Activating ESR1 mutations in hormone-resistant metastatic breast cancer

Abstract

Breast cancer is the most prevalent cancer in women, and over two-thirds of cases express estrogen receptor-α (ER-α, encoded by ESR1). Through a prospective clinical sequencing program for advanced cancers, we enrolled 11 patients with ER-positive metastatic breast cancer. Whole-exome and transcriptome analysis showed that six cases harbored mutations of ESR1 affecting its ligand-binding domain (LBD), all of whom had been treated with anti-estrogens and estrogen deprivation therapies. A survey of The Cancer Genome Atlas (TCGA) identified four endometrial cancers with similar mutations of ESR1. The five new LBD-localized ESR1 mutations identified here (encoding p.Leu536Gln, p.Tyr537Ser, p.Tyr537Cys, p.Tyr537Asn and p.Asp538Gly) were shown to result in constitutive activity and continued responsiveness to anti-estrogen therapies in vitro. Taken together, these studies suggest that activating mutations in ESR1 are a key mechanism in acquired endocrine resistance in breast cancer therapy.

Figure 1

Clinical timelines of the six index ER-positive metastatic breast cancer patients harboring ESR1 mutations. Shown are patients’ histories of clinical treatments from first diagnosis until the enrollment on the MI-ONCOSEQ study. Each bar represents the timeframe of a treatment.

Figure 2

Schematic representation of ESR1 mutations identified in this study. The structural domains of ESR1 are illustrated on top, including the transcription activation function-1 domain (AF-1), the DNA-binding domain (DBD), the hinge domain, and the ligand-binding domain (LBD/AF-2). Changed residues identified in mutants are marked in red, and the reference residues are bolded in the wild type sequence. Endometrium p.Tyr537Cys (Y537C) and p.Tyr537Asn (Y537N) are two mutations discovered in endometrial cancer from the TCGA study. Inv-mut-AA2 represents a ligand activity inversion mutant of ESR1 which renders the receptor with inverted response to anti-estrogen and estrogen. H11, helix 11; H12, helix 12.

Figure 3

Acquired ESR1 mutations are constitutively active. HEK-293T cells were co-transfected with an ERE-firefly luciferase reporter plasmid, a plasmid constitutively expressing Renilla luciferase as an internal control, and various ESR1 constructs as illustrated in Fig 2. Steroid hormone-deprived cells were either untreated (C) or stimulated with 5 nM of β-estradiol (E2) for 24 hrs. Firefly luciferase levels were normalized using corresponding Renilla luciferase levels for each condition. Fold change of ESR1 transcription activity was calculated using untreated wild type as control for each condition. Data shown are mean of triplicate. Amino acid mutations in respective ESR mutants are indicated. WT, wild-type ESR1.

Figure 4

Acquired ESR1 mutations maintain sensitivity to antiestrogen therapies. As described in Fig 3, HEK-293T cells were co-transfected with an ERE-firefly luciferase reporter plasmid, a plasmid constitutively expressing Renilla luciferase, and various ESR1 constructs as indicated. Steroid hormone-deprived cells were either untreated or treated with increasing doses of antiestrogen drugs tamoxifen (A) or fulvestrant (B) in the presence of 5 nM of β-estradiol (E2) for 24 hrs. Percentage change of ESR1 transcription activity was calculated using E2-treated cells as control for each tested construct. Data shown are mean of triplicate. Error bars indicate s.d. *, P values <0.001.