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1.
Nat Commun ; 11(1): 1836, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32296067

RESUMO

Studies on multisite phosphorylation networks of cyclin-dependent kinase (CDK) targets have opened a new level of signaling complexity by revealing signal processing routes encoded into disordered proteins. A model target, the CDK inhibitor Sic1, contains linear phosphorylation motifs, docking sites, and phosphodegrons to empower an N-to-C terminally directed phosphorylation process. Here, we uncover a signal processing mechanism involving multi-step competition between mutually diversional phosphorylation routes within the S-CDK-Sic1 inhibitory complex. Intracomplex phosphorylation plays a direct role in controlling Sic1 degradation, and provides a mechanism to sequentially integrate both the G1- and S-CDK activities while keeping S-CDK inhibited towards other targets. The competing phosphorylation routes prevent premature Sic1 degradation and demonstrate how integration of MAPK from the pheromone pathway allows one to tune the competition of alternative phosphorylation paths. The mutually diversional phosphorylation circuits may be a general way for processing multiple kinase signals to coordinate cellular decisions in eukaryotes.


Assuntos
Fase G1/fisiologia , Fase S/efeitos da radiação , Transdução de Sinais/fisiologia , Western Blotting , Divisão Celular/genética , Divisão Celular/fisiologia , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fase G1/genética , Imunoprecipitação , Espectrometria de Massas , Fosforilação , Fase S/genética , Transdução de Sinais/genética
2.
Am J Hum Genet ; 106(5): 717-725, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32330417

RESUMO

We identified three unrelated individuals with de novo missense variants in CDK19, encoding a cyclin-dependent kinase protein family member that predominantly regulates gene transcription. These individuals presented with hypotonia, global developmental delay, epileptic encephalopathy, and dysmorphic features. CDK19 is conserved between vertebrate and invertebrate model organisms, but currently abnormalities in CDK19 are not known to be associated with a human disorder. Loss of Cdk8, the fly homolog of CDK19, causes larval lethality, which is suppressed by expression of human CDK19 reference cDNA. In contrast, the CDK19 p.Tyr32His and p.Thr196Ala variants identified in the affected individuals fail to rescue the loss of Cdk8 and behave as null alleles. Additionally, neuronal RNAi-mediated knockdown of Cdk8 in flies results in semi-lethality. The few eclosing flies exhibit severe seizures and a reduced lifespan. Both phenotypes are fully suppressed by moderate expression of the CDK19 reference cDNA but not by expression of the two variants. Finally, loss of Cdk8 causes an obvious loss of boutons and synapses at larval neuromuscular junctions (NMJs). Together, our findings demonstrate that human CDK19 fully replaces the function of Cdk8 in the fly, the human disease-associated CDK19 variants behave as strong loss-of-function variants, and deleterious CDK19 variants underlie a syndromic neurodevelopmental disorder.


Assuntos
Encefalopatias/genética , Quinases Ciclina-Dependentes/genética , Epilepsia Generalizada/genética , Deficiência Intelectual/genética , Mutação de Sentido Incorreto/genética , Adulto , Sequência de Aminoácidos , Animais , Pré-Escolar , Quinase 8 Dependente de Ciclina/deficiência , Quinase 8 Dependente de Ciclina/genética , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Junção Neuromuscular , Doenças Raras/genética , Convulsões/genética , Síndrome , Adulto Jovem
3.
PLoS Genet ; 16(3): e1008584, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32176685

RESUMO

Progression through the cell cycle in eukaryotes is regulated on multiple levels. The main driver of the cell cycle progression is the periodic activity of cyclin-dependent kinase (CDK) complexes. In parallel, transcription during the cell cycle is regulated by a transcriptional program that ensures the just-in-time gene expression. Many core cell cycle regulators are widely conserved in eukaryotes, among them cyclins and CDKs; however, periodic transcriptional programs are divergent between distantly related species. In addition, many otherwise conserved cell cycle regulators have been lost and independently evolved in yeast, a widely used model organism for cell cycle research. For a better understanding of the evolution of the cell cycle regulation in opisthokonts, we investigated the transcriptional program during the cell cycle of the filasterean Capsaspora owczarzaki, a unicellular species closely related to animals. We developed a protocol for cell cycle synchronization in Capsaspora cultures and assessed gene expression over time across the entire cell cycle. We identified a set of 801 periodic genes that grouped into five clusters of expression over time. Comparison with datasets from other eukaryotes revealed that the periodic transcriptional program of Capsaspora is most similar to that of animal cells. We found that orthologues of cyclin A, B and E are expressed at the same cell cycle stages as in human cells and in the same temporal order. However, in contrast to human cells where these cyclins interact with multiple CDKs, Capsaspora cyclins likely interact with a single ancestral CDK1-3. Thus, the Capsaspora cyclin-CDK system could represent an intermediate state in the evolution of animal-like cyclin-CDK regulation. Overall, our results demonstrate that Capsaspora could be a useful unicellular model system for animal cell cycle regulation.


Assuntos
Ciclo Celular/genética , Quinases Ciclina-Dependentes/genética , Eucariotos/genética , Transcriptoma/genética , Células Cultivadas , Ciclinas/genética , Expressão Gênica/genética , Saccharomyces cerevisiae/genética , Transcrição Genética/genética
4.
Expert Opin Pharmacother ; 21(8): 941-952, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32164461

RESUMO

INTRODUCTION: Cyclin-dependent kinases (CDKs) are critical regulators of cell cycle progression in both normal and malignant cells, functioning through complex molecular interactions. Deregulation of CDK-dependent pathways is commonly found in both non-small cell and small cell lung cancer, and these derangements suggest vulnerabilities that can be exploited for clinical benefit. AREAS COVERED: In this review, the authors present an overview of the biology of CDKs in normal and malignant cells, with a focus on lung cancer, followed by an assessment of preclinical work that has demonstrated the vital role of CDKs in lung cancer development and progression, and the activity of CDK inhibitors in a variety of lung cancer models. Finally, the experience with clinical trials of CDK inhibitors in lung cancer is discussed along with the current status of these agents in cancer therapy. EXPERT OPINION: Despite strong biological rationale and promising preclinical studies, the results of clinical trials of CDK inhibitors in lung cancer have thus far been disappointing. Further clinical development of CDK inhibitors in lung cancer will depend on the identification of predictive biomarkers and the design of combination regimens that take advantage of the unique molecular alterations that drive lung cancer growth and survival.


Assuntos
Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Quinases Ciclina-Dependentes/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclo Celular/efeitos dos fármacos , Ensaios Clínicos como Assunto , Ciclinas/metabolismo , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Carcinoma de Pequenas Células do Pulmão/enzimologia , Carcinoma de Pequenas Células do Pulmão/patologia , Resultado do Tratamento
5.
Sci China Life Sci ; 63(3): 332-342, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32060861

RESUMO

Cellular senescence (CS) is a state of stable cell cycle arrest characterized by the production and secretion of inflammatory molecules. Early studies described oncogene-induced senescence (OIS) as a barrier to tumorigenesis, such that the therapeutic induction of CS might represent a rational anti-cancer strategy. Indeed, the validity of this approach has been borne out by the development and approval of the cyclin-dependent kinase (CDK) inhibitor palbociclib for the treatment of breast cancer. Apart from tumors, senescent cells have also been shown to accumulate during natural mammalian aging, where they produce detrimental effects on the physiology of surrounding tissues. Thus, pharmacological senescent cell depletion has been proposed as an approach to delay age-related functional decline; this has been formally demonstrated in animal models. In this review article, we describe the current mechanistic understanding of cellular senescence at the molecular level and how it informs the development of new therapeutic strategies to combat cancer and aging.


Assuntos
Envelhecimento/efeitos dos fármacos , Antineoplásicos/farmacologia , Senescência Celular/efeitos dos fármacos , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Animais , Antineoplásicos/metabolismo , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Descoberta de Drogas , Feminino , Humanos , Piperazinas/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Piridinas/metabolismo
6.
Nat Commun ; 11(1): 1032, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098961

RESUMO

The AMP-activated protein kinase (AMPK) is a master sensor of the cellular energy status that is crucial for the adaptive response to limited energy availability. AMPK is implicated in the regulation of many cellular processes, including autophagy. However, the precise mechanisms by which AMPK controls these processes and the identities of relevant substrates are not fully understood. Using protein microarrays, we identify Cyclin Y as an AMPK substrate that is phosphorylated at Serine 326 (S326) both in vitro and in cells. Phosphorylation of Cyclin Y at S326 promotes its interaction with the Cyclin-dependent kinase 16 (CDK16), thereby stimulating its catalytic activity. When expressed in cells, Cyclin Y/CDK16 is sufficient to promote autophagy. Moreover, Cyclin Y/CDK16 is necessary for efficient AMPK-dependent activation of autophagy. This functional interaction is mediated by AMPK phosphorylating S326 of Cyclin Y. Collectively, we define Cyclin Y/CDK16 as downstream effector of AMPK for inducing autophagy.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Análise Serial de Proteínas , Pironas/farmacologia , Serina/metabolismo , Tiofenos/farmacologia
7.
Nat Commun ; 11(1): 740, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029739

RESUMO

Primary and acquired drug resistance imposes a major threat to achieving optimized clinical outcomes during cancer treatment. Aberrant changes in epigenetic modifications are closely involved in drug resistance of tumor cells. Using BET inhibitor (BETi) resistant leukemia cells as a model system, we demonstrated herein that genome-wide enhancer remodeling played a pivotal role in driving therapeutic resistance via compensational re-expression of pro-survival genes. Capitalizing on the CRISPR interference technology, we identified the second intron of IncRNA, PVT1, as a unique bona fide gained enhancer that restored MYC transcription independent of BRD4 recruitment in leukemia. A combined BETi and CDK7 inhibitor treatment abolished MYC transcription by impeding RNAPII loading without affecting PVT1-mediated chromatin looping at the MYC locus in BETi-resistant leukemia cells. Together, our findings have established the feasibility of targeting enhancer plasticity to overcome drug resistance associated with epigenetic therapies.


Assuntos
Leucemia Experimental/tratamento farmacológico , Leucemia Experimental/genética , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Elementos Facilitadores Genéticos , Feminino , Genes myc/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Humanos , Células Jurkat , Células K562 , Leucemia Experimental/metabolismo , Camundongos , Modelos Genéticos , Fenilenodiaminas/administração & dosagem , Pirimidinas/administração & dosagem , RNA Polimerase II/metabolismo , RNA Longo não Codificante/genética
8.
J Enzyme Inhib Med Chem ; 35(1): 414-423, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31899991

RESUMO

A novel series of cyclin-dependent kinases (CDKs) inhibitors, which play critical roles in the cell cycle control and regulation of cell transcription, were synthesised. A systematic study of enzymatic and cellular assays led to the identification of compound X22 with a nanomolar potency against CDK4 and CDK9 and potent antiproliferative activities against a panel of tumour cell lines. X22 could induce cell cycle arrest and cell apoptosis in cancer cell lines. X22 dose-dependently inhibits signalling pathways downstream of CDKs in cancer cells. In vivo antitumor activity assays, oral administration of X22 led to significant tumour regression in mouse model without obvious toxicity. Superior anti-cancer efficacy in vitro and in vivo of X22 demonstrated combined depletion of cell cycle and transcriptional CDK all contributed to antitumor activity. Taken together, concomitant inhibition of cell cycle and transcriptional CDK activities provided valuable guide for further structural optimisation.


Assuntos
Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Transcrição Genética/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/enzimologia , Neoplasias/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Gynecol Oncol ; 156(1): 211-221, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31776040

RESUMO

OBJECTIVE: Cyclin-dependent kinase 7 (CDK7) engages tumor growth by acting as a direct link between the regulation of transcription and the cell cycle. Here, we investigated the clinical significance of CDK7 expression and its potential as a therapeutic target in epithelial ovarian cancer (EOC). METHODS: CDK7 expression was examined in 436 ovarian tissues including normal to metastatic ovarian tumors using immunohistochemistry, and its clinical implications were analyzed. Furthermore, we performed in vitro and in vivo experiments using CDK7 siRNA or a covalent CDK7 inhibitor (THZ1) to elucidate the effect of CDK7 inhibition on tumorigenesis in EOC cells. RESULTS: The patient incidence of high CDK7 expression (CDK7High) gradually increased from normal ovarian epithelium to EOC (P < 0.001). Moreover, CDK7High was associated with an advanced stage and high-grade histology (P = 0.035 and P = 0.011, respectively) in EOC patients and had an independent prognostic significance in EOC recurrence (P = 0.034). CDK7 inhibition with siRNA or THZ1 decreased cell proliferation and migration, and increased apoptosis in EOC cells, and this anti-cancer mechanism is caused by G0/G1 cell cycle arrest. In in vivo therapeutic experiments using cell-line xenograft and PDX models, CDK7 inhibition significantly decreased the tumor weight, which was mediated by cell proliferation and apoptosis. CONCLUSION: Mechanistic interrogation of CDK7 revealed that it is significantly associated with an aggressive phenotype of EOC, and it has independent prognostic power for EOC recurrence. Furthermore, CDK7 may be a potential therapeutic target for patients with EOC, whether platinum sensitive or resistant.


Assuntos
Carcinoma Epitelial do Ovário/enzimologia , Quinases Ciclina-Dependentes/biossíntese , Neoplasias Ovarianas/enzimologia , Animais , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/biossíntese , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Feminino , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/enzimologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Fenilenodiaminas/farmacologia , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia
10.
Artigo em Inglês | MEDLINE | ID: mdl-31678512

RESUMO

Lipid homeostasis allows cells to adjust membrane biophysical properties in response to changes in environmental conditions. In the yeast Saccharomyces cerevisiae, a downward shift in temperature from an optimal reduces membrane fluidity, which triggers a lipid remodeling of the plasma membrane. How changes in membrane fluidity are perceived, and how the abundance and composition of different lipid classes is properly balanced, remain largely unknown. Here, we show that the levels of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], the most abundant plasma membrane phosphoinositide, drop rapidly in response to a downward shift in temperature. This change triggers a signaling cascade transmitted to cytosolic diphosphoinositol phosphate derivatives, among them 5-PP-IP4 and 1-IP7, that exert regulatory functions on genes involved in the inositol and phospholipids (PLs) metabolism, and inhibit the activity of the protein kinase Pho85. Consistent with this, cold exposure triggers a specific program of neutral lipids and PLs changes. Furthermore, we identified Pho85 as playing a key role in controlling the synthesis of long-chain bases (LCBs) via the Ypk1-Orm2 regulatory circuit. We conclude that Pho85 orchestrates a coordinated response of lipid metabolic pathways that ensure yeast thermal adaptation.


Assuntos
Aclimatação/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Metabolismo dos Lipídeos/fisiologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Membrana Celular/metabolismo , Temperatura Baixa/efeitos adversos , Regulação Fúngica da Expressão Gênica/fisiologia , Quinase 3 da Glicogênio Sintase/metabolismo , Fluidez de Membrana/fisiologia , Redes e Vias Metabólicas/fisiologia , Transdução de Sinais/fisiologia
11.
Cell Mol Life Sci ; 77(2): 231-242, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31420702

RESUMO

Regulation of cell division is orchestrated by cyclins, which bind and activate their catalytic workmates, the cyclin-dependent kinases (CDKs). Cyclins have been traditionally defined by an oscillating (cyclic) pattern of expression and by the presence of a characteristic "cyclin box" that determines binding to the CDKs. Noteworthy, the Human Genome Sequence Project unveiled the existence of several other proteins containing the "cyclin box" domain. These potential "cyclins" have been named new, orphan or atypical, creating a conundrum in cyclins nomenclature. Moreover, although many years have passed after their discovery, the scarcity of information regarding these possible members of the family has hampered the establishment of criteria for systematization. Here, we discuss the criteria that define cyclins and we propose a classification and nomenclature update based on structural features, interactors, and phylogenetic information. The application of these criteria allows to systematically define, for the first time, the subfamily of atypical cyclins and enables the use of a common nomenclature for this extended family.


Assuntos
Ciclinas/genética , Animais , Divisão Celular/genética , Quinases Ciclina-Dependentes/genética , Genoma Humano/genética , Humanos , Filogenia
12.
Oncogene ; 39(1): 50-63, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31462705

RESUMO

Resistance of breast cancer to human epidermal growth factor receptor 2 (HER2) inhibitors involves reprogramming of the kinome through HER2/HER3 signaling via the activation of multiple tyrosine kinases and transcriptional upregulation. The heterogeneity of induced kinases prevents kinase targeting by a single kinase inhibitor and presents a major challenge to the treatment of therapeutically recalcitrant HER2-positive breast cancers (HER2+ BCs). As a result, there is a critical need for effective treatment that attacks the aberrant kinome activation associated with resistance to HER2-targeted therapy. Here, we describe a novel treatment strategy that targets cyclin-dependent kinase 7 (CDK7) in HER2 inhibitor-resistant (HER2iR) breast cancer. We show that both HER2 inhibitor-sensitive (HER2iS) and HER2iR breast cancer cell lines exhibit high sensitivity to THZ1, a newly identified covalent inhibitor of the transcription regulatory kinase CDK7. CDK7 promotes cell cycle progression through inhibition of transcription, rather than via direct phosphorylation of classical CDK targets. The transcriptional kinase activity of CDK7 is regulated by HER2, and by the receptor tyrosine kinases activated in response to HER2 inhibition, as well as by the downstream SHP2 and PI3K/AKT pathways. A low dose of THZ1 displayed potent synergy with the HER2 inhibitor lapatinib in HER2iR BC cells in vitro. Dual HER2 and CDK7 inhibition induced tumor regression in two HER2iR BC xenograft models in vivo. Our data support the utilization of CDK7 inhibition as an additional therapeutic avenue that blocks the activation of genes engaged by multiple HER2iR kinases.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Quinases Ciclina-Dependentes/genética , Fenilenodiaminas/farmacologia , Pirimidinas/farmacologia , Receptor ErbB-2/genética , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lapatinib/farmacologia , Proteína Oncogênica v-akt/genética , Fosfatidilinositol 3-Quinases/genética , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Receptor ErbB-2/antagonistas & inibidores , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética
13.
Genes Dev ; 34(1-2): 53-71, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31857346

RESUMO

Hippo signaling controls organ size and tumor progression through a conserved pathway leading to nuclear translocation of the transcriptional effector Yki/Yap/Taz. Most of our understanding of Hippo signaling pertains to its cytoplasmic regulation, but how the pathway is controlled in the nucleus remains poorly understood. Here we uncover an evolutionarily conserved mechanism by which CDK7 promotes Yki/Yap/Taz stabilization in the nucleus to sustain Hippo pathway outputs. We found that a modular E3 ubiquitin ligase complex CRL4DCAF12 binds and targets Yki/Yap/Taz for ubiquitination and degradation, whereas CDK7 phosphorylates Yki/Yap/Taz at S169/S128/S90 to inhibit CRL4DCAF12 recruitment, leading to Yki/Yap/Taz stabilization. As a consequence, inactivation of CDK7 reduced organ size and inhibited tumor growth, which could be reversed by restoring Yki/Yap activity. Our study identifies an unanticipated layer of Hippo pathway regulation, defines a novel mechanism by which CDK7 regulates tissue growth, and implies CDK7 as a drug target for Yap/Taz-driven cancer.


Assuntos
Carcinogênese/genética , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Animais , Antineoplásicos/farmacologia , Carcinogênese/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Quinases Ciclina-Dependentes/genética , Drosophila melanogaster/genética , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/fisiopatologia , Camundongos , Tamanho do Órgão/genética , Fenilenodiaminas/farmacologia , Proteólise , Pirimidinas/farmacologia
14.
Cancer Lett ; 469: 510-523, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31765738

RESUMO

The bromodomain and extra-terminal domain protein BRD4 has been recognized as a key oncogenic driver and a druggable target against cancer. However, these BRD4 inhibitors as monotherapy were moderate in efficacy in preclinical models. Here we utilized a small-scale drug synergy screen that combined the BRD4 inhibitor (JQ1) with 8 epigenetic or transcriptional targeted chemicals and identified THZ1 (a CDK7 inhibitor) acting synergistically with JQ1 against head neck squamous cell carcinoma (HNSCC). Combinational JQ1 and THZ1 treatment impaired cell proliferation, induced apoptosis and senescence, which were largely recapitulated by dual BRD4 and CDK7 knockdown. Combinational treatment inhibited tumor growth and progression in 4NQO-induced HNSCC and xenograft animal models. RNA-sequencing analyses identified hundreds of differentially expressed genes modulated by JQ1 and THZ1, which were significantly enriched in categories including cell cycle and apoptosis. Mechanistically, combinational treatment reduced H3K27ac enrichment in the super-enhancer region of YAP1, which inactivated its transcription and in turn induced anti-proliferative and pro-apoptotic effects. Combined BRD4 and CDK7 upregulation associated with worst prognosis in HNSCC patients. Collectively, our findings reveal a novel therapeutic strategy of pharmacological inhibitions of BRD4 and CDK7 against HNSCC.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Fatores de Transcrição/antagonistas & inibidores , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Azepinas/farmacologia , Proteínas de Ciclo Celular/genética , Quinases Ciclina-Dependentes/genética , Sinergismo Farmacológico , Humanos , Camundongos , Fenilenodiaminas/farmacologia , Pirimidinas/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Fatores de Transcrição/genética , Triazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Biomed Pharmacother ; 121: 109640, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31810114

RESUMO

Pulmonary artery smooth muscle cell (PASMC) proliferation contributes to pulmonary vascular remodeling, which ultimately leads to pulmonary arterial hypertension (PAH). Osthole has been previously shown to inhibit tumor cell growth. Our previous experiments demonstrated that osthole could prevent monocrotaline-induced PAH and pulmonary artery remodeling in rats and that its effects might be associated with inhibiting PASMC proliferation. However, the exact mechanism remains unclear. In this study, we observed the inhibitory effect of osthole on platelet-derived growth factor (PDGF)-BB-induced rat PASMC growth, cell cycle progression and proliferating cell nuclear antigen (PCNA) expression, as measured by CCK-8 assay, flow cytometric analysis and western blotting, respectively. We also detected the expression and activities of the cell cycle regulators cyclin D1/CDK4, cyclin E1/CDK2, p53, p27 and p21 and the TGF-ß1/Smad/p38 signaling pathways in rat PASMCs by western blotting. Our results show that osthole effectively suppressed PDGF-BB-stimulated proliferation, PCNA protein expression, and cell cycle progression in rat PASMCs in vitro. We further demonstrated that treatment with osthole significantly induced cell cycle arrest at the G0/G1 phase in PASMCs, which was supported by the finding that osthole significantly decreased cyclin D1/CDK4 and cyclin E1/CDK2 protein levels and increased p53, p27 and p21 protein levels. These effects may partly be attributed to the downregulation of TGF-ß1/Smad/p38 signaling pathway activation. Our findings suggest that osthole is a potential therapeutic candidate that warrants further investigation regarding its potential use for the treatment of PAH.


Assuntos
Cumarínicos/farmacologia , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/citologia , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Becaplermina/farmacologia , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo
16.
Cancer Genomics Proteomics ; 17(1): 91-100, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31882554

RESUMO

BACKGROUND/AIM: Circulating mRNA can be a useful source of cancer biomarkers. We took advantage of direct transcriptomic analysis in plasma RNA to identify novel mRNA markers for non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Plasma RNA from NSCLC patients and healthy individuals was profiled with cDNA-mediated annealing, selection, extension and ligation (DASL) microarrays. The microarray results were further validated in plasma RNA. RESULTS: Through RNA profiling and online database mining, four gene transcripts were filtered as candidate markers of NSCLC. After validation, the PCTAIRE-1 transcript was identified as a circulating mRNA marker. The diagnostic potential of PCTAIRE-1 was evaluated by receiver operating characteristic curve analysis, which gave a sensitivity and specificity of 60% and 85%, respectively. In addition, high plasma PCTK1 levels were also correlated with poor progression-free survival (p=0.008). CONCLUSION: Circulating mRNA can be profiled with the DASL assay. From the profile, PCTAIRE-1 RNA in the plasma we discovered as a novel diagnostic/prognostic biomarker and an indicator of poor survival in NSCLC patients.


Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Quinases Ciclina-Dependentes/sangue , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/diagnóstico , RNA Mensageiro/sangue , Transcriptoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Estudos de Casos e Controles , Quinases Ciclina-Dependentes/genética , Feminino , Seguimentos , Humanos , Biópsia Líquida , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/genética , Curva ROC , Taxa de Sobrevida
17.
Int J Cancer ; 146(2): 577-588, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31271443

RESUMO

The Mediator complex is a transcriptional regulator interacting with transcription factors and RNA-polymerase-II. Recently, we identified its subunit CDK19 to be specifically expressed in prostate cancer (PCa) and to be functionally implicated in PCa aggressiveness. Aim of our study was to comprehensively characterize the protein expression of CDK19 and its paralog CDK8 in PCa. We performed immunohistochemistry (IHC) for CDK19/CDK8 on a large cohort including needle biopsies from 202 patients, 799 primary tumor foci of radical prostatectomy specimens from 415 patients, 120 locally advanced tumor foci obtained by palliative transurethral resection, 140 lymph node metastases, 67 distant metastases and 82 benigns. Primary tumors were stained for the proliferation marker Ki67, androgen receptor (AR) and ERG. For 376 patients, clinic-pathologic data were available. Primary endpoint was disease-recurrence-free survival (DFS). Nuclear CDK19 and CDK8 expression increases during progression showing the highest intensity in metastatic and castration-resistant tumors. High CDK19 expression on primary tumors correlates with DFS independently from Gleason grade and PSA. Five-year-DFS rates of patients with primary tumors expressing no, moderate and high CDK19 are 73.7, 56.9 and 30.4%, respectively. CDK19 correlates with Gleason grade, T-stage, Ki67 proliferation-index, nuclear AR expression and ERG-status. Therapeutic options for metastatic and castration-resistant PCa remain limited. In the current study, we confirmed an important role of the Mediator subunit CDK19 in advanced PCa supporting current developments to target CDK19 and its paralog CDK8. Furthermore, CDK19 protein expression has the potential to predict disease recurrence independently from established biomarkers thus contributing to individual management for PCa patients.


Assuntos
Biomarcadores Tumorais/metabolismo , Quinase 8 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Recidiva Local de Neoplasia/diagnóstico , Neoplasias da Próstata/patologia , Biópsia , Núcleo Celular/metabolismo , Progressão da Doença , Intervalo Livre de Doença , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Masculino , Gradação de Tumores , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Prognóstico , Próstata/patologia , Próstata/cirurgia , Prostatectomia , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/cirurgia
18.
Phytochemistry ; 169: 112165, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31610323

RESUMO

Cyclin dependent kinase A; 1 (CDKA; 1) is essential in G1/S transition of cell cycle and its oxidation has been implicated in cell cycle arrest during plant abiotic stress. In the present study, an evaluation at the molecular level was performed to find possible sites of protein oxidative modifications. In vivo studies demonstrated that carbonylation of maize CDKA,1 is associated with a decrease in complex formation with maize cyclin D (CycD). Control and in vitro oxidized recombinant CDKA; 1 were sequenced by mass spectrometry. Proline at the PSTAIRE cyclin-binding motif was identified as the most susceptible oxidation site by comparative analysis of the resulted peptides. The specific interaction between CDKA; 1 and CycD6; 1, measured by surface plasmon resonance (SPR), demonstrated that the affinity and the kinetic of the interaction depended on the reduced-oxidized state of the CDKA; 1. CDKA; 1 protein oxidative modification would be in part responsible for affecting cell cycle progression, and thus producing plant growth inhibition under oxidative stress.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Prolina/metabolismo , Zea mays/enzimologia , Sequência de Aminoácidos , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Ciclinas/química , Modelos Moleculares , Oxirredução , Prolina/química , Alinhamento de Sequência
19.
Surgery ; 167(1): 80-86, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31648931

RESUMO

BACKGROUND: The cyclin-dependent-kinase inhibitor/retinoblastoma pathway has been implicated in sporadic medullary thyroid carcinoma tumorigenesis. Somatic CDKN2C loss has been associated with decreased overall survival in medullary thyroid carcinoma patients. We evaluated CDKN2C loss in a prospective clinical environment using a novel Clinical Laboratory Improvement Amendments-certified assay to confirm its association with aggressive disease and to interrogate response to targeted therapy. METHODS: Patients with advanced sporadic medullary thyroid carcinoma underwent tumor genotyping for the purpose of management of targeted therapy and prognostication. RESULTS: Of patients with informative CDKN2C assay results, 30 (51.8%) were haploinsufficient/1N and 28 (48.3%) were 2N. Forty patients (69.0%) had a somatic RET mutation, and 36.9% had alterations of both genes. Thirty patients (51.7%) were treated with systemic therapy. Presence of genetic alterations in CDKN2C or RET did not predict treatment response. Patients with 1N CDKN2C loss had significantly shorter time-to-distant-metastasis than patients with normal copy number (P = .03). CONCLUSION: This is the first evaluation in the clinical setting of CDKN2C haploinsufficiency in sporadic medullary thyroid carcinoma. Although a larger cohort and longer follow-up will be required, loss seems to be associated with more aggressive disease and may indicate patients that might receive benefit from treatment with a CDK inhibitor.


Assuntos
Carcinoma Neuroendócrino/tratamento farmacológico , Inibidor de Quinase Dependente de Ciclina p18/genética , Quinases Ciclina-Dependentes/antagonistas & inibidores , Técnicas de Genotipagem/métodos , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias da Glândula Tireoide/tratamento farmacológico , Adulto , Idoso , Carcinoma Neuroendócrino/genética , Carcinoma Neuroendócrino/patologia , Feminino , Seguimentos , Técnicas de Genotipagem/instrumentação , Haploinsuficiência , Humanos , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Prognóstico , Intervalo Livre de Progressão , Estudos Prospectivos , Inibidores de Proteínas Quinases/farmacologia , Estudos Retrospectivos , Medição de Risco/métodos , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Adulto Jovem
20.
Eur J Med Chem ; 187: 111952, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31846828

RESUMO

Cyclin-dependent kinase (CDK) family members are promising molecular targets in discovering potent inhibitors in disease settings, they function differentially. CDK2, CDK4 and CDK6, directly regulate the cell cycle, while CDK9 primarily modulates the transcription regulation. In discovering inhibitors of these CDKs, toxicity associated with off-target effect on other CDK homologs often posts as a clinical issue and hinders their further therapeutic development. To improve efficacy and reduce toxicity, here, using the Proteolysis Targeted Chimeras (PROTACs) approach, we design and further optimize small molecule degraders targeting multiple CDKs. We showed that heterobifunctional compound A9 selectively degraded CDK2. We also identified a dual-degrader, compound F3, which potently induced degradation of both CDK2 (DC50: 62 nM) and CDK9 (DC50: 33 nM). In human prostate cancer PC-3 cells, compound F3 potently inhibits cell proliferation by effectively blocking the cell cycle in S and G2/M phases. Our preliminary data suggests that PROTAC-oriented CDK2/9 degradation is potentially an effective therapeutic approach.


Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteólise/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , Relação Dose-Resposta a Droga , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Células MCF-7 , Modelos Moleculares , Estrutura Molecular , Células PC-3 , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas
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