RESUMEN
Current cardiac safety testing focuses on detecting drug-induced QTC prolongation as a surrogate for risk of Torsade de Pointes. The nonclinical strategy, described in International Conference on Harmonization (ICH) S7B, includes in vitro assessment of hERG block or ventricular repolarization delay and in vivo QT prolongation. Several studies have reported predictive values of ICH S7B results for clinical QTC outcomes for small molecules; none has examined peptides and proteins other than monoclonal antibodies. To address this knowledge gap, information for peptides and proteins submitted to the US Food and Drug Administration (FDA) was collected. Results of hERG assays, ventricular repolarization assays, and in vivo QT assessment were compared with clinical QTC study outcomes. The results show that 14% clinical QTC studies for approved and investigational products failed to exclude 10-ms QTC prolongation. Clinical QTC prolongation for these molecules lacked concentration-dependence which is expected for hERG block-mediated mechanism or QTC prolongation could not be excluded due to characterization in the clinical study. The hERG and ventricular repolarization assays do not identify clinical QTC prolongation potential for peptides and proteins. Lack of alignment between hERG and ventricular repolarization assay results and clinical QTC outcomes suggests that the mechanisms of QTC prolongation by some peptides and proteins are unrelated to direct cardiac ion channel block. Similar to large targeted proteins and monoclonal antibodies, peptides and proteins regardless of size have a low likelihood of direct cardiac ion channel interactions. This characteristic supports waiving the requirement for thorough QT assessment for products comprised of naturally occurring amino acids unless proarrhythmia potential is suggested by nonclinical or clinical data.
Asunto(s)
Síndrome de QT Prolongado , Torsades de Pointes , Humanos , Síndrome de QT Prolongado/inducido químicamente , Corazón , Torsades de Pointes/inducido químicamente , Péptidos/efectos adversos , Canales Iónicos , Anticuerpos Monoclonales/efectos adversos , ElectrocardiografíaRESUMEN
Tumor necrosis happens commonly in advanced solid tumors. We reported that necroptosis plays a major role in tumor necrosis. Although several key necroptosis regulators including receptor interacting protein kinase 1 (RIPK1) have been identified, the regulation of tumor necroptosis during tumor development remains elusive. Here, we report that Z-DNA-binding protein 1 (ZBP1), not RIPK1, mediates tumor necroptosis during tumor development in preclinical cancer models. We found that ZBP1 expression is dramatically elevated in necrotic tumors. Importantly, ZBP1, not RIPK1, deletion blocks tumor necroptosis during tumor development and inhibits metastasis. We showed that glucose deprivation triggers ZBP1-depedent necroptosis in tumor cells. Glucose deprivation causes mitochondrial DNA (mtDNA) release to the cytoplasm and the binding of mtDNA to ZBP1 to activate MLKL in a BCL-2 family protein, NOXA-dependent manner. Therefore, our study reveals ZBP1 as the key regulator of tumor necroptosis and provides a potential drug target for controlling tumor metastasis.
Asunto(s)
Neoplasias de la Mama/genética , Necroptosis/genética , Proteínas de Unión al ARN/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Células HEK293 , Humanos , Células MCF-7 , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Proteínas de Unión al ARN/metabolismo , Tratamiento con ARN de Interferencia/métodos , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Tumor necrosis factor (TNF) has a critical role in diverse cellular events including inflammation, apoptosis and necroptosis through different signaling complexes. However, little is known about how the transition from inflammatory signaling to the engagement of death pathways is modulated. Here we report that the cytoplasmic retinoic acid receptor gamma (RARγ) controls receptor-interacting protein kinase 1 (RIP1)-initiated cell death when cellular inhibitor of apoptosis (cIAP) activity is blocked. Through screening a short hairpin RNA library, we found that RARγ was essential for TNF-induced RIP1-initiated apoptosis and necroptosis. Our data suggests that RARγ initiates the formation of death signaling complexes by mediating RIP1 dissociation from TNF receptor 1. We demonstrate that RARγ is released from the nucleus to orchestrate the formation of the cytosolic death complexes. In addition, we demonstrate that RARγ has a similar role in TNF-induced necroptosis in vivo. Thus, our study suggests that nuclear receptor RARγ provides a key checkpoint for the transition from life to death.The molecular switch between how tumour necrosis factor (TNF) controls inflammation versus cell death is less well defined. Here, the authors show that the nuclear receptor retinoic acid receptor gamma is released from the nucleus to disrupt TNF initiated cell death complexes in the cytoplasm.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores de Ácido Retinoico/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Citoprotección/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones Noqueados , Modelos Biológicos , Receptores del Factor de Necrosis Tumoral/metabolismo , Proteína de Dominio de Muerte Asociada a Receptor de TNF/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Receptor de Ácido Retinoico gammaRESUMEN
NADPH oxidases (NOXs) are involved in inflammation, angiogenesis, tumor growth, and osteoclast differentiation. However, the role of NOX1 and NOX2 in macrophage differentiation and tumor progression is still elusive. Here we report that NOX1 and NOX2 are critical for the differentiation of monocytes to macrophages, the polarization of M2-type but not M1-type macrophages, and the occurrence of tumor-associated macrophages (TAMs). We found that deletion of both NOX1 and NOX2 led to a dramatic decrease in ROS production in macrophages and resulted in impaired efficiency in monocyte-to-macrophage differentiation and M2-type macrophage polarization. We further showed that NOX1 and NOX2 were critical for the activation of the MAPKs JNK and ERK during macrophage differentiation and that the deficiency of JNK and ERK activation was responsible for the failure of monocyte-to-macrophage differentiation, in turn affecting M2 macrophage polarization. Furthermore, we demonstrated that the decrease in M2 macrophages and TAMs, concomitant with the reduction of cytokine and chemokine secretion, contributed to the delay in wound healing and the inhibition of tumor growth and metastasis in NOX1/2 double knockout mice compared with WT mice. Collectively, these data provide direct evidence that NOX1 and NOX2 deficiency impairs macrophage differentiation and the occurrence of M2-type TAMs during tumor development.
Asunto(s)
Diferenciación Celular/inmunología , Macrófagos/inmunología , Glicoproteínas de Membrana/inmunología , Monocitos/inmunología , NADH NADPH Oxidorreductasas/inmunología , NADPH Oxidasas/inmunología , Especies Reactivas de Oxígeno/inmunología , Animales , Diferenciación Celular/genética , Quimiocinas/genética , Quimiocinas/inmunología , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/inmunología , Macrófagos/enzimología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Monocitos/enzimología , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasa 1 , NADPH Oxidasa 2 , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Intratumoral heterogeneity and treatment resistance drive breast cancer (BC) metastasis and recurrence. The RUNX2 transcription factor is upregulated in early stage luminal BC. However, the precise mechanism by which RUNX2 regulates an oncogenic phenotype in luminal BCs remains an enigma. We show that RUNX2 is predictive of poor overall survival in BC patients. RUNX2 associated with the TAZ transcriptional co-activator to promote a tumorigenic phenotype that was inhibited by knockdown of TAZ. RUNX2 increased endogenous TAZ translocation to the nucleus, which was prevented by inhibiting RUNX2. RUNX2/TAZ interaction was associated with ectodomain shedding of an oncogenic soluble E-Cadherin fragment (sE-Cad), which is known to cooperate with human epidermal growth factor receptor-2 (HER2/ErbB2) to increase BC growth. Neutralizing E-Cadherin antibodies or TAZ knockdown reduced the levels of sE-Cad in RUNX2-expressing BC cells and inhibited tumorsphere formation. RUNX2 expression also increased HER2-mediated tumorsphere size, which was reduced after treatment with the HER2-targeting agents Herceptin and lapatinib. These data support a novel role for RUNX2 in promoting an oncogenic phenotype in luminal BC in the context of TAZ, sE-Cad, and HER2. Using this signaling pathway to monitor BC cell oncogenic activity will accelerate the discovery of new therapeutic modalities to treat BC patients.
Asunto(s)
Cadherinas/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Transducción de Señal , Esferoides Celulares/metabolismo , Factores de Transcripción/metabolismo , Animales , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Doxiciclina/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Células MCF-7 , Ratones , Células 3T3 NIH , Unión Proteica , Interferencia de ARN , Receptor ErbB-2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Solubilidad , Esferoides Celulares/efectos de los fármacos , Transactivadores , Factores de Transcripción/genética , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZRESUMEN
Activation of genes promoting aerobic glycolysis and suppression of mitochondrial oxidative phosphorylation is one of the hallmarks of cancer. The RUNX2 transcription factor mediates breast cancer (BC) metastasis to bone and is regulated by glucose availability. But, the mechanisms by which it regulates glucose metabolism and promotes an oncogenic phenotype are not known. RUNX2 expression in luminal BC cells correlated with lower estrogen receptor-α (ERα) levels, anchorage-independent growth, expression of glycolytic genes, increased glucose uptake, and sensitivity to glucose starvation, but not to inhibitors of oxidative phosphorylation. Conversely, RUNX2 knockdown in triple-negative BC cells inhibited mammosphere formation and glucose dependence. RUNX2 knockdown resulted in lower LDHA, HK2, and GLUT1 glycolytic gene expression, but upregulation of pyruvate dehydrogenase-A1 (PDHA1) mRNA and enzymatic activity, which was consistent with lower glycolytic potential. The NAD-dependent histone deacetylase, SIRT6, a known tumor suppressor, was a critical regulator of these RUNX2-mediated metabolic changes. RUNX2 expression resulted in elevated pAkt, HK2, and PDHK1 glycolytic protein levels that were reduced by ectopic expression of SIRT6. RUNX2 also repressed mitochondrial oxygen consumption rates (OCR), a measure of oxidative phosphorylation (respiration). Overexpression of SIRT6 increased respiration in RUNX2-positive cells, but knockdown of SIRT6 in cells expressing low RUNX2 decreased respiration. RUNX2 repressed SIRT6 expression at both the transcriptional and post-translational levels and endogenous SIRT6 expression was lower in malignant BC tissues or cell lines that expressed high levels of RUNX2. These results support a hypothesis whereby RUNX2-mediated repression of the SIRT6 tumor suppressor regulates metabolic pathways that promote BC progression.
Asunto(s)
Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Glucosa/metabolismo , Sirtuinas/biosíntesis , Neoplasias de la Mama Triple Negativas/genética , Proliferación Celular/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glucólisis/genética , Humanos , Células MCF-7 , Proteínas de Neoplasias/biosíntesis , Fosforilación Oxidativa , Sirtuinas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.
Asunto(s)
Subunidad alfa 1 del Factor de Unión al Sitio Principal/análisis , ADN/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Factores de Transcripción/análisis , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Antineoplásicos/farmacología , Colorimetría , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/inmunología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , ADN/genética , ADN/metabolismo , Ensayos de Selección de Medicamentos Antitumorales/métodos , Regulación de la Expresión Génica , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
The fat-soluble prohormone cholecalciferol (Vitamin D3) is a precursor of the circulating 25-OH Vitamin D3, which is converted by 1α-hydroxylase to the biologically active 1,25-OH Vitamin D3. Active Vitamin D3 interacts with the Vitamin D receptor (VDR), a transcription factor that plays an important role in calcium mobilization and bone formation. RUNX2 is a DNA-binding transcription factor that regulates target genes important in bone formation, angiogenesis, and cancer metastasis. Using computer-assisted drug design (CADD) and a microtiter plate-based DNA-binding enzyme-linked immunosorbent assay (D-ELISA) to measure nuclear RUNX2 DNA binding, we have found that Vitamin D3 prohormones can modulate RUNX2 DNA binding, which was dose-dependent and sensitive to trypsin, salt, and phosphatase treatment. Unlabeled oligonucleotide or truncated, dominant negative RUNX2 proteins were competitive inhibitors of RUNX2 DNA binding. The RUNX2 heterodimeric partner, Cbfß, was detected in the binding complexes with specific antibodies. Evaluation of several RUNX2:DNA targeted small molecules predicted by CADD screening revealed a previously unknown biological activity of the inactive Vitamin D3 precursor, cholecalciferol. Cholecalciferol modulated RUNX2:DNA binding at nanomolar concentrations even in cells with low VDR. Cholecalciferol and 25-OH Vitamin D3 prohormones were selective inhibitors of RUNX2-positive endothelial, bone, and breast cancer cell proliferation, but not of cells lacking RUNX2 expression. These compounds may have application in modulating RUNX2 activity in an angiogenic setting, in metastatic cells, and to promote bone formation in disease-mediated osteoporosis. The combination CADD discovery and D-ELISA screening approaches allows the testing of other novel derivatives of Vitamin D and/or transcriptional inhibitors with the potential to regulate DNA binding and biological function.
Asunto(s)
Colecalciferol/farmacología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , ADN/metabolismo , Calcifediol/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Receptores de Calcitriol/metabolismoRESUMEN
Increased expression of PDGF-beta receptors is a landmark of hepatic stellate cell activation and transdifferentiation into myofibroblasts. However, the molecular mechanisms that regulate the fate of the receptor are lacking. Recent studies suggested that N-acetylcysteine enhances the extracellular degradation of PDGF-beta receptor by cathepsin B, thus suggesting that the absence of PDGF-beta receptors in quiescent cells is due to an active process of elimination and not to a lack of expression. In this communication we investigated further molecular mechanisms involved in PDGF-beta receptor elimination and reappearance after incubation with PDGF-BB. We showed that in culture-activated hepatic stellate cells there is no internal protein pool of receptor, that the protein is maximally phosphorylated by 5 min and completely degraded after 1 h by a lysosomal-dependent mechanism. Inhibition of receptor autophosphorylation by tyrphostin 1296 prevented its degradation, but several proteasomal inhibitors had no effect. We also showed that receptor reappearance is time and dose dependent, being more delayed in cells treated with 50 ng/ml (48 h) compared with 10 ng/ml (24 h).