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

Abstract

Many ERα-positive breast cancers develop resistance to endocrine therapy via mutation of estrogen receptors (ER) whose constitutive activation is associated with shorter patient survival. Because there is now a clinical need for new antiestrogens (AE) against these mutant ER, we describe here our development and characterization of three chemically novel AE that effectively suppress proliferation of breast cancer cells and tumors. Our AE are effective against wild type and Y537S and D538G ER, the two most commonly occurring constitutively active ER. The 3 new AE 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 to WT ER, mutants exhibited ~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-gamma 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 ER to different AE and make clear the value of having a toolkit of AE for treatment of endocrine therapy-resistant tumors driven by different constitutively active ER.

In silico screening for ERα downmodulators identifies thioridazine as an anti-proliferative agent in primary, 4OH-tamoxifen-resistant and Y537S ERα-expressing breast cancer cells

“…However, recently, it has been shown that a significant fraction of metastatic BC express a mutated, constitutively active variant of ERα (e.g., Y537S ERα), which confers to the disease hormone-independent growth, Tam insensitivity and resistance to ICIinduced ERα degradation [18]. In turn, new compounds that target ERα in metastatic BC as additional ET drugs are strongly required, and, indeed, new orally active antiestrogens that directly bind to and induce the degradation of the mutated ERα form [18,24,25] are being searched. Therefore, the above-described results prompted us to test the ability of thioridazine to influence the ERα intracellular content in two cellular models mimicking metastatic BC (i.e., Tam-1 4 resistant MCF-7 cells [12] and genome-edited MCF-7 cells expressing the Y537S ERα mutant [13]).…”

“…4OH-Tamoxifen resistance is a critical issue in BC management. Thus, novel drugs inducing ERα degradation in Tam-resistant (Tam-Res) metastatic BC are demandingly required [18]. Because we found that thioridazine reduces the ERα intracellular content and preferentially affects cancer Differentially expressed genes that were significantly upregulated (FDR<5%) in Y537S-MCF-7 compared with that in MCF-7 cells were further compared with those upregulated genes identified in two other published datasets [13,19,20].…”

“…Consequently, different lines of research are trying to identify novel molecules that induce ERα degradation in Tam-resistant metastatic BC [18,24,25] or new pathways that could be targeted alone or in combination with ICI [13, 19,20,26,27]. Therefore, we next asked whether the combination of ICI and thioridazine could exhibit anti-proliferative synergism in primary and metastatic BC cell lines.…”

Estrogen-independent molecular actions of mutant estrogen receptor alpha in endometrial cancer

“…The ESR1 LBD mutations occur in a region of the protein essential for ligand binding and interactions with co-regulatory proteins, with the majority of mutations found at residues D538 and Y537. Studies into the molecular and phenotypic consequences of ESR1 LBD mutations have been performed in breast cancer, revealing that the mutations confer estrogen-independent ESR1 activity, which drives gene regulation and cell proliferation in the absence of estrogens (Merenbakh-Lamin et al 2013;Robinson et al 2013;Toy et al 2013;Jeselsohn et al 2014;Bahreini 2017;Toy et al 2017;Zhao 2017;Jeselsohn 2018). Biochemical characterization of the mutations suggests that mutant ESR1 favors the activated conformation of the receptor irrespective of ligand, causing constitutive receptor activity (Merenbakh-Lamin et al 2013;Fanning 2016;Toy et al 2017;Zhao 2017;Katzenellenbogen 2018).…”

“…Studies into the molecular and phenotypic consequences of ESR1 LBD mutations have been performed in breast cancer, revealing that the mutations confer estrogen-independent ESR1 activity, which drives gene regulation and cell proliferation in the absence of estrogens (Merenbakh-Lamin et al 2013;Robinson et al 2013;Toy et al 2013;Jeselsohn et al 2014;Bahreini 2017;Toy et al 2017;Zhao 2017;Jeselsohn 2018). Biochemical characterization of the mutations suggests that mutant ESR1 favors the activated conformation of the receptor irrespective of ligand, causing constitutive receptor activity (Merenbakh-Lamin et al 2013;Fanning 2016;Toy et al 2017;Zhao 2017;Katzenellenbogen 2018). Gene expression analyses highlight the ability of mutant ESR1 to regulate canonical ESR1 target genes in the absence of estrogens (Merenbakh-Lamin et al 2013;Robinson et al 2013;Toy et al 2013;Jeselsohn et al 2014;Bahreini 2017;Toy et al 2017;Jeselsohn 2018;Katzenellenbogen 2018).…”