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BACKGROUND AND AIMS: HCC is the most common primary liver tumor, with an increasing incidence worldwide. HCC is a heterogeneous malignancy and usually develops in a chronically injured liver. The NF-κB signaling network consists of a canonical and a noncanonical branch. Activation of canonical NF-κB in HCC is documented. However, a functional and clinically relevant role of noncanonical NF-κB and its downstream effectors is not established. APPROACH AND RESULTS: Four human HCC cohorts (total n = 1462) and 4 mouse HCC models were assessed for expression and localization of NF-κB signaling components and activating ligands. In vitro , NF-κB signaling, proliferation, and cell death were measured, proving a pro-proliferative role of v-rel avian reticuloendotheliosis viral oncogene homolog B (RELB) activated by means of NF-κB-inducing kinase. In vivo , lymphotoxin beta was identified as the predominant inducer of RELB activation. Importantly, hepatocyte-specific RELB knockout in a murine HCC model led to a lower incidence compared to controls and lower maximal tumor diameters. In silico , RELB activity and RELB-directed transcriptomics were validated on the The Cancer Genome Atlas HCC cohort using inferred protein activity and Gene Set Enrichment Analysis. In RELB-active HCC, pathways mediating proliferation were significantly activated. In contrast to v-rel avian reticuloendotheliosis viral oncogene homolog A, nuclear enrichment of noncanonical RELB expression identified patients with a poor prognosis in an etiology-independent manner. Moreover, RELB activation was associated with malignant features metastasis and recurrence. CONCLUSIONS: This study demonstrates a prognostically relevant, etiology-independent, and cross-species consistent activation of a lymphotoxin beta/LTßR/RELB axis in hepatocarcinogenesis. These observations may harbor broad implications for HCC, including possible clinical exploitation.
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Bone morphogenetic protein (BMP)-9, a member of the TGFß-family of cytokines, is believed to be mainly produced in the liver. The serum levels of BMP-9 were reported to be reduced in newly diagnosed diabetic patients and BMP-9 overexpression ameliorated steatosis in the high fat diet-induced obesity mouse model. Furthermore, injection of BMP-9 in mice enhanced expression of fibroblast growth factor (FGF)21. However, whether BMP-9 also regulates the expression of the related FGF19 is not clear. Because both FGF21 and 19 were described to protect the liver from steatosis, we have further investigated the role of BMP-9 in this context. We first analyzed BMP-9 levels in the serum of streptozotocin (STZ)-induced diabetic rats (a model of type I diabetes) and confirmed that BMP-9 serum levels decrease during diabetes. Microarray analyses of RNA samples from hepatic and intestinal tissue from BMP-9 KO- and wild-type mice (C57/Bl6 background) pointed to basal expression of BMP-9 in both organs and revealed a down-regulation of hepatic Fgf21 and intestinal Fgf19 in the KO mice. Next, we analyzed BMP-9 levels in a cohort of obese patients with or without diabetes. Serum BMP-9 levels did not correlate with diabetes, but hepatic BMP-9 mRNA expression negatively correlated with steatosis in those patients that did not yet develop diabetes. Likewise, hepatic BMP-9 expression also negatively correlated with serum LPS levels. In situ hybridization analyses confirmed intestinal BMP-9 expression. Intestinal (but not hepatic) BMP-9 mRNA levels were decreased with diabetes and positively correlated with intestinal E-Cadherin expression. In vitro studies using organoids demonstrated that BMP-9 directly induces FGF19 in gut but not hepatocyte organoids, whereas no evidence of a direct induction of hepatic FGF21 by BMP-9 was found. Consistent with the in vitro data, a correlation between intestinal BMP-9 and FGF19 mRNA expression was seen in the patients' samples. In summary, our data confirm that BMP-9 is involved in diabetes development in humans and in the control of the FGF-axis. More importantly, our data imply that not only hepatic but also intestinal BMP-9 associates with diabetes and steatosis development and controls FGF19 expression. The data support the conclusion that increased levels of BMP-9 would most likely be beneficial under pre-steatotic conditions, making supplementation of BMP-9 an interesting new approach for future therapies aiming at prevention of the development of a metabolic syndrome and liver steatosis.
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Diabetes Mellitus Experimental , Hígado Graso , Humanos , Ratas , Ratones , Animales , Factor 2 de Diferenciación de Crecimiento/metabolismo , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Obesidad/complicaciones , Obesidad/metabolismo , Hígado/metabolismo , Hígado Graso/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , ARN Mensajero/metabolismoRESUMEN
Radiotherapy is a mainstay cancer therapy whose antitumor effects partially depend on T cell responses. However, the role of Natural Killer (NK) cells in radiotherapy remains unclear. Here, using a reverse translational approach, we show a central role of NK cells in the radiation-induced immune response involving a CXCL8/IL-8-dependent mechanism. In a randomized controlled pancreatic cancer trial, CXCL8 increased under radiotherapy, and NK cell positively correlated with prolonged overall survival. Accordingly, NK cells preferentially infiltrated irradiated pancreatic tumors and exhibited CD56dim-like cytotoxic transcriptomic states. In experimental models, NF-κB and mTOR orchestrated radiation-induced CXCL8 secretion from tumor cells with senescence features causing directional migration of CD56dim NK cells, thus linking senescence-associated CXCL8 release to innate immune surveillance of human tumors. Moreover, combined high-dose radiotherapy and adoptive NK cell transfer improved tumor control over monotherapies in xenografted mice, suggesting NK cells combined with radiotherapy as a rational cancer treatment strategy.
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Interleucina-8 , Células Asesinas Naturales , Neoplasias , Traslado Adoptivo , Animales , Humanos , Inmunidad , Interleucina-8/inmunología , Interleucina-8/metabolismo , Células Asesinas Naturales/inmunología , Ratones , Neoplasias/inmunología , Neoplasias/radioterapia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Increase of blood capillary density at the interface between normal and ischemic tissue after acute MI reduces infarct size and improves cardiac function. Cardiac injury triggers the production of the matricellular component TSP-1, but its role in angiogenesis is not clear, as both anti- and proangiogenic properties have been reported. It is unknown whether TSP-1 is modulated by other factors released during cardiac injury. Among these, Ado is a well-known promoter of angiogenesis. This study determined whether Ado modulates TSP-1 expression and the implication on angiogenesis. Ado dose dependently increased the production of TSP-1 by human macrophages. With the use of agonists and antagonists of AdoRs, coupled to RNA interference, we observed that this effect is mediated via A2AR and A2BR. The Ado effect was reproduced by cholera toxin (Gs protein activator) and forskolin (adenylate cyclase activator) and blocked by the PKA inhibitor H89. Conditioned medium from Ado-treated macrophages stimulated microvessel outgrowth from aortic ring explants by 400%, and induced vessel formation in matrigel plugs. Microvessel outgrowth and vessel formation were blocked completely by addition of anti-TSP-1 antibodies to conditioned medium. Chronic administration of Ado to rats after MI maintained long-term expression of TSP-1 in the infarct border zone, and this was associated with enhanced border-zone vascularization. Ado up-regulates TSP-1 production by macrophages, resulting in stimulation of angiogenesis. The mechanism involves A2AR and A2BR and is mediated through the cAMP/PKA pathway. This information may be important when designing Ado-based therapies of angiogenesis.
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Adenosina/metabolismo , Macrófagos/metabolismo , Infarto del Miocardio/metabolismo , Neovascularización Fisiológica/fisiología , Trombospondina 1/biosíntesis , Animales , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Microscopía Confocal , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Regulación hacia ArribaRESUMEN
It is now well accepted that multipotent Bone-Marrow Mesenchymal Stem Cells (BM-MSC) contribute to cancer progression through several mechanisms including angiogenesis. However, their involvement during the lymphangiogenic process is poorly described. Using BM-MSC isolated from mice of two different backgrounds, we demonstrate a paracrine lymphangiogenic action of BM-MSC both in vivo and in vitro. Co-injection of BM-MSC and tumor cells in mice increased the in vivo tumor growth and intratumoral lymphatic vessel density. In addition, BM-MSC or their conditioned medium stimulated the recruitment of lymphatic vessels in vivo in an ear sponge assay, and ex vivo in the lymphatic ring assay (LRA). In vitro, MSC conditioned medium also increased the proliferation rate and the migration of both primary lymphatic endothelial cells (LEC) and an immortalized lymphatic endothelial cell line. Mechanistically, these pro-lymphangiogenic effects relied on the secretion of Vascular Endothelial Growth Factor (VEGF)-A by BM-MSC that activates VEGF Receptor (VEGFR)-2 pathway on LEC. Indeed, the trapping of VEGF-A in MSC conditioned medium by soluble VEGF Receptors (sVEGFR)-1, -2 or the inhibition of VEGFR-2 activity by a specific inhibitor (ZM 323881) both decreased LEC proliferation, migration and the phosphorylation of their main downstream target ERK1/2. This study provides direct unprecedented evidence for a paracrine lymphangiogenic action of BM-MSC via the production of VEGF-A which acts on LEC VEGFR-2.
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Linfangiogénesis , Células Madre Mesenquimatosas/fisiología , Animales , Movimiento Celular , Proliferación Celular , Medios de Cultivo Condicionados , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Femenino , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos C57BL , Fosforilación , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The endothelial cell spheroid assay provides a suitable in vitro model to study (lymph) angiogenesis and test pro- and anti-(lymph) angiogenic factors or drugs. Usually, the extent of cell invasion, observed through optical microscopy, is measured. The present study proposes the spatial distribution of migrated cells as a new descriptor of the (lymph) angiogenic response. The utility of this novel method rests with its capacity to locally characterise spheroid structure, allowing not only the investigation of single and collective cell invasion but also the evolution of the spheroid core itself. Moreover, the proposed method can be applied to 2D-projected spheroid images obtained by optical microscopy, as well as to 3D images acquired by confocal microscopy. To validate the proposed methodology, endothelial cell invasion was evaluated under different experimental conditions. The results were compared with widely used global parameters. The comparison shows that our method prevents local spheroid modifications from being overlooked and leading to the possible misinterpretation of results.
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Rastreo Celular/métodos , Células Endoteliales/ultraestructura , Neovascularización Patológica/genética , Esferoides Celulares/ultraestructura , Inhibidores de la Angiogénesis/farmacología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Células Endoteliales/efectos de los fármacos , Humanos , Imagenología Tridimensional , Microscopía Confocal , Neovascularización Patológica/tratamiento farmacológico , Esferoides Celulares/efectos de los fármacos , Telomerasa/química , Telomerasa/aislamiento & purificación , Factor A de Crecimiento Endotelial Vascular/química , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: The lymphatic system controls tissue homeostasis by draining protein-rich lymph to the vascular system. Lymphangiogenesis, the formation of lymphatic vessels, is a normal event in childhood but promotes tumor spread and metastasis during adulthood. Blocking lymphangiogenesis may therefore be of therapeutic interest. Production of adenosine is enhanced in the tumor environment and contributes to tumor progression through stimulation of angiogenesis. In this study, we determined whether adenosine affects lymphangiogenesis. METHODS: Lymphatic endothelial cells (HMVEC-dLy) were cultured in presence of adenosine and their proliferation, migration and tube formation was assessed. Gelatin sponges embedded with the stable analogue of adenosine 2-chloro adenosine were implanted in mice ear and lymphangiogenesis was quantified. Mice were intravenously injected with adenoviruses containing expression vector for 5'-endonucleotidase, which plays a major role in the formation of adenosine. RESULTS: In vitro, we observed that adenosine decreased the proliferation of lymphatic endothelial cells, their migration and tube formation. However, in vivo, gelatin sponges containing 2-chloro adenosine and implanted in mice ear displayed an elevated level of lymphangiogenesis (2.5-fold, p<0.001). Adenovirus-mediated over-expression of cytosolic 5'-nucleotidase IA stimulated lymphangiogenesis and the recruitment of macrophages in mouse liver. Proliferation of lymphatic endothelial cells was enhanced (2-fold, p<0.001) when incubated in the presence of conditioned medium from murine macrophages. CONCLUSION: We have shown that adenosine stimulates lymphangiogenesis in vivo, presumably through a macrophage-mediated mechanism. This observation suggests that blockade of adenosine receptors may help in anti-cancer therapies.
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Adenosina/farmacología , Linfangiogénesis/efectos de los fármacos , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Expresión Génica , Humanos , Hígado/metabolismo , Linfangiogénesis/genética , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P1/metabolismoRESUMEN
BACKGROUND: Abnormal lymphatic vessel formation (lymphangiogenesis) is associated with different pathologies such as cancer, lymphedema, psoriasis and graft rejection. Lymphatic vasculature displays distinctive features than blood vasculature, and mechanisms underlying the formation of new lymphatic vessels during physiological and pathological processes are still poorly documented. Most studies on lymphatic vessel formation are focused on organism development rather than lymphangiogenic events occurring in adults. We have here studied lymphatic vessel formation in two in vivo models of pathological lymphangiogenesis (corneal assay and lymphangioma). These data have been confronted to those generated in the recently set up in vitro model of lymphatic ring assay. Ultrastructural analyses through Transmission Electron Microscopy (TEM) were performed to investigate tube morphogenesis, an important differentiating process observed during endothelial cell organization into capillary structures. RESULTS: In both in vivo models (lymphangiogenic corneal assay and lymphangioma), migrating lymphatic endothelial cells extended long processes exploring the neighboring environment and organized into cord-like structures. Signs of intense extracellular matrix remodeling were observed extracellularly and inside cytoplasmic vacuoles. The formation of intercellular spaces between endothelial cells led to tube formation. Proliferating lymphatic endothelial cells were detected both at the tips of sprouting capillaries and inside extending sprouts. The different steps of lymphangiogenesis observed in vivo are fully recapitulated in vitro, in the lymphatic ring assay and include: (1) endothelial cell alignment in cord like structure, (2) intracellular vacuole formation and (3) matrix degradation. CONCLUSIONS: In this study, we are providing evidence for lymphatic vessel formation through tunneling relying on extensive matrix remodeling, migration and alignment of sprouting endothelial cells into tubular structures. In addition, our data emphasize the suitability of the lymphatic ring assay to unravel mechanisms underlying lymphangiogenesis.