FGFR2-triggered autophagy and activation of Nrf-2 reduce breast cancer cell response to anti-ER drugs.

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.

Dual-target inhibitors based on ERα: Novel therapeutic approaches for endocrine resistant breast cancer.

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.

Ethanolic extract of Persea americana Mill. (Lauraceae) seeds induced antiestrogenic effects in young female Wistar rats.

OBJECTIVES: The ethanol extract of Persea americana seeds was found to inhibit the development of estrogen-dependent conditions in female Wistar rats, suggesting the ability of its secondary metabolites to interact with estrogen receptors (ERs), either as partial agonists or as antagonists. To test this hypothesis, the abovementioned extract was assessed for its ability to mimic and/or antagonize estradiol effects. METHODS: Two experiments were conducted in ovariectomized (OVX) rats: (1) animals were treated with estradiol valerate (E2V; 1 mg/kg) or P. americana at doses of 25 and 50 mg/kg; (2) animals were treated with E2V alone (0.75 mg/kg) or in combination with P. americana at the abovementioned doses. Treatments were given orally for 3 days and animals were sacrificed for biochemical and histological analyses of the uterus and vagina. RESULTS: When administered alone, P. americana did not change the histomorphology of both organs (uterus and vagina). In combination with E2V, P. americana decreased uterine weight [30 % decrease (p<0.001) at 25 mg/kg and 24 % (p<0.01) at 50 mg/kg] and epithelium height (37 % decrease). This was associated with decreased estradiol levels (at least 86 % decrease, p<0.001) in the uterus. Similarly, vagina epithelium height decreased by at least 34 % (p<0.05) when E2V was co-administered with P. americana. CONCLUSIONS: The seed extract of P. americana contains ER antagonist secondary metabolites accounting for its ability to inhibit the development of estrogen-dependent conditions in female rats.

Complete elimination of estrogen receptor α by PROTAC estrogen receptor α degrader ERD-148 in breast cancer cells.

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.

A novel scaffold long-acting selective estrogen receptor antagonist and degrader with superior preclinical profile against ER+ breast cancer.

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.

Hot-Spot Residue-Based Virtual Screening of Novel Selective Estrogen-Receptor Degraders for Breast Cancer Treatment.

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 (IC50 = 4.21 nM), favorable ERα binding affinity (Ki = 14.6 nM), and excellent ERα degradative ability (DC50 = 9.7 nM), which indicated its potential to evolve as a promising SERD for breast cancer treatment.

MATH: A Deep Learning Approach in QSAR for Estrogen Receptor Alpha Inhibitors.

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.

Identification of an Imidazopyridine-based Compound as an Oral Selective Estrogen Receptor Degrader for Breast Cancer Therapy.

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.

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 ERß 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.

Molecular basis and dual ligand regulation of tetrameric estrogen receptor α/14-3-3ζ protein complex.

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.

Design, synthesis and biological evaluation of fluorinated selective estrogen receptor degraders (FSERDs) --- A promising strategy for advanced ER positive breast cancer.

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.

Structure-guided identification of novel dual-targeting estrogen receptor α degraders with aromatase inhibitory activity for the treatment of endocrine-resistant breast cancer.

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.

Discovery of highly potent proapoptotic antiestrogens in a series of androst-5,16-dienes D-modified with imidazole-annulated pendants.

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 3ß-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 IC50 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.

Blocking the Hormone Receptors Modulates NLRP3 in LPS-Primed Breast Cancer Cells.

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.

A novel selective estrogen receptor degrader induces cell cycle arrest in breast cancer via ERα degradation and the autophagy-lysosome pathway.

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.

Apoptosis-modulatory miR-361-3p as a novel treatment target in endocrine-responsive and endocrine-resistant breast cancer.

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.

Novel pentacyclic derivatives and benzylidenes of the progesterone series cause anti-estrogenic and antiproliferative effects and induce apoptosis in breast cancer cells.

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 IC50 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.

The AF-2 cofactor binding region is key for the selective SUMOylation of estrogen receptor alpha by antiestrogens.

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 ERß, at different levels. Swapping domains between ERα and ERß 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 ERß. 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 ERß containing the H3-H4 region of ERα, but not of ERß. 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.

Allyl ether of mansonone G as a potential anticancer agent for colorectal cancer.

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.