Abstract 2014: Genitourinary cancer-derived cells produce microenvironment proteinases that regulate proteinase activated receptors (PARs) to drive oncogenic signaling

Abstract

Abstract Background: Thrombin-triggered activation of proteinase-activated receptor-1 (PAR1) is recognized as a key stimulus for driving epithelial malignancies (IUBMB life.63:397, 2011; PMID:21557443). However, the enzymes for regulating PAR1 in the tumour microenvironment are not known. Hypothesis: We hypothesize that prostate (PC) and urinary bladder cancer (UBC) cells can regulate microenvironment signaling to drive cancer progression via the secretion of proteinases that cleave/regulate proteinase-activated receptors (PARs). Aims: 1.) Visualize the cleavage status of N- and C-terminally-dual-labelled PARs in receptor-transfected UBC- and PC-derived cells. 2.) Measure PAR cleavage by UBC and PC-secreted proteinases. 3.) Identify the UBC & PC-derived proteinases that cleave/regulate PAR function. Methods: The cleavage status of dual-labeled PARs (N-terminus-mCherry/C-terminus-YFP) expressed in PC & UBC cells was determined by confocal image analysis as already described (JBC 288:32979, 2013; PMID: 26957205). Intact receptors look 'yellow' and cleaved receptors look 'green'. The cleavage of PARs in UBC and PC cells was studied for dual-tagged-PAR-expressing UBC and PC cells exposed or not to cell-derived culture supernatants, enzyme agonists for PAR1 and 2 (thrombin and trypsin, respectively), and proteinase inhibitors (e.g. for MMPs and other enzymes). The impact of CRISPR-mediated elimination of MMPs from PC3 cells on PAR1 cleavage status was also monitored. Results: UBC- and PC-produced enzymes can cleave PAR1 as efficiently as thrombin, an established PAR1 agonist. None of the UBC cell lines produce proteinases that cleave PAR2. Inhibition of MMP activity partially prevents PAR1 cleavage by UBC cells and completely prevents PAR1 cleavage in PC3-PC cells. Conclusions: We conclude that UBC and PC cells secrete PAR-regulating proteinases that can directly regulate PAR1 in the tumour microenvironment so as to drive cancer progression. Funding: Alberta Innovates CRIO Grant, Prostate Cancer Canada Discovery Grant, Motorcycle Ride for Dad, Johnson & Johnson Alberta Health Partnership and CIHR Note: This abstract was not presented at the meeting. Citation Format: Stacy G. Gibson, Koichiro Mihara, Andries Zijlstra, Matthew E. Hyndman, Morley D. Hollenberg. Genitourinary cancer-derived cells produce microenvironment proteinases that regulate proteinase activated receptors (PARs) to drive oncogenic signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2014. doi:10.1158/1538-7445.AM2017-2014