Abstract P4-04-08: Therapeutic strategy for ERα mutation driven-endocrine resistance in ER-positive breast cancers

Abstract

Abstract Background: Although estrogen receptor (ER)-positive breast cancers are treated with endocrine therapy, 25% of these patients are at risk of relapse and the development of acquired endocrine resistance. Recently mutations in the ER gene (ESR1) have been identified which induce resistance to endocrine therapy. The most frequent ESR1 mutation, Y537S, promotes ligand-independent ER activity. It is known that ER regulates the cell cycle in a ligand-dependent manner. In this study, we examined the effects of the Y537S ESR1 mutation on cell cycle signaling and therapeutic response to checkpoint inhibitor. Material and Methods: MCF-7 cells expressing the Y537S ESR1 mutation were generated by CRISPR-Cas9 knock-in techniques. Cells were incubated in phenol red minus medium containing 5% charcoal-dextran treated serum for 5 days to remove exogenous hormones. Cell cycle and apoptosis were examined by Flow cytometry and Annexin-V assays. Proliferation was analyzed by BrdU incorporation. Cell senescence was determined using beta-galactosidase assays. Cell cycle checkpoint kinases were examined by western blot analysis. Cell growth was analyzed using soft agar or MTT assays. Results: Levels of p53 and apoptosis pathway proteins were significantly elevated in Y537S ESR1 mutant cells using RNA expression and reverse-phase protein microarrays. The ATM/ATR and Chk1/Chk2 mediated checkpoint signaling, the upstream pathway of p53, was activated in ESR1 Y537S mutation, which was repressed with fulvestrant treatment. ESR1 Y537 mutant cells accumulated about 5 fold in S phase and 1.7 fold in G2/M phase compared to control cells in estrogen deprived (ED) condition. BrdU incorporation was also increased about 2.5 fold compared with parental cells in estrogen-free medium. In addition, ESR1 Y537 mutant cells expressed a DNA double-strand break marker, gamma-H2AX protein in ED condition. Apoptosis and senescence were observed in ESR1 Y537S mutant cells in regular medium, however, apoptosis was not shown in ED medium. Chk1 inhibitor, PF477736 sensitized MCF-7 expressing ESR1 Y537S mutation to endocrine treatments such as fulvestrant, tamoxifen and AZD9496. Conclusion: Combination therapy with cell cycle checkpoint kinase inhibitor may lead better prognosis in ER mutation driven-endocrine resistance in postmenopausal breast cancers. Citation Format: Kim J-A, Fuqua SAW. Therapeutic strategy for ERα mutation driven-endocrine resistance in ER-positive breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-04-08.