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

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.前列腺癌（Prostate cancer, PCa）是全球男性最常见的恶性肿瘤。多数前列腺癌病例并不致命，但当癌细胞扩散至其他器官时，预后极差。约80%发生原发性前列腺癌远处转移的患者，其转移靶器官为骨骼。本研究对PC3/nKR细胞的分泌组（secretome）进行了表征：该细胞系是原代PC3细胞的新亚株，最初从未经过抗自然杀伤（natural killer, NK）细胞处理、接种PC3细胞的裸鼠体内分离得到。划痕愈合实验与Transwell实验结果显示，相较于亲本PC3细胞，PC3/nKR细胞的迁移与侵袭能力显著增强，同时对NK细胞介导的细胞毒性具有更高的抵抗性。本研究采用液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry）联用结合2-plex串联质量标签（tandem mass tag, TMT）双标定量技术，对PC3/nKR与PC3细胞的分泌蛋白进行了定量分析。共计鉴定出598种分泌蛋白，可定量的蛋白共561种；其中PC3/nKR细胞相较于PC3细胞，分泌上调的蛋白有45种，分泌下调的蛋白有40种。为验证该定量结果，本研究通过蛋白质印迹（western blotting）法对衔接蛋白crk、丝氨酸蛋白酶抑制剂B3（serpin B3）以及半胱氨酸蛋白酶抑制剂M（cystatin-M）进行了验证分析。研究发现，PC3/nKR细胞可选择性分泌NKG2D配体2、HLA-A以及IL-6，这可能与其获得NK细胞毒性抵抗特性相关；同时该细胞系高分泌crk蛋白，这或许是其迁移与侵袭能力增强的分子基础。基于本次分泌组分析结果，我们认为PC3/nKR细胞可作为研究前列腺癌转移与进展的新型细胞模型。