Prostate cancer is a leading cause of male cancer-related deaths, worldwide. Kallikrein-related peptidase 4 (KLK4) is a serine protease which is over-expressed in prostate cancer, versus benign disease, and further increased in fatal bone metastatic prostate cancer. In vitro, KLK4 induces prostate cancer cell proliferation, migration and invasion, as well as promoting an epithelial-to-mesenchymal transition. However, KLK4 substrates in primary and metastatic prostate cancer are not well understood and must be identified to understand the mechanism of KLK4 action in prostate cancer.
To elucidate KLK4 targets on a proteome-wide scale, we employed a novel proteomic approach, called the PROtein Topography and Migration Analysis Platform (PROTOMAP1). Proteins secreted by PC-3 cells, derived from a patient with bone metastatic prostate cancer, were harvested, concentrated and treated with active, recombinant KLK4, or an inactive mutant KLK4 control. Protein digests were separated by SDS-PAGE and gel lanes sliced into 32 horizontal sections, prior to identification of proteins in each section by LC-MS/MS. Those proteins detected in gel sections of lower molecular weight in KLK4-treated samples, versus controls, were deemed KLK4 substrates.
In total, 452 PC-3 cell-secreted proteins were identified, of which 20 were potential novel KLK4 substrates. These included proteases, protease inhibitors, extracellular matrix proteins, cell adhesion molecules and cytokines. Of particular interest was matrix metalloproteinase-1 (MMP-1), as KLK4 was shown to activate MMP-1, a protein involved in prostate cancer growth and metastasis in vivo2. In addition, the anti-angiogenic thrombospondin-1 (TSP-1) was cleaved by KLK4, potentially as a mechanism to increase blood vessel formation, favouring tumour progression.
We are currently using 2- and 3-dimensional cell culture models of prostate cancer to delineate the biological consequences of KLK4 proteolysis of these and other novel targets identified in this study, with the aim of identifying pathways amenable to therapeutic intervention.