RESUMO
Metastasisation occurs through the acquisition of invasive and survival capabilities that allow tumour cells to colonise distant sites. While the role of multicellular aggregates in cancer dissemination is acknowledged, the mechanisms that drive the formation of multiclonal cell aggregates are not fully elucidated. Here, we show that cancer cells of different tissue of origins can perform collective directional migration and can actively form heteroclonal aggregates in 3D, through a proliferation-independent mechanism. Coalescence of distant cell clusters is mediated by subcellular actin-rich protrusions and multicellular outgrowths that extend towards neighbouring aggregates. Coherently, perturbation of cytoskeletal dynamics impairs collective migration while myosin II activation is necessary for multicellular movements. We put forward the hypothesis that cluster attraction is mediated by secreted soluble factors. Such a hypothesis is consistent with the abrogation of aggregation by inhibition of PI3K/AKT/mTOR and MEK/ERK, the chemoattracting activity of conditioned culture media and with a wide screening of secreted proteins. Our results present a novel collective migration model and shed light on the mechanisms of formation of heteroclonal aggregates in cancer.
Assuntos
Neoplasias , Fosfatidilinositol 3-Quinases , Humanos , Movimento Celular , Actinas/metabolismoRESUMO
OBJECTIVES: Somatic mosaicism of PIK3CA gene is currently recognized as the molecular driver of Klippel-Trenaunay syndrome. However, given the limitation of the current technologies, PIK3CA somatic mutations are detected only in a limited proportion of Klippel-Trenaunay syndrome cases and tissue biopsy remains an invasive high risky, sometimes life-threatening, diagnostic procedure. Next generation sequencing liquid biopsy using cell-free DNA has emerged as an innovative non-invasive approach for early detection and monitoring of cancer. This approach, overcoming the space-time profile constraint of tissue biopsies, opens a new scenario also for others diseases caused by somatic mutations. METHODS: In the present study, we performed a comprehensive analysis of seven patients (four females and three males) with Klippel-Trenaunay syndrome. Blood samples from both peripheral and efferent vein from malformation were collected and cell-free DNA was extracted from plasma. Tissue biopsies from vascular lesions were also collected when available. Cell-free DNA libraries were performed using Oncomine™ Pan-Cancer Cell-Free Assay. Ion Proton for sequencing and Ion Reporter Software for analysis were used (Life Technologies, Carlsbad, CA, USA). RESULTS: Cell-free circulating DNA analysis revealed pathogenic mutations in PIK3CA gene in all patients. The mutational load was higher in plasma obtained from the efferent vein at lesional site (0.81%) than in the peripheral vein (0.64%) leading to conclude for a causative role of the identified variants. Tissue analysis, available for one amputated patient, confirmed the presence of the mutation at the malformation site at a high molecular frequency (14-25%), confirming its causative role. CONCLUSIONS: Our data prove for the first time that the cell-free DNA-next generation sequencing-liquid biopsy, which is currently used exclusively in an oncologic setting, is indeed the most effective tool for Klippel-Trenaunay syndrome diagnosis and tailored personalized treatment.
Assuntos
Ácidos Nucleicos Livres/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Síndrome de Klippel-Trenaunay-Weber/diagnóstico , Mosaicismo , Mutação , Análise de Sequência de DNA , Adulto , Ácidos Nucleicos Livres/sangue , Tomada de Decisão Clínica , DNA/sangue , Feminino , Marcadores Genéticos , Humanos , Síndrome de Klippel-Trenaunay-Weber/sangue , Síndrome de Klippel-Trenaunay-Weber/genética , Síndrome de Klippel-Trenaunay-Weber/terapia , Biópsia Líquida , Masculino , Pessoa de Meia-Idade , Fenótipo , Projetos Piloto , Valor Preditivo dos Testes , PrognósticoRESUMO
Vascular physiology relies on the concerted dynamics of several cell types, including pericytes, endothelial, and vascular smooth muscle cells. The interactions between such cell types are inherently dynamic and are not easily described with static, fixed, experimental approaches. Pericytes are mural cells that support vascular development, remodeling, and homeostasis, and are involved in a number of pathological situations including cancer. The dynamic interplay between pericytes and endothelial cells is at the basis of vascular physiology and few experimental tools exist to properly describe and study it. Here we employ a previously developed ex vivo murine aortic explant to study the formation of new blood capillary-like structures close to physiological situation. We develop several mouse models to culture, identify, characterize, and follow simultaneously single endothelial cells and pericytes during angiogenesis. We employ microscopy and image analysis to dissect the interactions between cell types and the process of cellular recruitment on the newly forming vessel. We find that pericytes are recruited on the developing sprout by proliferation, migrate independently from endothelial cells, and can proliferate on the growing capillary. Our results help elucidating several relevant mechanisms of interactions between endothelial cells and pericytes.
Assuntos
Células Endoteliais/metabolismo , Neovascularização Fisiológica , Pericitos/metabolismo , Animais , Aorta/citologia , Aorta/metabolismo , Células Endoteliais/citologia , Camundongos , Camundongos Transgênicos , Pericitos/citologiaRESUMO
Prostate cancer (PCa) is one of the most common cancer in men. Although hormone-sensitive PCa responds to androgen-deprivation, there are no effective therapies for castration-resistant PCa. It has been recently suggested that proton pump inhibitors (PPIs) may increase the risk of certain cancers; however, association with PCa remains elusive. Here, we evaluated the tumorigenic activities of PPIs in vitro, in PCa cell lines and epithelial cells from benign prostatic hyperplasia (BPH) and in vivo, in PCa mice xenografts. PPIs increased survival and proliferation, and inhibited apoptosis in LNCaP cells. These effects were attenuated or absent in androgen-insensitive DU-145 and PC3 cells, respectively. Specifically, omeprazole (OME) promoted cell cycle progression, increased c-Myc expression, ErbB2 activity and PSA secretion. Furthermore, OME induced the phosphorylation of MAPK-ERK1/2, PI3K/Akt and GSK-3ß, and blunted the expression and activity of cellular prostatic acid phosphatase. OME also increased survival, proliferation and PSA levels in BPH cells. In vivo, OME promoted tumor growth in mice bearing LNCaP xenografts. Our results indicate that PPIs display tumorigenic activities in PCa cells, suggesting that their long-term administration in patients should be carefully monitored.
Assuntos
Fosfatase Ácida/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias Hormônio-Dependentes/enzimologia , Omeprazol/toxicidade , Fosfatidilinositol 3-Quinase/metabolismo , Neoplasias da Próstata/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Inibidores da Bomba de Prótons/toxicidade , Receptor ErbB-2/metabolismo , Fosfatase Ácida/metabolismo , Animais , Apoptose/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Hormônio-Dependentes/patologia , Células PC-3 , Fosforilação , Neoplasias da Próstata/patologia , Transdução de SinaisRESUMO
Extrusion of apoptotic cells from epithelial tissues requires orchestrated morphological rearrangements of the apoptotic cell and its neighbors. However, the connections between the apoptotic cascade and events leading to extrusion are not fully understood. Here, we characterize an apoptotic extrusion apical actin ring (EAAR) that is assembled within the apoptotic cell and drives epithelial extrusion. Caspase-mediated cleavage of myotonic dystrophy kinase-related CDC42-binding kinase-α (MRCKα) triggers a signaling pathway that leads to the assembly of EAAR that pulls actin bundles, resulting in the compaction and removal of the cell body. We provide a detailed portrait of the EAAR including F-actin flow, the contribution of myosin contraction, and actin polymerization at bundles' terminals when the product of MRCKα cleavage is expressed. These results add to our understanding of the mechanisms controlling the process of epithelial extrusion by establishing a causal relationship between the triggering events of apoptosis, the activation of MRCKα, and its subsequent effects on the dynamics of actomyosin cytoskeleton rearrangement.
Assuntos
Actomiosina/metabolismo , Apoptose/fisiologia , Caspases/metabolismo , Células Epiteliais/metabolismo , Miotonina Proteína Quinase/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Células CACO-2 , Miosinas Cardíacas/metabolismo , Linhagem Celular , Cães , Células HEK293 , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Centro Organizador dos Microtúbulos/fisiologia , Cadeias Leves de Miosina/metabolismo , Miosinas/metabolismo , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismoRESUMO
AGMA1, a prevailingly cationic, guanidine-bearing, linear, amphoteric polyamidoamine is an effective siRNA condensing agent. Here two AGMA1 samples of different molecular weight, i.e. AGMA1-5 and AGMA1-10 were evaluated as siRNA condensing agents and transfection promoters. AGMA1-10 formed stable polyplexes with a size lower than 50 nm and positive zeta potential. AGMA1-5 polyplexes were larger, about 100 nm in size. AGMA1-10 polyplexes, but not AGMA1-5 proved to be an effective intracellular siRNA carrier, able to trigger gene silencing in Hela and PC3 cell lines without eliciting cytotoxic effects. AGMA1-10 knocked down AKT-1 expression upon transfection with an AKT-1 specific siRNA. The polyplex entry mechanism was investigated and was mediated by macropinocytosis. In conclusion, AGMA1 has potential as an efficient, non-toxic tool for the intracellular delivery of siRNA and warrants further investigation.
Assuntos
Agmatina/análogos & derivados , Técnicas de Transferência de Genes , Poliaminas/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Agmatina/administração & dosagem , Agmatina/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Técnicas de Transferência de Genes/normas , Células HeLa , Humanos , Poliaminas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Dissecting the cellular signaling that governs the motility of eukaryotic cells is one of the fundamental tasks of modern cell biology, not only because of the large number of physiological processes in which cell migration is crucial, but even more so because of the pathological ones, in particular tumor invasion and metastasis. Cell migration requires the coordination of at least four major processes: polarization of intracellular signaling, regulation of the actin cytoskeleton and membrane extension, focal adhesion and integrin signaling and contractile forces generation and rear retraction. Among the molecular components involved in the regulation of locomotion, the phosphatidylinositol-3-kinase (PI3K) pathway has been shown to exert fundamental role. A pivotal node of such pathway is represented by the serine/threonine kinase 3-phosphoinositide-dependent protein kinase-1 (PDPK1 or PDK1). PDK1, and the majority of its substrates, belong to the AGC family of kinases (related to cAMP-dependent protein kinase 1, cyclic Guanosine monophosphate-dependent protein kinase and protein kinase C), and control a plethora of cellular processes, downstream either to PI3K or to other pathways, such as RAS GTPase-MAPK (mitogen-activated protein kinase). Interestingly, PDK1 has been demonstrated to be crucial for the regulation of each step of cell migration, by activating several proteins such as protein kinase B/Akt (PKB/Akt), myotonic dystrophy-related CDC42-binding kinases alpha (MRCKα), Rho associated coiled-coil containing protein kinase 1 (ROCK1), phospholipase C gamma 1 (PLCγ1) and ß3 integrin. Moreover, PDK1 regulates cancer cell invasion as well, thus representing a possible target to prevent cancer metastasis in human patients. The aim of this review is to summarize the various mechanisms by which PDK1 controls the cell migration process, from cell polarization to actin cytoskeleton and focal adhesion regulation, and finally, to discuss the evidence supporting a role for PDK1 in cancer cell invasion and dissemination.
RESUMO
BACKGROUND: Thymidylate synthase (TS), one of the key enzymes for thymidine synthesis, is a target of pemetrexed (PEM), a key agent for the systemic therapy of malignant pleural mesothelioma (MPM) and its overexpression has been correlated to PEM-resistance. In MPM, experimental data report activation of the c-SRC tyrosine kinase suggesting it as a potential target to be further investigated. RESULTS: MPM cell lines showed different sensitivity, being MSTO the most and REN the least sensitive to PEM. REN cells showed high levels of both TS and SRC: dasatinib inhibited SRC activation and suppressed TS protein expression, starting from 100 nM dose, blocking the PEM-induced up regulation of TS protein levels. Dasatinib treatment impaired cells migration, and both sequential and co-administration with PEM significantly increased apoptosis. Dasatinib pretreatment improved sensitivity to PEM, downregulated TS promoter activity and, in association with PEM, modulated the downstream PI3K-Akt-mTOR signaling. Cell lines and Methods: In three MPM cell lines (MPP89, REN and MSTO), the effects of c-SRC inhibition, in correlation with TS expression and PEM sensitivity, were evaluated. PEM and dasatinib, a SRC inhibitor, were administered as single agents, in combination or sequentially. Cell viability, apoptosis and migration, as well as TS expression and SRC activation have been assessed. CONCLUSIONS: These data indicate that dasatinib sensitizes mesothelioma cells to PEM through TS down-regulation.
Assuntos
Antineoplásicos/farmacologia , Dasatinibe/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Pemetrexede/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Interações Medicamentosas , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares , Mesotelioma , Mesotelioma Maligno , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/metabolismoRESUMO
One of the most important steps in tumor progression involves the transformation from a differentiated epithelial phenotype to an aggressive, highly motile phenotype, where tumor cells invade neighboring tissues. Invasion can occur either by isolated mesenchymal cells or by aggregates that migrate collectively and do not lose completely the epithelial phenotype. Here, we show that, in a three-dimensional cancer cell culture, collective migration of cells eventually leads to aggregation in large clusters. We present quantitative measurements of cluster velocity, coalescence rates, and proliferation rates. These results cannot be explained in terms of random aggregation. Instead, a model of chemotaxis-driven aggregation - mediated by a diffusible attractant - is able to capture several quantitative aspects of our results. Experimental assays of chemotaxis towards culture conditioned media confirm this hypothesis. Theoretical and numerical results further suggest an important role for chemotactic-driven aggregation in spreading and survival of tumor cells.
Assuntos
Quimiotaxia , Modelos Biológicos , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , HumanosRESUMO
The ability of cells to migrate is essential for different physiological processes including embryonic development, angiogenesis, tissue repair and immune response. In the context of cancer such abilities acquire dramatic implications, as they are exploited by tumor cells to invade neighboring or distant healthy tissues. 3-Phosphoinositide dependent protein kinase-1 (PDK1 or PDPK1) is an ancient serine-threonine kinase belonging to AGC kinase family. An increasing amount of data points at a pivotal role for PDK1 in the regulation of cell migration. PDK1 is a transducer of PI3K signaling and activates multiple downstream effectors, thereby representing an essential hub coordinating signals coming from extracellular cues to the cytoskeletal machinery, the final executor of cell movement. Akt, PAK1, ß3 integrin, ROCK1, MRCKα and PLCγ1 are, according to the literature, the signaling transducers through which PDK1 regulates cell migration. In addition, PDK1 contributes to tumor cell invasion by regulating invadopodia formation and both amoeboid and collective cancer cell invasion. This and other pieces of evidence, such as its reported overexpression across several tumor types, corroborate a PDK1 role tumor aggressiveness. Altogether, these findings indicate the possibility to rationally target PDK1 in human tumors in order to counteract cancer cell dissemination in the organism.
Assuntos
Movimento Celular/fisiologia , Citoesqueleto/fisiologia , Invasividade Neoplásica/patologia , Invasividade Neoplásica/fisiopatologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos , Modelos Biológicos , Piruvato Desidrogenase Quinase de Transferência de AcetilRESUMO
Cellular protrusions are highly dynamic structures involved in fundamental processes, including cell migration and invasion. For a cell to migrate, its leading edge must form protrusions, and then adhere or retract. The spatial and temporal coordination of protrusions and retraction is yet to be fully understood. The study of protrusion dynamics mainly relies on live-microscopy often coupled to fluorescent labeling. Here we report the use of an alternative, label-free, quantitative and rapid assay to analyze protrusion dynamics in a cell population based on the real-time recording of cell activity by means of electronic sensors. Cells are seeded on a plate covered with electrodes and their shape changes map into measured impedance variations. Upon growth factor stimulation the impedance increases due to protrusive activity and decreases following retraction. Compared to microscopy-based methods, impedance measurements are suitable to high-throughput studies on different cell lines, growth factors and chemical compounds. We present data indicating that this assay lends itself to dissect the biochemical signaling pathways controlling adhesive protrusions. Indeed, we show that the protrusion phase is sustained by actin polymerization, directly driven by growth factor stimulation. Contraction instead mainly relies on myosin action, pointing at a pivotal role of myosin in lamellipodia retraction.
Assuntos
Movimento Celular/fisiologia , Extensões da Superfície Celular/fisiologia , Impedância Elétrica , Pseudópodes/fisiologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Cetuximab/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Células HEK293 , Células HeLa , Fator de Crescimento de Hepatócito/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Miosinas/antagonistas & inibidores , Tiazolidinas/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The mechanism by which angiogenic endothelial cells break the physical barrier of the vascular basement membrane and consequently sprout to form new vessels in mature tissues is unclear. Here, we show that the angiogenic endothelium is characterized by the presence of functional podosome rosettes. These extracellular-matrix-degrading and adhesive structures are precursors of de novo branching points and represent a key feature in the formation of new blood vessels. VEGF-A stimulation induces the formation of endothelial podosome rosettes by upregulating integrin α6ß1. In contrast, the binding of α6ß1 integrin to the laminin of the vascular basement membrane impairs the formation of podosome rosettes by restricting α6ß1 integrin to focal adhesions and hampering its translocation to podosomes. Using an ex vivo sprouting angiogenesis assay, transgenic and knockout mouse models and human tumour sample analysis, we provide evidence that endothelial podosome rosettes control blood vessel branching and are critical regulators of pathological angiogenesis.
Assuntos
Estruturas da Membrana Celular/fisiologia , Células Endoteliais/fisiologia , Neoplasias/fisiopatologia , Neovascularização Patológica/fisiopatologia , Animais , Membrana Basal/metabolismo , Linhagem Celular Tumoral , Estruturas da Membrana Celular/efeitos dos fármacos , Estruturas da Membrana Celular/metabolismo , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Integrina alfa6beta1/genética , Integrina alfa6beta1/metabolismo , Laminina/metabolismo , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/fisiopatologia , Masculino , Metaloproteinase 14 da Matriz/metabolismo , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/metabolismo , Melanoma Experimental/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia de Fluorescência , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Interferência de RNA , Acetato de Tetradecanoilforbol/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Directional cell migration is of paramount importance in both physiological and pathological processes, such as development, wound healing, immune response, and cancer invasion. Here, we report that 3-phosphoinositide-dependent kinase 1 (PDK1) regulates epithelial directional migration and invasion by binding and activating myotonic dystrophy kinase-related CDC42-binding kinase α (MRCKα). We show that the effect of PDK1 on cell migration does not involve its kinase activity but instead relies on its ability to bind membrane phosphatidylinositol (3,4,5)-trisphosphate. Upon epidermal growth factor (EGF) stimulation, PDK1 and MRCKα colocalize at the cell membrane in lamellipodia. We demonstrate that PDK1 positively modulates MRCKα activity and drives its localization within lamellipodia. Likewise, the retraction phase of lamellipodia is controlled by PDK1 through an MRCKα-dependent mechanism. In summary, we discovered a functional pathway involving PDK1-mediated activation of MRCKα, which links EGF signaling to myosin contraction and directional migration.
Assuntos
Movimento Celular , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Pseudópodes/enzimologia , Domínio Catalítico , Membrana Celular/enzimologia , Ativação Enzimática , Fator de Crescimento Epidérmico/fisiologia , Células HeLa , Humanos , Miotonina Proteína Quinase , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Transporte Proteico , Pseudópodes/ultraestrutura , Piruvato Desidrogenase Quinase de Transferência de AcetilRESUMO
The intrinsic complexity of the process of vessel formation limits the efficacy of cellular assays for elucidation of its molecular and pharmacologic mechanisms. We developed an ex vivo three-dimensional (3D) assay of sprouting angiogenesis with arterial explants from human umbilical cords. In this assay, human arterial rings were embedded in basement membrane extract gel, leading to a network of capillarylike structures upon vascular endothelial growth factor (VEGF) A stimulation. The angiogenic outgrowth consisted of endothelial cells, which actively internalized acetylated-low-density lipoprotein, surrounded by pericytes. Computer-assisted quantification of this vascular network demonstrated considerable sensitivity of this assay to several angiogenic inhibitors, including kinase inhibitors and monoclonal antibodies. We also performed targeted gene knockdown on this model by directly infecting explanted umbilical arteries with lentiviruses carrying short-hairpin RNA. Downregulation of VEGFR2 resulted in a significant reduction of the sprouting capability, demonstrating the relevance of human vascular explants for functional genomics studies. Furthermore, a modification of this assay led to development of a 3D model of tumor-driven angiogenesis, in which angiogenic outgrowth was sustained by spheroids of prostate cancer cells in absence of exogenous growth factors. The human arterial ring assay bridges the gap between in vitro endothelial cell and animal model, and is a powerful system for identification of genes and drugs that regulate human angiogenesis.
Assuntos
Aorta/citologia , Técnicas de Cultura de Células/métodos , Neovascularização Patológica/patologia , Neovascularização Patológica/fisiopatologia , Neoplasias da Próstata/patologia , Artérias Umbilicais/citologia , Inibidores da Angiogênese/farmacologia , Animais , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Humanos , Imageamento Tridimensional/métodos , Lentivirus/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/tratamento farmacológico , Neoplasias da Próstata/irrigação sanguínea , Transdução Genética/métodos , Artérias Umbilicais/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
3-phosphoinositide-dependent protein kinase 1 (PDK1) is the pivotal element of the phosphatidylinositol 3 kinase (PI3K) signaling pathway because it phosphorylates Akt/PKB through interactions with phosphatidylinositol 3,4,5 phosphate. Recent data indicate that PDK1 is overexpressed in many breast carcinomas and that alterations of PDK1 are critical in the context of oncogenic PI3K activation. However, the role of PDK1 in tumor progression is still controversial. Here, we show that PDK1 is required for anchorage-independent and xenograft growth of breast cancer cells harboring either PI3KCA or KRAS mutations. In fact, PDK1 silencing leads to increased anoikis, reduced soft agar growth, and pronounced apoptosis inside tumors. Interestingly, these phenotypes are reverted by PDK1 wild-type but not kinase-dead mutant, suggesting a relevant role of PDK1 kinase activity, even if PDK1 is not relevant for Akt activation here. Indeed, the expression of constitutively active forms of Akt in PDK1 knockdown cells is unable to rescue the anchorage-independent growth. In addition, Akt down-regulation and pharmacological inhibition do not inhibit the effects of PDK1 overexpression. In summary, these results suggest that PDK1 may contribute to breast cancer, even in the absence of PI3K oncogenic mutations and through both Akt-dependent and Akt-independent mechanisms.
Assuntos
Neoplasias da Mama/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Anoikis/genética , Neoplasias da Mama/patologia , Proliferação de Células , Sobrevivência Celular/genética , Cromonas/farmacologia , Feminino , Humanos , Camundongos , Camundongos Nus , Morfolinas/farmacologia , Mutação , Proteínas Nucleares/genética , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras) , Pirazóis/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais/genética , Sulfonamidas/farmacologia , Fatores de Transcrição/genética , Transplante Heterólogo , Proteínas ras/genéticaRESUMO
Blood vessel formation depends on the highly coordinated actions of a variety of angiogenic regulators. Vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang-1) are both potent and essential proangiogenic factors with complementary roles in vascular development and function. Whereas VEGF is required for the formation of the initial vascular plexus, Ang-1 contributes to the stabilization and maturation of growing blood vessels. Here, we provide evidence of a novel microRNA (miRNA)-dependent molecular mechanism of Ang-1 signalling modulation aimed at stabilizing adult vasculature. MiRNAs are short non-coding RNA molecules that post-trascriptionally regulate gene expression by translational suppression or in some instances by cleavage of the respective mRNA target. Our data indicate that endothelial cells of mature vessels express high levels of miR-126, which primarily targets phosphoinositide-3-kinase regulatory subunit 2 (p85ß). Down-regulation of miR-126 and over-expression of p85ß in endothelial cells inhibit the biological functions of Ang-1. Additionally, knockdown of miR-126 in zebrafish resulted in vascular remodelling and maturation defects, reminiscent of the Ang-1 loss-of-function phenotype. Our findings suggest that miR-126-mediated phosphoinositide-3-kinase regulation, not only fine-tunes VEGF-signaling, but it strongly enhances the activities of Ang-1 on vessel stabilization and maturation.
Assuntos
Angiopoietina-1/metabolismo , Vasos Sanguíneos/embriologia , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , MicroRNAs/metabolismo , Morfogênese , Transdução de Sinais/genética , Angiopoietina-1/genética , Animais , Vasos Sanguíneos/metabolismo , Sobrevivência Celular , Regulação para Baixo/genética , Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Camundongos , MicroRNAs/genética , Neovascularização Fisiológica/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The integrin alpha6 subunit is part of the alpha6beta1 and alpha6beta4 integrin complexes, which are known to be receptors for laminins and to mediate several biological activities such as embryogenesis, organogenesis, and invasion of carcinoma cells. However, the precise role of alpha6 integrin in angiogenesis has not yet been addressed. We observed that both vascular endothelial growth factor-A and fibroblast growth factor-2 strongly upregulate alpha6 integrin in human endothelial cells. Moreover, alpha6 integrin was positively modulated in angiogenic vessels in pancreatic neuroendocrine carcinoma. In this transgenic mouse model of spontaneous tumorigenesis, alpha6 integrin expression increased in the angiogenic stage, while being expressed at low levels in normal and hyperplastic tissue. We studied the functional role of alpha6 integrin during angiogenesis by lentivirus-mediated gene silencing and blocking antibody. Cell migration and morphogenesis on basement membrane extracts, a laminin-rich matrix, was reduced in endothelial cells expressing low levels of alpha6 integrin. However, we did not observe any differences in collagen matrices. Similar results were obtained in the aortic ring angiogenesis assay. alpha6 integrin was required for vessel sprouting on basement membrane gels but not on collagen gels, as shown by stably silencing this integrin in the murine aorta. Finally, a neutralizing anti-alpha6 integrin antibody inhibited in vivo angiogenesis in chicken chorioallantoic membrane and transgenic tumor mouse model. In summary, we showed that the alpha6 integrin participated in vascular endothelial growth factor-A and fibroblast growth factor-2-driven angiogenesis in vitro and in vivo, suggesting that it might be an attractive target for therapeutic approaches in angiogenesis-dependent diseases such as tumor growth.
Assuntos
Membrana Basal/metabolismo , Células Endoteliais/metabolismo , Integrina alfa6/biossíntese , Animais , Membrana Basal/citologia , Movimento Celular/fisiologia , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Regulação para Baixo , Células Endoteliais/citologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Integrina alfa6/genética , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Regulação para CimaRESUMO
The bidirectional communication between integrin alphavbeta3 and vascular endothelial growth factor (VEGF) receptors acts to integrate and coordinate endothelial cell (EC) activity during angiogenesis. However, the molecular mechanisms involved in this signaling crosstalk are only partially revealed. We have found that protein kinase D1 (PKD1) was activated by VEGF-A, but not by other angiogenic factors, and associated with alphavbeta3 integrin. Moreover, knockdown of PKD1 increased endocytosis of alphavbeta3 and reduced its return from endosomes to the plasma membrane leading to accumulation of the integrin in Rab5- and Rab4-positive endosomes. Consistent with this, PKD1 knockdown caused defects in focal complex formation and reduced EC migration in response to VEGF-A. Moreover, knockdown of PKD1 reduced EC motility on vitronectin, whereas migration on collagen I was not PKD1 dependent. These results suggest that PKD1-regulated alphavbeta3 trafficking contributes to the angiogenesis process by integrating VEGF-A signaling with extracellular matrix interactions.
Assuntos
Movimento Celular/fisiologia , Células Endoteliais/fisiologia , Integrina alfaVbeta3/metabolismo , Proteína Quinase C/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Extensões da Superfície Celular/metabolismo , Células Cultivadas , Endocitose/fisiologia , Endossomos/metabolismo , Células Endoteliais/citologia , Adesões Focais/metabolismo , Humanos , Proteína Quinase C/genética , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Vitronectina/metabolismoRESUMO
The serine/threonine protein kinase phosphoinositide-dependent kinase 1 (PDK1) plays a central role in cellular signaling by phosphorylating members of the AGC family of kinases, including PKB/Akt. We now present evidence showing that PDK1 is essential for the motility of vascular endothelial cells (ECs) and that it is involved in the regulation of their chemotaxis. ECs differentiated from mouse embryonic stem cells lacking PDK1 completely lost their ability to migrate in vitro in response to vascular endothelial growth factor-A (VEGF-A). In addition, PDK1(-/-) embryoid bodies exhibit evident developmental and vascular defects that can be attributed to a reduced cell migration. Moreover, the overexpression of PDK1 increased the EC migration induced by VEGF-A. We propose a model of spatial distribution of PDK1 and Akt in which the synthesis of phosphatidylinositol 3,4,5 triphosphate at plasma membrane by activation of phosphoinositide 3-kinase recruits both proteins at the leading edge of the polarized ECs and promotes cell chemotaxis. These findings establish a mechanism for the spatial localization of PDK1 and its substrate Akt to regulate directional migration.
Assuntos
Movimento Celular/fisiologia , Células Endoteliais/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Células Cultivadas , Quimiotaxia/fisiologia , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/metabolismo , Células Endoteliais/citologia , Humanos , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Thrombospondin-1 (TSP-1), a natural inhibitor of angiogenesis, acts directly on endothelial cells (EC) via CD36 to inhibit their migration and morphogenesis induced by basic fibroblast growth factor. Here we show that CD36 triggered by TSP-1 inhibits in vitro angiogenesis stimulated by vascular endothelial growth factor-A (VEGF-A). To demonstrate that the TSP-1 inhibitory signal was mediated by CD36, we transduced CD36 in CD36-deficient endothelial cells. Both TSP-1 and the agonist anti-CD36 mAb SMO, which mimics TSP-1 activity, reduced the VEGF-A165-induced migration and sprouting of CD36-ECs. To address the mechanisms by which CD36 may exert its angiostatic function, we investigated the functional components of the C-terminal cytoplasmic tail by site-directed mutagenesis. Our results indicate that C464, R467, and K469 of CD36 are required for the inhibitory activity of TSP-1. In contrast, point mutation of C466 did not alter TSP-1 ability to inhibit EC migration and sprouting. Moreover, we show that activation of CD36 by TSP-1 down-modulates the VEGF receptor-2 (VEGFR-2) and p38 mitogen-associated protein kinase phosphorylation induced by VEGF-A165, and this effect was specifically abolished by point mutation at C464. These results identify specific amino acids of the C-terminal cytoplasmic tail of CD36 crucial for the in vitro angiostatic activity of TSP-1 and extend our knowledge of regulation of VEGFR-2-mediated biological activities on ECs.