Phosphorylation of the Mutant K303R Estrogen Receptor α at Serine 305 Impacts Aromatase Inhibitor Sensitivity

Aromatase inhibitors (AIs) are challenging tamoxifen as the treatment of choice for both early and advanced breast cancer in postmenopausal women with estrogen receptor (ER) α-positive diseases. However, resistance frequently occurs. Previously, we identified a lysine to arginine transition at residue 303 (K303R) in ERα in premalignant breast lesions and invasive breast cancers, which confers estrogen hypersensitivity and resistance to the AI Anastrozole (Ana) when transfected in MCF-7 breast cancer cells. To identify genes whose expression was associated with the development of aromatase inhibitor resistance (AIR), we performed microarray analysis, and found a marked increase in the gene expression of different IGF family members in the mutant cells. Immunoblot analysis also revealed elevated constitutive phosphorylation of the IGF-1R/IRS-1/Akt pathway in K303R-expressing cells. Treatment with IGF-1R and Akt inhibitors drastically inhibited proliferation of the K303R-mutant expressing cells, while wild-type ERα-expressing clones showed little reduction of growth by these inhibitors, confirming the increased IGF signaling activation in the mutant cells.Post-translational modifications of ERα, such as phosphorylation, tightly regulate its function, and the K303R mutation resides at a major post-translational modification site, serine (S) residue 305. We found that the mutant was more efficiently phosphorylated than the WT receptor by Akt in vitro, and that the mutant cells exhibited enhanced S305 phosphorylation in vivo. Mutation of S305 to alanine (A) to destroy this phosphorylation site prevented in vitro and in vivo Akt-mediated phosphorylation.The ERα S305 residue is an important site that modifies response to tamoxifen; thus, we questioned whether the S305 site could also influence AI response. To address the role of S305 phosphorylation on AIR, we generated pools of stable transfectants expressing exogenous WT, K303R, or K303R/S305A mutant receptors, and then evaluated for the effects of Ana in soft agar assays. Expression of the K303R/S305A mutant resulted in a significant reduction in the basal non-stimulated growth and sensitivity to Ana compared to K303R ERα-expressing pools, thus mimicking the same response profile of wild-type ERα clones. A selective blocking peptide (S305 peptide, residues 298-308) able to antagonize this phosphorylation reversed AIR. Blockade of S305 phosphorylation also resulted in a specific inhibition of IGF-1R/IRS-1/Akt activation, but only in the mutant cells.Our data suggest that the K303/S305 residues of the ERα mutation may be a novel determinant of aromatase inhibitor response in breast cancer, and blockade of S305 ERα phosphorylation represents a new therapeutic strategy to overcome resistance to hormonal therapies in tumors with the ERα mutation. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5053.