Introduction: In prostate cancer, cancer-associated fibroblasts (CAFs) promote tumor progression whereas non-malignant prostate fibroblasts (NPFs) do not. Resolving distinct signalling between CAFs and their NPF counterparts along with the dynamic interplay of CAFs with prostate tumour epithelium offers the potential to identify novel antineoplastic therapeutic targets.
Methods: The proteomes of patient-matched CAFs and NPFs (n=4) were analyzed by LC-MS/MS, with a hyper-reaction monitoring data-independent acquisition (HRM-DIA) workflow, to identify discriminating proteomic signatures . To investigate the intercellular communication between NPFs or CAFs and co-cultured prostate epithelial (PE) cells, cell type-specific labelling with amino acid precursors (CTAP) was used in conjunction with a synchronous precursor selection tandem MS/MS/MS (SPS-MS3) workflow. Specific culture parameters (i.e., cell types, monoculture, co-culture) were distinguishable with isobaric-labelled tandem mass tags (TMTs).
Major Findings: The CAF proteome exhibited a prominent interaction hub containing the fibrillar collagens COL1A1/2 and COL5A1; DDR2, a receptor for fibrillar collagens; and LOXL2, which promotes collagen crosslinking. Pharmacological inhibition of CAF-derived LOXL2 perturbed extracellular matrix (ECM) organization, decreased cell migration, and significantly impaired the motility of co-cultured RWPE-2 prostate tumor epithelial cells. Further interrogation of intercellular communication between NPFs or CAFs and BPH-1 immortalized PE cells revealed proteins exhibiting enhanced expression in BPH-1 cells upon co-culture with CAFs. These included the ECM protein FN1, the integrin family member ITGB4 along with its laminin ligands LAMC2 and LAMB3, cytoskeletal regulators CDC42 (a Rho family GTPase), TAGLN (an actin-binding protein) and VIM (an intermediate filament protein). Consistent with enhanced expression of CDC42, phosphoproteomics revealed perturbed phosphorylation of downstream PAK kinases in BPH-1 cells co-cultured with CAFs.
Conclusion: CAF-derived LOXL2 along with PE cell-expressed CDC42 and PAK kinases were indicated as important mediators of intercellular communication. These candidates may serve as an avenue for potential therapeutic strategies.