Estrogen-induced transcription at individual alleles is independent of receptor level and active conformation but can be modulated by coactivators activity.

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.

RON signalling promotes therapeutic resistance in ESR1 mutant breast cancer.

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.

ESR1 mutations in breast cancer.

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.

ESR1 mutations affect anti-proliferative responses to tamoxifen through enhanced cross-talk with IGF signaling.

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.

Androgen receptor promotes tamoxifen agonist activity by activation of EGFR in ERα-positive breast cancer.

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.

Targeting thyroid hormone receptor beta in triple-negative breast cancer.

The purpose of this study was to discover novel nuclear receptor targets in triple-negative breast cancer. Expression microarray, Western blot, qRT-PCR analyses, MTT growth assay, soft agar anchorage-independent growth assay, TRE reporter transactivation assay, and statistical analysis were performed in this study. We performed microarray analysis using 227 triple-negative breast tumors, and clustered the tumors into five groups according to their nuclear receptor expression. Thyroid hormone receptor beta (TRß) was one of the most differentially expressed nuclear receptors in group 5 compared to other groups. TRß low expressing patients were associated with poor outcome. We evaluated the role of TRß in triple-negative breast cancer cell lines representing group 5 tumors. Knockdown of TRß increased soft agar colony and reduced sensitivity to docetaxel and doxorubicin treatment. Docetaxel or doxorubicin long-term cultured cell lines also expressed decreased TRß protein. Microarray analysis revealed cAMP/PKA signaling was the only KEGG pathways upregulated in TRß knockdown cells. Inhibitors of cAMP or PKA, in combination with doxorubicin further enhanced cell apoptosis and restored sensitivity to chemotherapy. TRß-specific agonists enhanced TRß expression, and further sensitized cells to both docetaxel and doxorubicin. Sensitization was mediated by increased apoptosis with elevated cleaved PARP and caspase 3. TRß represents a novel nuclear receptor target in triple-negative breast cancer; low TRß levels were associated with enhanced resistance to both docetaxel and doxorubicin treatment. TRß-specific agonists enhance chemosensitivity to these two agents. Mechanistically enhanced cAMP/PKA signaling was associated with TRß's effects on response to chemotherapy.

Estrogen receptor (ER) α mutations in breast cancer: hidden in plain sight.

The idea that somatic ERα mutations could play an important role in the evolution of hormone-dependent breast cancers was proposed some years ago (Fuqua J Mammary Gland Biol Neoplasia 6(4):407-417, 2001; Dasgupta et al. Annu Rev Med 65:279-292, 2013), but has remained controversial until recently. A significant amount of new data has confirmed these initial observations and shown their significance, along with much additional work relevant to the treatment of breast cancer. Thus, it is the purpose of this review to summarize the research to date on the existence and clinical consequences of ERα mutations in primary and metastatic breast cancer.

Late-stage cascade of oxidation reactions during the biosynthesis of oxalicine B in Penicillium oxalicum.

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.

Four new chromone derivatives from the Arctic fungus Phoma muscivora CPCC 401424 and their antiviral activities.

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.

Oldenlandia diffusa extracts exert antiproliferative and apoptotic effects on human breast cancer cells through ERα/Sp1-mediated p53 activation.

Breast cancer is the most frequent tumor and a major cause of death among women. Estrogens play a crucial role in breast tumor growth, which is the rationale for the use of hormonal antiestrogen therapies. Unfortunately, not all therapeutic modalities are efficacious and it is imperative to develop new effective antitumoral drugs. Oldenlandia diffusa (OD) is a well-known medicinal plant used to prevent and treat many disorders, especially cancers. The aim of this study was to investigate the effects of OD extracts on breast cancer cell proliferation. We observed that OD extracts strongly inhibited anchorage-dependent and -independent cell growth and induced apoptosis in estrogen receptor alpha (ERα)-positive breast cancer cells, whereas proliferation and apoptotic responses of MCF-10A normal breast epithelial cells were unaffected. Mechanistically, OD extracts enhance the tumor suppressor p53 expression as a result of an increased binding of ERα/Sp1 complex to the p53 promoter region. Finally, we isolated ursolic and oleanolic acids as the bioactive compounds able to upregulate p53 expression and inhibit breast cancer cell growth. These acids were greatly effective in reducing tamoxifen-resistant growth of a derivative MCF-7 breast cancer cell line resistant to the antiestrogen treatment. Our results evidence how OD, and its bioactive compounds, exert antiproliferative and apoptotic effects selectively in ERα-positive breast cancer cells, highlighting the potential use of these herbal extracts as breast cancer preventive and/or therapeutic agents.

New dimeric chromanone derivatives from the mutant strains of Penicillium oxalicum and their bioactivities.

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.

Farnesoid X receptor, through the binding with steroidogenic factor 1-responsive element, inhibits aromatase expression in tumor Leydig cells.

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that regulates bile acid homeostasis. It is expressed in the liver and the gastrointestinal tract, but also in several non-enterohepatic tissues including testis. Recently, FXR was identified as a negative modulator of the androgen-estrogen-converting aromatase enzyme in human breast cancer cells. In the present study we detected the expression of FXR in Leydig normal and tumor cell lines and in rat testes tissue. We found, in rat Leydig tumor cells, R2C, that FXR activation by the primary bile acid chenodeoxycholic acid (CDCA) or a synthetic agonist GW4064, through a SHP-independent mechanism, down-regulates aromatase expression in terms of mRNA, protein levels, and its enzymatic activity. Transient transfection experiments, using vector containing rat aromatase promoter PII, evidenced that CDCA reduces basal aromatase promoter activity. Mutagenesis studies, electrophoretic mobility shift, and chromatin immunoprecipitation analysis reveal that FXR is able to compete with steroidogenic factor 1 in binding to a common sequence present in the aromatase promoter region interfering negatively with its activity. Finally, the FXR-mediated anti-proliferative effects exerted by CDCA on tumor Leydig cells are at least in part due to an inhibition of estrogen-dependent cell growth. In conclusion our findings identify for the first time the activators of FXR as negative modulators of the aromatase enzyme in Leydig tumor cell lines.

Pilot-scale waste activated sludge alkaline fermentation, fermentation liquid separation, and application of fermentation liquid to improve biological nutrient removal.

The use of sludge fermentative short-chain fatty acids (SCFA) as an additional carbon source of biological nutrient removal (BNR) has drawn much attention recently as it can reuse sludge organics, reduce waste activated sludge production, and improve BNR performance. Our previous laboratory study had shown that the SCFA production was significantly enhanced by controlling sludge fermentation at pH 10 with NaOH. This paper focused on a pilot-scale study of alkaline fermentation of waste activated sludge, separation of the fermentation liquid from the alkaline fermentation system, and application of the fermentation liquid to improve municipal biological nitrogen and phosphorus removal. NaOH and Ca(OH)(2) were used respectively to adjust the alkaline fermentation pH, and their effects on sludge fermentation and fermentation liquid separation were compared. The results showed that the use of Ca(OH)(2) had almost the same effect on SCFA production improvement and sludge volatile suspended solids reduction as that of NaOH, but it exhibited better sludge dewatering, lower chemical costs, and higher fermentation liquid recovery efficiency. When the fermentation liquids, adjusted with Ca(OH)(2) and NaOH respectively, were added continuously to an anaerobic-anoxic-aerobic municipal wastewater BNR system, both the nitrogen and phosphorus removals, compared with the control, were improved to the same levels. This was attributed to the increase of not only influent COD but also denitrifying phosphorus removal capability. It seems that the use of Ca(OH)(2) to control sludge fermentation at pH 10 for efficiently producing a carbon source for BNR is feasible.

Daldispones A and B, two new cyclopentenones from Daldinia sp. CPCC 400770.

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.

Optimizing granular anammox retention via hydrocycloning during two-stage deammonification of high-solid sludge anaerobic digester supernatant.

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.

New phenol and chromone derivatives from the endolichenic fungus Daldinia species and their antiviral activities.

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.

Microbial Transformation of neo-Clerodane Diterpenoid, Scutebarbatine F, by Streptomyces sp. CPCC 205437.

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.

Hormonal modulation of ESR1 mutant metastasis.

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.

Toxicity reduction of municipal wastewater by anaerobic-anoxic-oxic process.

OBJECTIVE: This study was conducted to optimize the operational parameters of anaerobic-anoxic-oxic (A²/O) processes to reduce the toxicity of municipal wastewater and evaluate its ability to reduce toxicity. METHODS: A luminescent bacterium toxicity bioassay was employed to assess the toxicity of influent and effluent of each reactor in the A²/O system. RESULTS: The optimum operational parameters for toxicity reduction were as follows: anaerobic hydraulic retention time (HRT) = 2.8 h, anoxic HRT = 2.8 h, aerobic HRT = 6.9 h, sludge retention time (SRT) = 15 days and internal recycle ratio (IRR) = 100%. An important toxicity reduction (%) was observed in the optimized A²/O process, even when the toluene concentration of the influent was 120.7 mg·L⁻¹. CONCLUSIONS: The toxicity of municipal wastewater was reduced significantly during the A²/O process. A²/O process can be used for toxicity reduction of municipal wastewater under toxic-shock loading.