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The Journal of Biological Chemistry Jul 2023Therapeutic strategies targeting nuclear receptors (NRs) beyond their endogenous ligand binding pocket have gained significant scientific interest driven by a need to...
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.
Topics: Humans; 14-3-3 Proteins; Estrogen Receptor alpha; Ligands; Tamoxifen; Protein Binding; Drug Discovery; Estrogen Antagonists
PubMed: 37224961
DOI: 10.1016/j.jbc.2023.104855 -
Angiogenesis Nov 2023Longitudinal mouse models of brain arteriovenous malformations (AVMs) are crucial for developing novel therapeutics and pathobiological mechanism discovery underlying...
BACKGROUND
Longitudinal mouse models of brain arteriovenous malformations (AVMs) are crucial for developing novel therapeutics and pathobiological mechanism discovery underlying brain AVM progression and rupture. The sustainability of existing mouse models is limited by ubiquitous Cre activation, which is associated with lethal hemorrhages resulting from AVM formation in visceral organs. To overcome this condition, we developed a novel experimental mouse model of hereditary hemorrhagic telangiectasia (HHT) with CreER-mediated specific, localized induction of brain AVMs.
METHODS
Hydroxytamoxifen (4-OHT) was stereotactically delivered into the striatum, parietal cortex, or cerebellum of R26; Alk1 (Alk1-iKO) littermates. Mice were evaluated for vascular malformations with latex dye perfusion and 3D time-of-flight magnetic resonance angiography (MRA). Immunofluorescence and Prussian blue staining were performed for vascular lesion characterization.
RESULTS
Our model produced two types of brain vascular malformations, including nidal AVMs (88%, 38/43) and arteriovenous fistulas (12%, 5/43), with an overall frequency of 73% (43/59). By performing stereotaxic injection of 4-OHT targeting different brain regions, Alk1-iKO mice developed vascular malformations in the striatum (73%, 22/30), in the parietal cortex (76%, 13/17), and in the cerebellum (67%, 8/12). Identical application of the stereotaxic injection protocol in reporter mice confirmed localized Cre activity near the injection site. The 4-week mortality was 3% (2/61). Seven mice were studied longitudinally for a mean (SD; range) duration of 7.2 (3; 2.3-9.5) months and demonstrated nidal stability on sequential MRA. The brain AVMs displayed microhemorrhages and diffuse immune cell invasion.
CONCLUSIONS
We present the first HHT mouse model of brain AVMs that produces localized AVMs in the brain. The mouse lesions closely resemble the human lesions for complex nidal angioarchitecture, arteriovenous shunts, microhemorrhages, and inflammation. The model's longitudinal robustness is a powerful discovery resource to advance our pathomechanistic understanding of brain AVMs and identify novel therapeutic targets.
Topics: Animals; Mice; Humans; Telangiectasia, Hereditary Hemorrhagic; Arteriovenous Malformations; Arteriovenous Fistula; Brain
PubMed: 37219736
DOI: 10.1007/s10456-023-09881-w -
Anticancer Research Mar 2023Monocarboxylate transporters (MCTs) transport short-chain monocarboxylates, such as lactate, and have been reported to be related to poor prognosis in breast cancer. Our...
BACKGROUND/AIM
Monocarboxylate transporters (MCTs) transport short-chain monocarboxylates, such as lactate, and have been reported to be related to poor prognosis in breast cancer. Our previous studies showed that a high glucose state altered MCT expression and changed the sensitivity of the tamoxifen active metabolite 4-hydroxytamoxifen (4-OHT) via hypoxia-inducible factor-1α (HIF-1α) protein expression. We hypothesized that MCT inhibitors affect 4-OHT-induced cytotoxicity under normal glucose conditions by decreasing HIF-1α protein expression. To test this hypothesis, we evaluated the combined effect of MCT inhibitor and 4-OHT using the estrogen receptor (ER)-positive breast cancer cell line MCF-7, under normal glucose conditions.
MATERIALS AND METHODS
Expression of MCTs and oxidative stress markers was evaluated by real-time PCR. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Reactive oxygen species (ROS) were measured using the cell permeability probe 2',7'-dichlorodihydrofluorescein diacetate.
RESULTS
MCT1 expression increased under normal glucose conditions. The MCT1 substrate/inhibitor, 5-oxoproline (5-OP), enhanced 4-OHT-induced cytotoxicity. Bindarit, a selective MCT4 inhibitor, decreased 4-OHT sensitivity, similar to results of our previous study under high glucose conditions. In contrast, the combination of 5-OP and 4-OHT decreased ATP levels compared with that by 4-OHT alone in MCF-7 cells. Furthermore, 5-OP significantly increased the ROS production induced by 4-OHT.
CONCLUSION
5-OP enhances 4-OHT-induced cytotoxicity in ER-positive breast cancer cells under normal glucose conditions.
Topics: Humans; Female; Breast Neoplasms; Pyrrolidonecarboxylic Acid; MCF-7 Cells; Reactive Oxygen Species; Tamoxifen; Oxidative Stress; Glucose
PubMed: 36854517
DOI: 10.21873/anticanres.16256 -
Molecular Pharmacology Feb 2023G protein-coupled receptors (GPCRs) constitute the most versatile family of pharmacological target proteins. For some "orphan" GPCRs, no ligand or drug-like modulator is...
G protein-coupled receptors (GPCRs) constitute the most versatile family of pharmacological target proteins. For some "orphan" GPCRs, no ligand or drug-like modulator is known. In this study, we have established and applied a parallelized assay to coscreen 29 different human GPCRs. Three compounds, chlorhexidine, Lys-05, and 9-aminoacridine, triggered transient Ca signals linked to the expression of GPR30. GPR30, also named G protein-coupled estrogen receptor 1 (GPER1), was reported to elicit increases in cAMP in response to 17-estradiol, 4-hydroxytamoxifen, or G-1. These findings could, however, not be reproduced by other groups, and the deorphanization of GPR30 is, therefore, intensely disputed. The unbiased screen and following experiments in transiently or stably GPR30-overexpressing HEK293 cells did not show responses to 17-estradiol, 4-hydroxytamoxifen, or G-1. A thorough analysis of the activated signaling cascade revealed a canonical G-coupled pathway, including phospholipase C, protein kinase C and ERK activation, receptor internalization, and sensitivity to the G inhibitor YM-254890. When expressed in different cell lines, the localization of a fluorescent GPR30 fusion protein appeared variable. An efficient integration into the plasma membrane and stronger functional responses were found in HEK293 and in MCF-7 cells, whereas GPR30 appeared mostly retained in endomembrane compartments in Cos-7 or HeLa cells. Thus, conflicting findings may result from the use of different cell lines. The newly identified agonists and the finding that GPR30 couples to G are expected to serve as a starting point for identifying physiologic responses that are controlled by this GPCR. SIGNIFICANCE STATEMENT: This study has identified and thoroughly characterized novel and reliably acting agonists of the G protein-coupled receptor GPER1/GPR30. Applying these agonists, this study demonstrates that GPR30 couples to the canonical G-phospholipase C pathway and is rapidly internalized upon continuous exposure to the agonists.
Topics: Humans; HEK293 Cells; HeLa Cells; Receptors, G-Protein-Coupled; Estradiol
PubMed: 36400433
DOI: 10.1124/molpharm.122.000580 -
Cancer Biology & Therapy Dec 2022Prolylcarboxypeptidase (PRCP) is a lysosomal serine protease that cleaves peptide substrates when the penultimate amino acid is proline. Previous studies have linked...
Prolylcarboxypeptidase (PRCP) is a lysosomal serine protease that cleaves peptide substrates when the penultimate amino acid is proline. Previous studies have linked PRCP to blood-pressure and appetite control through its ability to cleave peptide substrates such as angiotensin II and α-MSH. A potential role for PRCP in cancer has to date not been widely appreciated. Endocrine therapy resistance in breast cancer is an enduring clinical problem mediated in part by aberrant receptor tyrosine kinase (RTK) signaling. We previously found PRCP overexpression promoted 4-hydroxytamoxifen (4-OHT) resistance in estrogen receptor-positive (ER+) breast cancer cells. Currently, we tested the potential association between PRCP with breast cancer patient outcome and RTK signaling, and tumor responsiveness to endocrine therapy. We found high PRCP protein levels in ER+ breast tumors associates with worse outcome and earlier recurrence in breast cancer patients, including patients treated with TAM. We found a PRCP specific inhibitor (PRCPi) enhanced the response of ER+ PDX tumors and MCF7 tumors to endoxifen, an active metabolite of TAM in mice. We found PRCP increased IGF1R/HER3 signaling and AKT activation in ER+ breast cancer cells that was blocked by PRCPi. Thus, PRCP is an adverse prognostic marker in breast cancer and a potential target to improve endocrine therapy in ER+ breast cancers.
Topics: Animals; Mice; Carboxypeptidases; Drug Resistance, Neoplasm; Estrogen Receptor alpha; Neoplasm Recurrence, Local; Receptors, Estrogen; Tamoxifen; Breast Neoplasms
PubMed: 36332175
DOI: 10.1080/15384047.2022.2142008 -
Molecules (Basel, Switzerland) Oct 2022Utilizing McMurry reactions of 4,4'-dihydroxybenzophenone with appropriate carbonyl compounds, a series of 4-Hydroxytamoxifen analogues were synthesized. Their cytotoxic...
Utilizing McMurry reactions of 4,4'-dihydroxybenzophenone with appropriate carbonyl compounds, a series of 4-Hydroxytamoxifen analogues were synthesized. Their cytotoxic activity was evaluated in vitro on four human malignant cell lines (MCF-7, MDA-MB 231, A2058, HT-29). It was found that some of these novel Tamoxifen analogues show marked cytotoxicity in a dose-dependent manner. The relative ROS-generating capability of the synthetized analogues was evaluated by cyclic voltammetry (CV) and DFT modeling studies. The results of cell-viability assays, CV measurements and DFT calculations suggest that the cytotoxicity of the majority of the novel compounds is mainly elicited by their interactions with cellular targets including estrogen receptors rather than triggered by redox processes. However, three novel compounds could be involved in ROS-production and subsequent formation of quinone-methide preventing proliferation and disrupting the redox balance of the treated cells. Among the cell lines studied, HT-29 proved to be the most susceptible to the treatment with compounds having ROS-generating potency.
Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Electrons; Female; Humans; Reactive Oxygen Species; Receptors, Estrogen; Structure-Activity Relationship; Tamoxifen
PubMed: 36235291
DOI: 10.3390/molecules27196758 -
European Journal of Medicinal Chemistry Dec 2022In the last four decades, treatment of oestrogen receptor positive (ER+) breast cancer (BCa), has focused on targeting the estrogenic receptor signaling pathway. This...
In the last four decades, treatment of oestrogen receptor positive (ER+) breast cancer (BCa), has focused on targeting the estrogenic receptor signaling pathway. This signaling function is pivotal to sustain cell proliferation. Tamoxifen, a competitive inhibitor of oestrogen, has played a major role in therapeutics. However, primary and acquired resistance to hormone blockade occurs in a large subset of these cancers, and new approaches are urgently needed. Aromatase inhibitors and receptor degraders were approved and alternatively used. Yet, resistance appears in the metastatic setting. Here we report the design and synthesis of a series of proteolysis targeting chimeras (PROTACs) that induce the degradation of estrogen receptor alpha in breast cancer MCF-7 (ER+) cells at nanomolar concentration. Using a warhead based on 4-hydroxytamoxifen, bifunctional degraders recruiting either cereblon or the Von Hippel Lindau E3 ligases were synthesized. Our efforts resulted in the discovery of TVHL-1, a potent ERα degrader (DC: 4.5 nM) that we envisage as a useful tool for biological study and a platform for potential therapeutics.
Topics: Humans; Female; Receptors, Estrogen; Proteolysis; Von Hippel-Lindau Tumor Suppressor Protein; Chimera; Tamoxifen; Ubiquitin-Protein Ligases; Estrogen Receptor alpha; Breast Neoplasms
PubMed: 36148710
DOI: 10.1016/j.ejmech.2022.114770 -
Tissue & Cell Aug 2022Adipose-derived stromal cells (ASCs) are a promising cell source for novel tissue engineering approaches to breast reconstruction following cancer resection. However...
BACKGROUND AND AIM
Adipose-derived stromal cells (ASCs) are a promising cell source for novel tissue engineering approaches to breast reconstruction following cancer resection. However there is limited knowledge on the effect of adjuvant therapies such as hormonal therapy on ASCs, which may affect their efficacy in regenerative strategies. The present study aims to investigate the effects of Tamoxifen and its metabolites Afimoxifene (4-Hydroxy-Tamoxifen) and Endoxifen (N-desmethyl-4-hydroxytamoxifen) on patient-derived ASC viability, apoptosis, adipogenic differentiation and angiogenic potential.
METHODS
ASCs were isolated from fat harvested from female breast cancer patients undergoing breast reconstruction surgery or cosmetic procedures. Oestrogen receptor (ER α, β) expression was analysed using immunofluorescence. ASCs were then treated with various concentrations of Afimoxifene, Endoxifen and Tamoxifen (combination), and the impact on ASC viability and apoptosis determined. ASCs were cultured in adipogenic-differentiation media with or without tamoxifen and derivatives, and adipogenesis was measured using quantitative Real-time Polymerase chain reaction (qRT-PCR) and histological staining (Oil Red O). The effect on secreted VEGF levels was also quantified in ASC conditioned media RESULTS: ASCs were successfully isolated and characterised from human abdominal lipoaspirates or fat tissues (n = 8). ASCs subjected to varying doses of Tamoxifen and metabolites (up to 1000 nM) showed no decline in cell viability or increase in apoptosis, at physiological doses (upto 100 nM). Functional decline in adipogenic differentiation or gene expression was observed at supraphysiological concentrations of Tamoxifen (1000 nM). VEGF protein secretion in ASC-cell conditioned media was not significantly impacted irrespective of dosage.
CONCLUSION
At physiologically relevant doses, Tamoxifen treatment did not result in any deleterious effect on ASC survival and functionality and is unlikely to negatively impact ASC based breast reconstruction strategies for breast cancer patients receiving this adjuvant hormonal therapy.
Topics: Adipose Tissue; Breast Neoplasms; Cell Differentiation; Cells, Cultured; Culture Media, Conditioned; Female; Humans; Stromal Cells; Tamoxifen; Vascular Endothelial Growth Factor A
PubMed: 35777289
DOI: 10.1016/j.tice.2022.101858 -
International Journal of Molecular... Jan 2022Tamoxifen, a therapeutic agent for breast cancer, has been associated with genetic polymorphisms in the metabolism of ,-dialkylaminoethyl substituent, which plays an...
Tamoxifen, a therapeutic agent for breast cancer, has been associated with genetic polymorphisms in the metabolism of ,-dialkylaminoethyl substituent, which plays an important role in the expression of selective estrogen receptor modulator (SERM) activity. To solve this problem, we developed a novel estrogen receptor (ER) modulator, Az-01, on the basis of the aromaticity, dipole moment, and isopropyl group of guaiazulene. Az-01 showed four-fold lower binding affinity for ER than E2 but had similar ER-binding affinity to that of 4-hydroxytamoxifen (4-HOtam). Unlike tamoxifen, Az-01 acted as a partial agonist with very weak estrogenic activity at high concentrations when used alone, and it showed potent anti-estrogenic activity in the presence of E2. The cell proliferation and inhibition activities of Az-01 were specific to ER-expressing MCF-7 cells, and no effect of Az-01 on other cell proliferation signals was observed. These findings are important for the development of new types of SERMs without the ,-dialkylaminoethyl substituent as a privileged functional group for SERMs.
Topics: Azulenes; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Development; Drug Synergism; Estradiol; Estrogen Receptor Modulators; Female; Humans; MCF-7 Cells; Models, Molecular; Molecular Structure; Protein Binding; Protein Conformation; Receptors, Estrogen; Sesquiterpenes, Guaiane; Tamoxifen
PubMed: 35163039
DOI: 10.3390/ijms23031113 -
Gene May 2022The basic region leucin zipper (bZIP) protein c-Fos constitutes together with other bZIP proteins the AP-1 transcription factor complex. Expression of the c-Fos gene is...
The basic region leucin zipper (bZIP) protein c-Fos constitutes together with other bZIP proteins the AP-1 transcription factor complex. Expression of the c-Fos gene is regulated by numerous extracellular signaling molecules including mitogens, metabolites, and ligands for receptor tyrosine kinases, G protein-coupled receptors, and cytokine receptors. Here, we analyzed the effects of the stimulus-responsive MAP kinases ERK1/2 (extracellular signal-regulated protein kinase), JNK (c-Jun N-terminal protein kinase) and p38 protein kinase on transcription of the c-Fos gene. We used chromatin-integrated c-Fos promoter-luciferase reporter genes containing inactivating point mutations of DNA binding sites for distinct transcription factors. ERK1/2, JNK, and p38 protein kinases were specifically activated following expression of either a mutant of B-Raf, a truncated version of mitogen-activated/extracellular signal responsive kinase kinase kinase-1 (MEKK1), or a mutant of MAP kinase kinase-6 (MKK6), respectively. The results show that the DNA binding sites for serum response factor (SRF) and for the ternary complex factor (TCF) are of major importance for stimulating c-Fos promoter activity by MAP kinases. ERK1/2 and p38-induced stimulation of the c-Fos promoter additionally required the DNA binding site for the transcription factor AP-1. Mutation of the DNA binding site for STAT had no or only a small effect on c-Fos promoter activity. We conclude that MAP kinases do not activate distinct transcription factors involving distinct genetic elements. Rather, these kinases mainly target SRF and TCF proteins, leading to an activation of transcription of the c-Fos gene via the serum response element.
Topics: Gene Expression Regulation; HEK293 Cells; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Mutation; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Serum Response Element; Serum Response Factor; TCF Transcription Factors; Tamoxifen; Transcription, Genetic
PubMed: 35143939
DOI: 10.1016/j.gene.2022.146284