Estrogen receptor β2 induces proliferation and invasiveness of triple negative breast cancer cells: association with regulation of PHD3 and HIF-1α

Abstract

The two estrogen receptor (ER) subtypes, ERα and ERβ, belong to the nuclear receptor superfamily. The human ERβ variant ERβ2 is proposed to be expressed at higher levels than ERβ1 in many breast tumors and it has been suggested that ERβ2, in contrast to ERβ1, is associated with aggressive phenotypes of various cancers. However, the role of endogenous ERβ2 in breast cancer cells remains elusive. In this study, we identified that triple negative breast cancer (TNBC) cell lines express endogenous ERβ2, but not ERα or ERβ1. This allows novel studies of endogenous ERβ2 functions independent of ERα and ERβ1. We show that overexpression of ERβ2 in TNBC cells increased whereas knockdown of endogenous ERβ2 decreased cell proliferation and cell invasion. To elucidate the molecular mechanism responsible for these cellular phenotypes, we assayed ERβ2 dependent global gene expression profiles. We show that ERβ2 decreases prolyl hydroxylase 3 (PHD3) gene expression and further show that this is associated with increased hypoxia inducible factor 1α (HIF-1α) protein levels, thus providing a possible mechanism for the invasive phenotype. These results are further supported by analysing the expression of ERβ2 and PHD3 in breast tumor samples where a negative correlation between ERβ2 and PHD3 expression was observed. Together, we demonstrate that ERβ2 has an important role in enhancing cell proliferation and invasion, beyond modulation of ERβ and ERβ1 signalling which might contribute to the invasive characteristics of TNBC. The invasive phenotype could potentially be mediated through transcriptional repression of PHD3 and increased HIF-1α protein levels.

Keywords: ERβ isoform; ERβ2; PHD3; breast cancer; gene expression profile.

Figure 1. The TNBC cell lines BT549 and MDA-MB-231 express endogenous ERβ2

Analysis of ERβ2 mRNA levels in ER+/PR+/HER2- breast cancer cell lines (MCF-7, MDA-MB-175, ZR-751 and CAMA-1), ER-/PR-/HER2+ breast cancer cell lines (SK-BR-3 and HCC1569) and ER-/PR-/HER2- TNBC cell lines (Hs578T, MDA-MB-231, BT549 and BT20) by qPCR. mRNA levels are normalized to GAPDH, and mRNA levels are presented as means ± SD, relative to the expression level in MCF-7 cells.

Figure 2. Depletion of ERβ2 inhibits cellular proliferation and invasion

(A) ERβ2 siRNA down-regulates ERβ2 mRNA in BT549 cells. ERβ2 mRNA level was determined by qPCR after transfection with control siRNA or ERβ2 siRNA. Data are normalized to 36B4 and shown as relative fold change compared to control siRNA ± SD. *P < 0.05. (B) ERβ2 depletion reduces proliferation of the BT549 cell line. BT549 cells were transfected with control siRNA or ERβ2 siRNA. WST-1 assays as a measure of cellular proliferation were carried out at the indicated time points after siRNA transfection. Ratio of absorbance to day 1 is calculated. Data are shown as means ± SD. *P < 0.05. The experiment was repeated three times. One representative experiment is shown. (C) ERβ2 depletion reduces invasion of BT549 cell line. BT549 cells were transfected with control siRNA or ERβ2 siRNA, and cell invasion was evaluated by the BD Biocoat growth factor reduced Matrigel invasion chamber assay. Data represent means ± SD. **P < 0.01. Experiment was repeated twice. One representative experiment is shown. A, B, C, p values were calculated by t-test.

Figure 3. ERβ2 overexpression confers a more proliferative and invasive phenotype in vitro

(A) Western blot analysis showing increased protein level of ERβ2 after transient overexpression of ERβ2 protein. ERβ2 was detected by the PPZ0506 antibody. β-actin was used as a loading control. (B) ERβ2 overexpression promotes cell proliferation in the BT549 cells. WST-1 assays of cell proliferation were carried out at the indicated time points after transfection of ERβ2 or empty vector (EV). Ratio of absorbance to day 1 is calculated. Data are shown as means of relative absorbance ± SD. *P < 0.05, **P < 0.01. Experiments were repeated three times. One representative experiment is shown. (C) ERβ2 overexpression promotes cell invasion in the BT549 cell line. BT549 cells were transfected with ERβ2 or EV, and cell invasion was evaluated by the BD Biocoat growth factor reduced Matrigel invasion chamber assay. Data represent means ± SD. ***P < 0.001. Experiment was repeated twice. One representative experiment is shown. B,C, p values were calculated by t-test.

Figure 4. Validation of gene expression profiling data by qPCR

(A) Real-time PCR analysis for a subset of ERβ2 regulated genes identified by microarray analysis in BT549 cells. mRNA levels are normalized to 36B4. Data represent means ± SD. *P < 0.05. Fold change derived from microarray analysis is presented as numbers below the bars. (B) Real-time PCR analysis of selected genes in MDA-MB-231 cells. mRNA levels are normalized to 36B4. Data represent means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. A, B, p values were calculated by t-test relative to control siRNA-treated cells.

Figure 5. ERβ2 modulates levels of PHD3 and HIF-1α in TNBC cells

(A) ERβ2 knock-down increases PHD3 mRNA and protein levels. BT549 cells were transfected with control siRNA or ERβ2 siRNA. RNA was collected after 48 h while protein was collected after 72 h. PHD3 mRNA levels were determined by a qPCR assay and was normalized to 36B4, *P < 0.05 (top panel) and PHD3 protein levels were determined by Western blot analysis. β-actin was used as a loading control (bottom panel). (B) PHD3 knockdown promotes cell invasiveness for the BT549 cell line. BT549 cells were transfected with control siRNA or PHD3 siRNA. Data represent means ± SD. **P < 0.01. (C) BT549 cells were transfected with ERβ2 containing plasmid or a control EV. After 24 h, RNA was collected; qPCR was used to determine the PHD3 mRNA level, that was normalized to 36B4 (bar chart). Data represent means ± SD. **P < 0.01. BT549 cells were seeded onto microscope cover slide after ERβ2 overexpression; 24 h after plating cells, PHD3 protein level was detected by immunofluorescence. DAPI was used for nuclear staining (right figure). (D) BT549 cells were transfected with control siRNA or ERβ2 siRNA, and with empty vector or ERβ2 containing plasmid. Protein lysates were collected and HIF-1α levels were evaluated by both Western blot analysis (top panel) and HIF-1α ELISA (bottom graph). *P < 0.05, **P < 0.01. p values were calculated by t-test. (E) qPCR analysis of HIF-1α mRNA level after overexpression or knockdown of ERβ2. BT549 cells were transfected with control siRNA or ERβ2 siRNA and with EV or ERβ2 containing plasmid. mRNA levels were normalized to 36B4. Data represent means ± SD. *P < 0.05. A,B,C,D,E, p values were calculated by t-test, relative to control siRNA or empty vector transfected cells.

Figure 6. ERβ2 expression is high in breast tumor samples with low ERα expression and negatively correlates with PHD3 expression

(A) qPCR analysis of ERα mRNA level of 50 human breast cancer tissues determined ERα-low expression (n=20) and ERα-high expression (n = 30) breast tumors. Data represent means ± SD. ***P < 0.001. (B) qPCR analysis of ERβ2 mRNA level in ERα-low expression tumor samples compared to ERα-high expression breast tumor samples. Data represent means ± SD. *P < 0.05. (C) Negative correlation of PHD3 mRNA expression to ERβ2 mRNA expression in ERα-low expression breast tumors. *P < 0.05.