Autocrine extra-pancreatic trypsin 3 secretion promotes cell proliferation and survival in esophageal adenocarcinoma

Abstract

Trypsin or Tumor associated trypsin (TAT) activation of Protease-activated receptor 2 (PAR-2) promotes tumor cell proliferation in gastrointestinal cancers. The role of the trypsin/PAR-2 network in esophageal adenocarcinoma (EA) development has not yet been investigated. The aim of this study is to investigate the role of trypsin/PAR-2 activation in EA tumorogenesis and therapy. We found that esophageal adenocarcinoma cells (EACs) and Barrett's Metaplasia (BART) expressed high levels of type 3 extra-pancreatic trypsinogen (PRSS3), a novel type of TAT. Activity of secreted trypsin was detected in cultured media from EA OE19 and OE33 cultures but not from BART culture. Surface PAR-2 expression in BART and EACs was confirmed by both flow cytometry and immunofluorescence. Trypsin induced cell proliferation (~ 2 fold; P<0.01) in all tested cell lines at a concentration of 10 nM. Inhibition of PAR-2 activity in EACs via the PAR-2 antagonist ENMD (500 µM), anti-PAR2 antibody SAM-11 (2 µg/ml), or siRNA PAR-2 knockdown, reduced cell proliferation and increased apoptosis by up to 4 fold (P<0.01). Trypsin stimulation led to phosphorylation of ERK1/2, suggesting involvement of MAPK pathway in PAR-2 signal transduction. Inhibition of PAR-2 activation or siRNA PAR-2 knockdown in EACs prior to treatment with 5 FU reduced cell viability of EACs by an additional 30% (P<0.01) compared to chemotherapy alone. Our data suggest that extra-pancreatic trypsinogen 3 is produced by EACs and activates PAR-2 in an autocrine manner. PAR-2 activation increases cancer cell proliferation, and promotes cancer cell survival. Targeting the trypsin activated PAR-2 pathway in conjunction with current chemotherapeutic agents may be a viable therapeutic strategy in EA.

Figure 1. Cell immnunofluorescent staining for determination of PAR-2 surface expression.

(A) RNA expression profiling of PAR members by q-RT-PCR. (B) Contour plot of SSC vs. anti-PAR-2 antibody PE fluorescence. All cells subjected to analysis were first plotted by FSC (forward light scatter) vs. SSC (side light scatter) and gated to exclude debris and clumps. P2 is set for gating positive cells based on isotype control. Affiliated table shows percentage of positive cells (% of population) and MFI (mean fluorescent intensity, value subtracted from isotype background). (C) Representative image of immunofluorescence staining of PAR-2 on OE33 cell surface. Top picture shows the isotype control staining. Arrows in bottom picture indicate cell membrane staining for PAR-2 with anti-PAR-2 antibody N-19. Cell nuclei were stained with DAPI. Offset and gain values of the photomultiplier channel were regulated with respect to the setup selected for isotype negative control to make fluorescence intensity comparable across all samples.

Figure 2. Proliferative effect of trypsin on cell proliferation.

(A) BART cells were exposed to trypsin containing or control media. Day 1 after trypsin stimulation both 1 nM and 10 nM showed an increased ATP levels comparing to untreated cells (p<0.01). (B) Showing that trypsin induced OE33 cell proliferation at dose-dependent and time-dependent manner. OE33 cells were exposed to trypsin and left in serum-free medium for up to four days in the presence or absence of trypsin (1 nM or 10 nM). ATP levels in cell lysates were measured by Vialight® Plus Kit on day 0, day 2 and day 4. Stimulation with 10 nM trypsin showed a significant increase of ATP level comparing to untreated cells at day 4 post-treatment (p<0.01). (C) Depicting that 10 nM trypsin successfully stimulated cell growth in OE19, OE33 and BART cells in comparing with control groups, but failed to induce proliferation in FLO1 cells. Cell proliferation expressed as fold changes based on increase of arbitrary units of ATP chemiluninescent reading compared to untreated control group.

Figure 3. Erk/MAPK involvement in Trypsin Induced Signaling.

(A) Showing representative photographs for Western blot analysis of Erk1/2 phosphorylation status in BART and OE33 cells in the presence of or the absence of 10 nM trypsin at different time course as indicated. (B) Proliferation assay revealed that MEK inhibition abolished the proliferative responses of both BART and OE33 cells to 10 nM trypsin stimulation.

Figure 4. Effect of depletion or inactivation of PAR-2 on Erk1/2 phosphorylation and cell proliferation.

(A) Western blot analysis using anti-PAR-2 antibody N-19 showed that the siRNA against PAR-2 caused a significant decrease (55%) in PAR-2 expression in BART cells compared with scrambled treatment. (B) Flow cytometry analysis further confirmed that the knockdown of PAR-2 led to a 60% reduction in PAR-2 surface expression in comparing with scrambled control cells. (C) siRNA against PAR-2 abrogated trypsin-induced Erk1/2 phosphorylation in BART cells. (D) Showing that siRNA knockdown PAR-2 inhibited trypsin-induced cell proliferation in both BART and OE33 cells. Ctrl: non-transfection; Scmbl: scramble control; siPAR2: siRNA PAR-2 knockdown. (E) Inhibition of PAR-2 activity by PAR-2 antagonist ENMD or anti-PAR-2 antibody SAM-11 abolished trypsin-induced proliferation.

Figure 5. Effects of depletion and inactivation of PAR-2 on cell apoptosis.

(A) Representative counter plot of Annexin V v.s. 7-ADD staining for apoptotic cells measured by flow cytometric analysis upon siRNA PAR-2 knockdown in OE33 cells; (B) Apoptosis is expressed as a percentage of early apoptotic cells (right lower quadrant) in total cell population for OE19, OE33 and FLO1. (C) Effect of siRNA PAR-2 knockdown on cell viability measured by MTT assay in OE19 and OE33 cells. (D) PAR-2 inactivation with its antagonist ENMD or anti-PAR-2 antibody SAM -11 decreased cell viability in comparing with untreated control cells in OE19 and OE33 cell.

Figure 6. Profiling expression of trypsinogens and autocrine trypsin activity.

(A) Quantitative RT-PCR analysis of RNA expression of trypsinogens in EA cells and BART cells. Table shows the preferential transcriptional expression of type 3 trypsinogen (PRSS3). (B) Trypsin enzymatic activities in cultured media were determined by BioVision Trypsin Activity Assay Kit. Trypsin activities were calculated and converted into p-NA units and are presented as mean±SD (n = 12), three experiments of quadruplicates tested. (C) Trypsin activities in cultured media from OE19 and OE33 were detected by BioVision Trypsin Activity Assay Kit over up to 48 hrs time courses, curves indicating the kinetic secretion of trypsin of both cells.

Figure 7. Inactivation of PAR-2 confers a synergetic effect on 5FU.

(A) Dose-response curve of OE19 and OE33 cells to therapeutic agent 5FU. Cell viability measured by MTT assay. IC₅₀ = 23.3 µM for OE19 cells and IC₅₀ = 9.6 µM for OE33 cells. (B) In combination of blockade of PAR-2 activity by its antagonist ENMD (500 µM) or anti-PAR-2 antibody SAM-11 (2 µg/ml), 5FU (20 µM for OE19 and 10 µM for OE33) exerted higher cytotoxicity on OE19 and OE33 cells compared with 5FU alone (p<0.01). (C) Addition of 10 µg/ml leupeptin increases the effect of 5FU on OE33 cell viability.