RESUMO
Glioblastoma is the most malignant and most common form of brain tumor, still today associated with a poor 14-months median survival from diagnosis. Protein kinase A, particularly its regulatory subunit R2Alpha, presents a typical intracellular distribution in glioblastoma cells compared to the healthy brain parenchyma and this peculiarity might be exploited in a therapeutic setting. In the present study, a third-generation lentiviral system for delivery of shRNA targeting the regulatory subunit R2Alpha of protein kinase A was developed. Generated lentiviral vectors are able to induce an efficient and stable downregulation of R2Alpha in different cellular models, including non-stem and stem-like glioblastoma cells. In addition, our data suggest a potential correlation between silencing of the regulatory subunit of protein kinase A and reduced viability of tumor cells, apparently due to a reduction in replication rate. Thus, our findings support the role of protein kinase A as a promising target for novel anti-glioma therapies.
Assuntos
Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Glioblastoma/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Glioblastoma/genética , Glioblastoma/fisiopatologia , Glioma/genética , Glioma/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Transdução Genética/métodosRESUMO
Transforming growth factor ß (TGFß) signaling normally functions to regulate embryonic development and cellular homeostasis. It is increasingly recognized that TGFß signaling is regulated by cross-talk with other signaling pathways. We previously reported that TGFß activates protein kinase A (PKA) independent of cAMP through an interaction of an activated Smad3-Smad4 complex and the regulatory subunit of the PKA holoenzyme (PKA-R). Here we define the interaction domains of Smad4 and PKA-R and the functional consequences of this interaction. Using a series of Smad4 and PKA-R truncation mutants, we identified amino acids 290-300 of the Smad4 linker region as critical for the specific interaction of Smad4 and PKA-R. Co-immunoprecipitation assays showed that the B cAMP binding domain of PKA-R was sufficient for interaction with Smad4. Targeting of B domain regions conserved among all PKA-R isoforms and exposed on the molecular surface demonstrated that amino acids 281-285 and 320-329 were required for complex formation with Smad4. Interactions of these specific regions of Smad4 and PKA-R were necessary for TGFß-mediated increases in PKA activity, CREB (cAMP-response element-binding protein) phosphorylation, induction of p21, and growth inhibition. Moreover, this Smad4-PKA interaction was required for TGFß-induced epithelial mesenchymal transition, invasion of pancreatic tumor cells, and regulation of tumor growth in vivo.
