Twist-mediated Epithelial-mesenchymal Transition Promotes Breast Tumor Cell Invasion via Inhibition of Hippo Pathway

Abstract

Twist is a key transcription factor for Epithelial-mesenchymal transition (EMT), which is a cellular de-differentiation program that promotes invasion and metastasis, confers tumor cells with cancer stem cell (CSC)-like characteristics, and increases therapeutic resistance. However, the mechanisms that facilitate the functions of Twist remain unclear. Here we report that Twist overexpression increased expression of PAR1, an upstream regulator of the Hippo pathway; PAR1 promotes invasion, migration, and CSC-like properties in breast cancer by activating the transcriptional co-activator TAZ. Our study indicates that Hippo pathway inhibition is required for the increased migratory and invasiveness ability of breast cancer cells in Twist-mediated EMT.

Figure 1. Overexpression of Snail or Twist induces EMT in HMLE cells.

(A) Representative images show expression of E-cadherin and Vimentin in Snail- or Twist-expressing HMLE cells analyzed by immunofluorescent staining. Nuclei were visualized with DAPI staining (red). The morphologic changes associated with EMT are shown in the representative phase contrast images. Scale bars, 50 μm. (B) Expression of E-cadherin, N-cadherin and Vimentin in these cells was assessed by western blot analysis; actin served as a loading control.

Figure 2. Overexpression of Snail or Twist induces EMT in T47D and MCF7 cells.

(A) Representative images show expression of E-cadherin and ERα in Snail- or Twist-expressing T47D and MCF7 cells analyzed by immunofluorescent staining. Nuclei were visualized with DAPI staining (blue). The morphologic changes associated with EMT are shown in representative phase contrast images. Scale bars, 50 μm. (B) Expression of E-cadherin, N-cadherin and ERα in these cells was assessed by western blot analysis; actin served as a loading control. (C) Quantification of the relative mRNA levels of E-cadherin and ERα in Twist expressing T47D cells compared with vector-control cells using real-time PCR. Presented data are the mean ± SD from three separate experiments, with *and **indicate p < 0.01 in comparison with that of control.

Figure 3. Overexpression of Twist induces CSC-like properties in T47D cells.

(A) Graphic representation of cell growth rates by T47D cells stably expressing Twist or control vector. Cell counts were obtained daily over a 4 day period. Presented data are the mean ± SD from two independent experiments with triplicate samples. NS stands for statistically non-significant. (B) Tumorsphere formation was assessed in T47D cells overexpressing Twist under normoxic or hypoxic conditions. Representative images of tumorspheres are shown in the right panel. Scale bars, 100 μm. Left panel, are graphic representations of tumorsphere number. Presented data are the percentage of control vector values, with mean ± SD of three separate experiments performed in duplicate. ^#p <  0.05 and *p <  0.01 when vector control cells compared with their Twist-expressing clones, respectively.

Figure 4. Overexpression of Twist enhances cell migration and invasion of T47D cells.

(A) Graphic representation of the migratory capability of stably transfected T47D cells expressing either Twist or control vector assessed using a wound healing assay. A scratch (“wound”) was inflicted to a cell layer produced 48 hours post-plating, and culture continued for an additional 24 hrs. Wound closures were photographed at 0 and 24 hr. Presented data are the mean ± SD from three independent experiments, with *indicates p < 0.01 when comparing with control values. A representative experiment is shown in the right panel. Scale bars, 50 μm. (B) Graphic representation of the invasiveness of T47D cells stably expressing Twist or control vector using a modified Boyden Chamber invasion assay as described in the Materials and Methods. Presented data are the mean ± SD from three separate experiments, with *indicates p < 0.01 when comparing with control values. A representative experiment is shown in the right panel. Scale bars, 100 μm.

Figure 5. Overexpression of Twist induces the activation of PAR1 signaling.

(A) Graphic representation of the fold change in mRNA levels of Twist, F2R and F2RL2 in Twist-expressing T47D cells compared with control vector cells by real-time PCR. Presented data are the mean ± SD of three separate experiments, with *indicates p < 0.01 when comparing with control values. (B) Western blot analysis for p-TAZ, TAZ and CTGF expression in EMT-induced Twist-expressing T47D cells or T47D cells expressing control vector. Actin served as a loading control. (C) Effect of TAZ siRNA or NTC siRNA on CTGF promoter luciferase activity in Twist-overexpressing T47D cells and T47D cells expressing control vector. Assessments were made after 48 hours in culture. Presented data are mean ± SD of normalized luciferase activities determined from three separate experiments. *indicates p < 0.01 when control siRNA expressed in Twist-T47D cells compared with in vector control cells; and **indicates p < 0.01 when compared expression of TAZ siRNA and control siRNA in Twist-T47D cells. (D) Effect of TAZ siRNA on TAZ and Twist expression in EMT-induced Twist-expressing T47D cells and in T47D cells expressing control vector by western blot analysis. Actin served as a loading control.

Figure 6. Knockdown of TAZ suppresses Twist-induced cell migration and invasion of T47D cells.

(A) Effect of TAZ siRNA on cell migrating activity in Twist-overexpressing T47D cells and T47D cells expressing control vector using a wound healing assay. A scratch (“wound”) was inflicted to a cell layer produced 48 hours post-plating, and culture continued for an additional 24 hrs. Wound closures were photographed at 0 and 24 hr. Presented data are the mean ± SD from three independent experiments, with *and ^#indicating significant difference of p < 0.05 from control values. (B) Effect of TAZ siRNA on cell invasiveness in Twist-overexpressing T47D cells and T47D cells expressing control vector using a modified Boyden Chamber invasion assay as described in the Materials and Methods. Presented data are a graphic representation of the mean ± SD of percentage of invasive cells obtained from three separate experiments, with *and ^#indicating significant difference of p < 0.05 from control values. (C) Expression of Twist results in increased expression of PAR1, which promotes invasion, migration, and induces CSC-like properties in breast cancer cells by upregulating the expression of TAZ.