Metastasis-related Intracellular Proteins in Human Breast Cancer Cells

We have used a unique metastasis model system in combination with quantitative, comparative proteomics to identify novel markers associated with the ability of cancer cells to colonize and form metastasis, and subsequently analyzed the clinical relevance of these proteins using breast cancer biopsies from patients with known clinical outcome within a 10-year follow-up. The model system consists of two isogenic human breast cancer cell lines that are equally tumorigenic in mice, but one gives rise to metastasis while the other disseminates single cells that remain dormant in distant organs. Using stable isotopic labeling by amino acids in cell culture and subcellular fractionation, the nuclear, cytosol and mitochondria proteomes were analyzed by LC-MS/MS, identifying seven proteins that exhibited altered expression between the cell lines, including L-lactate dehydrogenase A, NADH-cytochrome b5 reductase 3 (CYB5R3), niemann-pick c1 protein, and nucleolar RNA helicase 2.

本研究采用独特的转移模型系统结合定量比较蛋白质组学技术，筛选与癌细胞定植并形成转移灶能力相关的新型标志物；随后针对10年随访期内具有明确临床结局的乳腺癌患者的活检组织样本，分析上述蛋白质的临床相关性。该模型系统包含两株同基因人乳腺癌细胞系，二者在小鼠体内的致瘤能力相当，但其中一株可形成转移灶，另一株则仅散播单个细胞并在远端器官中保持休眠状态。本研究采用细胞培养氨基酸稳定同位素标记技术结合亚细胞分级分离法，通过液相色谱-串联质谱（LC-MS/MS）对细胞核、细胞质与线粒体的蛋白质组进行分析，最终鉴定出7种在两株细胞系间表达存在差异的蛋白质，包括L-乳酸脱氢酶A、NADH-细胞色素b5还原酶3（CYB5R3）、尼曼-匹克C1蛋白以及核仁RNA解旋酶2。