Selective ERα Research Articles

Estrogen receptor α regulates the IKKs/NF-kB activity involved in the development of mechanical allodynia induced by REM sleep deprivation in rats

Several signaling pathways that converge in NF-kB activation have been linked to developing and maintaining different types of pathological pain. In addition, some mechanisms implied in the establishment of chronic pain have been demonstrated to have a sex-dependent correlation. This study aimed to determine if the IKKs/NF-kB signaling pathway is involved in establishing REM sleep deprivation (REMSD) induced mechanical allodynia in rats and its possible regulation depending on estradiol and estrogen receptors. Intrathecal administration of BMS-345541 or minocycline, two drugs that reduce the IKKs/NF-kB activity, avoided the development of mechanical allodynia in female but not in male rats subjected to 48 h of REMSD. Ovariectomy in female rats abolished the effect of BMS-345541 and minocycline. Meanwhile, the 17-β-estradiol restitution restored it. Intrathecal administration of MPP, a selective ERα antagonist, but not PHTPP, a selective ERβ antagonist, avoided the effect of BMS-345541 in female rats without hormonal manipulation. In addition, the transient run-down of ERα in female rats abolished the effect of BMS-345541. All data suggest an important role of ERα as a regulator of the IKKs/NF-kB activity. REMSD increased the ERα protein expression in the dorsal root ganglia and the dorsal spinal cord in females but not in male rats. Interestingly, ERα activation or ERα overexpression allowed the effect of BMS-345541 in male rats. Data suggest an important regulatory role of ERα in the IKKs/NF-kB activity on establishing mechanical allodynia induced by REMSD in female rats.

Identifying Potent Breast Cancer Inhibitors Against ERα Target Using Pharmacophore Model, 3D‐QSAR and MD Studies

Abstractthe current investigation, 109 known ERα inhibitors have been developed in the current research; pharmacophore modeling, Molecular docking, MM‐GBSA, and MD study have been performed to investigate the binding affinity of 9‐anilinoacridines with heterocyclic substitutes as selective ERα inhibitors for breast carcinoma. Pharmacophore model have been developed by Schrodinger suite 2019–2 phasemodule. To predict binding free energy of the ligands in complex with PDB and post docked energy minimization was performed by Prime, MM‐GB/SA module. The Induced fit docking studies were performed on the ligand modulated dynamic behaviour of the protein molecular dynamics study. A statistical substantial 3D ‐ QSAR design was created using the pharmacophore hypothesis. 109 known ERα inhibitors have been developed with pIC50 values between 4.0 and 6.0 and were used in ligand‐based pharmacophore modelling and 3D‐QSAR analysis. R2 (0.8294), Q2 (0.7~0.8), and F value (83.5) were used to statistically validate the developed five‐point hypothesis DHRRR1 employing a minimum square of four. Molecular dynamics simulations were run to comprehend the conformational changes and ligand stability at the protein active pocket. The predicted 3D‐QSAR model significantly correlated with experimentally reported in‐vitro antitumor activity. These in‐silico discoveries will help in the future search for potent ERα inhibitors with desirable pharmacophoric properties.

Abstract PO3-26-11: Enhancing endoplasmic reticulum stress for treating endocrine therapy resistant breast cancers driven by mutant estrogen receptors

Abstract Background: Estrogen receptor alpha (ERα) mutations are common (30-40%) in metastatic endocrine therapy-resistant breast cancers (ETR-BC), enable resistance to endocrine therapies and are the molecular drivers of ETR-BC. We had previously shown that an oligobenzamide, ERX-41, could enhance endoplasmic reticulum stress in ETR-BCs driven by mutant (MT) ERα, resulting in cancer cell death in vitro and and in vivo. To enable clinical translation of ERX-41, we performed lead optimization, followed by preclinical and IND-enabling studies. Methods: Over 2000 oligo-benzamides were designed, synthesized and tested in multiple BC models including those that express WT-ERα (MCF7, and ZR75) and BC models with acquired resistance (MCF7-Tam, and MCF7-LTLT) and engineered models that express MT-ERα (MCF7-ERα-D538G, MCF7-ERα-Y537S, ZR75-ERα-D538G, ZR75-ERα-Y537S). For our lead compound, mechanistic validation studies were performed using CRISPR LIPA mutants, RT-qPCR and Western blotting. Explants, organoids, cell line-(CDX) and patient-derived (PDX) xenografts were used to test the ex vivo and in vivo effectiveness of our lead compound as a monotherapy and in combination with abemaciclib. Results: Testing of >2000 synthesized oligo-benzamides identified a lead compound, ERX-315, that had broad and potent activity (IC50 between 20-100 nM) against both WT and MT (mutant) ERα-driven BC cells in in vitro assays. CRISPR KO of LIPA (which encodes lysosomal acid lipase (LAL) in BC cells abrogated cytotoxic response to ERX-315, validating LIPA as the molecular target of ERX-315. Ultrastructural and molecular studies confirm that ERX-315 induces significant endoplasmic reticulum stress, leading to a shutdown of de novo protein synthesis and apoptotic cell death in BC. Importantly, ERX-315 does not induce endoplasmic reticulum stress or cell death in normal cells and is non-toxic in animal models. We have shown that this capacity of ERX-315 to induce endoplasmic reticulum stress is unique among drugs targeting ERα, including selective ERα modulators and degraders, such as GDC-0180, AZD-9496 and fulvestrant. ERX-315 has potent anti-proliferative activity against MT-ERα-driven BC, as seen in genetically modified cell lines both grown as monolayer or spheroids in vitro, patient derived explants (PDEs) ex vivo and cell line derived (CDX) and patient-derived (PDX) xenografts in vivo. The combination of ERX-315 and CDK4/6 inhibitor abemaciclib was synergistic in decreasing the proliferation of both endocrine therapy-sensitive and endocrine therapy-resistant BC cells, in vitro, in xenograft models in vivo, and in primary patient-derived explants ex vivo. We are currently optimizing both the synthesis of ERX-315 using GMP process for kilogram scale production and validated methods for pharmacokinetic studies. We have developed formulations for both intravenous and oral administration with favorable pharmacokinetic parameters and proven utility of the formulated ERX-315 against CDX and PDX tumors in vivo. Toxicity studies in dogs and rodents have demonstrated >8-fold therapeutic to toxicity window. A phase I clinical trial is planned to be open to enrollment by Q1 2024. Conclusions: We have identified a lead compound ERX-315, which represents a novel class of agent that induce catastrophic levels of ER stress resulting in cancer cell death and that can effectively work against multiple forms of ETR-BC, including those driven by MT-ERα. Preclinical studies, GMP manufacturing, formulation and IND-enabling studies are being completed in time for the commencement of the phase I clinical trial by Q1 2024. Citation Format: Suryavathi Viswanadhapalli, Karla Parra, Tae-Kyung Lee, Rahul Gopalam, Kara Kassees, Tanner Reese, Gaurav Sharma, Xihui Liu, Xue Yang, ChiaYuan Chen, Carlos Roggero, Liping Chen, Sautam Bhattacharya, Uday Pratap, Russell Hayward, Sharron Gargosky, Jung-Mo Ahn, Ganesh V. Raj, Ratna Vadlamudi. Enhancing endoplasmic reticulum stress for treating endocrine therapy resistant breast cancers driven by mutant estrogen receptors [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 PO3-26-11.

The Estrogen Receptor Alpha Regulates the Sex-dependent Expression and Pronociceptive Role of Bestrophin-1 in Neuropathic Rats

Bestrophin-1, a calcium-activated chloride channel (CaCC), is involved in neuropathic pain; however, it is unclear whether it has a dimorphic role in female and male neuropathic rats. This study investigated if 17β-estradiol and estrogen receptor alpha (ERα) activation regulate bestrophin-1 activity and expression in neuropathic rats. Neuropathic pain was induced by L5-spinal nerve transection (SNT). Intrathecal administration of CaCCinh-A01 (.1–1 µg), a CaCC blocker, reversed tactile allodynia induced by SNT in female but not male rats. In contrast, T16Ainh-A01, a selective anoctamin-1 blocker, had an equal antiallodynic effect in both sexes. SNT increased bestrophin-1 protein expression in injured L5 dorsal root ganglia (DRG) in female rats but decreased bestrophin-1 protein in L5 DRG in male rats. Ovariectomy prevented the antiallodynic effect of CaCCinh-A01, but 17β-estradiol replacement restored it. The effect of CaCCinh-A01 was prevented by intrathecal administration of MPP, a selective ERα antagonist, in rats with and without prior hormonal manipulation. In female rats with neuropathy, ovariectomy prevented the increase in bestrophin-1 and ERα protein expression, while 17β-estradiol replacement allowed for an increase in both proteins in L5 DRG. Furthermore, ERα antagonism (with MPP) prevented the increase in bestrophin-1 and ERα protein expression. Finally, ERα activation with PPT, an ERα selective activator, induced the antiallodynic effect of CaCCinh-A01 in neuropathic male rats and prevented the reduction in bestrophin-1 protein expression in L5 DRG. In summary, data suggest ERα activation is necessary for bestrophin-1’s pronociceptive action to maintain neuropathic pain in female rats. PerspectiveThe mechanisms involved in neuropathic pain differ between male and female animals. Our data suggest that ERα is necessary for expression and function of bestrophin-1 in neuropathic female but not male rats. Data support the idea that a therapeutic approach to relieving neuropathic pain must be based on patient's gender.

Icariin attenuates the calcification of vascular smooth muscle cells through ERα - p38MAPK pathway.

To investigate the relationship between icariin and the osteoblastic differentiation of vascular smooth muscle cells (VSMCs) and the signal pathway involved. We applied a universally accepted calcification model of VSMCs induced by β glycerophosphate. Then the VSMCs calcification was observed by treatment with icariin and/or inhibitors of estrogen receptors (ERs) and p38-mitogen-activated protein kinase (MAPK) signaling. Icariin inhibited osteoblastic differentiation and mineralization of VSMCs due to decreased ALP activity and Runx2 expression. Further study demonstrated that icariin exerted this suppression effect through activating p38-MAPK but not extracellular-regulated kinase, JNK or Akt. An inhibitor of p38-MAPK partially reversed the inhibitory effects of icariin on osteoblastic differentiation. Interestingly, treatment of VSMCs with an ER antagonist ICI182780 and a selective ERα receptor antagonist PPT attenuated icariin-mediated inhibition effect of VSMCs calcification, associated with suppression of p38-MAPK phosphorylation. Icariin inhibited the osteoblastic differentiation of VSMCs, and that the inhibitory effects were mediated by p38-MAPK pathways through ERα.

OR11-01 A New Model for Estrogen Receptor-alpha Antagonist Mechanism of Action

Abstract Disclosure: J.C. Nwachukwu: None. J.W. Njeri: None. T. Venables: None. C. Seath: None. M.E. Pipkin: None. Y. Hou: None. B.S. Katzenellenbogen: None. J.A. Katzenellenbogen: None. K.W. Nettles: None. We recently developed Dual Mechanism Estrogen Receptor-α (ERα) Inhibitors (DMERI) with more antagonist efficacy than selective ERα modulators (SERMs) such as tamoxifen, and selective ERα degraders (SERDs) such as fulvestrant in certain breast cancer models. Current models of anti-estrogen action are largely based on genomics studies in growth arrested cells, where the effects primarily reversed estradiol-mediated gene expression. In that context, most ER antagonists, especially the SERDs, show very little gene activation. We were interested in understanding antagonist mechanism of action in asynchronous cells that were growing in intact medium that had not been charcoal-stripped to remove estradiol and growth factors. RNA-seq of asynchronous MCF-7 cells treated with 4-hydroxytamoxifen, fulvestrant, a SERM-like DMERI, and a SERD-like DMERI showed that all four classes of ligands equally up-regulated and down-regulated differentially expressed genes (DEGs), which included a core set of regulatory networks, as well as ligand-selective gene networks. The number of DEGs predicted efficacy, with tamoxifen regulating few genes, and the SERD-like DMERI regulating the most genes. The SERD-like DMERI also inhibited expression of many more genes that encode cell cycle regulators and key cancer drivers, such as ERα, AKT1, HRAS, and several growth factor receptors. Landscape in silico analysis based on chromatin immunoprecipitation (ChIP)-seq data suggests that recruitment of the ERα MegaTrans complex in collaboration with the oxysterol receptor, LXRα, predicts up-regulation, while cooperation with the retinoblastoma-associated proteins, RB1 and RBL1, predicts down-regulation of target genes. APEX2-mediated proximity labeling in asynchronous cells revealed that fulvestrant and the SERD-like DMERI labeled both coactivators and corepressors. Both compounds also increase labeling of STAG1 and other cohesin subunits that coordinate chromatin looping, while also associating with the nuclear matrix protein Lamin B1 and heterochromatin-associated proteins. These data suggest that antagonists have dual roles in genome organization. An shRNAmiR screen targeting over 300 chromatin regulatory factors revealed networks of shared and exclusive coregulators used by antiestrogens, including the heterochromatin protein, CBX1, which was exclusively required for growth inhibition by fulvestrant, and subunits of histone acetylase and methylase complexes such as KMT2A (the catalytic subunit of the MLL1 complex), which were broadly required. Thus, antiestrogen-driven growth suppression involves both common and ligand-selective transcriptional regulatory networks through utilization of both coactivators and corepressors. Supported by NIH R01CA220284 and BCRF-083 and BCRF-084 grants. Presentation Date: Friday, June 16, 2023

BDE-209 disturbed proliferation and differentiation of spermatogonia during mitotic process through estrogen receptor α.

Deca-bromodiphenyl ether (BDE-209) exposure caused spermatogenesis disorder resulting in poor sperm quality has become a public concern in recent years. Spermatogenesis refers to the process by which the division of spermatogonia stem cells (SSCs) produces haploid spermatozoa, including mitosis, meiosis, and spermiogenesis. However, the mechanism of mitosis including proliferation and differentiation of spermatogonia dysfunction induced by BDE-209 remains largely unclear. Here, our data showed that BDE-209 exposure caused a decline in sperm quality with seminiferous tubule structure disorder in rats. In addition, BDE-209 exposure damage spermatogonia proliferation and differentiation with decreasing level of PLZF and cKit in testis. Moreover, rats exposed to BDE-209 decreased the expression of ERα, whereas an elevated expression of Wnt3a and Wnt5a. Mechanistically, supplementation with propipyrazole triol (PPT, a selective ERα pathway agonist) rescued sperm quality and attenuated impairment of proliferation and differentiation of spermatogonia in BDE-209-induced rats. Therefore, ERα plays a crucial role in the proliferation and differentiation of spermatogonia during mitotic process. In conclusion, our study clarified the role of ERα in BDE-209-induced spermatogonia proliferation and differentiation in rats and provides a potential therapeutic application on poor sperm quality caused by BDE-209 exposure.

Discovery of a Novel Class of PROTACs as Potent and Selective Estrogen Receptor α Degraders to Overcome Endocrine-Resistant Breast Cancer In Vitro and In Vivo.

The estrogen receptor (ER) is a well-established target for endocrine therapies of ER-positive breast cancer (ER+ BC), but endocrine resistance limits the efficacy of clinical drugs. Using proteolysis targeting chimera (PROTAC) technology to degrade ERα may be an effective alternative to endocrine therapies. Herein, we disclose a novel series of potent and selective ERα PROTACs based on an oxabicycloheptane sulfonamide (OBHSA) scaffold, with no associated ERβ degradation. These PROTACs showed significant antiproliferation and ERα degradation activities against a broad spectrum of ER+ BC cells including tamoxifen-resistant and ERα mutant cell lines. Genomics analysis confirmed that these PROTACs inhibited the nascent RNA synthesis of ERα target genes and impaired genome-wide ERα binding. Compound ZD12 exhibited excellent antitumor potency and ERα degradation activity in both tamoxifen-sensitive and -resistant BC mice models, which are superior to fulvestrant. This study demonstrates the potential of these PROTACs as novel drug candidates for endocrine-resistant BC treatment.

Loureirin C, from Chinese Dragon's Blood (Dracaena cochinchinensis S.C. Chen), is a novel selective estrogen receptor α modulator with anti-Alzheimer's disease effects

As the incidence of Alzheimer's disease (AD) continues to rise in recent years, there are few therapeutic drugs for AD treatment with limited efficacy. AD occurs twice as often in women as that in men, partially due to the low estrogen level in women after menopause. Phytoestrogens (PEs), similar to endogenous estrogens in chemical structure with neuroprotection and fewer side effects, have good development and application prospects in AD-treatment. Loureirin C is an active ingredient isolated from Chinese Dragon's Blood (CDB) with a similar structure to 17β-E2. In our study, we found that loureirin C targeted to ERα and had partial-agonistic activity using molecular docking prediction and dual-luciferase reporter assay. However, it is still unclear whether loureirin C has estrogenic effects in body, and whether exerts anti-AD effect through ERα. In this paper, the ERα selective inhibitor MPP or ERα specific small interfering RNA (siERα) mediated gene silencing technology were used. Besides,E-SCREEN method were used to evaluate the estrogen effects of loureirin C in vivo and in vitro. MTT assay, Western blot, real-time PCR technology and behaver tests was used to investigate the neuroprotective effect, cognitive function and the underlying mechanism. We found that loureirin C possessed estrogenic activity, had neuroprotective effects in AD cells and improved cognitive impairment in AD mice via ERα. Loureirin C may be a potential candidate for AD.

Abstract PD10-04: PD10-04 Combination of the next generation oral SERD camizestrant (AZD9833) with CDK4/6 and mTOR/AKT inhibitors delivers robust efficacy in a broad range of ER+ breast tumors

Abstract Next-generation oral selective estrogen receptor degraders (ngSERDs) are aiming to become the backbone endocrine therapy (ET) for patients with estrogen receptor positive (ER+) breast cancer (BC) by achieving greater ER signaling blockade than current therapies and tackling key mechanisms of resistance. Camizestrant (AZD9833) is an ngSERD for the treatment of ER+ BC which has demonstrated selective ERα degradation, pure ER antagonism and significant anti-tumor activity in ESR1 wild-type (ESR1wt) and mutant (ESR1m) tumors, as well as encouraging clinical activity in early phase trials. ER+ BC is responsive to therapies targeting ER and CDK4/6 signaling in the adjuvant and metastatic settings. To explore camizestrant combination potential as a backbone ET, camizestrant was partnered with either palbociclib or abemaciclib in CDK4/6 inhibitor (CDK4/6i) naïve and resistant in vitro and in vivo models. Combination benefit was observed in vitro in 3 parental ER+ BC cell lines, moreover camizestrant plus abemaciclib showed activity in palbociclib-resistant cell lines including lines harboring CCNE1amp and RB1 loss. In vivo, combination of camizestrant and either CDK4/6i was well tolerated with the combinations promoting improved efficacy in ESR1wt and ESR1m PDX tumor models compared to monotherapy arms. ER+ BC has a high prevalence of alterations in the PI3K/AKT/PTEN pathway which provides opportunities for treatment with PI3K/AKT pathway inhibitors. Camizestrant delivered enhanced efficacy when combined with mTORC1 inhibitor everolimus and AKT inhibitor capivasertib in CTC-174, an ESR1m and PI3KCAm tumor model. Additionally, combination with capivasertib was more efficacious than single treatments, at clinically relevant dose and schedules, in both PI3Kwt and mutated pathway in PDX models. These data demonstrate the interplay between PI3K/AKT/PTEN pathway inhibition and camizestrant mechanism of action. Finally, we explored a potential triple combination of camizestrant, capivasertib and palbociclib in Palbociclib-resistant models representative of PI3KCA/AKT/PTEN wt and altered tumors. This strategy delivered robust efficacy across these models comparing to single treatments and double combinations. The triplet led to durable regressions at clinically achievable doses of all compounds, irrespective of genetic background and aligned to biomarker modulation of the three signaling axis. These preclinical data demonstrate the potential of camizestrant to become the backbone ET, with high combinability in vivo with inhibitors of CDK4/6, mTOR and AKT. These combinations demonstrate the opportunity to impact care of patients with early and metastatic ER+BC, delivering benefit to broad patient populations including those with ESR1wt or ESR1m tumors, independent of PI3K/AKT/PTEN pathway mutation status, and in patients with both CDK4/6i sensitive and resistant tumors. Citation Format: Larissa Carnevalli, Susana Ros, Jelena Urosevic, Natalie Cureton, Sophie Darcy, Mandy Lawson, Stuart Williamson, Pablo Morentin Gutierrez, Azadeh Bashi, Jennifer Moss, Christopher Morrow, Barry Simon, Teresa Klinowska. PD10-04 Combination of the next generation oral SERD camizestrant (AZD9833) with CDK4/6 and mTOR/AKT inhibitors delivers robust efficacy in a broad range of ER+ breast tumors. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-04.

Modification of oestrogen signalling pathways influences cough induced by citric acid but not capsaicin in the animal model of both sexes

To clarify the role of oestrogen signalling and the role of oestrogen receptor alpha (ERα) in the cough pathways we performed a study in which coughing was observed in both sexes animal models after the treatment by selective ERα degrader fulvestrant (ICI 182–780) and inhibitor of oestrogen synthesis danazol. Degradation of ERα with the normal plasma oestrogen levels induced by fulvestrant, significantly augments the cough response of female but not male guinea pigs. These changes were observed in citric acid-induced cough. Female guinea pigs responded with an increased count of cough expulsions per challenge time and we also detected shorter cough latency. The capsaicin-induced cough did not change. A similar response was observed after danazol treatment, which decreased the plasma oestrogen level. Our results indicate that the transient receptor potential vanilloid-1 (TRPV1) channel-mediated cough is resistant to the hypoestrous state, while the citric acid-mediated cough is oestrogen-dependent and hypersensitive during the hypoestrous state.

Estrogen receptor α activation modulates the gut microbiome and type 2 diabetes risk factors.

Estradiol and exercise can decrease risk factors associated with type 2 diabetes (T2D) including total body weight gain and abdominal fat gain. Estradiol functions through estrogen receptor (ER) α and ERβ. Some studies suggest that activation of ERα may provide protection against T2D. Female Wistar rats were ovariectomized and fed a high‐fat diet for 10 weeks and divided into the following 5 experimental groups: (1) no treatment (control), (2) exercise, (3) estradiol, (4) propylpyrazoletriyl (a selective ERα agonist), and (5) diarylpropionitrile (a selective ERβ agonist). ERα activation decreased the abundance of Firmicutes, and ERα and ERβ activation increased the abundance of Bacteroidetes. ERα activation decreased food consumption, and ERα and ERβ activation increased voluntary activity. Exercise was the only treatment to decrease the blood glucose and serum insulin levels. ERα activation, but not ERβ, increased hepatic protein expression of ACC and FAS and decreased hepatic protein expression of LPL. ERα activation also decreased hepatic mRNA expression of PPARα and PPARγ. This study elucidates the functions of estradiol by assessing specific activation of ERα and ERβ. As obesity increases the abundance of Firmicutes and decreases the abundance of Bacteroidetes, our study shows that ERα activation can restore the gut microbiome to non‐obese abundances. This study further provides novel insights into ERα’s role in hepatic fat metabolism via regulation of ACC, FAS, LPL, PPARα, and PPARγ.

Silibinin improves L-cell mass and function through an estrogen receptor-mediated antioxidative mechanism

BackgroundSilibinin, a major component of milk thistle extract silymarin, promotes hypoglycemia by activating estrogen receptor (ER) α and β-mediated pathways in pancreatic β-cells. Glucagon-like peptide-1 (GLP-1) is the enteroendocrine peptide produced in L-cells, and it controls glucose homeostasis through multiple pathways. The effect of silibinin on L-cell mass and function is still unknown. PurposeThe protective effect of silibinin on palmitate (PA)-treated intestinal L-cell line GLUTag cells and the SHRSP•Z-Leprfa/Izm-Dmcr (SP•ZF) diabetic rat model was investigated in current study. MethodsAfter pre-incubation with 50 μM silibinin for 4 h, GLUTag cells were treated with 0.125 mM PA. MTT, Annexin V/PI apoptosis, Hoechst 33342 staining, western blot, DCFH-DA, GLP-1 ELISA, qRT-PCR and immunofluorescence analyses were undertaken to determine ER-dependent protection of silibinin against PA-induced cellular damage. The differential protein expression of GLUTag cells under different treatments was examined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). The SP•ZF diabetic rat model was chosen for in vivo study. After 4 weeks of gastric gavage with 100 or 300 mg kg−1 of silibinin, the physiological indexes of the rats were measured. Cells expressing GLP-1, 8‑hydroxy-2′-deoxyguanosine (8-OHdG), ERα, and/or ERβ in duodenum tissues were detected by immunofluorescence. ResultsThe current study showed that the GLUTag cells preincubated with silibinin activated the transcription factor nuclear erythroid-2 like factor-2 (Nrf2)-antioxidant pathway, reduced reactive oxygen species (ROS) generation, and improved cell survival and GLP-1 content, while the antioxidative effect of silibinin was blocked by the selective ERα antagonist MPP or ERβ antagonist PHTPP in GLUTag cells. Our proteomics data further revealed that ERα or β inactivation reduced glutathione peroxide and proteins associated with endocytosis and reproduction, thus at least partially reversing the protective effect of silibinin. SP•ZF rats received silibinin treatment showed increased serum GLP-1 content and improved glucose homeostasis. Furthermore, silibinin upregulated ERα and β levels and reduced the level of 8-OHdG in GLP-1-positive cells. ConclusionsOur study showed that silibinin improved L-cell mass and function through an ER-mediated antioxidant pathway, and the proteomics analysis revealed for the first time the differential regulation of proteins by PA and silibinin in GLUTag cells.

Abstract P1-17-10: H3B-6545, a novel selective estrogen receptor covalent antagonist (SERCA), in estrogen receptor positive (ER+), human epidermal growth factor receptor 2 negative (HER2-) advanced breast cancer - A phase II study

Abstract Purpose: H3B-6545, a novel Selective ERα Covalent Antagonist (SERCA), inactivates both and wild-type and mutant ERα by targeting cysteine 530 and enforcing antagonist conformation. H3B-6545 demonstrated preclinical high activity in breast cancer models with constitutively active ESR1 mutations (Furman C, SABCS 2020) and clinical activity in pretreated women with ER+ breast cancer (Hamilton EP, ASCO 2021). Patients and Methods: The primary objective of the phase II is to estimate the objective response rate (ORR), clinical benefit rate (CBR), progression-free survival (PFS), and secondary objectives include safety. Results: 94 pts were treated with 450 mg daily, the recommended phase II dose: 11 in the phase I and 83 in the phase II parts of the trial. Patients and tumor characteristics were presented previously (Hamilton EP, ASCO, 2021). As of March 31, 2021, grade (gr) 2 or higher adverse events (AE) reported in ≥10% were anemia (19%), nausea (17%), fatigue (16%), and diarrhea (12%). Laboratory gr 2 or higher abnormalities reported in ≥10% pts were creatinine clearance decrease (39%), hemoglobin decrease (38%), Lymphocytes decrease (37%), ALT increase (14%), AST increase (13%), bilirubin increase (12%), and creatinine increase (12%). AE of gr 1 sinus bradycardia (asymptomatic) was reported in 35% and gr 2 (symptomatic, no intervention needed) was reported in 5%. Gr 2 and 3 QTcF prolongation were reported in 2 and 3 pts, respectively. There were no treatment-related deaths. Efficacy estimates are presented in Table 1. CI: Confidence interval. N: total number of pts in full analysis set, used in PFS analysis. n: number of response-evaluable pts, used in ORR and CBR analysis. Efficacy estimates were consistent across the various subgroups. Responses were demonstrated in heavily pretreated pts, pts with visceral metastases, pts with constitutionally active ESR1 Y537S mutations, and in pts who received chemotherapy in the metastatic setting. Numerically higher response rate and longer PFS were observed in pts with ESR1 Y537s. Conclusions: H3B-6545 has a manageable safety profile and demonstrated single-agent anti-tumor activity in heavily pretreated ER+, HER2- mBC patients. Clinical activity was consistent across the various subgroups. Tumors harboring the constitutionally active ESR1 Y537S mutation may present higher ERα activity, and consequently enrich for luminal A traits and demonstrate greater lineage dependence on ERα. Table 1.Consistency of H3B-6545 activity across the key subgroupsEfficacyClinical CharacteristicORR % (95% CI)CBR % (95% CI)Median PFS mo (95% CI)Overall population (N=94, n=72)16.7 (8.9, 27.3)40.3 (28.9, 52.5)5.1 (3.2, 6.2)Liver and/or lung metastases (N=76, n=62)17.7 (9.2, 29.5)41.9 (29.5, 55.2)5.4 (1.8, 7.2)≥3 prior regimens (N=49, n=39)20.5 (9.3, 36.5)48.7 (32.4, 65.2)5.4 (3.5, 7.3)Prior chemotherapy (N=47, n=35)17.1 (6.6, 33.6)51.4 (34.0, 68.6)5.5 (3.6, 7.3)PgR+ (N=38, n=32)15.6 (5.3, 32.8)50.0 (31.9, 68.1)5.4 (2.0, 8.8)ESR1 clonal Y537S (N=10, n=10)30.0 (6.7, 65.2)60.0 (26.2, 87.8)7.3 (0.8, 11.2)ESR1 clonal D538G (N=19, n=17)0.0 (0.0, 19.5)35.3 (14.2, 61.7)5.4 (1.7, 7.2) Citation Format: Erika P Hamilton, Judy S Wang, Timothy Pluard, Aki Morikawa, E Claire Dees, Robert H Jones, Barbara Haley, Anne Armstrong, Adam L Cohen, Pamela Munster, Gail S Wright, Fadi Kayali, Lisa Cantagallo, Manav Korpal, Jenny Long, Jianjun Xiao, Benoit Destenaves, Lei Gao, Tarek Sahmoud, Antonio Gualberto, Dejan Juric. H3B-6545, a novel selective estrogen receptor covalent antagonist (SERCA), in estrogen receptor positive (ER+), human epidermal growth factor receptor 2 negative (HER2-) advanced breast cancer - A phase II study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-17-10.

Abstract P1-17-03: H3B-6545 in combination with palbociclib in women with metastatic estrogen receptor-positive (ER+), human epidermal growth factor receptor 2 (HER2)-negative breast cancer, phase 1b study

Abstract Background: H3B-6545, a novel Selective ERα Covalent Antagonist (SERCA), inactivates both mutant and wild-type ERα by targeting cysteine 530 and enforcing antagonist conformation. It demonstrated a manageable safety profile and single-agent antitumor activity in heavily pretreated ER+, HER2- mBC patients (pts) (Hamilton et al, ASCO, 2021). Methods: The study aims at determining the recommended phase II doses of the combination of H3B-6545 and palbociclib in pts with ER+, HER2- mBC. Other endpoints include safety, pharmacokinetics, and preliminary efficacy. The escalation phase enrolls pts with at least 2 prior therapies in the metastatic setting. Up to one prior chemotherapy and up to one prior CDK4/6 inhibitor are allowed. Each cohort enrolled 6 pts to ensure availability of sufficient PK data. During the first cycle, H3B-6545 was added to palbociclib on day 9. Dose-limiting toxicities (DLTs) were assessed during the first 28 days starting from the 1st day of adding H3B-6545 to palbociclib (cycle 1 day 9 to cycle 2 day 8). Both drugs were started on day 1 of each of the subsequent cycles. Results: As of June 15, 2021, 14 pts were enrolled: 7 in Cohort 1 (H3B-6545 300 mg QD and palbociclib 100 mg QD) and 7 in Cohort 2 (H3B-6545 300 mg QD and palbociclib 125 mg QD). Median age was 61 years (range: 28-75 years), ECOG performance status was 0 in 7 pts (50%) and 1 in 7 pts (50%) and 9 pts (64%) had lung and/or liver metastases. Median number of prior therapies in the metastatic setting was 3 (range: 1-6). Prior therapy in the metastatic setting included fulvestrant (93%), CDK4/6 inhibitors (79%), aromatase inhibitors (64%), and chemotherapy (29%). One pt in each Cohort was not evaluable for dose limiting toxicity (DLT) assessment and no DLTs were observed in the first 2 Cohorts. Seven pts discontinued study treatment because of progression and 1 pt discontinued due to diagnosis of a second primary cancer during the first cycle. Non-hematological grade 2 or higher adverse events (AE), irrespective of causality, reported in ≥2 pts were: nausea, vomiting, abdominal pain, and bone pain, each observed in 2 pts (14%). Gr. 1 and 2 sinus bradycardia were reported in 6 (43%) and 1 pts (7%), respectively. One pt (7%) had grade 1 QT prolongation. No grade 4 AEs or treatment-related deaths were reported. For hematology and chemistry laboratory abnormalities: gr. 3 and 4 neutropenia in 7 pts (50%) and 2 pts (14%), respectively, gr. 3 thrombocytopenia in 1 pt (7%), gr. 3 anemia in 2 pts (14%). Grade 2 decrease in estimated glomerular filtration rate was observed in 7 pts (50%), all reported irrespective of causality. Co-administration of palbociclib had no meaningful impact on H3B-6545 exposure (15% and 21% increase in geometric means of AUC and Cmax, respectively). Co-administration of H3B-6545 modestly increased palbociclib exposure (49% and 36% increase in geometric means of AUC and Cmax, respectively). Among 6 response-evaluable pts in Cohort 1, 2 pts (33%) had confirmed partial responses and 4 pts (67%) had stable disease. Both responding pts received prior therapy with a CDK4/6 inhibitor and fulvestrant. Cohort 2 efficacy data is not yet mature. Recruitment is currently ongoing in Cohort 3 (H3B-6545 450 mg QD and palbociclib 125 mg QD). Conclusions: The combination of H3B-6545 (up to 300 mg dose) and palbociclib (up to 125 mg dose) was well-tolerated and demonstrated preliminary anti-tumor activity in heavily pretreated pts with ER+, HER2- mBC. ClinicalTrials.gov Identifier: NCT04288089. Citation Format: Stephen Johnston, Timothy J Pluard, Judy S Wang, Erika P Hamilton, Dejan Juric, Catherine R Scholz, Elizabeth Hnitecki, Sara Dar, Lei Gao, Lisa Cantagallo, Manav Korpal, Jianjun Alan Xiao, Lihua Yu, Tarek Sahmoud, Antonio Gualberto. H3B-6545 in combination with palbociclib in women with metastatic estrogen receptor-positive (ER+), human epidermal growth factor receptor 2 (HER2)-negative breast cancer, phase 1b study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-17-03.

ERα, but not ERβ and GPER, Mediates Estradiol-Induced Secretion of TSH in Mouse Pituitary.

Although estradiol (E2) plays a critical role in the promotion of pituitary development and in the regulation of various pituitary hormones, its effects on the thyroid-stimulating hormone (TSH) remain unaddressed. The actions of E2 are mediated by two classical nuclear estrogen receptors α (ERα) and β (ERβ) and the G protein-coupled estrogen receptor (GPER). However, the types of estrogen receptor involvement in the regulation of thyrotropes are still limited. In this study, we demonstrate that ERα, but not ERβ and GPER, is localized to thyrotropes in the pituitary of female mouse. In agreement with the presence of ERα in thyrotropes, E2 was shown to stimulate TSH release in vitro from primary culture of female mouse pituitary cells. PPT, a ERα-selective agonist, but not DPN (a ERβ-selective agonist) and G-1 (a GPER-selective agonist), was shown to stimulate TSH release in mouse pituitary cells. This effect could be prevented by the specific ER antagonist fulvestrant and the selective ERα antagonist MPP. The findings of this study suggest that E2 may bind to ERα to trigger TSH release and provide novel information on the differential regulation of multiple estrogen receptors in the pituitary.

Human Estrogen Receptor α Antagonists. Part 1: 3-D QSAR-Driven Rational Design of Innovative Coumarin-Related Antiestrogens as Breast Cancer Suppressants through Structure-Based and Ligand-Based Studies.

The estrogen receptor α (ERα) represents a 17β-estradiol-inducible transcriptional regulator that initiates the RNA polymerase II-dependent transcriptional machinery, pointed for breast cancer (BC) development via either genomic direct or genomic indirect (i.e., tethered) pathway. To develop innovative ligands, structure-based (SB) three-dimensional (3-D) quantitative structure-activity relationship (QSAR) studies have been undertaken from structural data taken from partial agonists, mixed agonists/antagonists (selective estrogen receptor modulators (SERMs)), and full antagonists (selective ERα downregulators (SERDs)) correlated with either wild-type or mutated ERα receptors. SB and ligand-based (LB) alignments allow us to rule out guidelines for the SB/LB alignment of untested compounds. 3-D QSAR models for ERα ligands, coupled with SB/LB alignment, were revealed to be useful tools to dissect the chemical determinants for ERα-based anticancer activity as well as to predict their potency. The herein developed protocol procedure was verified through the design and potency prediction of 12 new coumarin-based SERMs, namely, 3DQ-1a to 3DQ-1e, that upon synthesis turned to be potent ERα antagonists by means of either in vitro or in vivo assays (described in the second part of this study).

Human estrogen receptor α antagonists, part 2: Synthesis driven by rational design, in vitro antiproliferative, and in vivo anticancer evaluation of innovative coumarin-related antiestrogens as breast cancer suppressants

New twelve in silico designed coumarin-based ERα antagonists, namely 3DQ-1a to 3DQ-1е, were synthesized and confirmed as selective ERα antagonists, showing potencies ranging from single-digit nanomolar to picomolar. The hits were confirmed as selective estrogen receptor modulators and validated as antiproliferative agents using MCF-7 breast cancer cell lines exerting from picomolar to low nanomolar potency, at the same time showing no agonistic activity within endometrial cell lines. Their mechanism of action was inspected and revealed to be through the inhibition of the Raf-1/MAPK/ERK signal transduction pathway, preventing hormone-mediated gene expression on either genomic direct or genomic indirect level, and stopping the MCF-7 cells proliferation at G0/G1 phase. In vivo experiments, by means of the per os administration to female Wistar rats with pre-induced breast cancer, distinguished six derivatives, 3DQ-4a, 3DQ-2a, 3DQ-1a, 3DQ-1b, 3DQ-2b, and 3DQ-3b, showing remarkable potency as tumor suppressors endowed with optimal pharmacokinetic profiles and no significant histopathological profiles. The presented data indicate the new compounds as potential candidates to be submitted in clinical trials for breast cancer therapy.

Development of novel tetrahydroisoquinoline-hydroxamate conjugates as potent dual SERDs/HDAC inhibitors for the treatment of breast cancer

Concomitant inhibition of estrogen receptor alpha (ERα) and histone deacetylase (HDAC) signaling has been proven effective in endocrine-resistant ER+ breast cancers. Herein, a series of tetrahydroisoquinoline (THIQ)-hydroxamate conjugates were rationally designed and synthesized as dual SERDs/HDAC inhibitors by incorporating the hydroxamate, a known HDAC pharmacophore, into a privileged THIQ scaffold of selective ERα degraders (SERDs). Some of these THIQ-hydroxamate conjugates displayed remarkable HDAC6 inhibition and improved antiproliferative activity against MCF-7 cells. Particularly, the most potent HDAC inhibitor 19k also exhibits potent ERα binding affinity, good ERα degradation efficacy and the best antiproliferative activity. Besides, 19k displayed superior antitumor efficacy than the drug combination (Fulvestrant + SAHA) through promoting ERα degradation and histone acetylation in an MCF-7 xenograft model, without causing observable toxicity. Collectively, this study validates the therapeutic potential of a dual-acting compound with potent ERα degradation efficacy and HDAC6 inhibition in breast cancer.

Estradiol Inhibits Human Brain Vascular Pericyte Migration Activity: A Functional and Transcriptomic Analysis.

Stroke is the third leading cause of mortality in women and it kills twice as many women as breast cancer. A key role in the pathophysiology of stroke plays the disruption of the blood–brain barrier (BBB) within the neurovascular unit. While estrogen induces vascular protective actions, its influence on stroke remains unclear. Moreover, experiments assessing its impact on endothelial cells to induce barrier integrity are non-conclusive. Since pericytes play an active role in regulating BBB integrity and function, we hypothesize that estradiol may influence BBB by regulating their activity. In this study using human brain vascular pericytes (HBVPs) we investigated the impact of estradiol on key pericyte functions known to influence BBB integrity. HBVPs expressed estrogen receptors (ER-α, ER-β and GPER) and treatment with estradiol (10 nM) inhibited basal cell migration but not proliferation. Since pericyte migration is a hallmark for BBB disruption following injury, infection and inflammation, we investigated the effects of estradiol on TNFα-induced PC migration. Importantly, estradiol prevented TNFα-induced pericyte migration and this effect was mimicked by PPT (ER-α agonist) and DPN (ER-β agonist), but not by G1 (GPR30 agonist). The modulatory effects of estradiol were abrogated by MPP and PHTPP, selective ER-α and ER-β antagonists, respectively, confirming the role of ER-α and ER-β in mediating the anti-migratory actions of estrogen. To delineate the intracellular mechanisms mediating the inhibitory actions of estradiol on PC migration, we investigated the role of AKT and MAPK activation. While estradiol consistently reduced the TNFα-induced MAPK and Akt phosphorylation, only the inhibition of MAPK, but not Akt, significantly abrogated the migratory actions of TNFα. In transendothelial electrical resistance measurements, estradiol induced barrier function (TEER) in human brain microvascular endothelial cells co-cultured with pericytes, but not in HBMECs cultured alone. Importantly, transcriptomics analysis of genes modulated by estradiol in pericytes showed downregulation of genes known to increase cell migration and upregulation of genes known to inhibit cell migration. Taken together, our findings provide the first evidence that estradiol modulates pericyte activity and thereby improves endothelial integrity.