RESUMEN
Oxalicine B (1) is an α-pyrone meroterpenoid with a unique bispirocyclic ring system derived from Penicillium oxalicum. The biosynthetic pathway of 15-deoxyoxalicine B (4) was preliminarily reported in Penicillium canescens, however, the genetic base and biochemical characterization of tailoring reactions for oxalicine B (1) has remained enigmatic. In this study, we characterized three oxygenases from the metabolic pathway of oxalicine B (1), including a cytochrome P450 hydroxylase OxaL, a hydroxylating Fe(II)/α-KG-dependent dioxygenase OxaK, and a multifunctional cytochrome P450 OxaB. Intriguingly, OxaK can catalyze various multicyclic intermediates or shunt products of oxalicines with impressive substrate promiscuity. OxaB was further proven via biochemical assays to have the ability to convert 15-hydroxdecaturin A (3) to 1 with a spiro-lactone core skeleton through oxidative rearrangement. We also solved the mystery of OxaL that controls C-15 hydroxylation. Chemical investigation of the wild-type strain and deletants enabled us to identify 10 metabolites including three new compounds, and the isolated compounds displayed potent anti-influenza A virus bioactivities exhibiting IC50 values in the range of 4.0-19.9 µmol/L. Our studies have allowed us to propose a late-stage biosynthetic pathway for oxalicine B (1) and create downstream derivatizations of oxalicines by employing enzymatic strategies.
RESUMEN
The crude extract of the Arctic fungus Phoma muscivora CPCC 401424 displayed anti-influenza A virus activities which led us to investigated their secondary metabolites. Four new chromone derivatives, phomarcticones A-D (1-4) and five known chromone analogs (5-9) have been isolated from Arctic fungus Phoma muscivora CPCC 401424. Compounds 3 and 4 possess rare sulfoxide groups in chromone derivatives. Their structures and absolute configurations were elucidated by extensive analysis of spectroscopic data, electronic circular dichroism, and comparison with reported data. Compounds 3, 7, and 9 showed significant anti-influenza A virus activities with the IC50 values of 24.4, 4.2, and 2.7 µM, respectively.
Asunto(s)
Antivirales , Cromonas , Antivirales/química , Cromonas/farmacología , Cromonas/química , Hongos , Dicroismo Circular , Estructura MolecularRESUMEN
Three new chromanone dimer derivatives, paecilins F-H (1-3) and ten known compounds (4-13), were obtained from the mutant strains of Penicillium oxalicum 114-2. Their structures were elucidated by extensive analysis of spectroscopic data and comparison with reported data, and the configurations of 1-3 were resolved by quantum chemical calculations of NMR shifts and ECD spectra. Compounds 5 and 11 showed significant anti-influenza A virus activities with IC50 values of 5.6 and 6.9 µM, respectively. Compounds 8 and 9 displayed cytotoxic activities against the MIA-PaCa-2 cell line with IC50 values of 2.6 and 2.1 µM, respectively. Compound 10 exhibited antibacterial activities against Bacillus cereus with a MIC value of 4 µg mL-1.
RESUMEN
High-solid sludge anaerobic digestion leads to increased organic matters in digester supernatant, which promotes heterotrophic competition and reduces anaerobic ammonium oxidation (anammox) retention. This research demonstrated effective anammox retention by hydrocycloning during a two-stage deammonification. Anammox retention was evaluated by dividing large (>0.425 mm), medium (0.25 to 0.425 mm), and small (<0.25 mm) aggregate fractions via a sieve, whereby Candidatus Kuenenia abundance in each size aggregate was measured to be 16.8%, 5.0%, and 0.9% respectively. After hydrocyclone separation, large particles took up only 1.7% to 2.7% in the overflow discharge (upper discharge from the reactor), whereas its initial proportion was 19.4%, indicating limited anammox loss. The volume ratio change of large particles to total aggregates was defined for particle breakdown evaluation. Breakdown (23% to 32% large particles) occurred mainly during pumping (influenced by pump frequency and type), while little happened in the hydrocyclone. This research provided methods to use a sieve to evaluate anammox retention by hydrocyclone during high-solid sludge anaerobic digester supernatant deammonification, and information for reducing particle breakdown, pumping selection, and separation optimization.
Asunto(s)
Compuestos de Amonio , Aguas del Alcantarillado , Anaerobiosis , Bacterias , Reactores Biológicos , Nitrógeno , Oxidación-ReducciónRESUMEN
Biotransformation of the neo-clerodane diterpene, scutebarbatine F (1), by Streptomyces sp. CPCC 205437 was investigated for the first time, which led to the isolation of nine new metabolites, scutebarbatine F1-F9 (2-10). Their structures were determined by extensive high-resolution electrospray ionization mass spectrometry (HRESIMS) and NMR data analyses. The reactions that occurred included hydroxylation, acetylation, and deacetylation. Compounds 2-4 and 8-10 possess 18-OAc fragment, which were the first examples of 13-spiro neo-clerodanes with 18-OAc group. Compounds 7-10 were the first report of 13-spiro neo-clerodanes with 2-OH. Compounds 1-10 were biologically evaluated for the cytotoxic, antiviral, and antibacterial activities. Compounds 5, 7, and 9 exhibited cytotoxic activities against H460 cancer cell line with inhibitory ratios of 46.0, 42.2, and 51.1%, respectively, at 0.3 µM. Compound 5 displayed a significant anti-influenza A virus activity with inhibitory ratio of 54.8% at 20 µM, close to the positive control, ribavirin.
RESUMEN
Three new phenolic metabolites, daldispols A-C (1-3), two new chromone derivatives, (5R,7R)-5,7-dihydroxy-2-methyl-5,6,7,8-tetrahydro-4H-chromen-4-one (9) and (5R,7R)-5,7-dihydroxy-2-propyl-5,6,7,8-tetrahydro-4H-chromen-4-one (10), together with five known phenolic compounds (4-8) and two known chromone compounds (11 and 12) were isolated from the endolichenic fungus Daldinia sp. CPCC 400770. Their structures were elucidated on the basis of spectroscopic methods, electronic circular dichroism (ECD), and comparison with reported data. Compounds 1, 3, 4, 9, and 11 exhibited significant anti-influenza A virus (IAV) activities with IC50 values of 12.7, 6.4, 12.5, 16.1, and 9.0 µM, respectively, and compound 8 displayed significant anti-ZIKV activity with inhibitory ratio of 42.7% at 10 µM. The results demonstrated that the fungus Daldinia sp. CPCC 400770 might be a rich source for discovering anti-IAV secondary metabolites as potential novel leading compounds.
RESUMEN
Estrogen receptor alpha gene (ESR1) mutations occur frequently in ER-positive metastatic breast cancer, and confer clinical resistance to aromatase inhibitors. Expression of the ESR1 Y537S mutation induced an epithelial-mesenchymal transition (EMT) with cells exhibiting enhanced migration and invasion potential in vitro. When small subpopulations of Y537S ESR1 mutant cells were injected along with WT parental cells, tumor growth was enhanced with mutant cells becoming the predominant population in distant metastases. Y537S mutant primary xenograft tumors were resistant to the antiestrogen tamoxifen (Tam) as well as to estradiol (E2) withdrawal. Y537S ESR1 mutant primary tumors metastasized efficiently in the absence of E2; however, Tam treatment significantly inhibited metastasis to distant sites. We identified a nine-gene expression signature, which predicted clinical outcomes of ER-positive breast cancer patients, as well as breast cancer metastasis to the lung. Androgen receptor (AR) protein levels were increased in mutant models, and the AR agonist dihydrotestosterone significantly inhibited estrogen-regulated gene expression, EMT, and distant metastasis in vivo, suggesting that AR may play a role in distant metastatic progression of ESR1 mutant tumors.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Receptor alfa de Estrógeno/genética , Receptores Androgénicos/genética , Tamoxifeno/farmacología , Animales , Inhibidores de la Aromatasa/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Dihidrotestosterona/farmacología , Estradiol/metabolismo , Estrógenos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Mutación/genética , Metástasis de la Neoplasia , Receptores Androgénicos/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Oestrogen Receptor 1 (ESR1) mutations are frequently acquired in oestrogen receptor (ER)-positive metastatic breast cancer (MBC) patients who were treated with aromatase inhibitors (AI) in the metastatic setting. Acquired ESR1 mutations are associated with poor prognosis and there is a lack of effective therapies that selectively target these cancers. METHODS: We performed a proteomic kinome analysis in ESR1 Y537S mutant cells to identify hyperactivated kinases in ESR1 mutant cells. We validated Recepteur d'Origine Nantais (RON) and PI3K hyperactivity through phospho-immunoblot analysis, organoid growth assays, and in an in vivo patient-derived xenograft (PDX) metastatic model. RESULTS: We demonstrated that RON was hyperactivated in ESR1 mutant models, and in acquired palbociclib-resistant (PalbR) models. RON and insulin-like growth factor 1 receptor (IGF-1R) interacted as shown through pharmacological and genetic inhibition and were regulated by the mutant ER as demonstrated by reduced phospho-protein expression with endocrine therapies (ET). We show that ET in combination with a RON inhibitor (RONi) decreased ex vivo organoid growth of ESR1 mutant models, and as a monotherapy in PalbR models, demonstrating its therapeutic efficacy. Significantly, ET in combination with the RONi reduced metastasis of an ESR1 Y537S mutant PDX model. CONCLUSIONS: Our results demonstrate that RON/PI3K pathway inhibition may be an effective treatment strategy in ESR1 mutant and PalbR MBC patients. Clinically our data predict that ET resistance mechanisms can also contribute to CDK4/6 inhibitor resistance.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Femenino , Humanos , Ratones , Mutación , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Two new cyclopentenone derivatives, daldispones A (1) and B (2) were isolated from the fungus Daldinia sp. CPCC 400770. Their structures and absolute configurations were elucidated by extensive spectroscopic analyses and calculated electronic circular dichroism (ECD). Compounds 1 and 2 exhibited significant anti-influenza A virus activities with IC50 values of 16.0 and 7.4 µM, respectively. Compound 2 showed moderate antibacterial activities against Staphylococcus aureus, Enterococcus faecalis and Bacillus cereus.
Asunto(s)
Antibacterianos/farmacología , Antivirales/farmacología , Ciclopentanos/farmacología , Xylariales/metabolismo , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Antivirales/química , Antivirales/aislamiento & purificación , Dicroismo Circular , Ciclopentanos/química , Ciclopentanos/aislamiento & purificación , Concentración 50 Inhibidora , Análisis EspectralRESUMEN
Steroid hormones are pivotal modulators of pathophysiological processes in many organs, where they interact with nuclear receptors to regulate gene transcription. However, our understanding of hormone action at the single cell level remains incomplete. Here, we focused on estrogen stimulation of the well-characterized GREB1 and MYC target genes that revealed large differences in cell-by-cell responses, and, more interestingly, between alleles within the same cell, both over time and hormone concentration. We specifically analyzed the role of receptor level and activity state during allele-by-allele regulation and found that neither receptor level nor activation status are the determinant of maximal hormonal response, indicating that additional pathways are potentially in place to modulate cell- and allele-specific responses. Interestingly, we found that a small molecule inhibitor of the arginine methyltransferases CARM1 and PRMT6 was able to increase, in a gene specific manner, the number of active alleles/cell before and after hormonal stimulation, suggesting that mechanisms do indeed exist to modulate hormone receptor responses at the single cell and allele level.
Asunto(s)
Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Transcripción Genética , Estrógenos/metabolismo , Hormonas Esteroides Gonadales/genética , Histona Acetiltransferasas/genética , Humanos , Conformación Molecular , Proteínas Nucleares/antagonistas & inhibidores , Unión Proteica/genética , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Análisis de la Célula IndividualRESUMEN
The acquisition of ligand-independent ESR1 mutations during aromatase inhibitor therapy in metastatic estrogen receptor (ER)-positive breast cancer is a common mechanism of hormonal therapy resistance. Preclinical and clinical studies have demonstrated that ESR1 mutations can preexist in primary tumors and can be enriched during metastasis. Furthermore, ESR1 mutations express a unique transcriptional profile that favors tumor progression, suggesting that selected ESR1 mutations may influence metastasis. Several groups have used sensitive detection methods using patient liquid biopsies to track ESR1 or truncal somatic mutations to predict treatment outcome and tumor progression, and some of these techniques may eventually be used to guide sequential treatment options in patients. Further development and standardization of mutation tracking in circulating tumor DNA is ongoing. Clinically, patients with ESR1 mutations derive clinical benefit when treated with fulvestrant and CDK4/6-targeted therapies, but the development of more potent selective ER degraders and/or new targeted biotherapies are needed to overcome the endocrine-resistant phenotype of ESR1 mutant-bearing tumors. In this review, we discuss the mechanisms of resistance and dissemination of ESR1 mutations as well as the detection methods for ESR1 mutation tracking, newly discovered potential therapeutic targets, and the clinical implications and treatment options for treating patients with ESR1 mutant-bearing tumors.
Asunto(s)
Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Perfilación de la Expresión Génica/métodos , Mutación , Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Fulvestrant/uso terapéutico , Humanos , Biopsia Líquida , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Approximately 75% of breast cancers are estrogen receptor alpha (ERα)-positive and are treatable with endocrine therapies, but often patients develop lethal resistant disease. Frequent mutations (10-40%) in the ligand-binding domain (LBD) codons in the gene encoding ERα (ESR1) have been identified, resulting in ligand-independent, constitutively active receptors. In addition, ESR1 chromosomal translocations can occur, resulting in fusion proteins that lack the LBD and are entirely unresponsive to all endocrine treatments. Thus, identifying coactivators that bind to these mutant ERα proteins may offer new therapeutic targets for endocrine-resistant cancer. To define coactivator candidate targets, a proteomics approach was performed profiling proteins recruited to the two most common ERα LBD mutants, Y537S and D538G, and an ESR1-YAP1 fusion protein. These mutants displayed enhanced coactivator interactions as compared to unliganded wild-type ERα. Inhibition of these coactivators decreased the ability of ESR1 mutants to activate transcription and promote breast cancer growth in vitro and in vivo. Thus, we have identified specific coactivators that may be useful as targets for endocrine-resistant breast cancers.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/genética , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Humanos , Células MCF-7 , Unión Proteica/genética , Proteómica , Transcripción Genética/genética , Activación Transcripcional/genética , Translocación Genética/genéticaRESUMEN
After nearly 20 years of research, it is now established that mutations within the estrogen receptor (ER) gene, ESR1, frequently occur in metastatic breast cancer and influence response to hormone therapy. Though early studies presented differing results, sensitive sequencing techniques now show that ESR1 mutations occur at a frequency between 20 and 40% depending on the assay method. Recent studies have focused on several "hot spot mutations," a cluster of mutations found in the hormone-binding domain of the ESR1 gene. Throughout the course of treatment, tumor evolution can occur, and ESR1 mutations emerge and become enriched in the metastatic setting. Sensitive techniques to continually monitor mutant burden in vivo are needed to effectively treat patients with mutant ESR1. The full impact of these mutations on tumor response to different therapies remains to be determined. However, recent studies indicate that mutant-bearing tumors may be less responsive to specific hormonal therapies, and suggest that aromatase inhibitor (AI) therapy may select for the emergence of ESR1 mutations. Additionally, different mutations may respond discretely to targeted therapies. The need for more preclinical mechanistic studies on ESR1 mutations and the development of better agents to target these mutations are urgently needed. In the future, sequential monitoring of ESR1 mutational status will likely direct personalized therapeutic regimens appropriate to each tumor's unique mutational landscape.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Terapia Molecular Dirigida/métodos , Medicina de Precisión/métodos , Femenino , Humanos , MutaciónRESUMEN
In recent years, clinical trials investigating new drugs and therapeutic combinations have led to promising advances in breast cancer therapy. Subtyping breast cancers into hormone receptor (HR) positive, epidermal growth factor receptor (HER2) positive, and triple negative breast cancer (TNBC) is currently the basis of diagnosing and treating this disease. In addition to endocrine and HER2-targeted therapies in their respective subtypes, evidence from recent preclinical studies have shown several targetable pathways that overcome resistance in the clinical setting. The mTOR inhibitor everolimus and the CDK4/6 inhibitor palbociclib have been approved in HR-positive metastatic breast cancer (MBC) due to improved disease-free survival (DFS). Adding pertuzumab to trastuzumab in combination with taxanes further improves DFS in HER2-positive breast cancer. Targeted therapy to the heterogeneous group of TNBC is needed in combination with chemotherapy. However, patient selection and predictive biomarker development remains a big challenge for targeted therapy development in TNBC.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Terapia Molecular Dirigida , Antineoplásicos/administración & dosificación , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Supervivencia sin Enfermedad , Diseño de Fármacos , Resistencia a Antineoplásicos , Femenino , Humanos , Selección de PacienteRESUMEN
The purpose of this study was to address the role of ESR1 hormone-binding mutations in breast cancer. Soft agar anchorage-independent growth assay, Western blot, ERE reporter transactivation assay, proximity ligation assay (PLA), coimmunoprecipitation assay, silencing assay, digital droplet PCR (ddPCR), Kaplan-Meier analysis, and statistical analysis. It is now generally accepted that estrogen receptor (ESR1) mutations occur frequently in metastatic breast cancers; however, we do not yet know how to best treat these patients. We have modeled the three most frequent hormone-binding ESR1 (HBD-ESR1) mutations (Y537N, Y537S, and D538G) using stable lentiviral transduction in human breast cancer cell lines. Effects on growth were examined in response to hormonal and targeted agents, and mutation-specific changes were studied using microarray and Western blot analysis. We determined that the HBD-ESR1 mutations alter anti-proliferative effects to tamoxifen (Tam), due to cell-intrinsic changes in activation of the insulin-like growth factor receptor (IGF1R) signaling pathway and levels of PIK3R1/PIK3R3. The selective estrogen receptor degrader, fulvestrant, significantly reduced the anchorage-independent growth of ESR1 mutant-expressing cells, while combination treatments with the mTOR inhibitor everolimus, or an inhibitor blocking IGF1R, and the insulin receptor significantly enhanced anti-proliferative responses. Using digital drop (dd) PCR, we identified mutations at high frequencies ranging from 12 % for Y537N, 5 % for Y537S, and 2 % for D538G in archived primary breast tumors from women treated with adjuvant mono-tamoxifen therapy. The HBD-ESR1 mutations were not associated with recurrence-free or overall survival in response in this patient cohort and suggest that knowledge of other cell-intrinsic factors in combination with ESR1 mutation status will be needed determine anti-proliferative responses to Tam.
Asunto(s)
Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Mutación , Receptores de Somatomedina/genética , Tamoxifeno/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Modelos Genéticos , Receptor IGF Tipo 1 , Receptores de Estrógenos/metabolismo , Receptores de Somatomedina/metabolismo , Transducción de Señal , Tamoxifeno/uso terapéuticoRESUMEN
Wang and colleagues demonstrate that digital droplet PCR (ddPCR) identified ESR1 mutations in 7% of primary breast cancers. ESR1 mutations were also readily detected in metastatic tissues and circulating tumor DNA in the blood. These results suggest that ddPCR may be amendable for monitoring tumor burden, and to predict relapse. See related article by Wang et al., p. 1130.
Asunto(s)
Neoplasias de la Mama , Recurrencia Local de Neoplasia , ADN de Neoplasias/sangre , Humanos , MutaciónRESUMEN
Tamoxifen (Tam) resistance represents a significant clinical problem in estrogen receptor (ER) α-positive breast cancer. We previously showed that decreased expression of Rho guanine nucleotide dissociation inhibitor (Rho GDI) α, a negative regulator of the Rho GTPase pathway, is associated with Tam resistance. We now discover that androgen receptor (AR) is overexpressed in cells with decreased Rho GDIα and seek to determine AR's contribution to resistance. We engineered ERα-positive cell lines with stable knockdown (KD) of Rho GDIα (KD cells). Resistance mechanisms were examined using microarray profiling, protein-interaction studies, growth and reporter gene assays, and Western blot analysis combined with a specific AR antagonist and other signaling inhibitors. Tam-resistant tumors and cell lines with low Rho GDIα levels exhibited upregulated AR expression. Microarray of Rho GDIα KD cells indicated that activation of EGFR and ERα was associated with Tam treatment. When AR levels were elevated, interaction between AR and EGFR was detected. Constitutive and Tam-induced phosphorylation of EGFR and ERK1/2 was blocked by the AR antagonist Enzalutamide, suggesting that AR-mediated EGFR activation was a mechanism of resistance in these cells. Constitutive ERα phosphorylation and transcriptional activity was inhibited by Enzalutamide and the EGFR inhibitor gefitinib, demonstrating that AR-mediated EGFR signaling activated ERα. Tam exhibited agonist activity in AR overexpressing cells, stimulating ERα transcriptional activity and proliferation, which was blocked by Enzalutamide and gefitinib. We describe a novel model of AR-mediated Tam resistance through activation of EGFR signaling leading to ER activation in ERα-positive cells with low expression of Rho GDIα.