IGF-I mediated phosphorylation of ERαS167 occurs downstream of mTOR/p70S6K1 to impact nuclear localization and chromatin interaction.

Abstract

Abstract Abstract #6012 Crosstalk between the insulin-like growth factor (IGF) I receptor (IGF-IR) and estrogen receptor alpha (ERα) enhances breast cancer cell growth and survival. Interaction between these receptors has been extensively characterized in numerous in vitro and in vivo cell models. Activation of the IGF-IR results in the recruitment of the serine/threonine kinase Akt/PKB, which has been shown to directly phosphorylate ERα at Serine167. These findings were demonstrated in transient transfection model systems. To study if IGF-IR activation of Akt/PKB results in ERα phosphorylation, we used the MCF-7L breast cancer cell line expressing both IGF-IR and ERα. In these cells, IGF-I-induced activation of the IGF-IR/PI3K/Akt axis resulting in the subsequent phosphorylation of ERα at serine 167 as early as 10 minutes and was seen up to 24 hours indicating that this was not a transient phenomenon. In addition, this event was site-specific for ser167, as IGF-I exposure did not result in the phosphorylation of the estradiol (E2)/mitogen activated protein kinase (MAPK) regulated serine 118 site of ERα. Contrary to prior reports, we observed that IGF-I induced ERαS167 phosphorylation was abolished by rapamycin, an inhibitor of mammalian target of rapamycin (mTOR). As expected, rapamycin failed to block E2 stimulated ERαS118 phosphorylation. These events were further confirmed in two additional strains of MCF-7 cells (MVLN & MCF-7ATCC). In an effort to confirm that the events responsible for IGF-I induced ERαS167 phosphorylation were distal to mTOR activation, we targeted multiple additional signaling molecules in the IGF-IR/PI3K/Akt/mTOR pathway. Blockade of PI3K (LY294002), mTOR (rapamycin) and the downstream mTOR kinase p70S6K1 (H89) resulted in a dose-dependent ablation of ERαS167 phosphorylation. In addition to blocking ERα phosphorylation, H89 specifically also inhibited eukaryotic initiation factor (eIF) 4B (eIF4B), a known immediate downstream target of activated p70S6K1. Phosphorylation of ERαS118 did not require MAPK activation, as the MEK inhibitor U0126 ablated MAPK activation and did not affect ERαS167 phosphorylation. Furthermore, we find that siRNA-mediated knockdown of p70S6K1 results in a concordant attenuation of IGF-mediated ERαS167 phosphorylation independent of the ERαS118 site. Thus, we find that phosphorylation of ERαS167 in MCF-7L cells is regulated downstream of IGF-IR by p70S6K1, a kinase known to initiate the translation of multiple mRNA transcripts. Since modification of ERα by serine phosphorylation has been shown to impact DNA binding, transcription, dimerization and coactivator recruitment, we hypothesize that IGF-IR/PI3K/Akt initiation induces ERαS167 phosphorylation through an PI3K/mTOR-specific pathway and may alter classical nuclear ERα function. More importantly, IGF/ERα crosstalk occurs at the level of post-translational modification of ERα and may serve to promote the malignant phenotype in breast cancer cells. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6012.