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Volume 10 Supplement 2

Breast Cancer Research 2008

  • Poster presentation
  • Open Access

Insulin-like growth factor binding protein 3 modulates epidermal growth factor (EGF)-induced growth of breast epithelial cells by altering EGF receptor internalization

  • 1,
  • 1,
  • 1,
  • 1 and
  • 1
Breast Cancer Research200810 (Suppl 2) :P40

https://doi.org/10.1186/bcr1924

  • Published:

Keywords

  • Epidermal Growth Factor Receptor
  • Epidermal Growth Factor
  • T47D Cell
  • MCF10A Cell
  • Breast Epithelial Cell

Introduction

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.

Objective

To explore the potential interactions of IGFBP-3 with the EGFR/HER-2 pathways.

Methods

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.

Results

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.

Conclusion

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.

Authors’ Affiliations

(1)
Department of Clinical Sciences North Bristol, IGFs and Metabolic Endocrinology Group, The Medical School, Southmead Hospital, Bristol, UK

Copyright

© BioMed Central Ltd 2008

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