Estrogen Receptor Ligands Research Articles

Electrochemically Promoted Regioselective C3–H Trifluoro/Difluoromethylation of 2H-Indazoles at Room Temperature

AbstractA green and sustainable electrochemical approach is developed for the regioselective C3–H trifluoro/difluoromethylation of 2H-indazoles at room temperature. Relatively less expensive C-soft (+)/Ni-foam (–) electrodes are utilized to selectively functionalize the 2H-indazoles effectively by avoiding the use of any external oxidant and transition-metal salt. Moreover, along with the C3–H trifluoromethylation, for the very first time, direct C3–H difluoromethylation of 2-phenyl-2H-indazoles is accomplished. Diverse C3–H trifluoro/difluoromethylated 2H-indazoles having an array of functionalities are successfully synthesized in moderate to very good yields. As an application, a precursor of both an estrogen receptor ligand and an acetyl Co-A carboxylase inhibitor is synthesized. A plausible reaction mechanism is proposed based on control experiments and cyclic voltammetry studies.

The Aryl Hydrocarbon Receptor and Its Crosstalk: A Chemopreventive Target of Naturally Occurring and Modified Phytochemicals.

The aryl hydrocarbon receptor (AhR) is an environmentally sensitive transcription factor (TF) historically associated with carcinogenesis initiation via the activation of numerous carcinogens. Nowadays, the AhR has been attributed to multiple endogenous functions to maintain cellular homeostasis. Moreover, crosstalk, often reciprocal, has been found between the AhR and several other TFs, particularly estrogen receptors (ERs) and nuclear factor erythroid 2-related factor-2 (Nrf2). Adequate modulation of these signaling pathways seems to be an attractive strategy for cancer chemoprevention. Several naturally occurring and synthetically modified AhR or ER ligands and Nrf2 modulators have been described. Sulfur-containing derivatives of glucosinolates, such as indole-3-carbinol (I3C), and stilbene derivatives are particularly interesting in this context. I3C and its condensation product, 3,3'-diindolylmethane (DIM), are classic examples of blocking agents that increase drug-metabolizing enzyme activity through activation of the AhR. Still, they also affect multiple essential signaling pathways in preventing hormone-dependent cancer. Resveratrol is a competitive antagonist of several classic AhR ligands. Its analogs, with ortho-methoxy substituents, exert stronger antiproliferative and proapoptotic activity. In addition, they modulate AhR activity and estrogen metabolism. Their activity seems related to a number of methoxy groups introduced into the stilbene structure. This review summarizes the data on the chemopreventive potential of these classes of phytochemicals, in the context of AhR and its crosstalk modulation.

Estrogen Regulated Genes Compel Apoptosis in Breast Cancer Cells, Whilst Stimulate Antitumor Activity in Peritumoral Immune Cells in a Janus-Faced Manner.

Background: Breast cancer incidence and mortality exhibit a rising trend globally among both premenopausal and postmenopausal women, suggesting that there are serious errors in our preventive and therapeutic measures. Purpose: Providing a series of valuable, but misunderstood inventions highlighting the role of increasing estrogen signaling in prevention and therapy of breast cancer instead of its inhibition. Results: 1. Breast cells and breast cancer cells with germline BRCA1/2 mutations similarly show defects in liganded estrogen receptor (ER) signaling, demonstrating its role in genomic instability and cancer initiation. 2. In breast tumors, the increased expression of special receptor family maybe an effort for self-directed improvement of genomic defects, while the weakness or loss of receptors indicates a defect requiring medical repair. 3. ER overexpression in breast cancer cells is capable of strengthening estrogen signaling and DNA repair, while in ER negative tumors, HER2 overexpression tries to upregulate unliganded ER activation and genome stabilization. 4. ER-positive breast cancers responsive to endocrine therapy may show a compensatory ER overexpression resulting in a transient tumor response. Breast cancers non-responsive to antiestrogen treatment exhibit HER2-overexpression for compensating the complete inhibition of hormonal ER activation. 5. In breast tumors, somatic mutations serve upregulation of ER activation via liganded or unliganded pathway helping genome stabilization and apoptotic death. 6. The mutual communication between breast cancer and its inflammatory environment is a wonderful partnership among cells fighting for genome stabilization and apoptotic death of tumor. 7. In breast cancers, there is no resistance to genotoxic or immune blocker therapies, but rather, the nonresponsive tumor cells exhaust all compensatory possibilities against therapeutic damages. Conclusions: Understanding the behavior and ambition of breast cancer cells may achieve a turn in therapy via applying supportive care instead of genotoxic measures.

Receptor mediated biological activities of phytoestrogens

Phytoestrogens are plant-derived compounds that have chemical structures and functions similar to estrogen. Phytoestrogens act as ligand-inducible transcription factors involved in cellular growth by binding to estrogen receptors (ERs), specifically ER alpha (ERα) and beta (ERβ). Through this mechanism, phytoestrogens have a physiological function similar to that of the female hormone 17β-estradiol (E2), which can be useful in treating osteoporosis, cardiovascular disease, and cancer. Furthermore, phytoestrogens have been found to elicit various cellular responses depending on their affinity for ERs; in particular, they show a greater affinity with for ERβ. This study aimed to comprehensively analyze the mode of action of eight phytoestrogens, namely kaempferol, coumestrol, glycitein, apigenin, daidzein, genistein, equol, and resveratrol, by evaluating their estrogenic activity as ER ligands. Based on the bioluminescence resonance energy transfer (BRET)-based ER dimerization and transactivation assay results, all the phytoestrogens tested were identified as estrogen agonists by mediating ERα and ERβ dimerization. The specific binding and functions of ERα and ERβ were distinguished by differentiating between their dimerization activity. In addition, this study contributes to advancing our understanding of the overall mechanism of action involving both ERs.

Transcription decouples estrogen-dependent changes in enhancer-promoter contact frequencies and spatial proximity.

How enhancers regulate their target genes in the context of 3D chromatin organization is extensively studied and models which do not require direct enhancer-promoter contact have recently emerged. Here, we use the activation of estrogen receptor-dependent enhancers in a breast cancer cell line to study enhancer-promoter communication at two loci. This allows high temporal resolution tracking of molecular events from hormone stimulation to efficient gene activation. We examine how both enhancer-promoter spatial proximity assayed by DNA fluorescence in situ hybridization, and contact frequencies resulting from chromatin in situ fragmentation and proximity ligation, change dynamically during enhancer-driven gene activation. These orthogonal methods produce seemingly paradoxical results: upon enhancer activation enhancer-promoter contact frequencies increase while spatial proximity decreases. We explore this apparent discrepancy using different estrogen receptor ligands and transcription inhibitors. Our data demonstrate that enhancer-promoter contact frequencies are transcription independent whereas altered enhancer-promoter proximity depends on transcription. Our results emphasize that the relationship between contact frequencies and physical distance in the nucleus, especially over short genomic distances, is not always a simple one.

Estetrol/GPER/SERPINB2 transduction signaling inhibits the motility of triple-negative breast cancer cells

BackgroundEstetrol (E4) is a natural estrogen produced by the fetal liver during pregnancy. Due to its favorable safety profile, E4 was recently approved as estrogenic component of a new combined oral contraceptive. E4 is a selective ligand of estrogen receptor (ER)α and ERβ, but its binding to the G Protein-Coupled Estrogen Receptor (GPER) has not been described to date. Therefore, we aimed to explore E4 action in GPER-positive Triple-Negative Breast Cancer (TNBC) cells.MethodsThe potential interaction between E4 and GPER was investigated by molecular modeling and binding assays. The whole transcriptomic modulation triggered by E4 in TNBC cells via GPER was explored through high-throughput RNA sequencing analyses. Gene and protein expression evaluations as well as migration and invasion assays allowed us to explore the involvement of the GPER-mediated induction of the plasminogen activator inhibitor type 2 (SERPINB2) in the biological responses triggered by E4 in TNBC cells. Furthermore, bioinformatics analysis was aimed at recognizing the biological significance of SERPINB2 in ER-negative breast cancer patients.ResultsAfter the molecular characterization of the E4 binding capacity to GPER, RNA-seq analysis revealed that the plasminogen activator inhibitor type 2 (SERPINB2) is one of the most up-regulated genes by E4 in a GPER-dependent manner. Worthy, we demonstrated that the GPER-mediated increase of SERPINB2 is engaged in the anti-migratory and anti-invasive effects elicited by E4 in TNBC cells. In accordance with these findings, a correlation between SERPINB2 levels and a good clinical outcome was found in ER-negative breast cancer patients.ConclusionsOverall, our results provide new insights into the mechanisms through which E4 can halt migratory and invasive features of TNBC cells.

Abstract PO3-26-06: A self-replicating RNA Precision Immunotherapeutic for Overcoming Resistance to Endocrine Therapy in Estrogen Receptor Positive Breast Cancer (ER+BC)

Abstract Advanced or metastatic ER+HER2- breast cancer (ER+BC) is an incurable disease. Standard 1L therapy (SOC) utilizes endocrine blockade with a nonsteroidal aromatase inhibitor (NSAI) combined with a CDK4/6 inhibitor (CDK4/6i). While most patients respond and disease remains controlled for over 2 years (mPFS ~25-28 months), virtually all patients progress over time, requiring more toxic therapies with decreasing periods of disease control. Prolonging the time that the tumor remains under endocrine control is therefore of significant medical value. The most common molecular drivers of endocrine resistance are well characterized and include mutations in ESR1 and bypass mechanisms in select signaling pathways. RBI-1000 is a self-replicating RNA (srRNA) precision immunotherapeutic to generate robust immunity directed against acquired resistance mutations that develop in ER+ BC in response to endocrine blockade by NSAI. RBI-1000 includes 6 on-target mutations within the estrogen receptor ligand binding domain, and bypass mutations either in the form of activating mutations in PI3K kinase or amplifications of HER2/HER3. Precision immunotherapy (PIO), combined with SOC targeted therapy, educates the patient’s immune system to eliminate tumor cells that express acquired resistance mutations. Thus, the tumor is in a lose-lose situation where it is either eliminated by the targeted therapy, or if it develops acquired resistance mutations to the targeted therapy, it is killed by the PIO. Specifically for endocrine resistance, RBI-1000 PIO offers an attractive therapeutic approach to prevent/delay endocrine resistance specifically, by generating an effective immune response against those tumor cells that express the resistance-associated molecules that arise while on NSAI+CDK4/6i. The ability to encode multiple targets in a single therapeutic srRNA molecule, alongside the prolonged expression of srRNA in host cells (>49 days) creates a versatile and powerful cancer drug development platform. srRNA precision immunotherapeutics are delivered intramuscularly (i.e. a jab in the non-dominant deltoid) and generate an effective immune response via the same pathways and mechanisms as a traditional vaccine. We have demonstrated that this srRNA encapsulated in a lipid nanoparticle primes polyfunctional CD4 and CD8 T cells and antibody responses, leading to tumor growth inhibition and improved survival in a mouse model expressing the targeted acquired resistance mutations. Priming of T cells against acquired mutations is also confirmed in human HLA-transgenic mice. The immune cell-mediated elimination of clones expressing the acquired resistance mutations is predicted to prolong endocrine control of ER+BC, in an analogous manner to small molecule or monoclonal antibody targeted therapies, but with a more favorable dosing and adverse event profile due to precise immunologic targeting and no DDI. RBI-1000 is anticipated to enter clinical studies in the first half of 2024. Citation Format: Zelanna Goldberg, Christian Maine, Gabrielle Dailey, Christine Domingo, Gaelle Picarda, Hunter Little, Annie Chou, Jessica Sparks, Darina Spasova, Shiegeki Miyake-Stoner, Zachary C Hartman, Christopher Rabiola, Erika Crosby, Herbert Lyerly, Nathaniel Wang, Parinaz Aliahmad. A self-replicating RNA Precision Immunotherapeutic for Overcoming Resistance to Endocrine Therapy in Estrogen Receptor Positive Breast Cancer (ER+BC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-06.

Abstract CT075: A Phase 1 trial evaluating AC699, an orally bioavailable chimeric estrogen receptor degrader, in patients with advanced or metastatic breast cancer

Abstract Background The role of estrogen receptor (ER) signaling in driving breast carcinogenesis is well established, and endocrine therapy has been a mainstay in the treatment of patients with ER-positive advanced or metastatic breast cancer for decades. Aromatase inhibitors, selective estrogen receptor modulators, and selective ER degraders (SERDs) such as fulvestrant, and more recently elacestrant, are integral components of the therapeutic strategy for patients with ER-positive breast cancer. However, unmet medical need persists in this patient population, due to suboptimal clinical outcomes and the development of resistance to therapy. E3 ligase-engaged chimeric degraders represent a technological advance with the potential to induce potent and deeper ER degradation. Utilizing Accutar's proprietary Protein-Protein Interaction Targeting Chimeras (PPI-TAC) platform, AC699 was designed as a novel chimeric degrader targeting ERα. By effectively linking an ER ligand to an E3-ligase recruiting ligand, AC699 brings ERα in proximity to an E3 ligase, resulting in ubiquitination and subsequent degradation of ERα. Chimeric ER degraders possess the unique advantage of degrading the ER protein without the inherent risk of activating an ER signal. Moreover, these molecules are not degraded alongside the target protein, allowing for their efficient recycling within the cell. This direct mechanism enables chimeric ER degraders to achieve potent ER degradation with increased specificity, thereby potentially providing a higher therapeutic index compared to SERDs. Study Description The AC699-001 trial is a first in human Phase 1 dose escalation study (NCT05654532) which will enroll up to 60 patients with locally advanced or metastatic ER-positive, human epidermal growth factor receptor 2-negative, breast cancer. Patients must have received at least two prior lines of endocrine treatment, or at least one prior line, if combined with a CDK4/6 inhibitor. Prior chemotherapy is not required but must not exceed three prior lines of cytotoxic treatment. Patients must have at least one measurable lesion or at least one predominantly lytic bone lesion. AC699 is administered orally, once daily, with doses ranging from 100 mg to 600 mg in a standard 3+3 dose escalation design. The primary objective is evaluation of the safety and tolerability of AC699. Secondary objectives include assessment of preliminary anti-tumor activity and characterization of the pharmacokinetic profile of single and multiple doses of AC699. Enrollment began in December 2022 and a total of 5 sites have been activated in the United States. Citation Format: Rachel M. Layman, Manish R. Patel, David Cosgrove, Michael Danso, Nancy Mota, Marjorie E. Zettler, Katherine C. Pehlivan, Erika Hamilton. A Phase 1 trial evaluating AC699, an orally bioavailable chimeric estrogen receptor degrader, in patients with advanced or metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT075.

Rational Design of Hydroxylated Thiazole Orange Photocages for Green Light-Triggered DNA Recombination.

The precise control of DNA recombination enables the cell- or time-dependent regulation of gene expression in studies of gene function. Caged estrogen receptor ligands combined with a Cre-ERT2/loxP system are useful tools for light-triggered DNA recombination. However, the photolysis of most caged compounds requires ultraviolet or blue light, which is toxic and displays low tissue penetration. Although a cyanine-based photo-responsive protecting group (PPG) can release estrogen receptor ligands with longer-wavelength light, its low photolytic efficiency requires long illumination times. We developed a caged estrogen receptor ligand with improved green light-responsive PPGs. The rational modification of Hydroxylated Thiazole Orange (HTO) photocages using electron-donating groups (EDGs), such as dimethoxy (DiMeO)-substituted HTO, resulted in high photolytic efficiency (up to ϵΦ ≈320 M-1 cm-1 ). Theoretical calculations demonstrated that the enhanced photolytic efficiencies were derived from the increased intramolecular charge transfer by EDGs upon excitation. The efficient uncaging of estrogen receptor ligands enabled the control of gene recombination in a ligand-dependent Cre-ERT2/loxP system in live cells.

Synthesis and biological evaluation of novel D-ring fused steroidal N(2)-substituted-1,2,3-triazoles.

In this study, a series of 13 new D-ring fused steroidal N(2)-substituted-1,2,3-triazoles were synthesized, characterized and evaluated for their biological activities. The relative binding affinities of the synthesized compounds for the ligand-binding domains of estrogen receptors α and β, androgen receptor and glucocorticoid receptor demonstrated that androstane derivatives 3a and 3h and estratriene derivative 4e showed highly specific and strong binding affinity for estrogen receptor β, while 3b, 3e, 4a and 4b displayed high binding affinity for the glucocorticoid receptor. The synthesized compounds were tested for their ability to inhibit aldo-keto reductases 1C3 and 1C4 in vitro by monitoring NADPH consumption using fluorescence spectroscopy. The most potent aldo-keto reductase 1C3 inhibitors were compounds 3h (71.17%) and 3f (69.9%). Moreover, a molecular docking study was carried out for compounds 3f and 3h against aldo-keto reductase 1C3 and results showed that compounds 3h and 3f could bind in the same site and orientation as EM1404. However, polar atoms in the triazole group enable additional hydrogen bonding deeper in SP1 with Tyr319, Tyr216 and the NADP+ cofactor, which are not visible in the AKR1C3-EM1404 crystal structure. The synthesized compounds were screened for their anticancer activity against four cancer cell lines. Compound 3f demonstrated moderate toxic effects across various cancer types, while displaying lower toxicity towards the healthy cell line. In summary, our findings indicate that N(2)-substituted-1,2,3-triazoles are high-affinity ligands for estrogen receptor β and glucocorticoid receptor, inhibitors of aldo-keto reductase 1C3 enzyme, and exhibit antiproliferative effects against cancer cells, suggesting that they could serve as scaffolds for anticancer drug development.

Physicochemical characterization of B-hydroxyphenyl phosphine borane derivatives and their evaluation as nuclear estrogen receptor ligands.

Increasing the structural options in medicinal chemistry is a promising approach to develop new drug candidates. In this research, we designed and synthesized a series of B-hydroxyphenyl phosphine borane derivatives and investigated their structure-property and structure-activity relationships. The synthesized B-phenylphosphine borane derivatives exhibited sufficient stability in aqueous media, weaker hydrophobicity than the corresponding alkanes and silanes, and sufficient affinity for lipid membranes to enable permeability. Several B-hydroxyphenyl phosphine borane derivatives exhibited significant estrogen receptor (ER) agonistic activity with superior ligand-lipophilicity efficiency (LLE). The phosphine borane framework appears to be a promising option for structural development in drug discovery studies.

MiR-206 inhibits estrogen signaling and ovarian cancer cell migration without affecting GPER

AimsEstrogen-regulated pathways are involved in the etiology and progression of epithelial ovarian cancer (EOC), but the relative contribution of estrogen receptor isoforms is unclear. Only a subset of patients responds to antiestrogens including tamoxifen. Based on our previous evidence that miR-206 behaves as an oncosuppressor in EOC, we hypothesized that miR-206 would interfere with G protein-coupled estrogen receptor (GPER)-mediated signaling and cell motility. Main methodsPFKFB3 and FAK proteins from OC cells challenged with selective estrogen receptor agonist and antagonist were measured by Western blotting. Cell proliferation and motility were analyzed by MTT and Boyden chamber, respectively. Estrogen-dependent cells were transfected with miR-206 mimic or control using Lipofectamine. Key findingsThe migration of SKOV3 and OVCAR5 cells significantly increased following treatment with 17β-estradiol (E2) and the selective GPER agonist G1. However, tamoxifen failed to inhibit E2 effect and even promoted SKOV3 cell migration. Estrogen receptor ligands did not affect SKOV3 proliferation. The GPER antagonist G15 significantly prevented E2-mediated upregulation of PFKFB3 expression, while G1 concentration-dependently upregulated PFKFB3 levels. Consistent with the functional link between PFKFB3 and FAK activation, E2 and G1 increased FAK phosphorylation at Tyr397. Transfection with miR-206 abolished estrogen-induced EOC migration and down-regulated PFKFB3 protein levels. Notably, miR-206 transfection reduced ERα protein abundance, whereas GPER amount was unchanged. SignificanceBy blocking estrogen signaling and G1-induced EOC cell invasiveness with no direct interference with GPER levels, miR-206 mimics have the potential to act as pathway-selective antagonists and deserve further testing as RNA therapeutics in estrogen-dependent EOC.

Structural Optimization of Decoy Oligonucleotide-Based PROTAC That Degrades the Estrogen Receptor.

Proteolysis-targeting chimeras (PROTACs) have attracted attention as a chemical method of protein knockdown via the ubiquitin-proteasome system. Some oligonucleotide-based PROTACs have recently been developed for disease-related proteins that do not have optimal small-molecule ligands such as transcription factors. We have previously developed the PROTAC LCL-ER(dec), which uses a decoy oligonucleotide as a target ligand for estrogen receptor α (ERα) as a model transcription factor. However, LCL-ER(dec) has a low intracellular stability because it comprises natural double-stranded DNA sequences. In the present study, we developed PROTACs containing chemically modified decoys to address this issue. Specifically, we introduced phosphorothioate modifications and hairpin structures into LCL-ER(dec). Among the newly designed PROTACs, LCL-ER(dec)-H46, with a T4 loop structure at the end of the decoy, showed long-term ERα degradation activity while acquiring enzyme tolerance. These findings suggest that the introduction of hairpin structures is a useful modification of oligonucleotides in decoy oligonucleotide-based PROTACs.

Molecular Docking Studies and In-silico ADMET Profile Analysis of Triphala Plant constituents Morin and 9, 10-anthraquinone as Potential Inhibitors of human Estrogen Receptor Alpha

The expanding global burden of cancer demands novel treatment options. Herbal medicine offers a viable alternative to conventional cancer treatment. Breast cancer is frequently diagnosed cancer among women all over the world. Normal breast cells and specific breast cancer cells containestrogen and progesterone receptors. Estrogen and progesterone bind to receptors and stimulate cancer cell proliferation and expansion with growth factors (e.g., oncogenes and mutated gene suppressors). Tamoxifen, raloxifene, andtoremifene are among the most frequently used drugs for breast cancer, whereas estrogen continues to be produced in breast cancer cells. These medications primarily function against theestrogen binding to theestrogenreceptors on these cells. Protein-ligand interaction plays a major role in structural drug design. In this study, the molecular interactions between theHuman estrogen receptor alpha (PDB ID: 2IOK), and the two ligands isolated from Triphala Ayurvedic formulation morin, and 9,10-anthraquinone, an endogenous estrogen receptor ligand, estradiol, and the three economically available breast cancer drug, tamoxifen, raloxifene, and toremifene using the AutodockVina software tool. The ADMET properties of these substances were determinedusing popular web-based software tools that include preADMET, admetSAR, Molinspiration, and SwissADME.The present available anti-cancer drugs have so many side effects. The present work is aimed at determining the efficacy ofestrogen receptor inhibitors and the safety profile of morin, anthraquinone molecules isolated from Triphalapolyherbal formulation by in-silico methods. The binding energies (Kcal/mol) of ligands with human estrogen receptor were calculated as follows: morin (-9.0), 9,10-anthraquinone (-8.7), estradiol (-10.1), tamoxifen (-9.6), raloxifene (-9.8) and toremifene (-8.9). The resultssupported the drug-like properties of the molecules tested and they are likely to have a therapeutic effect.Further,in vivo and pre-clinical trials of the most active compound are also worthwhile for producing effective inhibitors.

FGF1 supports glycolytic metabolism through the estrogen receptor in endocrine-resistant and obesity-associated breast cancer

BackgroundObesity increases breast cancer risk and breast cancer-specific mortality, particularly for people with estrogen receptor (ER)-positive tumors. Body mass index (BMI) is used to define obesity, but it may not be the best predictor of breast cancer risk or prognosis on an individual level. Adult weight gain is an independent indicator of breast cancer risk. Our previous work described a murine model of obesity, ER-positive breast cancer, and weight gain and identified fibroblast growth factor receptor (FGFR) as a potential driver of tumor progression. During adipose tissue expansion, the FGF1 ligand is produced by hypertrophic adipocytes as a stimulus to stromal preadipocytes that proliferate and differentiate to provide additional lipid storage capacity. In breast adipose tissue, FGF1 production may stimulate cancer cell proliferation and tumor progression.MethodsWe explored the effects of FGF1 on ER-positive endocrine-sensitive and resistant breast cancer and compared that to the effects of the canonical ER ligand, estradiol. We used untargeted proteomics, specific immunoblot assays, gene expression profiling, and functional metabolic assessments of breast cancer cells. The results were validated in tumors from obese mice and breast cancer datasets from women with obesity.ResultsFGF1 stimulated ER phosphorylation independently of estradiol in cells that grow in obese female mice after estrogen deprivation treatment. Phospho- and total proteomic, genomic, and functional analyses of endocrine-sensitive and resistant breast cancer cells show that FGF1 promoted a cellular phenotype characterized by glycolytic metabolism. In endocrine-sensitive but not endocrine-resistant breast cancer cells, mitochondrial metabolism was also regulated by FGF1. Comparison of gene expression profiles indicated that tumors from women with obesity shared hallmarks with endocrine-resistant breast cancer cells.ConclusionsCollectively, our data suggest that one mechanism by which obesity and weight gain promote breast cancer progression is through estrogen-independent ER activation and cancer cell metabolic reprogramming, partly driven by FGF/FGFR. The first-line treatment for many patients with ER-positive breast cancer is inhibition of estrogen synthesis using aromatase inhibitors. In women with obesity who are experiencing weight gain, locally produced FGF1 may activate ER to promote cancer cell metabolic reprogramming and tumor progression independently of estrogen.

Rosetta Stone for Cancer Cure: Comparison of the Anticancer Capacity of Endogenous Estrogens, Synthetic Estrogens and Antiestrogens.

This work presents the history of the recognition of principal regulatory capacities of estrogen hormones having been mistakenly regarded as breast cancer promoting agents for more than 120years. Comprehensive analysis of the results of clinical, epidemiological, immunological and molecular studies justified that endogenous estrogens are the principal regulators of embryonic development, survival and reproduction via orchestrating appropriate expression and even edition of all genes in mammalians. Medical use of chemically modified synthetic estrogens caused toxic complications; thromboembolic events and increased cancer risk in female organs as they proved to be endocrine disruptors deregulating estrogen receptors (ERs) rather than their activators. Synthetic estrogen treatment exhibits ambiguous correlations with cancer risk at different sites, which may be attributed to an inhibition of the unliganded activation of estrogen receptors (ERs) coupled with compensatory liganded activation. The principle of estrogen induced breast cancer led to the introduction of antiestrogen therapies against this tumor; inhibition of the liganded activation of estrogen receptors and aromatase enzyme activity. The initial enthusiasm turned into disappointment as the majority of breast cancers proved to be primarily resistant to antiestrogens. In addition, nearly all patients showing earlier good tumor responses to endocrine therapy, later experienced secondary resistance leading to metastatic disease and fatal outcome. Studying the molecular events in tumors responsive and unresponsive to antiestrogen therapy, it was illuminated that a complete inhibition of liganded ER activation stimulates the growth of cancers, while a successful compensatory upregulation of estrogen signal may achieve DNA restoration, tumor regression and patient's survival. Recognition of the principal role of endogenous estrogens in gene expression, gene edition and DNA repair, estrogen treatment and stimulation of ER expression in patients may bring about a great turn in medical practice.

Abstract 3889: The role of CXCL1 in crosstalk between breast cancer cells with ESR1 mutations and lymphatic endothelial cells

Abstract Various endocrine therapies have been developed such as selective estrogen receptor modulation (SERM), selective estrogen receptor downregulator (SERD) and ligand deprivation using aromatase inhibitors (AI). Tamoxifen, fulvestrant, and AIs are the most used adjuvant treatment for pre- and postmenopausal women with early-stage estrogen receptor positive breast cancer. Tamoxifen and AIs have widespread anti-neoplastic and chemopreventive effects, yet despite this, many breast cancers that are initially receptive ultimately become resistant and recur. There are many known underlying molecular processes that result in resistance, but no overarching pattern has yet been identified. Our previous research demonstrates that the crucial molecular and cellular players secreted from a crosstalk between stromal cells and breast cancer cells can control the development of the tumor and the metastatic process in breast cancer. Breast cancer metastasis involves lymphatic dissemination in addition to hematogenous spreading. Although stromal lymphatic vessels (LVs) serve as initial metastatic routes, roles of organ-residing LVs are underinvestigated. In this report, we demonstrated that crosstalk between ESR1 mutant cells (Y537S and D538G), and lymphatic endothelial cells (LECs), a component of lymphatic vessels, promoted ESR mutant cell growth and migration. To identify a critical secreted factor in this crosstalk, we performed the cytokine array in secretome of crosstalk between ESR1 mutant cells and LECs, and found that CXCL1 is highly secreted from LECs. Knockdown of CXCR2 in ESR1 mutant cells attenuated ESR1 cell migration in the conditioned media of LECs crosstalked with ESR1 mutant cells. To investigate a potential impact given the current clinical landscape of endocrine resistant breast cancer with ESR1 mutations, we performed a cell viability assay and migration assay using reparixin, CXCR1/2 inhibitor in co-culture with ESR1 mutant cells and LECs. Significantly, reparixin decreased ESR1 mutant cell viability and migration compared to vehicle. We found that the combination treatment of reparixin and Palbociclib and ribociclib significantly inhibited ESR1 mutant cell growth and migration compared the single treatment. These findings implicate CXCL1-CXCR1/2 signaling as a critical event in the growth of breast cancer with ESR1 mutation and metastasis. Therefore, we have provided evidence that supports the hypothesis that functional inhibition of the CXCL1 and CDK4/6 signaling pathway has the potential to circumvent endocrine resistant tumor growth and metastasis. Citation Format: Daniel Galke, Kideok Jin. The role of CXCL1 in crosstalk between breast cancer cells with ESR1 mutations and lymphatic endothelial cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3889.

Abstract 4354: An exosome-based ESR1 monitoring RT-qPCR technology that rapidly and accurately detects circulating tumor acquired resistance variants at ≤0.1% frequency in liquid biopsy samples

Abstract Introduction: Hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) breast cancer is the most common type of breast cancer. Patients with HR+/HER2- metastatic breast cancer (mBC) often become resistant to aromatase inhibitors commonly used in endocrine therapy (ET). Estrogen receptor (ESR1) ligand binding domain mutations are frequently detected in HR+ mBC and have been reported to be associated with ET resistance, noting up to 60% patients with mBC will develop resistance to treatment via acquired resistance in ESR1. Recent studies have shown that monitoring of ESR1 mutations in plasma may serve as a predictive biomarker of acquired resistance to ET, showcasing a strong need for sensitive nucleic acid-based assays. We describe a comprehensive methodology for targeted clinical RT-qPCR monitoring of ESR1 mutations in plasma that utilizes both exosomal nucleic acids and cfDNA to report multiple mutations, provide a streamlined workflow, and accommodate a range of inputs from clinically relevant samples. Methods: Exosomal nucleic acids (exosomal DNA and RNA) and circulating cell-free DNA (cfDNA) were co-isolated using the ExoLution Plus Isolation Kit (Exosome Diagnostics). RT-qPCR-based target enrichment was performed using modified QuantideX reagents (Asuragen) and mutations were confirmed on the QuantStudio qPCR Platform (ThermoFisher). Bioinformatic analyses were conducted using custom software. Results: We developed novel technologies and tested greater than samples across key ET resistance mutations, utilizing contrived samples as needed due to availability. Preliminary studies demonstrated that ESR1 exosomal RNA + cfDNA ranged from approximately 9,500 copies to 64,000 copies in a set of 2mL female presumed normal plasma samples; these results suggest that even 2mL plasma could provide sufficient exosomal RNA/DNA + cfDNA for mutation detection at variant frequencies less than or equal to 0.01%. The ESR1 RT-qPCR showed greater than 90% analytical sensitivity (at less than or equal to 1% mutant allele frequency) and greater than90% analytical specificity. Further, the technology detected less than 10 mutant copies for most tested mutants, showing an LOD of at least 0.1% (5 mutant copies in a background of 5,000 WT copies). Conclusion: A fast, efficient, and sensitive ESR1 RT-qPCR panel approach was developed and evaluated, demonstrating the reliable and specific detection of rare variants in liquid biopsy specimens. Importantly, this prototype technology has potential to address several challenges associated with mutation monitoring in liquid biopsies by expanding detection of mutant analytes (exosomal RNA and DNA), improving analytical sensitivity (novel reagents and software), and increasing accessibility (qPCR instrument install base). Citation Format: Sarah Statt, Julie Thibert, Kurt Franzen, Aquiles Sanchez, Liangjing Chen, Megan Yociss, Elliot Hallmark, Melissa Church, Stela Filipovic-Sadic, Gary Latham, Johan Skog. An exosome-based ESR1 monitoring RT-qPCR technology that rapidly and accurately detects circulating tumor acquired resistance variants at ≤0.1% frequency in liquid biopsy samples. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4354.

Abstract 6403: A self-replicating RNA precision medicine approach to overcoming resistance to endocrine therapy in ER+BC

Abstract Drug resistance remains the major driving factor behind the clinical failure of targeted therapeutics. Current oncology precision medicine approaches rely on targeting known acquired resistance mutations, such as EGFR T790M or ALK/ROS mutations in NSCLC with 2nd and 3rd generation molecules designed to overcome or prevent resistance. These next generation targeted therapeutic approaches have increasingly long and complex drug development timelines and burdensome toxicities from off target effects (e.g. wild-type receptor targeting) or drug-drug interactions (DDI). The toxicities limit tolerability, compliance and combinability of different targeted therapeutics. RNA-based immunotherapy approaches offer an increasingly attractive alternative to next generation small molecule targeted therapeutics approaches: (1) RNA-based approaches only require a known acquired resistance sequence, (2) drug development timelines, cost and complexity can be meaningfully condensed, and (3) multiple acquired resistance mutations can be targeted with the same candidate. RBI-1000 is a candidate using a novel type of self-replicating RNA (srRNA) to generate robust immunity directed against acquired resistance mutations that develop in ER+ breast cancer (ER+ BC) in response to endocrine therapy. RBI-1000 includes on-target mutations within the estrogen receptor ligand binding domain, and bypass mutations either in the form of activating mutations in the PI3K kinase domain or amplifications of HER2/HER3. Here, we demonstrate that this srRNA encapsulated in a lipid nanoparticle primes polyfunctional CD4 and CD8 T cells leading to tumor growth inhibition and improved survival in a mouse model expressing the targeted acquired resistance mutation. Priming of T cells against acquired mutations is also confirmed in human HLA-transgenic mice. The immune cell-mediated elimination of clones expressing the acquired resistance mutations is predicted to prolong endocrine control of ER+BC, in an analogous manner to small molecule or monoclonal antibody targeted therapies, but with a more favorable dosing and adverse event profile due to precise immunologic targeting and no DDI. Citation Format: Zelanna Goldberg, Christian Maine, Gabrielle P. Dailey, Christine Domingo, Gaelle Picarda, Hunter Little, Annie Chou, Jessica Sparks, Darina Spasova, Shigeki Miyake-Stoner, Zachary C. Hartman, Christopher A. Rabiola, Erika J. Crosby, Herbert K. Lyerly, Nathaniel Wang, Parinaz Aliahmad. A self-replicating RNA precision medicine approach to overcoming resistance to endocrine therapy in ER+BC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6403.

THE CHEMICAL COMPOSITION OF PARABEN AND ITS EFFECTS ON HEALTH

Parabens are a group of alkyl esters of p-hydroxybenzoic acid and usually include methyl paraben, ethyl paraben, propyl paraben, butyl paraben, isobutyl paraben, isopropyl paraben and benzyl paraben. Parabens (or their salts) are widely used as preservatives in cosmetics and medicine due to their relatively low toxicity and long history of safe use. Testing of parabens has shown, to varying degrees, that individual paraben compounds have weak estrogenic activity in some in vitro screening tests, such as estrogen receptor ligand binding, regulation of CAT gene expression, and proliferation of MCF-7 cells. Reported in vivo effects include uterine weight gain (eg, butyl, isobutyl, and benzyl parabens) and male reproductive tract effects (eg, butyl and propyl parabens). However, in relation to estrogen as a control during in vivo studies, parabens are much less active than estrogen. While exposure to sufficient doses of exogenous estrogens can increase the risk of certain adverse effects, it is hypothesized that similar risks may arise from exposure to much weaker endocrine-active chemicals (EACs) is still speculation. In evaluating the possibility that exposure to weakly active EACs may be associated with adverse effects due to their endocrine mode of action, it is important to consider both the dose and potency of these compounds compared to estrogen. In this review, a comparative approach involving dose and potency is used to assess whether in utero or adult exposure to parabens may be associated with adverse effects due to their estrogen-modulating mode of action. Keywords: parabens, p-Hydroxybenzoates, PHBA, paraben applications, Methyl paraben.