IGF-IR cooperates with ERα to inhibit breast cancer cell aggressiveness by regulating the expression and localisation of ECM molecules

Abstract

IGF-IR is highly associated with the behaviour of breast cancer cells. In ERα-positive breast cancer, IGF-IR is present at high levels. In clinical practice, prolonged treatment with anti-estrogen agents results in resistance to the therapy with activation of alternative signaling pathways. Receptor Tyrosine Kinases, and especially IGF-IR, have crucial roles in these processes. Here, we report a nodal role of IGF-IR in the regulation of ERα-positive breast cancer cell aggressiveness and the regulation of expression levels of several extracellular matrix molecules. In particular, activation of IGF-IR, but not EGFR, in MCF-7 breast cancer cells results in the reduction of specific matrix metalloproteinases and their inhibitors. In contrast, IGF-IR inhibition leads to the depletion by endocytosis of syndecan-4. Global important changes in cell adhesion receptors, which include integrins and syndecan-4 triggered by IGF-IR inhibition, regulate adhesion and invasion. Cell function assays that were performed in MCF-7 cells as well as their ERα-suppressed counterparts indicate that ER status is a major determinant of IGF-IR regulatory role on cell adhesion and invasion. The strong inhibitory role of IGF-IR on breast cancer cells aggressiveness for which E2-ERα signaling pathway seems to be essential, highlights IGF-IR as a major molecular target for novel therapeutic strategies.

Figure 1. Evaluation of gene expression levels of MMPs and TIMPs after treatment with specific tyrosine kinase inhibitors and E2 in MCF-7 breast cancer cells.

The effect of IGF-IR or EGFR inhibition on the constitutive and the E2-mediated gene expression of (A) MT1-MMP, (B) MMP-9, (C) TIMP-1, and (D) TIMP-2 in MCF-7 breast cancer cells. The mRNA levels were assessed by Real Time PCR analysis. Cells were pre-treated with inhibitor of EGFR (AG1478, 1 μΜ) or IGF-IR (AG1024, 1 μΜ) for 30 min, followed by the introduction of E2 (10 nM), EGF (5 ng/mL) and IGF (15 ng/mL) where indicated. Total incubation time was 16 h. Statistically significant differences compared with control or E2-treated cells are shown with *(p < 0.05), **(p < 0.01), ***(p < 0.001) or ^##(p < 0.01), ^###(p < 0.001), respectively.

Figure 2. Investigation of IGF-IR and EGFR inhibition on syndecan expression levels and localisation.

Evaluation of (A) syndecan-1 and (B) syndecan-4 cell surface protein levels by flow cytometry. The experiments were performed following treatments with EGFR and IGF-IR inhibitors, in the presence or the absence of E2. Immunocytochemical localisation of (C) syndecan-1 and (D) syndecan-4 after treatment with EGFR and IGF-IR inhibitors, in the presence or the absence of E2. Bars = 25 μm. (E) Immunoprecipitation of syndecan-4 with IGF-IR from MCF-7 cell lysates.

Figure 3. Loss of syndecan-4 on ERα+ breast cancer cells resulting from IGF-IR inhibition is reduced at low temperature but not after MMP inhibition.

Time course of IGF-IR inhibition on cell surface syndecan-4 levels, in the presence or absence of E2, at 37 °C (A) or 15 °C (B). Control MCF-7 cultures were not exposed to AG1024 IGF-IR inhibitor. Lowering temperature to slow down internalisation reduced the loss of syndecan-4. (C) Similar FACS analysis of MCF-7 cells untreated or treated with IGF-IR inhibitor and/or EGF inhibitor in the presence or absence of E2 and the GM6001 general MMP inhibitor. Syndecan-4 levels were decreased despite the presence of sheddase inhibitor.

Figure 4. The role of IGF-IR on cell functions of MCF-7 breast cancer cells.

(A,B) Evaluation of MCF-7 cell adhesion to substrates coated with integrin-specific antibodies. Treatment with IGF-IR inhibitor markedly reduced adhesion to all four substrates comprised of antibodies to “RGD-binding” integrins. In contrast, adhesion to antibodies against the collagen-binding α2 subunit was increased. Data in A normalised to α5β1 antibody adhesion in the absence of inhibitor. (C) The effect of IGF-IR on both the constitutive and the E2-mediated cell adhesion to type I collagen substrates by MCF-7 breast cancer cells. Adhesion was increased by E2 treatment, particularly where IGF-IR was inhibited. (D) The blocking of α2 integrin reduced the adhesion ability on type I collagen substrates. The inhibition with both AG1024 and anti-α2 antibody led to the enhancement of this effect. (E) The effect of IGF-IR inhibition on type I collagen gel invasion by MCF-7 breast cancer cells, in the presence or absence of E2. Cells were incubated with AG1024 (inhibitor of IGF-IR) (1 μΜ) for 30 min, followed by the introduction of E2 (10 nm), where indicated. Total incubation time was 24 h. Invasion was increased following IGF-IR inhibition. (F) Treatment of MCF-7 cell with GM6001 (MMPs inhibitor) had no effect on invasion ability of these cells. Statistically significant differences compared with control or E2-treated cells are shown as *(p < 0.05), **(p < 0.01), ***(p < 0.001) or ^#(p < 0.05), ^##(p < 0.01), respectively.

Figure 5. Loss of ERα strongly augments aggressiveness of MCF-7 breast cancer cells.

(A) Confirmation of loss of ERα in MCF-7 SP10+ cells, which also led to almost complete loss of IGF-IR and syndecan-4 but to increased levels of EGFR. Gene expression levels were measured by Real-Time PCR and protein expression levels were measured by western blotting analysis. (B) The effect of IGF-IR on cell adhesion to collagen I substrate in the presence or absence of E2 of MCF-7 SP10+ breast cancer cells. (C) The blocking of α2β1 integrin had no effect on adhesion to collagen I of MCF-7 SP10+ cells. (D) The effect of IGF-IR on both the constitutive and the E2-mediated cell invasion on MCF-7 SP10+ breast cancer cells, respectively. Cell invasion was assessed by collagen type I assay. Cancer cells were incubated with the inhibitor of IGF-IR (AG1024, 1 μΜ) for 30 min, followed by the introduction of E2 (10 nM), where it was necessary. Total incubation time was 24 h. (E) The inhibition of MMPs, in the presence or the absence of IGF-IR inhibitor, led to suppression of invasiveness of MCF-7 SP10+ cells. Statistically significant differences compared with control or E2-treated cells are symbolized with *(p < 0.05), **(p < 0.01), ***(p < 0.001).