Contrasting activities of estrogen receptor beta isoforms in triple negative breast cancer

Abstract

Purpose: Triple negative breast cancer (TNBC), an aggressive subtype of breast cancer, lacks the three major receptors for predicting outcome or targeting therapy. Hence, our aim was to evaluate the potential of estrogen receptor beta (ERβ) as a possible endocrine therapy target in TNBC.

Methods: The expression and prognostic effect of ERβ isoforms were analyzed using TCGA breast tumor data, and the expression of ERβ isoform mRNA and protein in TNBC cell lines was assayed. Endogenous ERβ2 and ERβ5 were knocked down with siRNA, and ERβ2, ERβ5, and ERβ1 were upregulated using a doxycycline-inducible lentiviral system. Cell proliferation, migration and invasion, and specific gene expressions were evaluated.

Results: ERβ2 and ERβ5 were the predominant endogenous forms of ERβ in TNBC tumors and cell lines. High ERβ2 predicted worse clinical outcome. Knockdown of endogenous ERβ2/ERβ5 in cell lines suppressed proliferation, migration and invasion, and downregulated proto-oncogene survivin expression. ERβ2/ERβ5 upregulation did the reverse, increasing survivin and these cell activities. ERβ1 was barely detectable in TNBC cell lines, but its upregulation reduced survivin, increased tumor suppressor expression (E-cadherin and cystatins), and suppressed proliferation, migration and invasion in both ligand-independent and dependent manners, suggesting the possible translational benefit of ERβ ligands.

Conclusions: ERβ2/ERβ5 and ERβ1 exhibit sharply contrasting activities in TNBC cells. Our findings imply that delineating the absolute amounts and relative ratios of the different ERβ isoforms might have prognostic and therapeutic relevance, and could enable better selection of optimal approaches for treatment of this often aggressive form of breast cancer.

Keywords: Estrogen receptor beta (ERβ); Invasion; Migration; Proliferation; Triple negative breast cancer (TNBC).

Figure 1.. ERβ isoforms in human triple negative breast tumors and TNBC cell lines/

(a) Schematic of the human ERβ protein isoforms with their domain structure shown and their molecular weights indicated at the right (in kDa). (b) The positive rate of ERβ isoforms present in different human breast cancer subtypes, derived from the TSVdb and TCGA databases. (c) The relative mRNA expression level of different ERβ isoforms in breast cancer subtypes, derived by us from the TSVdb and TCGA databases. RSEM, RNA-Seq by Expectation Maximization. Red line indicates median value. **** p < 0.0001, unpaired t-test with Welch’s correction. (d) Relationship analysis of ERβ2 expression and DFS and OS and (e) similar analysis for ERβ5 in TNBC. Log rank p values are shown. (f) The expression of ERβ2 and ERβ5 mRNAs in different TNBC cell lines. (g) ERβ2 and ERβ5 proteins in TNBC cell lines (Bio-Rad, clone 5/25 antibody) with their nuclear versus cytoplasmic localization after cell fractionation followed by Western blotting. β-Actin localization is shown for comparison. (h) Expression of ERβ2 and ERβ5 protein in MDA-MB-231 cells treated with 50ng/ml doxycycline (Dox) for 7 days to induce expression of HA-ERβ2 or HA-ERβ5 in the absence or presence of 10 μM MG132 for 6 h. Protein detected on Western blot with HA antibody. β-Actin is shown as a loading control.

Figure 2.. Downregulation of ERβ2 and ERβ5 suppressed the proliferation, migration, and invasion of TNBC cells.

MDA-MB-231 cells were treated with 50 nM nontargeting siRNA (NS) or with ERβ2/β5 siRNA for 3 days. (a) ERβ2 and ERβ5 mRNAs were assayed using RT-PCR, and (b) ERβ2 and ERβ5 protein was monitored by Western blot and quantitated using LI-COR system software. (c) The proliferation of MDA-MB-231 cells was assayed using WST1 after treatment with NS or ERβ2/β5 siRNA for the indicated times. (d) Cell migration assays were performed with MDA-MB-231 and BT549 cells after treatment with NS or ERβ2/β5 siRNA for 3 days. Data are mean ± SEM. (e) Cell invasion assays were performed with MDA-MB-231 cells after treatment with NS or ERβ2/β5 siRNA for 3 days. Data are mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3.. Inducible expression of ERβ2 or ERβ5 in TNBC cells and effects on cellular activities.

MDA-MB-231 cells were infected with a Dox-inducible lentiviral expression vector for ERβ2 or ERβ5 shown in Supplementary Figure S2. MDA-MB-231/ERβ2 cells or MDA-MB-231/ERβ5 cells were maintained in media with or without 50 ng/ml Dox for 7 days, and then cellular activities were assayed in the continued absence or presence of Dox. (a and d) Proliferation using WST1 assay, (b and e) cell migration assays, and (c and f) invasion assays. Data are mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4.. Effects of ERβ1 on TNBC proliferation, migration, and invasion, and expression of E-cadherin, cystatins, and survivin.

(a) Western blot analysis of the expression of ERβ1 protein and E-cadherin after treatment of MDA-MB-231-ERβ1 cells with Dox (50 ng/ml for 7 days). (b) MDA-MB-231-ERβ1 cells were cultured in media with Dox for 7 days, and then cell proliferation was monitored after treatment with Vehicle, 10 nM E2, 10 nM LY500307, or 100 nM CLI for 72h in the continued absence or presence of Dox. Stars indicate significantly different from no Dox. ** p < 0.01. (c and d) Cell migration and invasion assays were conducted on cells treated as in panel b, with Dox for 7 days and then followed by veh or ligand treatment for 48h prior to assay. Pound sign indicates significantly different with Dox treatment vs no Dox. ##, p < 0.01. Stars indicate Dox+ligand treatment significantly different from Dox+veh. ** p < 0.01; *** p < 0.001; **** p < 0.0001. (e) The expression of cystatins (CSTs) was assayed using RT-PCR after cell treatment with veh, 10 nM E2, 10 nM LY500307, or 100 nM CLI for 2 days. Pound sign indicates significantly different with Dox treatment vs no Dox. ###, p<0.001; ####; p < 0.0001. Stars indicate Dox+ligand treatment significantly different from Dox+veh. ** p < 0.01; *** p < 0.001; **** p < 0.0001. (f) Survivin protein was monitored in Dox-inducible ERβ1-expressing MDA-MB-231 cells after treatment with Dox alone for 7 days or (g) with Dox for 7 days and then the addition of veh, 10 nM E2, 10 nM LY500307, or 100 nM CLI for 48 h prior to Western blot analysis. Numbers show quantitation of survivin and E-cadherin protein relative to β-actin as loading control. Control Veh is set at 1.0.

Figure 5.. Expression of survivin in human breast tumor subtypes and effects of ERβ2/β5 and ERβ1 on survivin in TNBC cells.

(a and b) Survivin mRNA expression in normal breast and in primary tumors, and in breast cancer subtypes, based on information from TCGA analysis using the UALCAN portal. (c) TNBC cells were treated with nontargeting siRNA (NS) or ERβ2/β5 siRNA for 3 days, and survivin mRNA was monitored, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; and (d) survivin protein was monitored in MDA-MB-231 cells by Western blot. (e and f) MDA-MB-231/ERβ2 or MDA-MB-231/ERβ5 cells were treated with Dox at the concentrations indicated, and survivin protein was monitored by Western blot. (g) MDA-MB-231 cells were infected with control AdGal or AdERβ1, and survivin protein was monitored by Western blot. β-Actin was used as a gel loading control.

Figure 6.. Impact of survivin knockdown or overexpression on cell proliferation, migration, and invasion.

MDA-MB-231 cells were treated with nontargeting siRNA (NS) or survivin/BIRC5 siRNA for 3 days, and survivin mRNA, and protein were assayed by (a) qPCR or (b) Western blot. MDA-MB-231 cells were treated with NS or survivin siRNA for 3 days, and (c) cell proliferation, (d) migration, and (e) invasion were monitored. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 7.. Schematic model of the contrasting effects of ERβ2 and ERβ5 versus ERβ1 in TNBC.

ERβ2 and ERβ5 show ligand-independent actions that increase the level of cell survivin and enhance cell migration, invasion and proliferation. By contrast, ERβ1 reduced survivin expression and elevated expression of E-cadherin and cystatins 1, 2, 4, and 5, associated with suppression of cell proliferation, and migration and invasion, effects that were further suppressed by treatment with ERβ ligands.