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.

Figure 1

Eribulin treatment of TNBC breast cancer cells in vitro eribulin reverses EMT and induces MET. (A) Schematic representation of treatment scheme. (B) Representative images of TNBC cells following treatment with indicated dose of eribulin for 1 week. Images taken at × 10 magnification. (C–E) Expression levels of EMT/MET-related marker genes in eribulin-treated (C) MX-1, (D) MDA-MB-157, and (E) Hs578T TNBC cells as measured by qPCR. Gene expression levels were normalised to GAPDH expression. (F) Protein expression of E-cadherin, N-cadherin, and vimentin as assessed by immunoblot analysis. β-Actin was used as a loading control. (G) Quantification of protein levels of E-cadherin (upper), N-cadherin (middle), and vimentin (lower) from the immunoblot analysis of (G). Bars show mean±s.e.m. (n=3). *P<0.05 vs control group (Dunnett multiple comparison test).

Figure 2

Eribulin reverses EMT and induces MET in MX-1 breast cancer xenografts in vivo. (A) Schematic representation of treatment scheme. (B) Representative IHC images of E-cadherin (upper), N-cadherin (middle), and ZEB1 (lower) in tumour specimens from animals treated with 0.3, 1, and 3 mg kg⁻¹ eribulin. Images taken at × 100 magnification. (C) Quantification of IHC staining of the markers shown in (B). Data for individual tumours are presented as points, with means±s.e.m. of the group shown by lines (n=10). ***P<0.001, ****P<0.0001 vs control group (Dunnett-type multiple comparison test).

Figure 3

Eribulin downregulates TGF-β/Smad pathway. (A) Schematic representation of TGF-β and eribulin treatment schedules for studies shown in (B–E). (B) Representative day 8 images of MCF10A cells following 7 days treatment with TGF-β (10 ng ml⁻¹). Images taken at × 10 magnification. (C) Expression levels of EMT-related markers in MCF10A cells treated with TGF-β (10 ng ml⁻¹) at day 8 as measured by qPCR. Gene expression levels were normalised to GAPDH expression. (D) Representative day 15 images of MCF10A cells preinduced to the EMT phenotype by TGF-β pretreatment, followed by additional 7 days treatment with eribulin. Images taken at × 10 magnification. (E) Expression levels of EMT-related markers in the same eribulin-treated MCF10A cells as in (D), as measured by qPCR. (F) Immunoblot analysis of phosphorylated Smad2, phosphorylated Smad3, and total Smad2/3 in MCF10A cells pretreated with 0.25 nM eribulin for 1 day followed by TGF-β treatment for 1 h. β-Actin was used as a loading control. * indicates non-specific band.

Figure 4

Eribulin treatment of MX-1 breast cancer cells in vitro decreases migration and invasiveness capacities. (A) Schematic representation of treatment scheme. (B) Inhibitory effects of eribulin or 5-FU on migration of MX-1 cells, expressed as percent changes in eribulin or 5-FU-treated cells compared with migration seen with untreated control cells. Data represent means±s.e.m. from three independent experiments. *P<0.05 vs vehicle control (Dunnett multiple comparison test). ^#P<0.05 vs 5-FU 10 μM (the Dunnett multiple comparison test except control). (C) Representative fields in the migration assay ( × 20 magnification). (D) Inhibitory effects of eribulin or 5-FU on invasion of MX-1 cells, expressed as percent changes in eribulin or 5-FU-treated cells compared with invasion seen with untreated control cells. Data represent means±s.e.m. from three independent experiments. *P<0.05 vs vehicle control (Dunnett multiple comparison test). ^#P<0.05 vs 5-FU 10 μM (the Dunnett multiple comparison test except control). (E) Representative fields in the invasion assay ( × 20 magnification).

Figure 5

Eribulin treatment reduces metastasis and increases survival in MX-1 in vivo experimental metastasis model. (A) Numbers of lung nodules at day 15 after tail vein injection of MX-1 cells that had been pretreated with eribulin, 5-FU, or DMSO (n=5–7). *P<0.01 by Kruskal–Wallis Dunn's multiple comparison test. (B) Representative lung images ( × 20 magnification) at day 15 after cell injection. (C) Survival of animals in the experimental metastasis assay (n=5–7).