Abstract P3-05-17: Haddow's paradox revisited: Anti-estrogen withdrawal induces ER-driven apoptosis in anti-estrogen-resistant cells

Anti-estrogen therapies that antagonize ER transcriptional activity have improved survival in many patients. However, resistance to anti-estrogen therapies is common, resulting in disease recurrence in ∼20% of patients within 10 years of follow-up. The current clinical strategy for managing ER+/HER2- breast cancer is treatment with anti-estrogens until disease progression, followed by subsequent lines of anti-estrogens and chemotherapies. However, before tamoxifen was approved in the 1970s, estrogens were used for treatment of breast cancer with response rates similar to those elicited by anti-estrogens in the advanced setting. Similarly, withdrawal of anti-estrogen therapy has shown anti-tumor effects in some cases. In 1970, Alexander Haddow summarized decades of observations that longer time intervals after menopause were associated with increased clinical benefit from estrogen therapy. These studies suggest that the return of ER signaling after a prolonged period of ER suppression (via menopause, or anti-estrogen therapy) may inhibit tumor growth. Preclinical studies have provided some insight on the mechanism underlying estrogen-induced apoptosis, but the anti-tumor effects of estrogen remain unclear, and biomarkers to identify patients that would benefit from estrogen therapy have not been identified. We found that MCF-7 cells with acquired resistance to the selective ER downregulator fulvestrant (MCF-7/FR) retain ER expression and harbor ESR1 (ER) gene amplification. Upon withdrawal of fulvestrant, these cells engage ER as demonstrated by increased luciferase transcriptional reporter activity and re-expression of select proteins encoded by ER-inducible genes. Following 8 days of fulvestrant withdrawal, MCF-7/FR cells show drastically decreased proliferation and increased apoptosis, concomitant with re-engagement of ER activity. Temporal cell cycle analysis revealed a reduction of cells in G0/G1 phase following 11 days of fulvestrant withdrawal, which paralleled decreases in cell cycle proteins E2F1 and phospho-Rb, as well as the anti-senescence protein FoxM1. Transcriptomic analyses confirmed that fulvestrant withdrawal progressively induces gene expression patterns indicative of stress and senescence. Similar effects were observed in long-term estrogen-deprived (LTED) MCF-7 cells treated with 17b-estradiol. In contrast, withdrawal of fulvestrant from T47D/FR cells, which do not retain ER expression, does not induce cell death or re-engage ER activity. Thus, re-engagement of ER in anti-estrogen-resistant cells may be required for anti-tumor effects of anti-estrogen withdrawal. We postulate that in MCF-7/FR cells relieved of fulvestrant, high ER levels drive aberrantly regulated transcription through ER hyperactivation, and altered ER/DNA binding patterns result in estrogen-induced apoptosis. Gene expression signatures suggest that fulvestrant withdrawal from MCF-7/FR cells induces downregulation of the transcription suppressive activity of the Polycomb Repressive Complex 2 (PRC2) that contains EZH2 methyltransferase. Ongoing studies are characterizing the roles of EZH2 and ER to determine therapeutic potential for anti-estrogen-resistant breast cancer. Citation Format: Sarah R Hosford, Todd W Miller. Haddow9s paradox revisited: Anti-estrogen withdrawal induces ER-driven apoptosis in anti-estrogen-resistant cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-17.