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
Phosphoinositide 3-kinase (PI3K) is activated in pancreatic cancer cells and plays a central role in their proliferation, survival, and drug resistance. Although the mechanism is unclear, PI3K activation in these cells could be due to physical interaction between its regulatory subunit (p85) and specific tyrosine kinases or their mediators. Consistent with this possibility, PI3K was precipitated with anti-phosphotyrosine antibodies and Akt phosphorylation was blocked by the tyrosine kinase inhibitors SU6656 and PD158780 in quiescent pancreatic cancer cells. Pull-down assays with a fusion protein (GST-p85NC-SH2), and coimmunoprecipitation studies, indicated that the insulin receptor substrate (IRS), and not the epidermal growth factor and insulin-like growth factor receptors or the Src tyrosine kinase, was physically associated with PI3K in these cells. Our data also indicated that SU6656 and PD158780 inhibited Akt activation in pancreatic cancer cells by interfering with the ability of IRS-1 to recruit PI3K. Furthermore, IRS-1 was phosphorylated on a p85-binding site (Y(612)), and IRS-specific small interfering RNA potently inhibited activation of PI3K and Akt in transfected cells. Taken together, these observations indicate that IRS is a mediator of PI3K activation in quiescent pancreatic cancer cells.