erbB3 recruitment of insulin receptor substrate 1 modulates insulin-like growth factor receptor signalling in oestrogen receptor-positive breast cancer cell lines

Abstract

Introduction: Recently we reported that insulin receptor substrate 1 (IRS-1), classically an adaptor protein for the insulin-like growth factor type I receptor (IGF-IR), associates with the epidermal growth factor receptor in oestrogen receptor (ER)-positive (ER+) tamoxifen-resistant breast cancer cells. In this study, we examined whether IRS-1 also associates with another erbB receptor family member, erbB3, and what impact this might have on IGF-IR signalling in three ER+ breast cancer cell lines.

Methods: Immunoprecipitation and Western blot analysis were utilised to examine the potential association between erbB3 and IRS-1 in MCF-7, T47D and BT-474 cells in the absence and presence of the erbB3/4 ligand heregulin β1 (HRGβ1). Subsequently, the impact of a selective IGF-IR/IR inhibitor 4-anilino-5-bromo-2-[4-(2-hydroxy-3-(N, N-dimethylamino)propoxy)anilino]pyrimidine on this association and HRGβ1 signalling was assessed in these cell lines. Immunohistochemical analysis of a small cohort of ER+ breast cancer patient samples was also performed to determine the potential clinical relevance of this novel interaction.

Results: Immunoprecipitation and Western blot analysis revealed an interaction between erbB3 and IRS-1 in MCF-7, T47D and BT-474 cells, with HRGβ1 significantly enhancing this recruitment and promoting IRS-1 phosphorylation at Y612. IRS-1 participates in erbB3 signalling in MCF-7 and T47D cells as IRS-1 knockdown impaired HRGβ1 signalling. Importantly, recruitment of IRS-1 by erbB3 reduced IRS-1 association with IGF-IR in MCF-7 and T47D cells, whilst blockade of IGF-IR-enhanced erbB3-IRS-1 interaction and sensitised both cell lines to HRGβ1, allowing HRGβ1 to override IGF-IR blockade. Consequently, suppression of IRS-1 signalling enhanced the effects of IGF-IR inhibition in these cells. This novel interaction may have clinical relevance, as immunohistochemical analysis of a small ER+ breast tumour series revealed significant positive correlations between phosphorylated IRS-1 Y612 expression and total erbB3, phosphorylated Akt and Ki-67 expression.

Conclusions: IRS-1 can be recruited to IGF-IR and erbB3 in ER+ breast cancer cells, and this provides an adaptive resistance mechanism when these receptors are targeted individually. Consequently, cotargeting IGF-IR and either erbB3 or IRS-1 should prove to be a more effective strategy for the treatment of ER+ breast cancer.

Figure 1

Western blot analysis. (a) Phosphorylated and total epidermal growth factor receptor (EGFR), erbB2, erbB3, Akt and extracellular-signal regulated kinase 1/2 (ERK1/2) and (b) phosphorylated and total insulin receptor substrate 1 (IRS-1) protein expression following treatment of MCF-7, T47D and BT-474 breast cancer cells with either heregulin β1 (HRGβ1) (10 ng/ml) or vehicle control for 5 minutes. (c) Western blot analysis of phosphorylated and total insulin-like growth factor type I receptor (IGF-IR) protein expression following treatment of MCF-7, T47D and BT-474 breast cancer cells with HRGβ1 (10 ng/ml), IGF-I (10 ng/ml) or vehicle control for 5 minutes. Data are representative of three separate experiments.

Figure 2

Western blot showing erbB3, EGFR, erbB2 and insulin receptor substrate 1 protein expression. (a) Immunoprecipitation (IP) with rabbit immunoglobulin G (IgG) (negative control), insulin receptor substrate 1 (IRS-1) or erbB3 antibody for MCF-7 cells treated with either heregulin β1 (HRGβ1) (10 ng/ml) or vehicle control for 5 minutes or up to 20 minutes, (b) immunoprecipitation with either IRS-1 or erbB3 antibody in T47D and BT-474 cells treated with either HRGβ1 (10 ng/ml) or vehicle control for 5 minutes. (c) Western blot analysis of IGF-IR and IRS-1 protein expression following immunoprecipitation with IRS-1 antibody in MCF-7, T47D and BT-474 cells treated with either HRGβ1 (10 ng/ml) or vehicle control for 5 minutes. (d) Densitometric analysis of the loss of IRS-1 association with IGF-IR following HRGβ1 treatment in MCF-7 and T47D cells. The results are expressed as means ± standard errors of the mean of at least three separate experiments. The y-axis represents arbitrary optical densitometric units. In MCF-7 cells * P ≤ 0.01 versus Control; In T47D cells * P ≤ 0.05 versus Control.

Figure 3

Western blot analysis. (a) Phosphorylated and total erbB3, insulin receptor substrate 1 (IRS-1), Akt, extracellular-signal regulated kinase 1/2 (ERK1/2) and β-actin protein expression in MCF-7 and T47D cells incubated in medium supplemented with (a) lipid and control siRNA (C si) mix (100 nM) or lipid and erbB3 siRNA (3 si) mix (100 nM) for 4 days and subsequently challenged with either heregulin β1 (HRGβ1) (10 ng/ml) or vehicle control alone for 5 minutes. Densitometric analysis of (b) phosphorylated IRS-1 Y612 and Y896 protein levels in MCF-7 and (c) T47D cells resulting from HRGβ1-primed erbB3 siRNA- versus control siRNA-treated groups. The results are expressed as means ± standard errors of the mean of at least three separate experiments. In MCF-7 cells * P ≤ 0.01 versus Control siRNA for both phosphorylation sites; In T47D cells * P ≤ 0.01 versus Control siRNA for pY612 IRS-1 and * P ≤ 0.001 versus Control siRNA for pY896 IRS-1.

Figure 4

Western blot analysis. (a) Phosphorylated and total insulin receptor substrate 1 (IRS-1), Akt, extracellular-signal regulated kinase 1/2 (ERK1/2) and β-actin protein expression in MCF-7 and T47D cells incubated in medium supplemented with either lipid and control siRNA (C si) mix (100 nM) or lipid and IRS si mix (100 nM) for 4 days and subsequently challenged with either heregulin β1 (HRGβ1) (10 ng/ml) or vehicle control alone for 5 minutes. Densitometric analysis of phosphorylated Akt and ERK1/2 protein levels in (b) MCF-7 and (c) T47D cells resulting from the HRGβ1-primed IRS-1 siRNA- versus control siRNA-treated groups. The results are expressed as means ± standard errors of the mean of at least three separate experiments. In MCF-7 cells * P ≤ 0.01 versus Control siRNA; In T47D cells * P ≤ 0.001 versus Control siRNA.

Figure 5

Western blot analysis. (a) Total and phosphorylated insulin-like growth factor type I receptor (IGF-IR), insulin receptor substrate 1 (IRS-1), erbB3, Akt and extracellular-signal regulated kinase 1/2 (ERK1/2) protein expression following incubation of MCF-7 and T47D cells in medium containing either the specific IGF-IR/IR tyrosine kinase inhibitor 4-anilino-5-bromo-2-[4-(2-hydroxy-3-(N, N-dimethylamino)propoxy)anilino]pyrimidine (ABDP) (1 μM) or appropriate vehicle control for 24 hours and subsequently challenged with increasing concentrations of heregulin β1 (HRGβ1) (0.1 to 10 ng/ml) or vehicle control for 5 minutes. Densitometric analysis of phosphorylated Akt and ERK1/2 protein levels in (b) MCF-7 and (c) T47D cells treated with HRGβ1 in the absence and presence of ABDP. The results are expressed as the means ± standard errors of the mean of at least three separate experiments from the 1 ng/ml HRGβ1-primed groups. (d) Western blot analysis of total IGF-IR, erbB3 and IRS-1 expression following immunoprecipitation (IP) with total IRS-1 antibody in MCF-7 and T47D cells incubated for 24 hours in medium containing ABDP (1 μM) or vehicle control. * P ≤ 0.05 versus HRGβ1 for both cell lines.

Figure 6

Western blot analysis. (a) Total and phosphorylated insulin-like growth factor type I receptor (IGF-IR), insulin receptor substrate 1 (IRS-1), Akt, extracellular signal-regulated kinase 1/2 (ERK1/2) and β-actin protein expression following incubation of MCF-7 and T47D cells in medium containing either lipid and control siRNA (C si) mix (100 nM) or lipid and IRS si mix (100 nM) in the absence or presence of 4-anilino-5-bromo-2-[4-(2-hydroxy-3-(N, N-dimethylamino)propoxy)anilino]pyrimidine (ABDP) (1 μM) for 2 days and subsequently challenged with heregulin β1 (HRGβ1) (10 ng/ml) for 5 minutes. Data are representative of at least three separate experiments. The effects of 4-day treatment with HRGβ1 (10 ng/ml), ABDP (0.1 μM) or a combination of HRGβ1 and ABDP on the growth of (b) MCF-7 cells or (c) T47D cells. The results are expressed as the means ± standard errors of the mean of triplicate wells and are representative of at least three separate experiments. *P ≤ 0.01 versus control, ^†P ≤ 0.001 versus ABDP.

Figure 7

Immunocytochemical staining. (a) Phosphorylated insulin receptor substrate 1 (IRS-1) Y612, insulin-like growth factor type I receptor (IGF-IR), Y1316, total erbB3, Akt and Ki-67 in paraffin-embedded primary clinical oestrogen-positive (ER+) breast cancer sections (original magnification, ×40). (b) Representative boxplot and scatterplot showing statistically significant correlations between immunostaining HScore values for phosphorylated membrane IRS-1 Y612 and total membrane and cytoplasmic erbB3 in the clinical ER+ breast cancer series.