P44 Functional study on beta-arrestins involvement in Ras induced transformation of mammalian cells

Background: Considerable cross-talk exists between the insulinlike growth factor (IGF) axis and estrogens; both are linked to breast cancer progression. Breast tumours expressing estrogen receptors (ERs) respond well to therapeutic strategies targeting this receptor. However, breast cancer progression is associated with loss of estrogen receptor expression, generally without deletion or mutation, but often due to epigenetic silencing. In screening tumours for aberrant DNA methylation, IGFBPs particularly IGFBP-3 have consistently been among the most prevalent genes identified. IGFBP-3 is the main IGFBP found in human serum and in addition to modulating the actions of the IGFs, IGFBP-3 can also act intrinsically in either a positive or negative manner depending on the context. In keeping with these in vitro data, epidemiology studies have also reported both positive and inverse associations of IGFBP-3 with pre-menopausal breast cancer. Aims: To treat cells with the demethylating agent, 5-Aza-2’deoxycytidine (AZA) to establish re-expression of the estrogen receptor with a view to assessing any coincident changes in cell growth and abundance of IGFBP-3 and any potential involvement of IGFBP-3 in the effects of AZA on the cells. Methods: ERa negative breast cancer cells, MDA-MB-231 were dosed with AZA (1mM) for 72 hrs +/− estradiol (10nM) for 24 hrs. DNA methylation status of the ERa was assessed using combined bisulfite restriction analysis (COBRA), a quantitative technique to determine DNA methylation levels at specific gene loci. Cell growth was evaluated by cell counting and tritiated thymidine incorporation. Abundance of ERa and IGFBP-3 were assessed using Western immunoblotting and concentrations of IGF-I, IGF-II and IGFBP-3 were measured by radioimmunoassay. Silencing of IGFBP-3 was achieved using siRNA with a non-silencing siRNA used as a control. Results: AZA caused a 36% decrease in methylation of the ERa promoter resulting in re-expression of ERa that was functional, as estradiol was then able to stimulate cell growth. The concentrations of IGF-I and IGF-II also increased slightly but despite these increases and the re-expression of ERa, AZA resulted in a 47% decrease in cell growth which was only partially negated by estradiol. AZA also caused an approximate 30% increase in IGFBP-3 abundance and preventing this increase using siRNA then negated the growth inhibitory effect of AZA. COBRA results indicated that the AZAinduced increase in IGFBP-3 was not due to DNA-demethylation, suggesting an indirect up-regulation of IGFBP-3 by AZA. In the presence of AZA, but with IGFBP-3 silenced, the cells responded even better to estradiol. Conclusion: AZA inhibited the growth of ER-negative breast cancer cells despite inducing the re-expression of a functional ERa and increases in IGF-I/-II. AZA also indirectly caused a coincident increase in the abundance of IGFBP-3, without affecting the methylation status of IGFBP-3. This increase in IGFBP-3 mediated the observed over-riding growth inhibition induced by AZA. P44 Functional study on beta-arrestins involvement in Ras induced transformation of mammalian cells H. Zheng, N. Natalishvili, C. Worrall, A. Girnita, L. Girnita. Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden