Somatic estrogen receptor α mutations that induce dimerization promote receptor activity and breast cancer proliferation
Seema Irani, Wuwei Tan, Qing Li, Weiyi Toy, Catherine Jones, Mayur Gadiya, Antonio Marra, John A. Katzenellenbogen, Kathryn E. Carlson, Benita S. Katzenellenbogen, Mostafa Karimi, Ramya Segu Rajappachetty, Isabella S. Del Priore, Jorge S. Reis-Filho, Yang Shen, Sarat Chandarlapaty
Seema Irani, Wuwei Tan, Qing Li, Weiyi Toy, Catherine Jones, Mayur Gadiya, Antonio Marra, John A. Katzenellenbogen, Kathryn E. Carlson, Benita S. Katzenellenbogen, Mostafa Karimi, Ramya Segu Rajappachetty, Isabella S. Del Priore, Jorge S. Reis-Filho, Yang Shen, Sarat Chandarlapaty
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Research Article Endocrinology Oncology

Somatic estrogen receptor α mutations that induce dimerization promote receptor activity and breast cancer proliferation

Abstract

Physiologic activation of estrogen receptor α (ERα) is mediated by estradiol (E2) binding in the ligand-binding pocket of the receptor, repositioning helix 12 (H12) to facilitate binding of coactivator proteins in the unoccupied coactivator binding groove. In breast cancer, activation of ERα is often observed through point mutations that lead to the same H12 repositioning in the absence of E2. Through expanded genetic sequencing of breast cancer patients, we identified a collection of mutations located far from H12 but nonetheless capable of promoting E2-independent transcription and breast cancer cell growth. Using machine learning and computational structure analyses, this set of mutants was inferred to act distinctly from the H12-repositioning mutants and instead was associated with conformational changes across the ERα dimer interface. Through both in vitro and in-cell assays of full-length ERα protein and isolated ligand-binding domain, we found that these mutants promoted ERα dimerization, stability, and nuclear localization. Point mutations that selectively disrupted dimerization abrogated E2-independent transcriptional activity of these dimer-promoting mutants. The results reveal a distinct mechanism for activation of ERα function through enforced receptor dimerization and suggest dimer disruption as a potential therapeutic strategy to treat ER-dependent cancers.

Authors

Seema Irani, Wuwei Tan, Qing Li, Weiyi Toy, Catherine Jones, Mayur Gadiya, Antonio Marra, John A. Katzenellenbogen, Kathryn E. Carlson, Benita S. Katzenellenbogen, Mostafa Karimi, Ramya Segu Rajappachetty, Isabella S. Del Priore, Jorge S. Reis-Filho, Yang Shen, Sarat Chandarlapaty

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Figure 1

Activating ESR1 mutations outside helix 12 of the LBD.

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Activating ESR1 mutations outside helix 12 of the LBD. (A) ESR1 mutation...
(A) ESR1 mutations (n = 649) in breast cancer samples from the MSK clinical sequencing cohort. NTD, N-terminal domain; DBD, DNA-binding domain; LBD, ligand-binding domain; CTD, C-terminal domain. (B) Mapping of a few of the ESR1 mutations onto the structure of the ERα-LBD (Protein Data Bank ID 1GWR). Residues in the vicinity of the dimer interface are shown in red (a red arrow indicates the disordered region having residue S463), and those closer to the H11–H12 loop are shown in blue. (C) Luciferase reporter assay of MCF7 cells transfected with the HA-tagged ESR1 mutants/WT or empty vector (EV), estrogen response element (ERE)–luciferase reporter, and Renilla luciferase reporter plasmids. The graph represents individual data points and mean ± SD (n = 3), with P values (Welch’s t test) for mutant versus WT indicated. (D) Cell viability of doxycycline-inducible (Dox-inducible) MCF7 cells and parental cells with or without 10 nM E2 growing in hormone-depleted medium supplemented with 0.5 μg/mL Dox; plotted as mean ± SD (n = 6), with P values calculated from Welch’s t test for mutant versus parental cells on the final day indicated. (E) Plot of the percentage increase in confluence from initial time point for the Dox-inducible MCF7 cells, growing in hormone-depleted medium with 0.5 μg/mL of Dox. Data are plotted as mean ± SEM (n = 6); statistical analysis was performed using 2-way ANOVA for mutant versus WT on the final day indicated. (F) MCF7 HA-ESR1 WT or mutant–expressing cell-derived xenograft tumor growth represented as mean ± SEM (n = 3–6 mice per group). The estrogen pellet was removed after the tumor volume reached 250 mm3, and mice were fed with Dox to induce HA-tagged ESR1 expression. Statistical analysis was performed using 2-way ANOVA on the final day indicated. F461V had 2 mice after day 16 and hence was not included in statistical analysis. (G and H) Growth inhibition of MCF7 cells expressing HA-ESR1 mutants or empty vector, as measured by cell viability assay, in the presence of fulvestrant (G) and elacestrant (H); EC50 values for sigmoidal fit are presented in Supplemental Tables 2 and 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.