Mutation site and context dependent effects of ESR1 mutation in genome-edited breast cancer cell models

Abstract

Background: Mutations in the estrogen receptor alpha (ERα) 1 gene (ESR1) are frequently detected in ER+ metastatic breast cancer, and there is increasing evidence that these mutations confer endocrine resistance in breast cancer patients with advanced disease. However, their functional role is not well-understood, at least in part due to a lack of ESR1 mutant models. Here, we describe the generation and characterization of genome-edited T47D and MCF7 breast cancer cell lines with the two most common ESR1 mutations, Y537S and D538G.

Methods: Genome editing was performed using CRISPR and adeno-associated virus (AAV) technologies to knock-in ESR1 mutations into T47D and MCF7 cell lines, respectively. Various techniques were utilized to assess the activity of mutant ER, including transactivation, growth and chromatin-immunoprecipitation (ChIP) assays. The level of endocrine resistance was tested in mutant cells using a number of selective estrogen receptor modulators (SERMs) and degraders (SERDs). RNA sequencing (RNA-seq) was employed to study gene targets of mutant ER.

Results: Cells with ESR1 mutations displayed ligand-independent ER activity, and were resistant to several SERMs and SERDs, with cell line and mutation-specific differences with respect to magnitude of effect. The SERD AZ9496 showed increased efficacy compared to other drugs tested. Wild-type and mutant cell co-cultures demonstrated a unique evolution of mutant cells under estrogen deprivation and tamoxifen treatment. Transcriptome analysis confirmed ligand-independent regulation of ERα target genes by mutant ERα, but also identified novel target genes, some of which are involved in metastasis-associated phenotypes. Despite significant overlap in the ligand-independent genes between Y537S and D538G, the number of mutant ERα-target genes shared between the two cell lines was limited, suggesting context-dependent activity of the mutant receptor. Some genes and phenotypes were unique to one mutation within a given cell line, suggesting a mutation-specific effect.

Conclusions: Taken together, ESR1 mutations in genome-edited breast cancer cell lines confer ligand-independent growth and endocrine resistance. These biologically relevant models can be used for further mechanistic and translational studies, including context-specific and mutation site-specific analysis of the ESR1 mutations.

Keywords: ESR1 mutations; Endocrine resistance; Genome-edited cells; Metastatic breast cancer; RNA-seq.

Fig. 1

Generation and characterization of ESR1 mutant, genome-edited MCF7 and T47D cell line models. a ESR1 mutation allele frequency in DNA and RNA was determined by digital droplet PCR. b T47D and MCF7 wild-type (WT) or mutant clones were pooled and treated with vehicle, 1 nM estradiol (E2) or 1 μM of fulvestrant (Ful) for 24 h, and lysates were immunoblotted as indicated. The blot is representative of three independent experiments. ER estrogen receptor. c T47D and MCF7 clones were pooled after hormone deprivation, transfected with ERE-TK, and relative light units (RLU) were determined (one-way analysis of variance (Anova), **p < 0.01). The experiment was repeated three times and the figure shows one representative experiment with two biological replicates. d Hormone-deprived T47D and MCF7 cells were treated with vehicle, 1 nM E2, 1 μM fulvestrant or 1 nM E2 with 1 μM fulvestrant for 12 h, and RNA was isolated, and RT-qPCR was performed (one-way Anova for comparison of basal level, Student’s t test for comparison of fulvestrant response in the presence of E2, *p < 0.05, **p < 0.01)

Fig. 2

ESR1 mutant cells exhibit ligand-independent growth. T47D (a) and MCF7 (b) wild-type (WT) or mutant clones were hormone-deprived for 3 days, pooled, treated with vehicle or 1 nM estradiol (E2) for up to 8 days, and cell numbers were quantified by the FluoResporter kit. Growth fold change (FC) was normalized to day 0: **p < 0.01, one-way analysis of variance, comparison of FC growth between WT and mutant cells on the last day. The experiment was repeated three times with six biological replicates, and similar results were obtained

Fig. 3

ESR1 mutant-cells display resistance against selective estrogen receptor modulators (SERMs) and selective estrogen receptor degraders (SERDs). Graphical (a) and tabular (b) presentation of half maximal inhibitory concentration (IC50) values that were determined in dose–response curves in wild-type (WT), Y537S and D538G cells treated with 20 pM estradiol (E2) plus varying doses of 4OHT, raloxifene (Ral), fulvestrant (Ful), and AZD9496 in T47D and MCF7 cell lines. One-way analysis of variance was performed to compare the IC50 values of mutants to WT within each cell line and drug (*p < 0.05, **p < 0.01). Each dot is representative of the mean of a single experiment with six biological replicates. The experiments were performed six times (T47D) or eight times (MCF-7). c Pooled T47D-WT and T47D-D538G cells were mixed at a ratio of 99:1 and grown in 10% FBS, 10% CSS, 10% CSS + 1 nM E2 + 100 nM 4OHT, or 10% CSS + 1 nM E2 + 30 nM fulvestrant. The mutation allele frequency was analyzed at each passage using digital droplet PCR

Fig. 4

Genome-wide transcriptomic analysis reveals regulation of ligand-independent estrogen receptor (ER) targets, and of novel target genes by ERα mutants in T47D and MC7 cells. a T47D and MCF7 cell lines were hormone-deprived for 3 days, treated with vehicle (veh) or 1 nM of estradiol (E2) for 24 h, RNA was isolated and RNA sequencing analysis was performed. The heat map shows normalized log2 fold change (FC) of genes differentially regulated in mutants vs wild-type (WT) in the absence of ligand (FC >2, p value <0.005). The genes are sorted based on E2 regulation in WT (red arrow ligand-independent E2 activated genes, blue arrow ligand-independent E2 downregulated genes, green circle ligand-independent non-E2 regulated genes, i.e. “novel target genes”). b Hormone-deprived T47D and MCF7 cells were treated with veh, 1 nM E2, 1 μM of fulvestrant (Ful) or 1 nM E2 plus 1 μM of Ful for 24 h. RNA was isolated, and GREB1 or insulin-like growth factor-binding protein 4 (IGFBP4) expression was assessed by quantitative RT-qPCR (one-way analysis of variance (Anova) for comparison of basal level, Student’s t test for comparison of Ful response in the presence of E2, *p < 0.05, **p < 0.01). The experiment was repeated twice with three biological replicates each time. c Cells were hormone-deprived, treated with 1 nM of E2 for 45 minutes, and chromatin-immunoprecipitation (ChIP) assays were performed on the ER binding sites on GREB1 and IGFBP4 promoters. The data are presented as fold enrichment compared to IgG control (one-way Anova, **p < 0.01). The experiment was repeated twice with three biological replicates each time and the figure shows a representative experiment. d The chi-square test was used to assess the statistical significance of overlaps in venn diagrams. Left panel overlap of E2-regulated genes in WT cells between the cell lines (chi-square test, **p < 0.01). Right panel overlap of ligand-independent target genes between different mutations within each cell line and between the two cell lines (chi-square test, **p < 0.01)