Oral Presentation 9th General Meeting of the International Proteolysis Society 2015

Global profiling of kallikrein-related peptidase 4 (KLK4) substrates and downstream regulated genes in prostate cancer (#16)

Ruth A Fuhrman-Luck 1 , Marcus L Hastie 2 , Bosco K Ho 3 , Thomas Stoll 2 , Carson R Stephens 1 , Melanie Lehman 1 , Anja Rockstroh 1 , Colleen Nelson 1 , Daniela Loessner 4 , Jeffrey J Gorman 2 , Oded Kleifeld 5 , Judith A Clements 1
  1. Australian Prostate Cancer Research Centre- Queensland, Institute of Health and Biomedical Innovation at the Translational Research Institute, Queensland University of Technology, Brisbane, Queensland, Australia
  2. Protein Discovery Centre, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia
  3. Monash Bioinformatics Platform, Monash University, Clayton, Victoria, Australia
  4. School of Biomedical Science, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia
  5. Faculty of Medicine, Nursing & Health Sciences, Monash University, Clayton, Victoria, Australia

Inhibiting molecular drivers of prostate cancer progression is essential to prevent end-stage disease, for which only palliative treatments exist. KLK4 is over-produced in prostate cancer versus benign disease, and promotes cancer cell proliferation and migration in vitro, and tumour growth in vivo. The proteolytic mechanism through which KLK4 drives prostate tumourigenesis is unknown, confounded by limited knowledge of KLK4 substrates and regulated genes. Thus, we sought to define the KLK4 substrate repertoire and downstream regulated genes in prostate cancer, to delineate the mechanism of action of KLK4 in prostate cancer.                                                        

To identify KLK4 substrates, conditioned media (CM) from prostate cancer or associated stroma was treated with active recombinant KLK4 (400:1, CM: active KLK4) or inactive mutant KLK4 (37 ºC/ 18 h). Substrates were identified by the PROtein TOpography and Migration Analysis Platform (PROTOMAP; [1]), which couples SDS-PAGE with in-gel trypsin digestion and LC-M/MS to detect shifts in protein mass following proteolysis. Terminal Amine Isotopic Labeling of Substrates (TAILS; [2]) was also performed, where KLK4-generated substrate N-termini were isolated prior to identification by LC-MS/MS. Gene expression in KLK4-treated (20 nM; 37 ºC/ 18 h) or control-treated prostate cancer or stromal cells was analysed using a custom Agilent 180K microarray.

57 novel putative KLK4 substrates were identified, in addition to established substrates, which validated the approach. Notably, KLK4 activated pro-matrix metalloproteinase-1, constituting a putative mechanism for KLK4-induced cell migration. KLK4 further liberated an N-terminal fragment with purported angiogenic activity from thrombospondin-1, where angiogenesis is essential to sustain the expanding tumour. Pathway analysis of KLK4-regulated genes predicted KLK4 to activate transforming growth factor β1 (TGFβ1) signalling in prostate stroma. As TGFβ1 signalling in prostate stroma supports tumour growth, our findings highlight KLK4 as a multi-functional regulator of tumour progression and a promising therapeutic target.

  1. Dix et al, Cell, 2008
  2. Kleifeld et al, Nat Biotechnol, 2010