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

Abstract

BackgroundMutations 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.MethodsGenome 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.ResultsCells 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.ConclusionsTaken 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-017-0851-4) contains supplementary material, which is available to authorized users.

Structural underpinnings of oestrogen receptor mutations in endocrine therapy resistance

“…The D538G mutation, observed in ~20% of patients with AI-treated metastatic breast cancers 9,12,20,22 , in the h11–12 loop of the ERα LBD, has constitutive activity comparable to or somewhat less than that of Y537S (Figure 2B) 24,27,28 . Structural data shows that the charged D538 residue in WT apo-ERα introduces a kink in the protein backbone driven by its strong preference for solvent exposure and electrostatic repulsion from other nearby acidic residues (e.g., D351), and it initiates the helical character at the start of h12 16 .…”

Structurally Novel Antiestrogens Elicit Differential Responses from Constitutively Active Mutant Estrogen Receptors in Breast Cancer Cells and Tumors

“…Thus, in T47D cells, Y537S elicited a more endocrine-resistant phenotype, whereas in the MCF7 cell background the Y537S and D538G mutant ERs were more similar to each other in their response to antiestrogens. This suggests that cell context, including alterations in genomic and cell signaling pathways, may work with ERs to confer different cell behaviors and responsiveness or resistance to treatment with different endocrine agents (23,31,32). This is not surprising, since it is known, for example, that among other differences, MCF7 and T47D cells carry different mutant forms of PI3K.…”

“…T47D cells with both mutant ERα alleles were generated by CRISPR-Cas9 technology and mutation status of the clonal cell lines was verified by genotyping as described before (21). MCF7 cells or T47D cells containing 50% mutant Y537S- or D538G-ERα and 50% wild type ERα, determined by DNA sequencing and digital drop PCR analyses as detailed previously (17,23), were generated by adenovirus associated viral infection (MCF7) or CRISPR-Cas-9 methodology (T47D) (17,21,23,24). These cells were cultured as described(17,21,23,24).…”

ESR1 mutations in breast cancer

“…[20][21][22] Using in vitro-derived ESR1 mutant cell line models generated using CRISPR/Cas9 technology, or through natural selection of cells in hormone-deprived conditions, Martin et al 23 showed that there was a high overlap between ER chromatin binding sites of estrogen-stimulated WT receptor and hormone-deprived mutant receptors. [20][21][22] Using in vitro-derived ESR1 mutant cell line models generated using CRISPR/Cas9 technology, or through natural selection of cells in hormone-deprived conditions, Martin et al 23 showed that there was a high overlap between ER chromatin binding sites of estrogen-stimulated WT receptor and hormone-deprived mutant receptors.…”

“…The transcriptomes of WT and mutant ERs have been described by several groups, showing there are shared, classical ER signaling signatures, as well as mutant-specific transcriptional regulation. [20][21][22] Using in vitro-derived ESR1 mutant cell line models generated using CRISPR/Cas9 technology, or through natural selection of cells in hormone-deprived conditions, Martin et al 23 showed that there was a high overlap between ER chromatin binding sites of estrogen-stimulated WT receptor and hormone-deprived mutant receptors. They further showed that estrogen treatment of both the WT and Y537S models exhibited a 74% concordance in ER binding sites.…”