Eribulin mesilate suppresses experimental metastasis of breast cancer cells by reversing phenotype from epithelial-mesenchymal transition (EMT) to mesenchymal-epithelial transition (MET) states.
Eribulin mesilate suppresses experimental metastasis of breast cancer cells by reversing phenotype from epithelial-mesenchymal transition (EMT) to mesenchymal-epithelial transition (MET) states.
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DOI：
10.1038/bjc.2014.80
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发表时间：
2014-03-18
影响因子：
8.8
通讯作者：
Matsui, J.
中科院分区：
文献类型：
--
作者：
Yoshida, T.;Ozawa, Y.;Kimura, T.;Sato, Y.;Kuznetsov, G.;Xu, S.;Uesugi, M.;Agoulnik, S.;Taylor, N.;Funahashi, Y.;Matsui, J.
甲磺酸埃立布林（Eribulin mesilate）是一种非紫杉烷微管动力学抑制剂，与临床晚期转移性乳腺癌患者的无进展生存期相比，它显示出更高的总生存期（OS）的趋势。这一发现表明，除了其已确定的抗有丝分裂活性外，伊瑞布林可能还有其他先前未被认识到的抗肿瘤机制。为了研究这种可能性，我们研究了伊瑞布林对人乳腺癌细胞上皮-间充质转化（EMT）和间充质-上皮转化（MET）平衡的影响。雌激素受体(ER−)/孕激素受体(PR−)/人上皮生长受体2 （HER2−）三阴性乳腺癌（TNBC）细胞为间充质表型，用艾瑞布林处理7天，然后分别用实时荧光定量PCR （qPCR）和免疫印迹法检测存活细胞中emt相关基因和蛋白的表达变化。此外，还对经布林处理的细胞进行了增殖、迁移和侵袭试验。为了研究瑞布林对TGF-β/Smad信号传导的影响，我们分析了Smad蛋白的磷酸化状态。在体内，采用qPCR、免疫印迹和免疫组织化学分析方法检测经伊瑞布林处理的小鼠TNBC异种移植物的EMT/MET状态。最后，研究人员利用实验性肺转移模型，在体内环境中测量经艾瑞伯林治疗的TNBC的转移活性。在体外用伊瑞bulin处理TNBC细胞导致了从间质表型向上皮表型转变的形态学变化。EMT标志物的表达分析显示，艾瑞布林处理导致几个间充质标记基因的表达降低，几个上皮标记基因的表达增加。在TGF-β诱导的EMT模型中，艾瑞布林治疗逆转EMT，与抑制Smad2和Smad3磷酸化一致。与这些变化一致的是，经伊瑞布林处理7天的TNBC细胞显示出体外迁移和侵袭能力下降。在体内异种移植物模型中，通过qPCR、免疫印迹和上皮和间充质标记蛋白的免疫组织化学分析评估，艾瑞布林治疗逆转了EMT并诱导了MET。最后，在体内实验转移模型中评估，用艾瑞布林体外预处理7天的TNBC存活细胞导致肺转移数量减少。在体外和体内实验中，艾力布林对人乳腺癌细胞中EMT/ met相关通路组分均有显著影响，这与从间质状态到上皮状态的表型转换相一致，并与在体外观察到的迁移和侵袭性降低以及体内实验转移相对应。这些临床前研究结果可能为临床观察通过抑制乳腺癌患者转移的进一步扩散而延长生存期提供了合理的科学依据。
Eribulin mesilate (eribulin), a non-taxane microtubule dynamics inhibitor, has shown trends towards greater overall survival (OS) compared with progression-free survival in late-stage metastatic breast cancer patients in the clinic. This finding suggests that eribulin may have additional, previously unrecognised antitumour mechanisms beyond its established antimitotic activity. To investigate this possibility, eribulin's effects on the balance between epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) in human breast cancer cells were investigated. Triple negative breast cancer (TNBC) cells, which are oestrogen receptor (ER−)/progesterone receptor (PR−)/human epithelial growth receptor 2 (HER2−) and have a mesenchymal phenotype, were treated with eribulin for 7 days, followed by measurement of EMT-related gene and protein expression changes in the surviving cells by quantitative real-time PCR (qPCR) and immunoblot, respectively. In addition, proliferation, migration, and invasion assays were also conducted in eribulin-treated cells. To investigate the effects of eribulin on TGF-β/Smad signalling, the phosphorylation status of Smad proteins was analysed. In vivo, the EMT/MET status of TNBC xenografts in mice treated with eribulin was examined by qPCR, immunoblot, and immunohistochemical analysis. Finally, an experimental lung metastasis model was utilised to gauge the metastatic activity of eribulin-treated TNBC in the in vivo setting. Treatment of TNBC cells with eribulin in vitro led to morphological changes consistent with transition from a mesenchymal to an epithelial phenotype. Expression analyses of EMT markers showed that eribulin treatment led to decreased expression of several mesenchymal marker genes, together with increased expression of several epithelial markers. In the TGF-β induced EMT model, eribulin treatment reversed EMT, coincident with inhibition of Smad2 and Smad3 phosphorylation. Consistent with these changes, TNBC cells treated with eribulin for 7 days showed decreased capacity for in vitro migration and invasiveness. In in vivo xenograft models, eribulin treatment reversed EMT and induced MET as assessed by qPCR, immunoblot, and immunohistochemical analyses of epithelial and mesenchymal marker proteins. Finally, surviving TNBC cells pretreated in vitro with eribulin for 7 days led to decreased numbers of lung metastasis when assessed in an in vivo experimental metastasis model. Eribulin exerted significant effects on EMT/MET-related pathway components in human breast cancer cells in vitro and in vivo, consistent with a phenotypic switch from mesenchymal to epithelial states, and corresponding to observed decreases in migration and invasiveness in vitro as well as experimental metastasis in vivo. These preclinical findings may provide a plausible scientific basis for clinical observations of prolonged OS by suppression of further spread of metastasis in breast cancer patients treated with eribulin.
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