RESUMO
Chronic activation of the phosphoinositide 3-kinase (PI3K)/PTEN signal transduction pathway contributes to metastatic cell growth, but up to now effectors mediating this response are poorly defined. By simulating chronic activation of PI3K signaling experimentally, combined with three-dimensional (3D) culture conditions and gene expression profiling, we aimed to identify novel effectors that contribute to malignant cell growth. Using this approach we identified and validated PKN3, a barely characterized protein kinase C-related molecule, as a novel effector mediating malignant cell growth downstream of activated PI3K. PKN3 is required for invasive prostate cell growth as assessed by 3D cell culture assays and in an orthotopic mouse tumor model by inducible expression of short hairpin RNA (shRNA). We demonstrate that PKN3 is regulated by PI3K at both the expression level and the catalytic activity level. Therefore, PKN3 might represent a preferred target for therapeutic intervention in cancers that lack tumor suppressor PTEN function or depend on chronic activation of PI3K.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Membrana Basal/enzimologia , Membrana Basal/metabolismo , Membrana Basal/patologia , Catálise , Divisão Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Modelos Animais de Doenças , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , Masculino , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase , Fosfatidilinositol 3-Quinases/genética , Neoplasias da Próstata/genética , Proteína Quinase C/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Fosfatases/deficiência , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Regulação para CimaRESUMO
Smad transcription factors mediate the growth inhibitory effect of transforming growth factor-beta (TGF-beta) in many cell types. Mutational inactivation of Smads has been correlated with loss of responsiveness to TGF-beta-mediated signal transduction. In this study, we compare the contribution of individual Smads to TGF-beta-induced growth inhibition and endogenous gene expression in isogenic cellular backgrounds. Smad2, Smad3 and Smad4 expression were selectively inhibited in differentiation-competent cells by using improved antisense molecules. We found that TGF-beta mediates its inhibitory effect on HaCaT keratinocyte cell growth predominantly through Smad3. Inhibition of Smad3 expression was sufficient to interfere with TGF-beta-induced cell cycle arrest and to induce or suppress endogenous cell cycle regulators. Inhibition of Smad4 expression exhibited a partial effect, whereas inhibition of Smad2 expression had no effect. By gene expression profiling, we identified TGF-beta-dependent genes that are differentially regulated by Smad2 and Smad3 under regular growth conditions on a genome-wide scale. We show that Smad2, Smad3 and Smad4 contribute to the regulation of TGF-beta responses to varying extents, and demonstrate, in addition, that these Smads exhibit distinct roles in different cell types.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica , Transdução de Sinais , Transativadores/fisiologia , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Western Blotting , Ciclo Celular , Linhagem Celular , Análise Mutacional de DNA , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Humanos , Immunoblotting , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos Antissenso/farmacologia , RNA/metabolismo , RNA Mensageiro/metabolismo , Proteína Smad2 , Proteína Smad3 , Proteína Smad4 , Transfecção , Fator de Crescimento Transformador beta/metabolismoRESUMO
The study of signal transduction processes using antisense oligonucleotides is often complicated by low intracellular stability of the antisense reagents or by nonspecific effects that cause toxicity. Here, we introduce a new class of antisense molecules, so-called GeneBlocs, which are characterized by improved stability, high target RNA specificity, and low toxicity. GeneBlocs allow for efficient downregulation of mRNA expression at nanomolar concentrations, and they do not interfere with cell proliferation. We demonstrate these beneficial properties using a positive readout system. GeneBloc-mediated inhibition of tumor suppressor PTEN (phosphatase and tension homologue detected on chromosome 10) expression leads to hyperactivation of the phosphatidylinositol (PI) 3-kinase pathway, thereby mimicking the loss of PTEN function and its early consequences observed in mammalian cancer cells. Specifically, cells treated with PTEN GeneBlocs show functional activation of Akt, a downstream effector of PI 3-kinase signaling, and exhibit enhanced proliferation when seeded on a basement membrane matrix. In addition, GeneBlocs targeting the catalytic subunit of PI 3-kinase, p110, specifically inhibit signal transduction of endogenous or recombinant PI 3-kinase. This demonstrates that GeneBlocs are powerful tools to analyze and to modulate signal transduction processes and, therefore, represent alternative reagents for the validation of gene function.