Background:The epithelial-to-mesenchymal transition (EMT) is a common feature in early cancer invasion. Increased vimentin is a canonical marker of the EMT; however, the role of vimentin in EMT remains unknown.Methods:To clarify this, we induced EMT in lung cancer cells with TGF-β1, followed by treatment with the vimentin-targeting drug ALD-R491, live-cell imaging, and quantitative proteomics.Results:We identified 838 proteins in the intermediate filament fraction of cells. TGF-β1 treatment increased the proportion of vimentin in this fraction and the levels of 24 proteins. Variants of fibronectin showed the most pronounced increase (137-fold), followed by regulators of the cytoskeleton, cell motility, and division, such as the mRNA-splicing protein SON. TGF-β1 increased cell spreading and cell migration speed, and changed a positive correlation between cell migration speed and persistence to negative. ALD-R491 reversed these mesenchymal phenotypes to epithelial and the binding of RNA-binding proteins, including SON.Conclusions:These findings present many new interactors of intermediate filaments, describe how EMT and vimentin filament dynamics influence the intermediate filament interactome, and present ALD-R491 as a possible EMT-inhibitor. The observations support the hypothesis that the dynamic turnover of vimentin filaments and their interacting proteins govern mesenchymal cell migration, EMT, cell invasion, and cancer metastasis.
背景：上皮-间质转化（EMT）是癌症早期侵袭的常见特征。波形蛋白表达增加是EMT的经典标志物，但波形蛋白在EMT中的作用机制尚未明确。 方法：为阐明该机制，本研究采用TGF-β1诱导肺癌细胞发生EMT，随后使用靶向波形蛋白药物ALD-R491进行处理，结合活细胞成像技术与定量蛋白质组学分析。 结果：我们在细胞中间丝组分中鉴定出838种蛋白质。TGF-β1处理显著提高了该组分中波形蛋白的比例及24种蛋白的表达水平。其中纤连蛋白变体增幅最为显著（达137倍），其次是细胞骨架调节因子、细胞运动与分裂相关蛋白（如mRNA剪接蛋白SON）。TGF-β1处理促进了细胞铺展并提高细胞迁移速度，同时将细胞迁移速度与持续性之间的正相关关系转变为负相关。ALD-R491能逆转这些间质表型向上皮表型转化，并影响包括SON在内的RNA结合蛋白的结合状态。 结论：本研究揭示了中间丝的大量新型相互作用蛋白，阐明了EMT与波形蛋白丝动态变化如何影响中间丝相互作用组，并提出ALD-R491可能作为EMT抑制剂。这些发现支持以下假说：波形蛋白丝及其相互作用蛋白的动态更替调控着间质细胞的迁移、EMT进程、细胞侵袭及癌症转移。
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