Estrogen Receptor-Alpha 36 Mediates Mitogenic Antiestrogen Signaling in ER-Negative Breast Cancer Cells

It is prevailingly thought that the antiestrogens tamoxifen and ICI 182, 780 are competitive antagonists of the estrogen-binding site of the estrogen receptor-alpha (ER-α). However, a plethora of evidence demonstrated both antiestrogens exhibit agonist activities in different systems such as activation of the membrane-initiated signaling pathways. The mechanisms by which antiestrogens mediate estrogen-like activities have not been fully established. Previously, a variant of ER-α, EP–α36, has been cloned and showed to mediate membrane-initiated estrogen and antiestrogen signaling in cells only expressing ER-α36. Here, we investigated the molecular mechanisms underlying the antiestrogen signaling in ER-negative breast cancer MDA-MB-231 and MDA-MB-436 cells that express high levels of endogenous ER-α36. We found that the effects of both 4-hydoxytamoxifen (4-OHT) and ICI 182, 780 (ICI) exhibited a non-monotonic, or biphasic dose response curve; antiestrogens at low concentrations, elicited a mitogenic signaling pathway to stimulate cell proliferation while at high concentrations, antiestrogens inhibited cell growth. Antiestrogens at l nM induced the phosphorylation of the Src-Y416 residue, an event to activate Src, while at 5 µM induced Src-Y527 phosphorylation that inactivates Src. Antiestrogens at 1 nM also induced phosphorylation of the MAPK/ERK and activated the Cyclin D1 promoter activity through the Src/EGFR/STAT5 pathways but not at 5 µM. Knock-down of ER-α36 abrogated the biphasic antiestrogen signaling in these cells. Our results thus indicated that ER-α36 mediates biphasic antiestrogen signaling in the ER-negative breast cancer cells and Src functions as a switch of antiestrogen signaling dependent on concentrations of antiestrogens through the EGFR/STAT5 pathway

Different concentrations of antiestrogens affect the association of ER-α36 and Src differently.

<p>Co-immunoprecipitation and Western blot analysis of HA-ER-α36 and Src in MDA-MB-231 cells. Cells transiently transfected with an expression of HA-tagged ER-α36 and treated with different concentrations of antiestrogens for 10 min were lysised and the cell lysates were immunoprecipitated with pre-immune and anti-HA antibodies. The immunoprecipitates were blotted by anti-HA and anti-Src antibodies.</p

STAT5 is involved in antiestrogen-induced Cyclin D1 promoter activity.

<p>(A). The involvement of STAT5 in antiestrogens-induced Cyclin D1 promoter activity. Cells were transfected with the luciferase reported plasmid Cyclin D1 pl-963 together with an empty expression vector or two dominant-negative STAT5a mutants, STAT5aΔ713 and STAT5aΔ740, respectively. Transfected cells were treated with vehicle (ethanol), 1 nM or 5 µM of antiestrogens. Columns: means of the relative luciferase activity from four independent experiments. Luciferase activity in the cells transfected with an empty expression vector and treated with vehicle is arbitrarily set as 1.0; bars, SE. *, p<0.05, for cells treated with vehicle (V) vs 1 nM of antiestrogens. (B). The GAS1 is involved in induction of the Cyclin D1 promoter activity by antiestrogens. Cells were transiently transfected with either the wild-type Cyclin D1 promoter (CycD1) or the same promoter construct containing mutated GAS1 (GAS1mut) or GAS2 (GAS2mut) sequence, respectively. Transfected cells were treated with vehicle or 1 nM of antiestrogens, and the luciferase activity was presented relative to the wild-type Cyclin D1 promoter-transfected cells treated with vehicle that is arbitrarily set as 1.0. *, p<0.05, for cells treated with vehicle (V) vs 1 nM of antiestrogens.</p

Antiestrogens induce biphasic STAT5 activities in ER-negative breast cancer cells.

<p>(A). ER-negative breast cancer cells were transfected with the luciferase reported plasmid 4XM67 TATA-TK-Luc that containing four copies of STAT-binding sites upstream of the minimal TK promoter. Transfected cells were treated with vehicle (ethanol), 1 nM or 5 µM of 4-OHT or ICI 182, 780. The luciferase activities were assayed and normalized using a cytomegalovirus-driven Renilla luciferase plasmid. Columns: means of the relative luciferase activity from four independent experiments. Luciferase activity in transfected cells treated with vehicle is arbitrarily set as 1.0; bars, SE. *, p<0.05, for cells treated with vehicle (V) vs 1 nM of antiestrogens. #, p<0.05, for cells treated with 5 µM vs 1 nM of antiestrogens. (B&C). Cells were transfected with the 4XM67 TATA-TK-Luc reporter together with an empty expression vector (vector) and the expression vectors of two dominant-negative STAT5a mutants carrying truncations at their C-terminal (STAT5aΔ713 and STAT5aΔ740) before treated with vehicle (ethanol), 1 nM or 5 µM of antiestrogens. Columns: means of the relative luciferase activity from three independent experiments. Luciferase activity of cells co-transfected with an empty expression vector and treated with vehicle is arbitrarily set as 1.0; bars, SE. *, p<0.05, for cells treated with vehicle (V) vs 1 nM of antiestrogens.</p

Src is involved in antiestrogen-induced Cyclin D1 expression.

<p>(A). Western blot analysis of Cyclin D1 expression in MDA-MB-231 and -436 cells. Cells were treated with vehicle (ethanol) and antiestrogens alone or together with the Src inhibitors PP2 and dasatinib, the EGFR inhibitor AG1478 and PI3K inhibitor LY294002. Cell lysates were analyzed with anti-Cyclin D1 antibody and anti-Acin antibody was used to ensure equal loading. The experiment was repeated three times, and the representative results are shown. (B). Src inhibitors inhibit antiestrogen-induced Cyclin D1 promoter activity. ER-negative breast cancer cells were transfected with the luciferase reported plasmid Cyclin D1 pl-963 that containing a luciferase gene driven by the Cyclin D1 promoter. Transfected cells were treated with vehicle (ethanol), 1 nM or 5 µM of antiestrogens, and 1 nM of antiestrogens together with different inhibitors. The luciferase activities were assayed and normalized using a cytomegalovirus promoter-driven Renilla luciferase plasmid. Columns: means of the relative luciferase activity in cells treated with vehicle that is arbitrarily set as 1.0 from four independent experiments; bars, SE. *, p<0.05, for cells treated with vehicle (V) vs 1 nM of antiestrogens, or vehicle (V) vs 1 nM of antiestrogens plus AG1478. (C). The involvement of Src in antiestrogen-induced Cyclin D1 promoter activity. Cells were transfected with the luciferase reported plasmid Cyclin D1 pl-963 together with an empty expression vector or Src mutants, a dominant-negative mutant (SrcK295R) and a constitutively active mutant (SrcY527F). Transfected cells were treated with vehicle (ethanol), 1 nM or 5 µM of antiestrogens. The luciferase activities were assayed and normalized using a cytomegalovirus-driven Renilla luciferase plasmid. Columns: means of the relative luciferase activity from four independent experiments. Luciferase activity in transfected cells treated with vehicle is arbitrarily set as 1.0; bars, SE. *, p<0.05, for cells treated with vehicle (V) vs 1 nM of antiestrogens. #, p<0.05, for cells treated with vehicle (V) vs 5 µM of antiestrogens.</p

ER-negative breast cancer cells exhibit biphasic antistrogen signaling.

<p>(A). The effects of 4-OHT and ICI 182, 780 on the proliferation rate of MDA-MB-231 and MDA-MB-436 cells. Cells maintained for three days in phenol red-free DMEM plus 2.5% dextran-charcoal-stripped fetal calf serum were treated with indicated concentrations of 4-OHT, ICI or ethanol vehicle as a control. The cell numbers were determined using an automatic cell counter after 12 days. Five dishes were used for each concentration and experiments were repeated more than three times. The mean cell number ± SE are shown. (B). The dose-dependent phosphorylation pattern of the MAPK/ERK1/2 in MDA-MB-231 and MDA-MB-436 cells treated with different concentrations of antiestrogens. Starved cells were treated with indicated doses of 4-OHT or ICI 182, 780 (ICI) for 10 min. Western blot analysis was performed to assess induction of ERK1/2 phosphorylation. The experiment was repeated more than three times. The representative results are shown. (C). The dose dependent induction Cyclin D1 by antiestrogens in MDA-MB-231 and MDA-MB-436 cells. The experiment was repeated more than three times. The representative results are shown.</p

Involvement of Estrogen Receptor Variant ER-α36, Not GPR30, in Nongenomic Estrogen Signaling

Accumulating evidence suggested that an orphan G protein-coupled receptor (GPR)30, mediates nongenomic responses to estrogen. The present study was performed to investigate the molecular mechanisms underlying GPR30 function. We found that knockdown of GPR30 expression in breast cancer SK-BR-3 cells down-regulated the expression levels of estrogen receptor (ER)-α36, a variant of ER-α. Introduction of a GPR30 expression vector into GPR30 nonexpressing cells induced endogenous ER-α36 expression, and cotransfection assay demonstrated that GPR30 activated the promoter activity of ER-α36 via an activator protein 1 binding site. Both 17β-estradiol (E2) and G1, a compound reported to be a selective GPR30 agonist, increased the phosphorylation levels of the MAPK/ERK1/2 in SK-BR-3 cells, which could be blocked by an anti-ER-α36-specific antibody against its ligand-binding domain. G1 induced activities mediated by ER-α36, such as transcription activation activity of a VP16-ER-α36 fusion protein and activation of the MAPK/ERK1/2 in ER-α36-expressing cells. ER-α36-expressing cells, but not the nonexpressing cells, displayed high-affinity, specific E2 and G1 binding, and E2- and G1-induced intracellular Ca2+ mobilization only in ER-α36 expressing cells. Taken together, our results demonstrated that previously reported activities of GPR30 in response to estrogen were through its ability to induce ER-α36 expression. The selective G protein-coupled receptor (GPR)30 agonist G1 actually interacts with ER-α36. Thus, the ER-α variant ER-α36, not GPR30, is involved in nongenomic estrogen signaling