A Suite of Activity-Based Probes To Dissect the KLK Activome in Drug-Resistant Prostate Cancer.

Scott Lovell,Elena De Vita,Anna Neodo,Maria Maneiro,Thomas Kryza,Elizabeth D Williams,Elisabeth Engelsberger,Alexander T Bakker,Reiko J Tanaka,Congyi Xu,Charlotte L Bevan,Edward W Tate,Leran Zhang,Nathalie Bock,Judith A Clements

doi:10.1021/jacs.1c03950

Abstract

Kallikrein-related peptidases (KLKs) are a family of secreted serine proteases, which form a network (the KLK activome) with an important role in proteolysis and signaling. In prostate cancer (PCa), increased KLK activity promotes tumor growth and metastasis through multiple biochemical pathways, and specific quantification and tracking of changes in the KLK activome could contribute to validation of KLKs as potential drug targets. Herein we report a technology platform based on novel activity-based probes (ABPs) and inhibitors enabling simultaneous orthogonal analysis of KLK2, KLK3, and KLK14 activity in hormone-responsive PCa cell lines and tumor homogenates. Importantly, we identifed a significant decoupling of KLK activity and abundance and suggest that KLK proteolysis should be considered as an additional parameter, along with the PSA blood test, for accurate PCa diagnosis and monitoring. Using selective inhibitors and multiplexed fluorescent activity-based protein profiling (ABPP), we dissect the KLK activome in PCa cells and show that increased KLK14 activity leads to a migratory phenotype. Furthermore, using biotinylated ABPs, we show that active KLK molecules are secreted into the bone microenvironment by PCa cells following stimulation by osteoblasts suggesting KLK-mediated signaling mechanisms could contribute to PCa metastasis to bone. Together our findings show that ABPP is a powerful approach to dissect dysregulation of the KLK activome as a promising and previously underappreciated therapeutic target in advanced PCa.

Highlights

Prostate cancer (PCa) is the most frequently diagnosed cancer among men in industrialized nations and remains a leading cause of cancer-related deaths.[1]
Inspired by multiplexed activity-based probes (ABPs) toolkits previously reported for the proteasome,[26,59] neutrophil serine proteases,[24] and caspases,[32] we developed the first probe set enabling simultaneous assessment of the activity of each PCa-relevant KLK in PCa samples
Development of selective ABPs for KLK2 and KLK14 was complicated by their similar trypsin-like activity; proteases with the same primary (P1) specificity are often coexpressed in tissues, and to generate selective tools, it is necessary to explore more complex specificity determinants.[60]

Summary

Introduction

Prostate cancer (PCa) is the most frequently diagnosed cancer among men in industrialized nations and remains a leading cause of cancer-related deaths.[1]. In CRPC, PCa cells evolve resistance to androgen-targeting therapies by restoring AR signaling through diverse mechanisms, and the majority of patients present with bone metastases with increased risk of morbidity and mortality due to alterations in skeletal integrity.[4] Mapping critical pathways involved in establishing CRPC may enable identification of novel therapeutic targets which can reduce disease recurrence.[5]. KLK2 can activate protease-activated receptors (PARs) on the surface of neighboring fibroblasts, which in turn release cytokines that stimulate PCa cell proliferation,[11] while in the bone micro-

Results

Discussion

Conclusion