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. Author manuscript; available in PMC: 2018 Mar 1.
Published in final edited form as: Cancer Discov. 2016 Dec 16;7(3):277–287. doi: 10.1158/2159-8290.CD-15-1523

Figure 1. Newly detected ESR1 mutations exhibit a range of estrogen-independent activities.

Figure 1

(A) Diagram of ESR1 Ligand Binding Domain with somatic mutations identified from 929 breast tumors analyzed. Height of the circles correlates to the number of cases with that specific mutation. The color codes of the circles are as follow: green for missense mutations, red for truncating mutations (Nonsense, Nonstop, Frameshift deletion, Frameshift insertion, Splice site) and black for in frame mutations. (B) Activation of ER reporter gene. ER+ MCF7 cells were transfected with empty vector, HA-ERα wild type (WT) or indicated ESR1 mutation, ERE-luciferase and Renilla luciferase reporter constructs in hormone-depleted medium with 10 nM of E2 added for 24 hours where indicated. Firefly luciferase activity shows increased activity in absence of E2 or presence of E2 for certain mutations. Graphs were plotted with the mean ± SD of three biological replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (C) Activation of ER target genes. MCF7 cells were transfected with empty vector, HA-ERα WT or mutant in hormone-depleted medium and harvested 48 hours post-transfection for qRT-PCR analysis. Bars represent mean ± SD of three technical replicates normalized to actin (ACTB) expression. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (D) Activation of ER phosphorylation in MCF7 cells. Expression level of the mutant HA-tagged ERs and their relative phosphorylation status at Serine118 and Serine 167, treated with or without 10 nM E2 for 24 hours by immunoblot analysis with specific antibodies as indicated. (E) Activation of hormone independent cell proliferation. Doxycycline inducible ER mutant receptors (E380Q, S463P, L536R and Y537S) expressing MCF7 cells were seeded in 96-well plates in hormone-depleted medium with or without the addition of doxycycline and proliferation was assayed using resazurin regeant. Data show sufficiency of these 4 mutants to promote cell growth in the absence of estradiol. Each point in the graph represented mean ± SD of 6 technical replicates. (F) Binding of the SRC3 NRD to Y537S, D538G, E380Q or S463P ERα LBD in the absence or presence of E2. SRC3 was titrated into a fixed amount of ER-LBD-biotin and time-resolved Förster resonance energy transfer (tr-FRET) indicated that only Y537S and D538G were able to recruit SRC3 in the absence of E2 but not E380Q and S463P. LBD, ligand-binding domain.