RESUMEN
Extracellular adenosine, produced through the activity of ecto-5'-nucleotidase CD73, elicits potent immunosuppressive effects, and its upregulation in tumor cells as well as in stromal and immune cell subsets within the tumor microenvironment is hypothesized to represent an important resistance mechanism to current cancer immunotherapies. Soluble CD73 (sCD73) enzymatic activity measured in patient serum or plasma at a baseline is reported to have prognostic as well as predictive relevance, with higher sCD73 activity associating with poor overall and progression-free survival in melanoma patients undergoing anti-PD1 monoclonal antibody treatment. Here, we report a novel NMR-based method that measures the ex-vivo kinetics of sCD73 activity with high specificity and reproducibility and is suitable for future high-throughput implementation. Unlike the existing assays, this method has the advantage of directly and simultaneously measuring the concentration of both the CD73 substrate and product with minimal sample manipulation or special reagents. We establish the utility of the assay for measuring the activity of sCD73 in human serum and show a strong linear correlation between sCD73 protein levels and enzyme activity. Together with our finding that sCD73 appears to be the predominant activity for the generation of adenosine in human blood, our results demonstrate a link between activity and protein levels that will inform future clinical application.
Asunto(s)
5'-Nucleotidasa/sangre , 5'-Nucleotidasa/química , Pruebas de Enzimas/métodos , Espectroscopía de Resonancia Magnética , Métodos Analíticos de la Preparación de la Muestra , Tampones (Química) , Humanos , Cinética , SolubilidadRESUMEN
Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S-adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)-specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Met-cycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co-regulated in models of cancer cell differentiation and co-expression is observed at the invasive front of human lung tumors. EZH2 knockdown or small-molecule inhibition leads to de-repression of RXRα resulting in reduced Lat1 expression. Our results describe a Lat1-EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA-mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target.
Asunto(s)
Aminoácidos/metabolismo , Epigénesis Genética/fisiología , Transportador de Aminoácidos Neutros Grandes 1/fisiología , Complejo Represivo Polycomb 2/fisiología , Animales , Transporte Biológico/genética , Proliferación Celular/genética , Proteína Potenciadora del Homólogo Zeste 2 , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Tumorales CultivadasRESUMEN
Cereblon (CRBN), a substrate receptor of the Cullin 4 RING E3 ubiquitin ligase complex, is the target of the immunomodulatory drugs lenalidomide and pomalidomide. Recently, it was demonstrated that binding of these drugs to CRBN promotes the ubiquitination and subsequent degradation of 2 common substrates, transcription factors Aiolos and Ikaros. Here we report that CC-122, a new chemical entity termed pleiotropic pathway modifier, binds CRBN and promotes degradation of Aiolos and Ikaros in diffuse large B-cell lymphoma (DLBCL) and T cells in vitro, in vivo, and in patients, resulting in both cell autonomous as well as immunostimulatory effects. In DLBCL cell lines, CC-122-induced degradation or short hairpin RNA-mediated knockdown of Aiolos and Ikaros correlates with increased transcription of interferon (IFN)-stimulated genes independent of IFN-α, -ß, and -γ production and/or secretion and results in apoptosis in both activated B-cell (ABC) and germinal center B-cell DLBCL cell lines. Our results provide mechanistic insight into the cell-of-origin independent antilymphoma activity of CC-122, in contrast to the ABC subtype selective activity of lenalidomide.
Asunto(s)
Antineoplásicos/farmacología , Linfocitos B/efectos de los fármacos , Factor de Transcripción Ikaros/genética , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Péptido Hidrolasas/genética , Piperidonas/farmacología , Quinazolinonas/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales , Animales , Antineoplásicos/química , Linfocitos B/metabolismo , Linfocitos B/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Factor de Transcripción Ikaros/metabolismo , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Interferones/genética , Interferones/metabolismo , Lenalidomida , Lentivirus/genética , Lentivirus/metabolismo , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Ratones , Ratones SCID , Imitación Molecular , Péptido Hidrolasas/metabolismo , Piperidonas/química , Proteolisis/efectos de los fármacos , Quinazolinonas/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal/genética , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Linfocitos T/patología , Talidomida/análogos & derivados , Talidomida/farmacología , Ubiquitina-Proteína Ligasas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cereblon (CRBN), the molecular target of lenalidomide and pomalidomide, is a substrate receptor of the cullin ring E3 ubiquitin ligase complex, CRL4(CRBN) . T cell co-stimulation by lenalidomide or pomalidomide is cereblon dependent: however, the CRL4(CRBN) substrates responsible for T cell co-stimulation have yet to be identified. Here we demonstrate that interaction of the transcription factors Ikaros (IKZF1, encoded by the IKZF1 gene) and Aiolos (IKZF3, encoded by the IKZF3 gene) with CRL4(CRBN) is induced by lenalidomide or pomalidomide. Each agent promotes Aiolos and Ikaros binding to CRL4(CRBN) with enhanced ubiquitination leading to cereblon-dependent proteosomal degradation in T lymphocytes. We confirm that Aiolos and Ikaros are transcriptional repressors of interleukin-2 expression. The findings link lenalidomide- or pomalidomide-induced degradation of these transcriptional suppressors to well documented T cell activation. Importantly, Aiolos could serve as a proximal pharmacodynamic marker for lenalidomide and pomalidomide, as healthy human subjects administered lenalidomide demonstrated Aiolos degradation in their peripheral T cells. In conclusion, we present a molecular model in which drug binding to cereblon results in the interaction of Ikaros and Aiolos to CRL4(CRBN) , leading to their ubiquitination, subsequent proteasomal degradation and T cell activation.
Asunto(s)
Factor de Transcripción Ikaros/metabolismo , Péptido Hidrolasas/metabolismo , Linfocitos T/efectos de los fármacos , Talidomida/análogos & derivados , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Inhibidores de la Angiogénesis/farmacología , Humanos , Factor de Transcripción Ikaros/genética , Factores Inmunológicos/farmacología , Lenalidomida , Péptido Hidrolasas/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Linfocitos T/metabolismo , Talidomida/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.
Asunto(s)
Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/genética , Animales , Dominio Catalítico/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Perros , Ensayos de Selección de Medicamentos Antitumorales/métodos , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-TransferidoraRESUMEN
We have identified TRB3, a human homologue of Drosophila tribbles, as a novel transcriptional target of phosphatidylinositol (PI) 3-kinase. TRB3 expression is remarkably reduced in prostate cancer PC-3 cells after inhibition of PI 3-kinase. TRB3 expression is furthermore controlled by nutrient supplies: Both the lack of glucose or amino acids results in a substantial increase in TRB3 protein levels in a PI 3-kinase-dependent manner. This increase is reversed by the addition of fresh nutrients. Stress stimuli, such as osmotic stress, hypoxia or serum starvation do not affect TRB3 expression. Thus, TRB3 may function as a nutrient sensor. Inhibition of TRB3 expression has no effect on growth of PC-3 cells under regular growth conditions. However, in the absence of glucose overexpression of TRB3 in PC-3 cells can interfere with apoptosis and restore growth on extracellular matrix. Taken together, our data point to an important role of TRB3 in sensing reduced nutrient supplies and in providing survival signals during these periods.
Asunto(s)
Aminoácidos Esenciales/deficiencia , Proteínas de Ciclo Celular/metabolismo , Glucosa/deficiencia , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Represoras/metabolismo , Aminoácidos Esenciales/farmacología , Proteínas de Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Glucosa/farmacología , Células HeLa , Humanos , Masculino , Fosfatidilinositol 3-Quinasas/farmacología , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/biosíntesis , Proteínas Represoras/efectos de los fármacos , Proteínas Represoras/genética , Transducción de Señal , Activación TranscripcionalRESUMEN
Cancer cells frequently evade apoptosis during tumorigenesis by acquiring mutations in apoptotic regulators. Chronic activation of the PI 3-kinase-Akt pathway through loss of the tumor suppressor PTEN is one mechanism by which these cells can gain increased protection against apoptosis. We report here that REDD1 (RTP801) can act as a transcriptional downstream target of PI 3-kinase signaling in human prostate cancer cells (PC-3). REDD1 expression is markedly reduced in PC-3 cells treated with LY294002 (LY) or Rapamycin and strongly induced under hypoxic conditions in a hypoxia-inducible factor-1 (HIF-1)-dependent manner. Loss of function studies employing antisense molecules or RNA interference indicate that REDD1 is essential for invasive growth of prostate cancer cells in vitro and in vivo. Reduced REDD1 levels can sensitize cells towards apoptosis, whereas elevated levels of REDD1 induced by hypoxia or overexpression desensitize cells to apoptotic stimuli. Taken together our data designate REDD1 as a novel target for therapeutic intervention in prostate cancer.
Asunto(s)
Fosfatidilinositol 3-Quinasas/fisiología , Neoplasias de la Próstata/metabolismo , Factores de Transcripción/fisiología , Apoptosis , Hipoxia de la Célula , Línea Celular Tumoral , Cromonas/farmacología , Cobalto/farmacología , Dimetilsulfóxido/farmacología , Expresión Génica/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Masculino , Morfolinas/farmacología , Invasividad Neoplásica/genética , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/inmunología , ARN sin Sentido/genética , Transducción de Señal , Sirolimus/farmacología , Factores de Transcripción/análisis , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
RNA interference (RNAi) is a RNA-mediated sequence-specific gene silencing mechanism. Recently, this mechanism has been used to down-regulate protein expression in mammalian cells by applying synthetic- or vector-generated small interfering RNAs (siRNAs). However, for the evaluation of this new knockdown technology, it is crucial to demonstrate biological consequences beyond protein level reduction. Here, we demonstrate that this new siRNA-based technology is suitable to analyse protein functions using the phosphatidylinositol (PI) 3-kinase signal transduction pathway as a model system. We demonstrate stable and transient siRNA-mediated knockdown of one of the PI 3-kinase catalytic subunits, p110beta, which leads to inhibition of invasive cell growth in vitro as well as in a tumour model system. Importantly, this result is consistent with loss-of-function phenotypes induced by conventional RNase H-dependent antisense molecules or treatment with the PI 3-kinase inhibitor LY294002. RNAi knockdown of the downstream kinases Akt1 and Akt2 does not reduce cell growth on extracellular matrix. Our data show that synthetic siRNAs, as well as vector-based expression of siRNAs, are a powerful new tool to interfere with signal transduction processes for the elucidation of gene function in mammalian cells.
Asunto(s)
Fosfatidilinositol 3-Quinasas/genética , ARN Interferente Pequeño/genética , Transducción de Señal , Animales , Dominio Catalítico/genética , Dominio Catalítico/fisiología , División Celular/genética , División Celular/fisiología , Expresión Génica , Células HeLa , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Conformación de Ácido Nucleico , Fosfohidrolasa PTEN , Fosfatidilinositol 3-Quinasas/fisiología , Monoéster Fosfórico Hidrolasas/genética , Regiones Promotoras Genéticas/genética , Interferencia de ARN , ARN Polimerasa III/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/metabolismo , Trasplante Heterólogo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Double-stranded short interfering RNAs (siRNA) induce post-transcriptional silencing in a variety of biological systems. In the present study we have investigated the structural requirements of chemically synthesised siRNAs to mediate efficient gene silencing in mammalian cells. In contrast to studies with Drosophila extracts, we found that synthetic, double-stranded siRNAs without specific nucleotide overhangs are highly efficient in gene silencing. Blocking of the 5'-hydroxyl terminus of the antisense strand leads to a dramatic loss of RNA interference activity, whereas blocking of the 3' terminus or blocking of the termini of the sense strand had no negative effect. We further demonstrate that synthetic siRNA molecules with internal 2'-O-methyl modification, but not molecules with terminal modifications, are protected against serum-derived nucleases. Finally, we analysed different sets of siRNA molecules with various 2'-O-methyl modifications for stability and activity. We demonstrate that 2'-O-methyl modifications at specific positions in the molecule improve stability of siRNAs in serum and are tolerated without significant loss of RNA interference activity. These second generation siRNAs will be better suited for potential therapeutic application of synthetic siRNAs in vivo.
Asunto(s)
Proteínas Proto-Oncogénicas , Interferencia de ARN , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Endorribonucleasas/metabolismo , Células HeLa , Humanos , Metilación , Oligonucleótidos Antisentido/genética , Fosfohidrolasa PTEN , Fosfatidilinositol 3-Quinasas/biosíntesis , Fosfatidilinositol 3-Quinasas/genética , Monoéster Fosfórico Hidrolasas/biosíntesis , Monoéster Fosfórico Hidrolasas/genética , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt , Estabilidad del ARN , ARN Bicatenario/química , ARN Bicatenario/genética , Transfección , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genéticaRESUMEN
RNA interference (RNAi) is a powerful tool to induce loss-of-function phenotypes by inhibiting gene expression post-transcriptionally. Synthetic short interfering RNAs (siRNAs) as well as vector-based siRNA expression systems have been used successfully to silence gene expression in a variety of biological systems. We describe the development of an inducible siRNA expression system that is based on the tetracycline repressor and eukaryotic RNA polymerase III promoters (U6 and 7SK). For proof of concept we selectively inhibited expression of two catalytic subunits of the phosphatidylinositol 3-kinase (PI 3-kinase), p110alpha and p110beta, by using vector-derived short hairpin RNAs (shRNAs). Stable pools of human prostate cancer cells (PC-3) exhibiting reduced levels of both PI 3-kinase catalytic subunits due to the expression of corresponding shRNAs in an inducible fashion were established and analyzed for their invasive potential in vitro as well as in an orthotopic metastatic mouse model. This inducible system for RNAi allows an unbiased and comparable analysis of loss-of-function phenotypes by comparing selected isogenic cell populations on the induced and non-induced level. In addition, conditional RNAi allows the study of essential and multifunctional genes involved in complex biological processes by preventing inhibitory and compensatory effects caused by constitutive knockdown.
Asunto(s)
Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Conformación de Ácido Nucleico , Neoplasias de la Próstata/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Animales , Dominio Catalítico , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas/biosíntesis , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/genética , Regiones Promotoras Genéticas/genética , Neoplasias de la Próstata/enzimología , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/genética , Interferencia de ARN , ARN Polimerasa III/genética , ARN Interferente Pequeño/química , Tetraciclina/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Transducción de Señal , Transactivadores/fisiología , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta/genética , Western Blotting , Ciclo Celular , Línea Celular , Análisis Mutacional de ADN , Regulación hacia Abajo , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Humanos , Immunoblotting , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos Antisentido/farmacología , ARN/metabolismo , ARN Mensajero/metabolismo , Proteína Smad2 , Proteína smad3 , Proteína Smad4 , Transfección , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The pharmaceutical industry is currently facing several challenges to identify and develop novel drug targets. Traditional drug discovery focussed on a small number of well-characterized gene products. Recently, this picture has changed with the completion of the draft sequence of the human genome, which has led to the identification of thousands of novel genes with unknown or poorly understood function. To cope with this overwhelming number of potential drug target candidates, new strategies for the elucidation of gene function, as well as their involvement in intracellular pathways, are required.
Asunto(s)
Bioensayo , Industria Farmacéutica/tendencias , Farmacología/tendencias , Transducción de Señal/efectos de los fármacos , Fenómenos Fisiológicos Celulares , Células/efectos de los fármacos , Industria Farmacéutica/métodos , Genómica , Humanos , Farmacología/métodosRESUMEN
The PKN (protein kinase N) family of Ser/Thr protein kinases regulates a diverse set of cellular functions, such as cell migration and cytoskeletal organization. Inhibition of tumour PKN activity has been explored as an oncology therapeutic approach, with a PKN3-targeted RNAi (RNA interference)-derived therapeutic agent in Phase I clinical trials. To better understand this important family of kinases, we performed detailed enzymatic characterization, determining the kinetic mechanism and lipid sensitivity of each PKN isoform using full-length enzymes and synthetic peptide substrate. Steady-state kinetic analysis revealed that PKN1-3 follows a sequential ordered Bi-Bi kinetic mechanism, where peptide substrate binding is preceded by ATP binding. This kinetic mechanism was confirmed by additional kinetic studies for product inhibition and affinity of small molecule inhibitors. The known lipid effector, arachidonic acid, increased the catalytic efficiency of each isoform, mainly through an increase in kcat for PKN1 and PKN2, and a decrease in peptide KM for PKN3. In addition, a number of PKN inhibitors with various degrees of isoform selectivity, including potent (Ki<10 nM) and selective PKN3 inhibitors, were identified by testing commercial libraries of small molecule kinase inhibitors. This study provides a kinetic framework and useful chemical probes for understanding PKN biology and the discovery of isoform-selective PKN-targeted inhibitors.
Asunto(s)
Adenosina Trifosfato/química , Ácido Araquidónico/química , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/química , Inhibidores de Proteínas Quinasas/química , Catálisis , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , CinéticaRESUMEN
PKN3 is an AGC-family protein kinase implicated in growth of metastatic prostate cancer cells with phosphoinositide 3-kinase pathway deregulation. The molecular mechanism, however, by which PKN3 contributes to malignant growth and tumorigenesis is not well understood. Using orthotopic mouse tumor models, we now show that inducible knockdown of PKN3 protein not only blocks metastasis, but also impairs primary prostate and breast tumor growth. Correspondingly, overexpression of exogenous PKN3 in breast cancer cells further increases their malignant behavior and invasiveness in-vitro. Mechanistically, we demonstrate that PKN3 physically interacts with Rho-family GTPases, and preferentially with RhoC, a known mediator of tumor invasion and metastasis in epithelial cancers. Likewise, RhoC predominantly associates with PKN3 compared to its closely related PKN family members. Unlike the majority of Rho GTPases and PKN molecules, which are ubiquitously expressed, both PKN3 and RhoC show limited expression in normal tissues and become upregulated in late-stage malignancies. Since PKN3 catalytic activity is increased in the presence of Rho GTPases, the co-expression and preferential interaction of PKN3 and RhoC in tumor cells are functionally relevant. Our findings provide novel insight into the regulation and function of PKN3 and suggest that the PKN3-RhoC complex represents an attractive therapeutic target in late-stage malignancies.
Asunto(s)
Neoplasias de la Próstata/metabolismo , Proteína Quinasa C/metabolismo , Proteínas ras/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Doxiciclina/uso terapéutico , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Masculino , Ratones , Metástasis de la Neoplasia/genética , Neoplasias de la Próstata/patología , Unión Proteica , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genética , ARN Interferente Pequeño , Proteínas ras/genética , Proteína rhoC de Unión a GTPRESUMEN
Smad2 and Smad3 are intracellular mediators of transforming growth factor beta (TGFbeta) signaling that share various biochemical properties, but data emerging from functional analyses in several cell types indicate that these two Smad proteins may convey distinct cellular responses. Therefore, we have investigated the individual roles of Smad2 and Smad3 in mediating the cytostatic and proapoptotic effects of TGFbeta as well as their function in epithelial-to-mesenchymal transition. For this purpose, we transiently depleted mouse mammary epithelial cells (Nme) of Smad2 and/or Smad3 mainly by a strategy relying on RNaseH-induced degradation of mRNA. The effect of such depletion on hallmark events of TGFbeta-driven epithelial-to-mesenchymal transition was analyzed, including dissolution of epithelial junctions, formation of stress fibers and focal adhesions, activation of metalloproteinases, and transcriptional regulation of acknowledged target genes. Furthermore, we investigated the effect of Smad2 and Smad3 knockdown on the TGFbeta-regulated transcriptome by microarray analysis. Our results identify Smad3 as a key factor to trigger TGFbeta-regulated events and ascribe tumor suppressor as well as oncogenic activities to this protein.
Asunto(s)
Células Epiteliales/metabolismo , Glándulas Mamarias Animales/citología , Transducción de Señal , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Apoptosis/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Inducción Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Uniones Intercelulares/efectos de los fármacos , Uniones Intercelulares/metabolismo , Metaloproteinasas de la Matriz/biosíntesis , Ratones , Oligonucleótidos Antisentido/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína smad3/genéticaRESUMEN
Peroxiredoxin 6 (Prdx6) is a cytoprotective enzyme with largely unknown in vivo functions. Here, we use Prdx6 knockout mice to determine its role in UV protection and wound healing. UV-mediated keratinocyte apoptosis is enhanced in Prdx6-deficient mice. Upon skin injury, we observe a severe hemorrhage in the granulation tissue of knockout animals, which correlates with the extent of oxidative stress. At the ultrastructural level endothelial cells appear highly damaged, and their rate of apoptosis is enhanced. Knock-down of Prdx6 in cultured endothelial cells also increases their susceptibility to oxidative stress, thus confirming the sensitivity of this cell type to loss of Prdx6. Wound healing studies in bone marrow chimeric mice demonstrate that Prdx6-deficient inflammatory and endothelial cells contribute to the hemorrhage phenotype. These results provide insight into the cross-talk between hematopoietic and resident cells at the wound site and the role of reactive oxygen species in this interplay.
Asunto(s)
Peroxiredoxina VI/fisiología , Piel/irrigación sanguínea , Piel/lesiones , Animales , Apoptosis/genética , Vasos Sanguíneos/citología , Vasos Sanguíneos/fisiología , Vasos Sanguíneos/ultraestructura , Células Cultivadas , Quimera/genética , Quimera/metabolismo , Células Endoteliales/patología , Células Endoteliales/ultraestructura , Endotelio Vascular/ultraestructura , Femenino , Técnica del Anticuerpo Fluorescente Directa , Hemorragia/etiología , Hemorragia/patología , Humanos , Inmunohistoquímica , Queratinocitos/enzimología , Queratinocitos/efectos de la radiación , Masculino , Ratones , Ratones Noqueados , Modelos Biológicos , Estrés Oxidativo , Peroxiredoxina VI/deficiencia , Peroxiredoxina VI/genética , Especies Reactivas de Oxígeno/metabolismo , Rayos Ultravioleta , Venas Umbilicales/citología , Irradiación Corporal Total , Cicatrización de Heridas/genéticaRESUMEN
The epidermis undergoes continuous self-renewal to maintain its protective function. Whereas growth factors are known to modulate overall skin homeostasis, the intracellular signaling pathways, which control the delicate balance between proliferation and differentiation in keratinocytes, are largely unknown. Here we show transient upregulation of the phosphoinositide 3-kinase (PI3K) catalytic subunits p110alpha and p110beta in differentiating keratinocytes in vitro, expression of these subunits in the epidermis of normal and wounded skin, and enhanced Akt phosphorylation in the hyperproliferative wound epidermis. Stimulation of PI3K activity in cultured keratinocytes by stable expression of an inducible, constitutively active PI3K mutant promoted cell proliferation and inhibited terminal differentiation in keratinocyte monocultures and induced the formation of a hyperplastic, disorganized and poorly differentiated epithelium in organotypic skin cultures. Activation of PI3K signaling also caused reorganization of the actin cytoskeleton and induced keratinocyte migration in vitro and in skin organ cultures. The identification of 122 genes, which are differentially expressed after induction of PI3K signaling provides insight into the molecular mechanisms underlying the observed effects of active PI3K on keratinocytes and indicates that hyperproliferation may be achieved at the expense of genome integrity. These results identify PI3K as an important intracellular regulator of epidermal homeostasis and repair.
Asunto(s)
Epidermis/fisiología , Homeostasis/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Cicatrización de Heridas/fisiología , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase I , Citoesqueleto/enzimología , Citoesqueleto/metabolismo , Epitelio/metabolismo , Humanos , Queloide/metabolismo , Queratinocitos/metabolismo , Ratones , Ratones Endogámicos BALB C , Técnicas de Cultivo de Órganos , Fosfatidilinositol 3-Quinasas/metabolismo , Activación Transcripcional , TransfecciónRESUMEN
We identified a novel human cDNA encoding a mitochondrial protein, MTP18 (mitochondrial protein, 18 kDa) as a transcriptional downstream target of phosphatidylinositol (PI) 3-kinase signaling. We demonstrate that MTP18 mRNA as well as protein expression is dependent on PI 3-kinase activity. Confocal microscopy and biochemical fractionation revealed a mitochondrial localization of MTP18. Loss-of-function analysis employing antisense molecules revealed that MTP18 is essential for cell viability in PC-3 and HaCaT cells. We show that knockdown of MTP18 protein level results in a cytochrome c release from mitochondria and consequently leads to apoptosis. In addition, HaCaT cells with reduced levels of MTP18 become more sensitive to apoptotic stimuli. This effect is accompanied by dramatic subcellular alterations. Reduction of MTP18 impairs mitochondrial morphology resulting in the formation of a highly interconnected mitochondrial reticulum in COS-7 cells. Conversely, overexpression of MTP18 induces a punctuate morphology of mitochondria suggesting also a functional role of MTP18 in maintaining the mitochondrial integrity. Hence, our data indicate an unexpected connection of PI 3-kinase signaling, apoptosis and the morphology of mammalian mitochondria.
Asunto(s)
Apoptosis/fisiología , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , División Celular , Línea Celular , ADN sin Sentido/genética , ADN Complementario/genética , Células HeLa , Humanos , Mitocondrias/ultraestructura , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
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.
Asunto(s)
División Celular/fisiología , Transformación Celular Neoplásica/genética , Técnicas Genéticas , Oligonucleótidos Antisentido/química , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Animales , Apoptosis/efectos de la radiación , Secuencia de Bases , División Celular/genética , Línea Celular , Activación Enzimática , Expresión Génica , Humanos , Oligonucleótidos Antisentido/farmacología , Fosfohidrolasa PTEN , Fosfatidilinositol 3-Quinasas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Ratas , Fase S/efectos de los fármacos , Transducción de Señal/genética , Proteínas Supresoras de Tumor/genética , Rayos UltravioletaRESUMEN
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.