RESUMEN
CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Modelos Biológicos , Factor de Transcripción TFIIH/metabolismo , Transcripción Genética/genética , Empalme Alternativo/genética , Supervivencia Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Activación Enzimática/genética , Células HL-60 , Humanos , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Based on one-year observation, the concentration, sources, and potential source areas of volatile organic compounds (VOCs) were comprehensively analyzed to investigate the pollution characteristics of ambient VOCs in Haikou, China. The results showed that the annual average concentration of total VOCs (TVOCs) was 11.4 ppbV, and the composition was dominated by alkanes (8.2 ppbV, 71.4%) and alkenes (1.3 ppbV, 20.5%). The diurnal variation in the concentration of dominant VOC species showed a distinct bimodal distribution with peaks in the morning and evening. The greatest contribution to ozone formation potential (OFP) was made by alkenes (51.6%), followed by alkanes (27.2%). The concentrations of VOCs and nitrogen dioxide (NO2) in spring and summer were low, and it was difficult to generate high ozone (O3) concentrations through photochemical reactions. The significant increase in O3 concentrations in autumn and winter was mainly related to the transmission of pollutants from the northeast. Traffic sources (40.1%), industrial sources (19.4%), combustion sources (18.6%), solvent usage sources (15.5%) and plant sources (6.4%) were identified as major sources of VOCs through the positive matrix factorization (PMF) model. The southeastern coastal areas of China were identified as major potential source areas of VOCs through the potential source contribution function (PSCF) and concentration-weighted trajectory (CWT) models. Overall, the concentration of ambient VOCs in Haikou was strongly influenced by traffic sources and long-distance transport, and the control of VOCs emitted from vehicles should be strengthened to reduce the active species of ambient VOCs in Haikou, thereby reducing the generation of O3.
Asunto(s)
Contaminantes Atmosféricos , Ozono , Compuestos Orgánicos Volátiles , Compuestos Orgánicos Volátiles/análisis , Emisiones de Vehículos/análisis , Contaminantes Atmosféricos/análisis , Monitoreo del Ambiente/métodos , Ozono/química , Alcanos/análisis , Alquenos , ChinaRESUMEN
It has been shown that the anterior cingulate cortex (ACC) and its dopamine system are crucial for decision making that requires physical/emotional effort, but not for all forms of cost-benefit decision making. Previous studies had mostly employed behavioral tasks with two competing cost-reward options that were preset by the experimenters. However, few studies have been conducted using scenarios in which the subjects have full control over the energy/time expenditure required to obtain a proportional reward. Here, we assessed the roles of the ACC and its dopamine system in cost-benefit decision making by utilizing a "do more get more" (DMGM) task and a time-reward trade-off (TRTO) task, wherein the animals were able to self-determine how much effort or time to expend at a nosepoke operandum for a proportional reward. Our results showed that (1) ACC inactivation severely impaired DMGM task performance, with a reduction in the rate of correct responses and a decrease in the effort expended, but did not affect the TRTO task; and (2) blocking ACC D2 receptors had no impact on DMGM task performance in the baseline cost-benefit scenario, but it significantly reduced the attempts to invest increased effort for a large reward when the benefit-cost ratio was reduced by half. In contrast, blocking ACC D1 receptors had no effect on DMGM task performance. These findings suggest that the ACC is required for self-paced effort-based but not for time-reward trade-off decision making. Furthermore, ACC dopamine D2 but not D1 receptors are involved in DMGM decision making.
Asunto(s)
Conducta de Elección , Toma de Decisiones , Giro del Cíngulo , Receptores Dopaminérgicos/fisiología , Animales , Ratas , RecompensaRESUMEN
The purpose of this study was to explore the hydrolytic ability of Lactobacillus helveticus CICC 22171 with regard to protein and the expression of enzyme genes during protein utilization. The results revealed that the strain hydrolyzed casein from the C-terminal, reached the maximum level in 6 h, and the number of amino acids in the hydrolyzed peptide was 7-33. The molecular weight was 652.4-3432.74 kDa. Hydrophobic peptides produced by hydrolysis were the source of ß-casein bitterness. Leucine and glutamine were the preferred cleavage points after 1 h; tyrosine and tryptophan subsequently increased. The first step of hydrolysis was controlled by PrtP and PrtM genes and coordinated with the action of PrtH1 and PrtH2. The transport system consisted of DtpT, OppB, OppD and OppF. The hydrolytic third step endopeptidase system consisted of the aminopeptidases (PepN, PepC, PepM and PepA), the endopeptidases (PepE, PepF and PepO); the dipeptidases (PepV and PepD), the tripeptidase PepT; the proline peptidases (PepX, PepP, PepQ, PepR and PepI). The expression of CEP genes was significantly different, and the expression level of genes related to the transport system significantly increased from 0 to 1 h. The specificity of the substrate and action site of endopeptidase was abundant.
RESUMEN
CDK7 has emerged as an exciting target in oncology due to its roles in two important processes that are misregulated in cancer cells: cell cycle and transcription. This report describes the discovery of SY-5609, a highly potent (sub-nM CDK7 Kd) and selective, orally available inhibitor of CDK7 that entered the clinic in 2020 (ClinicalTrials.gov Identifier: NCT04247126). Structure-based design was leveraged to obtain high selectivity (>4000-times the closest off target) and slow off-rate binding kinetics desirable for potent cellular activity. Finally, incorporation of a phosphine oxide as an atypical hydrogen bond acceptor helped provide the required potency and metabolic stability. The development candidate SY-5609 displays potent inhibition of CDK7 in cells and demonstrates strong efficacy in mouse xenograft models when dosed as low as 2 mg/kg.
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Neoplasias de la Mama , Ciclo Celular , Quinasas Ciclina-Dependientes , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas , Animales , Femenino , Humanos , Ratones , Apoptosis , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ratones Endogámicos BALB C , Ratones Desnudos , Inhibidores de Proteínas Quinasas/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lactobacillus helveticus is a homofermentative lactic acid bacterium. It is widely used in the fabrication of Swiss cheese and other dairy products. The aim of this study was to elucidate the mechanism by which L. helveticus utilizes protein. Lactobacillus helveticus CICC22171 were cultured in two different media with various nitrogen sources. The control contained 20 basic amino acids, while the experimental medium contained casein. De novo transcriptome and isobaric tags for relative and absolute quantification (iTRAQ) proteome analyses were applied to determine how L. helveticus utilizes protein. The casein underwent extracellular hydrolysis via ATP-binding cassette (ABC) transporter upregulation and Mn2+-associated cell envelope proteinase (CEP) downregulation. Sigma factors and EF-Tu were upregulated and Mg2+ was reduced in bacteria to accommodate DNA transcription and protein translation in preparation for proteolysis. Hydrolase activity was upregulated to digest intracellular polypeptides and control endopeptidase genes. In these bacteria, casein utilization affected glycolysis, trehalose phosphotransferase system (PTS), and key factors associated with aerobic respiration and reduced glucose consumption.
RESUMEN
Recent studies suggest that targeting transcriptional machinery can lead to potent and selective anticancer effects in cancers dependent on high and constant expression of certain transcription factors for growth and survival. Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the CDK-activating kinase complex. Its function is required for both cell-cycle regulation and transcriptional control of gene expression. CDK7 has recently emerged as an attractive cancer target because its inhibition leads to decreased transcript levels of oncogenic transcription factors, especially those associated with super-enhancers. Here, we describe a selective CDK7 inhibitor SY-1365, which is currently in clinical trials in populations of patients with ovarian and breast cancer (NCT03134638). In vitro, SY-1365 inhibited cell growth of many different cancer types at nanomolar concentrations. SY-1365 treatment decreased MCL1 protein levels, and cancer cells with low BCL2L1 (BCL-XL) expression were found to be more sensitive to SY-1365. Transcriptional changes in acute myeloid leukemia (AML) cell lines were distinct from those following treatment with other transcriptional inhibitors. SY-1365 demonstrated substantial antitumor effects in multiple AML xenograft models as a single agent; SY-1365-induced growth inhibition was enhanced in combination with the BCL2 inhibitor venetoclax. Antitumor activity was also observed in xenograft models of ovarian cancer, suggesting the potential for exploring SY-1365 in the clinic in both hematologic and solid tumors. Our findings support targeting CDK7 as a new approach for treating transcriptionally addicted cancers. SIGNIFICANCE: These findings demonstrate the molecular mechanism of action and potent antitumor activity of SY-1365, the first selective CDK7 inhibitor to enter clinical investigation.
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Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Neoplasias Ováricas/patología , Inhibidores de Proteínas Quinasas/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Moleculares , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/enzimología , Inhibidores de Proteínas Quinasas/química , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
KRAS is frequently mutated in lung cancers and is associated with aggressive biology and chemotherapy resistance. Therefore, innovative approaches are needed to treat these lung cancers. Prior work implicated the IFN-stimulated gene 15 (ISG15) deubiquitinase (DUB) USP18 as having antineoplastic activity by regulating lung cancer growth and oncoprotein stability. This study demonstrates that USP18 affects the stability of the KRAS oncoprotein. Interestingly, loss of USP18 reduced KRAS expression, and engineered gain of USP18 expression increased KRAS protein levels in lung cancer cells. Using the protein synthesis inhibitor cycloheximide, USP18 knockdown significantly reduced the half-life of KRAS, but gain of USP18 expression significantly increased its stability. Intriguingly, loss of USP18 altered KRAS subcellular localization by mislocalizing KRAS from the plasma membrane. To explore the biologic consequences, immunohistochemical (IHC) expression profiles of USP18 were compared in lung cancers of KrasLA2/+ versus cyclin E engineered mouse models. USP18 expression was higher in Kras-driven murine lung cancers, indicating a link between KRAS and USP18 expression in vivo To solidify this association, loss of Usp18 in KrasLA2/+ /Usp18-/- mice was found to significantly reduce lung cancers as compared with parental KrasLA2/+ mice. Finally, translational relevance was confirmed in a human lung cancer panel by showing that USP18 IHC expression was significantly higher in KRAS-mutant versus wild-type lung adenocarcinomas.Implications: Taken together, this study highlights a new way to combat the oncogenic consequences of activated KRAS in lung cancer by inhibiting the DUB USP18. Mol Cancer Res; 15(7); 905-14. ©2017 AACR.
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Adenocarcinoma/genética , Endopeptidasas/genética , Neoplasias Pulmonares/genética , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas p21(ras)/genética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Membrana Celular/genética , Ciclina E/genética , Cicloheximida/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Ratones Noqueados , Mutación , Ubiquitina TiolesterasaRESUMEN
Background: The first generation CDK2/7/9 inhibitor seliciclib (CYC202) causes multipolar anaphase and apoptosis in lung cancer cells with supernumerary centrosomes (known as anaphase catastrophe). We investigated a new and potent CDK2/9 inhibitor, CCT68127 (Cyclacel). Methods: CCT68127 was studied in lung cancer cells (three murine and five human) and control murine pulmonary epithelial and human immortalized bronchial epithelial cells. Robotic CCT68127 cell-based proliferation screens were used. Cells undergoing multipolar anaphase and inhibited centrosome clustering were scored. Reverse phase protein arrays (RPPAs) assessed CCT68127 effects on signaling pathways. The function of PEA15, a growth regulator highlighted by RPPAs, was analyzed. Syngeneic murine lung cancer xenografts (n = 4/group) determined CCT68127 effects on tumorigenicity and circulating tumor cell levels. All statistical tests were two-sided. Results: CCT68127 inhibited growth up to 88.5% (SD = 6.4%, P < .003) at 1 µM, induced apoptosis up to 42.6% (SD = 5.5%, P < .001) at 2 µM, and caused G1 or G2/M arrest in lung cancer cells with minimal effects on control cells (growth inhibition at 1 µM: 10.6%, SD = 3.6%, P = .32; apoptosis at 2 µM: 8.2%, SD = 1.0%, P = .22). A robotic screen found that lung cancer cells with KRAS mutation were particularly sensitive to CCT68127 ( P = .02 for IC 50 ). CCT68127 inhibited supernumerary centrosome clustering and caused anaphase catastrophe by 14.1% (SD = 3.6%, P < .009 at 1 µM). CCT68127 reduced PEA15 phosphorylation by 70% (SD = 3.0%, P = .003). The gain of PEA15 expression antagonized and its loss enhanced CCT68127-mediated growth inhibition. CCT68127 reduced lung cancer growth in vivo ( P < .001) and circulating tumor cells ( P = .004). Findings were confirmed with another CDK2/9 inhibitor, CYC065. Conclusions: Next-generation CDK2/9 inhibition elicits marked antineoplastic effects in lung cancer via anaphase catastrophe and reduced PEA15 phosphorylation.
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Adenosina/análogos & derivados , Anafase/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Pulmonares/tratamiento farmacológico , Fosfoproteínas/genética , Inhibidores de Proteínas Quinasas/farmacología , Adenosina/farmacología , Adenosina/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Centrosoma/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Pulmonares/genética , Masculino , Ratones , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/genética , Purinas/farmacología , Roscovitina , Transducción de Señal/efectos de los fármacosRESUMEN
Despite advances in targeted therapy, lung cancer remains the most common cause of cancer-related mortality in the United States. Chromosomal instability is a prominent feature in lung cancer and, because it rarely occurs in normal cells, it represents a potential therapeutic target. Our prior work discovered that lung cancer cells undergo anaphase catastrophe in response to inhibition of cyclin-dependent kinase 2 (CDK2), followed by apoptosis and reduced growth. In this study, the effects and mechanisms of the multi-CDK inhibitor dinaciclib on lung cancer cells were investigated. We sought to determine the specificity of CDK-dependent induction of anaphase catastrophe. Live cell imaging provided direct evidence that dinaciclib caused multipolar cell divisions resulting in extensive chromosome missegregation. Genetic knockdown of dinaciclib CDK targets revealed that repression of CDK2 and CDK1, but not CDK5 or CDK9, triggered anaphase catastrophe in lung cancer cells. Overexpression of CP110, which is a mediator of CDK2 inhibitor-induced anaphase catastrophe (and a CDK1 and 2 phosphorylation substrate), antagonized anaphase catastrophe and apoptosis following dinaciclib treatment. Consistent with our previous findings, acquisition of activated KRAS sensitized lung cancer cells to dinaciclib-mediated anaphase catastrophe and cell death. Combining dinaciclib with the mitotic inhibitor taxol augmented anaphase catastrophe induction and reduced cell viability of lung cancer cells. Thus, the multi-CDK inhibitor dinaciclib causes anaphase catastrophe in lung cancer cells and should be investigated as a potential therapeutic for wild-type and KRAS-mutant lung cancer, individually or in combination with taxanes. Mol Cancer Ther; 15(11); 2758-66. ©2016 AACR.
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Anafase/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proteína Quinasa CDC2/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Compuestos de Piridinio/farmacología , Animales , Proteínas de Ciclo Celular/metabolismo , División Celular/efectos de los fármacos , División Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Óxidos N-Cíclicos , Resistencia a Antineoplásicos/genética , Humanos , Indolizinas , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Taxoides/farmacologíaRESUMEN
Chromosomal instability (CIN) is a hallmark of solid tumor biology and is implicated in carcinogenesis. Preferentially eliminating malignant cells by targeting CIN and aneuploidy is an attractive antineoplastic strategy. We previously reported that CDK2 antagonism causes lung cancer cells to undergo anaphase catastrophe and apoptosis through inhibition of phosphorylation of the centrosomal protein CP110. Cells with activating KRAS mutations were particularly sensitive to CDK2 inhibition due to downregulation of CP110 protein levels. This study investigated mechanisms of CDK2 antagonism that mediate anaphase catastrophe via changes in CP110 protein expression and how activated KRAS affects CP110 levels in lung cancers. Site-directed mutagenesis revealed candidate CDK phosphorylation sites of CP110 (residues Ser 170 and Thr 194) critical for conferring anaphase catastrophe by altering centrosome clustering in mitosis. Intriguingly, KRAS mutation can promote CP110 protein degradation by upregulating the ubiquitin ligase SCF(cyclinF), which targets CP110 protein for destabilization. Finally, CDK2 inhibitor response was enhanced when combined with knockdown of the deubiquitinase USP33 that in turn accelerates CP110 protein degradation. Thus, this study provides molecular pharmacologic insights into how CP110 expression regulates response to CDK2 inhibition. An improved understanding of in vitro antineoplastic mechanisms of combining CDK2 antagonism with induced CP110 repression provides a rationale for exploring clinical consequences of this strategy. Taken together, preclinical findings obtained from combining CDK2 inhibition with USP33 repression have implications for treating patients with non-small cell lung cancers.
Asunto(s)
Anafase/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Ubiquitina Tiolesterasa/metabolismo , Anafase/genética , Animales , Sitios de Unión/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Centrosoma/efectos de los fármacos , Centrosoma/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Ciclinas/metabolismo , Humanos , Immunoblotting , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas Asociadas a Microtúbulos/genética , Mutación , Fosfoproteínas/genética , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Purinas/farmacología , Interferencia de ARN , Roscovitina , Serina/genética , Serina/metabolismo , Treonina/genética , Treonina/metabolismo , Ubiquitina Tiolesterasa/genéticaRESUMEN
Aneuploidy is frequently detected in human cancers and is implicated in carcinogenesis. Pharmacologic targeting of aneuploidy is an attractive therapeutic strategy, as this would preferentially eliminate malignant over normal cells. We previously discovered that CDK2 inhibition causes lung cancer cells with more than two centrosomes to undergo multipolar cell division leading to apoptosis, defined as anaphase catastrophe. Cells with activating KRAS mutations were especially sensitive to CDK2 inhibition. Mechanisms of CDK2-mediated anaphase catastrophe and how activated KRAS enhances this effect were investigated. Live-cell imaging provided direct evidence that following CDK2 inhibition, lung cancer cells develop multipolar anaphase and undergo multipolar cell division with the resulting progeny apoptotic. The siRNA-mediated repression of the CDK2 target and centrosome protein CP110 induced anaphase catastrophe of lung cancer cells. In contrast, CP110 overexpression antagonized CDK2 inhibitor-mediated anaphase catastrophe. Furthermore, activated KRAS mutations sensitized lung cancer cells to CDK2 inhibition by deregulating CP110 expression. Thus, CP110 is a critical mediator of CDK2 inhibition-driven anaphase catastrophe. Independent examination of murine and human paired normal-malignant lung tissues revealed marked upregulation of CP110 in malignant versus normal lung. Human lung cancers with KRAS mutations had significantly lower CP110 expression as compared with KRAS wild-type cancers. Thus, a direct link was found between CP110 and CDK2 inhibitor antineoplastic response. CP110 plays a mechanistic role in response of lung cancer cells to CDK2 inhibition, especially in the presence of activated KRAS mutations.