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Beyond their clinical use as selective estrogen receptor modulators (SERMs), raloxifene and tamoxifen have attracted recent attention for their favorable activity against a broad range of dangerous human pathogens. While consistently demonstrated to occur independently on classic estrogen receptors, the mechanisms underlying SERMs antimicrobial efficacy remain still poorly elucidated, but fundamental to benefit from repurposing strategies of these drugs. Macrophages are innate immune cells that protect from infections by rapidly reprogramming their metabolic state, particularly cholesterol disposal, which is at the center of an appropriate macrophage immune response as well as of the anabolic requirements of both the pathogen and the host cells. The microsomal antiestrogen binding site (AEBS) comprises enzymes involved in the last stages of cholesterol biosynthesis and is a high affinity off-target site for SERMs. We review here recent findings from our laboratory and other research groups in support of the hypothesis that AEBS multiprotein complex represents the candidate pre-genomic target of SERMs immunomodulatory activity. The cholesterol restriction resulting from SERMs-mediated AEBS inhibition may be responsible for boosting inflammatory and antimicrobial pathways that include inflammasome activation, modulation of Toll-like receptors (TLRs) responses, induction of interferon regulatory factor (IRF3) and nuclear factor erythroid 2-related factor 2 (NRF2)-mediated transcriptional programs and, noteworthy, the mitigation of excessive inflammatory and proliferative responses, leading to the overall potentiation of the macrophage response to infections., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Maria Candida Cesta and Marcello Allegretti are employees of Dompé farmaceutici S.p.A. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: Patients bearing estrogen receptor (ER)α-negative breast cancer tumors confront poor prognosis and are typically unresponsive to hormone therapy. Previous studies have shown that calcitriol, the active vitamin D metabolite, can induce ERα expression in ERα-negative cells. EB1089, a calcitriol analog with reduced calcemic effects, exhibits greater potency than calcitriol in inhibiting cancer cell growth. However, the impact of EB1089 on ERα expression in triple-negative breast cancer (TNBC) cells remains unexplored. This study aims to investigate whether EB1089 could induce functional ERα expression in TNBC cell lines, potentially enabling the antiproliferative effects of antiestrogens., Materials and Methods: TNBC cell lines HCC1806 and HCC1937 were treated with EB1089, and ERα expression was analyzed using real-time PCR and Western blots. The transcriptional activity of induced ERα was evaluated through a luciferase reporter assay. The antiproliferative effects of tamoxifen and fulvestrant antiestrogens were assessed using the sulforhodamine B assay in the EB1089-treated cells., Results: Our findings indicated that EB1089 significantly induced ERα mRNA and protein expression in TNBC cells. Moreover, EB1089-induced ERα exhibited transcriptional activity and effectively restored the inhibitory effects of antiestrogens, thereby suppressing cell proliferation in TNBC cells., Conclusion: EB1089 induced the expression of functional ERα in TNBC cells, restoring the antiproliferative effects of antiestrogens. These results highlight the potential of using EB1089 as a promising strategy for re-establishment of the antiproliferative effect of antiestrogens as a possible management for TNBC. This research lays the foundation for potential advancements in TNBC treatment, offering new avenues for targeted and effective interventions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.)

Background: Genetic abnormalities in the FGFR signalling occur in 40% of breast cancer (BCa) patients resistant to anti-ER therapy, which emphasizes the potential of FGFR-targeting strategies. Recent findings indicate that not only mutated FGFR is a driver of tumour progression but co-mutational landscapes and other markers should be also investigated. Autophagy has been recognized as one of the major mechanisms underlying the role of tumour microenvironment in promotion of cancer cell survival, and resistance to anti-ER drugs. The selective autophagy receptor p62/SQSTM1 promotes Nrf-2 activation by Keap1/Nrf-2 complex dissociation. Herein, we have analysed whether the negative effect of FGFR2 on BCa cell response to anti-ER treatment involves the autophagy process and/or p62/Keap1/Nrf-2 axis., Methods: The activity of autophagy in ER-positive MCF7 and T47D BCa cell lines was determined by analysis of expression level of autophagy markers (p62 and LC3B) and monitoring of autophagosomes' maturation. Western blot, qPCR and proximity ligation assay were used to determine the Keap1/Nrf-2 interaction and Nrf-2 activation. Analysis of 3D cell growth in Matrigel® was used to assess BCa cell response to applied treatments. In silico gene expression analysis was performed to determine FGFR2/Nrf-2 prognostic value., Results: We have found that FGFR2 signalling induced autophagy in AMPKα/ULK1-dependent manner. FGFR2 activity promoted dissociation of Keap1/Nrf-2 complex and activation of Nrf-2. Both, FGFR2-dependent autophagy and activation of Nrf-2 were found to counteract the effect of anti-ER drugs on BCa cell growth. Moreover, in silico analysis showed that high expression of NFE2L2 (gene encoding Nrf-2) combined with high FGFR2 expression was associated with poor relapse-free survival (RFS) of ER+ BCa patients., Conclusions: This study revealed the unknown role of FGFR2 signalling in activation of autophagy and regulation of the p62/Keap1/Nrf-2 interdependence, which has a negative impact on the response of ER+ BCa cells to anti-ER therapies. The data from in silico analyses suggest that expression of Nrf-2 could act as a marker indicating potential benefits of implementation of anti-FGFR therapy in patients with ER+ BCa, in particular, when used in combination with anti-ER drugs., (© 2024. The Author(s).)

Estrogen receptor alpha (ERα), a nuclear transcription factor, is a well-validated therapeutic target for more than 70% of all breast cancers (BCs). Antagonizing ERα either by selective estrogen receptor modulators (SERMs) or selective estrogen receptor degraders (SERDs) forms the foundation of endocrine therapy and has achieved great success in the treatment of ERα positive (ERα+) BCs. Unfortunately, despite initial effectiveness, endocrine resistance eventually emerges in up to 30% of ERα+ BC patients and remains a significant medical challenge. Several mechanisms implicated in endocrine resistance have been extensively studied, including aberrantly activated growth factor receptors and downstream signaling pathways. Hence, the crosstalk between ERα and another oncogenic signaling has led to surge of interest to develop combination therapies and dual-target single agents. This review briefly introduces the synergisms between ERα and another anticancer target and summarizes the recent advances of ERα-based dual-targeting inhibitors from a medicinal chemistry perspective. Accordingly, their rational design strategies, structure-activity relationships (SARs) and biological activities are also dissected to provide some perspectives on future directions for ERα-based dual target drug discovery in BC therapy., Competing Interests: Declaration of competing interest All co-authors have seen and agreed with the contents of the manuscript and there is no financial interest to report., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Breast cancer is one of the most common malignant tumors in women worldwide, with the majority of cases showing expression of estrogen receptors (ERs). Although drugs targeting ER have significantly improved survival rates in ER-positive patients, drug resistance remains an unmet clinical need. Fulvestrant, which overcomes selective estrogen receptor modulator (SERM) and AI (aromatase inhibitor) resistance, is currently the only long-acting selective estrogen receptor degrader (SERD) approved for both first and second-line settings. However, it fails to achieve satisfactory efficacy due to its poor solubility. Therefore, we designed and synthesized a series of novel scaffold (THC) derivatives, identifying their activities as ER antagonists and degraders. G-5b, the optimal compound, exhibited binding, antagonistic, degradation or anti-proliferative activities comparable to fulvestrant in ER + wild type and mutants breast cancer cells. Notably, G-5b showed considerably improved stability and solubility. Research into the underlying mechanism indicated that G-5b engaged the proteasome pathway to degrade ER, subsequently inhibiting the ER signaling pathway and leading to the induction of apoptosis and cell cycle arrest events. Furthermore, G-5b displayed superior in vivo pharmacokinetics and pharmacodynamics properties, coupled with a favorable safety profile in the MCF-7 tamoxifen-resistant (MCF-7/TR) tumor xenograft model. Collectively, G-5b has emerged as a highly promising lead compound, offering potent antagonistic and degradation activities, positioning it as a novel long-acting SERD worthy of further refinement and optimization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data will be made available on request., (Copyright © 2023. Published by Elsevier Masson SAS.)

Purpose: Estrogen Receptor α (ERα) is a well-established therapeutic target for Estrogen Receptor (ER)-positive breast cancers. Both Selective Estrogen Receptor Degraders (SERD) and PROTAC ER degraders are synthetic compounds suppressing the ER activity through the degradation of ER. However, the differences between SERD and PROTAC ER degraders are far from clear., Methods: The effect of PROTAC ER degrader ERD-148 and SERD fulvestrant on protein degradation was evaluated by western blot analysis. The cell proliferation was tested by WST-8 assays and the gene expressions were assessed by gene microarray and real-time RT-PCR analysis after the compound treatment., Results: ERD-148 is a potent and selective PROTAC ERα degrader. It degrades not only unphosphorylated ERα but also the phosphorylated ERα in the cells. In contrast, the SERD fulvestrant showed much-reduced degradation potency on the phosphorylated ERα. The more complete degradation of ERα by ERD-148 translates into a greater maximum cell growth inhibition. However, ERD-148 and fulvestrant share a similar gene regulation profile except for the variation of regulation potency. Further studies indicate that ERD-148 degrades the ERα in fulvestrant-resistant cells., Conclusion: PROTAC ER degrader has a different mechanism of action compared to SERD which may be used in treating fulvestrant-resistant cancers., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The estrogen-receptor alfa (ERα) is considered pivotal for breast cancer treatment. Although selective estrogen-receptor degraders (SERDs) have been developed to induce ERα degradation and antagonism, their agonistic effect on the uterine tissue and poor pharmacokinetic properties limit further application of ERα; thus, discovering novel SERDs is necessary. The ligand preferentially interacts with several key residues of the protein (defined as hot-spot residues). Improving the interaction with hot-spot residues of ERα offers a promising avenue for obtaining novel SERDs. In this study, pharmacophore modeling, molecular mechanics/generalized Born surface area (MM/GBSA), and amino-acid mutation were combined to determine several hot-spot residues. Focusing on the interaction with these hot-spot residues, hit fragments A1-A3 and A9 were virtually screened from two fragment libraries. Finally, these hit fragments were linked to generate compounds B1-B3 , and their biological activities were evaluated. Remarkably, compound B1 exhibited potent antitumor activity against MCF-7 cells (IC 50 = 4.21 nM), favorable ERα binding affinity ( K i = 14.6 nM), and excellent ERα degradative ability (DC 50 = 9.7 nM), which indicated its potential to evolve as a promising SERD for breast cancer treatment.

Breast cancer ranks as the second leading cause of death among women, but early screening and self-awareness can help prevent it. Hormone therapy drugs that target estrogen levels offer potential treatments. However, conventional drug discovery entails extensive, costly processes. This study presents a framework for analyzing the quantitative structure-activity relationship (QSAR) of estrogen receptor alpha inhibitors. Our approach utilizes supervised learning, integrating self-attention Transformer and molecular graph information, to predict estrogen receptor alpha inhibitors. We established five classification models for predicting these inhibitors in breast cancer. Among these models, our proposed MATH model achieved remarkable precision, recall, F1 score, and specificity, with values of 0.952, 0.972, 0.960, and 0.922, respectively, alongside an ROC AUC of 0.977. MATH exhibited robust performance, suggesting its potential to assist pharmaceutical and health researchers in identifying candidate compounds for estrogen alpha inhibitors and guiding drug discovery pathways.

The pro-oncogenic activities of estrogen receptor alpha (ERα) drive breast cancer pathogenesis. Endocrine therapies that impair the production of estrogen or the action of the ERα are therefore used to prevent primary disease metastasis. Although recent successes with ERα degraders have been reported, there is still the need to develop further ERα antagonists with additional properties for breast cancer therapy. We have previously described a benzothiazole compound A4B17 that inhibits the proliferation of androgen receptor-positive prostate cancer cells by disrupting the interaction of the cochaperone BAG1 with the AR. A4B17 was also found to inhibit the proliferation of estrogen receptor-positive (ER + ) breast cancer cells. Using a scaffold hopping approach, we report here a group of small molecules with imidazopyridine scaffolds that are more potent and efficacious than A4B17. The prototype molecule X15695 efficiently degraded ERα and attenuated estrogen-mediated target gene expression as well as transactivation by the AR. X15695 also disrupted key cellular protein-protein interactions such as BAG1-mortalin (GRP75) interaction as well as wild-type p53-mortalin or mutant p53-BAG2 interactions. These activities together reactivated p53 and resulted in cell-cycle block and the induction of apoptosis. When administered orally to in vivo tumor xenograft models, X15695 potently inhibited the growth of breast tumor cells but less efficiently the growth of prostate tumor cells. We therefore identify X15695 as an oral selective ER degrader and propose further development of this compound for therapy of ER + breast cancers., Significance: An imidazopyridine that selectively degrades ERα and is orally bioavailable has been identified for the development of ER + breast cancer therapeutics. This compound also activates wild-type p53 and disrupts the gain-of-function tumorigenic activity of mutant p53, resulting in cell-cycle arrest and the induction of apoptosis., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Therapeutic strategies targeting nuclear receptors (NRs) beyond their endogenous ligand binding pocket have gained significant scientific interest driven by a need to circumvent problems associated with drug resistance and pharmacological profile. The hub protein 14-3-3 is an endogenous regulator of various NRs, providing a novel entry point for small molecule modulation of NR activity. Exemplified, 14-3-3 binding to the C-terminal F-domain of the estrogen receptor alpha (ERα), and small molecule stabilization of the ERα/14-3-3ζ protein complex by the natural product Fusicoccin A (FC-A), was demonstrated to downregulate ERα-mediated breast cancer proliferation. This presents a novel drug discovery approach to target ERα; however, structural and mechanistic insights into ERα/14-3-3 complex formation are lacking. Here, we provide an in-depth molecular understanding of the ERα/14-3-3ζ complex by isolating 14-3-3ζ in complex with an ERα protein construct comprising its ligand-binding domain (LBD) and phosphorylated F-domain. Bacterial co-expression and co-purification of the ERα/14-3-3ζ complex, followed by extensive biophysical and structural characterization, revealed a tetrameric complex between the ERα homodimer and the 14-3-3ζ homodimer. 14-3-3ζ binding to ERα, and ERα/14-3-3ζ complex stabilization by FC-A, appeared to be orthogonal to ERα endogenous agonist (E2) binding, E2-induced conformational changes, and cofactor recruitment. Similarly, the ERα antagonist 4-hydroxytamoxifen inhibited cofactor recruitment to the ERα LBD while ERα was bound to 14-3-3ζ. Furthermore, stabilization of the ERα/14-3-3ζ protein complex by FC-A was not influenced by the disease-associated and 4-hydroxytamoxifen resistant ERα-Y537S mutant. Together, these molecular and mechanistic insights provide direction for targeting ERα via the ERα/14-3-3 complex as an alternative drug discovery approach., Competing Interests: Conflict of interest The authors declare the following competing interest(s): L. B. and C. O. are scientific co-founders of Ambagon Therapeutics. C. O. and E. S. are employees of Ambagon Therapeutics., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Heterocyclic derivatives of steroid hormones are potent anticancer agents, which are used in the chemotherapy of breast and prostate cancers. Here, we describe a novel series of androstenes, D-modified with imidazole-annulated pendants, with significant anticancer activity. Novel C17-linked imidazole-annulated heterocyclic derivatives of dehydropregnenolone acetate were synthesized by the cyclocondensation with amidines using 3β-acetoxy-21-bromopregna-5,16-dien-20-one as the substrate. The antiproliferative potency of all the synthesized compounds was evaluated against human prostate (22Rv1) and human breast (MCF7) cancer cell lines and cytochromes P450. The lead compound, imidazo[1,2-a]pyridine derivative 3h, was revealed to be a promising candidate for future anticancer drug design, particularly against ERα-positive breast cancer. Lead compound 3h was found to be selective against MCF7 cells with IC 50 of 0.1 μM and to act as both a potent selective agent blocking estrogen receptor α, which is involved in the stimulation of breast cancer growth, and an effective apoptosis inducer. The potential ability of compound 3h to bind to ERα was studded using molecular docking and molecular dynamics simulation. The selectivity analysis showed that lead steroid 3h produces no effects on cytochromes P450 CYP17A1, CYP7A1, and CYP21A2., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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.

Although endocrine therapies involving pharmaceuticals, such as tamoxifen and aromatase inhibitors, had initially demonstrated good responses in patients with estrogen receptor-positive (ER+) breast cancer, they often led to drug resistance. ER plays a vital role in the progression of metastatic diseases. Fulvestrant, a first generation selective estrogen receptor degrader (SERD), can effectively downregulate the ER protein and inhibit its downstream signaling pathways. However, as the drug needs to be intramuscularly injected, its widespread use is limited owing to poor patient compliance. Herein, we described a novel class of orally bioavailable fluorine-substituted SERDs that exhibit improved pharmacokinetic profiles. We substituted the hydroxyl group of clinical SERD candidate 6 with a fluorine atom to diminish phase II metabolism. The subsequent structure-activity relationship (SAR) investigation identified 22h and 27b, which can effectively degrade ER in a dose-dependent manner and exhibit considerable antiproliferative potency and efficacy in vitro and in vivo. The excellent pharmacokinetic profiles of 27b render it promising candidate of clinically useful oral SERD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Drug resistance is a major challenge in conventional endocrine therapy for estrogen receptor (ER) positive breast cancer (BC). BC is a multifactorial disease, in which simultaneous aromatase (ARO) inhibition and ERα degradation may effectively inhibit the signal transduction of both proteins, thus potentially overcoming drug resistance caused by overexpression or mutation of target proteins. In this study, guided by the X-ray structure of a hit compound 30a in complex with ER-Y537S, a structure-based optimization was performed to get a series of multiacting inhibitors targeting both ERα and ARO, and finally a novel class of potent selective estrogen receptor degraders (SERDs) based on a three-dimensional oxabicycloheptene sulfonamide (OBHSA) scaffold equipped with aromatase inhibitor (AI) activity were identified. Of these dual-targeting SERD-AI hybrids, compound 31q incorporating a 1H-1,2,4-triazole moiety showed excellent ERα degradation activity, ARO inhibitory activity and remarkable antiproliferative activity against BC resistant cells. Furthermore, 31q manifested efficient tumor suppression in MCF-7 tumor xenograft models. Taken together, our study reported for the first time the highly efficient dual-targeting SERD-AI hybrid compounds, which may lay the foundation of translational research for improved treatment of endocrine-resistant BC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Breast cancer (BC), a well-known estrogen-dependent cancer, is the most common cancer among women and the leading cause of cancer deaths. One of the most important therapeutic approaches for BC is endocrine therapy targeting estrogen receptor alpha (ERα) and thus blocking the estrogen receptor signaling pathway. Drugs, such as tamoxifen or fulvestrant, are developed based on this theory and have benefited numerous patients with BC for many years. However, many patients with advanced BC, such as tamoxifen-resistant BC, cannot benefit from these developed drugs anymore. Therefore, new drugs targeting ERα are urgently needed by patients with BC. Recently, elacestrant, a novel selective estrogen receptor degrader (SERD), was approved by the United States Food and Drug Administration (FDA), highlighting the importance of ERα degradation in endocrine therapy. Proteolysis targeting chimera (PROTAC) has been considered a powerful technique for targeting protein degradation (TPD). In this regard, we developed and studied a novel ERα degrader, which is a PROTAC-like SERD named 17e. We found that compound 17e can inhibit the growth of BC both in vitro and in vivo and induce the cell cycle arrest of BC. Importantly, 17e displayed no apparent toxicity toward healthy kidney and liver cells. Moreover, we observed that the presence of 17e led to a dramatic increase in the autophagy-lysosome pathway in an ERα-independent manner. Finally, we revealed that a decrease in MYC, a frequent deregulation oncogene in human cancers, was mediated by both ERα degradation and autophagy activation in the presence of 17e. Collectively, we discovered that compound 17e induced ERα degradation and exerts significant anti-cancer effects on BC mainly through promoting the autophagy-lysosome pathway and decreasing MYC level., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: I would like to declare that the work described was original that has not been published previously, and is not under consideration for publication elsewhere, in whole or in part. All the authors listed have approved the manuscript that is enclosed. No conflict of interest exists in the submission of this manuscript, and the manuscript is approved by all authors for publication., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

NOD-like receptor protein 3 (NLRP3) may contribute to the growth and propagation of breast cancer (BC). The effect of estrogen receptor-α (ER-α), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) on NLRP3 activation in BC remains unknown. Additionally, our knowledge of the effect of blocking these receptors on NLRP3 expression is limited. We used GEPIA, UALCAN, and the Human Protein Atlas for transcriptomic profiling of NLRP3 in BC. Lipopolysaccharide (LPS) and adenosine 5'-triphosphate (ATP) were used to activate NLRP3 in luminal A MCF-7 and in TNBC MDA-MB-231 and HCC1806 cells. Tamoxifen (Tx), mifepristone (mife), and trastuzumab (Tmab) were used to block ER-α, PR, and HER2, respectively, on inflammasome activation in LPS-primed MCF7 cells. The transcript level of NLRP3 was correlated with ER-ɑ encoding gene ESR1 in luminal A (ER-α + , PR + ) and TNBC tumors. NLRP3 protein expression was higher in untreated and LPS/ATP-treated MDA-MB-231 cells than in MCF7 cells. LPS/ATP-mediated NLRP3 activation reduced cell proliferation and recovery of wound healing in both BC cell lines. LPS/ATP treatment prevented spheroid formation in MDA-MB-231 cells but did not affect MCF7. HGF, IL-3, IL-8, M-CSF, MCP-1, and SCGF-b cytokines were secreted in both MDA-MB-231 and MCF7 cells in response to LPS/ATP treatment. Tx (ER-α inhibition) promoted NLRP3 activation and increased migration and sphere formation after LPS treatment of MCF7 cells. Tx-mediated activation of NLRP3 was associated with increased secretion of IL-8 and SCGF-b compared to LPS-only-treated MCF7 cells. In contrast, Tmab (Her2 inhibition) had a limited effect on NLRP3 activation in LPS-treated MCF7 cells. Mife (PR inhibition) opposed NLRP3 activation in LPS-primed MCF7 cells. We have found that Tx increased the expression of NLRP3 in LPS-primed MCF7. These data suggest a link between blocking ER-α and activation of NLRP3, which was associated with increased aggressiveness of the ER-α + BC cells.

Breast cancer (BC) is the most diagnosed cancer in women worldwide. In estrogen receptor (ER)-positive disease, anti-estrogens and aromatase inhibitors (AI) improve patient survival; however, many patients develop resistance. Dysregulation of apoptosis is a common resistance mechanism; thus, agents that can reinstate the activity of apoptotic pathways represent promising therapeutics for advanced drug-resistant disease. Emerging targets in this scenario include microRNAs (miRs). To identify miRs modulating apoptosis in drug-responsive and -resistant BC, a high-throughput miR inhibitor screen was performed, followed by high-content screening microscopy for apoptotic markers. Validation demonstrated that miR-361-3p inhibitor significantly increases early apoptosis and reduces proliferation of drug-responsive (MCF7), plus AI-/antiestrogen-resistant derivatives (LTED, TamR, FulvR), and ER- cells (MDA-MB-231). Importantly, proliferation-inhibitory effects were observed in vivo in a xenograft model, indicating the potential clinical application of miR-361-3p inhibition. RNA-seq of tumour xenografts identified FANCA as a direct miR-361-3p target, and validation suggested miR-361-3p inhibitor effects might be mediated in part through FANCA modulation. Moreover, miR-361-3p inhibition resulted in p53-mediated G1 cell cycle arrest through activation of p21 and reduced BC invasion. Analysis of publicly available datasets showed miR-361-3p expression is significantly higher in primary breast tumours vspaired normal tissue and is associated with decreased overall survival. In addition, miR-361-3p inhibitor treatment of BC patient explants decreased levels of miR-361-3p and proliferation marker, Ki67. Finally, miR-361-3p inhibitor showed synergistic effects on BC growth when combined with PARP inhibitor, Olaparib. Together, these studies identify miR-361-3p inhibitor as a potential new treatment for drug-responsive and -resistant advanced BC.

The promising antitumor effects of progesterone derivatives have been identified in many studies. However, the specific mechanism of action of this class of compounds has not been fully described. Therefore, in this study, we investigated the antiproliferative and (anti)estrogenic activities of novel pentacyclic derivatives and benzylidenes of the progesterone series. The antiproliferative effects of the compounds were evaluated on hormone-dependent MCF7 breast cancer cells using the MTT test. Estrogen receptor α (ERα) activity was assessed by a luciferase-based reporter assay. Immunoblotting was used to evaluate the expression of signaling proteins. All benzylidenes demonstrated inhibitory effects with IC 50 values below 10 µM, whereas pentacyclic derivatives were less active. These patterns may be associated with the lability of the geometry of benzylidene molecules, which contributes to an increase in the affinity of interaction with the receptor. The selected compounds showed significant anti-estrogenic potency. Benzylidene 1d ((8 S,9 S,10R,13 S,14 S,17 S)-17-[(2E)-3-(4-fluorophenyl)prop-2-enoyl]-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15-decahydrocyclopenta[a]phenanthren-3-one) was the most active in antiproliferative and anti-estrogenic assays. Apoptosis induced by compound 1d was accompanied by decreases in CDK4, ERα, and Cyclin D1 expression. Compounds 1d and 3d were characterized by high inhibitory potency against resistant breast cancer cells. Apoptosis induced by the leader compounds was confirmed by PARP cleavage and flow cytometry analysis. Compound 3d caused cell arrest in the G2/M phase. Further analysis of novel derivatives of the progesterone series is of great importance for medicinal chemistry, drug design, and oncology., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Antiestrogens (AEs) are used to treat all stages of estrogen receptor (ER)-positive breast cancer. Selective estrogen receptor modulators such as tamoxifen have tissue-specific partial agonist activity, while selective estrogen receptor downregulators such as fulvestrant (ICI182,780) display a more complete antiestrogenic profile. We have previously observed that fulvestrant-induced ERα SUMOylation contributes to transcriptional suppression, but whether this effect is seen with other AEs and is specific to ERα is unclear. Here we show that several AEs induce SUMOylation of ERα, but not ERβ, at different levels. Swapping domains between ERα and ERβ indicates that the ERα identity of the ligand-binding domain helices 3 and 4 (H3-H4 region), which contribute to the static part of the activation function-2 (AF-2) cofactor binding groove, is sufficient to confer fulvestrant-induced SUMOylation to ERβ. This region does not contain lysine residues unique to ERα, suggesting that ERα-specific residues in H3-H4 determine the capacity of the AE-bound ERα ligand-binding domain to recruit the SUMOylation machinery. We also show that the SUMO E3 ligase protein inhibitor of activated STAT 1 increases SUMOylation of ERα and of ERβ containing the H3-H4 region of ERα, but not of ERβ. Together, these results shed new light on the molecular basis for the differential capacity of selective estrogen receptor modulators and selective estrogen receptor downregulators to suppress transcription by ERα., Competing Interests: Conflict of interest The authors declare they have no conflict of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Mansonone G (MG), a 1,2-naphthoquinone isolated from the heartwood of Mansonia gagei Drumm, exhibited several pharmacological activities such as anti-bacterial, anti-estrogenic and anti-adipogenic effect. This study evaluated the cytotoxicity of MG and its derivatives as well as determined the mechanism(s) underlying the cytotoxic activity of the most potent MG derivative on two CRC cell lines, HCT-116 cells carrying p53 wild-type and HT-29 cells carrying p53 mutant. We found that MG and its derivatives could inhibit viability of HCT-116 and HT-29 cells in a concentration-dependent manner. Of all semi-synthetic derivatives of MG, allyl ether mansonone G (MG7) was the most potent cytotoxic agent toward cancer cells and less toxic to normal cells. MG7 could induce ROS generation which was associated with cytotoxicity and apoptosis in both HCT-116 and HT-29 cells. Western blot analysis revealed that MG7 downregulated the expression of Bcl-2 and Bcl-xL proteins in both CRC cell lines and upregulated the expression of BAK protein in HT-29 cells. Moreover, MG7 inhibited AKT signaling pathway in both CRC cell lines and modulated ERK1/2 signaling pathway by inhibiting ERK1/2 phosphorylation in HCT-116 cells and activating ERK1/2 phosphorylation in HT-29 cells. Molecular docking revealed that MG7 could bind to the ATP-binding pocket of AKT and ERK1 via hydrophobic interactions., (© 2022. The Author(s).)

Antiestrogen resistance of breast cancer has been related to enhanced growth factor receptor expression and activation. We have previously shown that ectopic expression and subsequent activation of the insulin-like growth factor-1 receptor (IGF1R) or the epidermal growth factor receptor (EGFR) in MCF7 or T47D breast cancer cells results in antiestrogen resistance. In order to identify novel therapeutic targets to prevent this antiestrogen resistance, we performed kinase inhibitor screens with 273 different inhibitors in MCF7 cells overexpressing IGF1R or EGFR. Kinase inhibitors that antagonized antiestrogen resistance but are not directly involved in IGF1R or EGFR signaling were prioritized for further analyses. Various ALK (anaplastic lymphoma receptor tyrosine kinase) inhibitors inhibited cell proliferation in IGF1R expressing cells under normal and antiestrogen resistance conditions by preventing IGF1R activation and subsequent downstream signaling; the ALK inhibitors did not affect EGFR signaling. On the other hand, MEK (mitogen-activated protein kinase kinase)1/2 inhibitors, including PD0325901, selumetinib, trametinib and TAK-733, selectively antagonized IGF1R signaling-mediated antiestrogen resistance but did not affect cell proliferation under normal growth conditions. RNAseq analysis revealed that MEK inhibitors PD0325901 and selumetinib drastically altered cell cycle progression and cell migration networks under IGF1R signaling-mediated antiestrogen resistance. In a group of 219 patients with metastasized ER + breast cancer, strong pMEK staining showed a significant correlation with no clinical benefit of first-line tamoxifen treatment. We propose a critical role for MEK activation in IGF1R signaling-mediated antiestrogen resistance and anticipate that dual-targeted therapy with a MEK inhibitor and antiestrogen could improve treatment outcome., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Patients with estrogen receptor-positive (ER+) breast cancer, the most common subtype, remain at risk for lethal metastatic disease years after diagnosis. Recurrence arises partly because tumor cells in bone marrow become resistant to estrogen-targeted therapy. Here, we utilized a co-culture model of bone marrow mesenchymal stem cells (MSCs) and ER+ breast cancer cells to recapitulate interactions of cancer cells in bone marrow niches. ER+ breast cancer cells in direct contact with MSCs acquire cancer stem-like (CSC) phenotypes with increased resistance to standard antiestrogenic drugs. We confirmed that co-culture with MSCs increased labile iron in breast cancer cells, a phenotype associated with CSCs and disease progression. Clinically approved iron chelators and in-house lysosomal iron-targeting compounds restored sensitivity to antiestrogenic therapy. These findings establish iron modulation as a mechanism to reverse MSC-induced drug resistance and suggest iron modulation in combination with estrogen-targeted therapy as a promising, translatable strategy to treat ER+ breast cancer., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: The selective estrogen receptor degrader (SERD) and full receptor antagonist provides an important therapeutic option for hormone receptor (HR)-positive breast cancer. Endocrine therapies include tamoxifen, a selective estrogen receptor modulator (SERM), that exhibits receptor agonist and antagonist activity, and aromatase inhibitors that block estrogen biosynthesis but which demonstrate acquired resistance. Fulvestrant, the only currently approved SERD, is limited by poor drug-like properties. A key focus for improving disease management has been development of oral SERDs with optimized target occupancy and potency and superior clinical efficacy., Areas Covered: Using PubMed, clinicaltrials.gov, and congress websites, this review explored the preclinical development and clinical pharmacokinetics from early phase clinical studies (2015 or later) of novel oral SERDs, including giredestrant, amcenestrant, camizestrant, elacestrant, and rintodestrant., Expert Opinion: Numerous oral SERDs are in clinical development, aiming to form the core endocrine therapy for HR-positive breast cancer. Through property- and structure-based drug design of estrogen receptor-binding, antagonism, degradation, anti-proliferation, and pharmacokinetic properties, these SERDs have distinct profiles which impact clinical dosing, efficacy, and safety. Assuming preliminary safety and activity data are confirmed in phase 3 trials, these promising agents could further improve the management, outcomes, and quality of life in HR-positive breast cancer.

Chemical manipulation of estrogen receptor alpha ligand binding domain structural mobility tunes receptor lifetime and influences breast cancer therapeutic activities. Selective estrogen receptor modulators (SERMs) extend estrogen receptor alpha (ERα) cellular lifetime/accumulation. They are antagonists in the breast but agonists in the uterine epithelium and/or in bone. Selective estrogen receptor degraders/downregulators (SERDs) reduce ERα cellular lifetime/accumulation and are pure antagonists. Activating somatic ESR1 mutations Y537S and D538G enable resistance to first-line endocrine therapies. SERDs have shown significant activities in ESR1 mutant setting while few SERMs have been studied. To understand whether chemical manipulation of ERα cellular lifetime and accumulation influences antagonistic activity, we studied a series of methylpyrollidine lasofoxifene (Laso) derivatives that maintained the drug's antagonistic activities while uniquely tuning ERα cellular accumulation. These molecules were examined alongside a panel of antiestrogens in live cell assays of ERα cellular accumulation, lifetime, SUMOylation, and transcriptional antagonism. High-resolution x-ray crystal structures of WT and Y537S ERα ligand binding domain in complex with the methylated Laso derivatives or representative SERMs and SERDs show that molecules that favor a highly buried helix 12 antagonist conformation achieve the greatest transcriptional suppression activities in breast cancer cells harboring WT/Y537S ESR1 . Together these results show that chemical reduction of ERα cellular lifetime is not necessarily the most crucial parameter for transcriptional antagonism in ESR1 mutated breast cancer cells. Importantly, our studies show how small chemical differences within a scaffold series can provide compounds with similar antagonistic activities, but with greatly different effects of the cellular lifetime of the ERα, which is crucial for achieving desired SERM or SERD profiles., Competing Interests: DH, SW, NL, MS, CJ, GH, ES, EN, RH, SC, ML, SM, GG No competing interests declared, SF In the interest of transparency, Dr. Fanning's laboratory receives sponsored research funds from Olema Oncology Inc. Olema was not involved in this study. This work has no impact on the company, (© 2022, Hosfield et al.)

Endocrine therapies in the treatment of early and metastatic estrogen receptor α positive (ERα+) breast cancer (BC) are greatly limited by de novo and acquired resistance. Selective estrogen receptor degraders (SERDs) like fulvestrant provide new strategies for endocrine therapy combinations due to unique mechanisms. Herein, we disclose our structure-based optimization of LSZ102 by replacing 6-hydroxybenzothiophene with 6 H -thieno[2,3- e ]indazole. Subsequent acrylic acid degron modifications led us to identify compound 40 as the preferred candidate. In general, compound 40 showed much better pharmacological profiles than the lead LSZ102, exhibiting growth inhibition of wild-type or tamoxifen-resistant MCF-7 cells, potent ERα degradation, together with superior pharmacokinetic properties, directional target tissue distribution including the brain, and robust antitumor efficacy in the mice breast cancer xenograft model. Currently, 40 is being evaluated in preclinical trials.

The classical estrogen receptor α (ERα) has been a clinical therapeutic target for decades. ERα-targeted drugs have shown great clinical success, in particular as antagonists for the treatment of ERα-positive breast cancers. However, ERα-targeted agonists have also been clinically useful (e.g., for the treatment of osteoporosis). The breast cancer field is regularly identifying novel ERα-binding compounds with the goal of identifying new potential ERα-targeted therapeutics. To determine whether such newly identified ERα-binding compounds have clinical potential, it is important to characterize the estrogenic activity (i.e., both receptor-mediated agonism and/or antagonism) of these compounds. This chapter focuses on methods that allow determination of whether an ERα-binding compound acts as an agonist or antagonist of the receptor and whether the compound induces degradation of the receptor., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Antigen-specific peripheral tolerance is crucial to prevent the development of organ-specific autoimmunity. However, its function decoupled from thymic tolerance remains unclear. We used desmoglein 3 (Dsg3), a pemphigus antigen expressed in keratinocytes, to analyze peripheral tolerance under physiological antigen-expression conditions. Dsg3-deficient thymi were transplanted into athymic mice to create a unique condition in which Dsg3 was expressed only in peripheral tissue but not in the thymus. When bone marrow transfer was conducted from high-avidity Dsg3-specific T cell receptor-transgenic mice to thymus-transplanted mice, Dsg3-specific CD4 + T cells developed in the transplanted thymus but subsequently disappeared in the periphery. Additionally, when Dsg3-specific T cells developed in Dsg3 -/- mice were adoptively transferred into Dsg3-sufficient recipients, the T cells disappeared in an antigen-specific manner without inducing autoimmune dermatitis. However, Dsg3-specific T cells overcame this disappearance and thus induced autoimmune dermatitis in Treg-ablated recipients but not in Foxp3 -mutant recipients with dysfunctional Tregs. The molecules involved in disappearance were sought by screening the transcriptomes of wild-type and Foxp3 -mutant Tregs. OX40 of Tregs was suggested to be responsible. Consistently, when OX40 expression of Tregs was constrained, Dsg3-specific T cells did not disappear. Furthermore, Tregs obtained OX40L from dendritic cells in an OX40 -dependent manner in vitro and then suppressed OX40L expression in dendritic cells and Birc5 expression in Dsg3-specific T cells in vivo. Lastly, CRISPR/Cas9-mediated knockout of OX40 signaling in Dsg3-specific T cells restored their disappearance in Treg-ablated recipients. Thus, Treg-mediated peripheral deletion of autoreactive T cells operates as an OX40 -dependent regulatory mechanism to avoid undesired autoimmunity besides thymic tolerance., Competing Interests: The authors declare no competing interest.

The selective estrogen receptor (ER) modulator, tamoxifen, is the only endocrine agent with approvals for both the prevention and treatment of premenopausal and postmenopausal estrogen-receptor positive breast cancer as well as for the treatment of male breast cancer. Endoxifen, a secondary metabolite resulting from CYP2D6-dependent biotransformation of the primary tamoxifen metabolite, N-desmethyltamoxifen (NDT), is a more potent antiestrogen than either NDT or the parent drug, tamoxifen. However, endoxifen's antitumor effects may be related to additional molecular mechanisms of action, apart from its effects on ER. In phase 1/2 clinical studies, the efficacy of Z-endoxifen, the active isomer of endoxifen, was evaluated in patients with endocrine-refractory metastatic breast cancer as well as in patients with gynecologic, desmoid, and hormone-receptor positive solid tumors, and demonstrated substantial oral bioavailability and promising antitumor activity. Apart from its potent anticancer effects, Z-endoxifen appears to result in similar or even greater bone agonistic effects while resulting in little or no endometrial proliferative effects compared with tamoxifen. In this review, we summarize the preclinical and clinical studies evaluating endoxifen in the context of breast and other solid tumors, the potential benefits of endoxifen in bone, as well as its emerging role as an antimanic agent in bipolar disorder. In total, the summarized body of literature provides compelling arguments for the ongoing development of Z-endoxifen as a novel drug for multiple indications., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The transforming growth factor beta (TGF-β) signaling pathway plays complex role in the regulation of cell proliferation, apoptosis and differentiation in breast cancer. TGF-β activation can lead to multiple cellular responses mediating the drug resistance evolution, including the resistance to antiestrogens. Tamoxifen is the most commonly prescribed antiestrogen that functionally involved in regulation of TGF-β activity. In this review, we focus on the role of TGF-β signaling in the mechanisms of tamoxifen resistance, including its interaction with estrogen receptors alfa (ERα) pathway and breast cancer stem cells (BCSCs). We summarize the current reported data regarding TGF-β signaling components as markers of tamoxifen resistance and review current approaches to overcoming tamoxifen resistance based on studies of TGF-β signaling., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Long non-coding RNA (lncRNA) FOXD3 antisense RNA 1 (FOXD3-AS1) has been reported to participate in multiple processes that contribute toward the development of cancer. The present study aimed to explore the effect of lncRNA FOXD3-AS1 on anti-estrogen resistance in breast cancer (BC) cells. FOXD3-AS1 was found to be highly expressed in BC cell lines. Moreover, FOXD3-AS1 was highly expressed in estrogen receptor-negative (ER - ) cells compared to the ER-positive (ER + ) cells. FOXD3-AS1 overexpression in T47D and MCF-7 (ER + ) cells enhanced the resistance of cells to tamoxifen (TMX), whereas FOX3-AS1 downregulation reduced the TMX resistance in MDA-MB-231 (ER - ) cells. Similar results were reproduced in vivo that FOXD3-AS1 inhibition reduced the growth of xenograft tumors formed by MDA-MB-231 cells following TMX treatment whereas FOXD3-AS1 overexpression in T47D cells facilitated tumor growth. The bioinformatic analysis and luciferase assays indicated that FOXD3-AS1 sponged microRNA-363 (miR-363) to restore expression of trefoil factor 1 (TFF1) mRNA. Overexpression of miR-363 reduced T47D cell proliferation induced by FOXD3-AS1, whereas overexpression of TFF1 restored growth of MDA-MB-231 cells reduced after FOXD3-AS1 silencing. The phosphorylation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) was increased by FOXD3-AS1 but attenuated by miR-363. Inhibition of PI3K/Akt blocked the role of FOXD3-AS1 and reduced the TMX resistance in T47D and MCF-7 cells. Taken together, the present study suggested that FOXD3-AS1 sponges miR-363 to upregulate TFF1 expression, leading to PI3K/Akt signaling activation and anti-estrogen resistance in BC cells.

Selective estrogen receptor modulators (SERMs) act as estrogen receptor (ERα) agonists or antagonists depending on the target issue. Tamoxifen (TAM) (a non-steroidal triphenylethylene derivative) was the first SERM approved as anti-estrogen for the treatment of metastatic breast cancer. On the hunt for novel SERMs with potential growth inhibitory activity on breast cancer cell lines yet no potential to induce endometrial carcinoma, we designed and synthesized 28 novel TAM analogs. The novel analogs bear a triphenylethylene scaffold. Modifications on rings A , B, and C aim to attenuate estrogenic/anti-estrogenic activities of the novel compounds so they can potentially inhibit breast cancer and provide positive, beneficial estrogenic effects on other tissues with no risk of developing endometrial hyperplasia. Compound 12 ( E/Z -1-(2-{4-[1-(4-Chloro-phenyl)-2-(4-methoxy-phenyl)-propenyl]-phenoxy}-ethyl)-piperidine) showed an appreciable relative ERα agonistic activity in a yeast estrogen screen (YES) assay. It successfully inhibited the growth of the MCF-7 cell line with GI 50 = 0.6 µM, and it was approximately three times more potent than TAM. It showed no potential estrogenicity on Ishikawa endometrial adenocarcinoma cell line via assaying alkaline phosphatase (AlkP) activity. Compound 12 was tested in vivo to assess its estrogenic properties in an uterotrophic assay in an ovariectomized rat model. Compared to TAM, it induced less increase in wet uterine wet weight and showed no uterotrophic effect. Compound 12 is a promising candidate for further development due to its inhibition activity on MCF-7 proliferation with moderate AlkP activity and no potential uterotrophic effects. The in vitro estrogenic activity encourages further investigations toward potential beneficial properties in cardiovascular, bone, and brain tissues.

Dry age-related macular degeneration (dAMD) is a chronic degenerative ophthalmopathy that leads to serious burden of visual impairment. Antioxidation in retinal pigment epithelium (RPE) cells is considered as a potential treatment for dAMD. Our previous studies have showed that naringenin (NAR) protects RPE cells from oxidative damage partly through SIRT1-mediated antioxidation. In this study, we tested the hypothesis that the Nrf2 signaling is another protective mechanism of NAR on dAMD. NaIO 3 -induced mouse retinopathy and ARPE-19 cell injury models were established. Immunochemical staining, immunofluorescence, and western blotting were performed to detect the protein expressions of Nrf2 and HO-1. In addition, ML385 (activity inhibitor of Nrf2) and zinc protoporphyrin (ZnPP, activity inhibitor of HO-1) were applied to explore the effect of NaIO 3 or NAR. The results showed that NAR increased the protein expressions of Nrf2 and HO-1 in the retinas in mice exposed to NaIO 3 at the early stage. NAR treatment also resulted in a stronger activation of Nrf2 at the early stage in NaIO 3 -treated ARPE-19 cells. Moreover, inhibition of HO-1 by ZnPP weakened the cytoprotective effect of NAR. The constitutive accumulation and activation of Nrf2 induced by NaIO 3 led to the death of RPE cells. However, NAR decreased the protein expressions of Nrf2 and HO-1 towards normal level in the mouse retinas and ARPE-19 cells exposed to NaIO 3 at the late stage. Our findings indicate that NAR protects RPE cells from oxidative damage via activating the Nrf2 signaling pathway., Competing Interests: The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article., (Copyright © 2021 Wenpei Chen et al.)

Breast Cancer (BC) is a leading cause of death in women, currently affecting 13% of female population worldwide. First-line clinical treatments against Estrogen Receptor positive (ER+) BC rely on suppressing estrogen production, by inhibiting the aromatase (AR) enzyme, or on blocking estrogen-dependent pro-oncogenic signaling, by targeting Estrogen Receptor (ER) α with selective Modulators/Degraders (SERMs/SERDs). The development of dual acting molecules targeting AR and ERα represents a tantalizing alternative strategy to fight ER + BC, reducing the incidence of adverse effects and resistance onset that limit the effectiveness of these gold-standard therapies. Here, in silico design, synthesis, biological evaluation and an atomic-level characterization of the binding and inhibition mechanism of twelve structurally related drug-candidates enable the discovery of multiple compounds active on both AR and ERα in the sub-μM range. The best drug-candidate 3a displayed a balanced low-nanomolar IC 50 towards the two targets, SERM activity and moderate selectivity towards a BC cell line. Moreover, most of the studied compounds reduced ERα levels, suggesting a potential SERD activity. This study dissects the key structural traits needed to obtain optimal dual acting drug-candidates, showing that multitarget compounds may be a viable therapeutic option to counteract ER + BC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Fibrosis is the final common pathology of most chronic diseases as seen in the heart, liver, lung, kidney, and skin and contributes to nearly half of death in the developed countries. Fibrosis, or scarring, is mainly characterized by the transdifferentiation of fibroblasts into myofibroblasts and the excessive accumulation of extracellular matrix (ECM) secreted by myofibroblasts. Despite immense efforts made in the field of organ fibrosis over the past decades and considerable understanding of the occurrence and development of fibrosis gained, there is still lack of an effective treatment for fibrotic diseases. Therefore, identifying a new therapeutic strategy against organ fibrosis is an unmet clinical need. Naringenin, a flavonoid that occurs naturally in citrus fruits, has been found to confer a wide range of pharmacological effects including antioxidant, anti-inflammatory, and anticancer benefits and thus potentially exerting preventive and curative effects on numerous diseases. In addition, emerging evidence has revealed that naringenin can prevent the pathogenesis of fibrosis in vivo and in vitro via the regulation of various pathways that involved signaling molecules such as transforming growth factor- β 1/small mother against decapentaplegic protein 3 (TGF- β 1/Smad3), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), sirtuin1 (SIRT1), nuclear factor-kappa B (NF- κ B), or reactive oxygen species (ROS). Targeting these profibrotic pathways by naringenin could potentially become a novel therapeutic approach for the management of fibrotic disorders. In this review, we present a comprehensive summary of the antifibrotic roles of naringenin in vivo and in vitro and their underlying mechanisms of action. As a food derived compound, naringenin may serve as a promising drug candidate for the treatment of fibrotic disorders., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2021 Yanfei Du et al.)

The representative morphological features of pyroptosis are excessive cell swelling and subsequent membrane rupture. However, the mechanism underlying the cell's inherent inability to regulate volume during the progression of pyroptosis is poorly understood. In the current study, we found that both volume-activated chloride currents (I cl, vol ) and the regulatory volume decrease (RVD) were markedly decreased in bone marrow-derived macrophages (BMDMs) undergoing pyroptosis induced by lipopolysaccharides (LPS) and nigericin. The inhibition of I Cl, vol and RVD by the chloride channel blockers, tamoxifen or DCPIB, led to the emergence of pyroptosis-like phenotypes such as activated-caspase-1, pyroptotic-body-like bubbles, and a fried-egg-like appearance. Moreover, the expression of the volume-activated chloride channel (VRAC) constituent protein Leucine-Rich Repeat-Containing 8A (LRRC8A) was significantly down-regulated in pyroptotic BMDMs treated with LPS and nigericin. The silencing of LRRC8A expression by small interfering RNA (si)-LRRC8A transfection not only reduced I Cl, vol and RVD, but also caused BMDMs to show pyroptosis-like manifestations such as activated-caspase-1, membrane bubbles, and have a fried-egg-like appearance. These results reveal a new mechanism for the loss of volume regulation in the process of pyroptotic cell swelling and strongly suggest that a functional deficiency of VRAC/LRRC8A plays a key role in this disorder., (Copyright © 2021 Elsevier Inc. All rights reserved.)

As a proinflammatory cytokine, tumor necrosis factor-α (TNF-α) is central to the female reproductive tract and affects various phases of follicular development and uterine cycles. High levels of TNF-α play a vital role in the pathogenesis of polycystic ovary syndrome (PCOS) in patients. Clinicians know that dexamethasone can inhibit the induction of androgen by suppressing the adrenal glands which improves the status of the endocrine system in PCOS patients. We hypothesize that dexamethasone has much more functionality and can exert a therapeutic effect by antagonizing TNF-α. We added TNF-α to the follicular culture medium to simulate the high TNF-α levels observed in the endocrine environment of PCOS patients. Dexamethasone was added to the medium to determine if it could counteract the inhibitory effect of TNF-α on follicular growth and 17β-estradiol (E 2 ) secretion. Follicular diameter, E 2 concentration, follicle survival, antral-like cavity formation, and ovulation were measured to assess the effects of dexamethasone. In our work, TNF-α inhibited in vitro follicular growth and E 2 secretion in a dose-dependent manner. Based on the results of the present research, we concluded that the addition of dexamethasone partially counteracts the repressive effect of TNF-α on follicle growth and E 2 secretion during in vitro culture of the preantral follicles of mice. Thus, the findings in this paper suggest that dexamethasone may act as a therapy by counteracting the effects of TNF-α in PCOS patients. These results provide a new foundation for exploring the treatment of PCOS patients with dexamethasone., (© 2021. The Author(s).)

Background: Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic kidney disorder that impairs renal functions progressively leading to kidney failure. The disease affects between 1:400 and 1:1000 ratio of the people worldwide. It is caused by the mutated PKD1 and PKD2 genes which encode for the defective polycystins. Polycystins mimic the receptor protein or protein channel and mediate aberrant cell signaling that causes cystic development in the renal parenchyma. The cystic development is driven by the increased cyclic AMP stimulating fluid secretion and infinite cell growth. In recent years, natural product-derived small molecules or drugs targeting specific signaling pathways have caught attention in the drug discovery discipline. The advantages of natural products over synthetic drugs enthusiast researchers to utilize the medicinal benefits in various diseases including ADPKD., Conclusion: Overall, this review discusses some of the previously studied and reported natural products and their mechanisms of action which may potentially be redirected into ADPKD., (© 2021. Japanese Society of Nephrology.)

Objective: To report a case of a testosterone deficient man desiring maintenance of spermatogenesis converting from clomiphene citrate (CC) to Natesto, who had a decrease in gonadotropins and semen parameter values after making this medication change. The data on men maintaining gonadotropins and semen parameter values after converting from CC to Natesto is also reported., Methods: A retrospective chart review was performed. Baseline hormones prior to treatment, and again on CC and Natesto, as well as semen parameters on CC and on Natesto were assessed., Results: A 32-year-old testosterone deficient man desiring to maintain future fertility potential who had a poor symptomatic response to CC despite an adequate serum testosterone response was converted to Natesto 11 mg twice daily. His gonadotropins diminished as did his semen parameter values but with dose titration of Natesto to 11 mg in the morning and 5.5 mg in the evening he had normalization of gonadotropins and a rise in semen parameter values back towards his values on CC with a continued satisfactory symptomatic response. The remainder of the 49 men to date converting from CC to Natesto revealed stability in gonadotropins and semen parameter values., Conclusion: Testosterone deficient men interested in maintaining spermatogenesis who convert from CC to Natesto seeking a more robust symptomatic response should be followed closely with repeat serum gonadotropins and semen parameters to confirm that spermatogenesis is not being suppressed. Dose titration of Natesto may be effective at optimizing gonadotropins, semen parameter values, testosterone levels, and symptomatic response to treatment., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Osteosarcoma is a highly malignant musculoskeletal tumor that is commonly noticed in adolescent children, young children, and elderly adults. Due to advances in surgery, chemotherapy and imaging technology, survival rates have improved to 70-80%, but chemical treatments do not enhance patient survival; in addition, the survival rate after chemical treatments is still low. The most obvious clinical feature of osteosarcoma is new bone formation, which is called "sun burst". Estrogen receptor alpha (ERα) is an essential feature of osteogenesis and regulates cell growth in various tumors, including osteosarcoma. In this study, we sought to investigate the role of ERα in osteosarcoma and to determine if ERα can be used as a target to facilitate the chemosensitivity of osteosarcoma to current treatments. The growth rate of each cell clone was assayed by MTT and trypan blue cell counting, and cell cycle analysis was conducted by flow cytometry. Osteogenic differentiation was induced by osteogenic induction medium and quantified by ARS staining. The effects of ERα on the chemoresponse of OS cells treated with doxorubicin were evaluated by colony formation assay. Mechanistic studies were conducted by examining the levels of proteins by Western blot. The role of ERα on OS prognosis was investigated by an immunohistochemical analysis of OS tissue array. The results showed an impaired growth rate and a decreased osteogenesis ability in the ERα-silenced P53(+) OS cell line U2OS, but not in P53(-) SAOS2 cells, compared with the parental cell line. Cotreatment with tamoxifen, an estrogen receptor inhibitor, increased the sensitivity to doxorubicin, which decreased the colony formation of P53(+) U2OS cells. Cell cycle arrest in the S phase was observed in P53(+) U2OS cells cotreated with low doses of doxorubicin and tamoxifen, while increased levels of apoptosis factors indicated cell death. Moreover, patients with ER-/P53(+) U2OS showed better chemoresponse rates (necrosis rate > 90%) and impaired tumor sizes, which were compatible with the findings of basic research. Taken together, ERα may be a potential target of the current treatments for osteosarcoma that can control tumor growth and improve chemosensitivity. In addition, the expression of ERα in osteosarcoma can be a prognostic factor to predict the response to chemotherapy.

Estrogen receptor α emerged as a well validated therapeutic target of breast cancer for decades. However, approximately 50% of patients who initially responding to standard-of-care (SoC), such as undergo therapy of Tamoxifen, generally inevitably progress to an endocrine-resistance ER+ phenotype. Recently, selective estrogen receptor covalent antagonists (SERCAs) targeted to ERα have been demonstrated as a therapeutic alternative. In the present study, series of novel 6-OH-benzothiophene (BT) derivatives targeting ERα and deriving from Raloxifene were designed, synthesized, and biologically evaluated as covalent antagonists. Driven by the antiproliferative efficacy in ER+ breast cancer cells, our chemical optimization finally led to compound 19d that with potent antagonistic activity in ER+ tumor cells while without agonistic activity in endometrial cells. Moreover, the docking simulation was carried out to elucidate the binding mode, revealing 19d as an antagonist and covalently binding to the cysteine residue at the 530 position of ER helix H11., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The human estrogen receptor alpha (ERα) is an important regulator in breast cancer development and progression. The frequent ERα mutations in the ligand-binding domain (LBD) can increase the resistance of antiestrogen drugs, highlighting the need to develop new drugs to target ERα-positive breast cancer. In this study, we combined molecular docking, molecular dynamics simulations and binding free energy calculations to develop a structure-based virtual screening workflow to identify hit compounds capable of interfering with the recognition of ERα by the specific response element of DNA. A druggable pocket on the DNA binding domain (DBD) of ERα was identified as the potential binding site. The hits binding modes were further analyzed to reveal the structural characteristics of the DBD-inhibitor complexes. The core structure of the lead molecules was synthesized and was found to inhibit the E2-induced cell proliferation in MCF-7 cell lines. These findings provide an insight into the structural basis of ligand-ERα for alternate sites beyond the LBD-based pocket. The core structure proposed in this study could potentially be used as the lead molecule for further rational optimization of the antiestrogen drug structure with stronger binding of DBD and higher activity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

As a promising flavonoid, naringenin has shown potential anti-inflammatory and antioxidant properties mainly in inflammatory pain models by oral administration. Therefore, we investigated the antinociceptive activity of this compound by intraperitoneally (i.p.) administration, as well as, associated mechanism of action considering the involvement of L-arginine/nitric oxide (NO)/cyclic GMP (cGMP)/potassium channel (KATP) pathway and opioid receptors. The antinociceptive effect of naringenin was evaluated in male NMRI mice using formalin test at early and late phases. To assess the involvement of L-arginine/NO/cGMP/KATP pathway and opioid receptors, mice were pretreated i.p. with L-arginine (NO precursor), S-nitroso-N-acetylpenicillamine (SNAP, NO donor), N(gamma)-nitro-L-arginine methyl ester (L-NAME, inhibitor of nitric oxide synthase), sildenafil (inhibitor of phosphodiesterase enzyme), glibenclamide (KATP channel blocker) and naloxone (an opioid receptor antagonist), respectively 20 min before administration of the most effective dose of naringenin. Naringenin showed a dose-dependent antinociceptive effect at both early and late phases of the formalin test. The dose of 100 mg/kg of naringenin was identified as the most effective dose and selected for further experiments. Our mechanistic evaluations showed that L-arginine, SNAP and sildenafil could enhance the antinociceptive effects of naringenin, revealing the critical role of NO and cGMP during its antinociceptive effect. On the other hand, glibenclamide and naloxone could mitigate the antinociceptive potential of naringenin at both phases of formalin test, which confirmed the associated role of KATP channels and opioid receptors. In conclusion, naringenin could be a promising antinociceptive agent acting through opioid receptors and L-arginine/NO/cGMP/KATP channel pathway., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

MAP/microtubule affinity-regulating kinase 4 (MARK4) is a member of serine/threonine kinase family and considered an attractive drug target for many diseases. Screening of Indian Medicinal Plants, Phytochemistry, and Therapeutics (IMPPAT) using virtual high-throughput screening coupled with enzyme assay suggested that Naringenin (NAG) could be a potent inhibitor of MARK4. Structure-based molecular docking analysis showed that NAG binds to the critical residues found in the active site pocket of MARK4. Furthermore, molecular dynamics (MD) simulation studies for 100 ns have delineated the binding mechanism of NAG to MARK4. Results of MD simulation suggested that binding of NAG further stabilizes the structure of MARK4 by forming a stable complex. In addition, no significant conformational change in the MARK4 structure was observed. Fluorescence binding and isothermal titration calorimetric measurements revealed an excellent binding affinity of NAG to MARK4 with a binding constant (K) = 0.13 × 10 6 M -1 obtained from fluorescence binding studies. Further, enzyme inhibition studies showed that NAG has an admirable IC 50 value of 4.11 µM for MARK4. Together, these findings suggest that NAG could be an effective MARK4 inhibitor that can potentially be used to treat cancer and neurodegenerative diseases., (© 2021 Wiley Periodicals LLC.)

Reproductive cancers in both genders represent serious health problems, whose incidence has significantly risen over the past decades. Although considerable differences among reproductive cancers exist, we aimed to identify similar signaling pathways and key molecular oncomarkers shared among six human reproductive cancers that can advance the current knowledge of cancer biology to propose new strategies for more effective therapies. Using a computational analysis approach, here we uncover aberrant miRNAs-mRNAs networks shared in six reproductive tumor types, and identify common molecular mechanisms strictly associated with cancer promotion and aggressiveness. Based on the fact that estrogenic and androgenic signaling pathways were most active in prostate and breast cancers, we further demonstrated that both androgen and estrogen deprivation therapy are capable of regulating the expression of the same key molecular sensors associated with endoplasmic reticulum dysfunction and cell cycle in these cancers. Overall, our data reveal a potential mechanistic framework of cellular processes that are shared among reproductive cancers, and particularly, highlight the importance of hormonal deprivation in breast and prostate cancers and potentially new biomarkers of response to these therapeutic approaches., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cancer cells have an increased need for glucose and, despite aerobic conditions, obtain their energy through aerobic oxidation and lactate fermentation, instead of aerobic oxidation alone. Glutamine is an essential amino acid in the human body. Glutaminolysis and glycolysis are crucial for cancer cell survival. In the therapy of estrogen receptor α (ERα)-positive breast cancer (BC), the focus lies on hormone sensitivity targeting therapy with selective estrogen receptor modulators (SERMs) such as 4-hydroxytamoxifen (4-OHT), although this therapy is partially limited by the development of resistance. Therefore, further targets for therapy improvement of ERα-positive BC with secondary 4-OHT resistance are needed. Hence, increased glucose requirement and upregulated glutaminolysis in BC cells could be used. We have established sublines of ERα-positive MCF7 and T47D BC cells, which were developed to be resistant to 4-OHT. Further, glycolysis inhibitor 2-Deoxy-D-Glucose (2-DG) and glutaminase inhibitor CB-839 were analyzed. Co-treatments using 4-OHT and CB-839, 2-DG and CB-839, or 4-OHT, 2-DG and CB-839, respectively, showed significantly stronger inhibitory effects on viability compared to single treatments. It could be shown that tamoxifen-resistant BC cell lines, compared to the non-resistant cell lines, exhibited a stronger reducing effect on cell viability under co-treatments. In addition, the tamoxifen-resistant BC cell lines showed increased expression of proto-oncogene c-Myc compared to the parental cell lines. This could be reduced depending on the treatment. Suppression of c-Myc expression using specific siRNA completely abolished resistance to 4OH-tamoxifen. In summary, our data suggest that combined treatments affecting the metabolism of BC are suitable depending on the cellularity and resistance status. In addition, the anti-metabolic treatments affected the expression of the proto-oncogene c-Myc, a key player in the regulation of cancer cell metabolism.

Mammalian cochlear outer hair cells (OHCs) are essential for hearing. Severe hearing impairment follows OHC degeneration. Previous attempts at regenerating new OHCs from cochlear supporting cells (SCs) have been unsuccessful, notably lacking expression of the key OHC motor protein, Prestin. Thus, regeneration of Prestin+ OHCs represents a barrier to restore auditory function in vivo. Here, we reported the successful in vivo conversion of adult mouse cochlear SCs into Prestin+ OHC-like cells through the concurrent induction of two key transcriptional factors known to be necessary for OHC development: Atoh1 and Ikzf2 . Single-cell RNA sequencing revealed the upregulation of 729 OHC genes and downregulation of 331 SC genes in OHC-like cells. The resulting differentiation status of these OHC-like cells was much more advanced than previously achieved. This study thus established an efficient approach to induce the regeneration of Prestin+ OHCs, paving the way for in vivo cochlear repair via SC transdifferentiation., Competing Interests: SS, SL, ZL, MR, SH, GW, ZL We filed an auditory hair cell regeneration patent based on the key findings of this manuscript., (© 2021, Sun et al.)

Apigenin, a flavonoid, is reported to act as an estrogen receptor (ER) agonist and inhibit aromatase enzyme. However, amentoflavone, a biflavonoid bearing two apigenin molecules, has not been evaluated for its endocrine modulatory effects. Besides, it is highly consumed by young people to build muscles, enhance mood and lose weight. In the present study, apigenin was used as a reference molecule and ER mediated as well as ER-independent estrogenic/antiestrogenic activity of amentoflavone was investigated. Antitumor activity of amentoflavone was also investigated in both ER positive (MCF-7 BUS) and triple-negative (MDA-MB-231) breast cancer cells and its cytotoxicity was evaluated in human breast epithelial cells (MCF-10A). Our data confirmed ER agonist, aromatase inhibitory and cytotoxic effects of apigenin in breast cancer cells, where no ER mediated estrogenic effect and physiologically irrelevant, slight, aromatase inhibition was found for amentoflavone. Although selective cytotoxicity of amentoflavone was found in MCF-7 BUS cells, it does not seem to be an alternative to the present cytotoxic drugs. Therefore, neither an adverse effect, mediated by an estrogenic/antiestrogenic effect of amentoflavone nor a therapeutical benefit would be expected from amentoflavone. Further studies could be performed to investigate its in vivo effects.

Polar cod (Boreogadus saida) is a key species in the arctic marine ecosystem vulnerable to effects of pollution, particularly from petroleum related activities. To facilitate studying the effects of those pollutants, we adapted a precision-cut liver slice culture protocol for this species. Using this system on board a research vessel, we studied gene expression in liver slice after exposure to the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP), ethynylestradiol (EE2), and their mixtures, to map their molecular targets and examine possible anti-estrogenic effects of BaP. The exposure experiments were performed with BaP alone (0.1, 1, and 10 μM) or in combination with low concentrations of EE2 (5 nM) to mimic physiological estradiol levels in early vitellogenic female fish. Transcriptome analysis (RNA-seq) was performed after 72 h exposure in culture to map the genes and cellular pathways affected. The results provide a view of global transcriptome responses to BaP and EE2, which resulted in enrichment of many pathways such as the aryl hydrocarbon (Ahr) and estrogen receptor pathways. In the mixture exposure, BaP resulted in anti-estrogenic effects, shown by attenuation of EE2 activated transcription of many estrogen target genes. The results from this ex vivo experiment suggest that pollutants that activate the Ahr pathway such as the PAH compound BaP can result in anti-estrogenic effects that may lead to endocrine disruption in polar cod., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Efforts to improve estrogen receptor-α (ER)-targeted therapies in breast cancer have relied upon a single mechanism, with ligands having a single side chain on the ligand core that extends outward to determine antagonism of breast cancer growth. Here, we describe inhibitors with two ER-targeting moieties, one of which uses an alternate structural mechanism to generate full antagonism, freeing the side chain to independently determine other critical properties of the ligands. By combining two molecular targeting approaches into a single ER ligand, we have generated antiestrogens that function through new mechanisms and structural paradigms to achieve antagonism. These dual-mechanism ER inhibitors (DMERIs) cause alternate, noncanonical structural perturbations of the receptor ligand-binding domain (LBD) to antagonize proliferation in ER-positive breast cancer cells and in allele-specific resistance models. Our structural analyses with DMERIs highlight marked differences from current standard-of-care, single-mechanism antiestrogens. These findings uncover an enhanced flexibility of the ER LBD through which it can access nonconsensus conformational modes in response to DMERI binding, broadly and effectively suppressing ER activity., Competing Interests: Competing interest statement: J.A.K. is a founder and stockholder of Radius Health Inc. and a consultant of Celcuity Inc. B.S.K. is a consultant of Celcuity Inc.