Oral Presentation Symposium on Proteases and the Tumouri Microenvironment 2017

Differential proteomic profiling of non-malignant prostate fibroblasts and prostate cancer-associated fibroblasts (CAFs) identifies specific CAF-associated signalling networks (#19)

Elizabeth V Nguyen 1 2 , Brooke A Pereira 2 3 , Natalie Lister 2 3 , Mitchell G Lawrence 2 3 , Birunthi Niranjan 2 3 , Gail P Risbridger 2 3 4 , Renea A Taylor 2 5 , Roger J Daly 1 2
  1. Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Melbourne, Vic
  2. Cancer Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia
  3. Department of Anatomy & Developmental Biology, Monash University, Clayton, Victoria, Australia
  4. Cancer Research Division, Peter MacCallum Cancer Centre, Victorian Comprehensive Cancer Centre, University of Melbourne, Parkville, Victoria, Australia
  5. Department of Physiology, Monash University, Clayton, Victoria, Australia

In prostate cancer (PC), cancer-associated fibroblasts (CAFs) exhibit contrasting biological properties to non-malignant prostate fibroblasts (NPFs), promote tumourigenesis  and modulate the response of PC cells to androgens and anti-androgens. Resolving intercellular signalling pathways between CAFs and prostate epithelial or PC cells may offer novel opportunities for research translation. In order to address this issue, we undertook integrated proteomic and phosphoproteomic profiling of CAFs and NPFs in order to identify signalling networks that characterize the CAF phenotype.

Characterization of the proteome of 4 patient-matched CAF and NPF lines by LC-MS/MS identified 4075 quantifiable proteins, with 67 exhibiting significantly differential expression (adjusted p-value <0.05). Functional analyses revealed significant enrichment for ‘Cell adhesion’ and the ‘Extracellular region’ in the CAF lines. Network analysis of proteins over-expressed in CAFs revealed a protein-protein interaction hub containing multiple collagens including the fibrillar types COL1A1/2 and COL5A1/2, the receptor tyrosine kinase DDR2 that acts as a receptor for fibrillar collagens, and lysyl oxidase-like 2 (LOXL2) that promotes collagen crosslinking. Marked upregulation of DDR2 and activation of FAK, an indicator of LOXL2-enhanced extracellular matrix stiffness, in CAFs was confirmed by Western blotting. These data implicate the collagen/DDR2 axis as a potential mechanism for CAF autocrine and paracrine signalling.

Characterization of the phosphoproteome of the CAF and NPF cell lines identified 10,714 quantifiable phosphopeptides mapping to 3,032 proteins. Network analysis of 444 proteins exhibiting enhanced phosphorylation (p<0.05) in CAFs resolved hubs related to focal adhesion signalling, cytoskeletal organization, mTOR pathway, and transcriptional repression mediated via HDAC1 and HDAC2. Overall, these data provide the first global characterization of signalling networks activated in CAFs, highlight interaction hubs likely to underpin key biological differences between CAFs and NPFs, and identify potential modes of intercellular communication between CAFs and prostate epithelial/cancer cells that may contribute to disease development and progression.