The K303R ERα mutation and aromatase inhibitor resistance.
Authors: Barone I , Cui Y , Herynk MH , Ando' S , Fuqua SA 
Published: December 2010

Abstract
Abstract #602
Aromatase inhibitors (AIs) are rapidly becoming the first choice for the endocrine treatment of steroid receptor positive breast cancer in postmenopausal women. However, de novo and acquired resistance is a significant problem. We have previously identified, in premalignant and invasive breast cancers, a somatic mutation at nucleotide 908 of ERα, which was significantly associated with postmenopausal status, larger tumor size, and axillary lymph node positivity. This mutation results in a lysine to arginine transition at residue 303 (K303R). We demonstrated that the K303R ERα mutation generated an estrogen hypersensitive phenotype in vitro and in vivo. Thus, we hypothesized that the mutant could provide a continuous mitogenic stimulus to the breast even during phases of low circulating hormone, such as in postmenopausal women, affording a proliferative advantage, especially during treatment with AIs. As preclinical models to directly test whether the mutation confers resistance to AIs, we generated MCF-7 parental and MCF-7-K303R-overexpressing cells stably transfected with the aromatase gene. Cells were stimulated with the aromatase substrate, androstenedione (AD), with or without the AI anastrazole (Ana). We found using MTT assays that Ana decreased AD-stimulated growth of WT vector control cells by 47%, but had no effect on AD-stimulated growth of MCF-7-K303R cells. Furthermore, in soft agar assays, the basal non-stimulated colony number of the mutant cells was increased 10-fold, and the AI was unable to inhibit the formation of mutant colonies. Aromatase activity of both cells was reduced by 95% by Ana, suggesting that this drug resistance cannot be explained by an intrinsic insensitivity of the aromatase enzyme itself to Ana.&#x2028; To examine the molecular mechanisms associated with the resistance of the mutant-expressing cells, we examined short-term nongenomic signaling, and found constitutively increased levels of phospho-IGF1R, IRS1 and AKT in the mutant cells. Interestingly, the hypersensitive mutant cells showed increased growth as well as pAkt activation with IGF1 treatment. Blockade of the IGF1R or PI3K/AKT pathways completely reversed the resistant mutant phenotype. To examine for effects on proliferation, immunoblot analyses were performed. K303R cells exhibited decreased levels of the CDK inhibitor p21 with AI treatment. Accordingly, apoptosis was decreased in the mutant cells with an increased ratio of bcl-2/Bax and decreased PARP cleavage.&#x2028; We conclude that the mutation indeed confers resistance to an AI. Potential mechanisms of resistance include cellular strategies to evade apoptosis and increase proliferation, possibly through enhanced cross-talk with growth factor pathways and reception of downstream signaling networks, such as AKT. Our results suggest that the mutation might be a new predictive marker of response to AIs in mutation-positive breast tumors, and highlight the importance of novel approaches toward the development of signal transduction inhibitors for treating patients with tumors resistant to hormone therapy.
Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 602.