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1.
Cell Cycle ; 12(6): 935-43, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23421998

RESUMO

HER2/neu oncogene is frequently deregulated in cancers, and the (PI3K)-Akt signaling is one of the major pathways in mediating HER2/neu oncogenic signal. p57 (Kip2) , an inhibitor of cyclin-depependent kinases, is pivotal in regulating cell cycle progression, but its upstream regulators remain unclear. Here we show that the HER2-Akt axis is linked to p57 (Kip2) regulation, and that Akt is a negative regulator of p57 (Kip2) . Ectopic expression of Akt can decrease the expression of p57 (Kip2) , while Akt inhibition leads to p57 (Kip2) stabilization. Mechanistic studies show that Akt interacts with p57 (Kip2) and causes cytoplasmic localization of p57 (Kip2) . Akt phosphorylates p57 on Ser 282 or Thr310. Akt activity results in destabilization of p57 by accelerating turnover rate of p57 and enhancing p57 ubiquitination. Importantly, the negative impact of HER2/Akt on p57 stability contributes to HER2-mediated cell proliferation, transformational activity and tumorigenicity. p57 restoration can attenuate these defects caused by HER2. Significantly, Kaplan-Meier analysis of tumor samples demonstrate that in tumors where HER2 expression was observed, high expression levels of p57 (Kip2) were associated with better overall survival. These data suggest that HER2/Akt is an important negative regulator of p57 (Kip2) , and that p57 restoration in HER2-overexpressing cells can reduce breast tumor growth. Our findings indicate the applicability of employing p57 regulation as a therapeutic intervention in HER2-overexpressing cancers.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Transformação Celular Neoplásica , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Células 3T3 , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Inibidor de Quinase Dependente de Ciclina p57/genética , Regulação para Baixo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transdução de Sinais , Ubiquitinação
2.
Cell Cycle ; 11(24): 4633-41, 2012 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-23187808

RESUMO

Subunit 6 of the COP9 signalosome complex, CSN6, is known to be critical to the regulation of the MDM2-p53 axis for cell proliferation and anti-apoptosis, but its many targets remain unclear. Here we show that p57 (Kip2) is a target of CSN6, and that CSN6 is a negative regulator of p57 (Kip2) . CSN6 associates with p57 (Kip2) , and its overexpression can decrease the steady-state expression of p57 (Kip2) ; accordingly, CSN6 deficiency leads to p57 (Kip2) stabilization. Mechanistic studies show that CSN6 associates with p57 (Kip2) and Skp2, a component of the E3 ligase, which, in turn, facilitates Skp2-mediated protein ubiquitination of p57 (Kip2) . Loss of Skp2 compromised CSN6-mediated p57 (Kip2) destabilization, suggesting collaboration between Skp2 and CSN6 in degradation of p57 (Kip2) . CSN6's negative impact on p57 (Kip2) elevation translates into cell growth promotion, cell cycle deregulation and potentiated transformational activity. Significantly, univariate Kaplan-Meier analysis of tumor samples demonstrates that high CSN6 expression or low p57 expression is associated with poor overall survival. These data suggest that CSN6 is an important negative regulator of p57 (Kip2) , and that overexpression of CSN6 in many types of cancer could lead to decreased expression of p57 (Kip2) and result in promoted cancer cell growth.


Assuntos
Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Complexos Multiproteicos/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Complexo do Signalossomo COP9 , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p57/genética , Humanos , Estimativa de Kaplan-Meier , Camundongos , Complexos Multiproteicos/genética , Células NIH 3T3 , Peptídeo Hidrolases/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia
3.
J Cell Biochem ; 103(4): 1219-30, 2008 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-17879958

RESUMO

The 5th subunit of COP9 signalosome (CSN5, also known as Jab1 or COPS5) is implicated in regulating p53 activity and is overexpressed in various tumors. However, the precise roles of CSN5 in p53 network and tumorigenesis are not well characterized. Here we show that CSN5 is a critical regulator of both p53 and MDM2. We show that curcumin, an important inhibitor of CSN-associated kinases, can downregulate not only CSN5 but also MDM2, which results in p53 stabilization. Importantly, CSN5 interacts with p53. CSN5 expression leads to p53 degradation, facilitating MDM2-mediated p53 ubiquitination, and promoting p53 nuclear export. Additionally, CSN5 expression results in stabilization of MDM2 through reducing MDM2 self-ubiquitination and decelerating turnover rate of MDM2. Significantly, we further show that CSN5 antagonizes the transcriptional activity of p53. These results demonstrate that CSN5 is a pivotal regulator for both p53 and MDM2. Our studies may pave the way for targeting CSN5 for anti-cancer drug development.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Peptídeo Hidrolases/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Transporte Ativo do Núcleo Celular , Complexo do Signalossomo COP9 , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Curcumina/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Ligação Proteica , Ubiquitinação
4.
Cancer Res ; 66(6): 3096-105, 2006 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-16540659

RESUMO

14-3-3 sigma is induced by tumor suppressor protein p53 in response to DNA damage. p53 can directly transactivate the expression of 14-3-3 sigma to cause a G(2) cell cycle arrest when cell DNA is damaged. The expression of 14-3-3 sigma protein is down-regulated in various tumors, but its function has not been fully established. Protein kinase B/Akt, a crucial regulator of oncogenic signal involved in cell survival and proliferation, is deregulated in many types of cancer. Akt activation can enhance p53 degradation, but its role in DNA damage response is not clear. Here, we show that Akt activation is diminished when p53 and 14-3-3 sigma is up-regulated in response to DNA damage. Evidence is provided that 14-3-3 sigma binds and inhibits Akt. In keeping with this concept, Akt-mediated cell survival is inhibited by 14-3-3 sigma. Significantly, we show that 14-3-3 sigma inhibits Akt-mediated cell growth, transformation, and tumorigenesis. Low expression of 14-3-3 sigma in human primary breast cancers correlates with Akt activation. These data provide an insight into Akt regulation and rational cancer gene therapy by identifying 14-3-3 sigma as a molecular regulator of Akt and as a potential anticancer agent for Akt-activated cancers.


Assuntos
Biomarcadores Tumorais/biossíntese , Transformação Celular Neoplásica/genética , Dano ao DNA/fisiologia , Exonucleases/biossíntese , Proteínas de Neoplasias/biossíntese , Proteína Oncogênica v-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteína Supressora de Tumor p53/biossíntese , Proteínas 14-3-3 , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Processos de Crescimento Celular/fisiologia , Transformação Celular Neoplásica/metabolismo , Ativação Enzimática , Exonucleases/genética , Exonucleases/metabolismo , Exorribonucleases , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Vison , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína Oncogênica v-akt/metabolismo , Ratos , Proteína Supressora de Tumor p53/genética
5.
J Cell Biochem ; 98(1): 128-38, 2006 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-16365885

RESUMO

Cyclin-dependent kinase (CDK) inhibitor p27 Kip1, a haplo-insufficient tumor suppressor, is downregulated by oncogenic signal of HER2, a receptor tyrosine kinase oncogene. HER2 promotes mitogenic growth and transformation of cancer cells. HER2 signaling can enhance p27 Kip1 ubiquitination, thereby promoting p27 degradation and subsequent activation of CDK activity. p27 ubiquitination and degradation is enhanced by JAB1 binding as well as by phosphorylation on Thr187. In this study, we generated modified p27 proteins, which are mutated at Thr 187 or deleted at JAB1 binding domain. We applied these modified p27 genes as novel anticancer agents for HER2-overexpressing cells under the control of a tetracycline (tet)-regulated gene expression system. Induction of p27 T187A and p27 T187A DeltaJAB inhibits HER2-activated cell growth, CDK2 activity, cell proliferation, and transformation. Significantly, a modified protein (p27 T187ADeltaJAB) reduced the tumor volume in a HER2-overexpressing tumor model efficiently. These findings demonstrate the applicability of employing modified p27 proteins as a therapeutic intervention in HER2-overexpressing cancers.


Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidores do Crescimento/fisiologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Substituição de Aminoácidos/genética , Animais , Complexo do Signalossomo COP9 , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Feminino , Deleção de Genes , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Nus , Mitógenos/fisiologia , Células NIH 3T3 , Peptídeo Hidrolases/genética , Ligação Proteica/genética , Receptor ErbB-2/biossíntese , Treonina/genética , Transfecção
6.
Oncogene ; 24(11): 1924-35, 2005 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-15688030

RESUMO

The FOXO family of Forkhead transcription factors, regulated by the phosphoinositide-3-kinase-Akt pathway, is involved in cell cycle regulation and apoptosis. Strong expression of HER2, a receptor tyrosine kinase oncogene, in cancers has been associated with a poor prognosis. Recently, FOXO4 was shown to regulate the transcription of the cyclin-dependent kinase inhibitor p27 Kip1 gene directly. Also, we have shown that HER2 promotes mitogenic growth and transformation of cancer cells by downregulation of p27 Kip1. Given the fact that FOXO4 mediates p27 transcription, we hypothesize that an Akt phosphorylation mutant of FOXO4 (FOXO4A3), which maintains the activity to transactivate p27 Kip1, may be used as an anticancer agent for HER2-overexpressing cancers. Here, we applied the FOXO4 gene as a novel anticancer agent for HER2-overexpressing cells under the control of a tetracycline (tet)-regulated gene expression system. Overexpression of FOXO4A3 inhibits HER2-activated cell growth. We found that FOXO4A3 inhibited the kinase activity of protein kinase B/Akt and reversed HER2-mediated p27 mislocation in the cytoplasm. FOXO4A3 expression also led to decreased levels of CSN5, a protein involved in p27 degradation. These data suggest that FOXO4A3 also can regulate p27 post-transcriptionally. In addition, we found that FOXO4A3 sensitized cells to apoptosis induced by the chemotherapeutic agent 2-methoxyestradiol. Most significantly for clinical application, FOXO4A3 expression in HER2-overexpressing cells can be regulated in vivo and reduces the tumor volume in a tumor model. These findings indicate the applicability of employing FOXO4 regulation as a therapeutic intervention in HER2-overexpressing cancers.


Assuntos
Proteínas de Transporte/metabolismo , Genes erbB-2/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Linhagem Celular , Transformação Celular Neoplásica , Ciclina D1/genética , Inibidor de Quinase Dependente de Ciclina p27 , Fatores de Transcrição Forkhead , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Rim , Camundongos , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt , Fatores de Transcrição/genética
7.
Oncogene ; 23(42): 7132-43, 2004 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-15273726

RESUMO

HER2/neu, a receptor tyrosine kinase oncogene, promotes mitogenic growth and antiapoptotic activity in cancer cells. Strong expression of HER2/neu in cancers has been associated with poor prognosis. Alternative reading frame protein (ARF), a tumor suppressor protein encoded by a gene located in the Ink4a/ARF gene locus, is frequently inactivated in human cancers. Little is known about the tumor suppressor role of ARF in HER2/neu-overexpressing cancers. Here, we applied the ARF gene as a tumor-suppressive agent for HER2/neu-overexpressing cells under the control of a tetracycline (tet)-regulated gene expression system. We found that ARF antagonized protein kinase B (PKB)/Akt-mediated p27Kip1 phosphorylation and increased p27 stability in HER2/neu-overexpressing cells. ARF expression also led to decreased levels of Cul1 and Skp2, two proteins involved in p27 degradation. We also found that ARF caused apoptosis in HER2/neu-overexpressing cells, and sensitized cells to apoptosis induced by the chemotherapeutic agents taxol and 2-methoxyestradiol. Most significantly for clinical application, we found that ARF inhibited HER2/neu-mediated cell growth, transformation, and tumorigenesis. These findings indicate that modulation of ARF activity may be a useful therapeutic intervention in HER2-overexpressing cancers.


Assuntos
Receptor ErbB-2/genética , Células 3T3 , Animais , Sequência de Bases , Divisão Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Clonagem Molecular , Primers do DNA , Genes Reporter , Humanos , Camundongos , Camundongos Knockout , Camundongos Nus , Camundongos Transgênicos , Receptor ErbB-2/deficiência , Transfecção , Transplante Heterólogo
8.
Int Immunopharmacol ; 4(5): 649-67, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15120650

RESUMO

The melanoma differentiation associated gene-7 (mda-7) cDNA was isolated by virtue of being induced during melanoma differentiation. Initial gene transfer studies convincingly demonstrated potent antitumor effects of mda-7. Further studies showed that the mechanism of antitumor activity was due to induction of apoptosis. Most striking was the tumor-selective killing by mda-7 gene transfer--normal cells were unaffected by Adenoviral delivery of mda-7 (Ad-mda7). A variety of molecules implicated in apoptosis and intracellular signaling are regulated by Ad-mda7 transduction. Different apoptosis effector proteins are regulated in different tumor types, suggesting that Ad-mda7 may regulate various signaling pathways. mda-7 encodes a secreted protein, MDA-7, which has now been designated as IL-24, and is a novel member of the IL-10 cytokine family. MDA-7/IL-24 protein is actively secreted from cells after mda-7 gene transfer. In human peripheral blood mononuclear cells (PBMC), STAT3 activation by MDA-7/IL-24 is followed by elaboration of secondary Th1 cytokines, demonstrating that MDA-7/IL-24 is a pro-Th1 cytokine. Furthermore, MDA-7/IL-24 is antagonized by the prototypic Th2 cytokine IL-10. MDA-7/IL-24 protein is endogenously expressed in cultured NK and B-cells and is also expressed in dendritic cells in tissues. MDA-7/IL-24 protein is expressed in nevi and melanoma primary tumors, to varying degrees, but is rarely expressed in malignant melanoma or other human tumors evaluated. Indeed, loss of MDA-7/IL-24 protein expression correlates strongly with melanoma tumor invasion and disease progression. The "bystander" effects proposed for MDA-7/IL-24 protein include immune stimulation, antiangiogenesis and receptor-mediated cytotoxicity. Thus, mda-7 is a unique multifunctional cytokine in the IL-10 family and may have potent antitumor utility in a clinical setting.


Assuntos
Citocinas/farmacologia , Genes Supressores de Tumor/efeitos dos fármacos , Interleucina-10/classificação , Interleucinas/classificação , Interleucinas/farmacologia , Citocinas/classificação , Citocinas/genética , Humanos , Interleucina-10/genética , Interleucina-10/farmacologia , Interleucinas/genética
9.
Mol Cell Biol ; 23(20): 7096-107, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14517281

RESUMO

The 14-3-3 sigma (sigma) protein, a negative regulator of the cell cycle, is a human mammary epithelium-specific marker that is downregulated in transformed mammary carcinoma cells. It has also been identified as a p53-inducible gene product involved in cell cycle checkpoint control after DNA damage. Although 14-3-3 sigma is linked to p53-regulated cell cycle checkpoint control, detailed mechanisms of how cell cycle regulation occurs remain unclear. Decreased expression of 14-3-3 sigma was recently reported in several types of carcinomas, further suggesting that the negative regulatory role of 14-3-3 sigma in the cell cycle is compromised during tumorigenesis. However, this possible tumor-suppressive role of 14-3-3 sigma has not yet been characterized. Here, we studied the link between 14-3-3 sigma activities and p53 regulation. We found that 14-3-3 sigma interacted with p53 in response to the DNA-damaging agent adriamycin. Importantly, 14-3-3 sigma expression led to stabilized expression of p53. In studying the molecular mechanism of this increased stabilization of p53, we found that 14-3-3 sigma antagonized the biological functions of Mdm2 by blocking Mdm2-mediated p53 ubiquitination and nuclear export. In addition, we found that 14-3-3 sigma facilitated the oligomerization of p53 and enhanced p53's transcriptional activity. As a target gene of p53, 14-3-3 sigma appears to have a positive feedback effect on p53 activity. Significantly, we also showed that overexpression of 14-3-3 sigma inhibited oncogene-activated tumorigenicity in a tetracycline-regulated 14-3-3 sigma system. These results defined an important p53 regulatory loop and suggested that 14-3-3 sigma expression can be considered for therapeutic intervention in cancers.


Assuntos
Biomarcadores Tumorais/metabolismo , Exonucleases/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas 14-3-3 , Transporte Ativo do Núcleo Celular , Animais , Antibacterianos/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Northern Blotting , Western Blotting , Bromodesoxiuridina/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Dano ao DNA , Regulação para Baixo , Doxiciclina/farmacologia , Exorribonucleases , Feminino , Genes Reporter , Glutationa Transferase/metabolismo , Humanos , Luciferases/metabolismo , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Modelos Biológicos , Células NIH 3T3 , Testes de Precipitina , Ligação Proteica , Fatores de Tempo , Ubiquitina/metabolismo
10.
Mol Ther ; 8(2): 207-19, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12907143

RESUMO

mda-7 is a novel tumor suppressor with cytokine properties. Adenoviral mda-7 (Ad-mda7) induces apoptosis and cell death selectively in tumor cells. The molecular mechanisms underlying the anti-tumor activity of Ad-mda7 in breast and lung cancer lines were investigated. Microarray analyses implicated both the beta-catenin and the PI3K signaling pathways. Ad-mda7 treatment increased protein expression from tumor suppressor genes, including E-cadherin, APC, GSK-3beta, and PTEN, and decreased expression of proto-oncogenes involved in beta-catenin and PI3K signaling. Ad-mda7 caused a redistribution of cellular beta-catenin from the nucleus to the plasma membrane, resulting in reduced TCF/LEF transcriptional activity, and upregulated the E-cadherin-beta-catenin adhesion complex in a tumor cell-specific manner. Expression of the PI3K pathway members (p85 PI3K, FAK, ILK-1, Akt, and PLC-gamma) was downregulated and expression of the PI3K antagonist PTEN was increased. Consistent with this result, pharmacological inhibition of PI3K by wortmannin did not abrogate killing by Ad-mda7. Killing of breast cancer cells by Ad-mda7 required both MAPK and MEK1/2 signaling pathways, whereas these pathways were not essential for MDA-7-mediated killing in lung cancer cells. Thus, in breast and lung tumor cells MDA-7 protein expression modulates cell-cell adhesion and intracellular signaling via coordinate regulation of the beta-catenin and PI3K pathways.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas do Citoesqueleto/metabolismo , Interleucinas/metabolismo , Neoplasias Pulmonares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Adenoviridae/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caderinas/metabolismo , Adesão Celular , Morte Celular , Linhagem Celular Tumoral , Movimento Celular , Proteínas do Citoesqueleto/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Interleucinas/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Inibidores de Fosfoinositídeo-3 Quinase , Ligação Proteica , Proto-Oncogenes/genética , Transativadores/antagonistas & inibidores , beta Catenina
11.
Cancer Metastasis Rev ; 22(4): 435-49, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12884917

RESUMO

The mammalian cell cycle can be divided into four phases: G1 (gap phase 1), S (DNA synthesis), G2 (gap phase 2), and M (mitosis). Progression through each phase of the cell cycle is delicately controled by the activity of different cyclin-dependent kinases (CDKs) and their regulatory subunits known as cyclins. CDK2, CDK4, CDK6 and their associated cyclins control the G1 to S phase transition. The association of CDK4 or CDK6 with D-type cyclins is critical for G1 phase progression, whereas the CDK2-cyclin E complex is important for initiation of the S phase. Cancer can originate from dysregulation of these regulators. A variety of intrinsic and extrinsic signals were recently identified to regulate these G1 or G1/S CDKs and cyclins. Here we discuss the regulators of these protein kinases at different mechanistic level with a hope that these insights can be applied to develop therapeutic strategies for cancer treatment.


Assuntos
Quinases Ciclina-Dependentes/fisiologia , Fase G1 , Regulação Neoplásica da Expressão Gênica , Neoplasias/enzimologia , Proteínas Proto-Oncogênicas , Quinases relacionadas a CDC2 e CDC28/metabolismo , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Progressão da Doença , Humanos , Modelos Biológicos , Neoplasias/metabolismo
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