Proteolytic Signal Crosstalk in the Prostate Cancer Microenvironment: PC3 Cell Metalloproteinases and Autocrine‐paracrine‐fibroblast Regulation of Proteinase‐activated Receptors (PARs)

Abstract

Tumour-associated fibroblasts(TAFs) and non-tumour epithelial cells (NT-EPCs) are known to respond to tumour-derived microenvironment agonists. We hypothesized that prostate cancer-derived PC3 cells can signal to TAFs and NT-EPCs by generating proteinases that can regulate proteinase-activated receptors(PARs). We thus expressed Dual-Tagged N-terminal-mCherry/C-terminal-YFP PAR1 in PC3 cells and in a non-tumour prostate epithelial cell line, RWPE. N-terminal N-luciferase(LUC)-tagged PARs 1,2 and 4 were expressed in WI38 fibroblasts. Upon live cell imaging, intact expressed dual-tagged PARs appear ‘yellow’; cleaved dual-tagged-PARs appear green (loss of N-terminal mCherry). Cleavage of N-LUC-PARs expressed in WI38 cells releases a N-LUC fluorescence signal into the supernatant. Imaging showed that PC3 cell-expressed dual-tagged PAR1 appeared ‘green’, demonstrating autocrine PAR cleavage. Addition of a general MMP inhibitor(GM6001) blocked PC3-expressed dual-tagged-PAR1 cleavage (yellow receptor) as did CRISPR-elimination of PC3-expressed MMP2. PC3-derived supernatants cleaved tagged PAR1 expressed in RWPE cells (green). PC3-derived supernatants also cleaved all of WI38 cell expressed N-LUC-PARs 1, 2 & 4, releasing fluorescence from the cells. We conclude that prostate cancer-derived PC3 cells produce PAR-regulating proteinases, including MMP2, that can regulate tumour and non-tumour cell PAR signalling by an autocrine and paracrine mechanism in a tumour microenvironment.