Alternate estrogen receptors promote invasion of inflammatory breast cancer cells via non-genomic signaling

Abstract

Although Inflammatory Breast Cancer (IBC) is a rare and an aggressive type of locally advanced breast cancer with a generally worst prognosis, little work has been done in identifying the status of non-genomic signaling in the invasiveness of IBC. The present study was performed to explore the status of non-genomic signaling as affected by various estrogenic and anti-estrogenic agents in IBC cell lines SUM149 and SUM190. We have identified the presence of estrogen receptor α (ERα) variant, ERα36 in SUM149 and SUM190 cells. This variant as well as ERβ was present in a substantial concentration in IBC cells. The treatment with estradiol (E2), anti-estrogenic agents 4-hydroxytamoxifen and ICI 182780, ERβ specific ligand DPN and GPR30 agonist G1 led to a rapid activation of p-ERK1/2, suggesting the involvement of ERα36, ERβ and GPR30 in the non-genomic signaling pathway in these cells. We also found a substantial increase in the cell migration and invasiveness of SUM149 cells upon the treatment with these ligands. Both basal and ligand-induced migration and invasiveness of SUM149 cells were drastically reduced in the presence of MEK inhibitor U0126, implicating that the phosphorylation of ERK1/2 by MEK is involved in the observed motility and invasiveness of IBC cells. We also provide evidence for the upregulation of p-ERK1/2 through immunostaining in IBC patient samples. These findings suggest a role of non-genomic signaling through the activation of p-ERK1/2 in the hormonal dependence of IBC by a combination of estrogen receptors. These findings only explain the failure of traditional anti-estrogen therapies in ER-positive IBC which induces the non-genomic signaling, but also opens newer avenues for design of modified therapies targeting these estrogen receptors.

Figure 1. Estrogen receptor expression profiling in IBC cell lines SUM149 and SUM190.

(A) Western blot analysis of ERα36, ERβ and GPR30 (B) Immunofluorescent localization of ERα36, ERβ and GPR30. Estrogen receptors (green) and DNA (blue) in these cells.

Figure 2. Estrogen receptor ligands induced ERK1/2 phosphorylation in SUM149 and SUM190 cells.

(A–C) The cells were maintained in 5% DCC for 48 hours and then treated with E2 (10 nM), DPN (10 nM) or G1 (10 nM) for the indicated times and then ERK1/2 phosphorylation was analyzed by western blotting. The expression level of total ERK was analyzed for normalization. Error bars indicate standard deviation.

Figure 3. Effect of traditional antiestrogen treatment in SUM149 and SUM190 cells.

(A) Effect of 4-hyroxytamoxifen (4OHT) and ICI 182780 (ICI) on E2-induced ERK1/2 phosphorylation. The cells were maintained in 5% DCC for 48 hours and then pretreated with or without ICI (100 nM) or 4OHT (100 nM) for1 hour before the treatment with or without E2 (10 nM). ERK1/2 phosphorylation was analyzed by western blotting. (B) Effect of dose dependent treatment of E2, 4OHT and DPN in SUM149 cells. The cells were maintained in 5% DCC for 48 hrs and then E2, 4OHT or DPN were treated at three concentrations (1, 10, 100 nM) of for 5 min. Error bars indicate standard deviation.

Figure 4. Effect of MEK inhibitor U0126 on p-ERK1/2 activation and cytoskeletal remodeling induced by estrogen receptor ligands and antiestrogens.

(A) Blocking of E2, DPN, 4OHT or G1 induced ERK1/2 phosphorylation in SUM149 cells by U0126. The cells were maintained in 5% DCC for 48 hours and then pretreated with or without U0126 (20 µM) for 15 min before treatment with or without E2 (10 nM), DPN (10 nM), 4OHT (10 nM) and G1 (10 nM) for 5 min. ERK1/2 phosphorylation was analyzed by western blotting. (B) Blocking of E2-induced cytoskeletal change in SUM149 cells by U0126. The cells were maintained for 48 hours in 5% DCC and then pretreated with U0126 (20 µM) before the treatment with or without E2 (10 nM) for 5 min. The cells were fixed and labeled with fluorescently conjugated phalloidin (for filamentous actin, red) and DAPI (for DNA). White-dotted areas in upper panels were magnified in lower panels.

Figure 5. E2-induced SUM149 cell migration and invasion.

(A) For migration, the cells (2 × 10⁴ cells) were added on the lower side of a Boyden chamber and incubated for 14 hours. Then the cells that passed through the filters were fixed, stained, and counted (left). For invasion, the cells (1 × 10⁵ cells) were added to the lower side of a matrigel coated Boyden chamber and incubated for 20 hours. Then the cells that invaded through the matrigel were fixed stained and counted (right). (B) The E2-induced migration and invasion was blocked by treatment with U0126. Error bars indicate standard deviation.

Figure 6. p-ERK1/2 expression of in IBC tissues.

(A) The expression of p-ERK1/2 was tested in tumor tissue samples from patients with IBC using the rabbit anti-p-ERK1/2 antibody. (B) Proposed working model for the stimulation of estrogen receptor ligand-induced non-genomic signaling pathway via p-ERK1/2 activation in IBC cells. Estrogen receptor ligand rapidly activates p-ERK1/2 phosphorylation, leading to the activation of estrogen non-genomic signaling including cell migration and invasiveness in inflammatory breast cancer.