GPER1-mediated IGFBP-1 induction modulates IGF-1-dependent signaling in tamoxifen-treated breast cancer cells

Abstract

Tamoxifen, a selective estrogen receptor modulator, is a commonly prescribed adjuvant therapy for estrogen receptor-α (ERα)-positive breast cancer patients. To determine if extracellular factors contribute to the modulation of IGF-1 signaling after tamoxifen treatment, MCF-7 cells were treated with IGF-1 in conditioned medium (CM) obtained from 4-OHT-treated MCF-7 cells and the accumulation of phospho-Akt (S473) was measured. CM inhibited IGF-1-dependent cell signaling and suggesting the involvement of extracellular factors (ie. IGFBPs). A significant increase in IGFBP-1 mRNA and extracellular IGFBP-1 protein was observed in 4-OHT-treated MCF-7 cells. Knockdown experiments demonstrated that both GPER1 and CREB mediate IGFBP-1 induction. Furthermore, experiments showed that 4-OHT-dependent IGFBP-1 transcription is downstream of GPER1-activation in breast cancer cells. Additionally, neutralization and knockdown experiments demonstrated a role for IGFBP-1 in the observed inhibition of IGF-1 signaling. These results suggested that 4-OHT inhibits IGF-1 signaling via GPER1 and CREB mediated extracellular IGFBP-1 accumulation in breast cancer cells.

Keywords: Breast cancer; GPER1; IGF-1; IGFBP-1; Tamoxifen.

Fig. 1

4-OHT-induced extracellular factor inhibits IGF-1-stimulated AKT phosphorylation. Conditioned medium (CM) from vehicle-treated MCF-7 cells (24 hours) was replaced with conditioned medium from 4-OHT-treated cells prior to IGF-1 stimulation (50 ng/mL) for 15 min. Total AKT and phospho-AKT were measured by immunoblot. *, p<0.05 compared to vehicle (V) control, and #, p<0.05 compared to IGF-1 treated sample. The results are representative of three independent experiments, and the error bars are SEM.

Fig. 2

4-OHT induced IGFBP-1 transcription and extracellular accumulation in MCF-7 cells. (A) Relative IGFBP-1 gene transcription determined by quantitative real-time PCR after 24 hours of treatment with vehicle (V), 100 nM 4-OHT, or 1μM 4-OHT. Relative transcription was normalized to the RPL30 transcript. (B) Immunoblot analysis of concentrated MCF-7 cell culture medium after 24 hours of treatment with vehicle (V), 100 nM 4-OHT, or 1μM 4-OHT. Results are representative of at least three independent experiments. *, p<0.05 compared to vehicle control, and the error bars are SEM.

Fig. 3

CREB was phosphorylated after treatment with 4-OHT and CREB knockdown partially inhibited 4-OHT-induced IGFBP-1 transcription in MCF-7 cells. (A) Immunoblot analysis of phospho-CREB (S133) after 2 hours of treatment with 4-OHT using the indicated doses. (B) Immunoblot analysis of CREB expression after transfection of CREB-targeting siRNA. (C) Relative IGFBP-1 gene transcription determined by quantitative real-time PCR in CREB knockdown and control cells (NT) after 24 hours of treatment with 4-OHT. *, p<0.05 compared to control, and #, p<0.05 compared to 4-OHT (1μM) treated sample. The results are representative of three independent experiments, and the error bars are SEM.

Fig. 4

GPER1-mediated CREB phosphorylation in MCF-7 cells. (A, B) The total ERα and GPER1 protein levels were reduced using siRNA knockdown. (C) Immunoblot analysis of CREB phosphorylation after 2 hours of treatment with 1μM 4-OHT subsequent to ERα or GPER1 knockdown compared to non-targeting siRNA (NT) cells. *, p<0.05 compared to control. The results are representative of three independent experiments, and the error bars are SEM.

Fig. 5

4-OHT-induced IGFBP-1 transcription and inhibition of IGF-1 stimulation is decreased after GPER1 knockdown in MCF-7 cells. (A) Relative IGFBP-1 gene transcription determined by quantitative real-time PCR in GPER1 knockdown and non-targeting control cells (NT) after 24 hours of treatment with vehicle or 1μM 4-OHT. (B) Immunoblot analysis p-IGF-1R, p-AKT and p-ERK1/2 after IGF-1 stimulation in control and GPER1 knockdown MCF-7 cells. (C) MCF-7 cell viability after treatment with 1μM 4-OHT with and without 1μM G36 treatment. *, p<0.05 compared to vehicle and #, p<0.05 compared to G36- treated sample. The results are representative of three independent experiments, and the error bars are SEM.

Fig. 6

4-OHT-induced IGFBP-1 transcription is mediated by PKA in MCF-7 cells. (A) Immunoblot analysis of CREB phosphorylation in PKA-selective inhibitor (H-89) and EGFR-selective inhibitor (AG-1478) followed by 4-OHT treatment for 2h. (B) Relative IGFBP-1 gene transcription determined by quantitative real-time PCR in 20 μM H-89 and 20 μM AG1478 treated MCF-7 cells after 24 hours of treatment with 1μM 4-OHT. *, p<0.05 compared to control. The results are representative of three independent experiments, and the error bars are SEM.

Fig. 7

Antibody-mediated IGFBP-1 neutralization or IGFBP-1 knockdown reverses the inhibition of IGF-1 stimulation in 4-OHT-treated MCF-7 cells. (A) Two hours prior to IGF-1 stimulation (15 minutes), control IgG or IGFBP-1 neutralizing antibody (10 ng/mL each) were added to MCF-7 cells treated with vehicle (V) or 4-OHT-treated for 24 hours. Total AKT and phospho-AKT levels were determined by immunoblot. (B) siRNA mediated reduction of IGFBP-1 protein expression. (C) After transfection with IGFBP-1 siRNAs or non-targeting control (NT), MCF-7 cells were treated with 1μM 4-OHT for 24 hours followed by 50 ng/mL IGF-1 stimulation for 15 minutes followed by the immunoblot analysis of p-IGF-1R (Y1135/1136), p-AKT (S473) and p-ERK1/2. (D) Extracellular IGFBP-1 protein concentration determined by ELISA. *, p<0.05 compared to vehicle, and #, p<0.05 compared to IGFBP-1 blocking Ab-treated. The results are representative of three independent experiments, and the error bars are SEM.