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

Abstract

Many estrogen receptor α (ERα)-positive breast cancers develop resistance to endocrine therapy via mutation of ERs whose constitutive activation is associated with shorter patient survival. Because there is now a clinical need for new antiestrogens (AE) against these mutant ERs, we describe here our development and characterization of three chemically novel AEs that effectively suppress proliferation of breast cancer cells and tumors. Our AEs are effective against wild-type and Y537S and D538G ERs, the two most commonly occurring constitutively active ERs. The three new AEs suppressed proliferation and estrogen target gene expression in WT and mutant ER-containing cells and were more effective in D538G than in Y537S cells and tumors. Compared with WT ER, mutants exhibited approximately 10- to 20-fold lower binding affinity for AE and a reduced ability to be blocked in coactivator interaction, likely contributing to their relative resistance to inhibition by AE. Comparisons between mutant ER-containing MCF7 and T47D cells revealed that AE responses were compound, cell-type, and ERα-mutant dependent. These new ligands have favorable pharmacokinetic properties and effectively suppressed growth of WT and mutant ER-expressing tumor xenografts in NOD/SCID-γ mice after oral or subcutaneous administration; D538G tumors were more potently inhibited by AE than Y537S tumors. These studies highlight the differential responsiveness of the mutant ERs to different AEs and make clear the value of having a toolkit of AEs for treatment of endocrine therapy-resistant tumors driven by different constitutively active ERs. Cancer Res; 77(20); 5602-13. ©2017 AACR.

Fig. 1. Proliferation of T47D cells harboring homozygous mutant ERα (introduced by CRISPR-Cas9) is suppressed by antiestrogens (AEs)

(A) Structures of the 3 AEs studied. T47D cells with WT-ERα, Y537S-ERα or D538G-ERα were cultured in (B) E2-deprived conditions or (C) with E2 (3×10⁻⁹ M) and compounds at 3×10⁻⁶ M for 6 days to evaluate the impact of compounds on cell viability. Values are the mean ± SD of 3 determinations from 3 separate experiments.

Fig. 2. Dose-dependent inhibition of T47D cell proliferation and gene expression by AEs: D538G-ERα is more effectively suppressed by AEs than Y537S-ERα

(A–E) T47D cells with WT- ERα, Y537S-ERα or D538G-ERα were cultured under estrogen-deprived conditions (in phenol red-free medium with 5% charcoal dextran-treated serum). They were treated with ligands at the concentrations indicated (3×10⁻¹¹ to 3×10⁻⁶ M), and cell proliferation was monitored after 6 days. Values are mean ± SD of 3 determinations from 3 separate experiments. (F, G) Effects of E2 and ligands on expression of the ER target genes GREB1 and PGR in T47D cells were monitored after treatments with Veh, E2 and ligands for 24 h, followed by RNA extraction and qPCR analysis. Black horizontal line indicates mRNA expression in Veh-treated WT T47D cells (set as 1), and dashed horizontal line indicates mRNA expression level in E2-treated cells. Values are the mean ± SD of 3 determinations from 2 separate experiments.

Fig. 3. Differences in the ability of compounds to induce the down-regulation of WT or mutant ERα protein in cells

T47D cells were treated with Vehicle Control, E2 or compounds (A, Fulv; B, 4-OHT; C, K-07; D, K-09; E, K-62) alone for 24 h at the concentrations indicated, and cells were subjected to in cell Western (ICW) blot analysis for evaluation of ERα protein levels.

Fig. 4. Pharmacokinetics and half-lives of new AEs in mice after sc or oral administration

(A) Pharmacokinetics (PK) of K-07 after single dose administration via sc injection (20mg/kg) or (B) oral gavage (20mg/kg). (C) PK of K-09 after single dose sc injection at 20 mg/kg or (D) oral gavage at 40 mg/kg. (E) PK of K-62 after single dose sc injection at 40 mg/kg and (F) oral gavage at 20 mg/kg. After compounds were administered, multiple plasma samples were collected from each mouse (n=4 for each experiment) over the course of 48 h. Compounds were quantified using LC-MS/MS. The data were fitted to a non-compartment PK model.

Fig. 5. New AEs show good growth suppression of MCF7 xenograft tumors and inhibition of estrogen target gene expression

NSG mice were supplemented with E2 (0.36 mg, 60-day release) pellets, injected with WT MCF7 cells to generate xenograft tumors, and dosed with 80 mg/kg of Fulv, K-07, K09 or K-62 by daily sc injection. (A) Tumor volume was monitored over time (2-way ANOVA, Bonferroni post-test, ****, P<0.0001, n=8–9 per group). (B) Harvested tumors were analyzed by qPCR for GREB1 and PGR RNA levels (1-way ANOVA, Tukey post-test, n=8–9/group). (C) E2-supplemented mice bearing MCF7 tumors were dosed daily with vehicle or 80 mg/kg of K-07 by oral gavage. Tumor volumes of Veh and K-07 treated tumors were monitored (2-way ANOVA, Bonferroni post-test, ****, P<0.0001, n=8 per group). (D) After 28 days, tumors were harvested at 24 h after the last control Veh or K-07 oral treatment for gene expression analysis (T-test, ****, P<0.0001, n=8 per group) and (E) Western blot examination of GREB1 and ERα protein in individual tumors. β-actin serves as the loading control. (F) Quantitation of ERα and GREB1 protein in Veh and K-07 treated tumors. (T-test, ****, P<0.0005 n=5 Veh and n=6 K-07 tumors).

Figure 6. Y537S ERα- and D538G ERα-containing tumors grow in the absence of estrogen and are arrested by K-07 treatment

NSG mice were ovariectomized and 3 weeks later received MCF7 cells containing half Y537S ER or D538G ER and half wild type ER. (A, B) Mice then received daily sc injection with Vehicle or 80 mg/kg of K-07 or Fulv, and tumor volumes were monitored over time (2-way ANOVA, Bonferroni post-test, ****, P<0.0001, n=8 per group). (C) Mice received daily treatment with oral vehicle or oral K-07 at 80 mg/kg, and tumor volumes were monitored over time (2-way ANOVA, Bonferroni post-test, P<0.0001, n=8 per group). (D, E) Tumors harvested at day 26 were analyzed for expression of the estrogen target genes GREB1 and PGR. In D, 1-way ANOVA, Tukey post-test, n = 8 per group. In E, T-test, ****, P<0.0001, n=8 per group. (F) Harvested tumors were also analyzed for ERα protein by Western blot of tumor lysates [some D538G tumors were too small for Western analyses so only 3 tumors are shown here]; and by (G) immunohistochemistry for ERα in tumor tissue sections.