Examination of proteases and protease-activated receptors (PARs) in Barrett's oesophagus and oesophageal adenocarcinoma

Abstract

Barrett’s oesophagus (BO), a metaplastic premalignant lesion, and gastroesophageal reflux disease (GORD) are two major risk factors for the development of oesophageal adenocarcinoma (OAC), an aggressive cancer the incidence of which is increasing faster than any other malignancy in the Western world. Proteases are known to be present in the refluxed contents of GORD patients and in inflamed BO and OAC tissue microenvironment. Yet, their role in these oesophageal pathologies is still poorly understood. Many (patho)-physiological responses induced by proteases can occur through their cell surface targets, the protease-activated receptors (PARs). Aberrant PAR expression and signalling have been reported in several malignancies. However, the tissue distribution patterns of PARs and their activating proteases as well as the effects of protease/PAR activation on oesophageal pathophysiology are largely unknown.

Therefore, the general aim of this thesis was to investigate the role of PARs and proteases in BO and OAC pathogenesis. More specifically, this thesis aims to: 1) characterise the expression of PAR family members (PAR1-PAR4) in a panel of normal oesophageal mucosa, BO, and OAC tissues, epithelial cell lines, and tissue-derived fibroblasts; 2) characterise the expression and localisation of candidate PAR activating proteases - MMP1, trypsin-1, KLK5, and KLK6 in normal oesophageal mucosa, BO, and OAC tissues; and 3) investigate the signalling and functional effects of PAR1 and PAR2 activation in oesophageal epithelial cells.

In Chapter 3, reverse transcription quantitative PCR (RT-qPCR) and immunohistochemical analyses revealed that PAR1 transcript and protein expression were significantly upregulated in BO and OAC tissues compared to normal oesophageal mucosa. PAR1 expression levels correlated significantly with disease progression. PAR3 and PAR4 mRNA overexpression were likewise detected; the former in BO and OAC tissues, and the later in tumour samples. PAR2 transcript and protein were found in all oesophageal tissue types with no statistically significant differences in expression levels. PAR1 and PAR2 expression within BO and OAC tissues were localised in various cell types but primarily in the epithelial cells.

While BO and OAC tissues showed significant increase in MMP1 and trypsin-1 expression, comparable levels of KLK5 protein was noted in all tissue types (Chapter 4). KLK6 transcript was markedly downregulated in BO tissues. Strikingly, MMP1, trypsin-1, and KLK5 were frequently coexpressed with PAR1 and PAR2 in OAC and BO tissues.

Analysis of the effects of PAR activation by the aforementioned proteases in a panel of representative epithelial cell lines revealed that PAR1- and PAR2-mediated signalling were dysregulated in OAC cells (Chapters 5 and 6). For instance, MMP1 but not thrombin induced Ca2+ signalling via PAR1 in OAC cells, whereas thrombin but not MMP1 evoked Ca2+ responses in normal and BO cells. In addition, while PAR2 activation triggered transient ERK1/2 signalling in normal and BO cells, ERK1/2 activation was sustained in OAC cells. Moreover, PAR1 and PAR2 stimulation activated PI3K/AKT signalling exclusively in OAC cells. Functionally, PAR1 and PAR2 activation promoted OAC cell invasion and chemotaxis, respectively. In all, these results highlight the PARs as potential players in OAC and BO pathogenesis and thus, potential therapeutic targets for these oesophageal pathologies.