RESUMEN
Despite the fact that AML is the most common acute leukemia in adults, patient outcomes are poor necessitating the development of novel therapies. We identified that inhibition of Thioredoxin Reductase (TrxR) is a promising strategy for AML and report a highly potent and specific inhibitor of TrxR, S-250. Both pharmacologic and genetic inhibition of TrxR impairs the growth of human AML in mouse models. We found that TrxR inhibition leads to a rapid and marked impairment of metabolism in leukemic cells subsequently leading to cell death. TrxR was found to be a major and direct regulator of metabolism in AML cells through impacts on both glycolysis and the TCA cycle. Studies revealed that TrxR directly regulates GAPDH leading to a disruption of glycolysis and an increase in flux through the pentose phosphate pathway (PPP). The combined inhibition of TrxR and the PPP led to enhanced leukemia growth inhibition. Overall, TrxR abrogation, particularly with S-250, was identified as a promising strategy to disrupt AML metabolism.
Asunto(s)
Vía de Pentosa Fosfato , Reductasa de Tiorredoxina-Disulfuro , Muerte Celular , Ciclo del Ácido Cítrico , Glucólisis , HumanosRESUMEN
BACKGROUND: Ovarian cancer is the leading cause of gynecologic cancer death in the United States despite effective first-line systemic chemotherapy. Cancer stem cells (CSCs) retain the ability to self-renew and proliferate and may be a means of harboring disease that evades standard treatment strategies. We previously performed a high-throughput screen to assess differential protein expression in ovarian CSCs compared to non-CSCs and observed that Thy-1 was more highly expressed in CSCs. Our primary aim was to validate Thy-1 (CD90) as a cancer stem cell (CSC) marker in epithelial ovarian cancer (EOC), correlate with clinical outcomes, and assess as a potential therapeutic target. RESULTS: Kaplan Meier (KM) Plotter data were correlated with survival outcomes. Quantitative real-time PCR, flow cytometry, and immunoblots assessed RNA and protein expression. Limiting dilution assays assessed self-renewal capacity and proliferation assays assessed proliferative capacity. RNA in-situ hybridization was performed on patient specimens to assess feasibility. Thy-1 (CD90) is more highly expressed in ovarian CSCs than non-CSCs, in EOC compared to benign ovarian epithelium (P < 0.001), and is highest in serous EOC (P < 0.05). Serous ovarian cancers with high Thy-1 expression have poorer outcomes (median PFS 15.8 vs. 18.3 months, P = 0 < 0.001; median OS 40.1 v. 45.8 months, P = 0.036). Endometrioid ovarian cancers with high Thy-1 have poorer PFS, but no difference in OS (upper quartile PFS 34 v. 11 months, P = 0.013; quartile OS not reached, P = 0.69). In vitro, Thy-1 expression is higher in CSCs versus non-CSCs. EOC cells with high Thy-1 expression demonstrate increased proliferation and self-renewal. Thy-1 knockdown in EOC cells decreases proliferative capacity and self-renewal capacity, and knockdown is associated with decreased expression of stem cell transcription factors NANOG and SOX2. RNA in situ hybridization is feasible in ovarian cancer tissue specimens. CONCLUSIONS: Thy-1 is a marker of ovarian CSCs. Increased expression of Thy-1 in EOC predicts poor prognosis and is associated with increased proliferative and self-renewal capacity. Thy-1 knockdown decreases proliferative and self-renewal capacity, and represents a potential therapeutic target.
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Biomarcadores de Tumor/metabolismo , Células Madre Neoplásicas/metabolismo , Neoplasias Ováricas/metabolismo , Antígenos Thy-1/metabolismo , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Autorrenovación de las Células , Quimioradioterapia Adyuvante , Femenino , Humanos , Estimación de Kaplan-Meier , Persona de Mediana Edad , Células Madre Neoplásicas/fisiología , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/terapia , Pronóstico , Antígenos Thy-1/genética , Resultado del TratamientoRESUMEN
The transmembrane protein, STRA6, functions as a vitamin A transporter and a cytokine receptor when activated by vitamin A-bound serum retinol binding protein 4 (RBP4). STRA6 activation transduces a JAK2-STAT3 signaling cascade and promotes tumorigenesis in a xenograft mouse model of colon cancer. We show here that RBP4 and STRA6 expression is associated with poor oncologic prognosis. Downregulating STRA6 or RBP4 in colon cancer cells decreased the fraction of cancer stem cells and their sphere and tumor initiation frequency. Furthermore, we show that high-fat diet (HFD) increases LGR5 expression and promotes tumor growth in a xenograft model independent of obesity. HFD increased STRA6 levels, and downregulation of STRA6 delays and impairs tumor initiation, tumor growth, and expression of stemness markers. Together, these data demonstrate a key role of STRA6 and RBP4 in the maintenance of colon cancer self-renewal and that this pathway is an important link through which consumption of HFD contributes to colon carcinogenesis.
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Transformación Celular Neoplásica/metabolismo , Neoplasias del Colon/etiología , Neoplasias del Colon/metabolismo , Proteínas de la Membrana/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas Plasmáticas de Unión al Retinol/metabolismo , Transducción de Señal , Animales , Línea Celular Tumoral , Autorrenovación de las Células/genética , Neoplasias del Colon/mortalidad , Neoplasias del Colon/patología , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Expresión Génica , Xenoinjertos , Humanos , Estimación de Kaplan-Meier , Masculino , Proteínas de la Membrana/genética , Ratones , Células Madre Neoplásicas/patología , Pronóstico , Proteínas Plasmáticas de Unión al Retinol/genéticaRESUMEN
Insulin stimulates glucose uptake through the translocation of the glucose transporter GLUT4 to the plasma membrane. The exocyst complex tethers GLUT4-containing vesicles to the plasma membrane, a process that requires the binding of the G protein (heterotrimeric guanine nucleotide-binding protein) RalA to the exocyst complex. We report that upon activation of RalA, the protein kinase TBK1 phosphorylated the exocyst subunit Exo84. Knockdown of TBK1 blocked insulin-stimulated glucose uptake and GLUT4 translocation; knockout of TBK1 in adipocytes blocked insulin-stimulated glucose uptake; and ectopic overexpression of a kinase-inactive mutant of TBK1 reduced insulin-stimulated glucose uptake in 3T3-L1 adipocytes. The phosphorylation of Exo84 by TBK1 reduced its affinity for RalA and enabled its release from the exocyst. Overexpression of a kinase-inactive mutant of TBK1 blocked the dissociation of the TBK1/RalA/exocyst complex, and treatment of 3T3-L1 adipocytes with specific inhibitors of TBK1 reduced the rate of complex dissociation. Introduction of phosphorylation-mimicking or nonphosphorylatable mutant forms of Exo84 blocked insulin-stimulated GLUT4 translocation. Thus, these data indicate that TBK1 controls GLUT4 vesicle engagement and disengagement from the exocyst, suggesting that exocyst components not only constitute a tethering complex for the GLUT4 vesicle but also act as "gatekeepers" controlling vesicle fusion at the plasma membrane.
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Adipocitos/efectos de los fármacos , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Células 3T3-L1 , Adipocitos/metabolismo , Animales , Transportador de Glucosa de Tipo 4/genética , Hipoglucemiantes/farmacología , Immunoblotting , Ratones , Mutación , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , Proteínas de Transporte Vesicular/genética , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismoRESUMEN
The ubiquitous Rho (Ras homology) GTPase Cdc42p can function in different settings to regulate cell polarity and cellular signaling. How Cdc42p and other proteins are directed to function in a particular context remains unclear. We show that the Cdc42p-interacting protein Bem4p regulates the mitogen-activated protein kinase (MAPK) pathway that controls filamentous growth in Saccharomyces cerevisiae. Bem4p controlled the filamentous-growth pathway but not other MAPK pathways (mating or high-osmolarity glycerol response [HOG]) that also require Cdc42p and other shared components. Bem4p associated with the plasma membrane (PM) protein, Sho1p, to regulate MAPK activity and cell polarization under nutrient-limiting conditions that favor filamentous growth. Bem4p also interacted with the major activator of Cdc42p, the guanine nucleotide exchange factor (GEF) Cdc24p, which we show also regulates the filamentous-growth pathway. Bem4p interacted with the pleckstrin homology (PH) domain of Cdc24p, which functions in an autoinhibitory capacity, and was required, along with other pathway regulators, to maintain Cdc24p at polarized sites during filamentous growth. Bem4p also interacted with the MAPK kinase kinase (MAPKKK) Ste11p. Thus, Bem4p is a new regulator of the filamentous-growth MAPK pathway and binds to general proteins, like Cdc42p and Ste11p, to promote a pathway-specific response.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Animales , Polaridad Celular/genéticaRESUMEN
Insulin-stimulated glucose uptake in fat and muscle is mediated by the major facilitative glucose transporter Glut4. Insulin controls the trafficking of Glut4 to the plasma membrane via regulation of a series of small G proteins, including RalA and Rab10. We demonstrate here that Rab10 is a bona fide target of the GTPase-activating protein AS160, which is inhibited after phosphorylation by the protein kinase Akt. Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2. Rab10 and RalA reside in the same pool of Glut4-storage vesicles in untreated cells, and, together with Rlf, they ensure maximal glucose transport. Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10. Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake.
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Transportador de Glucosa de Tipo 4/metabolismo , Insulina/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP ral/metabolismo , Células 3T3 , Adipocitos/metabolismo , Animales , Transporte Biológico , Células COS , Línea Celular , Chlorocebus aethiops , Activación Enzimática , Proteínas Activadoras de GTPasa/antagonistas & inhibidores , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Glucosa/metabolismo , Factores de Intercambio de Guanina Nucleótido , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Ratones , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Proteínas de Unión al GTP rab/agonistasRESUMEN
In the yeast Saccharomyces cerevisiae, the establishment and maintenance of silent chromatin at the telomere requires a delicate balance between opposing activities of histone modifying enzymes. Previously, we demonstrated that the protein arginine methyltransferase Hmt1 plays a role in the formation of yeast silent chromatin. To better understand the nature of the Hmt1 interactions that contribute to this phenomenon, we carried out a systematic reverse genetic screen using a null allele of HMT1 and the synthetic genetic array (SGA) methodology. This screen revealed interactions between HMT1 and genes encoding components of the histone deacetylase complex Rpd3L (large). A double mutant carrying both RPD3 and HMT1 deletions display increased telomeric silencing and Sir2 occupancy at the telomeric boundary regions, when comparing to a single mutant carrying Hmt1-deletion only. However, the dual rpd3/hmt1-null mutant behaves like the rpd3-null single mutant with respect to silencing behavior, indicating that RPD3 is epistatic to HMT1. Mutants lacking either Hmt1 or its catalytic activity display an increase in the recruitment of histone deacetylase Rpd3 to the telomeric boundary regions. Moreover, in such loss-of-function mutants the levels of acetylated H4K5, which is a substrate of Rpd3, are altered at the telomeric boundary regions. In contrast, the level of acetylated H4K16, a target of the histone deacetylase Sir2, was increased in these regions. Interestingly, mutants lacking either Rpd3 or Sir2 display various levels of reduction in dimethylated H4R3 at these telomeric boundary regions. Together, these data provide insight into the mechanism whereby Hmt1 promotes the proper establishment and maintenance of silent chromatin at the telomeres.
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Histona Desacetilasas/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Telómero/metabolismo , Acetilación , Inmunoprecipitación de Cromatina , Epistasis Genética , Silenciador del Gen , Genes Sintéticos/genética , Pruebas Genéticas , Genoma Fúngico/genética , Histona Desacetilasas/genética , Histonas/metabolismo , Metilación , Mutación/genética , Unión Proteica , Subunidades de Proteína/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Represoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2/metabolismoRESUMEN
In the budding yeast S. cerevisiae, nutrient limitation induces a MAPK pathway that regulates filamentous growth and biofilm/mat formation. How nutrient levels feed into the regulation of the filamentous growth pathway is not entirely clear. We characterized a newly identified MAPK regulatory protein of the filamentous growth pathway, Opy2. A two-hybrid screen with the cytosolic domain of Opy2 uncovered new interacting partners including a transcriptional repressor that functions in the AMPK pathway, Mig1, and its close functional homolog, Mig2. Mig1 and Mig2 coregulated the filamentous growth pathway in response to glucose limitation, as did the AMP kinase Snf1. In addition to associating with Opy2, Mig1 and Mig2 interacted with other regulators of the filamentous growth pathway including the cytosolic domain of the signaling mucin Msb2, the MAP kinase kinase Ste7, and the MAP kinase Kss1. As for Opy2, Mig1 overproduction dampened the pheromone response pathway, which implicates Mig1 and Opy2 as potential regulators of pathway specificity. Taken together, our findings provide the first regulatory link in yeast between components of the AMPK pathway and a MAPK pathway that controls cellular differentiation.
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Glucosa/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Fungal species exhibit diverse behaviors when presented with extracellular challenges. Pathogenic fungi can undergo cell differentiation and biofilm formation in response to fluctuating nutrient levels, and these responses are required for virulence. In the model fungal eukaryote Saccharomyces cerevisiae, nutrient limitation induces filamentous growth and biofilm/mat formation. Both responses require the same signal transduction (MAPK) pathway and the same cell adhesion molecule (Flo11) but have been studied under different conditions. We found that filamentous growth and mat formation are aspects of a related response that is regulated by the MAPK pathway. Cells in yeast-form mats differentiated into pseudohyphae in response to nutrient limitation. The MAPK pathway regulated mat expansion (in the plane of the XY-axis) and substrate invasion (downward in the plane of the Z-axis), which optimized the mat's response to extracellular nutrient levels. The MAPK pathway also regulated an upward growth pattern (in the plane of the Z-axis) in response to nutrient limitation and changes in surface rigidity. Upward growth allowed for another level of mat responsiveness and resembled a type of colonial chemorepulsion. Together our results show that signaling pathways play critical roles in regulating social behaviors in which fungal cells participate. Signaling pathways may regulate similar processes in pathogens, whose highly nuanced responses are required for virulence.
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Biopelículas , Sistema de Señalización de MAP Quinasas , Percepción de Quorum/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Adaptación Biológica , Adhesión Celular , Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/genética , Transducción de SeñalRESUMEN
Cell adhesion is a key feature in the regulation of many biological processes. In the budding yeast Saccharomyces cerevisiae, Flo11p is the major adhesion molecule that controls filamentous growth [1-3] and the expansion of interconnected cells in mats or biofilms [4]. We show here that Flo11p is shed from cells. Flo11p shedding attenuated adherence and contributed to the overall balance in adherence properties that was optimal for filamentous growth and mat formation. Shed Flo11p comprised an essential component of a fluid layer surrounding yeast mats that may be functionally analogous to the mucus secretions of higher eukaryotes. Genome-wide secretion profiling of Flo11p identified new regulatory proteins, including the furin protease Kex2p, which was required for cleavage and maturation of the Flo11p protein. Secreted mucin-like proteins may play unexpected roles in the adherence properties and virulence of microbial pathogens.
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Biopelículas/crecimiento & desarrollo , Glicoproteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Adhesión Celular , Procesamiento Proteico-PostraduccionalRESUMEN
The large amount of available genome sequencing data presents a huge challenge in the form of orphan sequences. This study reports the detailed functional characterization of one such orphan sequence in Schizosaccharomyces pombe. We identified this gene as a prominently upregulated 1.4 kb transcript in a screen for Cigarette smoke extract responsive genes in S. pombe and named it Stress Responsive Orphan 1 (Sro1). We report various functions of Sro1 in regulation of cellular behaviour under stress conditions. We show that this gene (Sro1) responds to a variety of stress conditions and that the expression of the gene is regulated mainly through the stress activated protein kinase (SAPK) Sty1 and its downstream transcription factor Atf1. Deletion of Sro1 also significantly alters the reactive oxygen species (ROS) generation profiles and the cell-cycle progression of S. pombe during stress conditions. The stress-specific alteration of the ROS generation profiles and checkpoint activation resulting from deletion of the gene suggest that Sro1 might be a key player in determining cellular responses/fate under stress conditions.
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Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/fisiología , Factor de Transcripción Activador 1/metabolismo , Antifúngicos/farmacología , Ciclo Celular/fisiología , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Peróxido de Hidrógeno/farmacología , Hidroxiurea/farmacología , Viabilidad Microbiana , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfoproteínas/metabolismo , Cloruro de Potasio/farmacología , Especies Reactivas de Oxígeno/metabolismo , Schizosaccharomyces/efectos de los fármacos , Proteínas de Schizosaccharomyces pombe/metabolismo , Humo , NicotianaRESUMEN
Translation initiation from the ribosomal P-site is the specialty of the initiator tRNAs (tRNA(fMet)). Presence of the three consecutive G-C base pairs (G29-C41, G30-C40 and G31-C39) in their anticodon stems, a highly conserved feature of the initiator tRNAs across the three kingdoms of life, has been implicated in their preferential binding to the P-site. How this feature is exploited by ribosomes has remained unclear. Using a genetic screen, we have isolated an Escherichia coli strain, carrying a G122D mutation in folD, which allows initiation with the tRNA(fMet) containing mutations in one, two or all the three G-C base pairs. The strain shows a severe deficiency of methionine and S-adenosylmethionine, and lacks nucleoside methylations in rRNA. Targeted mutations in the methyltransferase genes have revealed a connection between the rRNA modifications and the fundamental process of the initiator tRNA selection by the ribosome.