RESUMEN
Chemoresistance is a serious limitation of cancer treatment. Until recently, almost all the work done to study this limitation has been restricted to tumour cells. Here we identify a novel molecular mechanism by which endothelial cells regulate chemosensitivity. We establish that specific targeting of focal adhesion kinase (FAK; also known as PTK2) in endothelial cells is sufficient to induce tumour-cell sensitization to DNA-damaging therapies and thus inhibit tumour growth in mice. The clinical relevance of this work is supported by our observations that low blood vessel FAK expression is associated with complete remission in human lymphoma. Our study shows that deletion of FAK in endothelial cells has no apparent effect on blood vessel function per se, but induces increased apoptosis and decreased proliferation within perivascular tumour-cell compartments of doxorubicin- and radiotherapy-treated mice. Mechanistically, we demonstrate that endothelial-cell FAK is required for DNA-damage-induced NF-κB activation in vivo and in vitro, and the production of cytokines from endothelial cells. Moreover, loss of endothelial-cell FAK reduces DNA-damage-induced cytokine production, thus enhancing chemosensitization of tumour cells to DNA-damaging therapies in vitro and in vivo. Overall, our data identify endothelial-cell FAK as a regulator of tumour chemosensitivity. Furthermore, we anticipate that this proof-of-principle data will be a starting point for the development of new possible strategies to regulate chemosensitization by targeting endothelial-cell FAK specifically.
Asunto(s)
Daño del ADN , Resistencia a Antineoplásicos/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Citocinas/biosíntesis , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Resistencia a Antineoplásicos/genética , Células Endoteliales/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/deficiencia , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Humanos , Ratones , FN-kappa B/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Neoplasias/radioterapia , Fosforilación/efectos de los fármacosRESUMEN
The α6ß1-integrin is a major laminin receptor, and formation of a laminin-rich basement membrane is a key feature in tumour blood vessel stabilisation and pericyte recruitment, processes that are important in the growth and maturation of tumour blood vessels. However, the role of pericyte α6ß1-integrin in angiogenesis is largely unknown. We developed mice where the α6-integrin subunit is deleted in pericytes and examined tumour angiogenesis and growth. These mice had: (1) reduced pericyte coverage of tumour blood vessels; (2) reduced tumour blood vessel stability; (3) increased blood vessel diameter; (4) enhanced blood vessel leakiness, and (5) abnormal blood vessel basement membrane architecture. Surprisingly, tumour growth, blood vessel density and metastasis were not altered. Analysis of retinas revealed that deletion of pericyte α6-integrin did not affect physiological angiogenesis. At the molecular level, we provide evidence that pericyte α6-integrin controls PDGFRß expression and AKT-mTOR signalling. Taken together, we show that pericyte α6ß1-integrin regulates tumour blood vessels by both controlling PDGFRß and basement membrane architecture. These data establish a novel dual role for pericyte α6-integrin as modulating the blood vessel phenotype during pathological angiogenesis.
Asunto(s)
Vasos Sanguíneos/metabolismo , Integrina alfa6beta1/metabolismo , Neoplasias/irrigación sanguínea , Pericitos/metabolismo , Animales , Membrana Basal/efectos de los fármacos , Membrana Basal/metabolismo , Becaplermina , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Integrasas/metabolismo , Ratones , Metástasis de la Neoplasia , Neoplasias/metabolismo , Neoplasias/patología , Pericitos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-sis/farmacología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
The neuromuscular junction (NMJ) is the synapse between motoneurons and skeletal muscle, and is responsible for eliciting muscle contraction. Neurotransmission at synapses depends on the release of synaptic vesicles at sites called active zones (AZs). Various proteins of the extracellular matrix are crucial for NMJ development; however, little is known about the identity and functions of the receptors that mediate their effects. Using genetically modified mice, we find that integrin-α3 (encoded by Itga3), an adhesion receptor at the presynaptic membrane, is involved in the localisation of AZ components and efficient synaptic vesicle release. Integrin-α3 also regulates integrity of the synapse - mutant NMJs present with progressive structural changes and upregulated autophagy, features commonly observed during ageing and in models of neurodegeneration. Unexpectedly, we find instances of nerve terminal detachment from the muscle fibre; to our knowledge, this is the first report of a receptor that is required for the physical anchorage of pre- and postsynaptic elements at the NMJ. These results demonstrate multiple roles of integrin-α3 at the NMJ, and suggest that alterations in its function could underlie defects that occur in neurodegeneration or ageing.
Asunto(s)
Integrina alfa3/metabolismo , Unión Neuromuscular/metabolismo , Envejecimiento/metabolismo , Animales , Autofagia , Calcio/metabolismo , Desarrollo Embrionario , Ratones Endogámicos C57BL , Neuronas Motoras/metabolismo , Neuronas Motoras/ultraestructura , Desarrollo de Músculos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestructura , Mutación/genética , Unión Neuromuscular/ultraestructura , Transporte de Proteínas , Sinapsis/metabolismo , Sinapsis/ultraestructura , Transmisión Sináptica , Vesículas Sinápticas/metabolismoRESUMEN
Focal adhesion kinase (FAK) inhibitors have been developed as potential anticancer agents and are undergoing clinical trials. In vitro activation of the FAK kinase domain triggers autophosphorylation of Y397, Src activation, and subsequent phosphorylation of other FAK tyrosine residues. However, how FAK Y397 mutations affect FAK kinase-dead (KD) phenotypes in tumour angiogenesis in vivo is unknown. We developed three Pdgfb-iCreert -driven endothelial cell (EC)-specific, tamoxifen-inducible homozygous mutant mouse lines: FAK wild-type (WT), FAK KD, and FAK double mutant (DM), i.e. KD with a putatively phosphomimetic Y397E mutation. These ECCre+;FAKWT/WT , ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice were injected subcutaneously with syngeneic B16F0 melanoma cells. Tumour growth and tumour blood vessel functions were unchanged between ECCre+;FAKWT/WT and ECCre-;FAKWT/WT control mice. In contrast, tumour growth and vessel density were decreased in ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice, as compared with Cre - littermates. Despite no change in the percentage of perfused vessels or pericyte coverage in either genotype, tumour hypoxia was elevated in ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice. Furthermore, although ECCre+;FAKKD/KD mice showed reduced blood vessel leakage, ECCre+;FAKDM/DM and ECCre-;FAKDM/DM mice showed no difference in leakage. Mechanistically, fibronectin-stimulated Y397 autophosphorylation was reduced in Cre+;FAKKD/KD ECs as compared with Cre+;FAKWT/WT cells, with no change in phosphorylation of the known Src targets FAK-Y577, FAK-Y861, FAK-Y925, paxillin-Y118, p130Cas-Y410. Cre+;FAKDM/DM ECs showed decreased Src target phosphorylation levels, suggesting that the Y397E substitution actually disrupted Src activation. Reduced VE-cadherin-pY658 levels in Cre+;FAKKD/KD ECs were rescued in Cre+FAKDM/DM ECs, corresponding with the rescue in vessel leakage in the ECCre+;FAKDM/DM mice. We show that EC-specific FAK kinase activity is required for tumour growth, angiogenesis, and vascular permeability. The ECCre+;FAKDM/DM mice restored the KD-dependent tumour vascular leakage observed in ECCre+;FAKKD/KD mice in vivo. This study opens new fields in in vivo FAK signalling. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Permeabilidad Capilar/genética , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Melanoma/enzimología , Animales , Antineoplásicos Hormonales/farmacología , Permeabilidad Capilar/efectos de los fármacos , División Celular/genética , Hipoxia de la Célula/genética , Línea Celular Tumoral , Endotelio Vascular/enzimología , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/deficiencia , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Homocigoto , Melanoma/irrigación sanguínea , Melanoma/genética , Ratones , Mutación/genética , Trasplante de Neoplasias , Neovascularización Patológica/enzimología , Neovascularización Patológica/genética , Tamoxifeno/farmacologíaRESUMEN
BACKGROUND/OBJECTIVES: The vascular heterogeneity of pancreatic ductal adenocarcinoma (PDAC) has never been characterised. We analysed the heterogeneous vascular density of human PDAC along with its prognostic correlation. METHODS: Tissue Microarrays of 87 patients with different pancreatico-biliary pathologies were analysed in an automated manner (Ariol™) after CD31 staining to assess vascular density in juxta-tumoral and panstromal compartments. In vitro and ex vivo assays were carried out to assess the role of PSC. RESULTS: PDAC has a distinct vascular density and distribution of vessels compared to cholangiocarcinoma. The PDAC juxta-tumoral stroma was hypovascular and the normal adjacent rim was hypervascular compared to the panstromal compartment. These features adversely affected patient prognosis, suggesting a model for spatio-temporal PDAC evolution. Mice aortic rings and 3D organotypic cultures demonstrated pro- and anti-angiogenic signalling from activated PSC and cancer cells respectively. ATRA-induced quiescence suppressed the pro-angiogenic activity of PSC. CONCLUSION: Human PDAC has variable vascularity at microscopic level suggesting that novel stromal directed therapies would need to be determined by pathological characteristics.
Asunto(s)
Adenocarcinoma/patología , Vasos Sanguíneos/patología , Carcinoma Ductal Pancreático/patología , Células Estrelladas Pancreáticas/patología , Animales , Células Cultivadas , Colangiocarcinoma/patología , Humanos , Ratones , Análisis por Micromatrices , Microcirculación/efectos de los fármacos , Neovascularización Patológica/patología , Técnicas de Cultivo de Órganos , Pronóstico , Análisis de Supervivencia , Tretinoina/uso terapéutico , Microambiente TumoralRESUMEN
Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential for tumour development. It is initiated and regulated by growth factors via their surface receptors, which activate several intracellular signalling pathways in endothelial cells. Cell adhesion molecules, such as integrins, also regulate angiogenesis. Despite these facts, inhibitors of endothelial cell growth factor receptors or integrins have not been as effective as initially hoped in the long-term inhibition of angiogenesis in cancer patients. Signalling downstream of growth factor receptors and integrins converge on the ubiquitously expressed non-receptor tyrosine kinase focal adhesion kinase (FAK). FAK is involved in endothelial cell proliferation, migration and survival, is up-regulated in many cancers and has recently been shown to control tumour angiogenesis. Indeed, FAK inhibitors are presently being developed for the treatment of cancer. However, recent studies have indicated the complexities of understanding the precise role for FAK in angiogenesis. Here we have summarized some of the key features of FAK, addressed some of the apparently contradictory roles of this molecule in angiogenesis and provided some perspectives for future studies.
Asunto(s)
Proteína-Tirosina Quinasas de Adhesión Focal/fisiología , Neoplasias/irrigación sanguínea , Neovascularización Patológica/enzimología , Animales , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Proteína-Tirosina Quinasas de Adhesión Focal/química , Predicción , Eliminación de Gen , Humanos , Ratones , Mutación/genética , Neoplasias/enzimología , Neoplasias/terapiaRESUMEN
Some marketed antibiotics can cause mitochondria dysfunction via inhibition of the mitochondrial translation process. There is great interest in exploiting such effects within a cancer setting. To enhance accumulation of antibiotics within the mitochondria of cancer cells, and therefore delivery of a greater potency payload, a mitochondrial targeting group in the form of a triphenylphosphonium (TPP) cation was appended via an alkyl chain length consisting of 7 to 11 carbons to the ribosomal antibiotics azithromycin and doxycycline. Using MDA-MB-231 cells, the effects of each subseries on mitochondrial translation, mitochondrial bioenergetics, and cell viability are described.
RESUMEN
Small nucleolar RNAs play crucial roles in ribosome biogenesis. They guide folding, site-specific nucleotide modifications and participate in cleavage of precursor ribosomal RNAs. To better understand how the biogenesis of the box C/D small nucleolar RNPs (snoRNPs) occur in a cellular context, we used a new approach based on the possibility of relocalizing a given nuclear complex by adding an affinity tag for B23 to one component of this complex. We selectively delocalized each core box C/D protein, namely 15.5kD, Nop56, Nop58 and fibrillarin, and analyzed the effect of such changes on other components of the box C/D snoRNPs. We show that modifying the localization and the mobility of core box C/D proteins impairs their association with box C/D snoRNPs. In addition, we demonstrate that fibrillarin and Nop56 directly interact in vivo. This interaction, indispensable for the association of both proteins with the box C/D snoRNPs, does not involve the glycine- and arginine-rich domain or the RNA-binding domain but the alpha-helix domain of fibrillarin. In addition, no RNA seems required to maintain fibrillarin-Nop56 interaction.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Proteínas Nucleares/metabolismo , ARN Nucleolar Pequeño/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Cromosómicas no Histona/genética , Recuperación de Fluorescencia tras Fotoblanqueo , Células HeLa , Humanos , Hibridación Fluorescente in Situ , Proteínas Nucleares/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Nucleolar Pequeño/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Cell-cell fusion is a normal biological process playing essential roles in organ formation and tissue differentiation, repair and regeneration. Through cell fusion somatic cells undergo rapid nuclear reprogramming and epigenetic modifications to form hybrid cells with new genetic and phenotypic properties at a rate exceeding that achievable by random mutations. Factors that stimulate cell fusion are inflammation and hypoxia. Fusion of cancer cells with non-neoplastic cells facilitates several malignancy-related cell phenotypes, e.g., reprogramming of somatic cell into induced pluripotent stem cells and epithelial to mesenchymal transition. There is now considerable in vitro, in vivo and clinical evidence that fusion of cancer cells with motile leucocytes such as macrophages plays a major role in cancer metastasis. Of the many changes in cancer cells after hybridizing with leucocytes, it is notable that hybrids acquire resistance to chemo- and radiation therapy. One phenomenon that has been largely overlooked yet plays a role in these processes is polyploidization. Regardless of the mechanism of polyploid cell formation, it happens in response to genotoxic stresses and enhances a cancer cell's ability to survive. Here we summarize the recent progress in research of cell fusion and with a focus on an important role for polyploid cells in cancer metastasis. In addition, we discuss the clinical evidence and the importance of cell fusion and polyploidization in solid tumors.
RESUMEN
The overexpression of the protein tyrosine kinase, Focal adhesion kinase (FAK), in endothelial cells has implicated its requirement in angiogenesis and tumour growth, but how pericyte FAK regulates tumour angiogenesis is unknown. We show that pericyte FAK regulates tumour growth and angiogenesis in multiple mouse models of melanoma, lung carcinoma and pancreatic B-cell insulinoma and provide evidence that loss of pericyte FAK enhances Gas6-stimulated phosphorylation of the receptor tyrosine kinase, Axl with an upregulation of Cyr61, driving enhanced tumour growth. We further show that pericyte derived Cyr61 instructs tumour cells to elevate expression of the proangiogenic/protumourigenic transmembrane receptor Tissue Factor. Finally, in human melanoma we show that when 50% or more tumour blood vessels are pericyte-FAK negative, melanoma patients are stratified into those with increased tumour size, enhanced blood vessel density and metastasis. Overall our data uncover a previously unknown mechanism of tumour growth by pericytes that is controlled by pericyte FAK.
Asunto(s)
Proteína 61 Rica en Cisteína/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neovascularización Patológica , Pericitos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Aorta Torácica/patología , Carcinoma Pulmonar de Lewis/metabolismo , Adhesión Celular , Proliferación Celular , Femenino , Quinasa 1 de Adhesión Focal/genética , Humanos , Linfocinas/metabolismo , Masculino , Melanoma/irrigación sanguínea , Melanoma/metabolismo , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/patología , Factor de Crecimiento Placentario/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogénicas c-sis/metabolismo , Transducción de Señal , Microambiente Tumoral , Factor A de Crecimiento Endotelial Vascular/metabolismo , Tirosina Quinasa del Receptor AxlRESUMEN
PURPOSE: Neuroblastoma is a childhood malignancy originating from the sympathetic nervous system with a complex biology, prone to metastasize and relapse. High-risk, metastatic cases are explained in part by amplification or mutation of oncogenes, such as MYCN and ALK, and loss of tumor suppressor genes in chromosome band 1p. However, it is fundamental to identify other pathways responsible for the large portion of neuroblastomas with no obvious molecular alterations. EXPERIMENTAL DESIGN: Neuroblastoma cell lines were used for the assessment of tumor growth in vivo and in vitro Protein expression in tissues and cells was assessed using immunofluorescence and IHC. The association of promyelocytic leukemia (PML) expression with neuroblastoma outcome and relapse was calculated using log-rank and Mann-Whitney tests, respectively. Gene expression was assessed using chip microarrays. RESULTS: PML is detected in the developing and adult sympathetic nervous system, whereas it is not expressed or is low in metastatic neuroblastoma tumors. Reduced PML expression in patients with low-risk cancers, that is, localized and negative for the MYCN proto-oncogene, is strongly associated with tumor recurrence. PML-I, but not PML-IV, isoform suppresses angiogenesis via upregulation of thrombospondin-2 (TSP2), a key inhibitor of angiogenesis. Finally, PML-I and TSP2 expression inversely correlates with tumor angiogenesis and recurrence in localized neuroblastomas. CONCLUSIONS: Our work reveals a novel PML-I-TSP2 axis for the regulation of angiogenesis and cancer relapse, which could be used to identify patients with low-risk, localized tumors that might benefit from chemotherapy. Clin Cancer Res; 22(13); 3398-409. ©2016 AACR.
Asunto(s)
Recurrencia Local de Neoplasia/patología , Neovascularización Patológica/patología , Neuroblastoma/patología , Proteína de la Leucemia Promielocítica/metabolismo , Trombospondinas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/genética , Cresta Neural/embriología , Neuroblastoma/genética , Proteína de la Leucemia Promielocítica/genética , Isoformas de Proteínas/genética , Proto-Oncogenes Mas , Factores de Riesgo , Células Madre/citología , Sistema Nervioso Simpático/embriología , Trombospondinas/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Cellular ribosomal protein L29 (RPL29) is known to be important in protein synthesis, but its function during angiogenesis has never been described before. We have shown previously that mice lacking ß3-integrins support enhanced tumour angiogenesis and, therefore, deletion of endothelial αvß3 can provide a method for discovery of novel regulators of tumour angiogenesis. Here, we describe significant upregulation of RPL29 in ß3-null endothelial cells at both the mRNA and protein level. Ex vivo, we show that VEGF-stimulated microvessel sprouting was reduced significantly in Rpl29-heterozygous and Rpl29-null aortic ring assays compared with wild-type controls. Moreover, we provide in vivo evidence that RPL29 can regulate tumour angiogenesis. Tumour blood vessel density in subcutaneously grown Lewis lung carcinomas was reduced significantly in Rpl29-mutant mice. Additionally, depletion of Rpl29 using RNA interference inhibited VEGF-induced aortic ring sprouting, suggesting that anti-RPL29 strategies might have anti-angiogenic potential. Overall, our results identify that loss or depletion of RPL29 can reduce angiogenesis in vivo and ex vivo.
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Neovascularización Fisiológica/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/fisiología , Animales , Aorta Torácica/metabolismo , Aorta Torácica/patología , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Células Endoteliales/metabolismo , Expresión Génica , Integrina alfaVbeta3/deficiencia , Integrina alfaVbeta3/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/prevención & control , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Proteínas Ribosómicas/antagonistas & inhibidores , Proteínas Ribosómicas/deficiencia , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Genetic ablation of endothelial focal adhesion kinase (FAK) can inhibit pathological angiogenesis, suggesting that loss of endothelial FAK is sufficient to reduce neovascularization. Here we show that reduced stromal FAK expression in FAK-heterozygous mice unexpectedly enhances both B16F0 and CMT19T tumour growth and angiogenesis. We further demonstrate that cell proliferation and microvessel sprouting, but not migration, are increased in serum-stimulated FAK-heterozygous endothelial cells. FAK-heterozygous endothelial cells display an imbalance in FAK phosphorylation at pY397 and pY861 without changes in Pyk2 or Erk1/2 activity. By contrast, serum-stimulated phosphorylation of Akt is enhanced in FAK-heterozygous endothelial cells and these cells are more sensitive to Akt inhibition. Additionally, low doses of a pharmacological FAK inhibitor, although too low to affect FAK autophosphorylation in vitro, can enhance angiogenesis ex vivo and tumour growth in vivo. Our results highlight a potential novel role for FAK as a nonlinear, dose-dependent regulator of angiogenesis where heterozygous levels of FAK enhance angiogenesis.
Asunto(s)
Quinasa 1 de Adhesión Focal/metabolismo , Neoplasias/irrigación sanguínea , Neoplasias/enzimología , Neovascularización Patológica/enzimología , Animales , Proliferación Celular , Separación Celular , Supervivencia Celular , Células Endoteliales/patología , Heterocigoto , Inmunohistoquímica , Técnicas In Vitro , Ratones , Proteínas Mutantes/metabolismo , Neoplasias/patología , Neovascularización Patológica/patología , Paxillin/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Tejido Subcutáneo/patología , Talina/metabolismo , Carga Tumoral , Vinculina/metabolismoRESUMEN
Angiogenesis is essential for solid tumour growth, whilst the molecular profiles of tumour blood vessels have been reported to be different between cancer types. Although presently available anti-angiogenic strategies are providing some promise for the treatment of some cancers it is perhaps not surprisingly that, none of the anti-angiogenic agents available work on all tumours. Thus, the discovery of novel anti-angiogenic targets, relevant to individual cancer types, is required. Using Affymetrix microarray analysis of laser-captured, CD31-positive blood vessels we have identified 63 genes that are upregulated significantly (5-72 fold) in angiogenic blood vessels associated with human invasive ductal carcinoma (IDC) of the breast as compared with blood vessels in normal human breast. We tested the angiogenic capacity of a subset of these genes. Genes were selected based on either their known cellular functions, their enriched expression in endothelial cells and/or their sensitivity to anti-VEGF treatment; all features implicating their involvement in angiogenesis. For example, RRM2, a ribonucleotide reductase involved in DNA synthesis, was upregulated 32-fold in IDC-associated blood vessels; ATF1, a nuclear activating transcription factor involved in cellular growth and survival was upregulated 23-fold in IDC-associated blood vessels and HEX-B, a hexosaminidase involved in the breakdown of GM2 gangliosides, was upregulated 8-fold in IDC-associated blood vessels. Furthermore, in silico analysis confirmed that AFT1 and HEX-B also were enriched in endothelial cells when compared with non-endothelial cells. None of these genes have been reported previously to be involved in neovascularisation. However, our data establish that siRNA depletion of Rrm2, Atf1 or Hex-B had significant anti-angiogenic effects in VEGF-stimulated ex vivo mouse aortic ring assays. Overall, our results provide proof-of-principle that our approach can identify a cohort of potentially novel anti-angiogenic targets that are likley to be, but not exclusivley, relevant to breast cancer.
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Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Perfilación de la Expresión Génica , Neovascularización Patológica/genética , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Anticuerpos/inmunología , Anticuerpos/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Bevacizumab , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/irrigación sanguínea , Carcinoma Ductal de Mama/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Inmunohistoquímica , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones SCID , Microscopía Confocal , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Interferencia de ARN , Trasplante Heterólogo , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/inmunología , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Here we provide a protocol for quantitative three-dimensional ex vivo mouse aortic ring angiogenesis assays, in which developing microvessels undergo many key features of angiogenesis over a timescale similar to that observed in vivo. The aortic ring assay allows analysis of cellular proliferation, migration, tube formation, microvessel branching, perivascular recruitment and remodeling-all without the need for cellular dissociation-thus providing a more complete picture of angiogenic processes compared with traditional cell-based assays. Our protocol can be applied to aortic rings from embryonic stage E18 through to adulthood and can incorporate genetic manipulation, treatment with growth factors, drugs or siRNA. This robust assay allows assessment of the salient steps in angiogenesis and quantification of the developing microvessels, and it can be used to identify new modulators of angiogenesis. The assay takes 6-14 d to complete, depending on the age of the mice, treatments applied and whether immunostaining is performed.
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Aorta/fisiología , Neovascularización Fisiológica/fisiología , Técnicas de Cultivo de Tejidos , Animales , Aorta/citología , Movimiento Celular , Proliferación Celular , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Microscopía de Contraste de FaseRESUMEN
We report the characterization of a nucleolar localization sequence (NoLS) that targets the green fluorescent protein (GFP) into the granular component (GC) of nucleoli. This NoLS interacts in vitro specifically and directly with the major nucleolar protein B23 and more precisely with the region of B23 including the two acidic stretches. The affinity of NoLS for B23 is stronger than that of the HIV-1 Rev protein in vitro. Moreover, B23-NoLS interaction also occurs in vivo. Indeed, (1) NoLS confers on the GFP the behavior of B23 throughout the cell cycle, (2) the GFP-NoLS fusion and B23 remain colocalized after drug treatments, (3) a selective delocalization of B23 from nucleoli to nucleoplasm induces a concomitent delocalization of the GFP-NoLS fusion, and (4) the fusion of NoLS to fibrillarin makes it possible to colocalize fibrillarin and B23. Interestingly, by fusing NoLS to fibrillarin, both fibrillarin and the fibrillarin partner Nop56 are mislocalized in the GC of nucleoli. Similarly, by fusing the NoLS to MafG, part of the nuclear transcription factor NF-E2 composed of both MafG and p45 NF-E2, NF-E2 is redirected from the nucleoplasm to the nucleoli. Thus, we propose that the NoLS may be used as a tool to visualize and prove protein interactions in a cellular context.