Figure 3 from ESR1 F404 Mutations and Acquired Resistance to Fulvestrant in ESR1-Mutant Breast Cancer

F404 does not activate estrogen signaling. A, CRISPR clones of MCF7 cells expressing ESR1 F404L (1210T>C, CRISPR edit indicated by red arrows) or D538G (1613A>G; CRISPR edit indicated by black arrows) were identified by RT-PCR followed by Sanger sequencing (left-hand panels). Similarly, a second round of CRISPR was used to introduce ESR1 F404L (1210T>C) into a clone (D6C) that expressed D538G (1613A>G; right-hand panels). B, Estrogen-dependent growth was assessed in colony formation assay. Parental MCF7 cells and indicated ESR1-mutant models were grown in either the absence or presence of estradiol (1 nmol/L) for 14 days. C, Quantification of colony formation assays of ESR1-mutant models treated with and without estradiol (1 nmol/L). Sulforhodamine B (SRB)-stained colonies were dissolved, and absorbance at 565 nm was measured. Mean with SEM, n = 3 independent experiments, nonparametric one-way ANOVA with Dunn multiple comparisons test; **, P < 0.01. D, Expression of estrogen target genes, progesterone receptor (PgR), and trefoil factor-1 (TFF1), assessed by western blot in parental MCF7 cells and indicated ESR1-mutant models grown in either the absence or presence of estradiol (1 nmol/L) for 24 hours. E, MCF7 cells were transfected with ESR1 expression constructs with indicated ESR1 variants. Expression of ERα was determined by western blot. F, MCF7 cells were cotransfected with the indicated ESR1 expression constructs ERE-luciferase reporter and control construct. Cells were treated in either the absence or presence of estradiol (1 nmol/L) for 24 hours, and ERE-luciferase activity was assessed. Two-way repeated-measures ANOVA with Dunnett multiple comparisons test, n = 4 mean with SD; *, P < 0.05. WT, wild-type.