Elevated insulin-like growth factor 1 receptor signaling induces antiestrogen resistance through the MAPK/ERK and PI3K/Akt signaling routes

Abstract

Introduction: Insulin-like growth factor 1 (IGF-1) receptor (IGF-1R) is phosphorylated in all breast cancer subtypes. Past findings have shown that IGF-1R mediates antiestrogen resistance through cross-talk with estrogen receptor (ER) signaling and via its action upstream of the epidermal growth factor receptor and human epidermal growth factor receptor 2. Yet, the direct role of IGF-1R signaling itself in antiestrogen resistance remains obscure. In the present study, we sought to elucidate whether antiestrogen resistance is induced directly by IGF-1R signaling in response to its ligand IGF-1 stimulation.

Methods: A breast cancer cell line ectopically expressing human wild-type IGF-1R, MCF7/IGF-1R, was established by retroviral transduction and colony selection. Cellular antiestrogen sensitivity was evaluated under estrogen-depleted two-dimensional (2D) and 3D culture conditions. Functional activities of the key IGF-1R signaling components in antiestrogen resistance were assessed by specific kinase inhibitor compounds and small interfering RNA.

Results: Ectopic expression of IGF-1R in ER-positive MCF7 human breast cancer cells enhanced IGF-1R tyrosine kinase signaling in response to IGF-1 ligand stimulation. The elevated IGF-1R signaling rendered MCF7/IGF-1R cells highly resistant to the antiestrogens tamoxifen and fulvestrant. This antiestrogen-resistant phenotype involved mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase/protein kinase B pathways downstream of the IGF-1R signaling hub and was independent of ER signaling. Intriguingly, a MAPK/ERK-dependent agonistic behavior of tamoxifen at low doses was triggered in the presence of IGF-1, showing a mild promitogenic effect and increasing ER transcriptional activity.

Conclusions: Our data provide evidence that the IGF-1/IGF-1R signaling axis may play a causal role in antiestrogen resistance of breast cancer cells, despite continuous suppression of ER transcriptional function by antiestrogens.

Figure 1

Characterization of insulin-like growth factor 1 receptor overexpression, autophosphorylation, signaling transduction and proliferation of MCF7/insulin-like growth factor 1 receptor cells in response to insulin-like growth factor 1. (a) Immunofluorescence showing insulin-like growth factor 1 receptor (IGF-1R) overexpression in MCF7/IGF-1R cells compared to parental MCF7 cells. IGF-1R was probed with mouse monoclonal antibody against IGF-1Rβ (green). Cell nuclei were stained with 4',6-diamidino-2-phenylindole (blue). Original magnification, × 60. (b) IGF-1R autophosphorylation and signal transduction in time course exposure to IGF-1 (100 ng/mL) for 1, 5, 10, 30 and 60 minutes. ERα, estrogen receptor. (c) Proliferative response of MCF7 versus MCF7/IGF-1R cells to IGF-1 stimulation at the dose ranges indicated. Sulforhodamine B (SRB) absorbance values (optical density (OD) 510 nm) are representative of three independent experiments. Data are expressed as means ± SD. (d) IGF-1R autoactivation and signal transduction to IGF-1 exposure at various dose levels (1, 3, 10, 33 and 100 ng/mL) for two hours. Total IGF-1R levels were detected by IGF-1Rβ antibody at 97 kDa. IGF-1R triple tyrosine phosphorylation was determined by using phospho-IGF-1Rβ (Tyr1131) and phospho-IGF-1Rβ (Tyr1135/Tyr1136) rabbit antibodies. Phosphorylated extracellular signal-regulated kinase (p-ERK) and phosphorylated protein kinase B (p-Akt) were determined by using p-ERK1/2 and p-Akt antibodies, respectively. The status of the estrogen receptor (ER) was detected by rabbit anti-ERα antibody. Tubulin was detected as a control for protein loading.

Figure 2

Antiestrogen resistance of MCF7/IGF-1R cells is induced by high level of IGF-1R signaling. (a) Proliferative behavior of MCF7 versus MCF7/IGF-1R cells in response to 4-hydroxytamoxifen (TAM) in the dose range indicated in combination with estrogen 17β-estradiol (E2) (1 nM), IGF-1 (100 ng/mL) or E2 (1 nM) plus IGF-1 (100 ng/mL). Dimethyl sulfoxide (DMSO) was used as a control. (b) Proliferative behavior of MCF7/IGF-1R versus MCF7 cells in response to fulvestrant (FUL) with the same dose range and treatments as were used for TAM. Each treatment was carried out in triplicate. SRB absorbance values are representative of three independent experiments. Data are expressed as means ± SD.

Figure 3

IGF-1R signal-induced antiestrogen resistance in MCF7/IGF-1R cells is independent of ER status. (a) Differential ER levels in response to antiestrogens TAM and FUL. Cells were treated with 100 nM TAM or FUL for three days following two-day starvation. (-), untreated. (b) Knockdown of ER expression by small interfering RNA (siRNA). (c) Effect of ERα knockdown by siRNA on cell proliferation in response to TAM (1 μM), E2 (1 nM) or E2 (1 nM) plus TAM (1 μM). siCtrl, siRNA control; siERa, siRNA ERα; **P < 0.01. (d) Effect of ERα knockdown on cell proliferation in response to IGF-1 (100 ng/mL), IGF-1 (100 ng/mL) plus TAM (1 μM) or a combination of IGF-1 (100 ng/mL), E2 (1 nM) and TAM (1 μM). SRB proliferation OD values were normalized to the control DMSO. Data are representative of three individual experiments. Data are expressed as means ± SD. (e) Estrogen response element (ERE)-dependent luciferase (Luc) activity of MCF7/IGF-1R cells in the presence of TAM (1 μM), E2 (1 nM), E2 (1 nM) plus TAM (1 μM) and a combination of IGF-1 (100 ng/mL), E2 (1 nM) and TAM (1 μM). Luciferase activity was normalized to DMSO control. Three individual ERE-luciferase assays were performed. Error bars represent ± SD.

Figure 4

Involvement of mitogen-activated protein kinase/extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt in IGF-1R signal-mediated antiestrogen resistance of MCF7/IGF-1R cells. (a) Inhibitory effects of kinase inhibitors on IGF-1R autoactivation and its signaling transduction. (b) Inhibitory effects of kinase inhibitors on tamoxifen resistance of MCF7/IGF-1R cells in response to TAM (1 μM) individually or combined with E2 (1 nM) and IGF-1 (100 ng/mL) as indicated. (c) Inhibitory effects of kinase inhibitors on FUL resistance of MCF7/IGF-1R cells in response to FUL (1 μM) individually or combined with E2 (1 nM) and IGF-1 (100 ng/mL) as indicated. (-), no kinase inhibitor. SRB absorbance values are representative of three independent experiments. Error bars represent ± SD. ***P < 0.001.

Figure 5

The key IGF-1R signaling components in the regulation of TAM resistance of MCF7/IGF-1R cells. (a) Selected kinase inhibitors inhibiting IGF-1R signal-mediated TAM resistance. (b) Selected siRNA targeting genes (as named) involved in IGF-1R signal-mediated TAM resistance. Concentrations used: TAM (1 μM), E2 (1 nM) and IGF-1 (100 ng/mL). (-), no kinase inhibitor. siCtrl, nontargeting siRNA. Results are representative of duplicate experiments. Data are expressed as means ± SD. *P < 0.05. **P < 0.01. ***P < 0.001. (c) MetaCore Pathway Analysis showing the functional activities of the key players (dashed circles) in IGF-1R signal-mediated TAM resistance. Fold changes in cell proliferation as the result of inhibition of selected kinase inhibitors or knockdown of target genes are given in Additional file 3. 1, experiment 1 (exp 1) with kinase inhibitors. 2, experiment 2 (exp 2) with siRNA. Red bar, increase in proliferation. Blue bar, decrease in proliferation.

Figure 6

Three-dimensional responses of MCF7/IGF-1R cells to TAM (1 μM), E2 and IGF-1. Compared to parental MCF7 cells (a), MCF7/IGF-1R cells (b) in three-dimensional (3D) culture formed bigger acini in response to IGF-1 stimulation and displayed significant TAM resistance when treated with TAM (1 μM) + E2 + IGF-1, which was removable by kinase inhibitors BMS-536924, U0126 and BEZ235 (c). Cells (10,000/well) were seeded in 96-well plates. Acini were formed on 100% Matrigel and cultured for 14 days in starving medium containing 2% Matrigel and 5% charcoal/dextran-stripped fetal bovine serum with the treatments as indicated. Concentrations used: TAM (1 μM), E2 (1 nM) and IGF-1 (100 ng/mL). Confocal image original magnification, × 20. Red, rhodamine phalloidin (actin). Blue, Hoechst blue stain. Results are representative of two individual experiments.

Figure 7

TAM (10 nM) acts as an agonist in MCF7/IGF-1R stimulated by IGF-1. (a) Dose dependence of TAM resistance on IGF-1. (b) Antagonistic effect of 10 nM TAM on 1 nM E2-induced cell proliferation. (c) Antagonistic effect of 10 nM TAM on 1 nM E2-induced ERE-Luc activity. (d) Agonistic effect of 10 nM TAM on 100 ng/mL IGF-1-stimulated MCF7/IGF-1R cells. (e) ERE-Luc activity induced by agonistic TAM (10 nM) in IGF-1-stimulated MCF7/IGF-1R cells. (f) ER dependence of agonistic effect of TAM (10 nM) in MCF7/IGF-1R cells. siCtrl, siRNA control; siERα, siRNA ERα. Results are representative of three individual experiments. Luciferase activity was normalized to DMSO control. Error bars represent ± SD. *P < 0.05.

Figure 8

TAM (10 nM) agonistic behavior in MCF7/IGF-1R cells is not inhibited by phosphatidylinositol 3-kinase/Akt inhibitor BEZ235. (a) Inhibitory effects of kinase inhibitors BMS-536924, U0126 and BEZ235 on agonistic effect of TAM (10 nM) in the presence of IGF-1 (100 ng/mL). **P < 0.01. (b) Inhibitory effects of phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor BEZ235 at various dose ranges on IGF-1R signaling. (c) Agonistic behavior of TAM (10 nM) in response to BEZ235 kinase inhibitor. (-), no BEZ235. Results are representative of three independent experiments. Data are expressed as means ± SD.