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1.
Nat Commun ; 10(1): 5770, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852899

RESUMO

Autophagy is an essential cellular process affecting virus infections and other diseases and Beclin1 (BECN1) is one of its key regulators. Here, we identified S-phase kinase-associated protein 2 (SKP2) as E3 ligase that executes lysine-48-linked poly-ubiquitination of BECN1, thus promoting its proteasomal degradation. SKP2 activity is regulated by phosphorylation in a hetero-complex involving FKBP51, PHLPP, AKT1, and BECN1. Genetic or pharmacological inhibition of SKP2 decreases BECN1 ubiquitination, decreases BECN1 degradation and enhances autophagic flux. Middle East respiratory syndrome coronavirus (MERS-CoV) multiplication results in reduced BECN1 levels and blocks the fusion of autophagosomes and lysosomes. Inhibitors of SKP2 not only enhance autophagy but also reduce the replication of MERS-CoV up to 28,000-fold. The SKP2-BECN1 link constitutes a promising target for host-directed antiviral drugs and possibly other autophagy-sensitive conditions.


Assuntos
Autofagia/imunologia , Proteína Beclina-1/metabolismo , Infecções por Coronavirus/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Proteínas Quinases Associadas a Fase S/metabolismo , Animais , Autofagia/efeitos dos fármacos , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Proteólise/efeitos dos fármacos , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Proteínas Quinases Associadas a Fase S/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/imunologia , Células Vero
2.
Biomed Res Int ; 2019: 9648269, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31534970

RESUMO

Background/Aims: The molecular mechanism of dormancy initiation of cancer stem cells (CSCs) is not clear. This study was to explore the molecular mechanism by which CSCs switch from mitotic division to quiescence. Methods: MTT assays, flow cytometry, Western blotting, qRT-PCR, and immunofluorescence staining were used to test cell viability, cell cycle and expression of F-box and WD repeat domain-containing 7 (Fbxw7), c-myc, S phase kinase associated protein-2 (Skp2), cyclin-dependent kinase inhibitor 1B (p27), octamer-binding transcription factor 3/4 (Oct3/4), and ß catenin gene in 5-fluorouracil (5-FU)-treated A549 cells. Lung adenocarcinoma xenograft models were employed to detect the effects of Fbxw7 on tumor growth. Results: 5-FU inhibited the proliferation of A549 cells, with a median inhibitory concentration (IC50) of 200 µg/ml after 24 h treatment. 5-FU treatment increased the expressions of Oct3/4, Fbxw7, and p27 and increased the number of A549 cells at G0/G1. 5-FU treatment triggered nuclear translocation of ß-catenin, decreased the expression levels of c-myc and Skp2, and decreased the number of A549 cells at S phase. Release from 5-FU decreased the expressions of Oct3/4, Fbxw7 and p27; decreased the percentage of cells in the G0/G1 phase; increased the expressions of Skp2 and c-myc; and increased the proportion of cells in S phase. 5-FU treatment led to high expressions of Oct3/4, c-myc, and p27, with low expressions of Fbxw7 and Skp2. Knockdown of Fbxw7 augmented the expression of c-myc and decreased the proportion of A549 cells in Go/G1 phase. Skp2 siRNA increased the expression of p27 and the percentage of G0/G1 phase cells and reduced the proportion of S phase cells. Fbxw7 overexpression inhibited tumor growth in mouse lung adenocarcinoma xenograft models. When Fbxw7 expression was low, Skp2 expression was higher in lung adenocarcinoma tissues and associated with the differentiation of lung adenocarcinoma. Conclusion: 5-FU enriches the CSCs in lung adenocarcinoma cells via increasing Fbxw7 and decreasing Skp2 expression, followed by downregulation of c-myc and upregulation of p27, which switches cells to quiescence.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Proteína 7 com Repetições F-Box-WD/metabolismo , Neoplasias Pulmonares/metabolismo , Mitose , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Células A549 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Animais , Proteína 7 com Repetições F-Box-WD/genética , Fluoruracila/farmacologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/patologia , Proteínas Quinases Associadas a Fase S/genética , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Oncogene ; 38(40): 6696-6710, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31391550

RESUMO

Transcription factor PAX8 expression is upregulated in several types of cancers. However, little is known about the function of PAX8 in the progression of hepatoma and its regulatory mechanisms. Here, we show that PAX8 silencing inhibits the proliferation and clonogenicity of hepatoma cells and its growth in vivo. The HBV X protein (HBx) does not directly interacts, but stabilizes PAX8 by inhibiting proteasome-dependent ubiquitination and degradation. Furthermore, the E3 ubiquitin ligase complex component Skp2 through its LRR domain directly interacts with the Prd domain of PAX8 and targets PAX8 by recognizing its lysine 275 for ubiquitination and degradation in hepatoma cells. In addition, HBx directly interacts and is colocalized with Skp2 to inhibit its recognition and subsequent ubiquitination and degradation of PAX8 in hepatoma cells. Moreover, HBx upregulates the expression and phosphorylation of Aurora A, a serine-threonine kinase, which interacts with and phosphorylates PAX8 at S209 and T277, compromising the Skp2-recognized PAX8 ubiquitination and destabilization. Thus, HBx stabilizes PAX8 protein by inhibiting the Skp2 targeted PAX8 ubiquitination and enhancing the Aurora A-mediated its phosphorylation, contributing to the progression of hepatoma. Our findings suggest that PAX8 may a new target for design of therapies and uncover new insights into the pathogenesis of hepatoma.


Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Fator de Transcrição PAX8/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Progressão da Doença , Inativação Gênica , Células HEK293 , Humanos , Neoplasias Hepáticas/genética , Ligação Proteica , Proteínas Quinases Associadas a Fase S/metabolismo , Transativadores , Ubiquitinação
4.
Int J Mol Sci ; 20(16)2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426282

RESUMO

Though Pyrogallol, one of the natural polyphenols, was known to have anti-inflammatory and antitumor effects in breast and colon cancers, the underlying antitumor mechanisms of Pyrogallol, still remain unclear so far. Here, the antitumor mechanisms of Pyrogallol were elucidated in Hep3B and Huh7 hepatocellular carcinoma cells (HCCs). Pyrogallol showed significant cytotoxicity and reduced the number of colonies in Hep3B and Huh7 cells. Interestingly, Pyrogallol induced S-phase arrest and attenuated the protein expression of CyclinD1, Cyclin E, Cyclin A, c-Myc, S-phase kinase-associated protein 2 (Skp2), p-AKT, PI3K, increased the protein expression of p27, and also reduced the fluorescent expression of Cyclin E in Hep3B and Huh7 cells. Furthermore, Pyrogallol disturbed the interaction between Skp2, p27, and c-Myc in Huh7 cells. Notably, Pyrogallol upregulated miRNA levels of miR-134, and conversely, miR-134 inhibition rescued the decreased expression levels of c-Myc, Cyclin E, and Cyclin D1 and increased the expression of p27 by Pyrogallol in Huh7 cells. Taken together, our findings provide insight that Pyrogallol exerts antitumor effects in HCCs via miR-134 activation-mediated S-phase arrest and inhibition of PI3K/AKT/Skp2/cMyc signaling as a potent anticancer candidate.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , MicroRNAs/genética , Pirogalol/farmacologia , Antioxidantes/farmacologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fase S/efeitos dos fármacos , Proteínas Quinases Associadas a Fase S/metabolismo , Transdução de Sinais/efeitos dos fármacos
5.
Int J Mol Sci ; 20(13)2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31277523

RESUMO

The ubiquitin proteasome 26S system (UPS), involving monomeric and multimeric E3 ligases is one of the most important signaling pathways in many organisms, including plants. The SCF (SKP1/Cullin/F-box) multimeric complex is particularly involved in response to development and stress signaling. The SKP1 protein (S-phase kinase-associated protein 1) is the core subunit of this complex. In this work, we firstly identified 92 and 87 non-redundant Triticum aestivum SKP1-like (TaSKP) genes that were retrieved from the latest release of the wheat genome database (International Wheat Genome Sequencing Consortium (IWGSC) RefSeq v1.0) and the genome annotation of the TGAC v1 respectively. We then investigated the structure, phylogeny, duplication events and expression patterns of the SKP1-like gene family in various tissues and environmental conditions using a wheat expression platform containing public data. TaSKP1-like genes were expressed differentially in response to stress conditions, displaying large genomic variations or short insertions/deletions which suggests functional specialization within TaSKP1-like genes. Finally, interactions between selected wheat FBX (F-box) proteins and putative ancestral TaSKP1-like proteins were tested using the yeast two-hybrid (Y2H) system to examine the molecular interactions. These observations suggested that six Ta-SKP1 genes are likely to be ancestral genes, having similar functions as ASK1 and ASK2 in Arabidopsis, OSK1 and OSK20 in rice and PpSKP1 and PpSKP2 in Physcomitrella patens.


Assuntos
Genes de Plantas , Proteínas Quinases Associadas a Fase S/genética , Triticum/genética , Cromossomos de Plantas/genética , Duplicação Gênica , Regulação da Expressão Gênica de Plantas , Filogenia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Estresse Fisiológico/genética , Triticum/crescimento & desenvolvimento
6.
Zygote ; 27(3): 187-189, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31182173

RESUMO

SummaryWe report here the existence of bands of higher molecular weight after western blot analysis in three proteins - Skp1, p27 and IκBα in bovine preimplantation embryos. This finding is specific to preimplantation embryos (from the 2-cell stage to the blastocyst stage) and not differentiated fibroblast cells in which these bands were of expected molecular weight. We suggest that these bands of higher molecular weight represent a complex of proteins that are characteristic of preimplantation embryos.


Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário , Proteínas/metabolismo , Animais , Blastocisto/citologia , Western Blotting , Bovinos , Inibidor de Quinase Dependente de Ciclina p27/química , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Feminino , Peso Molecular , Inibidor de NF-kappaB alfa/química , Inibidor de NF-kappaB alfa/metabolismo , Proteínas/química , Proteínas Quinases Associadas a Fase S/química , Proteínas Quinases Associadas a Fase S/metabolismo
7.
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
8.
Biomed Res Int ; 2019: 9524561, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31139661

RESUMO

The incidence of spinal cord injury (SCI) continues to increase; however, the involved mechanisms remain unclear. Anaphase promoting complex (APC) and its regulatory subunit Cdh1 play important roles in the growth, development, and repair of the central nervous system (CNS). Cdh1 is involved in the pathophysiological processes of neuronal apoptosis and astrocyte-reactive proliferation after ischemic brain injury, whereas the role played by APC-Cdh1 in the proliferation and activation of oligodendrocyte precursor cells (OPCs) after SCI remains unresolved. Using primary cultures of spinal oligodendrocyte precursor cells, we successfully established an in vitro mechanical stretch injury model to simulate SCI. Cell viability and proliferation were determined by MTT assay and flow cytometric analysis of the cell cycle. Real-time fluorescent quantitative PCR and Western blot analysis determined the mRNA and protein expression levels of Cdh1 and its downstream substrates Skp2 and Id2. Mechanical stretch injury decreased the proliferative activity of OPCs and enhanced cellular Cdh1 expression. Dampened expression of Cdh1 in primary OPCs significantly promoted proliferation and activation of OPCs after SCI. In addition, the expression of the downstream substrates of Cdh1, Skp2, and Id2 was decreased following mechanical injury, whereas adenovirus-mediated Cdh1 RNA interference increased postinjury expression of Skp2 and Id2. These findings suggest that APC-Cdh1 might be involved in regulating the proliferation and activation of OPCs after mechanical SCI. Moreover, degraded ubiquitination of the downstream substrates Skp2 and Id2 might play an important role, at least in part, in the beneficial effects of OPCs activity following SCI.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdh1/metabolismo , Células Precursoras de Oligodendrócitos/metabolismo , Células Precursoras de Oligodendrócitos/patologia , Estresse Mecânico , Adenoviridae/metabolismo , Animais , Proliferação de Células , Vetores Genéticos/metabolismo , Proteína 2 Inibidora de Diferenciação/metabolismo , Ratos Sprague-Dawley , Proteínas Quinases Associadas a Fase S/metabolismo , Especificidade por Substrato
9.
Braz J Med Biol Res ; 52(5): e8412, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31038581

RESUMO

Multiple myeloma (MM) is a malignant neoplasm of plasma, and exhibits several harmful effects including osteolytic injuries, hypercalcemia, and immune dysfunction. Many patients with MM succumb to the underlying malignancy. An S-phase kinase-related protein 2 (Skp2) inhibitor, designated SKPin C1, has been developed and confirmed to have an inhibitory effect on metastatic melanoma cells. This study aimed to determine the effect of SKPin C1 on MM. Normal B lymphocytes, THP-1 cells, and MM U266 and RPMI 8226 cells were exposed to various dosages of SKPin C1 for 48 h. Cell proliferation was determined by MTT, EdU staining, and cell cycle assays. Western blot assays were performed to assess intracellular protein levels of Skp2, p27, and cleaved caspase-3. The amount of ubiquitin attached to p27 was determined using an immunoprecipitation assay. The viability of U266 and RPMI 8226 cells was significantly inhibited by 10 µM SKPin C1 and the inhibitory effect was enhanced with increasing doses of SKPin C1. In contrast, 50 µM SKPin C1 only marginally decreased viability of normal B lymphocytes in 12 h. Skp2 and p27 expression in U266 and RPMI 8226 cells was higher and lower, respectively, than that in the normal B lymphocytes. Treatment with SKPin C1 or Skp2 knockdown increased p27 protein levels in U266 and RPMI 8226 cells by preventing p27 from being ubiquitinated, which slowed the cell cycle, inhibited cell proliferation, and triggered apoptosis. Therefore, this study suggested SKPin C1 as a potent inhibitor against aberrant proliferation and immortalization of MM.


Assuntos
Apoptose , Proliferação de Células/fisiologia , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Mieloma Múltiplo/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/farmacologia , Humanos , Mieloma Múltiplo/fisiopatologia , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação/fisiologia
10.
Cell Commun Signal ; 17(1): 25, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30885218

RESUMO

BACKGROUND: Flavokawain B (FKB) has been identified from kava root extracts as a potent apoptosis inducer for inhibiting the growth of various cancer cell lines, including prostate cancer. However, the molecular targets of FKB in prostate cancer cells remain unknown. METHODS: An in vitro NEDD8 Initiation Conjugation Assay was used to evaluate the neddylation inhibitory activity of FKB. Molecular docking and a cellular thermal shift assay were performed to assess the direct interaction between FKB and the NEDD8 activating enzyme (NAE) complex. Protein neddylation, ubiqutination, stability and expression in cells were assessed with immunoprecipitation and Western blotting methods using specific antibodies. Deletion and site specific mutants and siRNAs were used to evaluate deep mechanisms by which FKB induces Skp2 degradation. Cell growth inhibition and apoptosis induction were measured by MTT, ELISA and Western blotting methods. RESULTS: FKB inhibits NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 neddylation in an in vitro assay. Molecular docking study and a cellular thermal shift assay reveal that FKB interacts with the regulatory subunit (i.e. APP-BP1) of the NAE. In addition, FKB causes Skp2 degradation in an ubiquitin and proteasome dependent manner. Overexpression of dominant-negative cullin1 (1-452), K720R mutant (the neddylation site) Cullin1 or the F-box deleted Skp2 that losses its binding to the Skp1/Cullin1 complex causes the resistance to FKB-induced Skp2 degradation, whereas siRNA knock-down of Cdh1, a known E3 ligase of Skp2 for targeted degradation, didn't attenuate the effect of FKB on Skp2 degradation. These results suggest that degradation of Skp2 by FKB is involved in a functional Cullin1. Furthermore, proteasome inhibitors Bortezomib and MG132 transcriptionally down-regulate the expression of Skp2, and their combinations with FKB result in enhanced inhibitory effects on the growth of prostate cancer cell lines via synergistic down-regulation of Skp2 and up-regulation of p27/Kip1 and p21/WAF1 protein expression. FKB also selectively inhibits the growth of RB deficient cells with high expression of Skp2. CONCLUSION: These findings provide a rationale for further investigating combination of FKB and Bortezomib for treatment of RB deficient, castration-resistant prostate cancer.


Assuntos
Antineoplásicos/farmacologia , Bortezomib/farmacologia , Flavonoides/farmacologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Proteínas Quinases Associadas a Fase S/metabolismo , Antígenos CD/metabolismo , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Bortezomib/uso terapêutico , Caderinas/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteínas Culina/metabolismo , Flavonoides/uso terapêutico , Humanos , Leupeptinas/farmacologia , Leupeptinas/uso terapêutico , Masculino , Proteína NEDD8/metabolismo , Células PC-3 , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo
11.
Cancer Res ; 79(9): 2195-2207, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30877106

RESUMO

Menin is a nuclear epigenetic regulator that can both promote and suppress tumor growth in a highly tissue-specific manner. The role of menin in colorectal cancer, however, remains unclear. Here, we demonstrate that menin was overexpressed in colorectal cancer and that inhibition of menin synergized with small-molecule inhibitors of EGFR (iEGFR) to suppress colorectal cancer cells and tumor xenografts in vivo in an EGFR-independent manner. Mechanistically, menin bound the promoter of SKP2, a pro-oncogenic gene crucial for colorectal cancer growth, and promoted its expression. Moreover, the iEGFR gefitinib activated endoplasmic reticulum calcium channel inositol trisphosphate receptor 3 (IP3R3)-mediated release of calcium, which directly bound menin. Combined inhibition of menin and iEGFR-induced calcium release synergistically suppressed menin-mediated expression of SKP2 and growth of colorectal cancer. Together, these findings uncover a molecular convergence of menin and the iEGFR-induced, IP3R3-mediated calcium release on SKP2 transcription and reveal opportunities to enhance iEGFR efficacy to improve treatments for colorectal cancer. SIGNIFICANCE: Menin acts as a calcium-responsive regulator of SKP2 expression, and small molecule EGFR inhibitors, which induce calcium release, synergize with Menin inhibition to reduce SKP2 expression and suppress colorectal cancer.


Assuntos
Cálcio/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Quimioterapia Combinada , Inibidores Enzimáticos/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Gefitinibe/farmacologia , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Camundongos Nus , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Tapsigargina/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
12.
J Exp Clin Cancer Res ; 38(1): 76, 2019 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760284

RESUMO

BACKGROUND: S-phase kinase-associated protein 2 (SKP2) is an oncogene and cell cycle regulator that specifically recognizes phosphorylated cell cycle regulator proteins and mediates their ubiquitination. Programmed cell death protein 4 (PDCD4) is a tumor suppressor gene that plays a role in cell apoptosis and DNA-damage response via interacting with eukaryotic initiation factor-4A (eIF4A) and P53. Previous research showed SKP2 may interact with PDCD4, however the relationship between SKP2 and PDCD4 is unclear. METHODS: To validate the interaction between SKP2 and PDCD4, mass spectrometric analysis and reciprocal co-immunoprecipitation (Co-IP) experiments were performed. SKP2 stably overexpressed or knockdown breast cancer cell lines were established and western blot was used to detect proteins changes before and after radiation. In vitro and in vivo experiments were performed to verify whether SKP2 inhibits cell apoptosis and promotes DNA-damage response via PDCD4 suppression. SMIP004 was used to test the effect of radiotherapy combined with SKP2 inhibitor. RESULTS: We found that SKP2 remarkably promoted PDCD4 phosphorylation, ubiquitination and degradation. SKP2 promoted cell proliferation, inhibited cell apoptosis and enhanced the response to DNA-damage via PDCD4 suppression in breast cancer. SKP2 and PDCD4 showed negative correlation in human breast cancer tissues. Radiotherapy combine with SKP2 inhibitor SMIP004 showed significant inhibitory effects on breast cancer cells in vitro and in vivo. CONCLUSIONS: We identify PDCD4 as an important ubiquitination substrate of SKP2. SKP2 promotes breast cancer tumorigenesis and radiation tolerance via PDCD4 degradation. Radiotherapy combine with SKP2-targeted adjuvant therapy may improve breast cancer patient survival in clinical medicine.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias da Mama/patologia , Carcinogênese/metabolismo , Tolerância a Radiação/fisiologia , Proteínas Quinases Associadas a Fase S/metabolismo , Animais , Neoplasias da Mama/metabolismo , Feminino , Humanos , Ubiquitinação
13.
Int J Cancer ; 145(2): 503-516, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-30628729

RESUMO

The intermediate conductance calcium-activated potassium channel (KCa3.1) plays an important role in maintaining intracellular calcium homeostasis and is involved in the tumorigenesis of many human cancers. However, it is unknown whether KCa3.1 plays a role in the genesis of hepatocellular carcinoma (HCC), one of the most common malignant tumors worldwide with a very poor prognosis. In our study, we found that the expression of KCa3.1 was significantly elevated in poorly differentiated HCC tissues compared to adjacent noncancerous tissues. In vitro and in vivo experiments showed that KCa3.1 could promote cell proliferation, migration, and invasion of HCC. Mechanistically, KCa3.1 promoted cell cycle progression and migration and invasion of HCC cells by activating S-phase protein kinase 2 (SKP2) to trigger the degradation of p21 and p27 and targeting Reelin (RELN) to induce epithelial-mesenchymal transition (EMT), respectively. Taken together, our results demonstrate that KCa3.1 plays an important role in the genesis and progression of HCC, implying that it might be a promising therapeutic target in HCC.


Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/secundário , Proteínas Quinases Associadas a Fase S/genética , Transdução de Sinais , Animais , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Transplante de Neoplasias , Prognóstico , Proteínas Quinases Associadas a Fase S/metabolismo , Ativação Transcricional , Regulação para Cima
14.
Pancreas ; 48(2): 267-274, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30629029

RESUMO

OBJECTIVES: Menin, a chromatin binding protein, interacts with various epigenetic regulators to regulate gene transcription, whereas forkhead box protein O1 (FOXO1) is a transcription factor that can be regulated by multiple signaling pathways. Both menin and FOXO1 are crucial regulators of ß-cell function and metabolism; however, whether or how they interplay to regulate ß cells is not clear. METHODS: To examine whether menin affects expression of FOXO1, we ectopically expressed menin complementary DNA and small hairpin RNA targeting menin via a retroviral vector in INS-1 cells. Western blotting was used to analyze protein levels. RESULTS: Our current work shows that menin increases the expression of FOXO1. Menin stabilizes FOXO1 protein level in INS-1 cells, as shown by increased half-life of FOXO1 by menin expression. Moreover, menin represses ubiquitination of FOXO1 protein and AKT phosphorylation, We found that menin stabilizes FOXO1 by repressing FOXO1 degradation mediated by S-phase kinase-associated protein 2 (Skp2), an E3 ubiquitin ligase, promoting caspase 3 activation and apoptosis. CONCLUSIONS: Because FOXO1 upregulates the menin gene transcription, our findings unravel a crucial menin and FOXO1 interplay, with menin and FOXO1 upregulating their expression reciprocally, forming a positive feedback loop to sustain menin and FOXO1 expression.


Assuntos
Proteína Forkhead Box O1/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Regulação para Cima , Animais , Linhagem Celular Tumoral , Proteína Forkhead Box O1/genética , Células HEK293 , Humanos , Fosforilação , Estabilidade Proteica , Proteólise , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Proteínas Quinases Associadas a Fase S/genética , Ubiquitinação
15.
J Biol Chem ; 294(11): 3920-3933, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30670587

RESUMO

Brain injury-mediated induction of reactive astrocytes often leads to glial scar formation in damaged brain regions. Activation of signal transducer and activator of transcription 3 (STAT3), a member of the STAT family of transcription factors, plays a pivotal role in inducing reactive astrocytes and glial scar formation. Endothelin-1 (ET-1) is a vasoconstrictor peptide, and its levels increase in brain disorders and promote astrocytic proliferation through ETB receptors. To clarify the mechanisms underlying ET-1-mediated astrocytic proliferation, here we examined its effects on STAT3 in cultured rat astrocytes. ET-1 treatment stimulated Ser-727 phosphorylation of STAT3 in the astrocytes, but Tyr-705 phosphorylation was unaffected, and ET-induced STAT3 Ser-727 phosphorylation was reduced by the ETB antagonist BQ788. ET-1 stimulated STAT3 binding to its consensus DNA-binding motifs. Monitoring G1/S phase cell cycle transition through bromodeoxyuridine (BrdU) incorporation, we found that ET-1 increases BrdU incorporation into the astrocytic nucleus, indicating cell cycle progression. Of note, STAT3 chemical inhibition (with stattic or 5,15-diphenyl-porphine (5,15-DPP)) or siRNA-mediated STAT3 silencing reduced ET-induced BrdU incorporation. Moreover, ET-1 increased astrocytic expression levels of cyclin D1 and S-phase kinase-associated protein 2 (SKP2), which were reduced by stattic, 5,15-DPP, and STAT3 siRNA. ChIP-based PCR analysis revealed that ET-1 promotes the binding of SAT3 to the 5'-flanking regions of rat cyclin D1 and SKP2 genes. Our results suggest that STAT3-mediated regulation of cyclin D1 and SKP2 expression underlies ET-induced astrocytic proliferation.


Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Ciclina D1/metabolismo , Endotelina-1/farmacologia , Proteínas Quinases Associadas a Fase S/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Astrócitos/citologia , Astrócitos/enzimologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ciclina D1/genética , Relação Dose-Resposta a Droga , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Wistar , Proteínas Quinases Associadas a Fase S/genética , Fator de Transcrição STAT3/antagonistas & inibidores , Relação Estrutura-Atividade
16.
New Phytol ; 222(3): 1458-1473, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30664234

RESUMO

P0 protein of some polerovirus members can target ARGONAUTE1 (AGO1) to suppress RNA silencing. Although P0 harbors an F-box-like motif reported to be essential for interaction with S phase kinase-associated protein 1 (SKP1) and RNA silencing suppression, it is the autophagy pathway that was shown to contribute to AGO1 degradation. Therefore, the role of P0-SKP1 interaction in silencing suppression remains unclear. We conducted global mutagenesis and comparative functional analysis of P0 encoded by Brassica yellows virus (BrYV) (P0Br ). We found that several residues within P0Br are required for local and systemic silencing suppression activities. Remarkably, the F-box-like motif mutant of P0Br , which failed to interact with SKP1, is destabilized in vivo. Both the 26S proteasome system and autophagy pathway play a role in destabilization of the mutant protein. Furthermore, silencing of a Nicotiana benthamiana SKP1 ortholog leads to the destabilization of P0Br . Genetic analyses indicated that the P0Br -SKP1 interaction is not directly required for silencing suppression activity of P0Br , but it facilitates stability of P0Br to ensure efficient RNA silencing suppression. Consistent with these findings, efficient systemic infection of BrYV requires P0Br . Our results reveal a novel strategy used by BrYV for facilitating viral suppressors of RNA silencing stability against degradation by plant cells.


Assuntos
Autofagia , Luteoviridae/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Inativação Gênica , Modelos Biológicos , Mutagênese/genética , Mutação/genética , Proteínas de Plantas/metabolismo , Estabilidade Proteica , Tabaco/metabolismo , Tabaco/virologia , Proteínas Virais/química
17.
J Biol Chem ; 294(8): 2961-2969, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30643022

RESUMO

Retinoblastoma is a childhood retinal tumor that develops from cone photoreceptor precursors in response to inactivating RB1 mutations and loss of functional RB protein. The cone precursor's response to RB loss involves cell type-specific signaling circuitry that helps to drive tumorigenesis. One component of the cone precursor circuitry, the thyroid hormone receptor ß2 (TRß2), enables the aberrant proliferation of diverse RB-deficient cells in part by opposing the down-regulation of S-phase kinase-associated protein 2 (SKP2) by the more widely expressed and tumor-suppressive TRß1. However, it is unclear how TRß2 opposes TRß1 to enable SKP2 expression and cell proliferation. Here, we show that in human retinoblastoma cells TRß2 mRNA encodes two TRß2 protein isoforms: a predominantly cytoplasmic 54-kDa protein (TRß2-54) corresponding to the well-characterized full-length murine Trß2 and an N-terminally truncated and exclusively cytoplasmic 46-kDa protein (TRß2-46) that starts at Met-79. Whereas TRß2 knockdown decreased SKP2 expression and impaired retinoblastoma cell cycle progression, re-expression of TRß2-46 but not TRß2-54 stabilized SKP2 and restored proliferation to an extent similar to that of ectopic SKP2 restoration. We conclude that TRß2-46 is an oncogenic thyroid hormone receptor isoform that promotes SKP2 expression and SKP2-dependent retinoblastoma cell proliferation.


Assuntos
Proteínas de Neoplasias/metabolismo , Retinoblastoma/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Receptores beta dos Hormônios Tireóideos/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Proteínas de Neoplasias/genética , Isoformas de Proteínas , Estabilidade Proteica , Retinoblastoma/genética , Retinoblastoma/patologia , Proteínas Quinases Associadas a Fase S/genética , Receptores beta dos Hormônios Tireóideos/genética
18.
Mol Cancer Res ; 17(1): 250-262, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30237296

RESUMO

Deregulated expression of the transcriptional coactivator with PDZ-binding motif (WWTR1/TAZ) is a common feature of basal-like breast cancer (BLBC). Yet, how oncogenic TAZ regulates cell-cycle progression and proliferation in breast cancer remains poorly understood, and whether TAZ is required for tumor maintenance has not been established. Here, using an integrative oncogenomic approach, TAZ-dependent cellular programs essential for tumor growth and progression were identified. Significantly, TAZ-driven tumor cells required sustained TAZ expression, given that its withdrawal impaired both genesis and maintenance of solid tumors. Moreover, temporal inhibition of TAZ diminished the metastatic burden in established macroscopic pulmonary metastases. Mechanistic investigation revealed that TAZ controls distinct gene profiles that determine cancer cell fate through cell-cycle networks, including a specific, causal role for S-phase kinase-associated protein 2 (SKP2) in mediating the neoplastic state. Together, this study elucidates the molecular events that underpin the role of TAZ in BLBC and link to SKP2, a convergent communication node for multiple cancer signaling pathways, as a key downstream effector molecule. IMPLICATIONS: Understanding the molecular role of TAZ and its link to SKP2, a signaling convergent point and key regulator in BLBC, represents an important step toward the identification of novel therapeutic targets for TAZ-dependent breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/antagonistas & inibidores , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Transativadores/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Doxiciclina/farmacologia , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos SCID , Proteínas Quinases Associadas a Fase S/metabolismo , Transdução de Sinais , Transativadores/antagonistas & inibidores , Transativadores/genética
19.
Biosci Rep ; 39(1)2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30341246

RESUMO

Introduction: F-box proteins are the substrate-recognizing subunits of SKP1 (S-phase kinase-associated protein 1)-cullin1-F-box protein (SCF) E3 ligase complexes that play pivotal roles in multiple cellular processes, including cell proliferation, apoptosis, angiogenesis, invasion, and metastasis. Dysregulation of F-box proteins may lead to an unbalanced proteolysis of numerous protein substrates, contributing to progression of human malignancies. However, the prognostic values of F-box members, especially at mRNA levels, in breast cancer (BC) are elusive. Methods: An online database, which is constructed based on the gene expression data and survival information downloaded from GEO (http://www.ncbi.nlm.nih.gov/geo/), was used to investigate the prognostic values of 15 members of F-box mRNA expression in BC. Results: We found that higher mRNA expression levels of FBXO1, FBXO31, SKP2, and FBXO5 were significantly associated with worse prognosis for BC patients. While FBXO4 and ß-TrCP1 were found to be correlated to better overall survival (OS). Conclusion: The associated results provide new insights into F-box members in the development and progression of BC. Further researches to explore the F-box protein-targetting reagents for treating BC are needed.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/genética , Proteínas F-Box/genética , RNA Mensageiro/genética , Proteínas Quinases Associadas a Fase S/genética , Proteínas Supressoras de Tumor/genética , Apoptose/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Bases de Dados Factuais , Progressão da Doença , Proteínas F-Box/metabolismo , Feminino , Humanos , Metástase Linfática , Invasividade Neoplásica , Prognóstico , RNA Mensageiro/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Análise de Sobrevida , Proteínas Supressoras de Tumor/metabolismo
20.
Virus Res ; 260: 67-77, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30472094

RESUMO

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac12 gene, which is conserved in ten other baculovirus, codes a predicted 217 amino acid protein of unknown function. In this study, we investigated the role of ac12 during baculovirus infection, by generating an ac12 knockout virus. The transfection of the recombinant genome in insect cells resulted in unaltered viral dispersion and occlusion body production when compared to the control bacmid. This finding demonstrates that ac12 is a non-essential gene. Transmission and scanning electron microscopy (SEM) analyses showed that ac12 knockout virus produced occlusion bodies morphologically similar to those obtained with the control and capable to occlude virions. However, a slight but significant size difference was detected by SEM observation of purified occlusion bodies. This difference suggests that ac12 may be involved in regulatory pathways of polyhedrin production or occlusion body assembly without affecting either viral occlusion or oral infectivity in Rachiplusia nu larvae. This was evidenced by bioassays that showed no significant differences in the conditions tested. A qPCR analysis of viral gene expression during infection evidenced regulatory effects of ac12 over some representative genes of different stages of the viral cycle. In this study, we also showed that ac12 is transcribed at early times after infection and remains detectable up to 72 hours post-infection. The mRNA is translated during the infection and results in a protein that encodes an F-box domain that interacts in vivo and in vitro with S phase kinase associated protein 1 (SKP1) adaptor protein, which is potentially involved in protein ubiquitination pathways.


Assuntos
Interações Hospedeiro-Patógeno , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Corpos de Inclusão Viral/ultraestrutura , Larva/virologia , Lepidópteros/virologia , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Ligação Proteica , Proteínas Virais/genética , Replicação Viral
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