR. In support of this, inhibition of Src kinase activity by PP1 or siRNA blocked PAR-2-induced EGFR signaling cascade and cell growth. Finally, PAR-2 was detectable in both normal and GC specimens, but its expression was more pronounced in GC than controls and correlated with activated EGFR. These data show that PAR-2 is overexpressed in GC and suggest a role of PAR-2 in EGFR trans-activation and cell growth.

Figure 1

A: Representative flow cytometry histograms showing the expression of PAR-2 in AGS and MKN28 cells. Cell phenotype analysis was carried out after incubation of these two cell lines with a monoclonal PAR-2 or isotype control antibody at 4°C for 45 minutes. Numbers above the lines indicate the percentage of positive cells within the designed gates. B: Stimulation of both AGS and MKN28 with PAR-2-activating peptide results in enhanced cell growth. Cells were cultured in the presence or absence of PAR-2-activating (PAR-2 AP, 20 μmol/L) or -inactivating (PAR-2 IP, 20 μmol/L) peptide for 48 hours, and BrdU was added during the last 4 hours of culture. PAR-2 AP significantly enhances the growth of both AGS (P = 0.002) and MKN28 (P = 0.04) cells. Data are expressed in arbitrary units and indicate mean ± SD of four separate experiments.

Figure 2

PAR-2 P stimulates EGFR trans-activation in AGS cells. A: Representative expression of p-EGFR (upper blot) and total EGFR (lower blot) protein in AGS cells cultured in the presence or absence of PAR-2-activating or -inactivating peptide (20 μmol/L) for the indicated time points. p-EGFR was examined by Western blotting using an antibody that recognizes phosphorylation of the receptor on tyrosine residue 1173. One of four representative blots is shown. B: Total extracts of AGS cells cultured with or without the initial addition of PAR-2 AP for 1 hour were immunoprecipitated using a monoclonal anti-human EGFR antibody and then incubated with a p-Tyr antibody. No band was seen when proteins were immunoprecipitated using a control isotype antibody (ve−). After detection of p-EGFR, bots were stripped and incubated with a second anti-EGFR to ascertain equivalent loading of the lanes (lower blot). C: Representative Western blots showing p-ERK1/2 (upper blot) and total ERK1/2 (lower blot) in AGS cells stimulated with PAR-2 activating or inactivating peptide (20 μmol/L) for the indicated time points. One of four representative experiments is shown. D: Representative Western blot showing p-EGFR (upper blot) and total EGFR (lower blot) in AGS cells cultured in the presence or absence of trypsin (TRYP (10 and 100 μmol/L) for the indicated time points. E: Effect of PAR-2P on p-EGFR in primary gastric epithelial cells. Cells were isolated from normal mucosal specimens and cultured in the presence or absence of PAR-2 AP for the indicated time points. At the end, total proteins were prepared and analyzed by Western blotting. One of three representative experiments is shown.

Figure 3

A: The tyrphostin AG1478, an inhibitor of EGFR tyrosine kinase activity, suppresses EGF-induced phosphorylation of EGFR in AGS cells. Representative Western blots showing p-EGFR (upper blot) and total EGFR (lower blot) protein in AGS cells preincubated with AG1478 (20 μmol/L) or DMSO (vehicle) for 1 hour, and then either left unstimulated or stimulated with EGF (200 ng/ml) for 10 minutes. B: AG1478 inhibits PAR-2 P-stimulated p-EGFR and p-ERK1/2. Representative Western blots showing p- and total EGFR, and p- and total ERK1/2 protein in AGS cells preincubated with AG1478 (20 μmol/L) or medium for 1 hour and then either left unstimulated or stimulated with PAR-2 P (20 μmol/L) for an additional hour. C: AG1478 suppresses the PAR-2-driven GC cell growth. AGS and MKN28 cells were preincubated with AG1478 (20 μmol/L) or DMSO for 1 hour and then stimulated with PAR-2 AP (20 μmol/L) for a further 48 hours, and cell proliferation was assessed as indicated in Material and Methods. Data are expressed as mean ± SD of four separate experiments and indicate the maximal response of growth of cells cultured in the presence of PAR-2 P with or without AG1478 or DMSO. D: Representative Western blot showing EGFR (upper blot) and β-actin (lower blot) in AGS cells transfected with either EGFR or control siRNA for 48 hours. E: Suppression of EGFR by siRNA prevents the PAR-2P-mediated growth of GC cells. Cells were transfected with either EGFR or control siRNA for 48 hours, then washed, and cultured in 96-well plates in the presence or absence or PAR-2AP or FBS, as indicated in Material and Methods, for 48 hours. BrdU was added during the last 4 hours of culture. PAR-2 P significantly enhances the growth of AGS transfected with control siRNA (P = 0.03) but not the proliferation of EGFR siRNA-treated cells. In contrast, FBS significantly enhances the growth of cells treated with either EGFR or control siRNA (P < 0.001). Data are expressed in arbitrary units and indicate mean ± SD of three experiments.

Figure 4

PAR-2 P-induced EGFR trans-activation is not mediated by EGF-like ligands. A and B: Representative expression of p-EGFR (upper blot) and total EGFR (lower blot) protein in AGS cells preincubated with graded doses of a neutralizing EGFR (aEGFR) or control (IgG) antibody for 1 hour and then stimulated or not with EGF (200 ng/ml [A]) or TGF-α (100 ng/ml [B]) for a further 10 minutes. C: The aEGFR is not effective in neutralizing the effect of PAR-2 P on p-EGFR. Representative Western blots showing p-EGFR and total EGFR in AGS cells preincubated with aEGFR for 1 hour and then stimulated with PAR-2 AP for further 60 minutes. D: Pretreatment of GC cells with aEGFR does not affect the PAR-2-driven cell growth. AGS cells were preincubated with aEGFR or control IgG for 1 hour and then stimulated with PAR-2 AP (20 μmol/L) for further 48 hours, and cell proliferation was assessed as indicated in Material and Methods. Data are expressed as mean ± SD of four separate experiments and indicate the maximal response of growth of cells cultured in the presence of PAR-2 P with or without aEGFR or IgG. E: Neither 1,10-phenanthroline nor GM6001 inhibit the PAR-2 P-induced p-EGFR. Representative Western blots showing p-EGFR and total EGFR in AGS cells preincubated with 1,10-phenanthroline (300 μmol/L) or GM6001 (5 μmol/L) for 1 hour and then stimulated with PAR-2 AP for a further 60 minutes. One of two separate experiments is shown.

Figure 5

A: Representative expression of p-Src (upper blot) and β-actin (lower blot) protein in AGS cells cultured in the presence or absence of PAR-2 activating peptide (PAR-2 AP) or inactivating peptide (PAR-2 IP) (20 μmol/L) for the indicated time points. p-Src was examined by Western blotting using an antibody that recognizes phosphorylation of Src on tyrosine residue 418. Lower inset: quantitative data of p-Src/β-actin as measured by densitometry scanning of the representative Western blot. Values are expressed in arbitrary units (a.u.). B: Total extracts of AGS cells cultured with or without the initial addition of PAR-2 AP for 1 hour were immunoprecipitated using a monoclonal anti-human Src antibody and then incubated with a p-EGFR antibody. No band was seen when proteins were immunoprecipitated using a control isotype antibody (ve−). After detection of Src-bound p-EGFR, blots were stripped and incubated with a second anti-Src to ascertain equivalent loading of the lanes (lower blot). C: Total extracts of AGS cells, cultured with or without the initial addition of PAR-2 AP for the indicated time points, were immunoprecipitated using a monoclonal anti-human EGFR antibody and then incubated with a p-Src antibody. No band was seen when proteins were immunoprecipitated using a control isotype antibody (ve−). After detection of p-Src-bound EGFR, blots were stripped and incubated with a second anti-EGFR to ascertain equivalent loading of the lanes (lower blot).

Figure 6

A: Representative Western blots showing p-Src and β-actin in total extracts from AGS cells cultured with or without the Src kinases inhibitor, PP1 (20 μmol/L) or DMSO (vehicle) for 1 hour. B: Pretreatment of AGS cells with PP1 prevents the PAR-2 P-induced phosphorylation of EGFR and ERK1/2. AGS cell cultures were preincubated with, medium, PP1, or DMSO for 1 hour before adding PAR-2 AP for an additional hour. One of three representative Western blots is shown. C: Treatment of GC cells with PP1 prevents the PAR-2-driven growth. AGS and MKN28 cells were preincubated with PP1 or DMSO for 1 hour and then stimulated with PAR-2 AP (20 μmol/L) for a further 48 hours, and cell proliferation was assessed as indicated in Material and Methods. Data are expressed as mean ± SD of three separate experiments and indicate the maximal response of growth of cells cultured in the presence of PAR-2 P with or without PP1 or DMSO. D: Representative Western blots showing Src (upper blot), p-EGFR (middle blot), and β-actin (lower blot) in AGS either transfected with either Src or control siRNA for 48 hours and then cultured in the presence or absence of PAR-2AP for an additional hour. One of three separate experiments is shown.

Figure 7

An immunohistochemical staining for PAR-2 in paraffin-embedded sections from GC and control tissue sections. A marked accumulation of PAR-2 is seen in GC cells (original magnification, ×400 [A], ×40 [B]). C: PAR-2 in gastric mucosa of a patient with Hp infection (original magnification, ×200). In D, control immunohistochemical reactions using an isotype IgG that corresponds to the sections in B are shown (original magnification, ×40). E–H: PAR-2 and activated EGFR in serial gastric sections taken from a patient with GC. (original magnification, ×200 [E–G]; original magnification ×40 [F–H]). The example is representative of four separate experiments, in which biopsies taken from 15 patients with GC, eight patients with Hp-related gastritis, and 12 Hp-negative subjects were analyzed.