Inhibition of skeletal metastasis by ectopic ERalpha expression in ERalpha-negative human breast cancer cell lines

Abstract

Some hormone-independent breast cancers lack functional estrogen receptors (ERs) and show evidence of a more aggressive metastatic phenotype. A protective role of the ER has also been suggested in hormone-resistant breast cancer progression. In this study, we have investigated the effect of the ectopic expression of human ERalpha on the bone-metastatic potential of highly metastatic ERalpha-negative human breast cancer MDA-MB-231 and MDA-MB-435-F-L cell lines in an experimental model of bone metastasis in nude mice. ERalpha overexpression had no effect on the growth of both cell lines but reduced the expression of integrin alpha(v)beta(3) and the receptor activator of NF-kappaB, which are known to promote bone metastasis. A significant reduction in the incidence of osteolytic bone metastasis was observed by X-ray imaging of the legs and arms of mice inoculated with ERalpha-expressing clones of MDA-MB-231 cells in comparison to controls. Ectopic expression of ERalpha in MDA-MB-435-F-L cells also reduced their widespread skeletal metastasis to the legs, arms, spine, and mandible, as detected by whole-mouse enhanced green fluorescent protein imaging. Our study indicates for the first time that stable reintroduction of functional ERalpha in ERalpha-negative human breast cancer cells can inhibit their aggressive bone-metastatic potential in an experimental bone metastasis model.

Figure 1

Ectopic expression of functional ERα in human breast cancer cells. (A) MDA-MB-231 and MDA-MB-435-F-L cells were transfected with either a retroviral ERα expression vector or an empty vector, which carried a puromycin-resistant gene. Puromycin-resistant clones were selected, and the expression of ERα was determined by Western blot analysis with cell lysates from empty vector-transfected controls (Con) and ERα clones of the MDA-MB-231 and MDA-MB-435-F-L cell lines. In a separate Western blot analysis, the ERα level in MDA-MB-231 Cl.15 was compared with that in ERα-positive human breast cancer MCF-7 cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were measured in cell lysates to show equal sample loading. (B and C) To confirm the expression of a functional ERα in ERα clones of MDA-MB-231 (B) and MDA-MB-435-F-L (C) cells, the control cells, ERα clones and MCF-7 cells, were transiently cotransfected with an estrogen-responsive promoter luciferase plasmid (ERE-thymidine kinase-Luciferase) and a β-gal expression plasmid. Cells were treated with 10^-7 M E₂. Luciferase and β-gal activities in cell lysates were determined. Luciferase activity normalized with β-gal activity is presented as the mean ± SEM for each treatment from triplicate transfections. *Significant difference (P < .05) from the corresponding control without E₂ treatment, by Student's t test. (D) To determine the effect of ERα expression on the growth of MDA-MB-231 and MDA-MB-435-F-L cells, the controls and two ERα clones were plated in a 96-well plate, and MTT assay was performed to obtain the relative cell number. Each point represents the mean ± SEM from four wells. The means of the three cells at each time point were not significantly different by ANOVA.

Figure 2

Inhibition of bone invasion and osteolytic metastasis by the ectopic expression of ERα. Control and ERα cells of MDA-MB-231 and MDA-MB-435-F-L were inoculated through the left cardiac ventricle of female nude mice at 0.1 x 10⁶ cells/mouse. Osteolytic bone metastasis induced by MDA-MB-231 cells was detected with X-ray radiographs, as indicated with an arrow in (A). At the termination of the experiment, the femora and tibiae were fixed in buffered formalin and decalcified. Paraffin-embedded sections were stained with hematoxylin, eosin, orange G, and phloxine. Representative pictures of the stained tibial sections of MDA-MB-231 (B) and MDA-MB-435-F-L (C) cell-inoculated mice are presented.

Figure 3

Inhibition of integrin α_vβ₃ and RANK protein expression in ERα-transfected clones. Western blot analyses were performed with cell lysates from the MDA-MB-435-F-L cells and their respective ERα clones for integrin α_v, integrin, β₃ and RANK protein expression, as shown in (A), and from MDA-MB-231 cells and their respective ERα clones for RANK protein expression, as shown in (B). The density of each band was quantified with Image-Pro (MediaCybernetics, Silver Spring, MD) and divided by that of the corresponding actin band. The ratio is presented under each blot after normalizing the values for controls as one unit.