RESUMEN
RATIONALE: Endothelial colony forming cells (ECFCs) or late blood outgrowth endothelial cells can be isolated from human cord or peripheral blood, display properties of endothelial progenitors, home into ischemic tissues and support neovascularization in ischemic disease models. OBJECTIVE: To assess the functions of CYTL1 (cytokine-like 1), a factor we found preferentially produced by ECFCs, in regard of vessel formation. METHODS AND RESULTS: We show by transcriptomic analysis that ECFCs are distinguished from endothelial cells of the vessel wall by production of high amounts of CYTL1. Modulation of expression demonstrates that the factor confers increased angiogenic sprouting capabilities to ECFCs and can also trigger sprouting of mature endothelial cells. The data further display that CYTL1 can be induced by hypoxia and that it functions largely independent of VEGF-A (vascular endothelial growth factor-A). By recombinant production of CYTL1 we confirm that the peptide is indeed a strong proangiogenic factor and induces sprouting in cellular assays and functional vessel formation in animal models comparable to VEGF-A. Mass spectroscopy corroborates that CYTL1 is specifically O-glycosylated on 2 neighboring threonines in the C-terminal part and this modification is important for its proangiogenic bioactivity. Further analyses show that the factor does not upregulate proinflammatory genes and strongly induces several metallothionein genes encoding anti-inflammatory and antiapoptotic proteins. CONCLUSIONS: We conclude that CYTL1 can mediate proangiogenic functions ascribed to endothelial progenitors such as ECFCs in vivo and may be a candidate to support vessel formation and tissue regeneration in ischemic pathologies.
Asunto(s)
Proteínas Angiogénicas/metabolismo , Comunicación Autocrina , Proteínas Sanguíneas/metabolismo , Neovascularización de la Córnea , Citocinas/metabolismo , Células Progenitoras Endoteliales/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Neovascularización Fisiológica , Comunicación Paracrina , Proteínas Angiogénicas/genética , Animales , Proteínas Sanguíneas/genética , Hipoxia de la Célula , Citocinas/genética , Modelos Animales de Enfermedad , Femenino , Glicosilación , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/trasplante , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones SCID , Vías Secretoras , Transducción de Señal , Esferoides Celulares , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Genetic deletion of the tyrosine kinase JAK2 or the downstream transcription factor STAT5 in liver impairs growth hormone (GH) signalling and thereby promotes fatty liver disease. Hepatic STAT5 deficiency accelerates liver tumourigenesis in presence of high GH levels. To determine whether the upstream kinase JAK2 exerts similar functions, we crossed mice harbouring a hepatocyte-specific deletion of JAK2 (JAK2Δhep) to GH transgenic mice (GHtg) and compared them to GHtgSTAT5Δhep mice. Similar to GHtgSTAT5Δhep mice, JAK2 deficiency resulted in severe steatosis in the GHtg background. However, in contrast to STAT5 deficiency, loss of JAK2 significantly delayed liver tumourigenesis. This was attributed to: (i) activation of STAT3 in STAT5-deficient mice, which was prevented by JAK2 deficiency and (ii) increased detoxification capacity of JAK2-deficient livers, which diminished oxidative damage as compared to GHtgSTAT5Δhep mice, despite equally severe steatosis and reactive oxygen species (ROS) production. The reduced oxidative damage in JAK2-deficient livers was linked to increased expression and activity of glutathione S-transferases (GSTs). Consistent with genetic deletion of Jak2, pharmacological inhibition and siRNA-mediated knockdown of Jak2 led to significant upregulation of Gst isoforms and to reduced hepatic oxidative DNA damage. Therefore, blocking JAK2 function increases detoxifying GSTs in hepatocytes and protects against oxidative liver damage.
Asunto(s)
Hígado Graso/patología , Eliminación de Gen , Hormona de Crecimiento Humana/genética , Janus Quinasa 2/genética , Hígado/patología , Animales , Hígado Graso/genética , Hígado Graso/metabolismo , Glutatión Transferasa/metabolismo , Metabolismo de los Lípidos , Hígado/metabolismo , Masculino , Ratones , Ratones Transgénicos , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent "gold standard". Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution), at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients.
Asunto(s)
GTP Fosfohidrolasas/genética , Melanoma/genética , Proteínas de la Membrana/genética , Mutación , Fosfohidrolasa PTEN/genética , Proteínas Proto-Oncogénicas B-raf/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Línea Celular Tumoral , Estudios de Cohortes , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN/métodos , ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Humanos , Melanoma/sangre , Melanoma/patología , Estadificación de Neoplasias , Polimorfismo de Nucleótido Simple , Reproducibilidad de los ResultadosRESUMEN
Human hepatocellular carcinoma (HCC) is a heterogeneous disease, driven by different risk factors and presenting diverse clinicopathological features and outcomes. Epidemiological and experimental data indicate that the damage-associated molecular pattern molecules S100A8 and S100A9, forming a heterodimer called calprotectin, might be critically involved in HCC development. However, deletion of S100a9 in an inflammation- and cirrhosis-driven mouse model did not show any impairment in liver tumorigenesis, most likely due to functional compensation by other inflammatory cytokines. Here, we investigated the effect of calprotectin ablation in mice treated with diethylnitrosamine, a carcinogen-driven HCC model mimicking cancer development caused by acute liver damage in the absence of prominent chronic inflammation and tissue damage. We found that tumor cell proliferation was diminished in the absence of S100A8/A9, leading to significant reduction of tumor size. Our results demonstrate that calprotectin is required for the progression of non-inflammation driven liver tumor and might represent a therapeutic target for the treatment of HCC formed in non-cirrhotic liver.
Asunto(s)
Calgranulina A/genética , Calgranulina B/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Animales , Carcinoma Hepatocelular/patología , Modelos Animales de Enfermedad , Humanos , Neoplasias Hepáticas/patología , Masculino , RatonesRESUMEN
UNLABELLED: Signal transducer and activator of transcription 3 (Stat3) is activated in a variety of malignancies, including hepatocellular carcinoma (HCC). Activation of Ras occurs frequently at advanced stages of HCC by aberrant signaling through growth factor receptors or inactivation of effectors negatively regulating Ras signaling. Here, we addressed the role of Stat3 in Ras-dependent HCC progression in the presence and absence of p19(ARF) /p14(ARF) . We show that constitutive active (ca) Stat3 is tumor suppressive in Ras-transformed p19(ARF-/-) hepatocytes, whereas the expression of Stat3 lacking Tyr(705) phosphorylation (U-Stat3) enhances tumor formation. Accordingly, Ras-transformed Stat3(Δhc) /p19(ARF-/-) hepatocytes (lacking Stat3 and p19(ARF) ) showed increased tumor growth, compared to those expressing Stat3, demonstrating a tumor-suppressor activity of Stat3 in cells lacking p19(ARF) . Notably, endogenous expression of p19(ARF) in Ras-transformed hepatocytes conveyed oncogenic Stat3 functions, resulting in augmented or reduced HCC progression after the expression of caStat3 or U-Stat3, respectively. In accord with these data, the knockdown of p14(ARF) (the human homolog of p19(ARF) ) in Hep3B cells was associated with reduced pY-Stat3 levels during tumor growth to circumvent the tumor-suppressive effect of Stat3. Inhibition of Janus kinases (Jaks) revealed that Jak causes pY-Stat3 activation independently of p14(ARF) levels, indicating that p14(ARF) controls the oncogenic function of pY-Stat3 downstream of Jak. CONCLUSION: These data show evidence that p19(ARF) /p14(ARF) determines the pro- or anti-oncogenic activity of U-Stat3 and pY-Stat3 in Ras-dependent HCC progression.
Asunto(s)
Carcinoma Hepatocelular/fisiopatología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/fisiología , Neoplasias Hepáticas/fisiopatología , Factor de Transcripción STAT3/fisiología , Animales , Carcinoma Hepatocelular/patología , Proliferación Celular , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/deficiencia , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Hepatocitos/patología , Quinasas Janus/fisiología , Neoplasias Hepáticas/patología , Ratones , Ratones Noqueados , Transducción de Señal/fisiologíaRESUMEN
Both RAS and transforming growth factor (TGF)-ß signaling cascades are central in tumorigenesis and show synergisms depending on tumor stage and tissue context. In this review we focus on the interaction of RAS subeffector proteins with signaling components of the TGF-ß family including those of TGF-ßs, activins and bone morphogenic proteins. Compelling evidence indicates that RAS signaling is essentially involved in the switch from tumor-suppressive to tumor-promoting functions of the TGF-ß family leading to enhanced cancer growth and metastatic dissemination of primary tumors. Thus, the interface of these signaling cascades is considered as a promising target for the development of novel cancer therapeutics. The current pharmacological anti-cancer concepts combating the molecular cooperation between RAS and TGF-ß family signaling during carcinoma progression are critically discussed.
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Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Proteínas ras/metabolismo , Ensayos Clínicos como Asunto , HumanosRESUMEN
BACKGROUND & AIMS: Signal transducer and activator of transcription 3 (Stat3) is the main mediator of interleukin-6-type cytokine signaling required for hepatocyte proliferation and hepatoprotection, but its role in sclerosing cholangitis and other cholestatic liver diseases remains unresolved. METHODS: We investigated the role of Stat3 in inflammation-induced cholestatic liver injury and used mice lacking the multidrug resistance gene 2 (mdr2(-/-)) as a model for SC. RESULTS: We show that conditional inactivation of Stat3 in hepatocytes and cholangiocytes (stat3(Deltahc)) of mdr2(-/-) mice strongly aggravated bile acid-induced liver injury and fibrosis. A similar phenotype was observed in mdr2(-/-) mice lacking interleukin-6 production. Biochemical and molecular characterization suggested that Stat3 exerts hepatoprotective functions in both hepatocytes and cholangiocytes. Loss of Stat3 led to increased expression of tumor necrosis factor alpha, which might reduce the barrier function of bile ducts. Moreover, Stat3-deficient hepatocytes displayed up-regulation of bile acid biosynthesis genes and down-regulation of hepatoprotective epidermal growth factor receptor and insulin-like growth factor 1 signaling pathways. Consistently, stat3(Deltahc) mice were more sensitive to cholic acid-induced liver damage than control mice. CONCLUSIONS: Our data suggest that Stat3 prevents cholestasis and liver damage in sclerosing cholangitis via regulation of pivotal functions in hepatocytes and cholangiocytes.
Asunto(s)
Colangitis Esclerosante/complicaciones , Citoprotección , Cirrosis Hepática Experimental/prevención & control , Factor de Transcripción STAT3/fisiología , Subfamilia B de Transportador de Casetes de Unión a ATP/fisiología , Animales , Ácidos y Sales Biliares/toxicidad , Proliferación Celular , Hígado/efectos de los fármacos , Regeneración Hepática , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
UNLABELLED: Growth hormone (GH) resistance and low serum levels of insulinlike growth factor 1 (IGF-1) are common features in human liver fibrosis and cirrhosis. Signal transducer and activator of transcription 5 (STAT5) controls several vital functions in the liver, including GH-mediated transcription of IGF-1. To investigate the role of STAT5 in liver fibrogenesis, we specifically deleted the Stat5a/b locus both in hepatocytes and cholangiocytes in the multidrug resistance gene 2 knockout (Mdr2(-/-)) mouse model of cholestasis. Double knockout mice develop an early and severe liver fibrosis phenotype, accompanied by perturbed expression of key regulators of bile acid homeostasis. Deletion of Stat5 resulted in GH resistance, and IGF-1 levels in serum were undetectable. We could observe reduced expression of important hepatoprotective genes, such as epidermal growth factor receptor (Egfr), hepatocyte nuclear factor 6 (Hnf6), prolactin receptor (Prlr), and leukemia inhibitory factor receptor (Lifr) as well as increased numbers of apoptotic hepatocytes. CONCLUSION: Our data suggest that loss of STAT5 sensitizes hepatocytes to bile acid-induced damage and apoptosis caused by disruption of GH-induced transcription of Igf-1 and down-regulation of hepatoprotective genes. These findings could contribute to the understanding of liver fibrosis and future treatment strategies for liver fibrosis.
Asunto(s)
Colestasis/complicaciones , Hormona del Crecimiento/fisiología , Factor I del Crecimiento Similar a la Insulina/fisiología , Cirrosis Hepática Experimental/etiología , Factor de Transcripción STAT5/fisiología , Subfamilia B de Transportador de Casetes de Unión a ATP/fisiología , Animales , Apoptosis , Modelos Animales de Enfermedad , Receptores ErbB/genética , Factor Nuclear 6 del Hepatocito/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Transducción de Señal , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Transforming growth factor-beta cooperates with oncogenic Ras to activate nuclear beta-catenin during the epithelial to mesenchymal transition of hepatocytes, a process relevant in the progression of hepatocellular carcinoma (HCC). In this study we investigated the role of beta-catenin in the differentiation of murine, oncogene-targeted hepatocytes and in 133 human HCC patients scheduled for orthotopic liver transplantation. Transforming growth factor-beta caused dissociation of plasma membrane E-cadherin/beta-catenin complexes and accumulation of nuclear beta-catenin in Ras-transformed, but otherwise normal hepatocytes in p19(ARF)-/- mice. Both processes were inhibited by Smad7-mediated disruption of transforming growth factor-beta signaling. Overexpression of constitutively active beta-catenin resulted in high levels of CK19 and M2-PK, whereas ablation of beta-catenin by axin overexpression caused strong expression of CK8 and CK18. Therefore, nuclear beta-catenin resulted in dedifferentiation of neoplastic hepatocytes to immature progenitor cells, whereas loss of nuclear beta-catenin led to a differentiated HCC phenotype. Poorly differentiated human HCC showed cytoplasmic redistribution or even loss of E-cadherin, suggesting epithelial to mesenchymal transition. Analysis of 133 HCC patient samples revealed that 58.6% of human HCC exhibited strong nuclear beta-catenin accumulation, which correlated with clinical features such as vascular invasion and recurrence of disease after orthotopic liver transplantation. These data suggest that activation of beta-catenin signaling causes dedifferentiation to malignant, immature hepatocyte progenitors and facilitates recurrence of human HCC after orthotopic liver transplantation.
Asunto(s)
Carcinoma Hepatocelular/patología , Núcleo Celular/metabolismo , Neoplasias Hepáticas/patología , Hígado/patología , Recurrencia Local de Neoplasia/metabolismo , Células Madre/patología , beta Catenina/metabolismo , Animales , Cadherinas/metabolismo , Carcinoma Hepatocelular/irrigación sanguínea , Carcinoma Hepatocelular/metabolismo , Diferenciación Celular , Membrana Celular/metabolismo , Epitelio/metabolismo , Epitelio/patología , Femenino , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Hígado/metabolismo , Neoplasias Hepáticas/irrigación sanguínea , Neoplasias Hepáticas/metabolismo , Trasplante de Hígado , Masculino , Mesodermo/metabolismo , Mesodermo/patología , Ratones , Neovascularización Patológica/complicaciones , Fenotipo , Transporte de Proteínas , Transducción de Señal , Proteína smad7/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The transition of epithelial cells to a mesenchymal phenotype is of paramount relevance for embryonic development and adult wound healing. During the past decade, the epithelial-mesenchymal transition (EMT) has been increasingly recognized to occur during the progression of various carcinomas such as hepatocellular carcinoma (HCC). Here, we focus on EMT in both experimental liver models and human HCC, emphasizing the underlying molecular mechanisms which show partial recurrence of embryonic programs such as TGF-beta and Wnt/ beta-catenin signaling, including collaboration with hepatitis viruses. We further discuss the differentiation repertoire of malignant hepatocytes with respect to the potential acquisition of stemness, and the involvement of the mesenchymal to epithelial transition, the reversal of EMT, in cancer dissemination and metastatic colonization. The strong evidence for EMT in HCC patients demands novel strategies in pathological assessments and therapeutic concepts to efficiently combat HCC progression.
Asunto(s)
Carcinoma Hepatocelular/patología , Diferenciación Celular/fisiología , Transformación Celular Neoplásica/patología , Epitelio/patología , Neoplasias Hepáticas/patología , Animales , Carcinoma Hepatocelular/genética , Desdiferenciación Celular/fisiología , Transformación Celular Neoplásica/genética , Humanos , Neoplasias Hepáticas/genética , Mesodermo/patología , MetaplasiaRESUMEN
In skeletal muscle, the cytolinker plectin is prominently expressed at Z-disks and the sarcolemma. Alternative splicing of plectin transcripts gives rise to more than eight protein isoforms differing only in small N-terminal sequences (5-180 residues), four of which (plectins 1, 1b, 1d, and 1f) are found at substantial levels in muscle tissue. Using plectin isoform-specific antibodies and isoform expression constructs, we show the differential regulation of plectin isoforms during myotube differentiation and their localization to different compartments of muscle fibers, identifying plectins 1 and 1f as sarcolemma-associated isoforms, whereas plectin 1d localizes exclusively to Z-disks. Coimmunoprecipitation and in vitro binding assays using recombinant protein fragments revealed the direct binding of plectin to dystrophin (utrophin) and beta-dystroglycan, the key components of the dystrophin-glycoprotein complex. We propose a model in which plectin acts as a universal mediator of desmin intermediate filament anchorage at the sarcolemma and Z-disks. It also explains the plectin phenotype observed in dystrophic skeletal muscle of mdx mice and Duchenne muscular dystrophy patients.