Role of Environmental Estrogens and Acquired Endocrine Resistance in Breast Cancer and Implications for Treatment with Novel Antiestrogens

Abstract

Breast cancer is the most diagnosed malignancy among women in the United States. Approximately 70% of breast tumors express estrogen receptor (ER)-alpha and are deemed ER-positive. ER-positive breast tumors depend upon endogenous estrogens to promote ER-mediated cellular proliferation. Although adjuvant endocrine therapy is an effective treatment option for ER-positive breast cancer, recurrence remains an unresolved issue. Studies suggest that ESR1 ligand binding domain (ESR1-LBD) mutations and exposure to alternative estrogens may serve as potential mechanisms of resistance to endocrine therapy.

Chapter II of this dissertation examined whether two oxidized metabolites of n-butylparaben and iso-butylparaben, discovered in human urine, bind to ER-alpha and promote estrogen signaling. The estrogenic properties of metabolites 3-hydroxy n-butyl 4-hydroxybenzoate (3OH) and 2-hydroxy iso-butyl 4-hydroxybenzoate (2OH) were determined using the ER-positive, estrogen-dependent breast cancer cell line MCF-7. The 3OH and 2OH metabolites induced cellular proliferation with EC50 of 8.2 and 2.2 µM, respectively. The expression of a pro-proliferative, estrogen-inducible gene (GREB1) was induced by these compounds and blocked by co-administration of an ER antagonist. The metabolites promoted ER-dependent transcriptional activity of an ERE-luciferase reporter construct.

Crystal structures exist for human, but not rodent, ER-alpha-LBD. Consequently, rodent studies involving binding of compounds to ER-alpha-LBD are limited in their molecular-level interpretation and extrapolation to humans. In chapter III, we used the human ER-alpha-LBD structure (PDB 3UUD) as a template to produce rat and mouse homology models which were employed to generate docking poses of 23 Group A ligands (estradiol, diethylstilbestrol, and 21 paraben analogs) in AutoDock Vina. Numbers of interspecies ligand-receptor residue contacts were highly similar: Sorensen Sc = 93.1 ± 7.5% (human-mouse) and 92.5 ± 7.1% (human-rat). Pyramid plots of numbers of ligand receptor atom contacts by residue exhibited high interspecies symmetry. Group B ligands were 15 3,5-disubstituted parabens shown to exhibit decreased binding to human ER-alpha and increased antimicrobial activity. Ligand efficiencies calculated from docking of Group B ligands into human ER-alpha-LBD were highly correlated with those derived from published experimental data.

The most common recurring ESR1-LBD mutations, D538G and Y537S, are detected in ~30% of patients with metastatic breast cancer who are resistant to endocrine therapy. In chapter IV, we used the MCF-7 cells to develop in vitro models that express the Y537S and D538G mutants using CRISPR knock-in and screened novel compounds that target ER-alpha for degradation. Results show that compound ERD-148 attenuated ER-dependent growth with IC50 values of 0.8, 10.5, and 6.1 nM in wildtype MCF-7, Y537S, and D538G cells respectively. MCF-7 cells treated with ERD-148 for 24 hours show lower levels of ER-alpha protein expression compared to mutant cells treated at 1 nM for 24 hours. GREB1 gene expression was downregulated at nanomolar concentrations in MCF-7 and the mutant cell lines treated with ERD-148 for 24 hours.

In summary, our results indicate that oxidized paraben metabolites exhibit behavior akin to weak estrogens. Our constructed rodent ER-alpha-LBD receptors interact with ligands in like manner to the human receptor, thus providing a high level of confidence in extrapolations of rodent to human ligand-receptor interactions. ERD-148 was shown to inhibit the growth of ER-positive breast cancer via antagonism of ER-alpha. Future studies are needed to determine whether exposure to estrogenic EDCs or the presence of ESR1 mutations contribute to a greater risk of recurrence or poorer clinical outcomes specifically among patients with advanced disease.