RESUMEN

Protein kinase D (PKD) family members are increasingly implicated in multiple normal and abnormal biological functions, including signaling pathways that promote mitogenesis in pancreatic cancer. However, nothing is known about the effects of targeting PKD in pancreatic cancer. Our PKD inhibitor discovery program identified CRT0066101 as a specific inhibitor of all PKD isoforms. The aim of our study was to determine the effects of CRT0066101 in pancreatic cancer. Initially, we showed that autophosphorylated PKD1 and PKD2 (activated PKD1/2) are significantly upregulated in pancreatic cancer and that PKD1/2 are expressed in multiple pancreatic cancer cell lines. Using Panc-1 as a model system, we showed that CRT0066101 reduced bromodeoxyuridine incorporation; increased apoptosis; blocked neurotensin-induced PKD1/2 activation; reduced neurotensin-induced, PKD-mediated Hsp27 phosphorylation; attenuated PKD1-mediated NF-kappaB activation; and abrogated the expression of NF-kappaB-dependent proliferative and prosurvival proteins. We showed that CRT0066101 given orally (80 mg/kg/d) for 24 days significantly abrogated pancreatic cancer growth in Panc-1 subcutaneous xenograft model. Activated PKD1/2 expression in the treated tumor explants was significantly inhibited with peak tumor concentration (12 micromol/L) of CRT0066101 achieved within 2 hours after oral administration. Further, we showed that CRT0066101 given orally (80 mg/kg/d) for 21 days in Panc-1 orthotopic model potently blocked tumor growth in vivo. CRT0066101 significantly reduced Ki-67-positive proliferation index (P < 0.01), increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic cells (P < 0.05), and abrogated the expression of NF-kappaB-dependent proteins including cyclin D1, survivin, and cIAP-1. Our results show for the first time that a PKD-specific small-molecule inhibitor CRT0066101 blocks pancreatic cancer growth in vivo and show that PKD is a novel therapeutic target in pancreatic cancer.

Asunto(s)

Carcinoma Ductal Pancreático/patología , Proliferación Celular/efectos de los fármacos , Neoplasias Pancreáticas/patología , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones Desnudos , Peso Molecular , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

VEGF (vascular endothelial growth factor) plays an essential role in angiogenesis during development and in disease largely mediated by signalling events initiated by binding of VEGF to its receptor, VEGFR2 (VEGF receptor 2)/KDR (kinase insert domain receptor). Recent studies indicate that VEGF activates PKD (protein kinase D) in endothelial cells to regulate a variety of cellular functions, including signalling events, proliferation, migration and angiogenesis. To better understand the role of PKD in VEGF-mediated endothelial function, we characterized the effects of a novel pyrazine benzamide PKD inhibitor CRT5 in HUVECs (human umbilical vein endothelial cells). The activity of the isoforms PKD1 and PKD2 were blocked by this inhibitor as indicated by reduced phosphorylation, at Ser916 and Ser876 respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB (cAMP-response-element-binding protein) and HSP27 (heat-shock protein 27) at Ser82, was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 Ser82 phosphorylation. Furthermore, phosphorylation of HSP27 at Ser78, which occurs solely via the p38 MAPK (mitogen-activated protein kinase) pathway, was also unaffected by CRT5. In vitro kinase assays show that CRT5 did not significantly inhibit several PKC isoforms expressed in endothelial cells. CRT5 also decreased VEGF-induced endothelial migration, proliferation and tubulogenesis, similar to effects seen when the cells were transfected with PKD siRNA (small interfering RNA). CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.

Asunto(s)

Endotelio Vascular/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Humanos , Neovascularización Fisiológica/efectos de los fármacos , Fosforilación , Especificidad por Sustrato , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

The human endothelial vascular endothelial growth factor receptor 2 (VEGFR2/kinase domain region, KDR/fetal liver kinase-1, Flk-1) tyrosine kinase receptor is essential for VEGF-mediated physiological responses including endothelial cell proliferation, migration and survival. How VEGFR2 kinase activation and trafficking are co-coordinated in response to VEGF-A is not known. Here, we elucidate a mechanism for endothelial VEGFR2 response to VEGF-A dependent on constitutive endocytosis co-ordinated with ligand-activated ubiquitination and proteolysis. The selective VEGFR kinase inhibitor, SU5416, blocked the endosomal sorting required for VEGFR2 trafficking and degradation. Inhibition of VEGFR2 tyrosine kinase activity did not block plasma membrane internalization but led to endosomal accumulation. Lysosomal protease activity was required for ligand-stimulated VEGFR2 degradation. Activated VEGFR2 codistributed with the endosomal hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)/signal-transducing adaptor molecule (STAM) complex in a ligand and time-dependent manner, implying a role for this factor in sorting of ubiquitinated VEGFR2. Increased tyrosine phosphorylation of the Hrs subunit in response to VEGF-A links VEGFR2 activation and Hrs/STAM function. In contrast, VEGFR2 in quiescent cells was present on both the endothelial plasma membrane and early endosomes, suggesting constitutive recycling between these two compartments. This pathway was clathrin-linked and dependent on the AP2 adaptor complex as the A23 tyrphostin inhibited VEGFR2 trafficking. We propose a mechanism whereby the transition of endothelial VEGFR2 from a constitutive recycling itinerary to a degradative pathway explains ligand-activated receptor degradation in endothelial cells. This study outlines a mechanism to control the VEGF-A-mediated response within the vascular system.

Asunto(s)

Células Endoteliales/enzimología , Ubiquitina/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Células Cultivadas , Humanos , Fragmentos de Péptidos/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología

RESUMEN

Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-A165 (VEGF-A165) in endothelial cells. To define the role of NP-1 in the biological functions of VEGF, we developed a specific peptide antagonist of VEGF binding to NP-1 based on the NP-1 binding site located in the exon 7- and 8-encoded VEGF-A165 domain. The bicyclic peptide, EG3287, potently (K(i) 1.2 microM) and effectively (>95% inhibition at 100 microM) inhibited VEGF-A165 binding to porcine aortic endothelial cells expressing NP-1 (PAE/NP-1) and breast carcinoma cells expressing only NP-1 receptors for VEGF-A, but had no effect on binding to PAE/KDR or PAE/Flt-1. Molecular dynamics calculations, a nuclear magnetic resonance structure of EG3287, and determination of stability in media, indicated that it constitutes a stable subdomain very similar to the corresponding region of native VEGF-A165. The C terminus encoded by exon 8 and the three-dimensional structure were both critical for EG3287 inhibition of NP-1 binding, whereas modifications at the N terminus had little effect. Although EG3287 had no direct effect on VEGF-A165 binding to KDR receptors, it inhibited cross-linking of VEGF-A165 to KDR in human umbilical vein endothelial cells co-expressing NP-1, and inhibited stimulation of KDR and PLC-gamma tyrosine phosphorylation, activation of ERKs1/2 and prostanoid production. These findings characterize the first specific antagonist of VEGF-A165 binding to NP-1 and demonstrate that NP-1 is essential for optimum KDR activation and intracellular signaling. The results also identify a key role for the C-terminal exon 8 domain in VEGF-A165 binding to NP-1.

Asunto(s)

Neuropilina-1/antagonistas & inhibidores , Neuropilina-1/metabolismo , Fragmentos de Péptidos/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Unión Proteica , Conformación Proteica , Transducción de Señal , Porcinos , Factor A de Crecimiento Endotelial Vascular/farmacología

RESUMEN

Vascular endothelial growth factor (VEGF)-D binds to VEGF receptors (VEGFR) VEGFR2/KDR and VEGFR3/Flt4, but the signaling mechanisms mediating its biological activities in endothelial cells are poorly understood. Here we investigated the mechanism of action of VEGF-D, and we compared the signaling pathways and biological responses induced by VEGF-D and VEGF-A in endothelial cells. VEGF-D induced KDR and phospholipase C-gamma tyrosine phosphorylation more slowly and less effectively than VEGF-A at early times but had a more sustained effect and was as effective as VEGF-A after 60 min. VEGF-D activated extracellular signal-regulated protein kinases 1 and 2 with similar efficacy but slower kinetics compared with VEGF-A, and this effect was blocked by inhibitors of protein kinase C and mitogen-activated protein kinase kinase. In contrast to VEGF-A, VEGF-D weakly stimulated prostacyclin production and gene expression, had little effect on cell proliferation, and stimulated a smaller and more transient increase in intracellular [Ca(2+)]. VEGF-D induced strong but more transient phosphatidylinositol 3-kinase (PI3K)-mediated Akt activation and increased PI3K-dependent endothelial nitric-oxide synthase phosphorylation and cell survival more weakly. VEGF-D stimulated chemotaxis via a PI3K/Akt- and endothelial nitric-oxide synthase-dependent pathway, enhanced protein kinase C- and PI3K-dependent endothelial tubulogenesis, and stimulated angiogenesis in a mouse sponge implant model less effectively than VEGF-A. VEGF-D-induced signaling and biological effects were blocked by the KDR inhibitor SU5614. The finding that differential KDR activation by VEGF-A and VEGF-D has distinct consequences for endothelial signaling and function has important implications for understanding how multiple ligands for the same VEGF receptors can generate ligand-specific biological responses.

Asunto(s)

Endotelio Vascular/fisiología , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Factor D de Crecimiento Endotelial Vascular/fisiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiología , Animales , Humanos , Indoles/farmacología , Ratones , Neovascularización Fisiológica/fisiología , Factor A de Crecimiento Endotelial Vascular/farmacología , Factor D de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/agonistas , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores

RESUMEN

Vascular endothelial growth factor (VEGF) displays neurotrophic and neuroprotective activities, but the mechanisms underlying these effects have not been defined. Neuropilin-1 (NP-1) is a receptor for VEGF165 and placental growth factor-2 (PlGF-2), but the role of NP-1 in VEGF-dependent neurotrophic actions is unclear. Dorsal root ganglion (DRG) neurons expressed high levels of NP-1 mRNA and protein, much lower levels of KDR, and no detectable Flt-1. VEGF165 and PlGF-2 promoted DRG growth cone formation with an effect similar to that of nerve growth factor, whereas the Flt-1-specific ligand, PlGF-1, and the KDR/Flt-4 ligand, VEGF-D, had no effect. The chemorepellent NP-1 ligand, semaphorin 3A, antagonized the response to VEGF and PlGF-2. The specific KDR inhibitor, SU5614, did not affect the anti-chemorepellent effects of VEGF and PlGF-2, whereas a novel, specific antagonist of VEGF binding to NP-1, called EG3287, prevented inhibition of growth cone collapse. VEGF stimulated prostacyclin and prostaglandin E2 production in DRG cultures that was blocked by inhibitors of cyclooxygenases; the anti-chemorepellent activities of VEGF and PlGF-2 were abrogated by cyclooxygenase inhibitors, and a variety of prostacyclin analogues and prostaglandins strikingly inhibited growth cone collapse. These findings support a specific role for NP-1 in mediating neurotrophic actions of VEGF family members and also identify a novel role for prostanoids in the inhibition of neuronal chemorepulsion.

Asunto(s)

Ganglios Espinales/metabolismo , Neuronas/metabolismo , Neuropilina-1/metabolismo , Proteínas Gestacionales/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandinas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , División Celular , Células Cultivadas , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Epoprostenol/metabolismo , Exones , Humanos , Indoles/farmacología , Factor de Crecimiento Placentario , ARN/metabolismo , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Factores de Tiempo , Venas Umbilicales/citología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

VEGF is an important mediator of pathological angiogenesis in the eye and is a target for the development of novel anti-angiogenic molecules. In a previous study we identified 12-amino acid peptides derived from exon 6 of VEGF that inhibited VEGF binding to its receptors in HUVECs, endothelial cell functions, and in vitro angiogenesis. Screening of a series of truncated peptides corresponding to the inhibitory region of exon 6 identified a seven amino acid residue peptide, RKRKKSR, as the minimum exon 6-encoded sequence which retains the ability to inhibit VEGF receptor binding and angiogenesis in vitro. The effect of the seven-residue peptide was examined in a mouse model of ischaemic retinal neovascularisation. Administration of the peptide caused a 50% inhibition of retinal neovascularisation and was as effective in inhibiting ischaemic angiogenesis as soluble Flt-1 adenovirus. These results demonstrate that a seven amino acid VEGF exon 6-derived peptide is an effective inhibitor of ocular neovascularisation in vivo, and may have applications in the treatment of pathophysiological ocular neovascularisation in human disease.

Asunto(s)

Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Isquemia/metabolismo , Linfocinas/genética , Linfocinas/metabolismo , Péptidos/metabolismo , Neovascularización Retiniana/metabolismo , Animales , Línea Celular , Medio de Cultivo Libre de Suero , Modelos Animales de Enfermedad , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Exones , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Péptidos/genética , Embarazo , Retina/citología , Retina/patología , Neovascularización Retiniana/genética , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Microarray analysis using sets of known human genes provides a powerful platform for identifying candidate oncogenes involved in DNA amplification events but suffers from the disadvantage that information can be gained only on genes that have been preselected for inclusion on the array. To address this issue, we have performed comparative genome hybridization (CGH) and expression analyses on microarrays of clones, randomly selected from a cDNA library, prepared from a cancer containing the DNA amplicon under investigation. Application of this approach to the BT474 breast carcinoma cell line, which contains amplicons at 20q13, 17q11-21, and 17q22-23, identified 50 amplified and expressed genes, including genes from these regions previously proposed as candidate oncogenes. When considered together with data from microarray expression profiles and Northern analyses, we were able to propose five genes as new candidate oncogenes where amplification in breast cancer cell lines was consistently associated with higher levels of RNA expression. These included the HB01 histone acetyl transferase gene at 17q22-23 and the TRAP100 gene, which encodes a thyroid hormone receptor-associated protein coactivator, at 17q11-21. The results demonstrate the utility of this microarray-based CGH approach in hunting for candidate oncogenes within DNA amplicons.

Asunto(s)

Neoplasias de la Mama/genética , ADN Complementario/genética , Amplificación de Genes/genética , Perfilación de la Expresión Génica , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Mapeo Cromosómico/métodos , Mapeo Cromosómico/estadística & datos numéricos , Cromosomas Humanos Par 17/genética , Cromosomas Humanos Par 20/genética , Clonación Molecular , ADN de Neoplasias/genética , Perfilación de la Expresión Génica/estadística & datos numéricos , Genes Relacionados con las Neoplasias/genética , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , Distribución Aleatoria