PMC

Altered apoptotic response in mutant cells. A-C. Immunoblotting showing PARP and Rho GDIα in cells treated with vehicle, E₂1nM (24h), AD10nM (12, 24 and 48h, A) or AD10nM and AD+Ana1µM (24h, B); Bcl-2, Bax and Rho GDIα in cells treated with vehicle, AD10nM±Ana1µM (24h, C). D. Quantitative analysis is the fold difference in Bcl-2/Bax ratio relative to vehicle-treated MCF-7 Arom 1-cells. E. Elisa cell detection assay in cells treated with AD10nM±Ana1µM. Columns, mean. Bars, SD. *P<0.05, **P<0.005, n.s.=nonsignificant AD versus Ana+AD.

Inhibition of PI3K/Akt pathway reversed AI^R. A. Immunoblotting showing phospho-Akt and Akt in cells treated with vehicle, LY10µM or PI-103 (1, 5, 10µM) or Akti1/2 (1, 5, 10µM) for 30min. B-D. Anchorage-independent growth assay in cells treated with vehicle, AD10nM, Ana1µM ±LY10µM (B) or Akti1/21µM (C) or PD98059 (PD10µM, D) or ICI1µM (D). Bars, SD. ***P<0.0005 LY, Akti1/2 and ICI-treated cells versus control cells and cells treated with AD or Ana+AD. E. Cell death detection assay in cells treated with AD10nM ±Ana1µM±LY. Columns, mean. Bars, SD. *P<0.05 versus AD in MCF-7 Arom 1 and AD and Ana+AD in K303R Arom 1.

K303R ERα mutation confers resistance to anastrozole. A. MTT growth assays in cells treated with vehicle, E₂1nM, AD10nM and/or anastrozole (Ana100nM, 1µM, 10µM). Cell proliferation is expressed as fold change relative to vehicle-treated cells. Data are representative of three independent experiments, performed in quadruplicate. Columns, mean. Bars, SD. **P<0.005, n.s.=nonsignificant AD versus Ana+AD. B. Anchorage-independent growth assay in cells treated with vehicle, E₂1nM, AD10nM±Ana1µM. Bars, SD. *P=0.01, n.s.=nonsignificant AD versus Ana+AD. C-D. Anchorage-independent growth assay in MCF-7 Arom P (C) and CHO P (D) pools treated with vehicle, AD10nM±Ana1µM. Bars, SD. **P<0.01, n.s.=nonsignificant AD versus Ana+AD. Upper panels for C-D showed ERα and Rho GDIα. Numbers below blots represent aromatase activity.

Characterization of K303R ERα-expressing cells. A. Immunoblotting showing ERα and Rho GDIα (loading control) in MCF-7 parental (MCF-7) and YFP-K303R ERα-cells (MCF-7 K303R). B. Anchorage-independent growth assay in cells treated with vehicle or 17β-Estradiol (E₂ 10⁻¹², 10⁻¹¹, 10⁻¹⁰, 10⁻⁹M). Bars, SD. n.s.=nonsignificant, **P<0.005, ***P<0.0005 versus vehicle. C. WT ERα and K303R ERα–cells were injected into mice (n=5/group) supplemented with E₂. Tumor growth was plotted as the time for tumor tripling (months[mos.]). D. Immunoblotting showing aromatase (Arom), ERα and Rho GDIα. Numbers below blots represent aromatase activity. E. Anchorage-independent growth assay in cells treated with vehicle or 4-Androstene-3,17-dione (AD1, 10 and 25nM). Bars, SD. *P<0.05, **P<0.01, ***P<0.0005: K303R Arom 1-group versus the respective treatment in MCF-7 Arom 1-group. F. MCF-7 Arom 1 and K303R Arom 1-cells were injected into mice, supplemented with AD. Tumor growth was plotted as the percentage of change in mean tumor volume/animal (n=5) compared to day 0 of treatment ±S.D. *P<0.05, **P<0.01 K303R Arom 1-group versus MCF-7 Arom 1-group.

Mutant cells exhibited constitutive pAkt. A-F. Immunoblotting showing phospho-Akt (pAkt), Akt and Rho GDIα in cells treated with vehicle or AD 1, 10, 25 and 50nM (1h, A) or AD 10, 30, 60, 120min (10nM, B) or heregulin2ng/ml (H 5min, B); in xenograft tumors extracts (C, numbers below blots represent the average fold change in pAkt levels of K303R Arom 1-samples versus MCF-7 Arom 1-samples); in cells treated with vehicle, AD10nM±Ana1µM (1h, D); in cells treated with vehicle and ICI1µM for 1, 5, 10 and 30min (E) or 1, 3, 24 and 48h (F). Quantitative analysis is the fold difference in pAkt/Akt/Rho GDIα ratio relative to vehicle-treated MCF-7 Arom 1-cells.

Mutant cells showed increased PI3K/Akt activation. A. Lysates from CHO cells transiently transfected with WT or K303R ERα were immunoprecipitated (IP) with anti-p85 or anti-ERα and immunoblotted for ERα or p85. Input: whole-cell lysates. B, C. In vitro PI3-kinase activity of p85 immunoprecipitates from CHO cells transiently transfected with YFP-WT or YFP-K303R ERα, and CHO WT P and CHO K303R P pools (B) or MCF-7 Arom 1 and K303R Arom 1-cells treated with vehicle or LY294002 (LY10µM) for 45min (C). The enzyme activity was expressed as amounts of PIP₃ (picomoles) produced by cells. Columns, mean. Bars, SD. Values are means from triplicate readings ±S.D. in two experiments. *P<0.05, **P=0.001. Upper panels for H-I showed p85 expression in immunoprecipitates. L. Immunoblotting of pAkt, Akt, phospho-Ser167-ERα (pS167), YFP-ERα and Rho GDIα in CHO cells transiently transfected with YFP-WT or YFP-K303R ERα, or CHO WT P and CHO K303R P pools. E. CHO cells were transiently transfected with YFP-WT or YFP-K303R ERα. plus an ERE-luciferase reporter, and treated with vehicle, LY10µM or ICI1µM. Luciferase activities were normalized to β-galactosidase for relative luciferase units. Data are representative of three independent experiments, performed in triplicate. Columns, mean. Bars, SD. ***P=0.0008 versus vehicle. ERα and Rho GDIα expression was determinated by immunoblot (right panel).