Estrogen Receptor Beta 1: A Potential Therapeutic Target for Female Triple Negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the absence of estrogen receptor alpha, progesterone receptor, and HER2. These receptors often serve as targets in breast cancer treatment. As a result, TNBCs are difficult to treat and have a high propensity to metastasize to distant organs. For these reasons, TNBCs are responsible for over 50% of all breast cancer mortalities while only accounting for 15% to 20% of breast cancer cases. However, estrogen receptor beta 1 (ERβ1), an isoform of the ESR2 gene, has emerged as a potential therapeutic target in the treatment of TNBCs. Using an in vivo xenograft preclinical mouse model with human TNBC, we found that expression of ERβ1 significantly reduced both primary tumor growth and metastasis. Moreover, TNBCs with elevated levels of ERβ1 showed reduction in epithelial to mesenchymal transition markers and breast cancer stem cell markers, and increases in the expression of genes associated with inhibition of cancer cell invasiveness and metastasis, suggesting possible mechanisms underlying the antitumor activity of ERβ1. Gene expression analysis by quantitative polymerase chain reaction and RNA-seq revealed that treatment with chloroindazole, an ERβ-selective agonist ligand, often enhanced the suppressive activity of ERβ1 in TNBCs in vivo or in TNBC cells in culture, suggesting the potential utility of ERβ1 and ERβ ligand in improving TNBC treatment. The findings enable understanding of the mechanisms by which ERβ1 impedes TNBC growth, invasiveness, and metastasis and consideration of ways by which treatments involving ERβ might improve TNBC patient outcome.

Keywords: breast cancer; cancer progression; estrogen receptor beta; gene expression; metastasis.

Figure 1.

Effect of ERβ1 and ERβ ligand treatment on tumor growth and metastasis of TNBC in vivo. (A) Schematic of the overall experimental design of these studies. (B) Growth of MDA-MB-231 tumors in NSG mice over time, without and with dox induction of ERβ1 and treatment with ERβ1 ligand, CLI. Tumor volume determined by caliper measurements. Values are mean ± SEM with n = 7 mice per group. (C) Body weights of mice in different treatment groups over time show no statistically significant differences. (D, E) IVIS bioluminescence measurement of average radiance per mouse in each group at day 48 for primary tumors (n = 7 per group) and metastatic lesions in mice (n = 7 per group). (F) qPCR analysis of ERβ1 mRNA in primary tumors collected at day 48 in the 5 different groups of mice. Values are mean ± SEM. *P < .05, ** P < .01, ***P < .001, and ****P < .0001. (G) ERβ1 protein in primary tumors in the 5 different groups of mice, determined from Western blot analyses. Band intensities are normalized relative to β-actin × 10⁻³. Values are mean ± SEM. **P < .01, and ****P < .0001. Full gel blots are shown elsewhere (Fig. S1 (28)).

Figure 2.

Expression for EMT and breast cancer stem cell marker genes in primary tumors and their modulation by ERβ1 and ligand. (A) qPCR analysis of mRNA expression of EMT markers in primary tumors expressing ERβ1 compared with WT tumors collected at day 48. (B) Gene expression analysis of breast cancer stem cell markers by qPCR in primary tumors. Values are mean ± SEM. Statistical analysis of individual genes was done by multiple t-test. *P < .05, **P < .01, ***P < .001, and ****P < .0001.

Figure 3.

RNA-seq analysis of the effects of ERβ1 alone and together with ERβ agonist ligand, CLI, treatment on gene expression in breast cancer cells. (A) Heat map showing downregulation or upregulation of gene expression in MDA-MB-231 cells in the presence of elevated ERβ1 (+dox, 100 ng/mL doxycycline) or elevated ERβ1 and CLI treatment (100 ng/mL dox + 100 nM CLI) for 24 hours. K-means clustering reveals downregulated gene expressions (Cluster A) and upregulated gene expressions (Cluster B). (B) The Hallmark pathways most enriched in Cluster B are listed.

Figure 4.

RNA-seq analysis of the effects of ERβ1 together with ERβ agonist ligand, CLI, treatment on gene expression in breast cancer cells. (A) Heat map showing downregulation or upregulation of gene expression in MDA-MB-231 cells in the presence of elevated ERβ1 and CLI treatment (100 ng/mL dox + 100 nM CLI) for 24 hours. K-means clustering of the 2000 most variable genes reveals 4 clusters (Clusters A-D) with distinct patterns of gene expression. (B) The Hallmark pathways enriched in Cluster C and D are listed. The genes comprising the 3 most significantly regulated Hallmark pathways (Estrogen Response Early; Estrogen Response Late; and KRAS Up) are listed elsewhere (Table S1 (28)). (C) Genes with expression upregulated by ERβ1 and more highly upregulated by ERβ1 with agonist ligand CLI treatment. MDA-MB-231 cells, WT or containing dox-inducible stable ERβ1 were exposed to 100 ng/mL doxycycline in the presence of control vehicle (+dox) or 100 nM CLI (+dox + CLI) for 24 hours. RNA was then harvested from the cells and analyzed by qRT-PCR for the genes indicated. The expression of ESR2 (top left) was monitored to show the marked increase in ESR2 RNA with doxycycline in the presence of vehicle or CLI. n = 3 per group (WT; +dox; +dox + CLI). Values are mean ± SEM (n = 3 per group) with *P < .05, **P < .01, ***P < .001, and ****P < .0001.

Figure 5.

Expression of important genes in TNBC primary tumors and their regulation by ERβ1 and the ERβ ligand CLI. qPCR analysis of target gene mRNA expression in primary tumors with or without dox inducible ERβ1 in the presence or absence of the ERβ agonist ligand CLI. Tumors were harvested at day 48. Values are mean ± SEM. *P < .05, **P < .01, ***P < .001, and ****P < .0001.

Figure 6.

Immunohistochemical analysis of breast cancer metastases in the lungs of host mice with WT or ERβ1 containing TNBC mammary tumors, and effects of ERβ1 and CLI ligand on expression of EMT and stem cell marker genes in breast cancer lung metastases. Animals received water with or without doxycycline for induction of increased ERβ1 and also treatment with control vehicle or ERβ agonist ligand CLI. Lungs were harvested at day 48 and prepared for immunohistochemistry with staining of tissues as described in “Materials and Methods.” Representative tissue sections are shown. (A) Vehicle-treated control lung from animals with lung metastases derived from mammary tumors with WT TNBC breast cancer cells show extensive metastatic lesions, whereas lung tissue from animals with breast tumors expressing ERβ1 + CLI shows greatly reduced metastatic lesions. Magnification is 100×. (B) Integrated density showing quantitative assessment of metastatic lesions in the different treatment groups. Overall quantitative analysis of multiple metastatic lesions in tissue sections (n = 6) and fields (n = 5/section) is presented. Values are mean ± SEM with n = 24-28 evaluations per group. *P < .05, ** P < .01, ***P < .001, and ****P < .0001 by multiple t-test. (C) q-PCR gene expression analyses of EMT and stem cell marker genes in lung metastases. These genes were also studied in the primary tumor (Fig. 2). Values are mean ± SEM (n = 7 per group). ****P < .0001 by multiple t-test.

Figure 7.

Schematic model depicting the suppressive effects of ERβ1 and the ERβ ligand CLI on TNBC tumor growth, EMT markers, the cancer stem cell–like population, and metastasis. Binding of CLI ligand to ERβ1 may change the shape of ERβ1, affecting receptor interaction with kinases, coregulators, and components of the transcription complex. ERβ1 activity reduces tumor growth and distant metastasis. CoR, coregulator; CSC, cancer stem cells; EMT, epithelial mesenchymal transition.