IGF-1 Receptor Modulates FoxO1-Mediated Tamoxifen Response in Breast Cancer Cells

Abstract

Tamoxifen is a common adjuvant treatment for estrogen receptor (ER)α-positive patients with breast cancer; however, acquired resistance abrogates the efficacy of this therapeutic approach. We recently demonstrated that G protein-coupled estrogen receptor 1 (GPER1) mediates tamoxifen action in breast cancer cells by inducing insulin-like growth factor-binding protein-1 (IGFBP-1) to inhibit IGF-1-dependent signaling. To determine whether dysregulation of IGFBP-1 induction is associated with tamoxifen resistance, IGFBP-1 transcription was measured in tamoxifen-resistant MCF-7 cells (TamR) after tamoxifen (Tam) treatment. IGFBP-1 transcription was not stimulated in tamoxifen-treated TamR cells whereas decreased expression of FoxO1, a known modulator of IGFBP-1, was observed. Exogenous expression of FoxO1 rescued the ability of tamoxifen to induce IGFBP-1 transcription in TamR cells. As decreased IGF-1R expression is observed in tamoxifen-resistant cells, the requirement for IGF-1R expression on tamoxifen-stimulated IGFBP-1 transcription was investigated. In TamR and SK-BR-3 cells, both characterized by low IGF-1R levels, exogenous IGF-1R expression increased FoxO1 levels and IGFBP-1 expression, whereas IGF-1R knockdown in MCF-7 cells decreased tamoxifen-stimulated IGFBP-1 transcription. Interestingly, both 17β-estradiol (E2)-stimulated ERα phosphorylation and progesterone receptor (PR) expression were altered in TamR. PR is a transcription factor known to modulate FoxO1 transcription. In addition, IGF-1R knockdown decreased FoxO1 protein levels in MCF-7 cells. Furthermore, IGF-1R or FoxO1 knockdown inhibited the ability of tamoxifen to induce IGFBP-1 transcription and tamoxifen sensitivity in MCF-7 cells. These data provide a molecular mechanistic connection between IGF-1R expression and the FoxO1-mediated mechanism of tamoxifen action in breast cancer cells.Implications: Loss of IGF-1R expression is associated with decreased tamoxifen efficacy in patients with breast cancer and the development of tamoxifen resistance. This contribution identifies potential molecular mechanisms of altered tamoxifen sensitivity in breast cancer cells resulting from decreased IGF-1R expression. Mol Cancer Res; 15(4); 489-97. ©2017 AACR.

Fig. 1. Tam-induced IGFBP-1 transcription and FoxO1 expression are dysregulated in TamR cells

A, Relative cell viability of MCF-7 and TamR cells after 5 days of 1 μM Tam treatment. B, Relative IGFBP-1 transcription in MCF-7 and TamR cells after 24 h after treatment with 1 μM Tam. C, FoxO1 and p21 expression in MCF-7 and TamR cells. D, Relative IGFBP-1 transcription in TamR cells after exogenous FoxO1 expression and 1 μM Tam treatment. * = p < 0.05 and results are average or representative of three independent experiments. Error bars are SEM.

Fig. 2. FoxO1 mediates Tam-stimulated IGFBP-1 transcription and inhibition of IGF-1-dependent cell signaling in MCF-7 cells

A, FoxO1 expression after siRNA knockdown MCF-7 cells. B, qPCR analysis of 1 µM Tam-induced IGFBP-1 transcription after FoxO1 knockdown in MCF-7 cells. C, Tam-dependent inhibition of IGF-1-stimulated phospho-AKT accumulation is decreased after FoxO1 knockdown in MCF-7 cells. D, CREB expression and accumulation of p-CREB after 1 µM Tam treatment after FoxO1 knockdown in MCF-7 cells. E, Relative qPCR analysis of FoxO1 transcription in 1 μM Tam-treated MCF-7 cells. * = p < 0.05 and results are average or representative of three independent experiments. Error bars are SEM.

Fig. 3. IGF-1R knockdown decreased Tam sensitivity MCF-7 cells

A, IGF-1R expression in MCF-7 and TamR cells. B, IGF-1R expression after IGF-1R knockdown in MCF-7 cells. C, qPCR analysis of 1 µM Tam-induced IGFBP-1 transcription after IGF-1R knockdown in MCF-7 cells. * = p < 0.05, ns = not significant, and results are average or representative of three independent experiments. Error bars are SEM.

Fig. 4. Exogenous IGF-1R expression restores FoxO1 and IGFBP-1 expression in TamR and SK-BR-3 cells

A, IGF-1R and FoxO1 expression after exogenous IGF-1R expression in TamR cells. B, qPCR analysis of IGFBP-1 transcription in TamR cells after exogenous expression of IGF-1R. C, IGF-1R and ERα expression in MCF-7 and SK-BR-3 cells. D, qPCR analysis of IGFBP-1 transcription in MCF-7 and SK-BR-3 cells. E, IGF-1R, FoxO1, and IGFBP-1 expression after exogenous IGF-1R expression in SK-BR-3 cells. * = p < 0.05, EV = empty vector, and results are average or representative of three independent experiments. Error bars are SEM.

Fig. 5. IGF-1R knockdown induces the accumulation decreased FoxO1 protein levels

A, FoxO1 expression over 48 h after IGF-1R knockdown in MCF-7. B, Accumulation of p-ERK1/2 after IGF-1R knockdown in MCF-7 cells. C, Accumulation of p-ERK1/2 in MCF-7 and TamR cells. * = p < 0.05 and results are average or representative of three independent experiments. Error bars are SEM.

Fig. 6. Altered ERα phosphorylation and loss of E2-induced progesterone receptor (PR) and FoxO1 expression in TamR cells

A, p-ERα (S118 and S167) in MCF-7 and TamR cells treated after 10 nM E2 or 50 ng/mL IGF-1 treatment. B, PRA and PRB expression in MCF-7 and TamR cells treated with 10 nM E2. C, FoxO1 and p21 expression in MCF-7 and TamR cells treated with 10 nM E2. * = p < 0.05 and results are average or representative of three independent experiments. Error bars are SEM.

Fig. 7. Decreased Tam sensitivity in MCF-7 cells after IGF-1R or FoxO1 knockdown

A, Cell viability on day 5 of 1 μM Tam-treated MCF-7 cells after FoxO1 knockdown. B, Cell viability on day 5 of 1 μM Tam-treated MCF-7 cells after IGF-1R knockdown. * = p < 0.05 and results are average or representative of three independent experiments. Error bars are SEM.