RESUMO
Activator protein-1 (AP-1) regulates the expression of several genes involved in human tumorigenesis. However, there is little known about this transcription factor in pancreatic ductal adenocarcinoma. We recently found high levels of AP-1-binding activities and multiple AP-1/DNA complexes containing c-Jun, JunD, Fra1, and Fra2 in pancreatic cancer cells. Transient transfection assays indicated that AP-1 was functional and capable of transactivating its gene targets. Furthermore, a c-Jun transactivation mutant inhibited anchorage-dependent and anchorage-independent proliferation, suggesting that AP-1 had an essential role in pancreatic cancer cells. Our study also uncovered a novel mechanism by which protein kinase Akt controls c-Jun activity in pancreatic cancer cells. Indeed, distinct from its known ability to induce c-fos and fra1 and to stabilize c-Jun, Akt appeared to directly regulate the transcriptional activity of c-Jun independently of the phosphorylation sites targeted by c-Jun NH(2)-terminal kinase (Ser(63)/Ser(73)) and glycogen synthase kinase-3 (Thr(239)). Our data also suggest that growth factors might use this Akt-regulated mechanism to potently induce c-Jun targets such as cyclin D1. Collectively, our findings indicate that AP-1 has an important function in pancreatic cancer cells and provide evidence for a previously unknown Akt-mediated mechanism of c-Jun activation.
Assuntos
Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Immunoblotting , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Luciferases/genética , Luciferases/metabolismo , Mutação , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/fisiopatologia , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/fisiologia , TransfecçãoRESUMO
Nasopharyngeal carcinoma (NPC) is a malignancy of epithelial origin that is prone to local invasion and early distant metastasis. Although concurrent chemotherapy and radiotherapy improves the 5-year survival outcomes, persistent or recurrent disease still occurs. Therefore, novel therapeutic targets are needed for NPC patients. MicroRNAs (miRNAs) play important roles in normal cell homeostasis, and dysregulations of miRNA expression have been implicated in human cancers. In NPC, studies have revealed that miRNAs are dysregulated and involved in tumorigenesis, metastasis, invasion, resistance to chemo- and radiotherapy, and other disease- and treatment-related processes. The advantage of miRNA-based treatment approaches is that miRNAs can concurrently target multiple effectors of pathways involved in tumor cell differentiation and proliferation. Thus, miRNA-based cancer treatments, alone or combined with standard chemotherapy and/or radiotherapy, hold promise to improve treatment response and cure rates. In this review, we will summarize the dysregulation of miRNAs in NPC initiation, progression, and treatment as well as NPC-related signaling pathways, and we will discuss the potential applications of miRNAs as biomarkers and therapeutic targets in NPC patients. We conclude that miRNAs might be potential promising therapeutic targets in nasopharyngeal carcinoma.
RESUMO
Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) resulting in aberrant expression of chimeric nucleophosmin-ALK. Previously, nucleophosmin-ALK has been shown to activate phosphatidylinositol 3-kinase (PI3K) and its downstream effector, the serine/threonine kinase AKT. In this study, we hypothesized that the mammalian target of rapamycin (mTOR) pathway, which functions downstream of AKT, mediates the oncogenic effects of activated PI3K/AKT in ALK+ ALCL. Here, we provide evidence that mTOR signaling phosphoproteins, including mTOR, eukaryotic initiation factor 4E-binding protein-1, p70S6K, and ribosomal protein S6, are highly phosphorylated in ALK+ ALCL cell lines and tumors. We also show that AKT activation contributes to mTOR phosphorylation, at least in part, as forced expression of constitutively active AKT by myristoylated AKT adenovirus results in increased phosphorylation of mTOR and its downstream effectors. Conversely, inhibition of AKT expression or activity results in decreased mTOR phosphorylation. In addition, pharmacologic inhibition of PI3K/AKT down-regulates the activation of the mTOR signaling pathway. We also show that inhibition of mTOR with rapamycin, as well as silencing mTOR gene product expression using mTOR-specific small interfering RNA, decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis in ALK+ ALCL cells. Cell cycle arrest was associated with modulation of G(1)-S-phase regulators, including the cyclin-dependent kinase inhibitors p21(waf1) and p27(kip1). Apoptosis following inhibition of mTOR expression or function was associated with down-regulation of antiapoptotic proteins, including c-FLIP, MCL-1, and BCL-2. These findings suggest that the mTOR pathway contributes to nucleophosmin-ALK/PI3K/AKT-mediated tumorigenesis and that inhibition of mTOR represents a potential therapeutic strategy in ALK+ ALCL.
Assuntos
Linfoma Difuso de Grandes Células B/metabolismo , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Quinase do Linfoma Anaplásico , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Cromonas/farmacologia , Regulação para Baixo , Ativação Enzimática , Humanos , Linfoma Difuso de Grandes Células B/enzimologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Quinases/genética , Proteínas Tirosina Quinases/biossíntese , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Receptores Proteína Tirosina Quinases , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , TransfecçãoRESUMO
The Jun activation domain-binding protein 1 (JAB1), aside from being an activator protein 1 coactivator, is involved in degradation of the cyclin-dependent kinase inhibitor p27. We examined JAB1 and p27 protein expression in invasive breast carcinoma specimens and the association of this expression with clinical outcome. JAB1 was detected immunohistochemically in 43 of 53 (81%) tumors; 32 (60%) breast carcinomas showed high JAB1 expression (>50% of cells positive) and reduced or absent p27 levels (P = 0.02, Mann-Whitney U test). Tumors with high p27 expression were rarely positive for JAB1. All eight patients with JAB1-negative tumors had no evidence of relapse or disease progression at a median follow-up of 70 months. Immunoblotting showed strong JAB1 expression in breast carcinoma samples but not in paired normal breast epithelial samples. Targeted overexpression of JAB1 by regulated adenovirus in breast cancer cell lines also reduced p27 levels by accelerating degradation of p27. Thus, the JAB1:p27 ratio may be a novel indicator of aggressive, high-grade tumor behavior, and control of JAB1 could be a novel target for experimental therapies.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/biossíntese , Fatores de Transcrição/biossíntese , Proteínas Supressoras de Tumor/metabolismo , Adenoviridae/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Complexo do Signalossomo COP9 , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/genética , Carcinoma Lobular/metabolismo , Carcinoma Lobular/patologia , Inibidor de Quinase Dependente de Ciclina p27 , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Feminino , Técnicas de Transferência de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Pessoa de Meia-Idade , Peptídeo Hidrolases , Prognóstico , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
Abnormal interaction between non-coding RNAs has been demonstrated to be a common molecular event in various human cancers, but its significance and underlying mechanisms have not been well documented. RNA-binding proteins (RBPs) are key regulators of RNA transcription and post-transcriptional processing. In this study, we found that RNA-binding protein 24 (RBM24) was frequently downregulated in nasopharyngeal carcinoma (NPC). The restoration of RBM24 expression suppressed NPC cellular proliferation, migration and invasion and impeded metastatic colonization in mouse models. Microarray analyses revealed that miR-25 expression was upregulated by RBM24 expression in NPC cells. Similarly, ectopic miR-25 expression suppressed NPC cellular growth and motility by targeting the pro-oncogenic lncRNA MALAT1, and the knockdown of MALAT1 expression exhibited similar effects as RBM24 restoration in NPC cells. Overall, these findings suggest a novel role of RBM24 as a tumor suppressor. Mechanistically, RBM24 acts at least in part through upregulating the expression of miR-25, which in turn targets MALAT1 for degradation.
Assuntos
Carcinoma/genética , Carcinoma/patologia , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/metabolismo , Regulação para Cima/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Carcinoma Nasofaríngeo , Invasividade Neoplásica , Metástase NeoplásicaRESUMO
PURPOSE: p27(Kip1)(p27) is a universal cyclin-dependent kinase inhibitor that inhibits cell cycle transition from G(1) to S phase and is primarily regulated at the post-transcriptional level via the ubiquitin-proteasome pathway. In vitro data suggest that p27 degradation may be accelerated by the c-Jun activation domain binding protein-1 (JAB1), originally identified as a coactivator of the gene regulatory AP-1 proteins. We assessed p27 and JAB1 in systemic anaplastic large cell lymphoma (ALCL), a group of tumors in which a substantial subset overexpresses anaplastic lymphoma kinase (ALK). EXPERIMENTAL DESIGN: The study included 5 ALK-positive ALCL cell lines, namely Karpas 299, JB-6, SR-786, SU-DHL1, and TG-S1, and 66 ALCL tumors (24 ALK positive and 42 ALK negative). The cell lines were analyzed by Western blot methods, and the tumors were assessed immunohistochemically. RESULTS: SU-DHL1 and TG-S1 cells were positive for p27 and negative for JAB1, whereas SR-786 and JB-6 cells were positive for JAB-1 but negative for p27. Karpas 299 expressed p27 at relatively low levels and JAB1 at high levels. Using a 10% cutoff, p27 was positive in 12 of 66 (18.2%) ALCL tumors (5 ALK positive and 7 ALK negative), whereas JAB1 was detected in 47 of 53 (88.7%) tumors (15 ALK positive and 32 ALK negative) assessed. p27 and JAB1 expression were inversely correlated (Spearman r = -0.27, P = 0.03). For 54 ALCL patients with complete follow-up, and in separate analyses of patients with ALK-positive or -negative tumors, p27 expression correlated with poorer prognosis. CONCLUSIONS: p27 is absent or expressed at low levels in most ALCL tumors and inversely correlates with JAB1. These findings suggest that JAB1-mediated degradation of p27, allowing cell cycle progression, may play a role in the pathogenesis of ALCL.
Assuntos
Proteínas de Ciclo Celular/biossíntese , Proteínas de Ligação a DNA/biossíntese , Linfoma Difuso de Grandes Células B/metabolismo , Fatores de Transcrição/biossíntese , Proteínas Supressoras de Tumor/biossíntese , Adulto , Quinase do Linfoma Anaplásico , Western Blotting , Complexo do Signalossomo COP9 , Ciclo Celular , Proteínas de Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p27 , Proteínas de Ligação a DNA/genética , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Pessoa de Meia-Idade , Peptídeo Hidrolases , Fosforilação , Prognóstico , Proteínas Tirosina Quinases/biossíntese , Receptores Proteína Tirosina Quinases , Fatores de Tempo , Fatores de Transcrição/genética , Resultado do Tratamento , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genéticaRESUMO
PURPOSE: The purpose is to evaluate expression levels of Jun activation domain-binding protein 1 (JAB1) in breast cancer tissue and adjacent normal tissue, to determine whether JAB1 expression is associated with p27(Kip1) expression in invasive breast carcinomas, and to evaluate the prognostic significance of JAB1 and p27(Kip1) in node-negative breast cancer. EXPERIMENTAL DESIGN: JAB1 levels were measured in 10 matched pairs of invasive breast tumor tissue and adjacent normal tissue using Western blot analysis. We also investigated the immunoreactivity of JAB1 and p27(Kip1) levels in paraffin-embedded tissue specimens from 220 patients with node-negative breast cancer who had not received adjuvant systemic therapy. The median follow-up was 15 years. RESULTS: JAB1 was expressed at higher levels in invasive tumors than in adjacent normal tissue (P = 0.01). JAB1 overexpression was observed in 57% of invasive breast cancers. Low levels of p27(Kip1) were noted in 70% of the tumor specimens. We found an inverse correlation between JAB1 and p27(Kip1) expression levels (P = 0.01). JAB1 overexpression was associated with patient age of at least 50 years (P = 0.03) and tumor size of =2 cm (P = 0.01). Elevated levels of p27(Kip1) were associated with low nuclear grade (P = 0.01). At 5 years of follow-up, neither JAB1 nor p27(Kip1) expression was related to disease-free survival. CONCLUSIONS: These data indicate that JAB1 is commonly overexpressed in invasive breast carcinomas. JAB1 overexpression is associated with low levels of p27(Kip1) in node-negative breast cancer. In this study, JAB1 and p27(Kip1) were not independent prognostic factors.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor/fisiologia , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Neoplasias da Mama/mortalidade , Complexo do Signalossomo COP9 , Inibidor de Quinase Dependente de Ciclina p27 , Feminino , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Metástase Linfática/genética , Pessoa de Meia-Idade , Peptídeo Hidrolases , Receptor ErbB-2/análise , Receptores de Estrogênio/análise , Análise de Sobrevida , Fatores de TempoRESUMO
The cyclin-dependent kinase inhibitor p27(Kip1) (p27) plays a pivotal role in controlling cell proliferation during development and tumorigenesis. p27 has been implicated in pituitary tumorigenesis in studies of knockout mice and in analyses of human pituitary tumor samples. In this study, we further explored the role of p27 in human pituitary tumors by measuring levels of phosphorylated p27 (P-p27), and also Jun activation domain-binding protein 1 (Jab1), which is thought to facilitate the phosphorylation and degradation of p27, in normal pituitary tissue (n = 21), pituitary adenomas (n = 75), and pituitary carcinomas (n = 10). The amount of p27 protein in corticotroph adenomas and pituitary carcinomas was much lower than that in normal pituitary tissue or other types of pituitary adenoma. Nuclear P-p27 protein levels were significantly decreased in the adenomas, compared with the normals, and were much lower in the carcinomas, compared with either normal pituitary tissue or pituitary adenomas. However, P-p27 levels in corticotroph adenomas were similar to normal pituitary tissue, thus demonstrating a greatly increased ratio of P-p27 to p27 specifically in corticotroph tumors. No difference was found in Jab1 protein levels in either corticotroph tumors or other pituitary adenomas, compared with normal tissue, but there was a small but significant increase in Jab1 levels in carcinomas. Corticotroph and metastatic tumors both showed a significantly higher Ki-67 labeling index than normal pituitary or other types of pituitary adenomas, and in general the Ki-67 labeling index was negatively correlated with p27 nuclear staining. The amount of p27 and Jab1 mRNA was positively correlated in all pituitary samples studied but did not correlate with the changes in immunostaining. Our findings suggest that in corticotroph tumors there is an accentuated phosphorylation of p27 into P-p27, possibly related to increased cyclin E expression, whereas both p27 and P-p27 are subject to increased degradation in pituitary carcinomas. Such variations in phosphorylation may play a role in pituitary tumorigenesis, but modulation of Jab1 is unlikely to be important in the pathogenesis of pituitary adenomas.
Assuntos
Adenoma/metabolismo , Carcinoma/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Antígeno Ki-67/metabolismo , Neoplasias Hipofisárias/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Complexo do Signalossomo COP9 , Proteínas de Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p27 , Proteínas de Ligação a DNA/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Peptídeo Hidrolases , Fosforilação , RNA Mensageiro/metabolismo , Coloração e Rotulagem , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Chemoresistance is a major impediment to the successful treatment of cancer. It involves various mechanisms, including defects in the apoptosis program that is induced by anticancer drugs. To further explore the mechanisms underlying the development of chemoresistance in ovarian carcinoma after cisplatin (CDDP) treatment, we compared the effect of CDDP on expression of X-linked inhibitor of apoptosis protein (XIAP), a direct inhibitor of caspase-3, -7, and -9, Fas, Fas-ligand (Fas-L), and pro- and antiapoptotic proteins in a CDDP-sensitive human ovarian carcinoma cell line (2008) and its CDDP-resistant subclone (2008C13). In this article, we show that cisplatin treatment led to a differential expression of distinct apoptotic targets in the CDDP-sensitive cell line (2008) and its CDDP-resistant subclone (2008C13). The acquisition of cisplatin resistance was associated with the ability of the treated cells to enhanced expression of XIAP, whereas the death inducer Fas-L was abrogated in 2008C13 following treatment with CDDP. However, the CDDP-sensitive cells failed to activate XIAP but increased Fas-L expression, indicating that distinct regulatory mechanisms are operative. These findings suggest that the expression of XIAP and downregulation of Fas-L are linked to chemoresistance in ovarian carcinoma cells and may represent one of the potential antiapoptotic mechanisms involved during this process.
Assuntos
Antineoplásicos/farmacologia , Apoptose/genética , Cisplatino/farmacologia , Resistência a Medicamentos/genética , Neoplasias Ovarianas/tratamento farmacológico , Proteínas/genética , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Cisplatino/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Proteína Ligante Fas , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Neoplasias Ovarianas/genética , Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Regulação para Cima/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
Fas-associated death domain protein is a key component of the extrinsic apoptotic pathway. In addition, in animal models, Fas-associated death domain protein phosphorylation at serine 194 has been shown to affect cell proliferation, especially in T lymphocytes. The importance of Fas-associated death domain protein phosphorylation at serine 194 for the proliferation of B lymphocytes, however, is uncertain. Here we show in reactive lymph nodes that serine 194 phosphorylated Fas-associated death domain protein is expressed predominantly in the dark (proliferative) zone of germinal centers. In B-cell non-Hodgkin lymphoma cell lines, serine 194 phosphorylated Fas-associated death domain protein levels are substantially higher in highly proliferating cells and lower in serum-starved cells. We also used immunohistochemical analysis to assess Fas-associated death domain protein phosphorylation at serine 194 expression in 122 B-cell non-Hodgkin-type lymphomas. The mean percentage of serine 194 phosphorylated Fas-associated death domain protein positive tumor cells was 81% in Burkitt lymphoma, 41% in diffuse large B-cell lymphoma, 18% in follicular lymphoma, 18% in plasma cell myeloma, 12% in extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue, 11% in mantle cell lymphoma, and 2% in chronic lymphocytic leukemia/small lymphocytic lymphoma (P < .0001, Kruskal-Wallis test). Furthermore, in chronic lymphocytic leukemia/small lymphocytic lymphoma, serine 194 phosphorylated Fas-associated death domain protein was detected predominantly in proliferation centers. In the entire study group, the percentage of cells positive for serine 194 phosphorylated Fas-associated death domain protein correlated significantly with the proliferation index Ki-67 (Spearman R = 0.9, P < .0001). These data provide evidence that serine 194 phosphorylated Fas-associated death domain protein is involved in the proliferation of normal and neoplastic B cells and has features of a novel proliferation marker.
Assuntos
Ciclo Celular/fisiologia , Proteína de Domínio de Morte Associada a Fas/metabolismo , Linfoma de Células B/patologia , Serina/metabolismo , Biomarcadores Tumorais/metabolismo , Contagem de Células , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Proliferação de Células , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Humanos , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Linfonodos/metabolismo , Linfonodos/patologia , Linfadenite/metabolismo , Linfadenite/patologia , Linfoma de Células B/metabolismo , Fosforilação , Tonsilite/metabolismo , Tonsilite/patologiaRESUMO
BACKGROUND: Less than 50% of ovarian cancers respond to paclitaxel. Effective strategies are needed to enhance paclitaxel sensitivity. METHODS: A library of silencing RNAs (siRNAs) was used to identify kinases that regulate paclitaxel sensitivity in human ovarian cancer SKOv3 cells. The effect of dasatinib, an inhibitor of Src and Abl kinases, on paclitaxel sensitivity was measured in ovarian cancer cells and HEY xenografts. The roles of p27(Kip1), Bcl-2, and Cdk1 in apoptosis induced by dasatinib and paclitaxel were assessed using a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, siRNA knockdown of gene expression, transfection with Bcl-2 and Cdk1 expression vectors, and flow cytometry. All statistical tests were two-sided. RESULTS: Src family and Abl kinases were identified as modulators of paclitaxel sensitivity in SKOv3 cells. The siRNA knockdown of Src, Fyn, or Abl1 enhanced paclitaxel-mediated growth inhibition in ovarian cancer cells compared with a control siRNA. HEY cells treated with dasatinib plus paclitaxel formed fewer colonies than did cells treated with either agent alone. Treatment of HEY xenograft-bearing mice with dasatinib plus paclitaxel inhibited tumor growth more than treatment with either agent alone (average tumor volume per mouse, dasatinib + paclitaxel vs paclitaxel: 0.28 vs. 0.81 cm3, difference = 0.53 cm3, 95% confidence interval [CI] = 0.44 to 0.62 cm3, P = .014); dasatinib + paclitaxel vs. dasatinib: 0.28 vs. 0.55 cm3, difference = 0.27 cm3, 95% CI = 0.21 to 0.33 cm3, P = .035). Combined treatment induced more TUNEL-positive apoptotic cells than did either agent alone. The siRNA knockdown of p27(Kip1) decreased dasatinib- and paclitaxel-induced apoptosis compared with a negative control siRNA (sub-G1 fraction, control siRNA vs. p27(Kip1) siRNA: 42.5% vs. 20.1%, difference = 22.4%, 95% CI = 20.1% to 24.7%, P = .017). Studies with forced expression and siRNA knockdown of Bcl-2 and Cdk1 suggest that dasatinib-mediated induction of p27(Kip1) enhanced paclitaxel-induced apoptosis by negatively regulating Bcl-2 and Cdk1 expression. CONCLUSION: Inhibition of Src family and Abl kinases with either siRNAs or dasatinib enhances paclitaxel sensitivity of ovarian cancer cells through p27(Kip1)-mediated suppression of Bcl-2 and Cdk1 expression.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Pirimidinas/farmacologia , Tiazóis/farmacologia , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Caspase 3/análise , Linhagem Celular Tumoral , Proliferação de Células , Fatores de Confusão Epidemiológicos , Inibidor de Quinase Dependente de Ciclina p27/genética , Dasatinibe , Sinergismo Farmacológico , Feminino , Citometria de Fluxo , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes bcl-1 , Humanos , Immunoblotting , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Nus , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/análise , RNA Interferente Pequeno/metabolismo , Projetos de Pesquisa , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina/metabolismo , Treonina/metabolismo , Tirosina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/antagonistas & inibidoresRESUMO
PURPOSE: To show the functional, clinical, and biological significance of c-Jun-NH(2)-kinase (JNK)-1 in ovarian carcinoma. EXPERIMENTAL DESIGN: Analysis of the impact of JNK on 116 epithelial ovarian cancers was conducted. The role of JNK in vitro and in experimental models of ovarian cancer was assessed. We studied the role of N-5-[4-(4-methyl piperazine methyl)-benzoylamido]-2-methylphenyl-4-[3-(4-methyl)-pyridyl]-2-pyrimidine amine (WBZ_4), a novel JNK inhibitor redesigned from imatinib based on targeting wrapping defects, in cell lines and in experimental models of ovarian cancer. RESULTS: We found a significant association of pJNK with progression-free survival in the 116 epithelial ovarian cancers obtained at primary debulking therapy. WBZ_4 led to cell growth inhibition and increased apoptosis in a dose-dependent fashion in four ovarian cancer cell lines. In vivo, whereas imatinib had no effect on tumor growth, WBZ_4 inhibited tumor growth in orthotopic murine models of ovarian cancer. The antitumor effect was further increased in combination with docetaxel. Silencing of JNK-1 with systemically administered siRNA led to significantly reduced tumor weights compared with nonsilencing siRNA controls, indicating that indeed the antitumor effects observed were due to JNK-1 inhibition. CONCLUSIONS: These studies identify JNK-1 as an attractive therapeutic target in ovarian carcinoma and that the redesigned WBZ_4 compound should be considered for further clinical development.
Assuntos
Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/enzimologia , Piperazinas/farmacologia , Pirimidinas/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Indutores da Angiogênese , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Neovascularização Patológica/tratamento farmacológico , RNA Interferente Pequeno/farmacologiaRESUMO
JunB is a member of the Jun family of proteins that are components of the AP-1 transcription factor complex. AP-1 is involved in cell proliferation and apoptosis. Recent evidence suggests that Hodgkin and Reed-Sternberg cells overexpress JunB and that JunB facilitates constitutive CD30 expression by binding to an AP-1 site in the CD30 promoter. In this study we surveyed JunB expression in a variety of CD30+ lymphoma types including 42 cases of anaplastic large cell lymphoma, 36 classical Hodgkin lymphoma, 15 cutaneous anaplastic large cell lymphoma, and 11 CD30+ diffuse large B-cell lymphoma. In addition, seven cases of nodular lymphocyte-predominant Hodgkin lymphoma and 42 diffuse large B-cell lymphoma, known to be CD30-, were analyzed. JunB expression was assessed using tissue microarrays, immunohistochemistry and a monoclonal antibody specific for JunB. Expression of JunB was observed in 41 of 42 cases of anaplastic large cell lymphoma, including all 21 cases positive for anaplastic lymphoma kinase and 20 of 21 (95%) negative for anaplastic lymphoma kinase. JunB was also expressed in all cases of classical Hodgkin lymphoma, cutaneous anaplastic large cell lymphoma and CD30+ diffuse large B-cell lymphoma, and in lymphomatoid papulosis. By contrast, all nodular lymphocyte-predominant Hodgkin lymphomas and diffuse large B-cell lymphomas that were CD30- were also JunB-. We conclude that JunB is expressed in virtually all CD30+ lymphomas and is a potential target for experimental therapy in patients with these tumors.
Assuntos
Antígeno Ki-1/metabolismo , Linfoma/metabolismo , Papulose Linfomatoide/metabolismo , Proteínas Proto-Oncogênicas c-jun/biossíntese , Anticorpos Monoclonais , Biomarcadores Tumorais/biossíntese , Humanos , Imuno-Histoquímica , Linfonodos/metabolismo , Proteínas Proto-Oncogênicas c-jun/imunologia , Análise Serial de TecidosRESUMO
Anaplastic large cell lymphoma (ALCL) is a highly proliferative neoplasm that frequently carries the t(2;5)(p23;q35) and aberrantly expresses nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). Previously, NPM-ALK had been shown to activate the phosphatidylinositol 3 kinase (PI3K)/Akt pathway. As the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) (p27) is usually not expressed in ALCL, we hypothesized that activated Akt (pAkt) phosphorylates p27 resulting in increased p27 proteolysis and cell cycle progression. Here we demonstrate that inhibition of pAkt activity in ALCL decreases p27 phosphorylation and degradation, resulting in increased p27 levels and cell cycle arrest. Using immunohistochemistry, pAkt was detected in 24 (57%) of 42 ALCL tumors, including 8 (44%) of 18 ALK-positive tumors and 16 (67%) of 24 ALK-negative tumors, and was inversely correlated with p27 levels. The mean percentage of p27-positive tumor cells was 5% in the pAkt-positive group compared with 26% in the pAkt-negative group (P = .0076). These findings implicate that Akt activation promotes cell cycle progression through inactivation of p27 in ALCL.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p27 , Citoplasma/metabolismo , Regulação para Baixo , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-aktRESUMO
Cyclin-dependent kinase (CDK) inhibitor p27Kip1 binds to the cyclin E.CDK2 complex and plays a major role in controlling cell cycle and cell growth. Our group and others have reported that anti-HER2 monoclonal antibodies exert inhibitory effects on HER2-overexpressing breast cancers through G1 cell cycle arrest associated with induction of p27Kip1 and reduction of CDK2. The role of p27Kip1 in anti-HER2 antibody-induced cell cycle arrest and growth inhibition is, however, still uncertain. Here we have provided several lines of evidence supporting a critical role for p27Kip1 in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition. Induction of p27Kip1 and G1 growth arrest by anti-HER2 antibody, murine 4D5, or humanized trastuzumab (Herceptin) are concentration-dependent, time-dependent, irreversible, and long-lasting. The magnitude of G1 cell cycle arrest induced by trastuzumab or 4D5 is well correlated with the level of p27Kip1 protein induced. Up-regulation of p27Kip1 and G1 growth arrest could no longer be removed with as little as 14 h of treatment with trastuzumab. Anti-HER2 antibody-induced p27Kip1 protein, G1 arrest, and growth inhibition persist at least 5 days after a single treatment. The magnitude of growth inhibition of breast cancer cells induced by anti-HER2 antibody closely parallels the level of p27Kip1 induced. Induced expression of exogenous p27Kip1 results in a p27Kip1 level-dependent G1 cell cycle arrest and growth inhibition similar to that obtained with anti-HER2 antibodies. Reducing p27Kip1 expression using p27Kip1 small interfering RNA blocks anti-HER2 antibody-induced p27Kip1 up-regulation and G1 arrest. Treatment with anti-HER2 antibody significantly increases the half-life of p27Kip1 protein. Inhibition of ubiquitin-proteasome pathway, but not inhibition of calpain and caspase activities, up-regulates p27Kip1 protein to a degree comparable with that obtained with anti-HER2 antibodies. We have further demonstrated that anti-HER2 antibody significantly decreases threonine phosphorylation of p27Kip1 protein at position 187 (Thr-187) and increases serine phosphorylation of p27Kip1 protein at position 10 (Ser-10). Expression of S10A and T187A mutant p27Kip1 protein increases the fraction of cells in G1 and reduces a further antibody-induced G1 arrest. Consequently, p27Kip1 plays an important role in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition through post-translational regulation. Regulation of the phosphorylation of p27Kip1 protein is one of the post-translational mechanisms by which anti-HER2 antibody upregulates the protein.