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.
Highlights
Barrett’s esophagus (BE) is a condition characterized by the development of intestinal metaplasia of the esophageal mucosa
With primers specific to each subtype of PAR genes, quantitative RT-PCR reveals the transcription of PAR-1 and Protease-activated receptor 2 (PAR-2) in all cells, while PAR-3 and PAR-4 transcription was found at a comparatively lower level
PAR-2 surface expression was found at high levels in both the premalignant Barrett’s Metaplasia (BART) cell line as well as in Barrett’s-derived adenocarcinoma cell line OE33
Summary
Barrett’s esophagus (BE) is a condition characterized by the development of intestinal metaplasia of the esophageal mucosa. The clinical importance of this relatively common condition relates to its role as a precursor lesion to esophageal adenocarcinoma (EAC), entailing a 100-fold increased risk of developing EAC [1]. The cellular and molecular mechanisms underlying the development Barrett’s esophagus and its progression to cancer remain unclear. Our previous work showed that bile salt glycochenodeoxycholic acid (GCDA) activates ERK/MAPK pathway to produce a pro-proliferative effect in a Barrett’s cell line [3]. It is unclear whether trypsin in refluxate contributes to promote cell proliferation in these metaplastic cells
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