ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is associated with extensive dysregulation of the epigenome and epigenetic regulators, such as bromodomain and extraterminal motif (BET) proteins, have been suggested as potential targets for therapy. However, single-agent BET inhibition has shown poor efficacy in clinical trials, and no epigenetic approaches are currently used in PDAC. To circumvent the limitations of the current generation of BET inhibitors, we developed the compound XP-524 as an inhibitor of the BET protein BRD4 and the histone acetyltransferase EP300/CBP, both of which are ubiquitously expressed in PDAC tissues and cooperate to enhance tumorigenesis. XP-524 showed increased potency and superior tumoricidal activity than the benchmark BET inhibitor JQ-1 in vitro, with comparable efficacy to higher-dose JQ-1 combined with the EP300/CBP inhibitor SGC-CBP30. We determined that this is in part due to the epigenetic silencing of KRAS in vitro, with similar results observed using ex vivo slice cultures of human PDAC tumors. Accordingly, XP-524 prevented KRAS-induced, neoplastic transformation in vivo and extended survival in two transgenic mouse models of aggressive PDAC. In addition to the inhibition of KRAS/MAPK signaling, XP-524 also enhanced the presentation of self-peptide and tumor recruitment of cytotoxic T lymphocytes, though these lymphocytes remained refractory from full activation. We, therefore, combined XP-524 with an anti-PD-1 antibody in vivo, which reactivated the cytotoxic immune program and extended survival well beyond XP-524 in monotherapy. Pending a comprehensive safety evaluation, these results suggest that XP-524 may benefit PDAC patients and warrant further exploration, particularly in combination with immune checkpoint inhibition.
Subject(s)
Antineoplastic Agents/pharmacology , E1A-Associated p300 Protein/antagonists & inhibitors , Immune Checkpoint Inhibitors/pharmacology , Proteins/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Disease Models, Animal , Drug Synergism , E1A-Associated p300 Protein/chemistry , Gene Expression Regulation , Humans , Kaplan-Meier Estimate , Mice , Models, Molecular , Molecular Conformation , Molecular Structure , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/chemistry , Structure-Activity Relationship , Transcription Factors/genetics , Transcription Factors/metabolism , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
Advanced stage neuroblastoma is a very aggressive pediatric cancer with limited treatment options and a high mortality rate. Glycogen synthase kinase-3ß (GSK-3ß) is a potential therapeutic target in neuroblastoma. Using immunohistochemical staining, we observed positive GSK-3ß expression in 67% of human neuroblastomas (34 of 51 cases). Chemically distinct GSK-3 inhibitors (AR-A014418, TDZD-8, and 9-ING-41) suppressed the growth of neuroblastoma cells, whereas 9-ING-41, a clinically relevant small-molecule GSK-3ß inhibitor with broad-spectrum preclinical antitumor activity, being the most potent. Inhibition of GSK-3 resulted in a decreased expression of the antiapoptotic molecule XIAP and an increase in neuroblastoma cell apoptosis. Mouse xenograft studies showed that the combination of clinically relevant doses of CPT-11 and 9-ING-41 led to greater antitumor effect than was observed with either agent alone. These data support the inclusion of patients with advanced neuroblastoma in clinical studies of 9-ING-41, especially in combination with CPT-11.
Subject(s)
Enzyme Inhibitors/pharmacology , Glycogen Synthase Kinase 3 beta/antagonists & inhibitors , Indoles/pharmacology , Maleimides/pharmacology , Neuroblastoma/drug therapy , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cell Growth Processes/drug effects , Cell Line, Tumor , Drug Synergism , Female , Glycogen Synthase Kinase 3 beta/metabolism , Humans , Indoles/administration & dosage , Irinotecan/administration & dosage , Irinotecan/pharmacology , Maleimides/administration & dosage , Mice , Mice, Nude , Neuroblastoma/enzymology , Neuroblastoma/pathology , Xenograft Model Antitumor AssaysABSTRACT
Mating yeast cells interpret complex pheromone gradients and polarize their growth in the direction of the closest partner. Chemotropic growth depends on both the pheromone receptor and its associated G-protein. Upon activation by the receptor, Gα dissociates from Gßγ and Gß is subsequently phosphorylated. Free Gßγ signals to the nucleus via a MAPK cascade and recruits Far1-Cdc24 to the incipient growth site. It is not clear how the cell establishes and stabilizes the axis of polarity, but this process is thought to require local signal amplification via the Gßγ-Far1-Cdc24 chemotropic complex, as well as communication between this complex and the activated receptor. Here we show that a mutant form of Gß that cannot be phosphorylated confers defects in directional sensing and chemotropic growth. Our data suggest that phosphorylation of Gß plays a role in localized signal amplification and in the dynamic communication between the receptor and the chemotropic complex, which underlie growth site selection and maintenance.
Subject(s)
Chemotaxis , GTP-Binding Protein alpha Subunits/metabolism , GTP-Binding Protein beta Subunits/metabolism , Saccharomyces cerevisiae/physiology , Aldehyde Oxidoreductases/metabolism , Cell Cycle Proteins/metabolism , Cell Polarity/genetics , Cyclin-Dependent Kinase Inhibitor Proteins/metabolism , GTP-Binding Protein beta Subunits/genetics , Guanine Nucleotide Exchange Factors/metabolism , MAP Kinase Signaling System/genetics , Mutation/genetics , Phosphorylation/genetics , Protein Binding , Receptors, Pheromone/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Alterations of cell monolayer integrity and increased vascular permeability are key to many pathologies, including atherosclerosis, stroke, lung injury, cancer, digestive disorders and others. Current approaches to probe cell permeability require specific culture conditions and provide an average estimation of trans-monolayer permeability, while analysis of regional monolayer permeability in static and mechanically challenged monolayer at a single-cell scale resolution remains unavailable. We describe a novel method for visualization and rapid quantification of trans-monolayer permeability based on high-affinity interactions between ligand (FITC-conjugated avidin) added in the culture medium, which permeates cell monolayer to reach substrate-bound acceptor (biotinylated gelatin or collagen). This approach was used to simultaneously evaluate general and local permeability responses by endothelial cell (EC) monolayer to a spectrum of barrier protective and barrier disruptive agonists and their combinations. The results revealed the paracellular pathway as the predominant mechanism of agonist-induced mass transport by pulmonary EC. We also detected for the first time, in a direct assay, a synergistic effect of pathologically relevant levels of cyclic stretch (CS) and edemagenic agent thrombin in the development of pulmonary EC hyper-permeability response observed in ventilator-induced lung injury. The reported novel assay provides unique information about local monolayer permeability changes induced by agonists, mechanical factors or molecular perturbations in single cells. However, the spectrum of substrates, assay formats and experimental conditions compatible with this assay suggest its broad application in the areas of endothelial and epithelial biology, cancer research and other fields.
Subject(s)
Avidin/pharmacokinetics , Capillary Permeability/physiology , Endothelial Cells/metabolism , Fluorescein-5-isothiocyanate/analogs & derivatives , Pulmonary Artery/cytology , Tissue Array Analysis/methods , Ventilator-Induced Lung Injury/physiopathology , Avidin/metabolism , Biological Transport/physiology , Biotinylation , Cell Culture Techniques , Collagen/metabolism , Culture Media/chemistry , Fluorescein-5-isothiocyanate/metabolism , Fluorescein-5-isothiocyanate/pharmacokinetics , Fluorescent Antibody Technique , Gelatin/metabolism , HumansABSTRACT
Afadin is a novel regulator of epithelial cell junctions assembly. However, its role in the formation of endothelial cell junctions and the regulation of vascular permeability remains obscure. We previously described protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) in the in vitro and in vivo models of lung endothelial barrier dysfunction and acute lung injury, which were mediated by Rac GTPase. This study examined a role of afadin in the OxPAPC-induced enhancement of interactions between adherens junctions and tight junctions as a novel mechanism of endothelial cell (EC) barrier preservation. OxPAPC induced Rap1-dependent afadin accumulation at the cell periphery and Rap1-dependent afadin interaction with adherens junction and tight junction proteins p120-catenin and ZO-1, respectively. Afadin knockdown using siRNA or ectopic expression of afadin mutant lacking Rap1 GTPase binding domain suppressed OxPAPC-induced EC barrier enhancement and abolished barrier protective effects of OxPAPC against thrombin-induced EC permeability. Afadin knockdown also abolished protective effects of OxPAPC against ventilator-induced lung injury in vivo. These results demonstrate for the first time a critical role of afadin in the regulation of vascular barrier function in vitro and in vivo via coordination of adherens junction-tight junction interactions.
Subject(s)
Catenins/metabolism , Endothelium/metabolism , Lung/anatomy & histology , Lung/metabolism , Membrane Proteins/metabolism , Microfilament Proteins/metabolism , Phospholipids/metabolism , Phosphoproteins/metabolism , Adherens Junctions/metabolism , Animals , Cell Adhesion/physiology , Cell Line , Cells, Cultured , Cytoskeleton/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Phospholipids/chemistry , Tight Junctions/metabolism , Zonula Occludens-1 Protein , rap1 GTP-Binding Proteins/genetics , rap1 GTP-Binding Proteins/metabolism , Delta CateninABSTRACT
Small GTPase Rac is important regulator of endothelial cell (EC) barrier enhancement by prostacyclin characterized by increased peripheral actin cytoskeleton and increased interactions between VE-cadherin and other adherens junction (AJ) proteins. This study utilized complementary approaches including siRNA knockdown, culturing in Ca(2+) -free medium, and VE-cadherin blocking antibody to alter VE-cadherin extracellular interactions to investigate the role of VE-cadherin outside-in signaling in modulation of Rac activation and EC barrier regulation by prostacyclin analog iloprost. Spatial analysis of Rac activation in pulmonary EC by FRET revealed additional spike in iloprost-induced Rac activity at the sites of newly formed cell-cell junctions. In contrast, disruption of VE-cadherin extracellular trans-interactions suppressed iloprost-activated Rac signaling and attenuated EC barrier enhancement and cytoskeletal remodeling. These inhibitory effects were associated with decreased membrane accumulation and activation of Rac-specific guanine nucleotide exchange factors (GEFs) Tiam1 and Vav2. Conversely, plating of pulmonary EC on surfaces coated with extracellular VE-cadherin domain further promoted iloprost-induced Rac signaling. In the model of thrombin-induced EC barrier recovery, blocking of VE-cadherin trans-interactions attenuated activation of Rac pathway during recovery phase and delayed suppression of Rho signaling and restoration of EC barrier properties. These results suggest that VE-cadherin outside-in signaling controls locally Rac activity stimulated by barrier protective agonists. This control is essential for maximal EC barrier enhancement and accelerated barrier recovery.
Subject(s)
Antigens, CD/metabolism , Cadherins/metabolism , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Iloprost/pharmacology , Lung/drug effects , Lung/metabolism , rac GTP-Binding Proteins/metabolism , Adherens Junctions/drug effects , Adherens Junctions/metabolism , Antibodies/pharmacology , Cadherins/antagonists & inhibitors , Capillary Permeability/drug effects , Cells, Cultured , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Endothelial Cells/cytology , Epoprostenol/pharmacology , Guanine Nucleotide Exchange Factors/metabolism , Humans , Intercellular Junctions/drug effects , Intercellular Junctions/metabolism , Lung/cytology , Proto-Oncogene Proteins c-vav/metabolism , Pulmonary Artery/drug effects , Pulmonary Artery/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , T-Lymphoma Invasion and Metastasis-inducing Protein 1ABSTRACT
Vascular endothelial cells lining the blood vessels form the interface between the bloodstream and the vessel wall and as such they are continuously subjected to shear and cyclic stress from the flowing blood in the lumen. Additional mechanical stimuli are also imposed on these cells in the form of substrate stiffness transmitted from the extracellular matrix components in the basement membrane, and additional mechanical loads imposed on the lung endothelium as the result of respiration or mechanical ventilation in clinical settings. Focal adhesions (FAs) are complex structures assembled at the abluminal endothelial plasma membrane which connect the extracellular filamentous meshwork to the intracellular cytoskeleton and hence constitute the ideal checkpoint capable of controlling or mediating transduction of bidirectional mechanical signals. In this review we focus on focal adhesion kinase (FAK), a component of FAs, which has been studied for a number of years with regards to its involvement in mechanotransduction. We analyzed the recent advances in the understanding of the role of FAK in the signaling cascade(s) initiated by various mechanical stimuli with particular emphasis on potential implications on endothelial cell functions.
Subject(s)
Endothelial Cells/enzymology , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Focal Adhesions/enzymology , Mechanotransduction, Cellular , Animals , Apoptosis , Capillary Permeability , Cell Movement , Cell Proliferation , Endothelial Cells/pathology , Focal Adhesion Protein-Tyrosine Kinases/chemistry , Focal Adhesion Protein-Tyrosine Kinases/genetics , Focal Adhesions/pathology , Humans , Neovascularization, PhysiologicABSTRACT
Products resulting from oxidation of cell membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) exhibit potent protective effects against lung endothelial cell (EC) barrier dysfunction caused by pathologically relevant mechanical forces and inflammatory agents. These effects were linked to enhancement of peripheral cytoskeleton and cell adhesion interactions mediated by small GTPase Rac and inhibition of Rho-mediated barrier-disruptive signaling. However, the mechanism of OxPAPC-induced, Rac-dependent Rho downregulation critical for vascular barrier protection remains unclear. This study tested the hypothesis that Rho negative regulator p190RhoGAP is essential for OxPAPC-induced lung barrier protection against ventilator-induced lung injury (VILI), and investigated potential mechanism of p190RhoGAP targeting to adherens junctions (AJ) via p120-catenin. OxPAPC induced peripheral translocation of p190RhoGAP, which was abolished by knockdown of Rac-specific guanine nucleotide exchange factors Tiam1 and Vav2. OxPAPC also induced Rac-dependent tyrosine phosphorylation and association of p190RhoGAP with AJ protein p120-catenin. siRNA-induced knockdown of p190RhoGAP attenuated protective effects of OxPAPC against EC barrier compromise induced by thrombin and pathologically relevant cyclic stretch (18% CS). In vivo, p190RhoGAP knockdown significantly attenuated protective effects of OxPAPC against ventilator-induced lung vascular leak, as detected by increased cell count and protein content in the bronchoalveolar lavage fluid, and tissue neutrophil accumulation in the lung. These results demonstrate for the first time a key role of p190RhoGAP for the vascular endothelial barrier protection in VILI.
Subject(s)
Guanine Nucleotide Exchange Factors/metabolism , Phospholipids/metabolism , Repressor Proteins/metabolism , Ventilator-Induced Lung Injury/physiopathology , Animals , Disease Models, Animal , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Cells/physiology , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , Guanine Nucleotide Exchange Factors/genetics , Humans , Mice , Oxidation-Reduction , Phosphatidylcholines/pharmacology , Repressor Proteins/geneticsABSTRACT
Antiviral agents that complement vaccination are urgently needed to end the COVID-19 pandemic. The SARS-CoV-2 papain-like protease (PLpro), one of only two essential cysteine proteases that regulate viral replication, also dysregulates host immune sensing by binding and deubiquitination of host protein substrates. PLpro is a promising therapeutic target, albeit challenging owing to featureless P1 and P2 sites recognizing glycine. To overcome this challenge, we leveraged the cooperativity of multiple shallow binding sites on the PLpro surface, yielding novel 2-phenylthiophenes with nanomolar inhibitory potency. New cocrystal structures confirmed that ligand binding induces new interactions with PLpro: by closing of the BL2 loop of PLpro forming a novel "BL2 groove" and by mimicking the binding interaction of ubiquitin with Glu167 of PLpro. Together, this binding cooperativity translates to the most potent PLpro inhibitors reported to date, with slow off-rates, improved binding affinities, and low micromolar antiviral potency in SARS-CoV-2-infected human cells.
Subject(s)
Antiviral Agents/pharmacology , COVID-19 Drug Treatment , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Binding Sites/drug effects , COVID-19/metabolism , Coronavirus Papain-Like Proteases/isolation & purification , Coronavirus Papain-Like Proteases/metabolism , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Humans , Microbial Sensitivity Tests , Microsomes, Liver/chemistry , Microsomes, Liver/metabolism , Models, Molecular , Pandemics , Surface Plasmon Resonance , Tumor Cells, CulturedABSTRACT
The recent renascence of phenotypic drug discovery (PDD) is catalyzed by its ability to identify first-in-class drugs and deliver results when the exact molecular mechanism is partially obscure. Acute respiratory distress syndrome (ARDS) is a severe, life-threatening condition with a high mortality rate that has increased in frequency due to the COVID-19 pandemic. Despite decades of laboratory and clinical study, no efficient pharmacological therapy for ARDS has been found. An increase in endothelial permeability is the primary event in ARDS onset, causing the development of pulmonary edema that leads to respiratory failure. Currently, the detailed molecular mechanisms regulating endothelial permeability are poorly understood. Therefore, the use of the PDD approach in the search for efficient ARDS treatment can be more productive than classic target-based drug discovery (TDD), but its use requires a new cell-based assay compatible with high-throughput (HTS) and high-content (HCS) screening. Here we report the development of a new plate-based image cytometry method to measure endothelial barrier function. The incorporation of image cytometry in combination with digital image analysis substantially decreases assay variability and increases the signal window. This new method simultaneously allows for rapid measurement of cell monolayer permeability and cytological analysis. The time-course of permeability increase in human pulmonary artery endothelial cells (HPAECs) in response to the thrombin and tumor necrosis factor α treatment correlates with previously published data obtained by transendothelial resistance (TER) measurements. Furthermore, the proposed image cytometry method can be easily adapted for HTS/HCS applications.
Subject(s)
COVID-19/diagnostic imaging , High-Throughput Screening Assays/methods , Image Cytometry/methods , Respiratory Distress Syndrome/diagnostic imaging , COVID-19/diagnosis , COVID-19/virology , Cell Membrane Permeability/genetics , Drug Discovery , Endothelial Cells/ultrastructure , Endothelial Cells/virology , Humans , Image Processing, Computer-Assisted , Pandemics/prevention & control , Phenotype , Pulmonary Artery/diagnostic imaging , Pulmonary Artery/pathology , Pulmonary Artery/virology , Pulmonary Edema/diagnosis , Pulmonary Edema/diagnostic imaging , Pulmonary Edema/virology , Respiratory Distress Syndrome/diagnosis , Respiratory Distress Syndrome/virology , Respiratory Insufficiency/diagnosis , Respiratory Insufficiency/diagnostic imaging , Respiratory Insufficiency/virology , SARS-CoV-2/pathogenicity , Thrombin/pharmacology , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
The calpain-cathepsin hypothesis posits a key role for elevated calpain-1 and cathepsin-B activity in the neurodegeneration underlying neurotrauma and multiple disorders including Alzheimer's disease (AD). AD clinical trials were recently halted on alicapistat, a selective calpain-1 inhibitor, because of insufficient exposure of neurons to the drug. In contrast to neuroprotection, the ability of calpain-1 and cathepsin-B inhibitors to protect the blood-brain barrier (BBB), is understudied. Since cerebrovascular dysfunction underlies vascular dementia, is caused by ischemic stroke, and is emerging as an early feature in the progression of AD, we studied protection of brain endothelial cells (BECs) by selective and nonselective calpain-1 and cathepsin-B inhibitors. We show these inhibitors protect both neurons and murine BECs from ischemia-reperfusion injury. Cultures of primary BECs from ALDH2 -/- mice that manifest enhanced oxidative stress were sensitive to ischemia, leading to reduced cell viability and loss of tight junction proteins; this damage was rescued by calpain-1 and cathepsin-B inhibitors. In ALDH2 -/- mice 24 h after mild traumatic brain injury (mTBI), BBB damage was reflected by significantly increased fluorescein extravasation and perturbation of tight junction proteins, eNOS, MMP-9, and GFAP. Both calpain and cathepsin-B inhibitors alleviated BBB dysfunction caused by mTBI. No clear advantage was shown by selective versus nonselective calpain inhibitors in these studies. The lack of recognition of the ability of calpain inhibitors to protect the BBB may have led to the premature abandonment of this therapeutic approach in AD clinical trials and requires further mechanistic studies of cerebrovascular protection by calpain-1 inhibitors.
ABSTRACT
Antiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50 < 10 lower case Greek µM). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a 'BL2 groove' that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.
ABSTRACT
BACKGROUND: Therapeutic agents with novel mechanisms of action are needed to combat the growing epidemic of type 2 diabetes (T2D) and related metabolic syndromes. Liver X receptor (LXR) agonists possess preclinical efficacy yet produce side effects due to excessive lipogenesis. Anticipating that many beneficial and detrimental effects of LXR agonists are mediated by ABCA1 and SREPB1c expression, respectively, we hypothesized that a phenotypic optimization strategy prioritizing selective ABCA1 induction would identify an efficacious lead compound with an improved side effect profile over existing LXRß agonists. METHODS: We synthesized and characterized a novel small molecule for selective induction of ABCA1 vs. SREBP1c in vitro. This compound was evaluated in both wild-type mice and a high-fat diet (HFD) mouse model of obesity-driven diabetes through functional, biochemical, and metabolomic analysis. FINDINGS: Six weeks of oral administration of our lead compound attenuated weight gain, glucose intolerance, insulin signaling deficits, and adiposity. Global metabolomics revealed suppression of gluconeogenesis, free fatty acids, and pro-inflammatory metabolites. Target identification linked these beneficial effects to selective LXRß agonism and PPAR/RXR antagonism. INTERPRETATION: Our observations in the HFD model, combined with the absence of lipogenesis and neutropenia in WT mice, support this novel approach to therapeutic development for T2D and related conditions.
Subject(s)
ATP Binding Cassette Transporter 1/agonists , Metabolome , Metabolomics , Obesity/etiology , Obesity/metabolism , Adiposity/drug effects , Animals , Biomarkers , Cytokines/metabolism , Diet, High-Fat , Disease Models, Animal , Disease Susceptibility , Drug Development , Glucose Intolerance , Inflammation Mediators/metabolism , Insulin Resistance , Lipids/blood , Lipogenesis , Liver X Receptors/agonists , Male , Metabolomics/methods , Mice , Molecular Targeted Therapy , Obesity/drug therapy , Peroxisome Proliferator-Activated Receptors/antagonists & inhibitors , RNA, Small Interfering/genetics , Retinoid X Receptors/antagonists & inhibitorsABSTRACT
Increased levels of atrial natriuretic peptide (ANP) in the models of sepsis, pulmonary edema, and acute respiratory distress syndrome (ARDS) suggest its potential role in the modulation of acute lung injury. We have recently described ANP-protective effects against thrombin-induced barrier dysfunction in pulmonary endothelial cells (EC). The current study examined involvement of the Rac effector p21-activated kinase (PAK1) in ANP-protective effects in the model of lung vascular permeability induced by bacterial wall LPS. C57BL/6J mice or ANP knockout mice (Nppa(-/-)) were treated with LPS (0.63 mg/kg intratracheal) with or without ANP (2 µg/kg iv). Lung injury was monitored by measurements of bronchoalveolar lavage protein content, cell count, Evans blue extravasation, and lung histology. Endothelial barrier properties were assessed by morphological analysis and measurements of transendothelial electrical resistance. ANP treatment stimulated Rac-dependent PAK1 phosphorylation, attenuated endothelial permeability caused by LPS, TNF-α, and IL-6, decreased LPS-induced cell and protein accumulation in bronchoalveolar lavage fluid, and suppressed Evans blue extravasation in the murine model of acute lung injury. More severe LPS-induced lung injury and vascular leak were observed in ANP knockout mice. In rescue experiments, ANP injection significantly reduced lung injury in Nppa(-/-) mice caused by LPS. Molecular inhibition of PAK1 suppressed the protective effects of ANP treatment against LPS-induced lung injury and endothelial barrier dysfunction. This study shows that the protective effects of ANP against LPS-induced vascular leak are mediated at least in part by PAK1-dependent signaling leading to EC barrier enhancement. Our data suggest a direct role for ANP in endothelial barrier regulation via modulation of small GTPase signaling.
Subject(s)
Atrial Natriuretic Factor/pharmacology , Capillary Permeability/drug effects , Lipopolysaccharides/pharmacology , Lung/blood supply , p21-Activated Kinases/metabolism , Animals , Atrial Natriuretic Factor/genetics , Bronchoalveolar Lavage Fluid/cytology , Cell Line , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Humans , Lung/cytology , Lung/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Random Allocation , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Acquired resistance to fulvestrant and palbociclib is a new challenge to treatment of estrogen receptor positive (ER+) breast cancer. ER is expressed in most resistance settings; thus, bromodomain and extra-terminal protein inhibitors (BETi) that target BET-amplified ER-mediated transcription have therapeutic potential. Novel pyrrolopyridone BETi leveraged novel interactions with L92/L94 confirmed by a cocrystal structure of 27 with BRD4. Optimization of BETi using growth inhibition in fulvestrant-resistant (MCF-7:CFR) cells was confirmed in endocrine-resistant, palbociclib-resistant, and ESR1 mutant cell lines. 27 was more potent in MCF-7:CFR cells than six BET inhibitors in clinical trials. Transcriptomic analysis differentiated 27 from the benchmark BETi, JQ-1, showing downregulation of oncogenes and upregulation of tumor suppressors and apoptosis. The therapeutic approach was validated by oral administration of 27 in orthotopic xenografts of endocrine-resistant breast cancer in monotherapy and in combination with fulvestrant. Importantly, at an equivalent dose in rats, thrombocytopenia was mitigated.
Subject(s)
Breast Neoplasms/pathology , Drug Resistance, Neoplasm/drug effects , Fulvestrant/pharmacology , Piperazines/pharmacology , Pyridines/pharmacology , Pyridones/chemistry , Pyridones/pharmacology , Transcription Factors/antagonists & inhibitors , Animals , Breast Neoplasms/metabolism , Humans , MCF-7 Cells , Mice , Models, Molecular , Protein Domains , Pyridones/pharmacokinetics , Receptors, Estrogen/metabolism , Tissue Distribution , Transcription Factors/chemistry , Xenograft Model Antitumor AssaysABSTRACT
The filamentous/invasive growth pathway is activated by nutrient limitation in the haploid form of the yeast Saccharomyces cerevisiae, whereas exposure to mating-pheromone causes cells to differentiate into gametes. Although these two pathways respond to very different stimuli and generate very different responses, they utilize many of the same signaling components. This implies the need for robust mechanisms to maintain signal fidelity. Dse1 was identified in an allele-specific suppressor screen for proteins that interact with the pheromone-responsive Gbetagamma, and found to bind both to a Gbetagamma-affinity column, and to the shared MEKK, Ste11. Although overexpression of Dse1 stimulated invasive growth and transcription of both filamentation and mating-specific transcriptional reporters, deletion of DSE1 had no effect on these outputs. In contrast, pheromone hyper-induced transcription of the filamentation reporter in cells lacking Dse1 and in cells expressing a mutant form of Gbeta that exhibits diminished interaction with Dse1. Thus, the interaction of Dse1 with both Gbeta and Ste11 may be designed to control cross talk between the pheromone and filamentation pathways.
Subject(s)
Carrier Proteins/physiology , GTP-Binding Protein beta Subunits/physiology , MAP Kinase Kinase Kinases/physiology , Protein Precursors/pharmacology , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/physiology , Binding Sites , Carrier Proteins/genetics , Cell Cycle/drug effects , Chromatography, Affinity , GTP-Binding Protein beta Subunits/genetics , Gene Knockdown Techniques , Genes, Reporter , Haploidy , MAP Kinase Kinase Kinases/genetics , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/genetics , MAP Kinase Signaling System/physiology , Protein Interaction Mapping , Recombinant Fusion Proteins/physiology , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/pharmacology , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/physiology , Two-Hybrid System TechniquesABSTRACT
Matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) imaging mass spectrometry has emerged as a powerful, label-free technique to visualize penetration of small molecules in vivo and in vitro, including in 3D cell culture spheroids; however, some spheroids do not grow sufficiently large to provide enough area for imaging mass spectrometry. Here, we describe an ex vivo method for visualizing unlabeled peptides and small molecules in tumor explants, which can be divided into pieces of desired size, thus circumventing the size limitations of many spheroids. As proof-of-concept, a small molecule drug (4-hydroxytamoxifen), as well as a peptide drug (cyclosporin A) and peptide chemical probe, can be visualized after in vitro incubation with tumor explants so that this technique may provide a solution to robing cell penetration by unlabeled peptides.
ABSTRACT
A growing body of evidence suggests that the inflammatory NFκB pathway is associated with the progression of ER+ tumors to more aggressive stages. However, it is unknown whether NFκB is a driver or a consequence of aggressive ER+ disease. To investigate this question, we developed breast cancer cell lines expressing an inducible, constitutively active form of IκB kinase ß (CA-IKKß), a key kinase in the canonical NFκB pathway. We found that CA-IKKß blocked E2-dependent cell proliferation in vitro and tumor growth in vivo in a reversible manner, suggesting that IKKß may contribute to tumor dormancy and recurrence of ER+ disease. Moreover, coactivation of ER and IKKß promoted cell migration and invasion in vitro and drove experimental metastasis in vivo Gene expression profiling revealed a strong association between ER and CA-IKKß-driven gene expression and clinically relevant invasion and metastasis gene signatures. Mechanistically, the invasive phenotype appeared to be driven by an expansion of a basal/stem-like cell population rather than EMT. Taken together, our findings suggest that coactivation of ER and the canonical NFκB pathway promotes a dormant, metastatic phenotype in ER+ breast cancer and implicates IKKß as a driver of certain features of aggressive ER+ breast cancer.Significance: The canonical NFκB pathway promotes expansion of stem/basal-like cells and a dormant, metastatic phenotype in ER+ breast cancer cells. Cancer Res; 78(4); 974-84. ©2017 AACR.
Subject(s)
Breast Neoplasms/genetics , I-kappa B Kinase/metabolism , Receptors, Estrogen/metabolism , Animals , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation , Female , Humans , Mice , Mice, Nude , Phenotype , Signal TransductionABSTRACT
Oncogenic mutations in two isocitrate dehydrogenase (IDH)-encoding genes (IDH1 and IDH2) have been identified in acute myelogenous leukemia, low-grade glioma, and secondary glioblastoma (GBM). Our in silico and wet-bench analyses indicate that non-mutated IDH1 mRNA and protein are commonly overexpressed in primary GBMs. We show that genetic and pharmacologic inactivation of IDH1 decreases GBM cell growth, promotes a more differentiated tumor cell state, increases apoptosis in response to targeted therapies, and prolongs the survival of animal subjects bearing patient-derived xenografts (PDXs). On a molecular level, diminished IDH1 activity results in reduced α-ketoglutarate (αKG) and NADPH production, paralleled by deficient carbon flux from glucose or acetate into lipids, exhaustion of reduced glutathione, increased levels of reactive oxygen species (ROS), and enhanced histone methylation and differentiation marker expression. These findings suggest that IDH1 upregulation represents a common metabolic adaptation by GBMs to support macromolecular synthesis, aggressive growth, and therapy resistance.
Subject(s)
Drug Resistance, Neoplasm , Glioblastoma/enzymology , Glioblastoma/pathology , Isocitrate Dehydrogenase/genetics , Molecular Targeted Therapy , Mutation/genetics , Animals , Apoptosis/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Disease Progression , Drug Resistance, Neoplasm/drug effects , Erlotinib Hydrochloride/pharmacology , Erlotinib Hydrochloride/therapeutic use , Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Glioblastoma/drug therapy , Glioblastoma/genetics , Histones/metabolism , Isocitrate Dehydrogenase/metabolism , Ketoglutaric Acids/metabolism , Lipids/biosynthesis , Methylation , Mice , Mice, SCID , NADP/metabolism , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/pathology , Protein Kinase Inhibitors/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolismABSTRACT
The histone deacetylases (HDACs) occur in 11 different isoforms, and these enzymes regulate the activity of a large number of proteins involved in cancer initiation and progression. The discovery of isoform-selective HDAC inhibitors (HDACIs) is desirable, as it is likely that such compounds would avoid some of the undesirable side effects found with the first-generation inhibitors. A series of HDACIs previously reported by us were found to display some selectivity for HDAC6 and to induce cell-cycle arrest and apoptosis in pancreatic cancer cells. In the present work, we show that structural modification of these isoxazole-based inhibitors leads to high potency and selectivity for HDAC6 over HDAC1-3 and HDAC10, while unexpectedly abolishing their ability to block cell growth. Three inhibitors with lower HDAC6 selectivity inhibit the growth of cell lines BxPC3 and L3.6pl, and they only induce apoptosis in L3.6pl cells. We conclude that HDAC6 inhibition alone is insufficient for disruption of cell growth, and that some degree of classâ 1 HDAC inhibition is required. Moreover, the highly selective HDAC6Is reported herein that are weakly cytotoxic may find use in cancer immune system reactivation.