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BACKGROUND: The development of single-cell technologies yields large datasets of information as diverse and multimodal as transcriptomes, immunophenotypes, and spatial position from tissue sections in the so-called 'spatial transcriptomics'. Currently however, user-friendly, powerful, and free algorithmic tools for straightforward analysis of spatial transcriptomic datasets are scarce. RESULTS: Here, we introduce Single-Cell Spatial Explorer, an open-source software for multimodal exploration of spatial transcriptomics, examplified with 9 human and murine tissues datasets from 4 different technologies. CONCLUSIONS: Single-Cell Spatial Explorer is a very powerful, versatile, and interoperable tool for spatial transcriptomics analysis.
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Programas Informáticos , Transcriptoma , Humanos , Animales , Ratones , Perfilación de la Expresión Génica , Análisis Espacial , Análisis de la Célula IndividualRESUMEN
Strategies based on intracellular expression of artificial binding domains present several advantages over manipulating nucleic acid expression or the use of small molecule inhibitors. Intracellularly-functional nanobodies can be considered as promising macrodrugs to study key signaling pathways by interfering with protein-protein interactions. With the aim of studying the RAS-related small GTPase RHOA family, we previously isolated, from a synthetic phage display library, nanobodies selective towards the GTP-bound conformation of RHOA subfamily proteins that lack selectivity between the highly conserved RHOA-like and RAC subfamilies of GTPases. To identify RHOA/ROCK pathway inhibitory intracellular nanobodies, we implemented a stringent, subtractive phage display selection towards RHOA-GTP followed by a phenotypic screen based on F-actin fiber loss. Intracellular interaction and intracellular selectivity between RHOA and RAC1 proteins was demonstrated by adapting the sensitive intracellular protein-protein interaction reporter based on the tripartite split-GFP method. This strategy led us to identify a functional intracellular nanobody, hereafter named RH28, that does not cross-react with the close RAC subfamily and blocks/disrupts the RHOA/ROCK signaling pathway in several cell lines without further engineering or functionalization. We confirmed these results by showing, using SPR assays, the high specificity of the RH28 nanobody towards the GTP-bound conformation of RHOA subfamily GTPases. In the metastatic melanoma cell line WM266-4, RH28 expression triggered an elongated cellular phenotype associated with a loss of cellular contraction properties, demonstrating the efficient intracellular blocking of RHOA/B/C proteins downstream interactions without the need of manipulating endogenous gene expression. This work paves the way for future therapeutic strategies based on protein-protein interaction disruption with intracellular antibodies.
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Anticuerpos de Dominio Único , Actinas/metabolismo , Guanosina Trifosfato , Transducción de Señal , Anticuerpos de Dominio Único/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas ras/metabolismoRESUMEN
The high cytotoxic activity of Vγ9Vδ2 T lymphocytes against tumor cells makes them useful candidates in anticancer therapies. However, the molecular mechanism of their activation by phosphoantigens (PAgs) is not completely known. Many studies have depicted the mechanism of Vγ9Vδ2 T-cell activation by PAg-sensed accessory cells, such as immune presenting cells or tumor cells. In this study, we demonstrated that pure resting Vγ9Vδ2 T lymphocytes can self-activate through exogenous PAgs, involving their TCR and the butyrophilins BTN3A1 and BTN2A1. This is the first time that these three molecules, concurrently expressed at the plasma membrane of Vγ9Vδ2 T cells, have been shown to be involved together on the same and unique T cell during PAg activation. Moreover, the use of probucol to stimulate the inhibition of this self-activation prompted us to propose that ABCA-1 could be implicated in the transfer of exogenous PAgs inside Vγ9Vδ2 T cells before activating them through membrane clusters formed by γ9TCR, BTN3A1 and BTN2A1. The self-activation of Vγ9Vδ2 T cells, which leads to self-killing, can therefore participate in the failure of γδ T cell-based therapies with exogenous PAgs and should be taken into account.
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Receptores de Antígenos de Linfocitos T gamma-delta , Linfocitos T , Antígenos CD/metabolismo , Butirofilinas/metabolismo , Activación de Linfocitos , Subgrupos de Linfocitos TRESUMEN
As key regulators of the actin cytoskeleton, RHO GTPase expression and/or activity are deregulated in tumorigenesis and metastatic progression. Nevertheless, the vast majority of experiments supporting this conclusion was conducted on cell lines but not on human tumor samples that were mostly studied at the expression level only. Up to now, the activity of RHO proteins remains poorly investigated in human tumors. In this article, we present the development of a robust nanobody-based ELISA assay, with a high selectivity that allows an accurate quantification of RHO protein GTP-bound state in the nanomolar range (1 nM; 20 µg/L), not only in cell lines after treatment but also in tumor samples. Of note, we present here a fine analysis of RHOA-like and RAC1 active state in tumor samples with the most comprehensive study of RHOA-GTP and RHOC-GTP levels performed on human breast tumor samples. We revealed increased GTP-bound RHOA and RHOC protein activities in tumors compared to normal tissue counterparts, and demonstrated that the RHO active state and RHO expression are two independent parameters among different breast cancer subtypes. Our results further highlight the regulation of RHO protein activation in tumor samples and the relevance of directly studying RHO GTPase activities involvement in molecular pathways.
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Neoplasias de la Mama , Proteína de Unión al GTP rhoA , Proteína rhoC de Unión a GTP , Transformación Celular Neoplásica , Femenino , Guanosina Trifosfato , Humanos , Proteína de Unión al GTP rhoA/metabolismo , Proteína rhoC de Unión a GTP/metabolismoRESUMEN
Tumor-associated macrophages (TAMs) in chronic lymphocytic leukemia (CLL) are also called nurse-like cells (NLC), and confer survival signals through the release of soluble factors and cellular contacts. While in most patient samples the presence of NLC in co-cultures guarantees high viability of leukemic cells in vitro, in some cases this protective effect is absent. These macrophages are characterized by an "M1-like phenotype". We show here that their reprogramming towards an M2-like phenotype (tumor-supportive) with IL-10 leads to an increase in leukemic cell survival. Inflammatory cytokines, such as TNF, are also able to depolarize M2-type protective NLC (decreasing CLL cell viability), an effect which is countered by IL-10 or blocking antibodies. Interestingly, both IL-10 and TNF are implied in the pathophysiology of CLL and their elevated level is associated with bad prognosis. We propose that the molecular balance between these two cytokines in CLL niches plays an important role in the maintenance of the protective phenotype of NLCs, and therefore in the survival of CLL cells.
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An efficient purification of recombinant proteins often requires a high ratio of recombinant to host proteins. In plants, Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant leaf protein, thus strongly impacting purification yield. Here, we describe a simple and robust purification procedure for recombinant proteins based on a differential precipitation of RuBisCO. In this context, four Legume lectin domains of Arabidopsis thaliana which belong to receptor-like kinases and cell wall proteins were produced from Nicotiana benthamiana leaves. The recombinant proteins exhibit a unique lectin domain consisting of around 250 amino acid residues with several predicted N-glycosylation sites and a six His-tag at the N-terminus. After ammonium sulphate precipitation of total soluble proteins, depletion of RuBisCO was obtained using citrate and succinate buffers during the salting-in step: this depletion was pH-dependent and the presence of di- or tri-carboxylic acids was required. The depleted protein extracts were then subjected to two chromatographic steps which were used in the negative mode to submit a protein fraction enriched as much as possible in recombinant lectin domains to a third chromatographic step (immobilized metal-ion chromatography). Three of the Legume lectin domains were purified near to homogeneity and revealed multiple N-glycosylation isoforms, particularly those from receptor-like kinases, which were characterised using specific lectins and deglycosylation enzymes. The production and purification of recombinant lectin domains will facilitate their biochemical characterisation in the context of cell-to-cell signalling and cell wall organisation.
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Fabaceae/química , Lectinas/biosíntesis , Nicotiana/metabolismo , Ribulosa-Bifosfato Carboxilasa , Glicosilación , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Isoformas de Proteínas/biosíntesis , Proteínas Recombinantes/biosíntesis , Ribulosa-Bifosfato Carboxilasa/metabolismo , Nicotiana/genéticaRESUMEN
Hypoxia, a major inducer of angiogenesis, triggers major changes in gene expression at the transcriptional level. Furthermore, under hypoxia, global protein synthesis is blocked while internal ribosome entry sites (IRES) allow specific mRNAs to be translated. Here, we report the transcriptome and translatome signatures of (lymph)angiogenic genes in hypoxic HL-1 mouse cardiomyocytes: most genes are induced at the translatome level, including all IRES-containing mRNAs. Our data reveal activation of (lymph)angiogenic factor mRNA IRESs in early hypoxia. We identify vasohibin1 (VASH1) as an IRES trans-acting factor (ITAF) that is able to bind RNA and to activate the FGF1 IRES in hypoxia, but which tends to inhibit several IRESs in normoxia. VASH1 depletion has a wide impact on the translatome of (lymph)angiogenesis genes, suggesting that this protein can regulate translation positively or negatively in early hypoxia. Translational control thus appears as a pivotal process triggering new vessel formation in ischemic heart.
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Proteínas de Ciclo Celular/metabolismo , Hipoxia/metabolismo , Sitios Internos de Entrada al Ribosoma/fisiología , Miocitos Cardíacos/metabolismo , Transactivadores/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Línea Celular , Humanos , Ratones , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , ARN Mensajero/metabolismo , Motivos de Unión al ARN , TranscriptomaRESUMEN
RHO (Ras HOmologous) GTPases are molecular switches that activate, in their state bound to Guanosine triphosphate (GTP), key signaling pathways, which involve actin cytoskeleton dynamics. Previously, we selected the nanobody RH12, from a synthetic phage display library, which binds the GTP-bound active conformation of RHOA (Ras Homologous family member A). However, when expressed as an intracellular antibody, its blocking effect on RHO signaling led to a loss of actin fibers, which in turn affected cell shape and cell survival. Here, in order to engineer an intracellular biosensor of RHOA-GTP activation, we screened the same phage nanobody library and identified another RHO-GTP selective intracellular nanobody, but with no apparent toxicity. The recombinant RH57 nanobody displays high affinity towards GTP-bound RHOA/B/C subgroup of small GTPases in vitro. Intracellular expression of the RH57 allowed selective co-precipitation with the GTP-bound state of the endogenous RHOA subfamily. When expressed as a fluorescent fusion protein, the chromobody GFP-RH57 was localized to the inner plasma membrane upon stimulation of the activation of endogenous RHO. Finally, the RH57 nanobody was used to establish a BRET-based biosensor (Bioluminescence Resonance Energy Transfer) of RHO activation. The dynamic range of the BRET signal could potentially offer new opportunities to develop cell-based screening of RHOA subfamily activation modulators.
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Follicular lymphoma (FL) is a common non Hodgkin's lymphoma subtype in which immune escape mechanisms are implicated in resistance to chemo-immunotherapy. Although molecular studies point to qualitative and quantitative deregulation of immune checkpoints, in depth cellular analysis of FL immune escape is lacking. Here, by functional assays and in silico analyses we show that a subset of FL patients displays a 'high' immune escape phenotype. These FL cases are characterized by abundant infiltration of PD1+ CD16+ TCRVγ9Vδ2 γδ T lymphocytes. In a 3D co-culture assay (MALC), γδ T cells mediate both direct and indirect (ADCC in the presence of anti-CD20 mAbs) cytolytic activity against FL cell aggregates. Importantly, PD-1, which is expressed by most FL-infiltrating γδ T lymphocytes with ADCC capacity, impairs these functions. In conclusion, we identify a PD1-regulated γδ T cell cytolytic immune component in FL. Our data provide a treatment rational by PD-1 blockade aimed at boosting γδ T cell anti-tumor functions in FL.
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Despite the clinical success of blocking inhibitory immune checkpoint receptors such as programmed cell death-1 (PD-1) in cancer, the mechanisms controlling the expression of these receptors have not been fully elucidated. Here, we identify a post-transcriptional mechanism regulating PD-1 expression in T cells. Upon activation, the PDCD1 mRNA and ribonucleoprotein complexes coalesce into stress granules that require microtubules and the kinesin 1 molecular motor to proceed to translation. Hence, PD-1 expression is highly sensitive to microtubule or stress granule inhibitors targeting this pathway. Evidence from healthy donors and cancer patients reveals a common regulation for the translation of CTLA4, LAG3, TIM3, TIGIT, and BTLA but not of the stimulatory co-receptors OX40, GITR, and 4-1BB mRNAs. In patients, disproportionality analysis of immune-related adverse events for currently used microtubule drugs unveils a significantly higher risk of autoimmunity. Our findings reveal a fundamental mechanism of immunoregulation with great importance in cancer immunotherapy.
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Inmunoterapia/métodos , Microtúbulos/metabolismo , Linfocitos T/inmunología , HumanosRESUMEN
Impaired adipose tissue insulin signalling is a critical feature of insulin resistance. Here we identify a pathway linking the lipolytic enzyme hormone-sensitive lipase (HSL) to insulin action via the glucose-responsive transcription factor ChREBP and its target, the fatty acid elongase ELOVL6. Genetic inhibition of HSL in human adipocytes and mouse adipose tissue results in enhanced insulin sensitivity and induction of ELOVL6. ELOVL6 promotes an increase in phospholipid oleic acid, which modifies plasma membrane fluidity and enhances insulin signalling. HSL deficiency-mediated effects are suppressed by gene silencing of ChREBP and ELOVL6. Mechanistically, physical interaction between HSL, independent of lipase activity, and the isoform activated by glucose metabolism ChREBPα impairs ChREBPα translocation into the nucleus and induction of ChREBPß, the isoform with high transcriptional activity that is strongly associated with whole-body insulin sensitivity. Targeting the HSL-ChREBP interaction may allow therapeutic strategies for the restoration of insulin sensitivity.
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Adipocitos/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Resistencia a la Insulina , Insulina/metabolismo , Esterol Esterasa/metabolismo , Tejido Adiposo/metabolismo , Animales , Biomarcadores , Elongasas de Ácidos Grasos/genética , Elongasas de Ácidos Grasos/metabolismo , Expresión Génica , Glucosa/metabolismo , Resistencia a la Insulina/genética , Fluidez de la Membrana/genética , Ratones , Ratones Transgénicos , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Transducción de SeñalRESUMEN
Protein-protein interactions are attractive targets because they control numerous cellular processes. In oncology, apoptosis regulating Bcl-2 family proteins are of particular interest. Apoptotic cell death is controlled via PPIs between the anti-apoptotic proteins hydrophobic groove and the pro-apoptotic proteins BH3 domain. In ovarian carcinoma, it has been previously demonstrated that Bcl-xL and Mcl-1 cooperate to protect tumor cells against apoptosis. Moreover, Mcl-1 is a key regulator of cancer cell survival and is a known resistance factor to Bcl-2/Bcl-xL pharmacological inhibitors making it an attractive therapeutic target. Here, using a structure-guided design from the oligopyridine lead Pyridoclax based on Noxa/Mcl-1 interaction we identified a new derivative, active at lower concentration as compared to Pyridoclax. This new derivative selectively binds to the Mcl-1 hydrophobic groove and releases Bak and Bim from Mcl-1 to induce cell death and sensitize cancer cells to Bcl-2/Bcl-xL targeting strategies.
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Diseño de Fármacos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Piridinas/farmacología , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Estructura Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/química , Piridinas/síntesis química , Piridinas/química , Relación Estructura-Actividad , Células Tumorales Cultivadas , Proteína bcl-X/antagonistas & inhibidores , Proteína bcl-X/metabolismoRESUMEN
Dendrogenin A (DDA) is a newly discovered cholesterol metabolite with tumor suppressor properties. Here, we explored its efficacy and mechanism of cell death in melanoma and acute myeloid leukemia (AML). We found that DDA induced lethal autophagy in vitro and in vivo, including primary AML patient samples, independently of melanoma Braf status or AML molecular and cytogenetic classifications. DDA is a partial agonist on liver-X-receptor (LXR) increasing Nur77, Nor1, and LC3 expression leading to autolysosome formation. Moreover, DDA inhibited the cholesterol biosynthesizing enzyme 3ß-hydroxysterol-Δ8,7-isomerase (D8D7I) leading to sterol accumulation and cooperating in autophagy induction. This mechanism of death was not observed with other LXR ligands or D8D7I inhibitors establishing DDA selectivity. The potent anti-tumor activity of DDA, its original mechanism of action and its low toxicity support its clinical evaluation. More generally, this study reveals that DDA can direct control a nuclear receptor to trigger lethal autophagy in cancers.
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Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Colestanoles/farmacología , Imidazoles/farmacología , Leucemia Mieloide Aguda , Receptores X del Hígado/efectos de los fármacos , Melanoma , Animales , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Agonismo Parcial de Drogas , Expresión Génica/efectos de los fármacos , Células HEK293 , Células HL-60 , Humanos , Técnicas In Vitro , Receptores X del Hígado/metabolismo , Melanoma Experimental , Proteínas de Transporte de Membrana/efectos de los fármacos , Proteínas de Transporte de Membrana/genética , Ratones , Proteínas Asociadas a Microtúbulos/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/efectos de los fármacos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genéticaRESUMEN
Breast cancer (BC) remains the primary cause of death from cancer among women worldwide. Cholesterol-5,6-epoxide (5,6-EC) metabolism is deregulated in BC but the molecular origin of this is unknown. Here, we have identified an oncometabolism downstream of 5,6-EC that promotes BC progression independently of estrogen receptor α expression. We show that cholesterol epoxide hydrolase (ChEH) metabolizes 5,6-EC into cholestane-3ß,5α,6ß-triol, which is transformed into the oncometabolite 6-oxo-cholestan-3ß,5α-diol (OCDO) by 11ß-hydroxysteroid-dehydrogenase-type-2 (11ßHSD2). 11ßHSD2 is known to regulate glucocorticoid metabolism by converting active cortisol into inactive cortisone. ChEH inhibition and 11ßHSD2 silencing inhibited OCDO production and tumor growth. Patient BC samples showed significant increased OCDO levels and greater ChEH and 11ßHSD2 protein expression compared with normal tissues. The analysis of several human BC mRNA databases indicated that 11ßHSD2 and ChEH overexpression correlated with a higher risk of patient death, highlighting that the biosynthetic pathway producing OCDO is of major importance to BC pathology. OCDO stimulates BC cell growth by binding to the glucocorticoid receptor (GR), the nuclear receptor of endogenous cortisol. Interestingly, high GR expression or activation correlates with poor therapeutic response or prognosis in many solid tumors, including BC. Targeting the enzymes involved in cholesterol epoxide and glucocorticoid metabolism or GR may be novel strategies to prevent and treat BC.
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Neoplasias de la Mama/metabolismo , Carcinógenos/metabolismo , Colesterol/metabolismo , Receptores de Glucocorticoides/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Colesterol/análogos & derivados , Epóxido Hidrolasas/metabolismo , Receptor alfa de Estrógeno/metabolismo , Femenino , Células HEK293 , Humanos , Células MCF-7 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , ARN Mensajero/metabolismoRESUMEN
In the tumoral micro-environment (TME) of chronic lymphocytic leukemia (CLL), nurse-like cells (NLC) are tumor-associated macrophages which play a critical role in the survival and chemoresistance of tumoral cells. This pro-survival activity is known to involve soluble factors, but few data are available on the relative role of cells cross-talk. Here, we used a transcriptome-based approach to systematically investigate the expression of various receptor/ligand pairs at the surface of NLC/CLL cells. Their relative contribution to CLL survival was assessed both by fluorescent microscopy to identify cellular interactions and by the use of functional tests to measure the impact of uncoupling these pairs with blocking monoclonal antibodies. We found for the first time that lymphocyte function-associated antigen 3 (LFA-3), expressed in CLL at significantly higher levels than in healthy donor B-cells, and CD2 expressed on NLC, were both key for the specific pro-survival signals delivered by NLC. Moreover, we found that NLC/CLL interactions induced the shedding of soluble LFA-3. Importantly, in an exploratory cohort of 60 CLL patients receiving frontline immunochemotherapy, increased levels of soluble LFA-3 were found to correlate with shorter overall survival. Altogether, these data suggest that LFA-3/CD2 interactions promote the survival of CLL cells in the tumor microenvironment.
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In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications.
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Anticuerpos Monoclonales Humanizados , Biología Molecular/métodos , Biblioteca de Péptidos , Anticuerpos de Dominio Único , Animales , Camélidos del Nuevo Mundo , HumanosRESUMEN
The vascular endothelial growth factor VEGF-D promotes metastasis by inducing lymphangiogenesis and dilatation of the lymphatic vasculature, facilitating tumor cell extravasion. Here we report a novel level of control for VEGF-D expression at the level of protein translation. In human tumor cells, VEGF-D colocalized with eIF4GI and 4E-BP1, which can program increased initiation at IRES motifs on mRNA by the translational initiation complex. In murine tumors, the steady-state level of VEGF-D protein was increased despite the overexpression and dephosphorylation of 4E-BP1, which downregulates protein synthesis, suggesting the presence of an internal ribosome entry site (IRES) in the 5' UTR of VEGF-D mRNA. We found that nucleolin, a nucleolar protein involved in ribosomal maturation, bound directly to the 5'UTR of VEGF-D mRNA, thereby improving its translation following heat shock stress via IRES activation. Nucleolin blockade by RNAi-mediated silencing or pharmacologic inhibition reduced VEGF-D translation along with a subsequent constriction of lymphatic vessels in tumors. Our results identify nucleolin as a key regulator of VEGF-D expression, deepening understanding of lymphangiogenesis control during tumor formation. Cancer Res; 76(15); 4394-405. ©2016 AACR.
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Linfangiogénesis/genética , Fosfoproteínas/genética , Proteínas de Unión al ARN/genética , Factor D de Crecimiento Endotelial Vascular/metabolismo , Animales , Humanos , Ratones , Transfección , NucleolinaRESUMEN
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with a mortality that is almost identical to incidence. Because early detected PDAC is potentially curable, blood-based biomarkers that could detect currently developing neoplasia would improve patient survival and management. PDAC develops from pancreatic intraepithelial neoplasia (PanIN) lesions, graded from low grade (PanIN1) to high grade (PanIN3). We made the hypothesis that specific proteomic signatures from each precancerous stage exist and are detectable in plasma. METHODS: We explored the peptide profiles of microdissected PanIN cells and of plasma samples corresponding to the different PanIN grade from genetically engineered mouse models of PDAC using capillary electrophoresis coupled to mass spectrometry (CE-MS) and Chip-MS/MS. RESULTS: We successfully characterised differential peptides profiles from PanIN microdissected cells. We found that plasma from tumor-bearing mice and age-matched controls exhibit discriminative peptide signatures. We also determined plasma peptide signatures corresponding to low- and high-grade precancerous step present in the mice pancreas using the two mass spectrometry technologies. Importantly, we identified biomarkers specific of PanIN3. CONCLUSIONS: We demonstrate that benign and advanced PanIN lesions display distinct plasma peptide patterns. This strongly supports the perspectives of developing a non-invasive screening test for prediction and early detection of PDAC.
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Biomarcadores de Tumor/sangre , Carcinoma in Situ/sangre , Carcinoma Ductal Pancreático/sangre , Proteínas de Neoplasias/sangre , Neoplasias Pancreáticas/sangre , Péptidos/sangre , Lesiones Precancerosas/sangre , Animales , Biomarcadores de Tumor/análisis , Carcinoma in Situ/química , Carcinoma in Situ/patología , Carcinoma Ductal Pancreático/química , Modelos Animales de Enfermedad , Ratones , Proteínas de Neoplasias/análisis , Neoplasias Pancreáticas/química , Péptidos/análisis , Lesiones Precancerosas/química , Lesiones Precancerosas/patología , Análisis por Matrices de Proteínas , Proteoma/análisisRESUMEN
N-Glycans are widely distributed in living organisms but represent only a small fraction of the carbohydrates found in plants. This probably explains why they have not previously been considered as substrates exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, possesses a specific system for GlcNAc utilization expressed during host plant infection. This system encompasses a cluster of eight genes (nixE to nixL) encoding glycoside hydrolases (GHs). In this paper, we have characterized the enzymatic activities of these GHs and demonstrated their involvement in sequential degradation of a plant N-glycan using a N-glycopeptide containing two GlcNAcs, three mannoses, one fucose, and one xylose (N2M3FX) as a substrate. The removal of the α-1,3-mannose by the α-mannosidase NixK (GH92) is a prerequisite for the subsequent action of the ß-xylosidase NixI (GH3), which is involved in the cleavage of the ß-1,2-xylose, followed by the α-mannosidase NixJ (GH125), which removes the α-1,6-mannose. These data, combined to the subcellular localization of the enzymes, allowed us to propose a model of N-glycopeptide processing by X. campestris pv. campestris. This study constitutes the first evidence suggesting N-glycan degradation by a plant pathogen, a feature shared with human pathogenic bacteria. Plant N-glycans should therefore be included in the repertoire of molecules putatively metabolized by phytopathogenic bacteria during their life cycle.
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Brassica/genética , Enfermedades de las Plantas/genética , Polisacáridos/genética , Xanthomonas campestris/enzimología , Brassica/enzimología , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Humanos , Enfermedades de las Plantas/microbiología , Polisacáridos/metabolismo , Xanthomonas campestris/genética , Xanthomonas campestris/patogenicidad , Xilosidasas/genética , Xilosidasas/metabolismo , alfa-Manosidasa/genética , alfa-Manosidasa/metabolismoRESUMEN
This phase 1 trial was aimed to determine the safety, pharmacokinetics, and preliminary clinical activity of CYL-02, a nonviral gene therapy product that sensitizes pancreatic cancer cells to chemotherapy. CYL-02 was administrated using endoscopic ultrasound in 22 patients with pancreatic cancer that concomitantly received chemotherapy (gemcitabine). The maximum-tolerated dose (MTD) exceeded the maximal feasible dose of CYL-02 and was not identified. Treatment-related toxicities were mild, without serious adverse events. Pharmacokinetic analysis revealed a dose-dependent increase in CYL-02 DNA exposure in blood and tumors, while therapeutic RNAs were detected in tumors. No objective response was observed, but nine patients showed stable disease up to 6 months following treatment and two of these patients experienced long-term survival. Panels of plasmatic microRNAs and proteins were identified as predictive of gene therapy efficacy. We demonstrate that CYL-02 nonviral gene therapy has a favorable safety profile and is well tolerated in patients. We characterize CYL-02 biodistribution and demonstrate therapeutic gene expression in tumors. Treated patients experienced stability of disease and predictive biomarkers of response to treatment were identified. These promising results warrant further evaluation in phase 2 clinical trial.