P03-24 IGFBP-2 is a novel regulator of the estrogen receptor in breast epithelial cells

Abstract

Background: IGFBP-2 is associated with breast cancer development and many studies have indicated a positive association between estrogen receptor (ERa) status and IGFBP-2 both in vitro and in vivo. It is well known that ERa can regulate expression of IGF family members including IGFBP-2. However, a role for IGFBP-2 in affecting ER levels has not been examined. Aims: To determine the effects of IGFBP-2 on ERa expression in breast epithelial cells. Methods: The relatively normal breast epithelial cells (MCF10A) and the ER-a positive breast cancer cells (T47D) were examined. MCF10A and T47D cells were treated with IGFBP-2 (0–600ng/ml and 0–100ng/ml respectively) in the presence or absence of a disintegrin, RGD (100ng/ml) an IGF-1R blocking antibody, aIR3 (1mg/ml), NBI-31772 peptide (1mM), that blocks the formation of IGF:IGFBP complexes, a PTEN inhibitor Dipotassium Bisperoxo(picolinato) oxovanadate (V) (bpV, 0.1mM) or an inhibitor of PI3K LY294002. IGFBP-2 silencing was achieved by using siRNA technology. Protein abundance of ER-a, IGFBP-2 and Sp1 was determined by western immunoblotting. Quantitative RT-PCR was used to examine mRNA expression. Association between Sp1 and the ERa gene was measured using a Chromatin Immunoprecipitation Assay (ChIP). Results: Adding exogenous IGFBP-2 to the T47D cells and the nonmalignant breast epithelial cells MCF10A induced an up-regulation of ER-a abundance, at both protein (3.5 and 2 fold increase respectively) and mRNA level. This effect of IGFBP-2 was blocked in the presence of a disintegrin in both cell types. With the cancer cells, the increase in ER-a required activation of the IGF-1R, a reduction in PTEN and activation of PI3K. Whereas in the normal cells, IGFBP-2 did not require activation of the IGF-1R and its effect on ER-a was independent of PI3K signalling. In both cell models, the up-regulation of ER-a was mediated by an increase in the association of Sp1 with the ER-a gene. Conclusion: The majority of newly diagnosed breast cancers are hormone-dependent with first line treatment being anti-estrogen therapy. These data suggest that IGFBP-2 could be an important regulator of ER-a and as such may be contributing to breast cancer development and progression. Acknowledgements: We thank the Breast Cancer Campaign for funding this work.