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Insulin-like growth factor binding protein 3 modulates epidermal growth factor (EGF)-induced growth of breast epithelial cells by altering EGF receptor internalization
Breast Cancer Research volume 10, Article number: P40 (2008)
Insulin-like growth factor binding protein 3 (IGFBP-3) is the most abundant insulin-like growth factor binding protein in human serum and is able to modulate cell proliferation independently of its ability to bind insulin-like growth factor. Tumour-associated increases in IGFBP-3 levels relate to upregulation of epidermal growth factor receptor (EGFR) and HER-2 with increasing oestrogen independence. Remodelling of the extra-cellular matrix with increased fibronectin expression in poor prognostic tumours further enhances EGFR levels and signalling.
To explore the potential interactions of IGFBP-3 with the EGFR/HER-2 pathways.
Normal breast epithelial cells (MCF-10A) and breast cancer cells (T47D) were dosed with EGF (5 ng/ml and 10 ng/ml), IGFBP-3 (100 ng/ml), an EGFR/HER-2 tyrosine kinase inhibitor, (Iressa, 0.25 μm) and a ROCK inhibitor (Y-27632, 5 μM) either alone or in combinations on either plastic, laminin or fibronectin (0.25 μg/ml). Cell growth was evaluated by cell counting and tritiated thymidine incorporation. Internalisation of the EGFR and ER-2 was assessed by biotinylation and affinity purification using a Pin Point Cell Surface Isolation Kit (Pierce, Northumberland, UK) on whole cell lysates followed by western immunoblotting for the EGFR and HER-2. Statistical significance was determined using ANOVA.
On plastic and laminin with MCF10A cells, EGF and IGFBP-3 each increased cell proliferation alone (by 55.2%, P < 0.001 and 31.7%, P < 0.01, respectively), and together there was a synergistic increase of 278% (P < 0.001). In addition, the proliferative effect of IGFBP-3 alone, like that of EGF, was completely abrogated in the presence of Iressa. With T47D cells, EGF increased cell proliferation (by 3.39%, P < 0.001), IGFBP-3 alone had no effect, but in combination, in contrast to the normal cells, IGFBP-3 completely blocked EGF-induced growth (P > 0.01). These actions of IGFBP-3 on EGF-induced growth were reversed when the cells were cultured on fibronectin. Furthermore, we found that the modulation of EGF-induced proliferation by IGFBP-3 was not mediated by changes in the phosphorylation status of EGFR or HER-2. It was, however, associated with modulation of the internalisation of the EGFR and activation of Rho.
We found that IGFBP-3 had differential, matrix-dependent effects on EGF-mediated proliferation in normal and breast cancer cells, which was achieved through modulation of EGFR internalisation and the activation of Rho. Breast tumour levels of IGFBP-3 may determine their dependence on EGFR/HER-2 activity and their response to therapies targeting these receptors.
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Dennison, G., Holly, J., McIntosh, J. et al. Insulin-like growth factor binding protein 3 modulates epidermal growth factor (EGF)-induced growth of breast epithelial cells by altering EGF receptor internalization. Breast Cancer Res 10, P40 (2008). https://doi.org/10.1186/bcr1924
- Epidermal Growth Factor Receptor
- Epidermal Growth Factor
- T47D Cell
- MCF10A Cell
- Breast Epithelial Cell