Secretome profiling of PC3/nKR cells, a novel highly migrating prostate cancer subline derived from PC3 cells

Abstract

Prostate cancer (PCa) is the most common cancer among men worldwide. Most PCa cases are not fatal; however, the outlook is poor when PCa spreads to another organ. Bone is the target organ in about 80% of patients who experience metastasis from a primary PCa tumor. In the present study, we characterized the secretome of PC3/nKR cells, which are a new subline of PC3 cells that were originally isolated from nude mice that were implanted with PC3 cells without anti-natural killer (NK) cell treatment. Wound healing and Transwell assays revealed that PC3/nKR cells had increased migratory and invasive activities in addition to a higher resistance to NK cells-induced cytotoxicity as compared to PC3 cells. We quantitatively profiled the secreted proteins of PC3/nKR and PC3 cells by liquid chromatography-tandem mass spectrometry analysis coupled with 2-plex tandem mass tag labeling. In total, 598 secretory proteins were identified, and 561 proteins were quantified, among which 45 proteins were secreted more and 40 proteins were secreted less by PC3/nKR cells than by PC3 cells. For validation, the adapter molecule crk, serpin B3, and cystatin-M were analyzed by western blotting. PC3/nKR cells showed the selective secretion of NKG2D ligand 2, HLA-A, and IL-6, which may contribute to their NK cell-mediated cytotoxicity resistance, and had a high secretion of crk protein, which may contribute to their high migration and invasion properties. Based on our secretome analysis, we propose that PC3/nKR cells represent a new cell system for studying the metastasis and progression of PCa.

sBioSITe enables sensitive identification of the cell surface proteome through direct enrichment of biotinylated peptides

“…Although plectin is not annotated as a surface protein, it has been shown to have aberrant localization to the cell surface in some cancers [ 44 ]. Several histone proteins including H1.3, H1.2, H1.4 and H1.5 were also identified with biotinylated peptides, consistent with reports that histones are secreted in several cancers including prostate cancer [ 45 , 46 ]. Nucleolin, a nucleolar protein, was identified by 18 biotinylated peptides; it has been shown to be expressed on the cell surface in association with the actin cytoskeleton in some cancers [ 47 , 48 ].…”