Abstract
Growth factor activation of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway has been shown to activate the estrogen receptor (ER) α and to mediate tamoxifen resistance in breast cancer. Here, we investigated the regulation of the transcriptional activity of the newer ERβ by PI3K-AKT signaling. Tissue arrays of breast cancer specimens showed a positive association between the expressions of AKT and ERβ in the clinical setting. Reporter gene assays using pharmacologic and molecular inhibitors of AKT and constitutively active AKT revealed for the first time the ability of AKT to (a) potentiate ERβ activity and (b) target predominantly the activation function-2 (AF2) domain of the receptor, with a requirement for residue K269. Given the importance of coactivators in ER transcriptional activity, we further investigated the possible involvement of steroid receptor coactivator 1 (SRC1) and glucocorticoid receptor-interacting protein 1 (GRIP1) in AKT regulation of ERβ. Mammalian two-hybrid assays revealed that AKT enhanced both SRC1 and GRIP1 recruitment to the ERβ-AF2 domain, and reporter gene analyses revealed that AKT and GRIP1 cooperatively potentiated ERβ-mediated transcription to a level much greater than either factor alone. Investigations into AKT regulation of GRIP with mammalian one-hybrid assays showed that AKT potentiated the activation domains of GRIP1 itself, and in vitro kinase assays revealed that AKT directly phosphorylated GRIP1. The cross-talk between the PI3K-AKT and ERβ pathways, as revealed by the ability of AKT to regulate several components of ERβ-mediated transcription, may represent an important aspect that may influence breast cancer response to endocrine therapy.
