Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan.
J Proteomics. 2013 Dec 6;94:186-201. doi: 10.1016/j.jprot.2013.09.013. Epub 2013 Sep 27.
Cancer cell secretome profiling has been shown to be a promising strategy for identifying potential body fluid-accessible cancer biomarkers and therapeutic targets. However, very few reports have investigated low-molecular-mass (LMr) proteins (<15kDa) in the cancer cell secretome. In the present study, we applied tricine-SDS-gel-assisted fractionation in conjunction with LC-MS/MS to systemically identify LMr proteins in the secretomes of three nasopharyngeal carcinoma (NPC) cell lines. We examined two NPC tissue transcriptome datasets to identify LMr genes/proteins that are highly upregulated in NPC tissues and also secreted/released from NPC cells, obtaining 35 candidates. We verified the overexpression of four targets (LSM2, SUMO1, RPL22, and CCL5) in NPC tissues by immunohistochemistry and demonstrated elevated plasma levels of two targets (S100A2 and CCL5) in NPC patients by ELISA. Notably, plasma CCL5 showed good power (AUC 0.801) for discriminating NPC patients from healthy controls. Additionally, functional assays revealed that CCL5 promoted migration of NPC cells, an effect that was effectively blocked by CCL5-neutralizing antibodies and maraviroc, a CCL5 receptor antagonist. Collectively, our data indicate the feasibility of the tricine-SDS-gel/LC-MS/MS approach for efficient identification of LMr proteins from cancer cell secretomes, and suggest that CCL5 is a potential plasma biomarker and therapeutic target for NPC.
Both LMr proteome and cancer cell secretome represent attractive reservoirs for discovery of cancer biomarkers and therapeutic targets. Our present study provides evidence for the practicality of using the tricine-SDS-PAGE/LC-MS/MS approach for in-depth identification of LMr proteins from the NPC cell secretomes, leading to the discovery of CCL5 as a potential plasma biomarker and therapeutic target for NPC. We believe that the modified GeLC-MS/MS approach used here can be further applied to explore extremely low-abundance, extracellular LMr proteins with important biological functions in other cell lines and biospecimens.
癌细胞分泌组分析已被证明是一种很有前途的策略，可以用于鉴定潜在的体液可及性癌症生物标志物和治疗靶点。然而，很少有报道研究癌细胞分泌组中的低分子量（LMr）蛋白（<15kDa）。在本研究中，我们应用三辛基膦-SDS-凝胶辅助分级分离与 LC-MS/MS 相结合，系统地鉴定了三种鼻咽癌（NPC）细胞系分泌组中的 LMr 蛋白。我们检查了两个 NPC 组织转录组数据集，以鉴定在 NPC 组织中高度上调且从 NPC 细胞分泌/释放的 LMr 基因/蛋白，得到 35 个候选物。我们通过免疫组织化学验证了四个靶标（LSM2、SUMO1、RPL22 和 CCL5）在 NPC 组织中的过表达，并通过 ELISA 证明了 NPC 患者血浆中两个靶标（S100A2 和 CCL5）的升高水平。值得注意的是，血浆 CCL5 对区分 NPC 患者与健康对照具有很好的分辨力（AUC 0.801）。此外，功能测定表明 CCL5 促进 NPC 细胞的迁移，该作用可被 CCL5 中和抗体和 CCL5 受体拮抗剂 maraviroc 有效阻断。总之，我们的数据表明 tricine-SDS-凝胶/LC-MS/MS 方法可有效地从癌细胞分泌组中鉴定 LMr 蛋白，并且表明 CCL5 是 NPC 的潜在血浆生物标志物和治疗靶点。
LMr 蛋白质组和癌细胞分泌组都是发现癌症生物标志物和治疗靶点的有吸引力的资源库。本研究为使用 tricine-SDS-PAGE/LC-MS/MS 方法从 NPC 细胞分泌组中深入鉴定 LMr 蛋白提供了证据，发现 CCL5 是 NPC 的潜在血浆生物标志物和治疗靶点。我们相信，这里使用的改良 GeLC-MS/MS 方法可进一步应用于探索其他细胞系和生物样本中具有重要生物学功能的极少量、细胞外 LMr 蛋白质。
