RON signalling promotes therapeutic resistance in ESR1 mutant breast cancer.

BACKGROUND: Oestrogen Receptor 1 (ESR1) mutations are frequently acquired in oestrogen receptor (ER)-positive metastatic breast cancer (MBC) patients who were treated with aromatase inhibitors (AI) in the metastatic setting. Acquired ESR1 mutations are associated with poor prognosis and there is a lack of effective therapies that selectively target these cancers. METHODS: We performed a proteomic kinome analysis in ESR1 Y537S mutant cells to identify hyperactivated kinases in ESR1 mutant cells. We validated Recepteur d'Origine Nantais (RON) and PI3K hyperactivity through phospho-immunoblot analysis, organoid growth assays, and in an in vivo patient-derived xenograft (PDX) metastatic model. RESULTS: We demonstrated that RON was hyperactivated in ESR1 mutant models, and in acquired palbociclib-resistant (PalbR) models. RON and insulin-like growth factor 1 receptor (IGF-1R) interacted as shown through pharmacological and genetic inhibition and were regulated by the mutant ER as demonstrated by reduced phospho-protein expression with endocrine therapies (ET). We show that ET in combination with a RON inhibitor (RONi) decreased ex vivo organoid growth of ESR1 mutant models, and as a monotherapy in PalbR models, demonstrating its therapeutic efficacy. Significantly, ET in combination with the RONi reduced metastasis of an ESR1 Y537S mutant PDX model. CONCLUSIONS: Our results demonstrate that RON/PI3K pathway inhibition may be an effective treatment strategy in ESR1 mutant and PalbR MBC patients. Clinically our data predict that ET resistance mechanisms can also contribute to CDK4/6 inhibitor resistance.

Correction to: Targeted degradation of activating estrogen receptor α ligand-binding domain mutations in human breast cancer.

In the original publication of the article, the spelling of the sixth author's given name was incorrect. The corrected author name should read as "Wadie David". The original article has been corrected.

Targeted degradation of activating estrogen receptor α ligand-binding domain mutations in human breast cancer.

PURPOSE: Studies have identified several estrogen receptor α (ERα) ligand-binding domain (LBD) somatic mutations in endocrine therapy resistant, metastatic ER-positive breast cancers. The most common mutations, Tyr537Ser (Y537S) and Asp538Gly (D538G), are detected in ~ 30% of endocrine resistant metastatic breast cancer patients. These ESR1 mutations induce the agonist conformation of ERα, confer an estrogen-independent phenotype, and promote drug resistance to antiestrogens. METHODS: ER-positive, estrogen-dependent MCF-7 cells were engineered to express either the Y537S or D538G mutants using CRISPR knock-in (cY537S and cD538G). These cells were used to screen several estrogen receptor degrader (ERD) compounds synthesized using the Proteolysis Targeting Chimeras (PROTAC) method to induce degradation of ERα via the ubiquitin-proteasome pathway. RESULTS: Wild-type MCF-7 and ERα LBD mutant cells were treated with ERD-148 (10 pM-1 µM) and assayed for cellular proliferation using the PrestoBlue cell viability assay. ERD-148 attenuated ER-dependent growth with IC50 values of 0.8, 10.5, and 6.1 nM in MCF-7, cY537S, and cD538G cells, respectively. Western blot analysis showed that MCF-7 cells treated with 1 nM ERD-148 for 24 h exhibited reduced ERα protein expression as compared to the mutants. The ER-regulated gene, GREB1, demonstrated significant downregulation in parental and mutant cells after 24 h of ERD-148 treatment at 10 nM. Growth of the ER-negative, estrogen-independent MDA-MB-231 breast cancer cells was not inhibited by ERD-148 at the ~ IC90 observed in the ER-positive cells. CONCLUSION: ERD-148 inhibits the growth of ER-positive breast cancer cells via downregulating ERα with comparable potency to Fulvestrant with marginal non-specific toxicity.

Implication of environmental estrogens on breast cancer treatment and progression.

Breast cancer is the most diagnosed malignancy among women in the United States. Approximately 70% of breast tumors express estrogen receptor alpha and are deemed ER-positive. ER-positive breast tumors depend upon endogenous estrogens to promote ER-mediated cellular proliferation. Decades of research have led to a fundamental understanding of the role ER signaling in this disease and this knowledge has led to significant advancements in the clinical use of antiestrogens for breast cancer treatment. However, adjuvant breast cancer recurrence and metastatic disease progression due to endocrine therapy resistance are prominent and unresolved issues. The established role that estrogens play in breast cancer pathogenesis explains why some patients initially respond to endocrine therapy but also why a significant number of patients become refractory to antiestrogen treatment. It is been hypothesized that exposure to environmental steroid hormone mimics and/or acquired mechanisms of resistance may explain why endocrine therapy fails in a subset of breast cancer patients. This review will highlight: 1) the relationship between ER signaling and breast cancer pathogenesis, 2) the implication of environmental exposures on steroid hormone regulated processes including breast cancer, and 3) the unresolved issue of endocrine therapy resistance.

Homology models of mouse and rat estrogen receptor-α ligand-binding domain created by in silico mutagenesis of a human template: molecular docking with 17ß-estradiol, diethylstilbestrol, and paraben analogs.

Crystal structures exist for human, but not rodent, estrogen receptor-α ligand-binding domain (ERα-LBD). Consequently, rodent studies involving binding of compounds to ERα-LBD are limited in their molecular-level interpretation and extrapolation to humans. Because the sequences of rodent and human ERα-LBDs are > 95% identical, we expected their 3D structures and ligand binding to be highly similar. To test this hypothesis, we used the human ERα-LBD structure (PDB 3UUD) as a template to produce rat and mouse homology models. Employing the rodent models and human structure, we generated docking poses of 23 Group A ligands (17ß-estradiol, diethylstilbestrol, and 21 paraben analogs) in AutoDock Vina for interspecies comparisons. Ligand RMSDs (Å) (median, 95% CI) were 0.49 (0.21-1.82) (human-mouse) and 1.19 (0.22-1.82) (human-rat), well below the 2.0-2.5 Å range for equivalent docking poses. Numbers of interspecies ligand-receptor residue contacts were highly similar, with Sorensen Sc (%) = 96.8 (90.0-100) (human-mouse) and 97.7 (89.5-100) (human-rat). Likewise, numbers of interspecies ligand-receptor residue contacts were highly correlated: Pearson r = 0.913 (human-mouse) and 0.925 (human-rat). Numbers of interspecies ligand-receptor atom contacts were even more tightly correlated: r = 0.979 (human-mouse) and 0.986 (human-rat). Pyramid plots of numbers of ligand-receptor atom contacts by residue exhibited high interspecies symmetry and had Spearman r s = 0.977 (human-mouse) and 0.966 (human-rat). Group B ligands included 15 ring-substituted parabens recently shown experimentally to exhibit decreased binding to human ERα and to exert increased antimicrobial activity. Ligand efficiencies calculated from docking ligands into human ERα-LBD were well correlated with those derived from published experimental data (Pearson partial r p = 0.894 and 0.918; Groups A and B, respectively). Overall, the results indicate that our constructed rodent ERα-LBDs 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.

Metabolites of n-Butylparaben and iso-Butylparaben Exhibit Estrogenic Properties in MCF-7 and T47D Human Breast Cancer Cell Lines.

Two oxidized metabolites of n-butylparaben (BuP) and iso-butylparaben (IsoBuP) discovered in human urine samples exhibit structural similarity to endogenous estrogens. We hypothesized that these metabolites bind to the human estrogen receptor (ER) and promote estrogen signaling. We tested this using models of ER-mediated cellular proliferation. The estrogenic properties of 3-hydroxy n-butyl 4-hydroxybenzoate (3OH) and 2-hydroxy iso-butyl 4-hydroxybenzoate (2OH) were determined using the ER-positive, estrogen-dependent human breast cancer cell lines MCF-7, and T47D. The 3OH metabolite induced cellular proliferation with EC50 of 8.2 µM in MCF-7 cells. The EC50 for 3OH in T47D cells could not be reached. The 2OH metabolite induced proliferation with EC50 of 2.2 µM and 43.0 µM in MCF-7 and T47D cells, respectively. The EC50 for the parental IsoBuP and BuP was 0.30 and 1.2 µM in MCF-7 cells, 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 (ICI 182, 780), confirming the ER-dependence of these effects. The metabolites promoted significant ER-dependent transcriptional activity of an ERE-luciferase reporter construct at 10 and 20 µM for 2OH and 10 µM for 3OH. Computational docking studies showed that the paraben compounds exhibited the potential for favorable ligand-binding domain interactions with human ERα in a manner similar to known x-ray crystal structures of 17ß-estradiol in complex with ERα. We conclude that the hydroxylated metabolites of BuP and IsoBuP are weak estrogens and should be considered as additional components of potential endocrine disrupting effects upon paraben exposure.