title: Eribulin mesilate suppresses experimental metastasis of breast cancer cells by reversing phenotype from epithelial-mesenchymal transition (EMT) to mesenchymal-epithelial transition (MET) states.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Discovery Biology, Oncology Product Creation Unit, Eisai Co., Ltd, Tsukuba, Ibaraki 300-2635, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Yoshida
authors.affiliation: Discovery Biology, Oncology Product Creation Unit, Eisai Co., Ltd, Tsukuba, Ibaraki 300-2635, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Ozawa
authors.affiliation: Biomarkers and Personalized Medicine, Core Function Unit, Eisai Co., Ltd, Tsukuba, Ibaraki 300-2635, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Kimura
authors.affiliation: Biomarkers and Personalized Medicine, Core Function Unit, Eisai Co., Ltd, Tsukuba, Ibaraki 300-2635, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Sato
authors.affiliation: Biomarkers and Personalized Medicine, Core Function Unit, Eisai Inc., Andover, MA 01810, USA.
authors.forename: G
authors.initials: G
authors.lastname: Kuznetsov
authors.affiliation: Biomarkers and Personalized Medicine, Core Function Unit, Eisai Inc., Andover, MA 01810, USA.
authors.forename: S
authors.initials: S
authors.lastname: Xu
authors.affiliation: Biomarkers and Personalized Medicine, Core Function Unit, Eisai Co., Ltd, Tsukuba, Ibaraki 300-2635, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Uesugi
authors.affiliation: Biomarkers and Personalized Medicine, Core Function Unit, Eisai Inc., Andover, MA 01810, USA.
authors.forename: S
authors.initials: S
authors.lastname: Agoulnik
authors.affiliation: Biomarkers and Personalized Medicine, Core Function Unit, Eisai Inc., Andover, MA 01810, USA.
authors.forename: N
authors.initials: N
authors.lastname: Taylor
authors.affiliation: Biomarkers and Personalized Medicine, Core Function Unit, Eisai Inc., Andover, MA 01810, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Funahashi
authors.affiliation: Discovery Biology, Oncology Product Creation Unit, Eisai Co., Ltd, Tsukuba, Ibaraki 300-2635, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Matsui
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D018572:Disease-Free Survival; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D005260:Female; D005663:Furans / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D007659:Ketones / Q000494:pharmacology*; D008175:Lung Neoplasms / Q000188:drug therapy / Q000556:secondary; D051379:Mice; D008819:Mice, Nude; D009362:Neoplasm Metastasis; D010641:Phenotype; D064726:Triple Negative Breast Neoplasms / Q000188:drug therapy* / Q000473:pathology; D023041:Xenograft Model Antitumor Assays; D000095662:Polyether Polyketides
chemical_list: D005663:Furans; D007659:Ketones; C490954:eribulin; D000095662:Polyether Polyketides