Abstract
We hypothesized that microenvironment proteases generated by urothelial carcinoma (UC) cells dictate cell invasion & metastasis by signaling via Proteinase-Activated Receptors (PARs) and evaluated this hypothesis in UC cell lines in in vitro monolayer & organotypic 3D cultures UC cell lines: RT4, SW780, T24, 5637 In vitro: PAR1/2 function was assessed via calcium signalling assays. 2 methods detected UC-secreted proteases that cleave PAR1/2: 1) N-luciferase tag fused to PAR1/2 in a non-UC cell line released upon cleavage (paracrine proteinase activity); 2) N-terminal mCherry/RFP;C-terminal eYFP-tagged PAR construct transfected into UC cells to visualize receptor autocrine cleavage (intact receptor=yellow; cleaved receptor=green). The PAR1/2 effect on UC cell migration & invasion was assessed in monolayer wound healing assays with & without PAR1/2 agonism. Organotypic Model: UC cells were cultured in a bladder-derived fibroblast-containing 3D collagen biomatrix model that maintains epithelial phenotype & supports the growth of UC cell lines. UC cells were evaluated for proliferation, apoptosis, differentiation, & invasion in this model with/without PAR activation/inhibition. UC cell lines express PAR1/2 & secrete proteases & protease inhibitors that induce &/or affect signaling. PAR1/2 activating peptides stimulated migration & invasion of UC cells in monolayer cultures with inhibition by a PAR1 antagonist. In the organotypic setting neither PAR1 or 2 agonism caused significant proliferation, apoptosis, or invasion. Uniquely in the organotypic model we observed induction of differentiation in non-invasive cells (SW780 & RT4) & increased cytokeratin CK5-6 expression switching in the cell lines induced by PAR1 agonism & antagonism. Functional PAR1/2 are expressed by UC cells. Activation of these receptors increases invasion & migration in monolayer cultures, but not in organotypic cultures. Our data illustrate significant differences between monolayer & organotypic cultures & suggest PAR1 impacts cytokeratin expression & UC cell differentiation.
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