Effects of Estetrol on Migration and Invasion in T47-D Breast Cancer Cells through the Actin Cytoskeleton

Abstract

Estetrol (E4) is a natural human estrogen present at high concentrations during pregnancy. Due to its high oral bioavailability and long plasma half-life, E4 is particularly suitable for therapeutic applications. E4 acts as a selective estrogen receptor (ER) modulator, exerting estrogenic actions on the endometrium or the central nervous system, while antagonizing the actions of estradiol in the breast. We tested the effects of E4 on its own or in the presence of 17β-estradiol (E2) on T47-D ER+ breast cancer cell migration and invasion of three-dimensional matrices. E4 administration to T47-D cells weakly stimulated migration and invasion. However, E4 decreased the extent of movement and invasion induced by E2. Breast cancer cell movement requires a remodeling of the actin cytoskeleton. During exposure to E4, a weak, concentration-dependent, re-distribution of actin fibers toward the cell membrane was observed. However, when E4 was added to E2, an inhibition of actin remodeling induced by E2 was seen. Estrogens stimulate ER+ breast cancer cell movement through the ezrin-radixin-moesin family of actin regulatory proteins, inducing actin and cell membrane remodeling. E4 was a weak inducer of moesin phosphorylation on Thr(558), which accounts for its functional activation. In co-treatment with E2, E4 blocked the activation of this actin controller in a concentration-related fashion. These effects were obtained through recruitment of estrogen receptor-α. In conclusion, E4 acted as a weak estrogen on breast cancer cell cytoskeleton remodeling and movement. However, when E2 was present, E4 counteracted the stimulatory actions of E2. This contributes to the emerging hypothesis that E4 may be a naturally occurring ER modulator in the breast.

Keywords: actin cytoskeleton; breast cancer; cancer progression; estetrol; estrogen.

Figure 1

Estetrol regulates T47-D cell horizontal migration. Steroid-deprived, growth synchronized ER+ T47-D cells (A,B) or human umbilical vein endothelial cells (HUVEC) (C,D) were exposed to 10⁻⁹ M E2 or to increasing concentrations of E4, or to the combination of the steroids. Cells were scraped from the culture dish and the number of cells migrated over the start line was assayed after 48 h. (A,C) Representative images from triplicate experiments are shown. (B,D) Graphical representations of the mean number of migrated cells ± SD are provided. Quantification was obtained by pooling the results from three separate experiments. In each experiments, 10 random microscopical fields were selected and the number of migrated cells was assessed. *p < 0.05 vs. control. **p < 0.05 vs. E2.

Figure 2

Estetrol regulates ER+ breast cancer cell invasion of three-dimensional matrices. Steroid-deprived, growth synchronized ER+ T47-D cells were exposed to 10⁻⁹ M E2 or to increasing concentrations of E4, or to the combination of the steroids. Breast cancer cell invasion through matrigel was assayed by using invasion chambers. Invading cells were counted in the central field of triplicate membranes. (A) Representative images in chambers with matrigel are shown. (B) A graphical representation of the invasion indexes in the different conditions is provided. Quantification was obtained by pooling the results from three separate experiments. In each experiments, 10 random microscopical fields were selected and the number of invading cells counted. Invasion indexes were calculated as detailed in the Section “Materials and Methods.” *p < 0.05 vs. control. **p < 0.05 vs. E2.

Figure 3

Estetrol modulates the remodeling of the actin cytoskeleton and of cell membrane in T47-D breast cancer cells. T47-D cells were treated with E2 (10⁻⁹ M) or with different concentrations of E4, or with a combination of the two steroids. Actin fibers were stained with phalloidin linked to Texas Red (red labeling) and nuclei were counterstained with DAPI (blue labeling). Immunofluorescent analysis reveals the dynamic modifications of actin fibers through the time-course and the formation of specialized cell membrane structures. Images are representative of triplicate experiments. The box on top of the cells display sample areas of measurement (one per cell, indicated as the yellow line), showing the intensity of the signal throughout the measure. The results in the table are derived from the sampling of five areas of the cell membrane from 50 different random cells. The areas of minimum and maximum cell membrane thickness were always included. The results are the mean ± SD of the measurements. The graph plots the membrane/cytosol actin intensity ratio. *p < 0.05 vs. control. **p < 0.05 vs. E2.

Figure 4

Estetrol modulates the activation of the actin-binding protein, moesin. T47-D ER+ breast cancer cells were treated for 10 min with different amounts of E4, E2, or the combination (A,B). Other cells were treated with E4 (10⁻⁷ M) for different times (C). Lastly, T47-D cells were treated with E4 (10⁻⁷ M) in the presence or absence of the pure ER antagonist ICI 182,780 (D) or of siRNAs toward ERβ (E). Protein extracts were assayed with western analysis for their overall content of wild type moesin (moesin), Thr⁵⁵⁸-phosphorylated moesin (P-moesin), and ERβ. Images are representative of triplicate experiments. The bar graphs show the mean ± SD WB band intensity out of the three independent experiments. *p < 0.05 vs. control. **p < 0.05 vs. E2.