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.

Fig 1. The characteristics of PC3 and PC3/nKR cells.

(A) The relative wound area in wound scratch assays PC-3 and PC-3/nKR cells scratched by a tip were observed under light microscopy at intervals of 12 h and the results were normalized to those for PC-3 cells. (B) Representative images of cells that had invaded into a Transwell chamber are shown. (C) Effects of incubating PC-3/PC-3/nKR cells with the conditioned media (CM) from PC-3/nKR cells for 48 h, respectively. All the experiments were conducted in triplicate (**P < 0.01, ***P < 0.001). Relative migration is defined as the reciprocal of the relative distance when the cell is restored after scratching. (D) The representative pictures of wound healing assay for effect on condition media (CM) of PC-3/nKR on PC-3 cells and PC-3 on PC-3/nKR for 48 hrs, respectively.

Fig 2. The experimental strategy for the quantitative analysis of the secretomes of PC3 and PC3/nKR cells.

(A) The integrated workflow for the comparative proteomics analysis of proteins secreted from PC3 and PC3/nKR cells. (B) A schematic showing the processes for protein identification and quantification based on the results of the proteomic analysis. (C) A Venn diagram indicating that 94 of the 598 proteins that we identified were characterized as newly secretory based upon our results, as determined by comparison with The Human Protein Atlas and Plasma Proteome Database.

Fig 3. Validation of quantitative proteomics result using immunoblots.

(A) Western blotting analysis of proteins with increased (Crk, SerpinB3, Cystatin-M), decreased (ULBP2), and unchanged secretion (Follistatin) in conditioned media (CM) and cell extract (CE) samples from PC3/nKR cells as compared with those from PC3 cells. (B) Densitometry results of Crk, SerpinB3, Cystatin-M, ULBP2 and Follistatin.

Fig 4. Functional analysis of proteins that were secreted at higher levels by PC3/nKR cells than by PC3 cells.

(A) GO-based enrichment analysis and (B) KEGG pathway-based enrichment analysis of the proteins that were secreted at higher levels by PC3/nKR cells than by PC3 cells.

Fig 5. Protein-protein interaction analysis of highly secreted proteins in PC3/nKR cells by STRING.