Palma Camila de Souza, Grassi Mariana Lopes, Thomé Carolina Hassibe, Ferreira Germano Aguiar, Albuquerque Daniele, Pinto Mariana Tomazini, Ferreira Melo Fernanda Ursoli, Kashima Simone, Covas Dimas Tadeu, Pitteri Sharon J, Faça Vitor M
From the Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP/Brazil; the Cell-based Therapy Center, Ribeirao Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP/Brazil; and.
the Cell-based Therapy Center, Ribeirao Preto Blood Center, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP/Brazil; and.
Mol Cell Proteomics. 2016 Mar;15(3):906-17. doi: 10.1074/mcp.M115.052910. Epub 2016 Jan 13.
Epithelial to mesenchymal transition (EMT)(1) occurs naturally during embryogenesis, tissue repair, cancer progression, and metastasis. EMT induces cellular and microenvironmental changes resulting in loss of epithelial and acquisition of mesenchymal phenotypes, which promotes cellular invasive and migratory capabilities. EMT can be triggered by extracellular factors, including TGF-β, HGF, and EGF. Overexpression of transcription factors, such as SNAIL, SLUG, ZEB1/2, and TWIST1, also induces EMT and is correlated to cancer aggressiveness. Here, the breast adenocarcinoma cell line MCF7 was transduced with SNAIL to identify specific mechanisms controlled by this transcription factor during EMT. Overexpression of SNAIL led to EMT, which was thoroughly validated by molecular, morphological, and functional experiments. Subcellular proteome enrichment followed by GEL-LC-MS/MS was performed to provide extensive protein fractionation and in-depth proteomic analysis. Quantitative analysis relied on a SILAC strategy, using the invasive breast cancer cell line MDA-MB-231 as a reference for quantitation. Subsets of proteins enriched in each subcellular compartment led to a complementary list of 4289 proteins identified with high confidence. A subset of differentially expressed proteins was validated by Western blot, including regulation in specific cellular compartments, potentially caused by protein translocation. Protein network analysis highlighted complexes involved in cell cycle control and epigenetic regulation. Flow cytometry analysis indicated that SNAIL overexpression led to cell cycle arrest in G0/G1 phases. Furthermore, down-regulation of HDAC1 was observed, supporting the involvement of epigenetic processes in SNAIL-induced EMT. When HDAC1 activity was inhibited, MCF7 not only apparently initiated EMT but also up-regulated SNAIL, indicating the cross-talk between these two proteins. Both HDAC1 inhibition and SNAIL overexpression activated the AKT pathway. These molecular mechanisms appear to be essential to EMT and therefore for cancer metastasis. Specific control of such epigenetic processes might then represent effective approaches for clinical management of metastatic cancer.
上皮-间质转化（EMT）（1）在胚胎发育、组织修复、癌症进展和转移过程中自然发生。EMT诱导细胞和微环境变化，导致上皮表型丧失和间质表型获得，从而促进细胞的侵袭和迁移能力。EMT可由细胞外因子触发，包括转化生长因子-β（TGF-β）、肝细胞生长因子（HGF）和表皮生长因子（EGF）。转录因子如SNAIL、SLUG、ZEB1/2和TWIST1的过表达也可诱导EMT，并与癌症侵袭性相关。在此，用SNAIL转导乳腺癌细胞系MCF7，以确定该转录因子在EMT过程中控制的特定机制。SNAIL的过表达导致EMT，这通过分子、形态学和功能实验得到了充分验证。进行亚细胞蛋白质组富集，随后进行凝胶液相色谱-串联质谱（GEL-LC-MS/MS）分析，以提供广泛的蛋白质分级分离和深入的蛋白质组学分析。定量分析依赖于稳定同位素标记氨基酸细胞培养（SILAC）策略，使用侵袭性乳腺癌细胞系MDA-MB-231作为定量参考。在每个亚细胞区室中富集的蛋白质子集产生了一份高可信度鉴定的4289种蛋白质的补充清单。通过蛋白质印迹法验证了差异表达蛋白质的一个子集，包括特定细胞区室中的调节，这可能是由蛋白质易位引起的。蛋白质网络分析突出了参与细胞周期控制和表观遗传调控的复合物。流式细胞术分析表明，SNAIL的过表达导致细胞周期停滞在G0/G1期。此外，观察到组蛋白去乙酰化酶1（HDAC1）的下调，支持表观遗传过程参与SNAIL诱导的EMT。当HDAC1活性受到抑制时，MCF7不仅明显启动了EMT，而且上调了SNAIL，表明这两种蛋白质之间存在相互作用。HDAC1抑制和SNAIL过表达均激活了AKT信号通路。这些分子机制似乎对EMT至关重要，因此对癌症转移也至关重要。对这种表观遗传过程的特异性控制可能代表了转移性癌症临床管理的有效方法。
