Proteinases, Proteinase‐Activated Receptors (PARs) and Transient Receptor Potential (TRP) Ion Channels: Driving Tumorigenesis in the Bladder Cancer Microenvironment
Authors: Gibson Stacy , Mihara Koichiro , El‐Daly Mahmoud , Saifedine Mahmoud , Hollenberg Morley D. , Hyndman Matthew Eric 
Published: November 2023

Overview and hypothesesWe propose that bladder cancer initiation and progression involves proteinases that become elevated in the tumour microenvironment and that signal in part by cleaving and activating proteinase‐activated receptors (PARs). Because proteinases activate PARs through the cleavage of the N‐terminal cell surface domain of this subgroup of the G protein coupled receptor family, this mechanism would release the cleaved peptide along with the activating enzyme into the urine. We hypothesise that bladder cancer cells respond to PAR activation that can also involve signaling by transient receptor potential cation channels (TRPV4/TRPM8) along with the secretion of PAR‐regulating proteinases.AimsTo test this hypothesis, our aims were therefore to: (1) explore the expression and function of proteinase activated receptors (PARs 1, 2 & 4) within bladder cancer‐derived cell lines, (2) determine the expression and function of transient receptor potential (TRP) channels known to be affected by the activation of PARs (TRPV4, TRPM8) and (3) analyze the ability of bladder cancer cell lines to secrete PAR‐regulating proteinases into their supernatant.MethodsThe functionality of PARs (1, 2 and 4), TRPV4 and TRPM8 in bladder cancer cell lines (T24, HTB‐9) was evaluated by monitoring calcium signaling (JPET 288:358, 1999) stimulated by increasing concentrations of target‐selective agonists for (1) The PARs: PAR1 (TFLLR‐NH2), PAR2 (2fLIGRLO‐NH2) and PAR4 (AYPGKF‐NH2), (2) TRPV4 (GSK101) and (3) TRPM8 (Icilin). The relative abundance of T24 and HTB‐9 PAR and TRP (V4, M8) channel mRNA was evaluated using semi quantitative PCR, normalized to an actin signal. Production of PAR‐regulating proteinases by T24 and HTB‐9 cells was evaluated by a new novel PAR cleavage assay that monitors the release of an N‐terminal luciferase tag from cell surface‐expressed PARs 1 and 2.ResultsBladder tumour‐derived HTB‐9 & T24 cells possess (assessed through PCR) functional (assessed through calcium signaling) PARs 1 & 2 but with a differential sensitivity towards PAR2 (HTB‐9 > T24) vs PAR1 activation (HTB‐9 = T24). PAR4 is not expressed. These cell lines also showed the expression of functional TRPV4 channels, but not TRPM8. Both cell lines secrete PAR‐regulating proteinases that cleave PARs 1 and 2.ConclusionFunctional PARs 1 & 2 and TRPV4 channels which are present in bladder cancer cells may drive tumour progression. Further, tumour cell PAR‐regulating proteinase secretion can act via autocrine and paracrine mechanisms to enhance tumorigenesis. This mechanism may represent a therapeutic target for bladder cancer. Further, the secretion into the urine of the upregulated PAR‐regulating proteinases along with PAR N‐terminal peptide fragments may serve as a biomarker for bladder cancer progression.Support or Funding InformationAIHS CRIO Grant, Prostate Cancer Canada Discovery Grant, Motorcycle Ride for Dad and CIHR