RESUMO
Herein, we show that the hematopoietic-specific GEF VAV1 is ectopically expressed in primary pancreatic adenocarcinomas due to demethylation of the gene promoter. Interestingly, VAV1-positive tumors had a worse survival rate compared to VAV1-negative tumors. Surprisingly, even in the presence of oncogenic KRAS, VAV1 RNAi abrogates neoplastic cellular proliferation in vitro and in vivo, thus identifying Vav1 as a growth-stimulatory protein in this disease. Vav1 acts synergistically with the EGF receptor to stimulate pancreatic tumor cell proliferation. Mechanistically, the effects of Vav1 require its GEF activity and the activation of Rac1, PAK1, and NF-kappaB and involve cyclin D1 upregulation. Thus, the discovery of prooncogenic pathways regulated by Vav1 makes it an attractive target for therapeutic intervention.
Assuntos
Adenocarcinoma/metabolismo , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Ciclina D1/genética , Ciclina D1/metabolismo , Metilação de DNA , Fator de Crescimento Epidérmico/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/fisiopatologia , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-vav , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Taxa de Sobrevida , Quinases Ativadas por p21 , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Although all three Vav family members are expressed in T lymphocytes, the role that Vav3 plays in T cell activation is poorly defined. Here we show that, like Vav1, Vav3 undergoes rapid tyrosine phosphorylation after T cell receptor (TCR) cross-linkage and interacts with the adaptor molecules SLP76 and 3BP2 in a SH2-dependent manner. However, depletion of Vav1 but not Vav3 protein by RNA interference affects TCR-mediated IL-2 promoter activity. In contrast, Vav3 function is specifically required for coupling TCR stimulation to serum response element-mediated gene transcription. These data indicate that, although both Vav proteins are biochemically coupled to the TCR, they regulate distinct molecular pathways leading to defined gene transcriptional events.
Assuntos
Proteínas de Ciclo Celular , Proteínas Oncogênicas/imunologia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Regulação da Expressão Gênica/imunologia , Fatores de Troca do Nucleotídeo Guanina , Humanos , Células Jurkat , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas c-vav , Transcrição Gênica/imunologiaRESUMO
The discovery of the rules governing the inhibition of the various HDAC isoforms is likely to be key to identifying improved therapeutics that act as epigenetic modulators of gene transcription. Herein we present results on the modification of the CAP region of a set of triazolylphenyl-based HDACIs, and show that the nature of substitution on the phenyl ring plays a role in their selectivity for HDAC1 versus HDAC6, with low to moderate selectivity (2-51-fold) being achieved. In light of the valuable selectivity and potency that were identified for the triazolylphenyl ligand 6b in the inhibition of HDAC6 (IC50 = 1.9 nM), this compound represents a valuable research tool and a candidate for further chemical modifications. Lastly, these new HDACIs were studied for both their anticancer and antimalarial activity, which serve to validate the superior activity of the HDACI 10c.
Assuntos
Antimaláricos/síntese química , Antineoplásicos/síntese química , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/síntese química , Plasmodium falciparum/efeitos dos fármacos , Triazóis/síntese química , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Resistência a Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Isoenzimas/antagonistas & inibidores , Neoplasias Pancreáticas , Testes de Sensibilidade Parasitária , Plasmodium falciparum/enzimologia , Triazóis/química , Triazóis/farmacologiaRESUMO
NF-kappaB is an ubiquitous transcription factor that is a key in the regulation of the immune response and inflammation. T-cell receptor (TCR) cross-linking leads to NF-kappaB activation, an IkappaB kinase (IKK)-dependent process. However, the upstream kinases that regulate IKK activity following TCR activation remain to be fully characterized. Herein, we demonstrate using genetic analysis, pharmacological inhibition, and RNA interference (RNAi) that the conventional protein kinase C (PKC) isoform PKCalpha, but not PKCbeta1, is required for the activation of the IKK complex following T-cell activation triggered by CD3/CD28 cross-linking. We find that in the presence of Ca(2+) influx, the catalytically active PKCalphaA25E induces IKK activity and NF-kappaB-dependent transcription; which is abrogated following the mutations of two aspartates at positions 246 and 248, which are required for Ca(2+) binding to PKCalpha and cell membrane recruitment. Kinetic studies reveal that an early phase (1 to 5 min) of IKK activation following TCR/CD28 cross-linking is PKCalpha dependent and that a later phase (5 to 25 min) of IKK activation is PKCtheta dependent. Activation of IKK- and NF-kappaB-dependent transcription by PKCalphaA25E is abrogated by the PKCtheta inhibitor rottlerin or the expression of the kinase-inactive form of PKCtheta. Taken together, our results suggest that PKCalpha acts upstream of PKCtheta to activate the IKK complex and NF-kappaB in T lymphocytes following TCR activation.
Assuntos
Isoenzimas/metabolismo , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Linfócitos T/metabolismo , Acetofenonas/farmacologia , Ácido Aspártico/genética , Benzopiranos/farmacologia , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Cálcio/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Humanos , Quinase I-kappa B , Interleucina-2/genética , Isoenzimas/efeitos dos fármacos , Células Jurkat , Ativação Linfocitária , Mutação Puntual , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C-alfa , Proteína Quinase C-theta , Linfócitos T/enzimologia , Transcrição Gênica , Dedos de ZincoRESUMO
Recent studies using glycogen synthase kinase-3beta (GSK-3beta)-deficient mouse embryonic fibroblasts suggest that GSK-3beta positively regulates nuclear factor kappaB (NFkappaB)-mediated gene transcription. Because NFkappaB is suggested to participate in cell proliferation and survival pathways in pancreatic cancer, we investigated the role of GSK-3beta in regulating these cellular processes. Herein, we show that pancreatic cancer cells contain a pool of active GSK-3beta and that pharmacologic inhibition of GSK-3 kinase activity using small molecule inhibitors or genetic depletion of GSK-3beta by RNA interference leads to decreased cancer cell proliferation and survival. Mechanistically, we show that GSK-3beta influences NFkappaB-mediated gene transcription at a point distal to the Ikappa kinase complex, as only ectopic expression of the NFkappaB subunits p65/p50, but not an Ikappa kinase beta constitutively active mutant, could rescue the decreased cellular proliferation and survival associated with GSK-3beta inhibition. Taken together, our results simultaneously identify a previously unrecognized role for GSK-3beta in cancer cell survival and proliferation and suggest GSK-3beta as a potential therapeutic target in the treatment of pancreatic cancer.
Assuntos
Quinase 3 da Glicogênio Sintase/fisiologia , NF-kappa B/fisiologia , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Transcrição Gênica/fisiologia , Ureia/análogos & derivados , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Neoplasias Pancreáticas/patologia , Tiazóis/farmacologia , Ureia/farmacologiaRESUMO
Our triazole-based histone deacetylase inhibitor (HDACI), octanedioic acid hydroxyamide[3-(1-phenyl-1H-[1,2,3]triazol-4-yl)phenyl]amide (4a), suppresses pancreatic cancer cell growth in vitro with the lowest IC(50) value of 20 nM against MiaPaca-2 cell. In this study, we continued our efforts to develop triazol-4-ylphenyl bearing hydroxamate analogues by embellishing the terminal phenyl ring of 4a with different substituents. The isoform inhibitory profile of these hydroxamate analogues was similar to those of 4a. All of these triazol-4-ylphenyl bearing hydroxamates are pan-HDACIs like SAHA. Moreover, compounds 4h and 11a were found to be very effective inhibitors of cancer cell growth in the HupT3 (IC(50) = 50 nM) and MiaPaca-2 (IC(50) = 40 nM) cancer cell lines, respectively. Compound 4a was found to reactivate the expression of CDK inhibitor proteins and to suppress pancreatic cancer cell growth in vivo. Taken together, these data further support the value of the triazol-4-ylphenyl bearing hydroxamates in identifying potential pancreatic cancer therapies.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Triazóis/síntese química , Triazóis/farmacologia , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/farmacologia , Feminino , Inibidores de Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/química , Concentração Inibidora 50 , Camundongos , Camundongos Nus , Modelos Moleculares , Relação Estrutura-Atividade , Triazóis/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The histone deacetylases (HDACs) are able to regulate gene expression, and inhibitors of the HDACs (HDACIs) hold promise in the treatment of cancer as well as a variety of neurodegenerative diseases. To investigate the potential for isoform selectivity in the inhibition of HDACs, we prepared a small series of 2,4'-diaminobiphenyl ligands functionalized at the para-amino group with an appendage containing either a hydroxamate or a mercaptoacetamide group and coupled to an amino acid residue at the ortho-amino group. A smaller series of substituted phenylthiazoles was also explored. Some of these newly synthesized ligands show low-nanomolar potency in HDAC inhibition assays and display micromolar to low-nanomolar IC(50) values in tests against five pancreatic cancer cell lines. The isoform selectivity of these ligands for class I HDACs (HDAC1-3 and 8) and class IIb HDACs (HDAC6 and 10) together with QSAR studies of their correlation with lipophilicity are presented. Of particular interest is the selectivity of the mercaptoacetamides for HDAC6.
Assuntos
Acetamidas/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Compostos de Sulfidrila/farmacologia , Acetamidas/síntese química , Compostos Bicíclicos com Pontes/síntese química , Linhagem Celular Tumoral , Inibidores Enzimáticos/síntese química , Humanos , Ácidos Hidroxâmicos/síntese química , Concentração Inibidora 50 , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologia , Relação Quantitativa Estrutura-Atividade , Compostos de Sulfidrila/síntese químicaRESUMO
Actin reorganization at the immunological synapse is required for the amplification and generation of a functional immune response. Using small interfering RNA, we show here that dynamin 2 (Dyn2), a large GTPase involved in receptor-mediated internalization, did not alter antibody-mediated T cell receptor internalization but considerably affected T cell receptor-stimulated T cell activation by regulating multiple biochemical signaling pathways and the accumulation of F-actin at the immunological synapse. Moreover, Dyn2 interacted directly with the Rho family guanine nucleotide exchange factor Vav1, and this interaction was required for T cell activation. These data identify a functionally important interaction between Dyn2 and Vav1 that regulates actin reorganization and multiple signaling pathways in T lymphocytes.