Journal: Breast cancer research : BCR

Article Title: ERbeta1 represses basal breast cancer epithelial to mesenchymal transition by destabilizing EGFR.

doi: 10.1186/bcr3358

Figure Lengend Snippet: Figure 3 EGFR promotes EMT and its down-regulation is involved in ERb1-induced E-cadherin expression. (A) EGFR, total ERK1/2 and phospho-ERK1/2 levels in control (Lenti), ERa- and ERb1-expressing MDA-MB-231 cells following incubation with or without E2 for 24 h. (B) EGFR protein levels in control (Lenti) and ERb1-expressing Hs578T cells. (C) EGFR protein levels in MDA-MB-231 cells transiently transfected with control or ERb siRNA (3#). (D) ERb1-expressing MDA-MB-231 cells were incubated in absence or presence of 5 ng/ml TGF-b1 or 10 ng/ml EGF for 24 h and photographed (scale bars, 50 μm). (E) ERb1-expressing MDA-MB-231 cells were incubated in absence or presence of 10 ng/ml EGF for 24 h and analyzed for the expression of EGFR, total ERK1/2 and phospho-ERK1/2 by immunoblotting. Note that the decreased EGFR levels following EGF treatment is due to increased degradation. (F) miR-200a, miR-200b and miR-429 levels in control (Lenti) and ERb1-expressing MDA-MB-231 cells following incubation with 5 ng/ml TGF-b1 or 10 ng/ml EGF for 24 h. The graph shows the data as fold change compared with the untreated Lenti cells (mean of three separate experiments (± SEM) with P-value (*) ≤0.05%). (G) E-cadherin mRNA and protein levels in ERb1-expressing MDA-MB-231 cells following incubation with 5 ng/ml TGF-b1 or 10 ng/ml EGF for 24 h. The graph shows the mean of three separate experiments with SEM and P-value (*) ≤0.05% indicated. (H) ERb1-expressing MDA-MB-231 cells were stably co-transfected with an empty pBABE vector (ERb1:pBABE cells) or the pBABE-EGFR plasmid (ERb1:EGFR cells), photographed and analyzed for EGFR, E-cadherin and ERb1 expression by immunoblotting (scale bars, 50 μm).

Article Snippet: For the expression of wild-type EGFR, cells were stably transfected with the pBABE-EGFR construct (Addgene, plasmid # 11011, Cambridge, MA USA), using the empty pBABE vector (Addgene, plasmid # 1764) as a control.

Techniques: Expressing, Control, Incubation, Transfection, Western Blot, Stable Transfection, Plasmid Preparation

Journal: Breast cancer research : BCR

Article Title: ERbeta1 represses basal breast cancer epithelial to mesenchymal transition by destabilizing EGFR.

doi: 10.1186/bcr3358

Figure Lengend Snippet: Figure 4 ERb1 induces ubiquitylation and degradation of EGFR. (A) Control (Lenti) and ERb1-expressing MDA-MB-231 cells were incubated in the presence of 100 μM cycloheximide and 10 ng/ml EGF for the indicated times and analyzed for EGFR expression by immunoblotting. Treatment with EGF induces phosphorylation of EGFR and this accounts for the retarded electrophoretic mobility of EGFR at times 0.5 to 2. Lower panel: the graph represents the quantification of EGFR protein abundance from three independent experiments with SEM and P-value (*) ≤0.05% indicated. (B) Control (Lenti) and ERb1-expressing MDA-MB-231 cells were incubated in absence or presence of 1 μM MG-132 for 6 h and analyzed for EGFR expression by immunoblotting. Lower panel: the bar graph represents the quantification of EGFR protein levels with SEM and P-value (*) ≤0.05% indicated. (C) Control (Lenti) and ERb1-expressing MDA-MB-231 and Hs578T cells were incubated in the presence of 10 ng/ml EGF for 20 minutes. Lysates were immunoprecipitated with anti-EGFR antibody, followed by immunoblotting with the indicated antibodies. The bottom panel is the input control of cell lysates. (D) MDA-MB-231 cells were transiently transfected with control or ERb siRNA (3#). 72 h after the transfection, cells were incubated with 10 ng/ml EGF for 20 minutes and analyzed as described in C. (E) Control (Lenti) and ERb1-expressing MDA-MB-231 cells were serum starved, challenged with 10 ng/ml EGF for the indicated times and lysed under nondenaturing conditions. EGFR immunoprecipitates were probed with antibodies against EGFR and c-Cbl. The bottom panel is the input control of cell lysates.

Article Snippet: For the expression of wild-type EGFR, cells were stably transfected with the pBABE-EGFR construct (Addgene, plasmid # 11011, Cambridge, MA USA), using the empty pBABE vector (Addgene, plasmid # 1764) as a control.

Techniques: Control, Expressing, Incubation, Western Blot, Phospho-proteomics, Quantitative Proteomics, Immunoprecipitation, Transfection

Journal: Breast cancer research : BCR

Article Title: ERbeta1 represses basal breast cancer epithelial to mesenchymal transition by destabilizing EGFR.

doi: 10.1186/bcr3358

Figure Lengend Snippet: Figure 6 ERb1 levels positively correlate with E-cadherin in breast cancers. (A) Pearson’s correlation of ERb1 expression with expression of E-cadherin. N equals the number of patients for whom data were available. (B) Representative images of ERb1 and E-cadherin expression in two serial sections of the same tumor from two cases. Scale bars represent 200 μM. (C) ERb1 and E-cadherin were box-plotted in the 208 breast cancer patients. The patients were divided into three groups based on ERb1 expression scores in the tumors, representing low, medium and high expression of ERb1. Any outliers were marked with a circle and extreme cases with an asterisk. Data were analyzed using one-way ANOVA test with Games-Howell’s correction. (D) The percentage of E-cadherin-positive tumors was analyzed in the three groups of patients as described in C. Data were analyzed using Pearson’s c2 test. (E) Proposed mechanism for how ERb1 regulates EMT and influences invasion in breast cancer. EGFR promotes EMT in basal cells by activating ERK1/2, which in turn, by inducing the expression of ZEB1/2, results in the down-regulation of E- cadherin. This process requires repression of the expression of members of miR-200 family. By inducing the degradation of EGFR, ERb1 sustains ERK1/2 inactive, up-regulates miR200a-b and miR-429, down-regulates ZEB1/2 and induces the expression of E-cadherin.

Article Snippet: For the expression of wild-type EGFR, cells were stably transfected with the pBABE-EGFR construct (Addgene, plasmid # 11011, Cambridge, MA USA), using the empty pBABE vector (Addgene, plasmid # 1764) as a control.

Techniques: Expressing