Proteinase-activated receptor 2 promotes TGF-β-dependent cell motility in pancreatic cancer cells by sustaining expression of the TGF-β type I receptor ALK5

// Franziska Zeeh 1,* , David Witte 1,* , Thomas Gadeken 1 , Bernhard H. Rauch 2 , Evelin Grage-Griebenow 1 , Nadja Leinung 1 , Sofie Joline Fromm 1 , Stephanie Stolting 1 , Koichiro Mihara 3 , Roland Kaufmann 4 , Utz Settmacher 4 , Hendrik Lehnert 1 , Morley D. Hollenberg 3 and Hendrik Ungefroren 1 1 First Department of Medicine, University of Lubeck, Lubeck, Germany 2 Department of General Pharmacology, Institute of Pharmacology, University Medicine Greifswald, Greifswald, Germany 3 Department of Physiology & Pharmacology and Department of Medicine, Inflammation Research Network-Snyder Institute for Chronic Diseases, University of Calgary, Cumming School of Medicine, Calgary, AB, Canada 4 Department of General, Visceral and Vascular Surgery, Jena University Hospital, Jena, Germany * These authors have contributed equally to this work Correspondence to: Hendrik Ungefroren, email: // Keywords : TGF-β; PAR2; ALK5; signalling; cell motility Received : April 20, 2016 Accepted : May 14, 2016 Published : May 27, 2016 Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by high expression of transforming growth factor (TGF)-β and the G protein-coupled receptor proteinase-activated receptor 2 (PAR2), the latter of which functions as a cell-surface sensor for serine proteinases asscociated with the tumour microenvironment. Since TGF-β and PAR2 affect tumourigenesis by regulating migration, invasion and metastasis, we hypothesized that there is signalling crosstalk between them. Depleting PDAC and non-PDAC cells of PAR2 by RNA interference strongly decreased TGF-β1-induced activation of Smad2/3 and p38 mitogen-activated protein kinase, Smad dependent transcriptional activity, expression of invasion associated genes, and cell migration/invasion in vitro . Likewise, the plasminogen activator-inhibitor 1 gene in primary cultures of aortic smooth muscle cells from PAR2 -/- mice displayed a greatly attenuated sensitivity to TGF-β1 stimulation. PAR2 depletion in PDAC cells resulted in reduced protein and mRNA levels of the TGF-β type I receptor activin receptor-like kinase 5 (ALK5). Forced expression of wild-type ALK5 or a kinase-active ALK5 mutant, but not a kinase-active but Smad-binding defective ALK5 mutant, was able to rescue TGF-β1-induced Smad3 activation, Smad dependent transcription, and cell migration in PAR2-depleted cells. Together, our data show that PAR2 is crucial for TGF-β1-induced cell motility by its ability to sustain expression of ALK5. Therapeutically targeting PAR2 may thus be a promising approach in preventing TGF-β-dependent driven metastatic dissemination in PDAC and possibly other stroma-rich tumour types.