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1.
Proc Natl Acad Sci U S A ; 119(36): e2205608119, 2022 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-36037385

RESUMO

Cop9 signalosome (CSN) regulates the function of cullin-RING E3 ubiquitin ligases (CRLs) by deconjugating the ubiquitin-like protein NEDD8 from the cullin subunit. To understand the physiological impact of CSN function on the CRL network and cell proliferation, we combined quantitative mass spectrometry and genome-wide CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screens to identify factors that modulate cell viability upon inhibition of CSN by the small molecule CSN5i-3. CRL components and regulators strongly modulated the antiproliferative effects of CSN5i-3, and in addition we found two pathways involved in genome integrity, SCFFBXO5-APC/C-GMNN and CUL4DTL-SETD8, that contribute substantially to the toxicity of CSN inhibition. Our data highlight the importance of CSN-mediated NEDD8 deconjugation and adaptive exchange of CRL substrate receptors in sustaining CRL function and suggest approaches for leveraging CSN inhibition for the treatment of cancer.


Assuntos
Replicação do DNA , Ubiquitina-Proteína Ligases , Azepinas/metabolismo , Complexo do Signalossomo COP9/antagonistas & inibidores , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/metabolismo , Sobrevivência Celular , Proteínas Culina/genética , Proteínas Culina/metabolismo , Imidazóis/metabolismo , Proteína NEDD8/metabolismo , Pirazóis/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
2.
Cell Rep ; 34(10): 108822, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33691110

RESUMO

MED1 (mediator subunit 1) co-amplifies with HER2, but its role in HER2-driven mammary tumorigenesis is still unknown. Here, we generate MED1 mammary-specific overexpression mice and cross them with mouse mammary tumor virus (MMTV)-HER2 mice. We observe significantly promoted onset, growth, metastasis, and multiplicity of HER2 tumors by MED1 overexpression. Further studies reveal critical roles for MED1 in epithelial-mesenchymal transition, cancer stem cell formation, and response to anti-HER2 therapy. Mechanistically, RNA sequencing (RNA-seq) transcriptome analyses and clinical sample correlation studies identify Jab1, a component of the COP9 signalosome complex, as the key direct target gene of MED1 contributing to these processes. Further studies reveal that Jab1 can also reciprocally regulate the stability and transcriptional activity of MED1. Together, our findings support a functional cooperation between these co-amplified genes in HER2+ mammary tumorigenesis and their potential usage as therapeutic targets for the treatment of HER2+ breast cancers.


Assuntos
Neoplasias Mamárias Experimentais/patologia , Subunidade 1 do Complexo Mediador/metabolismo , Receptor ErbB-2/metabolismo , Animais , Antineoplásicos/uso terapêutico , Complexo do Signalossomo COP9/antagonistas & inibidores , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/metabolismo , Movimento Celular , Transição Epitelial-Mesenquimal , Feminino , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Lapatinib/uso terapêutico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Vírus do Tumor Mamário do Camundongo/genética , Subunidade 1 do Complexo Mediador/genética , Camundongos , Camundongos Nus , Metástase Neoplásica , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Fenótipo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ativação Transcricional
3.
BMC Mol Cell Biol ; 21(1): 36, 2020 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-32384871

RESUMO

BACKGROUND: The SMC5/6 complex, cohesin and condensin are the three mammalian members of the structural maintenance of chromosomes (SMC) family, large ring-like protein complexes that are essential for genome maintenance. The SMC5/6 complex is the least characterized complex in mammals; however, it is known to be involved in homologous recombination repair (HRR) and chromosome segregation. RESULTS: In this study, a yeast two-hybrid screen was used to help elucidate novel interactions of the kleisin subunit of the SMC5/6 complex, NSMCE4A. This approach discovered an interaction between NSMCE4A and GPS1, a COP9 signalosome (CSN) component, and this interaction was further confirmed by co-immunoprecipitation. Additionally, GPS1 and components of SMC5/6 complex colocalize during interphase and mitosis. CSN is a cullin deNEDDylase and is an important factor for HRR. Depletion of GPS1, which has been shown to negatively impact DNA end resection during HRR, caused an increase in SMC5/6 levels at sites of laser-induced DNA damage. Furthermore, inhibition of the dennedylation function of CSN increased SMC5/6 levels at sites of laser-induced DNA damage. CONCLUSION: Taken together, these data demonstrate for the first time that the SMC5/6 and CSN complexes interact and provides evidence that the CSN complex influences SMC5/6 functions during cell cycle progression and response to DNA damage.


Assuntos
Complexo do Signalossomo COP9/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Complexo do Signalossomo COP9/antagonistas & inibidores , Complexo do Signalossomo COP9/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Citoplasma/metabolismo , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Reparo do DNA , Humanos , Interfase/genética , Mitose/genética , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno , Técnicas do Sistema de Duplo-Híbrido
4.
Exp Cell Res ; 391(1): 112004, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32289284

RESUMO

Deubiquitinase (DUB) can reverse the ubiquitin signal, and participate in virtually all aspects of cancer progression. Thus, DUB represents an attractive target for development of anticancer drugs. However, little is known about DUB which can be used as drug targets. Here, we found that the constitutive photomorphogenic 9 (COP9) signalosome complex subunit 6 (COPS6/CSN6), a DUB belongs to JAMM/MPN domain-associated metallopeptidases(JAMMs) class, was highly expressed in pancreatic adenocarcinoma(PAAD) tissues. High expression of CSN6 was associated with tumor TNM stage and metastasis in PAAD patients. Moreover, we demonstrated that CSN6 promoted invasion and metastasis through regulating forkhead box protein A1 (FOXA1) in PAAD cells. Re-expression of FOXA1 rescued the decreased invasion and metastasis caused by CSN6 knockdown, whereas inhibition of FOXA1 alleviated the pro-metastasis effect induced by CSN6 overexpression. Further, CSN6 regulated the expression of FOXA1 via c-Fos in PAAD cells. Mechanistically, CSN6 stabilized c-Fos protein by binding to it and decreasing its ubiquitination. Our work identified CSN6 as a targeting-permissible deubiquitinase, and CSN6 inhibition maybe a potential treatment strategy for PAAD.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma/genética , Complexo do Signalossomo COP9/genética , Regulação Neoplásica da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Idoso , Complexo do Signalossomo COP9/antagonistas & inibidores , Complexo do Signalossomo COP9/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Fator 3-alfa Nuclear de Hepatócito/antagonistas & inibidores , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Leupeptinas/farmacologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Cell Prolif ; 52(5): e12654, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31222857

RESUMO

OBJECTIVES: Despite of the aberrant expression of 14-3-3ζ in head and neck squamous cell carcinoma (HNSCC), little is known about the role of 14-3-3ζ in the regulation of senescence in HNSCC. This study was performed to investigate whether 14-3-3ζ is implicated in senescence evasion of Hep-2 laryngeal cancer cells. METHODS: The expression of 14-3-3ζ was suppressed using RNA interference strategy. Senescence induction was determined by senescence-associated ß-galactosidase staining and the numbers of promyelocytic leukaemia nuclear body. Real-time PCR, western blotting and immunohistochemistry were applied for the expression of corresponding proteins. Xenograft experiment was performed to show in vivo effect of 14-3-3ζ silencing on tumour growth. RESULTS: 14-3-3ζ silencing significantly induced senescence phenotypes via 27 accumulations. Subsequently, we demonstrated that p27 accumulation is linked to inactivation of SCFSkp2 complex activity, probably due to the deneddylation of cullin-1 (Cul-1) as follows. (a) Neddylated Cul-1 is decreased by 14-3-3ζ silencing. (b) Blocking neddylation using MLN4924 reproduces senescence phenotypes. (c) Knockdown of CSN5, which functions as a deneddylase, was shown to restore the senescence phenotypes induced by 14-3-3ζ depletion. Finally, we demonstrated that 14-3-3ζ depletion effectively hindered the proliferation of Hep-2 cells implanted into nude mice. CONCLUSION: 14-3-3ζ negatively regulates senescence in Hep-2 cells, suggesting that 14-3-3ζ targeting may serve to suppress the expansion of laryngeal cancer via induction of senescence through the Cul-1/SCFSkp2 /p27 axis.


Assuntos
Proteínas 14-3-3/metabolismo , Proteínas Culina/metabolismo , Proteínas F-Box/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas 14-3-3/antagonistas & inibidores , Proteínas 14-3-3/genética , Animais , Complexo do Signalossomo COP9/antagonistas & inibidores , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Senescência Celular , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Laríngeas/metabolismo , Neoplasias Laríngeas/patologia , Masculino , Camundongos , Camundongos Nus , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Quinases Associadas a Fase S/genética
6.
Leukemia ; 33(1): 171-180, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30026574

RESUMO

Immunomodulatory drugs (IMiDs) including lenalidomide and pomalidomide bind cereblon (CRBN) and activate the CRL4CRBN ubiquitin ligase to trigger proteasomal degradation of the essential transcription factors IKZF1 and IKZF3 and multiple myeloma (MM) cytotoxicity. We have shown that CRBN is also targeted for degradation by SCFFbxo7 ubiquitin ligase. In the current study, we explored the mechanisms underlying sensitivity of MM cells to IMiDs using genome-wide CRISPR-Cas9 screening. We validate that CSN9 signalosome complex, a deactivator of Cullin-RING ubiquitin ligase, inhibits SCFFbxo7 E3 ligase-mediated CRBN degradation, thereby conferring sensitivity to IMiDs; conversely, loss of function of CSN9 signalosome activates SCFFbxo7 complex, thereby enhancing degradation of CRBN and conferring IMiD resistance. Finally, we show that pretreatment with either proteasome inhibitors or NEDD8 activating enzyme (NAE) inhibitors can abrogate degradation and maintain levels of CRBN, thereby enhancing sensitivity to IMiDs. These studies therefore demonstrate that CSN9 signalosome complex regulates sensitivity to IMiDs by modulating CRBN expression.


Assuntos
Complexo do Signalossomo COP9/metabolismo , Sistemas CRISPR-Cas , Fator de Transcrição Ikaros/metabolismo , Fatores Imunológicos/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Peptídeo Hidrolases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Bortezomib/farmacologia , Complexo do Signalossomo COP9/antagonistas & inibidores , Complexo do Signalossomo COP9/genética , Ciclopentanos/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Fator de Transcrição Ikaros/genética , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Peptídeo Hidrolases/genética , Prognóstico , Proteólise , Pirimidinas/farmacologia , Células Tumorais Cultivadas , Ubiquitina-Proteína Ligases , Ubiquitinação
7.
Cancer Res Treat ; 51(3): 886-900, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30282449

RESUMO

PURPOSE: Jab1 is a coactivator of c-Jun that enhances the transcriptional function of c-Jun. Jab1 is frequently overexpressed in various cancers and is associatedwith poor prognosis of cancer patients. Thus, Jab1 could be a potential therapeutic target in cancer. However, the role of Jab1 in biliary tract cancer (BTC) has not been studied. MATERIALS AND METHODS: We performed in vitro and in vivo experiments to evaluate the therapeutic potential ofJab1 inhibition in BTC. RESULTS: Among 8 BTC cell lines, many showed higher Jab1 expression levels. In addition, Jab1 silencing by siRNA increased p27 expression levels. SNU478 and HuCCT-1 cells exhibited profound Jab1 knockdown and increased p27 expression by Jab1-specific siRNA transfection. Jab1 silencing induced anti-proliferative and anti-migratory effects and resulted in G1 cell cycle arrest in SNU478 and HuCCT-1 cells. In addition, Jab1 silencing potentiated the anti-proliferative and anti-migratory effects of cisplatin by increasing DNA damage. Interestingly,Jab1 knockdown increased PTEN protein half-life, resulting in increased PTEN expression. In the HuCCT-1 mouse xenograft model, stable knockdown of Jab1 by shRNA also showed anti-proliferative effects in vivo, with decreased Ki-67 expression and AKT phosphorylation and increased Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and p27 expression. CONCLUSION: Jab1 knockdown demonstrated anti-proliferative and anti-migratory effects in BTC cells by increasing DNA damage and stabilizing PTEN, resulting in G1 cell cycle arrest. In addition, Jab1 silencing potentiated the anti-proliferative effects of cisplatin. Our data suggest that Jab1 may be a potential therapeutic target in BTC that is worthy of further investigations.


Assuntos
Antineoplásicos/administração & dosagem , Neoplasias do Sistema Biliar/metabolismo , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/metabolismo , Cisplatino/administração & dosagem , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Animais , Antineoplásicos/farmacologia , Neoplasias do Sistema Biliar/tratamento farmacológico , Neoplasias do Sistema Biliar/genética , Complexo do Signalossomo COP9/antagonistas & inibidores , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Camundongos , PTEN Fosfo-Hidrolase/química , PTEN Fosfo-Hidrolase/metabolismo , Estabilidade Proteica/efeitos dos fármacos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacologia , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Exp Cell Res ; 371(2): 379-388, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30180991

RESUMO

Up-regulation of mammalian COP9 signalosome subunit 6 (CSN6) and consequent reduction of SCF ubiquitin ligase substrate receptor ß-transduction repeat-containing protein (ß-TrCP) have been shown to be associated with cancer cells proliferation. However, it is unclear whether CSN6 and ß-TrCP are also involved in PDGF-induced pulmonary arterial smooth muscle cells (PASMCs) proliferation. This study aims to address this issue and further explore its potential mechanisms. Our results indicated that PDGF phosphorylated Akt, stimulated PASMCs proliferation; while inhibition of PDGF receptor (PDGFR) by imatinib prevented these effects. PDGF further up-regulated CSN6 protein expression, this was accompanied with ß-TrCP reduction and increase of Cdc25A. Inhibition of PDGFR/PI3K/Akt signaling pathway reversed PDGF-induced such changes and cell proliferation. Prior transfection of CSN6 siRNA blocked PDGF-induced ß-TrCP down-regulation, Cdc25A up-regulation and cell proliferation. Furthermore, pre-treatment of cells with MG-132 also abolished PDGF-induced ß-TrCP reduction, Cdc25A elevation and cell proliferation. In addition, pre-depletion of Cdc25A by siRNA transfection suppressed PDGF-induced PASMCs proliferation. Taken together, our study indicates that up-regulation of CSN6 by PDGFR/PI3K/Akt signaling pathway decreases ß-TrCP by increasing its ubiquitinated degradation, and thereby increases the expression of Cdc25A, which promotes PDGF-induced PASMCs proliferation.


Assuntos
Complexo do Signalossomo COP9/genética , Miócitos de Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Animais , Complexo do Signalossomo COP9/antagonistas & inibidores , Complexo do Signalossomo COP9/metabolismo , Proliferação de Células/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Regulação da Expressão Gênica , Mesilato de Imatinib/farmacologia , Leupeptinas/farmacologia , Masculino , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/metabolismo , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Proteínas Contendo Repetições de beta-Transducina/genética , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismo
9.
Cell Signal ; 49: 79-86, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29885455

RESUMO

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. It has been previously reported that CSN5 depletion is an effective method in human HCC. In the current study, we aimed to uncover gene signatures and key pathways during HCC. Gene expression profiles of GSE26485 were downloaded from GEO database. Totally, 101 differentially expressed genes (DEGs) were up-regulated and 146 ones were down-regulated. Biological processes (BP) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) analysis showed that the DEGs were mainly enriched in regulation of cell growth, oxidation-reduction process, mitotic cytokinesis, negative regulation of macroautophagy, endosome organization, lysosome, biosynthesis of antibiotics, small cell lung cancer and glutathione metabolism and so on (P < 0.05). Protein-protein interaction (PPI) network, Kaplan-Meier, log-rank method, western blot, immunohistochemistry and encyclopedia of DNA elements (ENCODE) analysis showed that CSN5 depletion took effects through down-regulation of SMAD5-related pathways which include EXO1, CENPA and NCAPG, resulting in the inactivation of H3K4me3 and H3K36me3. Those genes represent the promising targets for therapeutic intervention in HCC patients.


Assuntos
Complexo do Signalossomo COP9/genética , Biologia Computacional/métodos , Redes Reguladoras de Genes , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeo Hidrolases/genética , Complexo do Signalossomo COP9/antagonistas & inibidores , Complexo do Signalossomo COP9/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Peptídeo Hidrolases/metabolismo , Regiões Promotoras Genéticas , Mapas de Interação de Proteínas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/genética , Transcriptoma
10.
Cell Signal ; 34: 38-46, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28229932

RESUMO

The COP9 signalosome (CSN) is a multi-protein complex that is highly conserved in eukaryotes. Due to its regulatory impact on processes such as cell cycle, DNA damage response and apoptosis, the CSN is essential for mammalian cells. One of the best-studied functions of the CSN is the deNEDDylation of cullin-RING ligases (CRLs) via its catalytically active subunit CSN5/JAB1, thereby triggering the degradation of various target proteins. CSN5 was found to be overexpressed in many human cancer entities, including colon adenocarcinoma. Overactivation of WNT signaling is known as a key step in colon cancer development. Recently, we found that depletion of CSN5 in colorectal cancer (CRC) cells affects WNT signaling by downregulation of ß-catenin. To investigate changes in gene expression associated with the CSN5 knockdown, we performed a microarray using cDNA from the CRC cell line SW480. We found the WNT ligand WNT6 and the WNT inhibitors DKK1 and DKK4 differentially regulated in CSN5 knockdown cells. DKK1 expression and DKK1 protein levels depended on CSN5 in different CRC cell lines. In addition, DKK1 secretion was increased following CSN5 knockdown, affecting WNT signaling in SW480 cells. Consequently, blocking of secreted DKK1 in cell-conditioned media abolished ß-catenin downregulation in SW480 cells, while treatment with recombinant DKK1 mimicked the CSN5 knockdown effect. Furthermore, knockdown of DKK1 was able to rescue the proliferative deficiency of CSN5 knockdown cells. We conclude that downregulation of WNT signaling in colorectal cancer cells resulting from CSN5 knockdown is mediated, at least in part, by elevated DKK1 secretion. Moreover, experiments with the NEDDylation inhibitor MLN-4924 indicated that DKK1 expression is regulated by a so far unidentified repressor, the stability of which could be controlled by a CSN-regulated CRL.


Assuntos
Complexo do Signalossomo COP9/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeo Hidrolases/metabolismo , Complexo do Signalossomo COP9/antagonistas & inibidores , Complexo do Signalossomo COP9/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ciclopentanos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeo Hidrolases/genética , Pirimidinas/farmacologia , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo
11.
Angew Chem Int Ed Engl ; 56(5): 1294-1297, 2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-27981705

RESUMO

CSN5 is the zinc metalloprotease subunit of the COP9 signalosome (CSN), which is an important regulator of cullin-RING E3 ubiquitin ligases (CRLs). CSN5 is responsible for the cleavage of NEDD8 from CRLs, and blocking deconjugation of NEDD8 traps the CRLs in a hyperactive state, thereby leading to auto-ubiquitination and ultimately degradation of the substrate recognition subunits. Herein, we describe the discovery of azaindoles as a new class of CSN5 inhibitors, which interact with the active-site zinc ion of CSN5 through an unprecedented binding mode. The best compounds inhibited CSN5 with nanomolar potency, led to degradation of the substrate recognition subunit Skp2 in cells, and reduced the viability of HCT116 cells.


Assuntos
Complexo do Signalossomo COP9/antagonistas & inibidores , Indóis/metabolismo , Zinco/metabolismo , Sítios de Ligação , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/metabolismo , Domínio Catalítico , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Células HCT116 , Humanos , Indóis/química , Indóis/farmacologia , Simulação de Acoplamento Molecular , Proteína NEDD8/química , Proteína NEDD8/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Quinases Associadas a Fase S/química , Proteínas Quinases Associadas a Fase S/metabolismo , Zinco/química
12.
Nat Commun ; 7: 13166, 2016 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-27774986

RESUMO

The COP9 signalosome (CSN) is a central component of the activation and remodelling cycle of cullin-RING E3 ubiquitin ligases (CRLs), the largest enzyme family of the ubiquitin-proteasome system in humans. CRLs are implicated in the regulation of numerous cellular processes, including cell cycle progression and apoptosis, and aberrant CRL activity is frequently associated with cancer. Remodelling of CRLs is initiated by CSN-catalysed cleavage of the ubiquitin-like activator NEDD8 from CRLs. Here we describe CSN5i-3, a potent, selective and orally available inhibitor of CSN5, the proteolytic subunit of CSN. The compound traps CRLs in the neddylated state, which leads to inactivation of a subset of CRLs by inducing degradation of their substrate recognition module. CSN5i-3 differentially affects the viability of tumour cell lines and suppresses growth of a human xenograft in mice. Our results provide insights into how CSN regulates CRLs and suggest that CSN5 inhibition has potential for anti-tumour therapy.


Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Complexo do Signalossomo COP9/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Pirazóis/farmacologia , Ubiquitina-Proteína Ligases/genética , Animais , Antineoplásicos/síntese química , Azepinas/síntese química , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/metabolismo , Feminino , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma Anaplásico de Células Grandes/patologia , Camundongos , Camundongos SCID , Terapia de Alvo Molecular , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise/efeitos dos fármacos , Pirazóis/síntese química , Células THP-1 , Carga Tumoral/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Sci Rep ; 6: 26804, 2016 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-27226076

RESUMO

The COP9 signalosome has been implicated in pluripotency maintenance of human embryonic stem cells. Yet, the mechanism for the COP9 signalosome to regulate pluripotency remains elusive. Through knocking down individual COP9 subunits, we demonstrate that Cops2, but not the whole COP9 signalosome, is essential for pluripotency maintenance in mouse embryonic stem cells. Down-regulation of Cops2 leads to reduced expression of pluripotency genes, slower proliferation rate, G2/M cell cycle arrest, and compromised embryoid differentiation of embryonic stem cells. Cops2 also facilitates somatic cell reprogramming. We further show that Cops2 binds to Nanog protein and prevent the degradation of Nanog by proteasome. Moreover, Cops2 functions as transcriptional corepressor to facilitate pluripotency maintenance. Altogether, our data reveal the essential role and novel mechanisms of Cops2 in pluripotency maintenance.


Assuntos
Complexo do Signalossomo COP9/metabolismo , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteína Homeobox Nanog/metabolismo , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/genética , Sequência de Aminoácidos , Animais , Complexo do Signalossomo COP9/antagonistas & inibidores , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/fisiologia , Autorrenovação Celular , Técnicas de Reprogramação Celular , Corpos Embrioides , Células-Tronco Embrionárias/metabolismo , Técnicas de Silenciamento de Genes , Camundongos , Proteína Homeobox Nanog/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Fator 3 de Transcrição de Octâmero/antagonistas & inibidores , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/fisiologia , Estabilidade Proteica , Proteólise , Interferência de RNA , RNA Interferente Pequeno/genética , Fatores de Transcrição SOXC/antagonistas & inibidores , Fatores de Transcrição SOXC/genética , Fatores de Transcrição SOXC/fisiologia , Ovinos/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética
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