Identification and characterization of novel proteolytic interactions of prostate cancer- expressed kallikrein-related peptidases, type II transmembrane serine proteases and matrix metalloproteinases Janet C. Reid Bachelor of Science, Master of Science Qualifying, Graduate Diploma of Biotechnology Institute of Health and Biomedical Innovation Mater Medical Research Institute Translational Research Institute School of Biomedical Sciences, Queensland University of Technology A thesis submitted to the Queensland University of Technology in fulfillment of the requirements of a Doctor of Philosophy Jan 2015 Key Words Activity Based Probe (ABP), Calcium Signalling, Cell Surface, Hepatocyte Growth Factor (HGF), Hepatocyte Growth Factor Activator Inhibitor-1 (HAI-1), Kallikrein- Related Peptidase (KLK), Kidney Proximal Tubule Cells, Matrix Metalloproteinase (MMP), Prostate Cancer, Protease-Activated Receptor (PAR), Proteolytic Cascade, Recombinant Proteins, Serine Protease Inhibitor, Type II Transmembrane Serine Protease (TTSP). i Abstract In the tumour microenvironment pericellular proteolytic activity affects cells by cleavage of extracellular matrix proteins, activation of cell-surface receptors such as the protease activated receptors (PAR), and processing of mitogenic growth factors such as hepatocyte growth factor (HGF). As proteolysis is irreversible in physiological settings, protease activity is tightly regulated by a number of factors including localization, zymogen activation and protease inhibitors. However, overexpression of proteases in the dysregulated prostate cancer microenvironment has the potential to facilitate disease progression. The secreted serine proteases kallikrein-related peptidase (KLK)14 and KLK4 as well as the type II transmembrane serine proteases (TTSPs) hepsin and transmembrane protease serine 2 (TMPRSS2) have been implicated in the progression of prostate cancer as all four are upregulated this malignancy. For instance, increased KLK14 expression has been correlated with aggressive, late stage prostate cancer and decreased progression-free survival. KLK4 also has increased expression; however, this is seen in early stages of prostate cancer, with diminished KLK4 levels in late stages. Hepsin has also been implicated as expression increase through all prostate cancer stages to bone metastasis. Further, in a non-metastasising mouse model, hepsin overexpression has been implicated in invasion/extravasion and metastasis of primary prostate tumour cells through disorganisation of the basement membrane. TMPRSS2 also has increased expression in localized prostate cancer. Further, TMPRSS2 has dysregulated cellular localization in prostate cancer, moving from the apical cellular localization of normal epithelial prostate cells to the entire cell surface with intracellular accumulation in prostate cancer cells. Critically, KLK14, KLK4 and TMPRSS2 induce intracellular signalling pathways via proteolytic activation of PARs, and modulation of PAR- mediated signalling has been associated with cells acquiring a more cancerous phenotype. While hepsin and TMPRSS2 auto-activate, the endogenous activators of KLK14 and KLK4 are unknown. It was recently determined that matrix metalloproteinase MMP3 activates KLK4 in vitro and so may be an endogenous activator. Moreover, it is prostate expressed and has also been implication in the progression of several cancer types including prostate. In turn, MMP3 is activated by auto-activating TTSP family member ii matriptase, thereby potentially forming an activation cascade from matriptase to MMP3 to KLK4. As auto-activating proteases, hepsin and TMPRSS2 also have the potential to function in proteolytic activation cascades, activating either the KLKs or MMP3. In turn, the activated KLKs may induce PAR-mediated intracellular signalling or activate HGF, the MET receptor ligand. The goal of this study was to examine pericellular proteolytic activity. In particular, it focussed on proteolytic interactions of KLK14 and KLK4 with hepsin and TMPRSS2. To a lesser extent, interactions of hepsin and TMPRSS2 with MMPs, MMP3 and MMP9 were also examined. This study also examined HGF activation by KLK14 and KLK4, and inhibition of KLK14 by hepatocyte growth factor activator inhibitor-1 (HAI-1). Further, activation of PAR family members PAR1, PAR2 and PAR4 by KLK14, hepsin and matriptase, and PAR2 by KLK4, was examined. For the first part of this study recombinant pro-KLK14 was generated in insect cells and characterized. It was determined that while pro-KLK14 from this system is N- glycosylated, the protease produced by Cos-7 cells lack this modification. This indicates the potential for cell-type differential N-glycosylation of KLK14. Active recombinant KLK14 was also shown to activate pro-HGF, while KLK4 primarily degrades it. Examination of KLK14 inhibition by HAI-1A and HAI-1B isoforms indicated that this inhibitor is a KLK14 substrate rather than inhibitor as both isoforms were degraded by KLK14. In comparison, hepsin and HAI-1A formed the stable complexes characteristic of inhibition. The second part of this study examined the interactions between KLK4 and KLK14, the hepsin and TMPRSS2, and to a lesser extent matriptase, MMP3 and MMP9. The data from these studies suggest complex pericellular interactions between these proteases and can be summarized as follows: KLK4 and, to a lesser extent, KLK14 are proteolysed by hepsin and TMPRSS2, and the shortened forms of these KLKs were mainly detected in association with the cell Proteolysis of KLK4 by hepsin and TMPRSS2 does not appear to lead to activation; however, it is not known if cell-associated forms of KLK14 are activated by these TTSPs iii KLK4 cleaves the stem region of hepsin and TMPRSS2; however, these cleaved forms remain cell membrane-bound through disulphide links in the stem While KLK4 and KLK14 co-immunoprecipitate with hepsin and TMPRSS2, KLK14 and active KLK4 are located on the cell surface independently of these TTSPs MMP3 and MMP9 are activated by hepsin and TMPRSS2 (and matriptase) MMP3 and MMP9 co-immunoprecipitate with hepsin and TMPRSS2. The third and final part of the study focussed on PAR1, PAR2 and PAR4 activation by KLK14, matriptase and hepsin, as well as inhibition of KLK4 activation of PAR2 using the KLK4-specific inhibitor SFTI-FCQR. PAR2 activation by KLK4 was investigated further using human primary kidney proximal tubule cells (PTC), which have previously been described as co-expressing PAR2 and KLK4. It was determined that while KLK14 activated PAR1, PAR2 and PAR4, and matriptase activated PAR2-mediated signalling, in the form of intracellular Ca2+ mobilization, there was negligible activation of PAR1, PAR2 or PAR4 by hepsin. Further, KLK4 activation of PAR2 could be abrogated by the KLK4-specific inhibitor SFTI-FCQR. Interestingly, further examination of KLK4 activation of PAR2 showed that while it activates PAR2 in a number of cell types, it elicited minimal PAR2 activation and Ca2+ mobilization in human kidney PTCs. This data suggests cell-type biased agonism of PAR2 by KLK4 potentially initiating alternative intracellular signalling pathways, or alternatively, a KLK4 competitive substrate on the surface of these cells. In this final section LNCaP cells overexpressing hepsin or active site mutated hepsin were also generated as a tool to examine the participation of hepsin with other serine proteases in proteolytic cascades resulting in PAR activation. However, overexpression of hepsin in these cells resulted in minimal plasma membrane localization in contrast to catalytically inactive hepsin which readily localized to the plasma membrane. Importantly, this is the first report of KLK14 activation of pro-HGF. Further, this is the first time KLK14 degradation of HAI-1, and formation of hepsin-HAI-1 complexes, have been reported. It is also the first time cell-surface localization of KLK4 and KLK14 has been demonstrated. In addition, this is the first time active KLK4 has been isolated from the cell surface. iv The data in this study suggest that KLK14 may have a role in regulation of MET receptor/HGF signalling axis through HGF activation and degradation of key protease inhibitors, HAI-1A and HAI-1B. Further, the data set up the premise for a proteolytic cascade at the plasma membrane involving TMPRSS2 and hepsin activation of MMP3 and MMP9, with MMP3 then activating KLK4. Finally, the cell surface localization of KLK14 and KLK4 may facilitate activation of PARs, degradation of HAI-1 isoforms and pericellular activation of HGF. Based on the results of this study a number of exciting and interesting avenues have been proposed to further explore the role of KLK14, and these other prostate-expressed proteases, in modulation of the pericellular environment. v Table of Contents Key Words i Abstract ii Table of Contents vi List of Figures xi List of Tables xv List of Abbreviations xvi Publications contributed to during PhD xx Statement of Original Authorship xxi Acknowledgments xxii CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW ------------------------ 1 1.1 Prostate cancer 2 1.2 Proteases 3 1.3 Serine proteases 5 1.4 Kallikrein-related peptidases (KLKs) 7 1.4.1 KLK4 11 1.4.2 KLK14 13 1.4.3 KLKs in proteolytic cascades 14 1.4.4 KLKs in prostate cancer 17 1.5 Type II transmembrane serine proteases (TTSPs) 19 1.5.1 Hepsin 21 1.5.2 TMPRSS2 22 1.5.3 Matriptase 24 1.5.4 TTSPs in proteolytic cascades 25 1.5.5 TTSPs in prostate cancer 26 1.6 Hepatocyte growth factor 28 1.7 Hepatocyte growth factor activator