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Approximately 25% of cancers are preceded by chronic inflammation that occurs at the site of tumor development. However, whether this multifactorial oncogenic process, which commonly occurs in the intestines, can be initiated by a specific immune cell population is unclear. Here, we show that an intestinal T cell subset, derived from interleukin-17 (IL-17)-producing helper T (TH17) cells, induces the spontaneous transformation of the intestinal epithelium. This subset produces inflammatory cytokines, and its tumorigenic potential is not dependent on IL-17 production but on the transcription factors KLF6 and T-BET and interferon-γ. The development of this cell type is inhibited by transforming growth factor-ß1 (TGFß1) produced by intestinal epithelial cells. TGFß signaling acts on the pretumorigenic TH17 cell subset, preventing its progression to the tumorigenic stage by inhibiting KLF6-dependent T-BET expression. This study therefore identifies an intestinal T cell subset initiating cancer.
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Netrin-1 is upregulated in cancers as a protumoural mechanism1. Here we describe netrin-1 upregulation in a majority of human endometrial carcinomas (ECs) and demonstrate that netrin-1 blockade, using an anti-netrin-1 antibody (NP137), is effective in reduction of tumour progression in an EC mouse model. We next examined the efficacy of NP137, as a first-in-class single agent, in a Phase I trial comprising 14 patients with advanced EC. As best response we observed 8 stable disease (8 out of 14, 57.1%) and 1 objective response as RECIST v.1.1 (partial response, 1 out of 14 (7.1%), 51.16% reduction in target lesions at 6 weeks and up to 54.65% reduction during the following 6 months). To evaluate the NP137 mechanism of action, mouse tumour gene profiling was performed, and we observed, in addition to cell death induction, that NP137 inhibited epithelial-to-mesenchymal transition (EMT). By performing bulk RNA sequencing (RNA-seq), spatial transcriptomics and single-cell RNA-seq on paired pre- and on-treatment biopsies from patients with EC from the NP137 trial, we noted a net reduction in tumour EMT. This was associated with changes in immune infiltrate and increased interactions between cancer cells and the tumour microenvironment. Given the importance of EMT in resistance to current standards of care2, we show in the EC mouse model that a combination of NP137 with carboplatin-paclitaxel outperformed carboplatin-paclitaxel alone. Our results identify netrin-1 blockade as a clinical strategy triggering both tumour debulking and EMT inhibition, thus potentially alleviating resistance to standard treatments.
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Neoplasias Endometriales , Transición Epitelial-Mesenquimal , Netrina-1 , Animales , Femenino , Humanos , Ratones , Biopsia , Carboplatino/administración & dosificación , Carboplatino/farmacología , Carboplatino/uso terapéutico , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/genética , Neoplasias Endometriales/inmunología , Neoplasias Endometriales/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Perfilación de la Expresión Génica , Netrina-1/antagonistas & inhibidores , Paclitaxel/administración & dosificación , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , RNA-Seq , Análisis de Expresión Génica de una Sola Célula , Microambiente Tumoral/efectos de los fármacosRESUMEN
Both identity and plasticity of CD4 T helper (Th) cells are regulated in part by epigenetic mechanisms. However, a method that reliably and readily profiles DNA base modifications is still needed to finely study Th cell differentiation. Cytosine methylation in CpG context (5mCpG) and cytosine hydroxymethylation (5hmCpG) are DNA modifications that identify stable cell phenotypes, but their potential to characterize intermediate cell transitions has not yet been evaluated. To assess transition states in Th cells, we developed a method to profile Th cell identity using Cas9-targeted single-molecule nanopore sequencing. Targeting as few as 10 selected genomic loci, we were able to distinguish major in vitro polarized murine T cell subtypes, as well as intermediate phenotypes, by their native DNA 5mCpG patterns. Moreover, by using off-target sequences, we were able to infer transcription factor activities relevant to each cell subtype. Detection of 5mCpG and 5hmCpG was validated on intestinal Th17 cells escaping transforming growth factor ß control, using single-molecule adaptive sampling. A total of 21 differentially methylated regions mapping to the 10-gene panel were identified in pathogenic Th17 cells relative to their nonpathogenic counterpart. Hence, our data highlight the potential to exploit native DNA methylation profiling to study physiological and pathological transition states of Th cells.
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Metilación de ADN , Epigénesis Genética , Animales , Ratones , Citosina , ADN/metabolismo , Células Th17/metabolismoRESUMEN
We report the identification of a cell population that shares pericyte, stromal and stemness features, does not harbor the KrasG12D mutation and drives tumoral growth in vitro and in vivo. We term these cells pericyte stem cells (PeSCs) and define them as CD45- EPCAM- CD29+ CD106+ CD24+ CD44+ cells. We perform studies with p48-Cre;KrasG12D (KC), pdx1-Cre;KrasG12D ;Ink4a/Arffl/fl (KIC) and pdx1-Cre;KrasG12D ;p53R172H (KPC) and tumor tissues from PDAC and chronic pancreatitis patients. We also perform single-cell RNAseq analysis and reveal a unique signature of PeSC. Under steady-state conditions, PeSCs are barely detectable in the pancreas but present in the neoplastic microenvironment both in humans and mice. The coinjection of PeSCs and tumor epithelial cells leads to increased tumor growth, differentiation of Ly6G+ myeloid-derived suppressor cells, and a decreased amount of F4/80+ macrophages and CD11c+ dendritic cells. This population induces resistance to anti-PD-1 immunotherapy when coinjected with epithelial tumor cells. Our data reveal the existence of a cell population that instructs immunosuppressive myeloid cell responses to bypass PD-1 targeting and thus suggest potential new approaches for overcoming resistance to immunotherapy in clinical settings.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Humanos , Ratones , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/patología , Neoplasias Pancreáticas/genética , Pericitos , Proteínas Proto-Oncogénicas p21(ras) , Células Madre , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
BACKGROUND & AIMS: Transcription termination fine-tunes gene expression and contributes to the specification of RNA function in eukaryotic cells. Transcription termination of HBV is subject to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. However, the regulation of this cPAS and its impact on viral gene expression and replication is currently unknown. METHODS: To unravel the regulation of HBV transcript termination, we implemented a 3' RACE (rapid amplification of cDNA ends)-PCR assay coupled to single molecule sequencing both in in vitro-infected hepatocytes and in chronically infected patients. RESULTS: The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated with HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of the cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication. CONCLUSIONS: Our findings identify DDX5 and DDX17 as crucial determinants of HBV transcriptional fidelity and as host restriction factors for HBV replication. IMPACT AND IMPLICATIONS: HBV covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail for the achievement of HBV cure. Transcriptional fidelity is a cornerstone in the regulation of gene expression. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit recognition of the HBV polyadenylation signal and thereby transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in patients chronically infected with HBV suggests a role for these helicases in HBV persistence in vivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription.
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Beyond their critical role in hemostasis, platelets physically interact with neutrophils to form neutrophil-platelet aggregates (NPAs), enhancing neutrophil effector functions during inflammation. NPAs may also promote disease worsening in various inflammatory diseases. However, characterization of NPAs in cancer remains totally unexplored. Using ImageStreamX (ISX) imaging flow cytometer, we were not only allowed able to detect CD15+ CD14- CD36+ ITGA2B+ NPAs in both healthy donors' (HDs) and cancer patients' bloods, but we also showed that NPAs result from the binding of platelets preferentially to low-density neutrophils (LDNs) as opposed to normal-density neutrophils (NDNs). By reanalyzing two independent public scRNAseq data of whole blood leukocytes from cancer patients and HDs, we could identify a subset of neutrophils with high platelet gene expression that may correspond to NPAs. Moreover, we showed that cancer patients' derived NPAs possessed a distinct molecular signature compared to the other neutrophil subsets, independently of platelet genes. Gene ontology (GO) term enrichment analysis of this NPAs-associated neutrophil transcriptomic signature revealed a significant enrichment of neutrophil degranulation, chemotaxis and trans-endothelial migration GO terms. Lastly, using The Cancer Genome Atlas (TCGA), we could show by multivariate Cox analysis that the NPAs-associated neutrophil transcriptomic signature was associated with a worse patient prognosis in several cancer types. These results suggest that neutrophils from NPAs are systemically primed by platelets empowering them with cancer progression capacities once at tumor site. NPAs may therefore hold clinical utility as novel noninvasive blood prognostic biomarker in cancer patients with solid tumors.
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Neoplasias , Neutrófilos , Plaquetas , Citometría de Flujo , Humanos , Neoplasias/patología , Neutrófilos/patología , PronósticoRESUMEN
Merkel cell polyomavirus (MCPyV) is the first human polyomavirus etiologically associated with Merkel cell carcinoma (MCC), a rare and aggressive form of skin cancer. Similar to other polyomaviruses, MCPyV encodes early T antigen genes, viral oncogenes required for MCC tumor growth. To identify the unique oncogenic properties of MCPyV, we analyzed the gene expression profiles in human spontaneously immortalized keratinocytes (NIKs) expressing the early genes from six distinct human polyomaviruses (PyVs), including MCPyV. A comparison of the gene expression profiles revealed 28 genes specifically deregulated by MCPyV. In particular, the MCPyV early gene downregulated the expression of the tumor suppressor gene N-myc downstream-regulated gene 1 (NDRG1) in MCPyV gene-expressing NIKs and hTERT-MCPyV gene-expressing human keratinocytes (HK) compared to their expression in the controls. In MCPyV-positive MCC cells, the expression of NDRG1 was downregulated by the MCPyV early gene, as T antigen knockdown rescued the level of NDRG1. In addition, NDRG1 overexpression in hTERT-MCPyV gene-expressing HK or MCC cells resulted in a decrease in the number of cells in S phase and cell proliferation inhibition. Moreover, a decrease in wound healing capacity in hTERT-MCPyV gene-expressing HK was observed. Further analysis revealed that NDRG1 exerts its biological effect in Merkel cell lines by regulating the expression of the cyclin-dependent kinase 2 (CDK2) and cyclin D1 proteins. Overall, NDRG1 plays an important role in MCPyV-induced cellular proliferation.IMPORTANCE Merkel cell carcinoma was first described in 1972 as a neuroendocrine tumor of skin, most cases of which were reported in 2008 to be caused by a PyV named Merkel cell polyomavirus (MCPyV), the first PyV linked to human cancer. Thereafter, numerous studies have been conducted to understand the etiology of this virus-induced carcinogenesis. However, it is still a new field, and much work is needed to understand the molecular pathogenesis of MCC. In the current work, we sought to identify the host genes specifically deregulated by MCPyV, as opposed to other PyVs, in order to better understand the relevance of the genes analyzed on the biological impact and progression of the disease. These findings open newer avenues for targeted drug therapies, thereby providing hope for the management of patients suffering from this highly aggressive cancer.
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Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimiento Celular/genética , Proliferación Celular/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Poliomavirus de Células de Merkel/genética , Poliomavirus de Células de Merkel/fisiología , Antígenos Virales de Tumores/genética , Antígenos Virales de Tumores/metabolismo , Carcinogénesis/genética , Carcinoma de Células de Merkel/virología , Línea Celular , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Humanos , Queratinocitos/virología , Infecciones por Polyomavirus/virología , Piel/patología , Neoplasias Cutáneas/genética , Transcriptoma , Infecciones Tumorales por Virus/virologíaRESUMEN
Background: DNA methylation is an epigenetic control mechanism that may be altered by environmental exposures. We have previously reported that in utero exposure to the mycotoxin and liver carcinogen aflatoxin B1 from the maternal diet, as measured using biomarkers in the mothers' blood, was associated with differential DNA methylation in white blood cells of 6-month-old infants from The Gambia. Methods: Here we examined aflatoxin B1-associated differential DNA methylation in white blood cells of 24-month-old children from the same population (n = 244), in relation to the child's dietary exposure assessed using aflatoxin albumin biomarkers in blood samples collected at 6, 12 and 18 months of age. HM450 BeadChip arrays were used to assess DNA methylation, with data compared to aflatoxin albumin adduct levels using two approaches; a continuous model comparing aflatoxin adducts measured in samples collected at 18 months to DNA methylation at 24 months, and a categorical time-dose model that took into account aflatoxin adduct levels at 6, 12 and 18 months, for comparison to DNA methylation at 24 months. Results: Geometric mean (95% confidence intervals) for aflatoxin albumin levels were 3.78 (3.29, 4.34) at 6 months, 25.1 (21.67, 29.13) at 12 months and 49.48 (43.34, 56.49) at 18 months of age. A number of differentially methylated CpG positions and regions were associated with aflatoxin exposure, some of which affected gene expression. Pathway analysis highlighted effects on genes involved with with inflammatory, signalling and growth pathways. Conclusions: This study provides further evidence that exposure to aflatoxin in early childhood may impact on DNA methylation.
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Aflatoxina B1/efectos adversos , Metilación de ADN/efectos de los fármacos , Exposición a Riesgos Ambientales/efectos adversos , Experiencias Adversas de la Infancia , Aflatoxinas/efectos adversos , Aflatoxinas/análisis , Aflatoxinas/sangre , Albúminas/análisis , Preescolar , ADN/metabolismo , Metilación de ADN/genética , Epigénesis Genética/genética , Epigenómica/métodos , Femenino , Gambia/epidemiología , Humanos , Lactante , Leucocitos/metabolismo , MasculinoRESUMEN
The histone modifier lysine (K)-specific demethylase 2B (KDM2B) plays a role in the differentiation of hematopoietic cells, and its expression appears to be deregulated in certain cancers of hematological and lymphoid origins. We have previously found that the KDM2B gene is differentially methylated in cell lines derived from Epstein-Barr virus (EBV)-associated endemic Burkitt lymphoma (eBL) compared with that in EBV-negative sporadic Burkitt lymphoma-derived cells. However, whether KDM2B plays a role in eBL development has not been previously investigated. Oncogenic viruses have been shown to hijack the host cell epigenome to complete their life cycle and to promote the transformation process by perturbing cell chromatin organization. Here, we investigated whether EBV alters KDM2B levels to enable its life cycle and promote B-cell transformation. We show that infection of B cells with EBV leads to downregulation of KDM2B levels. We also show that LMP1, one of the main EBV transforming proteins, induces increased DNMT1 recruitment to the KDM2B gene and augments its methylation. By altering KDM2B levels and performing chromatin immunoprecipitation in EBV-infected B cells, we show that KDM2B is recruited to the EBV gene promoters and inhibits their expression. Furthermore, forced KDM2B expression in immortalized B cells led to altered mRNA levels of some differentiation-related genes. Our data show that EBV deregulates KDM2B levels through an epigenetic mechanism and provide evidence for a role of KDM2B in regulating virus and host cell gene expression, warranting further investigations to assess the role of KDM2B in the process of EBV-mediated lymphomagenesis.IMPORTANCE In Africa, Epstein-Barr virus infection is associated with endemic Burkitt lymphoma, a pediatric cancer. The molecular events leading to its development are poorly understood compared with those leading to sporadic Burkitt lymphoma. In a previous study, by analyzing the DNA methylation changes in endemic compared with sporadic Burkitt lymphoma cell lines, we identified several differential methylated genomic positions in the proximity of genes with a potential role in cancer, and among them was the KDM2B gene. KDM2B encodes a histone H3 demethylase already shown to be involved in some hematological disorders. However, whether KDM2B plays a role in the development of Epstein-Barr virus-mediated lymphoma has not been investigated before. In this study, we show that Epstein-Barr virus deregulates KDM2B expression and describe the underlying mechanisms. We also reveal a role of the demethylase in controlling viral and B-cell gene expression, thus highlighting a novel interaction between the virus and the cellular epigenome.
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Epigénesis Genética , Infecciones por Virus de Epstein-Barr/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Herpesvirus Humano 4/fisiología , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Adolescente , Adulto , Linfocitos B/virología , Linfoma de Burkitt/metabolismo , Línea Celular , Niño , Preescolar , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Metilación de ADN , Regulación hacia Abajo , Infecciones por Virus de Epstein-Barr/genética , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Tumor cell invasion is one of the key processes during cancer progression, leading to life-threatening metastatic lesions in melanoma. As methylation of cancer-related genes plays a fundamental role during tumorigenesis and may lead to cellular plasticity which promotes invasion, our aim was to identify novel epigenetic markers on selected invasive melanoma cells. Using Illumina BeadChip assays and Affymetrix Human Gene 1.0 microarrays, we explored the DNA methylation landscape of selected invasive melanoma cells and examined the impact of DNA methylation on gene expression patterns. Our data revealed predominantly hypermethylated genes in the invasive cells affecting the neural crest differentiation pathway and regulation of the actin cytoskeleton. Integrative analysis of the methylation and gene expression profiles resulted in a cohort of hypermethylated genes (IL12RB2, LYPD6B, CHL1, SLC9A3, BAALC, FAM213A, SORCS1, GPR158, FBN1 and ADORA2B) with decreased expression. On the other hand, hypermethylation in the gene body of the EGFR and RBP4 genes was positively correlated with overexpression of the genes. We identified several methylation changes that can have role during melanoma progression, including hypermethylation of the promoter regions of the ARHGAP22 and NAV2 genes that are commonly altered in locally invasive primary melanomas as well as during metastasis. Interestingly, the down-regulation of the methylcytosine dioxygenase TET2 gene, which regulates DNA methylation, was associated with hypermethylated promoter region of the gene. This can probably lead to the observed global hypermethylation pattern of invasive cells and might be one of the key changes during the development of malignant melanoma cells.
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Metilación de ADN , Melanoma/genética , Melanoma/secundario , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Dioxigenasas , Epigénesis Genética , Proteínas Activadoras de GTPasa/genética , Expresión Génica/genética , Perfilación de la Expresión Génica , Humanos , Invasividad Neoplásica/genética , Metástasis de la Neoplasia/genética , Fenotipo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genéticaRESUMEN
The interaction between the (epi)genetic makeup of an individual and his/her environmental exposure record (exposome) is accepted as a determinant factor for a significant proportion of human malignancies. Recent evidence has highlighted the key role of epigenetic mechanisms in mediating gene-environment interactions and translating exposures into tumorigenesis. There is also growing evidence that epigenetic changes may be risk factor-specific ("fingerprints") that should prove instrumental in the discovery of new biomarkers in cancer. Here, we review the state of the science of epigenetics associated with environmental stimuli and cancer risk, highlighting key developments in the field. Critical knowledge gaps and research needs are discussed and advances in epigenomics that may help in understanding the functional relevance of epigenetic alterations. Key elements required for causality inferences linking epigenetic changes to exposure and cancer are discussed and how these alterations can be incorporated in carcinogen evaluation and in understanding mechanisms underlying epigenome deregulation by the environment.
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Exposición a Riesgos Ambientales/efectos adversos , Epigénesis Genética , Epigenómica , Interacción Gen-Ambiente , Neoplasias/etiología , Animales , Metilación de ADN , Humanos , Neoplasias/patología , Factores de RiesgoRESUMEN
Previous studies have revealed a robust association between exposure to asbestos and human lung cancer. Accumulating evidence has highlighted the role of epigenome deregulation in the mechanism of carcinogen-induced malignancies. We examined the impact of asbestos on DNA methylation. Our genome-wide studies (using Illumina HumanMethylation450K BeadChip) of lung cancer tissue and paired normal lung from 28 asbestos-exposed or non-exposed patients, mostly smokers, revealed distinctive DNA methylation changes. We identified a number of differentially methylated regions (DMR) and differentially variable, differentially methylated CpGs (DVMC), with individual CpGs further validated by pyrosequencing in an independent series of 91 non-small cell lung cancer and paired normal lung. We discovered and validated BEND4, ZSCAN31 and GPR135 as significantly hypermethylated in lung cancer. DMRs in genes such as RARB (FDR 1.1 × 10-19 , mean change in beta [Δ] -0.09), GPR135 (FDR 1.87 × 10-8 , mean Δ -0.09) and TPO (FDR 8.58 × 10-5 , mean Δ -0.11), and DVMCs in NPTN, NRG2, GLT25D2 and TRPC3 (all with p <0.05, t-test) were significantly associated with asbestos exposure status in exposed versus non-exposed lung tumors. Hypomethylation was characteristic to DVMCs in lung cancer tissue from asbestos-exposed subjects. When DVMCs related to asbestos or smoking were analyzed, 96% of the elements were unique to either of the exposures, consistent with the concept that the methylation changes in tumors may be specific for risk factors. In conclusion, we identified novel DNA methylation changes associated with lung tumors and asbestos exposure, suggesting that changes may be present in causal pathway from asbestos exposure to lung cancer.
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Amianto/efectos adversos , Biomarcadores de Tumor/genética , Metilación de ADN , Estudio de Asociación del Genoma Completo , Neoplasias Pulmonares/etiología , Estudios de Casos y Controles , Islas de CpG , Epigénesis Genética , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/patología , Masculino , PronósticoRESUMEN
BACKGROUND: Naturally occurring polyphenolic compounds from fruits, particularly from blueberries, have been reported to be significantly involved in cancer chemoprevention and chemotherapy. Biotransformation of blueberry juice by Serratia vaccinii increases its polyphenolic content and endows it with anti-inflammatory properties. METHODS: This study evaluated the effect of a polyphenol-enriched blueberry preparation (PEBP) and its non-fermented counterpart (NBJ), on mammary cancer stem cell (CSC) development in in vitro, in vivo and ex vivo settings. Effects of PEBP on cell proliferation, mobility, invasion, and mammosphere formation were measured in vitro in three cell lines: murine 4T1 and human MCF7 and MDA-MB-231. Ex vivo mammosphere formation, tumor growth and metastasis observations were carried out in a BALB/c mouse model. RESULTS: Our research revealed that PEBP influence cellular signaling cascades of breast CSCs, regulating the activity of transcription factors and, consequently, inhibiting tumor growth in vivo by decreasing metastasis and controlling PI3K/AKT, MAPK/ERK, and STAT3 pathways, central nodes in CSC inflammatory signaling. PEBP significantly inhibited cell proliferation of 4T1, MCF-7 and MDA-MB-231. In all cell lines, PEBP reduced mammosphere formation, cell mobility and cell migration. In vivo, PEBP significantly reduced tumor development, inhibited the formation of ex vivo mammospheres, and significantly reduced lung metastasis. CONCLUSIONS: This study showed that polyphenol enrichment of a blueberry preparation by fermentation increases its chemopreventive potential by protecting mice against tumor development, inhibiting the formation of cancer stem cells and reducing lung metastasis. Thus, PEBP may represent a novel complementary alternative medicine therapy and a source for novel therapeutic agents against breast cancer.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Quimioprevención , Inflamación/patología , Células Madre Neoplásicas/patología , Polifenoles/uso terapéutico , Animales , Arándanos Azules (Planta)/química , Neoplasias de la Mama/complicaciones , Neoplasias de la Mama/enzimología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Fermentación , Humanos , Inflamación/complicaciones , Ratones , Ratones Endogámicos BALB C , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Invasividad Neoplásica , Metástasis de la Neoplasia , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/enzimología , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Polifenoles/farmacología , Transducción de Señal/efectos de los fármacos , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patologíaRESUMEN
Although Epstein-Barr virus (EBV) infection is widely distributed, certain EBV-driven malignancies are geographically restricted. EBV-associated Burkitt's lymphoma (eBL) is endemic in children living in sub-Saharan Africa. This population is heavily exposed to food contaminated with the mycotoxin aflatoxin B1 (AFB1). Here, we show that exposure to AFB1 in in vitro and in vivo models induces activation of the EBV lytic cycle and increases EBV load, two events that are associated with an increased risk of eBL in vivo. AFB1 treatment leads to the alteration of cellular gene expression, with consequent activations of signaling pathways, e.g. PI3K, that in turn mediate reactivation of the EBV life cycle. Finally, we show that AFB1 triggers EBV-driven cellular transformation both in primary human B cells and in a humanized animal model. In summary, our data provide evidence for a role of AFB1 as a cofactor in EBV-mediated carcinogenesis.
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Aflatoxina B1/toxicidad , Linfocitos B/virología , Linfoma de Burkitt/virología , Exposición a Riesgos Ambientales , Infecciones por Virus de Epstein-Barr/patología , Herpesvirus Humano 4/efectos de los fármacos , Animales , Linfocitos B/patología , Linfoma de Burkitt/inducido químicamente , Carcinogénesis/efectos de los fármacos , Carcinogénesis/metabolismo , Transformación Celular Neoplásica/efectos de los fármacos , Células Cultivadas , Femenino , Herpesvirus Humano 4/fisiología , Humanos , Masculino , Ratones Endogámicos NOD , Ratones SCID , Transducción de Señal , Activación Viral , Replicación Viral/efectos de los fármacosRESUMEN
Many studies have proved that oncogenic viruses develop redundant mechanisms to alter the functions of the tumor suppressor p53. Here we show that Epstein-Barr virus (EBV), via the oncoprotein LMP-1, induces the expression of ΔNp73α, a strong antagonist of p53. This phenomenon is mediated by the LMP-1 dependent activation of c-Jun NH2-terminal kinase 1 (JNK-1) which in turn favours the recruitment of p73 to ΔNp73α promoter. A specific chemical inhibitor of JNK-1 or silencing JNK-1 expression strongly down-regulated ΔNp73α mRNA levels in LMP-1-containing cells. Accordingly, LMP-1 mutants deficient to activate JNK-1 did not induce ΔNp73α accumulation. The recruitment of p73 to the ΔNp73α promoter correlated with the displacement of the histone-lysine N-methyltransferase EZH2 which is part of the transcriptional repressive polycomb 2 complex. Inhibition of ΔNp73α expression in lymphoblastoid cells (LCLs) led to the stimulation of apoptosis and up-regulation of a large number of cellular genes as determined by whole transcriptome shotgun sequencing (RNA-seq). In particular, the expression of genes encoding products known to play anti-proliferative/pro-apoptotic functions, as well as genes known to be deregulated in different B cells malignancy, was altered by ΔNp73α down-regulation. Together, these findings reveal a novel EBV mechanism that appears to play an important role in the transformation of primary B cells.
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Linfocitos B/metabolismo , Proteínas de Unión al ADN/genética , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/genética , Proteínas Nucleares/genética , Proteínas Supresoras de Tumor/genética , Proteínas de la Matriz Viral/genética , Apoptosis , Linfocitos B/virología , Transformación Celular Viral/genética , Transformación Celular Viral/fisiología , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Epigénesis Genética , Herpesvirus Humano 4/fisiología , Interacciones Huésped-Patógeno , Humanos , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Análisis de Secuencia de ARN , Transcripción Genética , Activación Transcripcional , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia Arriba , Proteínas de la Matriz Viral/metabolismoRESUMEN
Recent advances in laboratory sciences hold a promise for a 'leap forward' in understanding the aetiology of complex human diseases, notably cancer, potentially providing an evidence base for prevention. For example, remarkable advances in epigenomics have an important impact on our understanding of biological phenomena and importance of environmental stressors in complex diseases. Environmental and lifestyle factors are thought to be implicated in the development of a wide range of human cancers by eliciting changes in the epigenome. These changes, thus, represent attractive targets for biomarker discovery intended for the improvement of exposure and risk assessment, diagnosis and prognosis and provision of short-term outcomes in intervention studies. The epigenome can be viewed as an interface between the genome and the environment; therefore, aberrant epigenetic events associated with environmental exposures are likely to play an important role in the onset and progression of different human diseases. The advent of powerful technologies for analysing epigenetic patterns in both cancer tissues and normal cells holds promise that the next few years will be fundamental for the identification of critical cancer- and exposure-associated epigenetic changes and for their evaluation as new generation of biomarkers. Here, we discuss new opportunities in the current age of 'omics' technologies for studies with prospective design and associated biospecimens that represent exciting potential for characterising the epigenome as a key component of the fetal exposome and for understanding causal pathways and robust predictors of cancer risk and associated environmental determinants during in utero life. Such studies should improve our knowledge concerning the aetiology of childhood cancer and identify both novel biomarkers and clues to causation, thus, providing an evidence base for cancer prevention.
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Exposición a Riesgos Ambientales/efectos adversos , Epigénesis Genética , Epigenómica , Exposición Materna/efectos adversos , Neoplasias/etiología , Efectos Tardíos de la Exposición Prenatal , Factores de Edad , Biomarcadores , Niño , Preescolar , Metilación de ADN , Susceptibilidad a Enfermedades , Epigenómica/métodos , Femenino , Sangre Fetal/citología , Perfilación de la Expresión Génica , Humanos , Lactante , Recién Nacido , Modelos Estadísticos , Neoplasias/epidemiología , Embarazo , Factores de Riesgo , TranscriptomaRESUMEN
BACKGROUND: Distinct subpopulations of neoplastic cells within tumors, including hepatocellular carcinoma (HCC), display pronounced ability to initiate new tumors and induce metastasis. Recent evidence suggests that signals from transforming growth factor beta (TGF-ß) may increase the survival of these so called tumor initiating cells leading to poor HCC prognosis. However, how TGF-ß establishes and modifies the key features of these cell subpopulations is not fully understood. RESULTS: In the present report we describe the differential DNA methylome of CD133-negative and CD133-expressing liver cancer cells. Next, we show that TGF-ß is able to increase the proportion of CD133+ cells in liver cancer cell lines in a way that is stable and persistent across cell division. This process is associated with stable genome-wide changes in DNA methylation that persist through cell division. Differential methylation in response to TGF-ß is under-represented at promoter CpG islands and enriched at gene bodies, including a locus in the body of the de novo DNA methyl-transferase DNMT3B gene. Moreover, phenotypic changes induced by TGF-ß, including the induction of CD133, are impaired by siRNA silencing of de novo DNA methyl-transferases. CONCLUSIONS: Our study reveals a self-perpetuating crosstalk between TGF-ß signaling and the DNA methylation machinery, which can be relevant in the establishment of cellular phenotypes. This is the first indication of the ability of TGF-ß to induce genome-wide changes in DNA methylation, resulting in a stable change in the proportion of liver cancer cell subpopulations.
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Antígenos CD/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Metilación de ADN/efectos de los fármacos , Glicoproteínas/metabolismo , Neoplasias Hepáticas/patología , Péptidos/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Antígeno AC133 , Animales , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genoma Humano , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Ratones , Células 3T3 NIH , Células Madre Neoplásicas/patología , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Neonatal dried blood spots (DBS) represent an inexpensive method for long-term biobanking worldwide and are considered gold mines for research for several human diseases, including those of metabolic, infectious, genetic and epigenetic origin. However, the utility of DBS is restricted by the limited amount and quality of extractable biomolecules (including DNA), especially for genome wide profiling. Degradation of DNA in DBS often occurs during storage and extraction. Moreover, amplifying small quantities of DNA often leads to a bias in subsequent data, particularly in methylome profiles. Thus it is important to develop methodologies that maximize both the yield and quality of DNA from DBS for downstream analyses. RESULTS: Using combinations of in-house-derived and modified commercial extraction kits, we developed a robust and efficient protocol, compatible with methylome studies, many of which require stringent bisulfite conversion steps. Several parameters were tested in a step-wise manner, including blood extraction, cell lysis, protein digestion, and DNA precipitation, purification and elution. DNA quality was assessed based on spectrophotometric measurements, DNA detectability by PCR, and DNA integrity by gel electrophoresis and bioanalyzer analyses. Genome scale Infinium HumanMethylation450 and locus-specific pyrosequencing data generated using the refined DBS extraction protocol were of high quality, reproducible and consistent. CONCLUSIONS: This study may prove useful to meet the increased demand for research on prenatal, particularly epigenetic, origins of human diseases and for newborn screening programs, all of which are often based on DNA extracted from DBS.
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Metilación de ADN , ADN/sangre , Pruebas con Sangre Seca , Bancos de Muestras Biológicas , Línea Celular , Análisis por Conglomerados , ADN/aislamiento & purificación , Humanos , Recién Nacido , EspectrofotometríaRESUMEN
Our previous studies on cutaneous beta human papillomavirus 38 (HPV38) E6 and E7 oncoproteins highlighted a novel activity of IκB kinase beta (IKKß) in the nucleus of human keratinocytes, where it phosphorylates and stabilizes ΔNp73α, an antagonist of p53/p73 functions. Here, we further characterize the role of the IKKß nuclear form. We show that IKKß nuclear translocation and ΔNp73α accumulation are mediated mainly by HPV38 E7 oncoprotein. Chromatin immunoprecipitation (ChIP)/Re-ChIP experiments showed that ΔNp73α and IKKß are part, together with two epigenetic enzymes DNA methyltransferase 1 (DNMT1) and the enhancer of zeste homolog 2 (EZH2), of a transcriptional regulatory complex that inhibits the expression of some p53-regulated genes, such as PIG3. Recruitment to the PIG3 promoter of EZH2 and DNMT1 resulted in trimethylation of histone 3 on lysine 27 and in DNA methylation, respectively, both events associated with gene expression silencing. Decreases in the intracellular levels of HPV38 E7 or ΔNp73α strongly affected the recruitment of the inhibitory transcriptional complex to the PIG3 promoter, with consequent restoration of p53-regulated gene expression. Finally, the ΔNp73α/IKKß/DNMT1/EZH2 complex appears to bind a subset of p53-regulated promoters. In fact, the complex is efficiently recruited to several promoters of genes encoding proteins involved in DNA repair and apoptosis, whereas it does not influence the expression of the prosurvival factor Survivin. In summary, our data show that HPV38 via E7 protein promotes the formation of a multiprotein complex that negatively regulates the expression of several p53-regulated genes.
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Proteínas de Unión al ADN/metabolismo , Queratinocitos/virología , Proteínas Nucleares/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Regiones Promotoras Genéticas/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Western Blotting , Células Cultivadas , Inmunoprecipitación de Cromatina , Ensayo de Unidades Formadoras de Colonias , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteína Potenciadora del Homólogo Zeste 2 , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/virología , Técnica del Anticuerpo Fluorescente , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Inmunoprecipitación , Queratinocitos/citología , Queratinocitos/metabolismo , Luciferasas/metabolismo , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Papillomaviridae/genética , Proteínas E7 de Papillomavirus/genética , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/patología , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genéticaRESUMEN
Chromatin states are believed to play a key role in distinct patterns of gene expression essential for self-renewal and pluripotency of embryonic stem cells (ESCs); however, the genes governing the establishment and propagation of the chromatin signature characteristic of pluripotent cells are poorly understood. Here, we show that conditional deletion of the histone acetyltransferase cofactor Trrap in mouse ESCs triggers unscheduled differentiation associated with loss of histone acetylation, condensation of chromatin into distinct foci (heterochromatization), and uncoupling of H3K4 dimethylation and H3K27 trimethylation. Trrap loss results in downregulation of stemness master genes Nanog, Oct4, and Sox2 and marked upregulation of specific differentiation markers from the three germ layers. Chromatin immunoprecipitation-sequencing analysis of genome-wide binding revealed a significant overlap between Oct4 and Trrap binding in ESCs but not in differentiated mouse embryonic fibroblasts, further supporting a functional interaction between Trrap and Oct4 in the maintenance of stemness. Remarkably, failure to downregulate Trrap prevents differentiation of ESCs, suggesting that downregulation of Trrap may be a critical step guiding transcriptional reprogramming and differentiation of ESCs. These findings establish Trrap as a critical part of the mechanism that restricts differentiation and promotes the maintenance of key features of ESCs.