Estrogen utilization of IGF-1-R and EGF-R to signal in breast cancer cells

As breast cancer cells develop secondary resistance to estrogen deprivation therapy, they increase their utilization of non-genomic signaling pathways. Our prior work demonstrated that estradiol causes an association of ER alpha with Shc, Src and the IGF-1-R. In cells developing resistance to estrogen deprivation (surrogate for aromatase inhibition) and to the anti-estrogens tamoxifen, 4-OH-tamoxifen, and fulvestrant, an increased association of ER alpha with c-Src and the EGF-R occurs. At the same time, there is a translocation of ER alpha out of the nucleus and into the cytoplasm and cell membrane. Blockade of c-Src with the Sic kinase inhibitor, PP-2 causes relocation of ER alpha into the nucleus. While these changes are not identical in response to each anti-estrogen, ER alpha binding to the EGF-R is increased in response to 4-OH-tamoxifen when compared with tamoxifen. The changes in EGF-R interactions with ER alpha impart an enhanced sensitivity of tamoxifen-resistant cells to the inhibitory properties of the specific EGF-R tyrosine kinase inhibitor, AG 1478. However, with long term exposure of tamoxifen-resistant cells to AG 1478, the cells begin to re-grow but can now be inhibited by the IGF-R tyrosine kinase inhibitor, AG 1024. These data suggest that the IGF-R system becomes the predominant signaling mechanism as an adaptive response to the EGF-R inhibitor. Taken together, this information suggests that both the EGF-R and IGF-R pathways can mediate ER alpha signaling. To further examine the effects of fulvestrant on ER alpha function, we examined the acute effects of fulvestrant, on non-genomic functionality. Fulvestrant enhanced ER alpha association with the membrane IGF-1-receptor (IGF-1-R). Using siRNA or expression vectors to knock-down or knock-in selective proteins, we further demonstrated that the ER alpha/IGF-1-R association is Src-dependent. Fulvestrant rapidly induced IGF-1-R and MAPK phosphorylation. The Src inhibitor PP2 and IGF-1-R inhibitor AG1024 greatly blocked fulvestrant-induced ERet/IGF-1-R interaction leading to a further depletion of total cellular ER alpha induced by fulvestrant and further enhanced fulvestrant-induced cell growth arrest. More dramatic was the translocation of ER alpha to the plasma membrane in combination with the IGF-1-R as shown by confocal microscopy. Taken in aggregate, these studies suggest that secondary resistance to hormonal therapy results in usage of both IGF-R and EGF-R for non-genomic signaling. (C) 2009 Elsevier Ltd. All rights reserved.