Nongenomic oestrogen signalling in oestrogen receptor negative breast cancer cells: a role for the angiotensin II receptor AT1

Abstract

Introduction: Oestrogens can mediate some of their cell survival properties through a nongenomic mechanism that involves the mitogen-activated protein kinase (MAPK) pathway. The mechanism of this rapid signalling and its dependence on a membrane bound oestrogen receptor (ER), however, remains controversial. The role of G-protein-coupled receptor and epidermal growth factor (EGF) receptor in an ER-independent signalling pathway modulated by oestrogen was investigated.

Methods: ER-positive and ER-negative breast cancer cell lines (MCF-7 and SKBR3) and primary breast cancer cell cultures were used in this study. Cell proliferation was assessed using standard MTT assays. Protein and cAMP levels were detected by Western blotting and ELISA, respectively. Antigen localization was performed by immunocytochemistry, immunohistochemistry and immunofluorescence. Protein knockdown was achieved using small interfering RNA technologies.

Results: EGF and oestrogen, alone and in combination, induced cell proliferation and phosphorylation of MAPK proteins Raf and ERK (extracellular signal regulated kinase)1/2 in both ER-negative SKBR3 and ER-positive MCF-7 human breast cancer cell lines. Increased Raf phosphorylation was also observed in primary human breast cultures derived from ER-positive and ER-negative breast tumours. Oestrogen induced an increase in intracellular cAMP in ER-negative SKBR3 human breast cancer cells. Oestrogen-mediated cell growth and phosphorylation of MAPK was modified by the EGF receptor antagonist AG1478, the G-protein antagonist pertussis toxin, and the angiotensin II receptor antagonist saralasin. Knockdown of angiotensin II type 1 receptor (AT1) protein expression with small interfering RNA attenuated oestrogen-induced Raf phosphorylation in ER-negative cells. AT1 receptor was found to be expressed in the cell membrane of breast tumour epithelial cells.

Conclusion: These findings provide evidence that, in breast cancer cells, oestrogen can signal through AT1 to activate early cell survival mechanisms in an ER-independent manner.

Figure 1

Effect of 17β-oestradiol and EGF on cell proliferation and induction of MAPK protein expression in breast cancer cells. (a) Cell proliferation findings. SKBR3 and MCF-7 human breast cancer cells, and ER-negative and ER-positive primary breast cultures derived from patient tumours were treated with EGF (10 ng/ml) and 17β-oestradiol (10^-8 mol/l) alone and in combination for 24 hours. Cell proliferation assays were carried out using MTT thiozolyl blue. Proliferative index of the control group is standardised to 1. The results shown are expressed as mean ± standard error (n = 9). Statistical analysis was performed using Mann Whitney U test (*P < 0.02 versus control). (b) Western blot analysis of phospho-Raf and total Raf protein expression. SKBR3 and MCF-7 human breast cancer cells, and ER-negative and ER-positive primary breast cultures derived from patient tumours were treated with EGF (10 ng/ml) and 17β-oestradiol (10^-8 mol/l) alone and in combination for 10 minutes. (c) Phospo-ERK1/2 and total ERK1/2 protein expression. SKBR3 and MCF-7 human breast cancer cells were treated with 5, 10 and 50 ng/ml EGF and 17β-oestradiol (10^-8 mol/l) alone and in combination for 10 minutes. Results are representative of those obtained in three separate experiments. (d) Immunocytochemical localisation of phospho-ERK in ER-negative SKBR3 cells treated with EGF (10 ng/ml) and 17β-oestradiol (10^-8 mol/l) alone and in combination for 30 minutes. The slides were counter stained with Mayer's haematoxylin solution and viewed under a light microscope at 200× magnification. Positive cells stained cells stained brown against a blue background. Results are representative of those obtained in three separate experiments. E2, 17β-oestradiol; EGF, epidermal growth factor; ER, oestrogen receptor; ERK, extracellular signal regulated kinase; MAPK, mitogen-activated protein kinase; PI, proliferative index.

Figure 2

17β-estradiol and EGF induced cell proliferation and raf phosphorylation is mediated through EGFR. (a) SKBR3 and MCF-7 breast cancer cells were pretreated with or without the EGFR antagonist AG1478 (150 nmol/l) 1 hour before treatment with EGF (10 ng/ml) and 17β-oestradiol (10^-8 mol/l) alone and in combination for 24 hours. Cell proliferation assays were carried out using MTT thiozolyl blue. Proliferative index of the control group is standardized to 1. The results shown are expressed as mean ± standard error (n = 9). Statistical analysis was performed using Mann-Whitney U test (*P < 0.02 versus without AG1478). (b) Western blot analysis of phospho-Raf and total Raf in SKBR3 and MCF-7 breast cancer cells pretreated with or without the EGFR antagonist AG1478 (150 nmol/l) for 1 hour before 10 minutes of incubation with EGF (10 ng/ml) and 17β-oestradiol (10^-8 mol/l) alone and in combination. Results are representative of those obtained in three separate experiments. E2, 17β-oestradiol; EGFR, epidermal growth factor receptor; PI, proliferative index.

Figure 3

Effect of GPCR antagonism on 17β-oestradiol and EGF induced cell proliferation, Raf phosphorylation and cAMP production in breast cancer cells. (a) SKBR3 and MCF-7 breast cancer cells pretreated with or without the GPCR antagonist PTX (50 ng/ml) 1 hour before 24 hours of incubation EGF (10 ng/ml) and 17β-oestradiol (10^-8 mol/l) alone and in combination. Cell proliferation assays were carried out using MTT thiozolyl blue. Proliferative index of the control group is standardized to 1. The results shown are expressed as mean ± standard error (n = 9). Statistical analysis was performed using Mann Whitney U test (*P < 0.02 versus without PTX). (b) Western blot analysis of phospho-Raf and total Raf in SKBR3 and MCF-7 cells pretreated with or without the GPCR antagonist pertussis toxin (50 ng/ml) for 1 hour before 10 minutes of incubation with EGF (10 ng/ml) and 17β-oestradiol (10^-8 mol/l) alone and in combination. Results are representative of those obtained in three separate experiments. (c) Intracellular cAMP levels in SKBR3 cells treated with EGF (10 ng/ml) and 17β-oestradiol (10^-8 mol/l) alone and in combination. Results are expressed as mean ± standard error (n = 9). Statistical analysis was performed using Mann-Whitney U test (*P < 0.02 versus control). E2, 17β-oestradiol; EGFR, epidermal growth factor receptor; GPCR, G-protein-coupled receptor; PI, proliferative index.

Figure 4

The role of the AT1 receptor in 17β-oestradiol and EGF-mediated cell proliferation and Raf phosphorylation in breast cancer cells. (a) SKBR3 and MCF-7 breast cancer cells pretreated with or without angiotensin II receptor antagonist saralasin (10^-6 mol/l) 1 hour before 24 hours of incubation with EGF (10 ng/ml) and 17β-oestradiol (10^-8 mol/l) alone and in combination. Cell proliferation assays were carried out using MTT thiozolyl blue. Proliferative index of the control group is standardized to 1. The results shown are expressed as mean ± standard error (n = 9). Statistical analysis was performed using Mann-Whitney U test (*P < 0.02 versus without saralasin). (b) Western blot analysis of phospho-Raf and total Raf in SKBR3 cells pretreated with or without the AT1 antagonist saralasin (10^-6 mol/l) for 1 hour before 10 minutes of incubation with EGF (10 ng/ml) and 17β-oestradiol (10^-8 mol/l) alone and in combination. Results are representative of those obtained in three separate experiments. (c) Western bolt analysis of AT1 and phospho-Raf in SKRB3 cells transiently transfected with GAPDH siRNA, scrambled siRNA, or AT1 (#1 or #2) siRNA before treatment with 17β-oestradiol (10^-8 mol/l) for 10 minutes. Results are representative of those obtained in three separate experiments. Optical density readings of control values were normalized to 1 and experimental groups were expressed as a ratio. Values are expressed as mean ± standard error (n = 3). (d) Immunohistochemistry was carried out for AT1 receptor on primary human breast cancer tissue (7 μm). Negative control was matched IgG. The slides were counterstained with Mayer's haematoxylin solution and were viewed under a light microscope at 40× and 200× magnifications. Positive cells stained cells stained brown against a blue background. (e) Immunofluorescence studies of AT1 receptor in human primary tumour breast cancer tissue (7 μm) and SKBR3 breast cancer cells. Slides were viewed under a confocal microscope at 630×. AT1, angiotensin II type 1; E2, 17β-oestradiol; EGFR, epidermal growth factor receptor; siRNA, small interfering RNA.