METHODS: The isobaric tag for relative and absolute quantitation (iTRAQ)-based LC-MS/MS proteomic approach was applied to identify novel membrane-associated proteins in the lung-tropic metastatic cells. Public domain datasets were used to assess the clinical relevance of the candidate proteins. Cell-based and mouse models were used for biochemical and functional characterization of the protein molecule Sciellin (SCEL) identified by iTRAQ to elucidate its role and underlying mechanism in promoting lung colonization of TNBC cells.
RESULTS: The iTRAQ-based LC-MS/MS proteomic approach identified a membrane-associated protein SCEL that was overexpressed in the lung-tropic metastatic cells, and its high expression was significantly correlated with the late-stage TNBC and the shorter survival of the patients. Downregulation of SCEL expression significantly impaired the 3D colony-forming ability but not the migration and invasion ability of the lung colonization (LC) cells. Knockdown of SCEL reduced TNF-α-induced activation of the NF-κB/c-FLIP pro-survival and Akt/Erk1/2 growth signaling pathways in the LC cells. Specifically, knockdown of SCEL expression switched TNF-α-mediated cell survival to the caspase 3-dependent apoptosis. Conversely, ectopic expression of SCEL promoted TNF-α-induced activation of NF-κB/c-FLIP pro-survival and Akt/Erk1/2 pro-growth signaling pathway. The result of co-immunoprecipitation (Co-IP) and GST pull-down assay showed that SCEL could interact with TNFR1 to promote its protein stability. The xenograft mouse model experiments revealed that knockdown of SCEL resulted in increase of caspase-3 activity, and decrease of ki67 and TNFR1 expression as well as increase of tumor-associated macrophages in the metastatic lung lesions. Clinically, SCEL expression was found to be positively correlated with TNFR1 in TNBC tissues. Lastly, we showed that blocking TNF-α-mediated cell survival signaling by adalimumab effectively suppressed the lung colonization of the SCEL-positive, but not the SCEL-downregulated LC cells in the tail-vein injection model.
CONCLUSIONS: Our findings indicate that SCEL plays an essential role in the metastatic lung colonization of TNBC by promoting the TNF-α/TNFR1/NF-κB/c-FLIP survival and Akt/Erk1/2 proliferation signaling. Thus, SCEL may serve as a biomarker for adalimumab treatment of TNBC patients.
方法:基于相对和绝对定量(iTRAQ)的LC-MS/MS蛋白质组学方法的等量异位标签用于鉴定肺向转移性细胞中的新型膜相关蛋白。公共领域数据集用于评估候选蛋白的临床相关性。基于细胞的模型和小鼠模型用于通过iTRAQ鉴定的蛋白质分子Sciellin(SCEL)的生物化学和功能表征,以阐明其在促进TNBC细胞的肺定植中的作用和潜在机制。
结果:基于iTRAQ的LC-MS/MS蛋白质组学方法鉴定了一种在嗜肺转移细胞中过表达的膜相关蛋白SCEL,其高表达与晚期TNBC和患者生存期短显著相关。SCEL表达的下调显着损害了3D集落形成能力,但不损害肺定植(LC)细胞的迁移和侵袭能力。SCEL的敲低降低了TNF-α诱导的LC细胞中NF-κB/c-FLIP促存活和Akt/Erk1/2生长信号通路的激活。具体来说,SCEL表达的敲低将TNF-α介导的细胞存活转变为caspase3依赖性细胞凋亡。相反,SCEL的异位表达可促进TNF-α诱导的NF-κB/c-FLIP促存活和Akt/Erk1/2促生长信号通路的激活。免疫共沉淀(Co-IP)和GST下拉实验结果表明,SCEL可以与TNFR1相互作用以提高其蛋白稳定性。异种移植小鼠模型实验表明,SCEL敲低导致caspase-3活性增加,在转移性肺病变中ki67和TNFR1表达减少以及肿瘤相关巨噬细胞增加。临床上,在TNBC组织中发现SCEL表达与TNFR1呈正相关。最后,我们表明,通过阿达木单抗阻断TNF-α介导的细胞存活信号可有效抑制SCEL阳性的肺定植,而不是尾静脉注射模型中SCEL下调的LC细胞。
结论:我们的研究结果表明,SCEL通过促进TNF-α/TNFR1/NF-κB/c-FLIP的存活和Akt/Erk1/2信号的增殖在TNBC的转移性肺定植中起重要作用。因此,SCEL可作为阿达木单抗治疗TNBC患者的生物标志物。