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1.
Gene ; 766: 145163, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32980450

RESUMO

BACKGROUND: Cardia adenocarcinoma (CA) is a distinct form of gastric cancer, and the optimal means of treating it remains controversial. At present, the role of the condensation complex gene non-SMC condensin I complex subunit G (NCAPG) in CA is uncertain, and as such the present study was designed to elucidate its importance in this oncogenic context. METHODS: We first used bioinformatics approaches to assess NCAPG expression profiles in CA using public databases. Protein profiling was also used to examine the expression of this protein in CA tumors and adjacent tissues from 20 patients. Then the expression of NCAPG in CA samples was quantified via qRT-PCR and Western blotting. NCAPG knockdown and overexpression in the SGC-7901 and AGS cell lines were subsequently performed, after which the expression of key proteins associated with epithelial-mesenchymal transition (EMT; E-cadherin, vimentin, N-cadherin, Snail, Slug) and the regulation of apoptotic responses (caspase-3, Bax, Bcl-2) was measured. The mechanistic role of NCAPG in CA was additionally studied by analyzing proteins associated with Wnt/ß-catenin signaling including Wnt1, phosphorylated GSK3ß, ß-catenin, and phosphorylated ß- catenin. The impact of NCAPG on the migration, survival, and invasion of CA cells was further examined. RESULTS: CA samples exhibited high NCAPG expression. When this gene was overexpressed in cell lines, it reduced caspase-3, Bax, and E-cadherin levels whereas it elevated Bcl-2, vimentin, N-cadherin, Snail, and Slug levels. NCAPG overexpression also resulted in the enhanced expression of Wnt1, phosphorylated GSK3ß, and total ß-catenin and the reduced expression of phosphorylated ß-catenin. The knockdown of NCAPG, in contrast, yielded the opposite phenotype. At a functional level, the overexpression of NCAPG improved the apoptotic resistance of CA cells while driving them to undergo EMT and to become more invasive and migratory. CONCLUSIONS: NCAPG overexpression can promote EMT and suppress tumor cell apoptosis via the activation of Wnt/ß-catenin signaling.


Assuntos
Adenocarcinoma/genética , Apoptose/genética , Cárdia/patologia , Proteínas de Ciclo Celular/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias Gástricas/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Oncogenes/genética , Neoplasias Gástricas/patologia , Vimentina/genética
2.
Mol Cell ; 80(3): 377-378, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33157011

RESUMO

Li et al. (2020) elucidate the resistance mechanisms to small-molecule inhibitors targeting the G2/M cell cycle checkpoint kinase, CHK1, in a variety of non-small cell lung cancer cell lines using CRISPR-mediated genetic approaches and identify biomarkers of response.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Fosforilação , Proteínas Quinases/metabolismo
3.
Nat Commun ; 11(1): 5320, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087730

RESUMO

MicroRNAs (miRNAs) are endogenous small RNAs of ∼21 nt that regulate multiple biological pathways in multicellular organisms. They derive from longer transcripts that harbor an imperfect stem-loop structure. In plants, the ribonuclease type III DICER-LIKE1 assisted by accessory proteins cleaves the precursor to release the mature miRNA. Numerous studies highlight the role of the precursor secondary structure during plant miRNA biogenesis; however, little is known about the relevance of the precursor sequence. Here, we analyzed the sequence composition of plant miRNA primary transcripts and found specifically located sequence biases. We show that changes in the identity of specific nucleotides can increase or abolish miRNA biogenesis. Most conspicuously, our analysis revealed that the identity of the nucleotides at unpaired positions of the precursor plays a crucial role during miRNA biogenesis in Arabidopsis.


Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , MicroRNAs/biossíntese , MicroRNAs/genética , RNA de Plantas/biossíntese , RNA de Plantas/genética , Proteínas de Arabidopsis/metabolismo , Pareamento Incorreto de Bases , Proteínas de Ciclo Celular/metabolismo , Magnoliopsida/genética , Magnoliopsida/metabolismo , MicroRNAs/química , MicroRNAs/metabolismo , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , Processamento Pós-Transcricional do RNA , RNA de Plantas/química , Ribonuclease III/metabolismo
4.
Anticancer Res ; 40(11): 6265-6271, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33109564

RESUMO

BACKGROUND/AIM: Colorectal cancer (CRC) is one of the most common malignant tumors in the world. This study aimed to investigate the anticancer effect of the combination treatment of Ribociclib (LEE011) and 5-Fluorouracil (5-FU) on CRC cells. MATERIALS AND METHODS: HT-29 and SW480 cells were treated with LEE011, 5-FU, or the combination of LEE011 and 5-FU. Cell viability and cycle were investigated through 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and flow cytometry. The expression of cell cycle-related proteins was determined through western blot. RESULTS: The combined treatment of LEE011 with 5-FU synergistically reduced cell viability in HT-29 and SW480 cells. Specifically, it induced cell cycle arrest at the G1 phase, down-regulated the phosphorylation of retinoblastoma protein and the expression of p53. CONCLUSION: LEE011 exhibited potential as an effective therapeutic inhibitor for the combination treatment of CRC patients.


Assuntos
Aminopiridinas/farmacologia , Neoplasias Colorretais/patologia , Fluoruracila/farmacologia , Purinas/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos
5.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(10): 1422-1431, 2020 Oct 30.
Artigo em Chinês | MEDLINE | ID: mdl-33118511

RESUMO

OBJECTIVE: To screen the key genes related to the prognosis of lung adenocarcinoma through big data analysis and explore their clinical value and potential mechanism. METHODS: We analyzed GSE18842, GSE27262, and GSE33532 gene expression profile data obtained from the Gene Expression Omnibus (GEO). Bioinformatics methods were used to screen the differentially expressed genes in lung adenocarcinoma tissues and KEGG and GO enrichment analysis was performed, followed by PPI interaction network analysis, module analysis, differential expression analysis, and prognosis analysis. The expressions of MAD2L1 and TTK by immunohistochemistry were verified in 35 non-small cell lung cancer specimens and paired adjacent tissues. RESULTS: We identified a total of 256 genes that showed significant differential expressions in lung adenocarcinoma, including 66 up-regulated and 190 down-regulated genes. Thirty-two up-regulated core genes were screened by functional analysis, and among them 29 were shown to significantly correlate with a poor prognosis of patients with lung adenocarcinoma. All the 29 genes were highly expressed in lung adenocarcinoma tissues compared with normal lung tissues and were mainly enriched in cell cycle pathways. Seven of these key genes were closely related to the spindle assembly checkpoint (SAC) complex and responsible for regulating cell behavior in G2/M phase. We selected SAC-related proteins TTK and MAD2L1 to test their expressions in clinical tumor samples, and detected their overexpression in lung adenocarcinoma tissues as compared with the adjacent tissues. CONCLUSIONS: Seven SAC complex-related genes, including TTK and MAD2L1, are overexpressed in lung adenocarcinoma tissues with close correlation with the prognosis of the patients.


Assuntos
Adenocarcinoma de Pulmão , Proteínas de Ciclo Celular/genética , Neoplasias Pulmonares , Proteínas Mad2/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Adenocarcinoma de Pulmão/genética , Big Data , Biologia Computacional , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Pontos de Checagem da Fase M do Ciclo Celular
6.
Nucleic Acids Res ; 48(19): 10924-10939, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33010171

RESUMO

NBS1 is a critical component of the MRN (MRE11/RAD50/NBS1) complex, which regulates ATM- and ATR-mediated DNA damage response (DDR) pathways. Mutations in NBS1 cause the human genomic instability syndrome Nijmegen Breakage Syndrome (NBS), of which neuronal deficits, including microcephaly and intellectual disability, are classical hallmarks. Given its function in the DDR to ensure proper proliferation and prevent death of replicating cells, NBS1 is essential for life. Here we show that, unexpectedly, Nbs1 deletion is dispensable for postmitotic neurons, but compromises their arborization and migration due to dysregulated Notch signaling. We find that Nbs1 interacts with NICD-RBPJ, the effector of Notch signaling, and inhibits Notch activity. Genetic ablation or pharmaceutical inhibition of Notch signaling rescues the maturation and migration defects of Nbs1-deficient neurons in vitro and in vivo. Upregulation of Notch by Nbs1 deletion is independent of the key DDR downstream effector p53 and inactivation of each MRN component produces a different pattern of Notch activity and distinct neuronal defects. These data indicate that neuronal defects and aberrant Notch activity in Nbs1-deficient cells are unlikely to be a direct consequence of loss of MRN-mediated DDR function. This study discloses a novel function of NBS1 in crosstalk with the Notch pathway in neuron development.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neurogênese , Neurônios/metabolismo , Receptores Notch/metabolismo , Hidrolases Anidrido Ácido/metabolismo , Animais , Células Cultivadas , Dano ao DNA , Reparo do DNA , Embrião de Mamíferos , Fibroblastos , Proteína Homóloga a MRE11/metabolismo , Camundongos , Neurônios/citologia
7.
Nucleic Acids Res ; 48(19): 11097-11112, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33035348

RESUMO

The microprocessor complex cleaves the primary transcript of microRNA (pri-miRNA) to initiate miRNA maturation. Microprocessor is known to consist of RNase III DROSHA and dsRNA-binding DGCR8. Here, we identify Enhancer of Rudimentary Homolog (ERH) as a new component of Microprocessor. Through a crystal structure and biochemical experiments, we reveal that ERH uses its hydrophobic groove to bind to a conserved region in the N-terminus of DGCR8, in a 2:2 stoichiometry. Knock-down of ERH or deletion of the DGCR8 N-terminus results in a reduced processing of suboptimal pri-miRNAs in polycistronic miRNA clusters. ERH increases the processing of suboptimal pri-miR-451 in a manner dependent on its neighboring pri-miR-144. Thus, the ERH dimer may mediate 'cluster assistance' in which Microprocessor is loaded onto a poor substrate with help from a high-affinity substrate in the same cluster. Our study reveals a role of ERH in the miRNA biogenesis pathway.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Células HCT116 , Células HEK293 , Humanos , Células K562 , MicroRNAs/metabolismo , Ligação Proteica , Conformação Proteica
8.
Nat Commun ; 11(1): 5455, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-33116140

RESUMO

The expansion of the white adipose tissue (WAT) in obesity goes along with increased mechanical, metabolic and inflammatory stress. How adipocytes resist this stress is still poorly understood. Both in human and mouse adipocytes, the transcriptional co-activators YAP/TAZ and YAP/TAZ target genes become activated during obesity. When fed a high-fat diet (HFD), mice lacking YAP/TAZ in white adipocytes develop severe lipodystrophy with adipocyte cell death. The pro-apoptotic factor BIM, which is downregulated in adipocytes of obese mice and humans, is strongly upregulated in YAP/TAZ-deficient adipocytes under HFD, and suppression of BIM expression reduces adipocyte apoptosis. In differentiated adipocytes, TNFα and IL-1ß promote YAP/TAZ nuclear translocation via activation of RhoA-mediated actomyosin contractility and increase YAP/TAZ-mediated transcriptional regulation by activation of c-Jun N-terminal kinase (JNK) and AP-1. Our data indicate that the YAP/TAZ signaling pathway may be a target to control adipocyte cell death and compensatory adipogenesis during obesity.


Assuntos
Adipócitos Brancos/metabolismo , Adipócitos Brancos/patologia , Obesidade/metabolismo , Obesidade/patologia , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adipogenia , Animais , Proteína 11 Semelhante a Bcl-2/metabolismo , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Morte Celular , Células Cultivadas , Dieta Hiperlipídica , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Transativadores/deficiência , Transativadores/genética , Transativadores/metabolismo
9.
Cell Prolif ; 53(11): e12910, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33047378

RESUMO

OBJECTIVES: The mechanisms responsible for the postnatal loss of mammalian cardiac regenerative capacity are not fully elucidated. The aim of the present study is to investigate the role of progesterone in cardiac regeneration and explore underlying mechanism. MATERIALS AND METHODS: Effect of progesterone on cardiomyocyte proliferation was analysed by immunofluorescent staining. RNA sequencing was performed to screen key target genes of progesterone, and yes-associated protein (YAP) was knocked down to demonstrate its role in pro-proliferative effect of progesterone. Effect of progesterone on activity of YAP promoter was measured by luciferase assay and interaction between progesterone receptor and YAP promoter by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Adult mice were subjected to myocardial infarction, and then, effects of progesterone on adult cardiac regeneration were analysed. RESULTS: Progesterone supplementation enhanced cardiomyocyte proliferation in a progesterone receptor-dependent manner. Progesterone up-regulated YAP expression and knockdown of YAP by small interfering RNA reduced progesterone-mediated cardiomyocyte proliferative effect. Progesterone receptor interacted with the YAP promoter, determined by ChIP and EMSA; progesterone increased luciferase activity of YAP promoter and up-regulated YAP target genes. Progesterone administration also promoted adult cardiomyocyte proliferation and improved cardiac function in myocardial infarction. CONCLUSION: Our data uncover a role of circulating progesterone withdrawal as a novel mechanism for the postnatal loss of mammalian cardiac regenerative potential. Progesterone promotes both neonatal and adult cardiomyocyte proliferation by up-regulating YAP expression.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Cardiotônicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Progesterona/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Cardiotônicos/uso terapêutico , Proteínas de Ciclo Celular/genética , Células Cultivadas , Masculino , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/genética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Progesterona/uso terapêutico , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacos
10.
Int J Nanomedicine ; 15: 8109-8119, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33116521

RESUMO

Background: Cancer is a complex heterogeneous disease to which singular modes of treatment mostly fail to produce a desired therapeutic efficacy. Targeting different cellular pathways using combinational therapies has been gaining popularity in cancer treatment, with the added benefit of reducing dosage and side effects. Methods: A gold nanoparticle-mediated drug delivery nanoplatform was developed for co-delivery of doxorubicin and polo-like kinase 1 (PLK1) siRNA. Gold nanoparticles were coated with polyethyleneimine to facilitate assembly of PLK1 on the surface. Doxorubicin was loaded on nanoparticles through a pH-sensitive linker with a thiol group at one terminal end for controlled release. Results: The therapeutic efficiency of this co-delivery system was evaluated in 2D and 3D cultured systems. The reduced IC50 value clearly demonstrated the synergistic effect of combined drug and gene delivery over their individual delivery in a cancer treatment model. Conclusion: This study may provide an adaptable, facile platform to investigate drug-siRNA combinations for cancer inhibition.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias da Mama/terapia , Portadores de Fármacos/química , Terapia Genética/métodos , Ouro/química , Nanopartículas Metálicas/química , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/genética , Terapia Combinada , Doxorrubicina/química , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Humanos , Concentração de Íons de Hidrogênio , Polietilenoimina/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética
11.
Nat Commun ; 11(1): 5111, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037216

RESUMO

The nascent polypeptide exit tunnel (NPET) is a major functional center of 60S ribosomal subunits. However, little is known about how the NPET is constructed during ribosome assembly. We utilized molecular genetics, biochemistry, and cryo-electron microscopy (cryo-EM) to investigate the functions of two NPET-associated proteins, ribosomal protein uL4 and assembly factor Nog1, in NPET assembly. Structures of mutant pre-ribosomes lacking the tunnel domain of uL4 reveal a misassembled NPET, including an aberrantly flexible ribosomal RNA helix 74, resulting in at least three different blocks in 60S assembly. Structures of pre-ribosomes lacking the C-terminal extension of Nog1 demonstrate that this extension scaffolds the tunnel domain of uL4 in the NPET to help maintain stability in the core of pre-60S subunits. Our data reveal that uL4 and Nog1 work together in the maturation of ribosomal RNA helix 74, which is required to ensure proper construction of the NPET and 60S ribosomal subunits.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Modelos Moleculares , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Domínios Proteicos , Estabilidade de RNA , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
12.
PLoS One ; 15(10): e0226464, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33035223

RESUMO

Metaplastic breast carcinoma (MBC) is a clinically aggressive and rare subtype of breast cancer, with similar features to basal-like breast cancers. Due to rapid growth rates and characteristic heterogeneity, MBC is often unresponsive to standard chemotherapies; and novel targeted therapeutic discovery is urgently needed. Histone deacetylase inhibitors (DACi) suppress tumor growth and metastasis through regulation of the epithelial-to-mesenchymal transition axis in various cancers, including basal-like breast cancers. We utilized a new MBC patient-derived xenograft (PDX) to examine the effect of DACi therapy on MBC. Cell morphology, cell cycle-associated gene expressions, transwell migration, and metastasis were evaluated in patient-derived cells and tumors after treatment with romidepsin and panobinostat. Derivations of our PDX model, including cells, spheres, organoids, explants, and in vivo implanted tumors were treated. Finally, we tested the effects of combining DACi with approved chemotherapeutics on relative cell biomass. DACi significantly suppressed the total number of lung metastasis in vivo using our PDX model, suggesting a role for DACi in preventing circulating tumor cells from seeding distal tissue sites. These data were supported by our findings that DACi reduced cell migration, populations, and expression of mesenchymal-associated genes. While DACi treatment did affect cell cycle-regulating genes in vitro, tumor growth was not affected compared to controls. Importantly, gene expression results varied depending on the cellular or tumor system used, emphasizing the importance of using multiple derivations of cancer models in preclinical therapeutic discovery research. Furthermore, DACi sensitized and produced a synergistic effect with approved oncology therapeutics on inherently resistant MBC. This study introduced a role for DACi in suppressing the migratory and mesenchymal phenotype of MBC cells through regulation of the epithelial-mesenchymal transition axis and suppression of the CTC population. Preliminary evidence that DACi treatment in combination with MEK1/2 inhibitors exerts a synergistic effect on MBC cells was also demonstrated.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Depsipeptídeos/administração & dosagem , Inibidores de Histona Desacetilases/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Panobinostat/administração & dosagem , Animais , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Depsipeptídeos/farmacologia , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Neoplasias Pulmonares/genética , Camundongos , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/efeitos dos fármacos , Panobinostat/farmacologia , Modelagem Computacional Específica para o Paciente , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia
13.
PLoS One ; 15(10): e0235103, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33075068

RESUMO

PCNA sliding clamp binds factors through which histone deposition, chromatin remodeling, and DNA repair are coupled to DNA replication. PCNA also directly binds Eco1/Ctf7 acetyltransferase, which in turn activates cohesins and establishes cohesion between nascent sister chromatids. While increased recruitment thus explains the mechanism through which elevated levels of chromatin-bound PCNA rescue eco1 mutant cell growth, the mechanism through which PCNA instead worsens cohesin mutant cell growth remains unknown. Possibilities include that elevated levels of long-lived chromatin-bound PCNA reduce either cohesin deposition onto DNA or cohesin acetylation. Instead, our results reveal that PCNA increases the levels of both chromatin-bound cohesin and cohesin acetylation. Beyond sister chromatid cohesion, PCNA also plays a critical role in genomic stability such that high levels of chromatin-bound PCNA elevate genotoxic sensitivities and recombination rates. At a relatively modest increase of chromatin-bound PCNA, however, fork stability and progression appear normal in wildtype cells. Our results reveal that even a moderate increase of PCNA indeed sensitizes cohesin mutant cells to DNA damaging agents and in a process that involves the DNA damage response kinase Mec1(ATR), but not Tel1(ATM). These and other findings suggest that PCNA mis-regulation results in genome instabilities that normally are resolved by cohesin. Elevating levels of chromatin-bound PCNA may thus help target cohesinopathic cells linked that are linked to cancer.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , DNA Fúngico/genética , Instabilidade Genômica , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Acetilação , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
14.
Nat Commun ; 11(1): 5495, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33127907

RESUMO

Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances its stability, thereby aiding TIM localization to replication forks and the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ciclo Celular/genética , Quinase 1 do Ponto de Checagem/metabolismo , Estruturas Cromossômicas/metabolismo , Dano ao DNA , Reparo do DNA , Replicação do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Instabilidade Genômica/fisiologia , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Domínios Proteicos
15.
Mol Cell ; 79(6): 1008-1023.e4, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32871104

RESUMO

TMPRSS2-ERG gene fusion occurs in approximately 50% of cases of prostate cancer (PCa), and the fusion product is a key driver of prostate oncogenesis. However, how to leverage cellular signaling to ablate TMPRSS2-ERG oncoprotein for PCa treatment remains elusive. Here, we demonstrate that DNA damage induces proteasomal degradation of wild-type ERG and TMPRSS2-ERG oncoprotein through ERG threonine-187 and tyrosine-190 phosphorylation mediated by GSK3ß and WEE1, respectively. The dual phosphorylation triggers ERG recognition and degradation by the E3 ubiquitin ligase FBW7 in a manner independent of a canonical degron. DNA damage-induced TMPRSS2-ERG degradation was abolished by cancer-associated PTEN deletion or GSK3ß inactivation. Blockade of DNA damage-induced TMPRSS2-ERG oncoprotein degradation causes chemotherapy-resistant growth of fusion-positive PCa cells in culture and in mice. Our findings uncover a previously unrecognized TMPRSS2-ERG protein destruction mechanism and demonstrate that intact PTEN and GSK3ß signaling are essential for effective targeting of ERG protein by genotoxic therapeutics in fusion-positive PCa.


Assuntos
Proteínas de Ciclo Celular/genética , Glicogênio Sintase Quinase 3 beta/genética , Proteínas de Fusão Oncogênica/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/genética , Proteínas Tirosina Quinases/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Tratamento Farmacológico , Proteína 7 com Repetições F-Box-WD/genética , Xenoenxertos , Humanos , Masculino , Camundongos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
16.
Anticancer Res ; 40(9): 4961-4968, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878784

RESUMO

BACKGROUND/AIM: Despite advances in treatment modalities, the visual prognosis of retinoblastoma still remains unsatisfactory, underscoring the need to develop novel therapeutic approaches. MATERIALS AND METHODS: The effect on the growth of six human retinoblastoma cell lines and a normal human fibroblast cell line of CEP1347, a small-molecule kinase inhibitor originally developed for the treatment of Parkinson's disease and therefore with a known safety profile in humans, was examined. The role of the P53 pathway in CEP1347-induced growth inhibition was also investigated. RESULTS: CEP1347 selectively inhibited the growth of retinoblastoma cell lines expressing murine double minute 4 (MDM4), a P53 inhibitor. Furthermore, CEP1347 reduced the expression of MDM4 and activated the P53 pathway in MDM4-expressing retinoblastoma cells, which was required for the inhibition of their growth by CEP1347. CONCLUSION: We propose CEP1347 as a promising candidate for the treatment of retinoblastomas, where functional inactivation of P53 as a result of MDM4 activation is reportedly common.


Assuntos
Antineoplásicos/farmacologia , Carbazóis/farmacologia , Retinoblastoma/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reposicionamento de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Retinoblastoma/metabolismo , Retinoblastoma/patologia
17.
Biol Res ; 53(1): 41, 2020 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958054

RESUMO

BACKGROUND: Tumor angiogenesis is an essential event for tumor growth and metastasis. It has been showed that REC8, a component of the meiotic cohesion complex, played a vital role in Epithelial-Mesenchymal Transition (EMT) in gastric cancer. However, the role of REC8 in gastric cancer angiogenesis remains to be identified. RESULTS: Inhibition of REC8 expression in gastric cancer cells contributed to tumor angiogenesis in the gastric cancer microenvironment. The clinical analysis demonstrated that the loss of REC8 in gastric cancer with enrichment of MVD. Depletion of REC8 expression in gastric cancer cells significantly increased tube formation of human umbilical vein endothelial cells (HUVECs), which is attributed to enhancement of vascular endothelial growth factor (VEGF) secretion caused by REC8 slicing. While addition of neutralizing antibody targeted VEGF into supernatant drastically reversed the effect of REC8 loss in gastric cancer cells on tube formation. Mechanistic analyses indicated that ablation of REC8 promotes nuclear factor-κB (NF-κB) p65 activity and its downstream gene VEGF expression, leading to tube formation. CONCLUSIONS: These results demonstrated a novel REC8 function that suppressed tumor angiogenesis and progression by attenuation of VEGF in gastric cancer microenvironment.


Assuntos
Proteínas de Ciclo Celular/genética , NF-kappa B/genética , Neovascularização Patológica/genética , Neoplasias Gástricas/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Linhagem Celular Tumoral , Células Endoteliais da Veia Umbilical Humana , Humanos , Neoplasias Gástricas/irrigação sanguínea , Microambiente Tumoral
18.
Trends Pharmacol Sci ; 41(10): 684-686, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32893006

RESUMO

In a recent study, Saraswat and colleagues identified a novel proteolysis targeting chimera (PROTAC), ARV-825 (ARV), that efficiently degrades bromodomain-containing protein 4 (BRD4) to drug the 'undruggable' MYC in pancreatic cancer. ARV-loaded polyethylene glycol-poly lactic acid-co-glycolic acid (PLGA-PEG) polymeric nanoparticles (ARV-NPs) showed promising anticancer activity in both 2D cell culture and 3D multicellular tumor spheroid models of pancreatic cancer. This study demonstrates a unique therapeutic strategy in which targeting BRD4 for degradation via the E3 ubiquitin ligase cereblon (CRBN) pathway leads to sustained inhibition of oncogenic MYC expression for effective treatment of pancreatic cancer.


Assuntos
Proteínas Nucleares , Neoplasias Pancreáticas , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quimera/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Peptídeo Hidrolases , Proteólise , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases
19.
Nat Commun ; 11(1): 4676, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938922

RESUMO

Translation efficiency varies considerably between different mRNAs, thereby impacting protein expression. Translation of the stress response master-regulator ATF4 increases upon stress, but the molecular mechanisms are not well understood. We discover here that translation factors DENR, MCTS1 and eIF2D are required to induce ATF4 translation upon stress by promoting translation reinitiation in the ATF4 5'UTR. We find DENR and MCTS1 are only needed for reinitiation after upstream Open Reading Frames (uORFs) containing certain penultimate codons, perhaps because DENR•MCTS1 are needed to evict only certain tRNAs from post-termination 40S ribosomes. This provides a model for how DENR and MCTS1 promote translation reinitiation. Cancer cells, which are exposed to many stresses, require ATF4 for survival and proliferation. We find a strong correlation between DENR•MCTS1 expression and ATF4 activity across cancers. Furthermore, additional oncogenes including a-Raf, c-Raf and Cdk4 have long uORFs and are translated in a DENR•MCTS1 dependent manner.


Assuntos
Fator 4 Ativador da Transcrição/genética , Fatores de Iniciação em Eucariotos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Fator 4 Ativador da Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Códon , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Neoplasias/genética , Proteínas Oncogênicas/genética , Oncogenes , Fases de Leitura Aberta , RNA Mensageiro , RNA de Transferência/genética , RNA de Transferência/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genética
20.
PLoS One ; 15(9): e0237268, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32886660

RESUMO

Endosomal sorting complexes required for transport proteins (ESCRT) catalyze the fission of cellular membranes during budding of membrane away from the cytosol. Here we have used Total Internal Reflection Fluorescence (TIRF) microscopy to visualize the recruitment of ESCRTs specifically, ALIX, CHMP4b and VPS4 onto the budding HIV Gag virus-like particles (VLPs). We imaged the budding VLPs with 200 millisecond time resolution for 300 frames. Our data shows three phases for ESCRT dynamics: 1) recruitment in which subunits of ALIX, CHMP4b and VPS4 are recruited with constant proportions on the budding sites of HIV Gag virus like particles for nearly 10 seconds, followed by 2) disassembly of ALIX and CHMP4b while VPS4 signal remains constant for nearly 20 seconds followed by 3) disassembly of VPS4. We hypothesized that the disassembly observed in step 2 was catalyzed by VPS4 and powered by ATP hydrolysis. To test this hypothesis, we performed ATP depletion using (-) glucose medium, deoxyglucose and oligomycin. Imaging ATP depleted cells, we show that the disassembly of CHMP4b and ALIX observed in step 2 is ATP dependent. ATP depletion resulted in the recruitment of approximately 2-fold as many subunits of all ESCRTs. Resuming ATP production in cells, resulted in disassembly of the full ESCRT machinery which had been locked in place during ATP depletion. With some caveats, our experiments provide insight into the formation of the ESCRT machinery at the budding site of HIV during budding.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , HIV-1/fisiologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Liberação de Vírus , Células HEK293 , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Células HeLa , Humanos , Ligação Proteica , Vírion/fisiologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
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