RESUMEN
Bile acids (BA) have been found to promote coagulation by increasing tissue factor (TF) activity. The contribution of elevated BA levels and cholestasis to TF decryption within the liver parenchyma and the role of farnesoid X receptor (FXR) in this process remain unclear. We investigated the effects of BA on TF activity and thrombin generation in hepatocytes and correlated these effects with activation of FXR-dependent signaling and apoptosis. HepG2 cells and primary hepatocytes were incubated with chenodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), ursodeoxycholic acid (UCDA), or the synthetic FXR agonist GW4064 for 24 h. MTT tests demonstrated cell viability throughout experiments. TF activity was tested via factor Xa generation and thrombin generation was measured by calibrated automated thrombography. Increased TF activity alongside enhanced thrombin generation was observed with CDCA and GW4064 but not with GCDCA and UDCA. TF activity was substantially reduced when FXR activation was blocked with the antagonist DY 268. Quantitative polymerase chain reaction revealed upregulation of FXR target genes only by CDCA and GW4064. Western blot analysis and fluorescence microscopy showed no TF overexpression arguing for TF decryption. Caspase 3 activity measurements and flow cytometric analysis of Annexin V binding showed no signs of apoptosis. Long-term exposure of hepatocytes to nontoxic BA may cause intracellular FXR overstimulation, triggering TF decryption irrespective of the amphiphilic properties of BA. The effect of BA on TF activation correlates with the molecule's ability to enter the cells and activate FXR. TF decryption occurs independently of apoptotic mechanisms.
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Ácidos y Sales Biliares/metabolismo , Hepatocitos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Tromboplastina/metabolismo , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ácido Desoxicólico/farmacología , Células Hep G2 , Humanos , Isoxazoles/farmacología , Hígado/metabolismo , Transducción de Señal/efectos de los fármacos , Trombina/metabolismoRESUMEN
BACKGROUND & AIMS: Cholestasis comprises a spectrum of liver diseases characterized by the accumulation of bile acids. Bile acids and activation of the farnesoid X receptor (FXR) can inhibit autophagy, a cellular self-digestion process necessary for cellular homeostasis and regeneration. In mice, autophagy appears to be impaired in cholestasis and induction of autophagy may reduce liver injury. METHODS: Herein, we explored autophagy in human cholestasis in vivo and investigated the underlying molecular mechanisms in vitro. FXR chromatin immunoprecipitation-sequencing and qPCR were performed in combination with luciferase promoter studies to identify functional FXR binding targets in a human cholestatic liver sample. RESULTS: Autophagic processing appeared to be impaired in patients with cholestasis and in individuals treated with the FXR ligand obeticholic acid (OCA). In vitro, chenodeoxycholic acid and OCA inhibited autophagy at the level of autophagosome to lysosome fusion in an FXR-dependent manner. Rubicon, which inhibits autophago-lysosomal maturation, was identified as a direct FXR target that is induced in cholestasis and by FXR-agonistic bile acids. Genetic inhibition of Rubicon reversed the bile acid-induced impairment of autophagic flux. In contrast to OCA, ursodeoxycholic acid (UDCA), which is a non-FXR-agonistic bile acid, induced autophagolysosome formation independently of FXR, enhanced autophagic flux and was associated with reduced Rubicon levels. CONCLUSION: In models of human cholestasis, autophagic processing is impaired in an FXR-dependent manner, partly resulting from the induction of Rubicon. UDCA is a potent inducer of hepatic autophagy. Manipulating autophagy and Rubicon may represent a novel treatment concept for cholestatic liver diseases. LAY SUMMARY: Autophagy, a cellular self-cleansing process, is impaired in various forms of human cholestasis. Bile acids, which accumulate in cholestatic liver disease, induce Rubicon, a protein that inhibits proper execution of autophagy. Ursodeoxycholic acid, which is the first-line treatment option for many cholestatic liver diseases, induces hepatic autophagy along with reducing Rubicon.
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Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia/genética , Colestasis/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal/genética , Autofagosomas/metabolismo , Autofagia/efectos de los fármacos , Proteínas Relacionadas con la Autofagia/genética , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/metabolismo , Ácido Quenodesoxicólico/farmacología , Ácido Quenodesoxicólico/uso terapéutico , Colestasis/tratamiento farmacológico , Citotoxinas , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Hígado/metabolismo , Hígado/patología , Lisosomas/metabolismo , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Estudios Retrospectivos , Transfección , Ácido Ursodesoxicólico/metabolismo , Ácido Ursodesoxicólico/farmacologíaRESUMEN
BACKGROUND & AIMS: The nuclear farnesoid X receptor (FXR) agonist obeticholic acid (OCA) has been developed for the treatment of liver diseases. We aimed to determine whether OCA treatment increases the risk of gallstone formation. METHODS: Twenty patients awaiting laparoscopic cholecystectomy were randomized to treatment with OCA (25â¯mg/day) or placebo for 3â¯weeks until the day before surgery. Serum bile acids (BAs), the BA synthesis marker C4 (7α-hydroxy-4-cholesten-3-one), and fibroblast growth factor 19 (FGF19) were measured before and after treatment. During surgery, biopsies from the liver and the whole bile-filled gallbladder were collected for analyses of gene expression, biliary lipids and FGF19. RESULTS: In serum, OCA increased FGF19 (from 95.0⯱â¯8.5 to 234.4⯱â¯35.6â¯ng/L) and decreased C4 (from 31.4⯱â¯22.8 to 2.8⯱â¯4.0â¯nmol/L) and endogenous BAs (from 1,312.2⯱â¯236.2 to 517.7⯱â¯178.9â¯nmol/L; all p <0.05). At surgery, BAs in gallbladder bile were lower in patients that received OCA than in controls (OCA, 77.9⯱â¯53.6â¯mmol/L; placebo, 196.4⯱â¯99.3â¯mmol/L; p <0.01), resulting in a higher cholesterol saturation index (OCA, 2.8⯱â¯1.1; placebo, 1.8⯱â¯0.8; p <0.05). In addition, hydrophobic OCA conjugates accounted for 13.6⯱â¯5.0% of gallbladder BAs after OCA treatment, resulting in a higher hydrophobicity index (OCA, 0.43⯱â¯0.09; placebo, 0.34⯱â¯0.07, p <0.05). Gallbladder FGF19 levels were 3-fold higher in OCA patients than in controls (OCA, 40.3⯱â¯16.5â¯ng/L; placebo, 13.5⯱â¯13.1â¯ng/ml; p <0.005). Gene expression analysis indicated that FGF19 mainly originated from the gallbladder epithelium. CONCLUSIONS: Our results show for the first time an enrichment of FGF19 in human bile after OCA treatment. In accordance with its murine homolog FGF15, FGF19 might trigger relaxation and filling of the gallbladder which, in combination with increased cholesterol saturation and BA hydrophobicity, would enhance the risk of gallstone development. LAY SUMMARY: Obeticholic acid increased human gallbladder cholesterol saturation and bile acid hydrophobicity, both decreasing cholesterol solubility in bile. Together with increased hepatobiliary levels of fibroblast growth factor 19, our findings suggest that pharmacological activation of the farnesoid X receptor increases the risk of gallstone formation. Clinical trial number: NCT01625026.
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Ácido Quenodesoxicólico/análogos & derivados , Cálculos Biliares/inducido químicamente , Cálculos Biliares/cirugía , Hepatopatías/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/agonistas , Adulto , Ácidos y Sales Biliares/sangre , Ácidos y Sales Biliares/genética , Biopsia , Proteínas Portadoras/genética , Ácido Quenodesoxicólico/efectos adversos , Ácido Quenodesoxicólico/farmacología , Colestenonas/sangre , Método Doble Ciego , Femenino , Factores de Crecimiento de Fibroblastos/sangre , Vesícula Biliar/patología , Vesícula Biliar/cirugía , Cálculos Biliares/sangre , Expresión Génica , Humanos , Hígado/patología , Hepatopatías/sangre , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
BACKGROUND AND AIMS: Bile acids are now accepted as central signalling molecules for the regulation of glucose, amino acid and lipid metabolism. Adrenal gland cortex cells express the bile acid receptors farnesoid X receptor (FXR), the G protein-coupled bile acid receptor (TGR5) and the sphingosine-1-phosphate receptor 2 (S1PR2). We aimed to determine the effects of cholestasis and more specifically of bile acids on cortisol production. METHODS: FXR and TGR5 knockout mice and controls were subjected to common bile duct ligation (CBDL) or chenodeoxycholic acid (CDCA) feeding to model cholestasis. Human adrenocortical H295R cells were challenged with bile acids for mechanistic studies. RESULTS: We found that CBDL and CDCA feeding increased the levels of corticosterone, the rodent equivalent to human cortisol and mRNA and protein levels of steroidogenesis-related enzymes in adrenals independent of FXR and TGR5. Taurine-conjugated CDCA (TCDCA) significantly stimulated cortisol secretion, phosphorylation of extracellular signal-regulated kinase (ERK) and expression of steroidogenesis-related genes in human adrenocortical H295R cells. FXR and TGR5 agonists failed to induce cortisol secretion in H295R cells. S1PR2 inhibition significantly abolished TCDCA-induced cortisol secretion, lowered phosphorylation of ERK and abrogated enhanced transcription of steroidogenesis-related genes in H295R cells. Likewise, siRNA S1PR2 treatment reduced the phosphorylation of ERK and cortisol secretion. Steroidogenic factor-1 (SF-1) transactivation activity was increased upon TCDCA treatment suggesting that bile acid signalling is linked to SF-1. Treatment with SF-1 inverse agonist AC45594 also reduced TCDCA-induced steroidogenesis. CONCLUSIONS: Our findings indicate that supraphysiological bile acid levels as observed in cholestasis stimulate steroidogenesis via an S1PR2-ERK-SF-1 signalling pathway.
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Ácidos y Sales Biliares/metabolismo , Colestasis/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hidrocortisona/biosíntesis , Receptores de Esfingosina-1-Fosfato/metabolismo , Factor Esteroidogénico 1/metabolismo , Animales , Línea Celular , Ácido Quenodesoxicólico/farmacología , Glucosa/metabolismo , Humanos , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de SeñalRESUMEN
UNLABELLED: Balance of labile methyl groups (choline, methionine, betaine, and folate) is important for normal liver function. Quantitatively, a significant use of labile methyl groups is in the production of phosphatidylcholines (PCs), which are ligands for the nuclear liver receptor homolog-1 (LRH-1). We studied the role of LRH-1 in methyl-pool homeostasis and determined its metabolic effects using the methionine and choline-deficient (MCD) diet, which depletes methyl groups and results in a deleterious decrease in the PC-to-phosphatidylethanolamine ratio. We found that MCD diet-fed, liver-specific LRH-1 knockout mice (Lrh-1(-/-) ) do not show the expected decreased methyl-pool and PC/phosphatidylethanolamine ratio and are resistant to the hepatitis and fibrosis normally induced by the diet. Adaptive responses observed in wild-type mice on the MCD diet were also observed in Lrh-1(-/-) mice on a normal diet. This includes reduced expression of the highly active glycine-n-methyltransferase and the biliary phospholipid floppase multidrug-resistance protein 2 (Mdr2/Abcb4), resulting in reduced consumption of methyl groups and biliary PC secretion. In vitro studies confirm that Gnmt and Mdr2 are primary LRH-1 target genes. Additional similarities between hepatic gene expression profiles in MCD diet-fed wild-type and untreated Lrh-1(-/-) mice suggest that methyl-pool deficiency decreases LRH-1 activity, and this was confirmed by in vitro functional results in cells maintained in MCD medium. CONCLUSION: LRH-1 is a novel transcriptional regulator of methyl-pool balance; when the methyl-pool is depleted, decreased LRH-1 transactivation suppresses expression of key genes to minimize loss of labile methyl groups. (Hepatology 2016;63:95-106).
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Hígado/metabolismo , Metilación , Receptores Citoplasmáticos y Nucleares/fisiología , Animales , Masculino , Ratones , Ratones NoqueadosRESUMEN
Despite recent developments in treatment strategies, castration-resistant prostate cancer (CRPC) is still the second leading cause of cancer-associated mortality among American men, the biological underpinnings of which are not well understood. To this end, we measured levels of 150 metabolites and examined the rate of utilization of 184 metabolites in metastatic androgen-dependent prostate cancer (AD) and CRPC cell lines using a combination of targeted mass spectrometry and metabolic phenotyping. Metabolic data were used to derive biochemical pathways that were enriched in CRPC, using Oncomine concept maps (OCM). The enriched pathways were then examined in-silico for their association with treatment failure (i.e., prostate specific antigen (PSA) recurrence or biochemical recurrence) using published clinically annotated gene expression data sets. Our results indicate that a total of 19 metabolites were altered in CRPC compared to AD cell lines. These altered metabolites mapped to a highly interconnected network of biochemical pathways that describe UDP glucuronosyltransferase (UGT) activity. We observed an association with time to treatment failure in an analysis employing genes restricted to this pathway in three independent gene expression data sets. In summary, our studies highlight the value of employing metabolomic strategies in cell lines to derive potentially clinically useful predictive tools.
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Metabolómica , Orquiectomía , Neoplasias de la Próstata/metabolismo , Línea Celular Tumoral , Cromatografía Liquida , Expresión Génica , Glucuronosiltransferasa/metabolismo , Humanos , Masculino , Espectrometría de Masas , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genéticaRESUMEN
BACKGROUND: Bile, which contains bile acids, the natural ligands for farnesoid x receptor (FXR), moves from the liver to the intestine through bile ducts. Ductular reaction often occurs during biliary obstruction. A subset of patients with erythropoietic protoporphyria, an inherited genetic mutation in heme biosynthetic enzyme ferrochelatase, accumulate porphyrin-containing bile plugs, leading to cholestasis. Here, we examined the link between FXR, bile plug formation, and how heme biosynthesis relates to this connection. METHODS: We treated female and male wild-type and global and tissue-specific Fxr knockout mice with a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine, an inhibitor of ferrochelatase, and examined the expression of heme biosynthetic genes. We mined FXR mouse ChIP-Seq data, performed biochemical and histological analysis, and tested HepG2 and primary human hepatocytes after treatment with obeticholic acid, an FXR agonist. RESULTS: We observed that hepatic but not intestinal Fxr loss resulted in reduced bile plugs and ductular reaction in the liver. Then, we examined if FXR plays a regulatory role in heme biosynthesis and found significantly lower porphyrin accumulation in 3,5-diethoxycarbonyl-1, 4-dihydrocollidine-fed Fxr knockout mice. Gene expression and FXR mouse ChIP-Seq atlas analysis revealed that FXR orchestrates the expression of multiple heme biosynthetic enzymes. Finally, human HepG2 cells and primary human hepatocytes treated with obeticholic acid, showed increased expression of several heme biosynthetic genes. CONCLUSIONS: Overall, our data show that hepatic Fxr is necessary to maintain ductular reaction and accumulation of bile plugs. FXR can direct the expression of multiple heme biosynthetic genes. Thus, modulating FXR activity in EPP patients may help alleviate its associated liver disease.
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Colestasis , Porfirinas , Animales , Femenino , Humanos , Masculino , Ratones , Ferroquelatasa , Hemo , HígadoRESUMEN
The bile acid receptor FXR has emerged as a bona fide drug target for chronic cholestatic and metabolic liver diseases, ahead of all non-alcoholic fatty liver disease (NAFLD). FXR is highly expressed in the liver and intestine and activation at both sites differentially contributes to its desired metabolic effects. Unrestricted FXR activation, however, also comes along with undesired effects such as a pro-atherogenic lipid profile, pruritus and hepatocellular toxicity under certain conditions. Several pre-clinical studies have confirmed the potency of FXR activation for cholestatic and metabolic liver diseases, but overall it remains still open whether selective activation of intestinal FXR is advantageous over pan-FXR activation and whether restricted or modulated FXR activation can limit some of the side effects. Even more, FXR antagonist also bear the potential as intestinal-selective drugs in NAFLD models. In this review we will discuss the molecular prerequisites for FXR activation, pan-FXR activation and intestinal FXR in/activation from a therapeutic point of view, different steroidal and non-steroidal FXR agonists, ways to restrict FXR activation and finally what we have learned from pre-clinical models and clinical trials with different FXR therapeutics.
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Enfermedades Metabólicas , Enfermedad del Hígado Graso no Alcohólico , Ácidos y Sales Biliares , Humanos , Intestinos , Enfermedades Metabólicas/metabolismoRESUMEN
The liver is the central metabolic hub which coordinates nutritional inputs and metabolic outputs. Food intake releases bile acids which can be sensed by the bile acid receptor FXR in the liver and the intestine. Hepatic and intestinal FXR coordinately regulate postprandial nutrient disposal in a network of interacting metabolic nuclear receptors. In this review we summarize and update the "classical roles" of FXR as a central integrator of the feeding state response, which orchestrates the metabolic processing of carbohydrates, lipids, proteins and bile acids. We also discuss more recent and less well studied FXR effects on amino acid, protein metabolism, autophagic turnover and inflammation. In addition, we summarize the recent understanding of how FXR signaling is affected by posttranslational modifications and by different FXR isoforms. These modifications and variations in FXR signaling might be considered when FXR is targeted pharmaceutically in clinical applications.
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Inflamación/patología , Metabolismo de los Lípidos , Hígado/fisiopatología , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Humanos , Inflamación/etiología , Inflamación/metabolismo , Transducción de SeñalRESUMEN
Autophagy is a lysosomal degradation pathway in which the cell self-digests its own components to provide nutrients in harsh environmental conditions. It also represents an opportunity to rid the cell of superfluous and damaged organelles, misfolded proteins or invaded microorganisms. Liver autophagy contributes to basic hepatic functions such as lipid, glycogen and protein turnover. Deregulated hepatic autophagy has been linked to many liver diseases including alpha-1-antitrypsin deficiency, alcoholic and non-alcoholic fatty liver diseases, hepatitis B and C infections, liver fibrosis as well as liver cancer. Recently, bile acids and the bile acid receptor FXR have been implicated in the regulation of hepatic autophagy, which implies a role of autophagy also for cholestatic liver diseases. This review summarizes the current evidence of bile acid mediated effects on autophagy and how this affects cholestatic liver diseases. Although detailed studies are lacking, we suggest a concept that the activity of autophagy in cholestasis depends on the disease stage, where autophagy may be induced at early stages ("cholestophagy") but may be impaired in prolonged cholestatic states ("cholestopagy").
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Autofagia/fisiología , Ácidos y Sales Biliares/metabolismo , Colestasis/patología , Enfermedad del Hígado Graso no Alcohólico/patología , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Autofagosomas/efectos de los fármacos , Autofagosomas/metabolismo , Autofagia/efectos de los fármacos , Conductos Biliares/citología , Conductos Biliares/metabolismo , Colagogos y Coleréticos/farmacología , Colagogos y Coleréticos/uso terapéutico , Colestasis/tratamiento farmacológico , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Ácidos Fíbricos/farmacología , Ácidos Fíbricos/uso terapéutico , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/citología , Hígado/efectos de los fármacos , Hígado/patología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Farnesoid X receptor (FXR) is a nuclear receptor that controls gene regulation of different metabolic pathways and represents an upcoming drug target for various liver diseases. Several data sets on genome-wide FXR binding in different species and conditions exist. We have previously reported that these data sets are heterogeneous and do not cover the full spectrum of potential FXR binding sites. Here, we report the first meta-analysis of all publicly available FXR chromatin immunoprecipitation sequencing (ChIP-seq) data sets from mouse, rat, and human across different conditions using a newly generated analysis pipeline. All publicly available single data sets were biocurated in a standardized manner and compared on every relevant level from raw reads to affected functional pathways. Individual murine data sets were then virtually merged into a single unique "FXR binding atlas" spanning all potential binding sites across various conditions. Comparison of the single biocurated data sets showed that the overlap of FXR binding sites between different species is modest and ranges from 48% (mouse-human) to 55% (mouse-rat). Moreover, in vivo data among different species are more similar than human in vivo data compared to human in vitro data. The consolidated murine global FXR binding atlas virtually increases sequencing depth and allows recovering more and novel potential binding sites and signaling pathways that were missed in the individual data sets. The FXR binding atlas is publicly searchable (https://fxratlas.tugraz.at). Conclusion: Published single FXR ChIP-seq data sets and large-scale integrated omics data sets do not cover the full spectrum of FXR binding. Combining different individual data sets and creating an "FXR super-binding atlas" enhances understanding of FXR signaling capacities across different conditions. This is important when considering the potential wide spectrum for drugs targeting FXR in liver diseases.
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Secuenciación de Inmunoprecipitación de Cromatina , Agregación de Datos , Bases de Datos Genéticas , Receptores Citoplasmáticos y Nucleares/genética , Animales , Sitios de Unión/genética , Regulación de la Expresión Génica/genética , Humanos , Ratones , Unión Proteica/genética , Ratas , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Ventricular tachycardia (VT) is a major cause of sudden cardiac death (SCD). Clinical investigations can sometimes fail to identify the underlying cause of VT and the event is classified as idiopathic (iVT). VT contributes significantly to the morbidity and mortality in patients with coronary artery disease (CAD) and dilated cardiomyopathy (DCM). Since mutations in arrhythmia-associated genes frequently determine arrhythmia susceptibility screening for disease-predisposing variants could improve VT diagnostics and prevent SCD in patients. METHODS: Ninety-two patients diagnosed with coronary heart disease (CHD), DCM, or iVT were included in our study. We evaluated genetic profiles and variants in known cardiac risk genes by targeted next generation sequencing (NGS) using a newly designed custom panel of 96 genes. We hypothesized that shared morphological and phenotypical features among these subgroups may have an overlapping molecular base. To our knowledge, this was the first study of the deep sequencing of 96 targeted cardiac genes in Kazakhstan. The clinical significance of the sequence variants was interpreted according to the guidelines developed by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) in 2015. The ClinVar and Varsome databases were used to determine the variant classifications. RESULTS: Targeted sequencing and stepwise filtering of the annotated variants identified a total of 307 unique variants in 74 genes, totally 456 variants in the overall study group. We found 168 mutations listed in the Human Genome Mutation Database (HGMD) and another 256 rare/unique variants with elevated pathogenic potential. There was a predominance of high- to intermediate pathogenicity variants in LAMA2, MYBPC3, MYH6, KCNQ1, GAA, and DSG2 in CHD VT patients. Similar frequencies were observed in DCM VT, and iVT patients, pointing to a common molecular disease association. TTN, GAA, LAMA2, and MYBPC3 contained the most variants in the three subgroups which confirm the impact of these genes in the complex pathogenesis of cardiomyopathies and VT. The classification of 307 variants according to ACMG guidelines showed that nine (2.9%) variants could be classified as pathogenic, nine (2.9%) were likely pathogenic, 98 (31.9%) were of uncertain significance, 73 (23.8%) were likely benign, and 118 (38.4%) were benign. CHD VT patients carry rare genetic variants with increased pathogenic potential at a comparable frequency to DCM VT and iVT patients in genes related to sarcomere function, nuclear function, ion flux, and metabolism. CONCLUSIONS: In this study we showed that in patients with VT secondary to coronary artery disease, DCM, or idiopathic etiology multiple rare mutations and clinically significant sequence variants in classic cardiac risk genes associated with cardiac channelopathies and cardiomyopathies were found in a similar pattern and at a comparable frequency.
RESUMEN
Lung cancer is one of the deadliest cancers worldwide. Late diagnosis at an advanced, inoperable stage makes chemotherapy a treatment of choice, yet, with low response rates. The hedgehog signaling pathway (HHSP) is often reactivated in cancer. We identified miR-182-5p as a regulator of GLI2, a transcriptional regulator of the HHSP, and explored the role of the miR-182-5p/GLI2 axis in carcinogenesis and cisplatin resistance of lung adenocarcinoma (LADC). Expression of miRNAs and target genes was analyzed by RT-qPCR, expression of the GLI-protein family in LADC and adjacent lung tissue (nâ¯=â¯27 pairs) by immunohistochemistry. MiR-182-5p was manipulated, and data were generated by immunoblotting, immunofluorescence, apoptosis, proliferation/viability, dual-luciferase-, and colony forming assays. MiR-182-5p was down-regulated in cisplatin-resistant LADC cells and directly targeted GLI2. Interference with miR-182-5p or GLI2 silencing resulted in modulation of cell proliferation, clonogenic potential, and cisplatin-sensitivity. HHSP was markedly reactivated in LADC tissue compared to adjacent non-malignant lung tissue. Our results indicate that the miR-182-5p/GLI2 axis modulates tumorigenesis and cisplatin-resistance in LADC cells, by influencing the HHSP. Therefore, this axis might be considered as a potential biomarker and future therapeutic target in LADC patients.
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Adenocarcinoma del Pulmón/tratamiento farmacológico , Cisplatino/farmacología , MicroARNs/genética , Proteínas Nucleares/genética , Proteína Gli2 con Dedos de Zinc/genética , Células A549 , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/genética , Carcinogénesis/genética , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cisplatino/efectos adversos , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Hedgehog/genética , Humanos , Masculino , Persona de Mediana Edad , Transducción de Señal/efectos de los fármacosRESUMEN
INTRODUCTION: HULC (highly upregulated in liver cancer) is a long non-coding RNA (lncRNA) which is, as its name suggests, highly upregulated in hepatocellular carcinoma and in several other cancers. Increased HULC expression levels are strongly associated with clinicopathologic features such as tumor stages and overall survival and is a driver of tumor proliferation, migration, and invasion. Areas covered: This review addresses the discovery of HULC and discusses the consequences of HULC deregulation in cancer, the underlying molecular mechanisms and the potential of HULC as a biomarker and therapeutic target. Expert opinion: HULC is a promising candidate as a therapeutic target in cancer; however, more studies are necessary to further elucidate the underlying molecular mechanism(s), especially in cancer types other than hepatocellular carcinomas. Future studies that focus on an optimized HULC-targeting approach are necessary to clarify the best strategy to target this lncRNA in vivo and in patients.
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Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , ARN Largo no Codificante/genética , Animales , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/genética , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Terapia Molecular Dirigida , Regulación hacia ArribaRESUMEN
BACKGROUND: ChIP-seq is a method to identify genome-wide transcription factor (TF) binding sites. The TF FXR is a nuclear receptor that controls gene regulation of different metabolic pathways in the liver. OBJECTIVES: To re-analyze, standardize and combine all publicly available FXR ChIP-seq data sets to create a global FXR signaling atlas. METHODS: All data sets were (re-)analyzed in a standardized manner and compared on every relevant level from raw reads to affected functional pathways. RESULTS: Public FXR data sets were available for mouse, rat and primary human hepatocytes in different treatment conditions. Standardized re-analysis shows that the data sets are surprisingly heterogeneous concerning baseline quality criteria. Combining different data sets increased the depth of analysis and allowed to recover more peaks and functional pathways. CONCLUSION: Published single FXR ChIP-seq data sets do not cover the full spectrum of FXR signaling. Combining different data sets and creating a "FXR super-signaling atlas" enhances understanding of FXR signaling capacities.
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Análisis de Datos , Regulación de la Expresión Génica , Genoma , Receptores Citoplasmáticos y Nucleares , Animales , Sitios de Unión , Inmunoprecipitación de Cromatina , Bases de Datos Factuales , Humanos , Ratones , RatasRESUMEN
The precise molecular alterations driving castration-resistant prostate cancer (CRPC) are not clearly understood. Using a novel network-based integrative approach, here, we show distinct alterations in the hexosamine biosynthetic pathway (HBP) to be critical for CRPC. Expression of HBP enzyme glucosamine-phosphate N-acetyltransferase 1 (GNPNAT1) is found to be significantly decreased in CRPC compared with localized prostate cancer (PCa). Genetic loss-of-function of GNPNAT1 in CRPC-like cells increases proliferation and aggressiveness, in vitro and in vivo. This is mediated by either activation of the PI3K-AKT pathway in cells expressing full-length androgen receptor (AR) or by specific protein 1 (SP1)-regulated expression of carbohydrate response element-binding protein (ChREBP) in cells containing AR-V7 variant. Strikingly, addition of the HBP metabolite UDP-N-acetylglucosamine (UDP-GlcNAc) to CRPC-like cells significantly decreases cell proliferation, both in-vitro and in animal studies, while also demonstrates additive efficacy when combined with enzalutamide in-vitro. These observations demonstrate the therapeutic value of targeting HBP in CRPC.
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Hexosaminas/biosíntesis , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Línea Celular , Humanos , Masculino , Ratones , Ratones SCID , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Although alterations in xenobiotic metabolism are considered causal in the development of bladder cancer, the precise mechanisms involved are poorly understood. In this study, we used high-throughput mass spectrometry to measure over 2,000 compounds in 58 clinical specimens, identifying 35 metabolites which exhibited significant changes in bladder cancer. This metabolic signature distinguished both normal and benign bladder from bladder cancer. Exploratory analyses of this metabolomic signature in urine showed promise in distinguishing bladder cancer from controls and also nonmuscle from muscle-invasive bladder cancer. Subsequent enrichment-based bioprocess mapping revealed alterations in phase I/II metabolism and suggested a possible role for DNA methylation in perturbing xenobiotic metabolism in bladder cancer. In particular, we validated tumor-associated hypermethylation in the cytochrome P450 1A1 (CYP1A1) and cytochrome P450 1B1 (CYP1B1) promoters of bladder cancer tissues by bisulfite sequence analysis and methylation-specific PCR and also by in vitro treatment of T-24 bladder cancer cell line with the DNA demethylating agent 5-aza-2'-deoxycytidine. Furthermore, we showed that expression of CYP1A1 and CYP1B1 was reduced significantly in an independent cohort of bladder cancer specimens compared with matched benign adjacent tissues. In summary, our findings identified candidate diagnostic and prognostic markers and highlighted mechanisms associated with the silencing of xenobiotic metabolism. The metabolomic signature we describe offers potential as a urinary biomarker for early detection and staging of bladder cancer, highlighting the utility of evaluating metabolomic profiles of cancer to gain insights into bioprocesses perturbed during tumor development and progression.
Asunto(s)
Biomarcadores de Tumor/orina , Metabolómica/métodos , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/orina , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Western Blotting , Línea Celular Tumoral , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1 , Metilación de ADN , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Estadificación de Neoplasias , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vejiga Urinaria/metabolismo , Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/genética , Adulto JovenRESUMEN
BACKGROUND & AIMS: Recent studies have highlighted the role of noncoding RNAs (ncRNAs) in carcinogenesis, and suggested that this class of genes might be used as biomarkers in cancer. We searched the human genome for novel genes including ncRNAs related to hepatocellular carcinoma (HCC). METHODS: An HCC-specific gene library was generated and screened for deregulated genes with 46 HCCs, 4 focal nodular hyperplasias, and 7 cirrhoses utilizing cDNA arrays. Sequencing of library clones identified a novel ncRNA as the most up-regulated gene in HCC. This gene was also cloned from different monkeys and characterized by quantitative RT-PCR, Northern blot analysis and in situ hybridization. Structural and functional studies included comparative sequence and protein expression analyses, quantitative RT-PCR of polysomal preparations, and siRNA-mediated knockdown experiments. RESULTS: The most up-regulated gene in HCC named highly up-regulated in liver cancer (HULC) was characterized as a novel mRNA-like ncRNA. HULC RNA is spliced and polyadenlyated, and resembles the mammalian LTR transposon 1A. It does not contain substantial open reading frames, and no native translation product was detected. HULC is present in the cytoplasm, where it copurifies with ribosomes. siRNA-mediated knockdown of HULC RNA in 2 HCC cell lines altered the expression of several genes, 5 of which were known to be affected in HCC, suggesting a role for HULC in post-transcriptional modulation of gene expression. CONCLUSIONS: HULC is the first ncRNA with highly specific up-regulation in HCC. Because HULC was detected in blood of HCC patients, a potential use as novel biomarker can be envisaged.