Abstract 2293: Weak or partial agonism of ERα during long term estrogen deprivation drives unique mechanisms of estrogen-independent growth
Authors: Sikora Matthew J. , Johnson Michael D. , Rae James M. 
Published: June 2012

Abstract
Endocrine therapy remains the cornerstone of treatment of estrogen-receptor positive breast cancer, with aromatase inhibitors (AIs) the current front-line therapy in post-menopausal women. Despite extending disease-free survival, 15-20% of patients receiving adjuvant AIs will relapse within 5 years. Long term estrogen deprivation (LTED) of breast cancer cells in culture has been successfully used to mimic AI-induced estrogen depletion to dissect mechanisms of AI resistance, implicating several pathways in estrogen-independent growth. However, patients treated with the AI anastrozole on the ATAC trial were reported to have average serum estrogen (E2) concentrations of 3pM. Importantly, in vitro growth assays with MCF-7 cells show that 3pM E2 is sufficient to induce growth. Further, 5α-androstane-3β,17β-diol (3βAdiol) is an androgen metabolite generated independent of aromatase, and thus is insensitive to AI therapy. We have demonstrated that 3βAdiol is an ERα agonist and can induce the growth of breast cancer cells. We sought to expand upon established LTED models to account for these observations. We hypothesize that weak or partial agonism of ERα during LTED by low concentrations of E2 or by 3βAdiol may result in the development of estrogen-independence different to that seen during complete estrogen deprivation.
To model the effects of long term low concentrations of E2 or 3βAdiol, MCF-7 cells were grown in medium with charcoal stripped serum supplemented with defined hormone concentrations. Cells were selected in the EC10 and EC90 of E2 (1pM (LE) and 50pM (HE), respectively) or 3βAdiol (50pM (L3) and 1nM (H3), respectively), or estrogen deprived (ED – with 0.05% ethanol vehicle) for 7 months. Estrogen-independence was evaluated throughout selection by assessing cell growth in the absence or presence of E2 or 3βAdiol.
ED cells developed estrogen-independence at 9-12 weeks, and are minimally responsive to E2 or 3βAdiol. H3, L3 and LE cells developed the ability to grow in estrogen-free conditions by 3-4 months, 4-5 months, and 6-7 months, respectively. L3 and LE cells are minimally responsive to E2/3βAdiol, while H3 cells remain fully responsive to E2/3βAdiol compared to parental MCF-7. HE cells continue to mimic parental cells after selection. Estrogen-free growth is blocked by anti-estrogen in LE and L3 cells; ED and H3 cells are anti-estrogen resistant. Western blot analysis demonstrated that LE and L3 cells maintain ERα activation (via ERα phosphorylation) in the absence of hormone, correlating with activated MAPK and Akt. cDNA expression analysis suggests distinct mechanisms of estrogen-independence in ED versus H3 cells. The presence of low concentrations of E2 or 3βAdiol selected unique mechanisms of estrogen-independence versus traditional LTED. These long term selections may represent novel and clinically relevant models of AI resistance.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2293. doi:10.1158/1538-7445.AM2011-2293