RESUMEN
Phenotypic screening identified an arylsulfonamide compound with activity against Trypanosoma cruzi, the causative agent of Chagas' disease. Comprehensive mode of action studies revealed that this compound primarily targets the T. cruzi proteasome, binding at the interface between ß4 and ß5 subunits that catalyze chymotrypsin-like activity. A mutation in the ß5 subunit of the proteasome was associated with resistance to compound 1, while overexpression of this mutated subunit also reduced susceptibility to compound 1. Further genetically engineered and in vitro-selected clones resistant to proteasome inhibitors known to bind at the ß4/ß5 interface were cross-resistant to compound 1. Ubiquitinated proteins were additionally found to accumulate in compound 1-treated epimastigotes. Finally, thermal proteome profiling identified malic enzyme as a secondary target of compound 1, although malic enzyme inhibition was not found to drive potency. These studies identify a novel pharmacophore capable of inhibiting the T. cruzi proteasome that may be exploitable for anti-chagasic drug discovery.
Asunto(s)
Enfermedad de Chagas , Trypanosoma cruzi , Enfermedad de Chagas/tratamiento farmacológico , Descubrimiento de Drogas , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Trypanosoma cruzi/químicaRESUMEN
Available treatments for Chagas' disease and visceral leishmaniasis are inadequate, and there is a pressing need for new therapeutics. Drug discovery efforts for both diseases principally rely upon phenotypic screening. However, the optimization of phenotypically active compounds is hindered by a lack of information regarding their molecular target(s). To combat this issue we initiate target deconvolution studies at an early stage. Here, we describe comprehensive genetic and biochemical studies to determine the targets of three unrelated phenotypically active compounds. All three structurally diverse compounds target the Qi active-site of cytochrome b, part of the cytochrome bc1 complex of the electron transport chain. Our studies go on to identify the Qi site as a promiscuous drug target in Leishmania donovani and Trypanosoma cruzi with a propensity to rapidly mutate. Strategies to rapidly identify compounds acting via this mechanism are discussed to ensure that drug discovery portfolios are not overwhelmed with inhibitors of a single target.
Asunto(s)
Antiparasitarios/farmacología , Citocromos b/antagonistas & inhibidores , Descubrimiento de Drogas , Leishmania donovani/efectos de los fármacos , Leishmania donovani/genética , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/genética , Antiparasitarios/química , Antiparasitarios/aislamiento & purificación , Enfermedad de Chagas/tratamiento farmacológico , Citocromos b/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Leishmaniasis Visceral/tratamiento farmacológicoRESUMEN
Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the ß5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the ß4 and ß5 proteasome subunits. This induced pocket exploits ß4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.
Asunto(s)
Antiprotozoarios/administración & dosificación , Leishmania donovani/efectos de los fármacos , Leishmania infantum/efectos de los fármacos , Leishmaniasis Visceral/diagnóstico por imagen , Inhibidores de Proteasoma/administración & dosificación , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Antiprotozoarios/química , Sitios de Unión , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Leishmania donovani/química , Leishmania donovani/enzimología , Leishmania infantum/química , Leishmania infantum/enzimología , Leishmaniasis Visceral/parasitología , Masculino , Ratones , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/química , Conformación Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismoRESUMEN
Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and L. infantum, is responsible for â¼30â¯000 deaths annually. Available treatments are inadequate, and there is a pressing need for new therapeutics. N-Myristoyltransferase (NMT) remains one of the few genetically validated drug targets in these parasites. Here, we sought to pharmacologically validate this enzyme in Leishmania. A focused set of 1600 pyrazolyl sulfonamide compounds was screened against L. major NMT in a robust high-throughput biochemical assay. Several potent inhibitors were identified with marginal selectivity over the human enzyme. There was little correlation between the enzyme potency of these inhibitors and their cellular activity against L. donovani axenic amastigotes, and this discrepancy could be due to poor cellular uptake due to the basicity of these compounds. Thus, a series of analogues were synthesized with less basic centers. Although most of these compounds continued to suffer from relatively poor antileishmanial activity, our most potent inhibitor of LmNMT (DDD100097, K i of 0.34 nM) showed modest activity against L. donovani intracellular amastigotes (EC50 of 2.4 µM) and maintained a modest therapeutic window over the human enzyme. Two unbiased approaches, namely, screening against our cosmid-based overexpression library and thermal proteome profiling (TPP), confirm that DDD100097 (compound 2) acts on-target within parasites. Oral dosing with compound 2 resulted in a 52% reduction in parasite burden in our mouse model of VL. Thus, NMT is now a pharmacologically validated target in Leishmania. The challenge in finding drug candidates remains to identify alternative strategies to address the drop-off in activity between enzyme inhibition and in vitro activity while maintaining sufficient selectivity over the human enzyme, both issues that continue to plague studies in this area.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Antiprotozoarios/farmacología , Descubrimiento de Drogas , Leishmania donovani/efectos de los fármacos , Pirazoles/química , Pirazoles/farmacología , Animales , Cósmidos , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Leishmaniasis Visceral/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , Carga de Parásitos , Proteoma/análisis , ProteómicaRESUMEN
The lack of information regarding the mechanisms of action (MoA) or specific molecular targets of phenotypically active compounds can prove a barrier to their development as chemotherapeutic agents. Here, we report the results of our orthogonal genetic, molecular, and biochemical studies to determine the MoA of a novel 7-substituted 8-hydroxy-1,6-naphthyridine (8-HNT) series that displays promising activity against Trypanosoma brucei and Leishmania donovani High-throughput loss-of-function genetic screens in T. brucei highlighted two probable zinc transporters associated with resistance to these compounds. These transporters localized to the parasite Golgi apparatus. Directed by these findings, the role of zinc and other divalent cations in the MoA of these compounds was investigated. 8-HNT compounds were found to directly deplete intracellular levels of Zn2+, while the addition of exogenous Zn2+ and Fe2+ reduced the potency of compounds from this series. Detailed biochemical analyses confirmed that 8-HNT compounds bind directly to a number of divalent cations, predominantly Zn2+, Fe2+, and Cu2+, forming 2:1 complexes with one of these cations. Collectively, our studies demonstrate transition metal depletion, due to chelation, as the MoA of the 8-HNT series of compounds. Strategies to improve the selectivity of 8-HNT compounds are discussed.
Asunto(s)
Antiprotozoarios/farmacología , Proteínas de Transporte de Catión/genética , Quelantes/farmacología , Naftiridinas/farmacología , Proteínas Protozoarias/genética , Zinc/metabolismo , Antiprotozoarios/síntesis química , Proteínas de Transporte de Catión/metabolismo , Cationes Bivalentes , Quelantes/síntesis química , Cobre/metabolismo , Expresión Génica , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Hierro/metabolismo , Leishmania donovani/efectos de los fármacos , Leishmania donovani/genética , Leishmania donovani/crecimiento & desarrollo , Leishmania donovani/metabolismo , Mutación , Naftiridinas/síntesis química , Pruebas de Sensibilidad Parasitaria , Proteínas Protozoarias/metabolismo , Relación Estructura-Actividad , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/metabolismoRESUMEN
Mechanisms regulating protein degradation ensure the correct and timely expression of transcription factors such as hypoxia inducible factor (HIF). Under normal O2 tension, HIFα subunits are targeted for proteasomal degradation, mainly through vHL-dependent ubiquitylation. Deubiquitylases are responsible for reversing this process. Although the mechanism and regulation of HIFα by ubiquitin-dependent proteasomal degradation has been the object of many studies, little is known about the role of deubiquitylases. Here, we show that expression of HIF2α (encoded by EPAS1) is regulated by the deubiquitylase Cezanne (also known as OTUD7B) in an E2F1-dependent manner. Knockdown of Cezanne downregulates HIF2α mRNA, protein and activity independently of hypoxia and proteasomal degradation. Mechanistically, expression of the HIF2α gene is controlled directly by E2F1, and Cezanne regulates the stability of E2F1. Exogenous E2F1 can rescue HIF2α transcript and protein expression when Cezanne is depleted. Taken together, these data reveal a novel mechanism for the regulation of the expression of HIF2α, demonstrating that the HIF2α promoter is regulated by E2F1 directly and that Cezanne regulates HIF2α expression through control of E2F1 levels. Our results thus suggest that HIF2α is controlled transcriptionally in a cell-cycle-dependent manner and in response to oncogenic signalling.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Puntos de Control del Ciclo Celular/genética , Factor de Transcripción E2F1/genética , Endopeptidasas/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Hipoxia de la Célula/genética , Factor de Transcripción E2F1/biosíntesis , Endopeptidasas/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Regiones Promotoras Genéticas , ProteolisisRESUMEN
The transcription factor HIF-1α is essential for cells to rapidly adapt to low oxygen levels (hypoxia). HIF-1α is frequently deregulated in cancer and correlates with poor patient prognosis. Here, we demonstrate that the deubiquitinase Cezanne regulates HIF-1α homeostasis. Loss of Cezanne decreases HIF-1α target gene expression due to a reduction in HIF-1α protein levels. Surprisingly, although the Cezanne-regulated degradation of HIF-1α depends on the tumour suppressor pVHL, hydroxylase and proteasome activity are dispensable. Our data suggest that Cezanne is essential for HIF-1α protein stability and that loss of Cezanne stimulates HIF-1α degradation via proteasome-independent routes, possibly through chaperone-mediated autophagy.
Asunto(s)
Endopeptidasas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Apoptosis/fisiología , Línea Celular , Endopeptidasas/genética , Células HeLa , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inmunoprecipitación , Ratones , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Cell cycle progression is an energy demanding process and requires fine-tuned metabolic regulation. Cells must overcome an energy restriction checkpoint before becoming committed to progress through the cell cycle. Aerobic organisms need oxygen for the metabolic conversion of nutrients into energy. As such, environmental oxygen is a critical signalling molecule regulating cell fate. The Hypoxia Inducible Factors (HIFs) are a family of transcription factors that respond to changes in environmental oxygen and cell energy and coordinate a transcriptional program which forms an important part of the cellular response to a hostile environment. A significant proportion of HIF-dependent transcriptional target genes, code for proteins that are involved in energy homeostasis. In this review we discuss the role of the HIF system in the regulation of energy homeostasis in response to changes in environmental oxygen and the impact on cell cycle control, and address the implications of the deregulation of this effect in cancer.
Asunto(s)
Ciclo Celular/fisiología , Hipoxia de la Célula/fisiología , Metabolismo Energético , Factores de Transcripción/metabolismo , Animales , Homeostasis , Humanos , Ratones , Modelos Biológicos , Neoplasias/metabolismo , Transducción de Señal , Factores de Transcripción/químicaRESUMEN
Hypoxia is an important developmental cue for multicellular organisms but it is also a contributing factor for several human pathologies, such as stroke, cardiovascular diseases and cancer. In cells, hypoxia activates a major transcriptional program coordinated by the Hypoxia Inducible Factor (HIF) family. HIF can activate more than one hundred targets but not all of them are activated at the same time, and there is considerable cell type variability. In this report we identified the paired-like homeodomain pituitary transcription factor (PITX1), as a transcription factor that helps promote specificity in HIF-1α dependent target gene activation. Mechanistically, PITX1 associates with HIF-1ß and it is important for the induction of certain HIF-1 dependent genes but not all. In particular, PITX1 controls the HIF-1α-dependent expression of the histone demethylases; JMJD2B, JMJD2A, JMJD2C and JMJD1B. Functionally, PITX1 is required for the survival and proliferation responses in hypoxia, as PITX1 depleted cells have higher levels of apoptotic markers and reduced proliferation. Overall, our study identified PITX1 as a key specificity factor in HIF-1α dependent responses, suggesting PITX1 as a protein to target in hypoxic cancers.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factores de Transcripción Paired Box/metabolismo , Translocador Nuclear del Receptor de Aril Hidrocarburo/química , Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Hipoxia de la Célula , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Células HEK293 , Células HeLa , Histona Demetilasas/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Factores de Transcripción Paired Box/antagonistas & inhibidores , Factores de Transcripción Paired Box/genética , Fotoblanqueo , Unión Proteica , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Transcripción GenéticaRESUMEN
The gene encoding protein kinase WNK2 was recently identified to be silenced by promoter hypermethylation in gliomas and meningiomas, suggesting a tumour-suppressor role in these brain tumours. Following experimental depletion in cell lines, WNK2 was further found to control GTP-loading of Rac1, a signalling guanosine triphosphatase involved in cell migration and motility. Here we show that WNK2 promoter methylation also occurs in 17.5% (29 out of 166) of adult gliomas, whereas it is infrequent in its paediatric forms (1.6%; 1 out of 66). Re-expression of WNK2 in glioblastoma cells presenting WNK2 gene silencing reduced cell proliferation in vitro, tumour growth in vivo and also cell migration and invasion, an effect correlated with reduced activation of Rac1. In contrast, when endogenous WNK2 was depleted from glioblastoma cells with unmethylated WNK2 promoter, changes in cell morphology, an increase in invasion and activation of Rac1 were observed. Together, these results validate the WNK2 gene as a recurrent target for epigenetic silencing in glia-derived brain tumours and provide first mechanistic evidence for a tumour-suppressing role of WNK2 that is related to Rac1 signalling and tumour cell invasion and proliferation.
Asunto(s)
Neoplasias Encefálicas/genética , Metilación de ADN , Glioblastoma/genética , Invasividad Neoplásica/genética , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/genética , Proteína de Unión al GTP rac1/fisiología , Adulto , Neoplasias Encefálicas/patología , División Celular , Línea Celular Tumoral , Silenciador del Gen , Glioblastoma/patología , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
Cystic fibrosis (CF), a major life-limiting genetic disease leading to severe respiratory symptoms, is caused by mutations in CF transmembrane conductance regulator (CFTR), a chloride (Cl(-)) channel expressed at the apical membrane of epithelial cells. Absence of functional CFTR from the surface of respiratory cells reduces mucociliary clearance, promoting airways obstruction, chronic infection, and ultimately lung failure. The most frequent mutation, F508del, causes the channel to misfold, triggering its premature degradation and preventing it from reaching the cell surface. Recently, novel small-molecule correctors rescuing plasma membrane localization of F508del-CFTR underwent clinical trials but with limited success. Plausibly, this may be due to the mutant intrinsic plasma membrane (PM) instability. Herein, we show that restoration of F508del-CFTR PM localization by correctors can be dramatically improved through a novel pathway involving stimulation of signaling by the endogenous small GTPase Rac1 via hepatocyte growth factor (HGF). We first show that CFTR anchors to apical actin cytoskeleton (via Ezrin) upon activation of Rac1 signaling through PIP5K and Arp2/3. We then found that such anchoring retains pharmacologically rescued F508del-CFTR at the cell surface, boosting functional restoration by correctors up to 30% of wild-type channel levels in human airway epithelial cells. Our findings reveal that surface anchoring and retention is a major target pathway for CF pharmacotherapy, namely, to achieve maximal restoration of F508del-CFTR in patients in combination with correctors. Moreover, this approach may also translate to other disorders caused by trafficking-deficient surface proteins.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Factor de Crecimiento de Hepatocito/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismo , Animales , Células Cultivadas , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Ratones , Modelos Biológicos , Estructura Molecular , Mutación , Proteína de Unión al GTP rac1/genéticaRESUMEN
Members of the WNK (with-no-lysine [K]) subfamily of protein kinases regulate various ion channels involved in sodium, potassium, and chloride homeostasis by either inducing their phosphorylation or regulating the number of channel proteins expressed at the cell surface. Here, we describe findings demonstrating that the cell surface expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is also regulated by WNK4 in mammalian cells. This effect of WNK4 is independent of the presence of kinase and involves interaction with and inhibition of spleen tyrosine kinase (Syk), which phosphorylates Tyr512 in the first nucleotide-binding domain 1 (NBD1) of CFTR. Transfection of catalytically active Syk into CFTR-expressing baby hamster kidney cells reduces the cell surface expression of CFTR, whereas that of WNK4 promotes it. This is shown by biotinylation of cell surface proteins, immunofluorescence microscopy, and functional efflux assays. Mutation of Tyr512 to either glutamic acid or phenylalanine is sufficient to alter CFTR surface levels. In human airway epithelial cells, downregulation of endogenous Syk and WNK4 confirms their roles as physiologic regulators of CFTR surface expression. Together, our results show that Tyr512 phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and that WNK4 and Syk perform an antagonistic role in this process.
Asunto(s)
Membrana Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Cricetinae , Cricetulus , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Quinasa Syk , Tirosina/metabolismoRESUMEN
One mechanism by which mammalian cells regulate the uptake of glucose is the number of glucose transporter proteins (GLUT) present at the plasma membrane. In insulin-responsive cells types, GLUT4 is released from intracellular stores through inactivation of the Rab GTPase activating protein Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4) (also known as AS160). Here we describe that TBC1D4 forms a protein complex with protein kinase WNK1 in human embryonic kidney (HEK293) cells. We show that WNK1 phosphorylates TBC1D4 in vitro and that the expression levels of WNK1 in these cells regulate surface expression of the constitutive glucose transporter GLUT1. WNK1 was found to increase the binding of TBC1D4 to regulatory 14-3-3 proteins while reducing its interaction with the exocytic small GTPase Rab8A. These effects were dependent on the catalytic activity because expression of a kinase-dead WNK1 mutant had no effect on binding of 14-3-3 and Rab8A, or on surface GLUT1 levels. Together, the data describe a pathway regulating constitutive glucose uptake via GLUT1, the expression level of which is related to several human diseases.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Regulación de la Expresión Génica , Transportador de Glucosa de Tipo 1/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas 14-3-3/metabolismo , Transporte Biológico , Exocitosis , Silenciador del Gen , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Antígenos de Histocompatibilidad Menor , Oligonucleótidos/química , ARN Interferente Pequeño/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
The subfamily of WNK protein kinases is composed of four human genes and is characterised by a typical sequence variation within the conserved catalytic domain. Although most research has focussed on the role of WNK1, WNK3 and WNK4 in regulating different ion transporters in both the kidney and extrarenal tissues, there is growing evidence for additional roles of WNK kinases in various signalling cascades related to cancer. Here, we review the connection between WNK kinases and tumorigenesis and describe existing experimental evidence as well as potential new links to major aspects of tumour biology. In particular, we discuss their role in G1/S cell cycle progression, metabolic tumour cell adaptation, evasion of apoptosis and metastasis.
Asunto(s)
Neoplasias/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
WNK protein kinases form a kinase subfamily expressed in multi-cellular organisms and the human genome encodes four distinct WNK genes. Human WNK2 has been recently identified as a cell growth regulator that modulates activation of the ERK1/2 protein kinase and is epigenetically silenced in gliomas. Here we provide mechanistic insight into how WNK2 affects ERK activation. We found that WNK2 depletion decreased RhoA activation and promoted GTP-loading of Rac1, leading to stimulation of the Rac1-effector PAK1, which is the kinase responsible for subsequent phosphorylation of MEK1 at serine 298, thereby increasing MEK affinity towards ERK1/2. We propose that WNK2 controls a RhoA-mediated cross-talk mechanism that regulates the efficiency with which MEK1 can activate ERK1/2 upon growth factor stimulation.
Asunto(s)
MAP Quinasa Quinasa 1/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Línea Celular , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Silenciador del Gen , Humanos , Fosfoserina/metabolismo , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Recombinantes/metabolismo , Quinasas p21 Activadas/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Recently, a translocation t(2;3)(q13;p25), leading to the formation of a chimeric PAX8-peroxisome proliferator-activated receptor (PPAR)gamma 1 oncogene, was detected in follicular thyroid carcinomas (FTC), but not in follicular thyroid adenomas (FTA), papillary thyroid carcinomas (PTC), or multinodular hyperplasias. However, previous cytogenetic studies have identified the t(2;3)(q13;p25) translocation also in some cases of FTA. In this study, we have combined RT-PCR with primers in exons 4-8 of PAX8 and in exon 1 of PPAR gamma 1 with PPAR gamma immunohistochemistry to study PAX8-PPAR gamma 1 oncogene activation in FTC (n = 9), FTA (n = 16), PTC (n = 9), anaplastic thyroid carcinomas (n = 4), and multinodular hyperplasias (n = 2). PAX8-PPAR gamma 1 rearrangements were detected by RT-PCR in 5 of 9 (56%) FTC and in 2 of 16 (13%) FTA. By contrast, all cases of PTC, anaplastic thyroid carcinomas, and multinodular hyperplasia were RT-PCR-negative. Diffuse nuclear immunoreactivity for PPAR gamma was observed in 7 of 9 (78%) FTC, 5 of 16 FTA (31%), and 1 of 9 PTC (11%). Positivity was focal in 3 cases (1 FTC, 1 PTC, and 1 multinodular hyperplasia). Diffuse nuclear staining for PPAR gamma was present in RT-PCR- negative cases of FTC (n = 3), FTA (n = 3), and PTC (n = 1), suggesting that a different PAX8-PPAR gamma 1 breakpoint, a rearrangement between PPAR gamma 1 and a non-PAX8 partner, or overexpression of the native protein might be present. Our findings that PAX8-PPAR gamma 1 rearrangements are present in both follicular carcinomas and adenomas suggest that this oncogene is not a reliable marker to differentiate between FTC and FTA in fine-needle aspiration biopsies of follicular neoplasms of the thyroid.
Asunto(s)
Adenoma/genética , Proteínas de Unión al ADN/genética , Reordenamiento Génico , Proteínas Nucleares , Receptores Citoplasmáticos y Nucleares/genética , Neoplasias de la Tiroides/genética , Transactivadores/genética , Factores de Transcripción/genética , Adenoma/química , Biomarcadores de Tumor/genética , Carcinoma/química , Carcinoma/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Cariotipificación , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box , Receptores Citoplasmáticos y Nucleares/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Neoplasias de la Tiroides/química , Factores de Transcripción/análisis , Translocación GenéticaRESUMEN
OBJECTIVE: X-chromosome inactivation analysis was performed in order to assess the clonal origin of non-medullary thyroid tumours and to distinguish between multicentricity and multifocality in multiple papillary thyroid carcinoma (PTC). METHODS: One hundred and thirteen tumour samples from 31 patients with isolated PTC, 16 patients with multinodular PTC, 14 patients with follicular thyroid adenoma (FTA) and 15 patients with follicular thyroid carcinoma (FTC) were collected. The corresponding normal thyroid tissues were analysed, and in 14 cases, tumour-surrounding tissue was also studied. Genomic DNA was digested with HpaII and HhaI previous to PCR amplification of the polymorphic CAG repeat, on exon 1 of the human androgen receptor gene (HUMARA). PCR products were analysed by denaturing gel electrophoresis, silver staining and densitometric analysis. PCR products were also used to determine the number of CAG repeats of patients with isolated PTC, FTA, FTC and of 41 healthy volunteers. RESULTS: Heterozygosity for the HUMARA polymorphism was found in 64/76 (84%) cases. Lyonization of the thyroid was observed in 15/76 (20%) cases, which were excluded from clonal analysis. Except for two cases of isolated PTC, all tumour samples studied presented monoclonal X-inactivation patterns, while normal thyroid tissue was polyclonal. Monoclonal patterns were also found in 4/14 tumour-surrounding tissues. No difference was found in the length of CAG alleles between patients and controls. Of eight informative cases of multinodular PTC, three showed evidence of multicentricity and five revealed patterns consistent with multifocality. CONCLUSIONS: Both isolated and multinodular PTC as well as FTA and FTC are of monoclonal origin. Our results also suggest that approximately one-third of multiple PTC have an independent origin for the different nodules (multicentricity). Monoclonality was also found in tissues surrounding some PTC nodules. No association was found between the length of CAG alleles and thyroid malignancies.
Asunto(s)
Carcinoma Papilar/genética , ADN de Neoplasias/genética , Compensación de Dosificación (Genética) , Neoplasias de la Tiroides/genética , Alelos , Carcinoma Papilar/patología , ADN de Neoplasias/aislamiento & purificación , Densitometría , Electroforesis en Gel de Poliacrilamida , Exones/genética , Femenino , Humanos , Células Madre Neoplásicas/patología , Adhesión en Parafina , Receptores Androgénicos/genética , Secuencias Repetitivas de Ácidos Nucleicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Tiroides/patologíaRESUMEN
Os autores acompanharam uma gravida com estenose mitral severa com insuficiencia cardiaca na classe funcional Classe IV ("New York Heart Association"), que nao respondeu ao tratamento clinico. Ela foi submetida a uma valvuloplastia mitral com 16 semanas de gestacao, com melhora significativa de seu estado geral. Os autores concluem que o procedimento e simples e seguro, o que pode ser atestado pelos resultados positivos na literatura nacional e internacional