RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) develops through step-wise genetic and molecular alterations including Kras mutation and inactivation of various apoptotic pathways. Here, we find that development of apoptotic resistance and metastasis of KrasG12D-driven PDAC in mice is accelerated by deleting Plk3, explaining the often-reduced Plk3 expression in human PDAC. Importantly, a 41-kDa Plk3 (p41Plk3) that contains the entire kinase domain at the N-terminus (1-353 aa) is activated by scission of the precursor p72Plk3 at Arg354 by metalloendopeptidase nardilysin (NRDC), and the resulting p32Plk3 C-terminal Polo-box domain (PBD) is removed by proteasome degradation, preventing the inhibition of p41Plk3 by PBD. We find that p41Plk3 is the activated form of Plk3 that regulates a feed-forward mechanism to promote apoptosis and suppress PDAC and metastasis. p41Plk3 phosphorylates c-Fos on Thr164, which in turn induces expression of Plk3 and pro-apoptotic genes. These findings uncover an NRDC-regulated post-translational mechanism that activates Plk3, establishing a prototypic regulation by scission mechanism.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Ratones , Animales , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/patología , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismoRESUMEN
The term M-phase supershift denotes the phosphorylation-dependent substantial increase in the apparent molecular weight of numerous proteins of varied biological functions during M-phase induction. Although the M-phase supershift of multiple key mitotic regulators has been attributed to the multisite phosphorylation catalyzed by the Cdk1/cyclin B/Cks complex, this view is challenged by multiple lines of paradoxical observations. To solve this problem, we reconstituted the M-phase supershift of Xenopus Cdc25C, Myt1, Wee1A, APC3, and Greatwall in Xenopus egg extracts and characterized the supershift-producing phosphorylations. Our results demonstrate that their M-phase supershifts are each due to simultaneous phosphorylation of a considerable portion of S/T/Y residues in a long intrinsically disordered region that is enriched in both S/T residues and S/TP motifs. Although the major mitotic kinases in Xenopus egg extracts, Cdk1, MAPK, Plx1, and RSK2, are able to phosphorylate the five mitotic regulators, they are neither sufficient nor required to produce the M-phase supershift. Accordingly, inhibition of the four major mitotic kinase activities in Xenopus oocytes did not inhibit the M-phase supershift in okadaic acid-induced oocyte maturation. These findings indicate that the M-phase supershift is produced by a previously unrecognized category of mitotic phosphorylation that likely plays important roles in M-phase induction.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Xenopus , Animales , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina B/metabolismo , Mitosis , Ácido Ocadaico/metabolismo , Oocitos/metabolismo , Fosforilación , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMEN
The functional role of long noncoding RNAs (lncRNAs) in inherited metabolic disorders, including phenylketonuria (PKU), is unknown. Here, we demonstrate that the mouse lncRNA Pair and human HULC associate with phenylalanine hydroxylase (PAH). Pair-knockout mice exhibited excessive blood phenylalanine (Phe), musty odor, hypopigmentation, growth retardation, and progressive neurological symptoms including seizures, which faithfully models human PKU. HULC depletion led to reduced PAH enzymatic activities in human induced pluripotent stem cell-differentiated hepatocytes. Mechanistically, HULC modulated the enzymatic activities of PAH by facilitating PAH-substrate and PAH-cofactor interactions. To develop a therapeutic strategy for restoring liver lncRNAs, we designed GalNAc-tagged lncRNA mimics that exhibit liver enrichment. Treatment with GalNAc-HULC mimics reduced excessive Phe in Pair -/- and Pah R408W/R408W mice and improved the Phe tolerance of these mice.
Asunto(s)
Fenilalanina Hidroxilasa/metabolismo , Fenilalanina/metabolismo , Fenilcetonurias/genética , ARN Largo no Codificante/genética , Acetilgalactosamina , Animales , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Biopterinas/uso terapéutico , Dieta , Modelos Animales de Enfermedad , Femenino , Hepatocitos/metabolismo , Humanos , Hígado/embriología , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Conformación de Ácido Nucleico , Fenilalanina/administración & dosificación , Fenilalanina Hidroxilasa/deficiencia , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/tratamiento farmacológico , Fenilcetonurias/metabolismo , Unión Proteica , ARN Largo no Codificante/química , ARN Largo no Codificante/metabolismo , ARN Largo no Codificante/uso terapéuticoRESUMEN
BACKGROUND: Exercise training is well established as the most effective way to enhance muscle performance and muscle building. The composition of skeletal muscle fiber type affects systemic energy expenditures, and perturbations in metabolic homeostasis contribute to the onset of obesity and other metabolic dysfunctions. Long noncoding RNAs (lncRNAs) have been demonstrated to play critical roles in diverse cellular processes and diseases, including human cancers; however, the functional importance of lncRNAs in muscle performance, energy balance, and obesity remains elusive. We previously reported that the lncRNA H19 regulates the poly-ubiquitination and protein stability of dystrophin (DMD) in muscular dystrophy. METHODS: Here, we identified mouse/human H19-interacting proteins using mouse/human skeletal muscle tissues and liquid chromatography-mass spectrometry (LC-MS). Human induced pluripotent stem-derived skeletal muscle cells (iPSC-SkMC) from a healthy donor and Becker Muscular Dystrophy (BMD) patients were utilized to study DMD post-translational modifications and associated proteins. We identified a gain-of-function (GOF) mutant of H19 and characterized the effects on myoblast differentiation and fusion to myotubes using iPSCs. We then conjugated H19 RNA gain-of-function oligonucleotides (Rgof) with the skeletal muscle enrichment peptide agrin (referred to as AGR-H19-Rgof) and evaluated AGR-H19-Rgof's effects on skeletal muscle performance using wild-type (WT) C57BL/6 J mice and its anti-obesity effects using high-fat diet (HFD)- and leptin deficiency-induced obese mouse models. RESULTS: We demonstrated that both human and mouse H19 associated with DMD and that the H19 GOF exhibited enhanced interaction with DMD compared to WT H19. DMD was found to associate with serine/threonine-protein kinase MRCK alpha (MRCKα) and α-synuclein (SNCA) in iPSC-SkMC derived from BMD patients. Inhibition of MRCKα and SNCA-mediated phosphorylation of DMD antagonized the interaction between H19 and DMD. These signaling events led to improved skeletal muscle cell differentiation and myotube fusion. The administration of AGR-H19-Rgof improved the muscle mass, muscle performance, and base metabolic rate of WT mice. Furthermore, mice treated with AGR-H19-Rgof exhibited resistance to HFD- or leptin deficiency-induced obesity. CONCLUSIONS: Our study suggested the functional importance of the H19 GOF mutant in enhancing muscle performance and anti-obesity effects.
Asunto(s)
Diferenciación Celular/genética , Mutación con Ganancia de Función , Desarrollo de Músculos/genética , Músculo Esquelético/metabolismo , Obesidad/terapia , ARN Largo no Codificante/genética , Animales , Biomarcadores , Proteínas Portadoras , Células Cultivadas , Manejo de la Enfermedad , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Distrofina/genética , Distrofina/metabolismo , Técnica del Anticuerpo Fluorescente/métodos , Terapia Genética , Humanos , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Noqueados , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Obesidad/diagnóstico , Obesidad/etiología , Obesidad/metabolismo , Fosforilación , Unión ProteicaRESUMEN
Dystrophin proteomic regulation in muscular dystrophies (MDs) remains unclear. We report that a long noncoding RNA (lncRNA), H19, associates with dystrophin and inhibits E3-ligase-dependent polyubiquitination at Lys 3584 (referred to as Ub-DMD) and its subsequent protein degradation. In-frame deletions in BMD and a DMD non-silent mutation (C3340Y) resulted in defects in the ability of the protein to interact with H19, which caused elevated Ub-DMD levels and dystrophin degradation. Dmd C3333Y mice exhibited progressive MD, elevated serum creatine kinase, heart dilation, blood vessel irregularity and respiratory failure with concurrently reduced dystrophin and increased Ub-DMD status. H19 RNA oligonucleotides conjugated with agrin (AGR-H19) and nifenazone competed with or inhibited TRIM63. Dmd C3333Y animals, induced-pluripotent-stem-cell-derived skeletal muscle cells from patients with Becker MD and mdx mice subjected to exon skipping exhibited inhibited dystrophin degradation, preserved skeletal and cardiac muscle histology, and improved strength and heart function following AGR-H19 or nifenazone treatment. Our study paves the way for meaningful targeted therapeutics for Becker MD and for certain patients with Duchenne MD.
Asunto(s)
Músculo Esquelético/metabolismo , Distrofias Musculares/prevención & control , Oligonucleótidos/administración & dosificación , ARN Largo no Codificante/metabolismo , Animales , Antipirina/administración & dosificación , Antipirina/análogos & derivados , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Cardiomiopatías/prevención & control , Línea Celular , Modelos Animales de Enfermedad , Distrofina/genética , Distrofina/metabolismo , Inhibidores Enzimáticos/administración & dosificación , Femenino , Semivida , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Ratones Mutantes , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/metabolismo , Fuerza Muscular , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Mutación , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Niacinamida/administración & dosificación , Niacinamida/análogos & derivados , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Estabilidad Proteica , Proteolisis , ARN Largo no Codificante/genética , Proteínas de Motivos Tripartitos/antagonistas & inhibidores , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
A recent in vitro characterization of a recombinant pyruvate kinase (PK) from Aedes aegypti mosquitoes demonstrated that the enzyme is uniquely regulated by multiple allosteric effectors. Here, we further explored PK gene and protein expression, and enzymatic activity in key metabolic tissues of mosquitoes maintained under different nutritional conditions. We also studied the metabolic effects of PK depletion using several techniques including RNA interference and mass spectrometry-based stable-isotope tracing. Transcriptional analysis showed a dynamic post-feeding PK mRNA expression pattern within and across mosquito tissues, whereas corresponding protein levels remained stable throughout the time course analyzed. Nevertheless, PK activity significantly differed in the fat body of sucrose-, blood-fed, and starved mosquitoes. Genetic silencing of PK did not alter survival in blood-fed females maintained on sucrose. However, an enhanced survivorship was observed in PK-deficient females maintained under different nutritional regimens. Our results indicate that mosquitoes overcame PK deficiency by up-regulating the expression of genes encoding NADP-malic enzyme-1, phosphoenolpyruvate carboxykinase-1, phosphoglycerate dehydrogenase and glutamate dehydrogenase, and by decreasing glucose oxidation and metabolic pathways associated with ammonia detoxification. Taken together, our data demonstrate that PK confers to A. aegypti a metabolic plasticity to tightly regulate both carbon and nitrogen metabolism.
Asunto(s)
Aedes/genética , Isótopos de Carbono/análisis , Expresión Génica , Proteínas de Insectos/genética , Piruvato Quinasa/genética , Aedes/enzimología , Aedes/metabolismo , Animales , Proteínas de Insectos/deficiencia , Proteínas de Insectos/metabolismo , Espectrometría de Masas , Piruvato Quinasa/deficiencia , Piruvato Quinasa/metabolismo , Interferencia de ARNRESUMEN
Disseminated tumor cells (DTCs) undergo a dormant state in the distant metastatic site(s) before becoming overt metastatic diseases. In prostate cancer (PCa), bone metastasis can occur years after prostatectomy, suggesting that bone may provide dormancy-inducing factors. To search for these factors, we prepared conditioned media (CM) from calvariae. Using live-cell imaging, we found that Calvarial-CM treatment increased cellular quiescence in C4-2B4 PCa cells. Mass spectrometry analysis of Calvarial-CM identified 132 secreted factors. Western blot and ELISA analyses confirmed the presence of several factors, including DKK3, BMP1, neogenin and vasorin in the Calvarial-CM. qRT-PCR analysis of total calvariae versus isolated osteoblasts showed that DKK3, BMP1, vasorin and neogenin are mainly expressed by osteoblasts, while MIA, LECT1, NGAL and PEDF are expressed by other calvarial cells. Recombinant human DKK3, BMP1, vasorin, neogenin, MIA and NGAL treatment increased cellular quiescence in both C4-2b and C4-2B4 PCa cells. Mechanistically, DKK3, vasorin and neogenin, but not BMP1, increased dormancy through activating the p38MAPK signaling pathway. Consistently, DKK3, vasorin and neogenin failed to induce dormancy in cells expressing dominant-negative p38αMAPK while BMP1 remained active, suggesting that BMP1 uses an alternative dormancy signaling pathway. Thus, bone secretes multiple dormancy-inducing factors that employ distinct signaling pathways to induce DTC dormancy in bone.
Asunto(s)
Proteína Morfogenética Ósea 1/genética , Neoplasias Óseas/genética , Medios de Cultivo Condicionados/farmacología , Neoplasias de la Próstata/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Línea Celular Tumoral , Medios de Cultivo Condicionados/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Proteínas de la Membrana/genética , Metástasis de la Neoplasia , Osteoblastos/metabolismo , Osteoblastos/patología , Próstata/metabolismo , Próstata/patología , Prostatectomía , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Transducción de Señal/efectos de los fármacos , Cráneo/metabolismo , Cráneo/patologíaRESUMEN
How tumor cells genetically lose antigenicity and evade immune checkpoints remains largely elusive. We report that tissue-specific expression of the human long noncoding RNA LINK-A in mouse mammary glands initiates metastatic mammary gland tumors, which phenotypically resemble human triple-negative breast cancer (TNBC). LINK-A expression facilitated crosstalk between phosphatidylinositol-(3,4,5)-trisphosphate and inhibitory G-protein-coupled receptor (GPCR) pathways, attenuating protein kinase A-mediated phosphorylation of the E3 ubiquitin ligase TRIM71. Consequently, LINK-A expression enhanced K48-polyubiquitination-mediated degradation of the antigen peptide-loading complex (PLC) and intrinsic tumor suppressors Rb and p53. Treatment with LINK-A locked nucleic acids or GPCR antagonists stabilized the PLC components, Rb and p53, and sensitized mammary gland tumors to immune checkpoint blockers. Patients with programmed ccll death protein-1(PD-1) blockade-resistant TNBC exhibited elevated LINK-A levels and downregulated PLC components. Hence we demonstrate lncRNA-dependent downregulation of antigenicity and intrinsic tumor suppression, which provides the basis for developing combinational immunotherapy treatment regimens and early TNBC prevention.
Asunto(s)
Presentación de Antígeno/inmunología , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Neoplasias/inmunología , Oncogenes , ARN Largo no Codificante/genética , Escape del Tumor/genética , Escape del Tumor/inmunología , Adenoma/genética , Adenoma/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Ratones , Neoplasias/metabolismo , Neoplasias/patología , Fosforilación , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitinación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mistletoe (Viscum album) is a type of parasitic plant reported to have anticancer activity including in hepatocellular carcinoma (HCC). However, the mechanism of mistletoe's anticancer activity, and its effectiveness in treating HCC are not fully understood. We report here that mistletoe extracts, including Fraxini (grown on ash trees) and Iscador Q and M (grown on oak and maple trees), exert strong antiproliferative activity in Hep3B cells, with median inhibitory concentrations (IC50) of 0.5 µg/mL, 7.49 µg/mL, and 7.51 µg/mL, respectively. Results of Reversed Phase Proteomic Array analysis (RPPA) suggests that Fraxini substantially down-regulates c-Myc expression in Hep3B cells. Fraxini-induced growth inhibition (at a concentration of 1.25 µg/ml) was less pronounced in c-Myc knockdown Hep3B cells than in control cells. Furthermore, in the Hep3B xenograft model, Fraxini-treated (8 mg/kg body weight) mice had significantly smaller tumors (34.6 ± 11.9 mm3) than control mice (161.6 ± 79.4 mm3, p < 0.036). Similarly, c-Myc protein expression was reduced in Fraxini treated Hep3B cell xenografts compared to that of control mice. The reduction of c-Myc protein levels in vitro Hep3B cells appears to be mediated by the ubiquitin-proteasome system. Our results suggest the importance of c-Myc in Fraxini's antiproliferative activity, which warrants further investigation.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/tratamiento farmacológico , Lectinas de Plantas/farmacología , Proteínas Proto-Oncogénicas c-myc/genética , Viscum album/química , Animales , Antineoplásicos Fitogénicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Ratones Desnudos , Extractos Vegetales/química , Lectinas de Plantas/aislamiento & purificación , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal , Carga Tumoral/efectos de los fármacos , Ubiquitina/genética , Ubiquitina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Epithelial-mesenchymal transition (EMT) contributes significantly to interstitial matrix deposition in diabetic kidney disease (DKD). However, detection of EMT in kidney tissue is impracticable, and anti-EMT therapies have long been hindered. We reported that phosphatase and tensin homolog (PTEN) promoted transforming growth factor beta 1 (TGF-ß), sonic hedgehog (SHH), connective tissue growth factor (CTGF), interleukin 6 (IL-6), and hyperglycemia-induced EMT when PTEN was modified by a MEX3C-catalyzed K27-linked polyubiquitination at lysine 80 (referred to as PTENK27-polyUb). Genetic inhibition of PTENK27-polyUb alleviated Col4a3 knockout-, folic acid-, and streptozotocin-induced (STZ-induced) kidney injury. Serum and urine PTENK27-polyUb concentrations were negatively correlated with glomerular filtration rate (GFR) for diabetic patients. Mechanistically, PTENK27-polyUb facilitated dephosphorylation and protein stabilization of TWIST, SNAI1, and YAP in renal epithelial cells, leading to enhanced EMT. We identified that a small molecule, triptolide, inhibited MEX3C-catalyzed PTENK27-polyUb and EMT of renal epithelial cells. Treatment with triptolide reduced TWIST, SNAI1, and YAP concurrently and improved kidney health in Col4a3 knockout-, folic acid-injured disease models and STZ-induced, BTBR ob/ob diabetic nephropathy models. Hence, we demonstrated the important role of PTENK27-polyUb in DKD and a promising therapeutic strategy that inhibited the progression of DKD.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Nefropatías Diabéticas/metabolismo , Transición Epitelial-Mesenquimal , Riñón/metabolismo , Fosfohidrolasa PTEN/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/patología , Humanos , Riñón/patología , Ratones , Ratones Noqueados , Fosfohidrolasa PTEN/genética , Factores de Transcripción de la Familia Snail/genética , Factores de Transcripción de la Familia Snail/metabolismo , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Despite the structural conservation of PTEN with dual-specificity phosphatases, there have been no reports regarding the regulatory mechanisms that underlie this potential dual-phosphatase activity. Here, we report that K27-linked polyubiquitination of PTEN at lysines 66 and 80 switches its phosphoinositide/protein tyrosine phosphatase activity to protein serine/threonine phosphatase activity. Mechanistically, high glucose, TGF-ß, CTGF, SHH, and IL-6 induce the expression of a long non-coding RNA, GAEA (Glucose Aroused for EMT Activation), which associates with an RNA-binding E3 ligase, MEX3C, and enhances its enzymatic activity, leading to the K27-linked polyubiquitination of PTEN. The MEX3C-catalyzed PTENK27-polyUb activates its protein serine/threonine phosphatase activity and inhibits its phosphatidylinositol/protein tyrosine phosphatase activity. With this altered enzymatic activity, PTENK27-polyUb dephosphorylates the phosphoserine/threonine residues of TWIST1, SNAI1, and YAP1, leading to accumulation of these master regulators of EMT. Animals with genetic inhibition of PTENK27-polyUb, by a single nucleotide mutation generated using CRISPR/Cas9 (PtenK80R/K80R), exhibit inhibition of EMT markers during mammary gland morphogenesis in pregnancy/lactation and during cutaneous wound healing processes. Our findings illustrate an unexpected paradigm in which the lncRNA-dependent switch in PTEN protein serine/threonine phosphatase activity is important for physiological homeostasis and disease development.
Asunto(s)
Transición Epitelial-Mesenquimal/fisiología , Fosfohidrolasa PTEN/metabolismo , ARN Largo no Codificante/fisiología , Animales , Línea Celular , Femenino , Humanos , Masculino , Ratones , UbiquitinaciónRESUMEN
Inactivating mutations in the control of virulence two-component regulatory system (covRS) often account for the hypervirulent phenotype in severe, invasive group A streptococcal (GAS) infections. As CovR represses production of the anti-phagocytic hyaluronic acid capsule, high level capsule production is generally considered critical to the hypervirulent phenotype induced by CovRS inactivation. There have recently been large outbreaks of GAS strains lacking capsule, but there are currently no data on the virulence of covRS-mutated, acapsular strains in vivo. We investigated the impact of CovRS inactivation in acapsular serotype M4 strains using a wild-type (M4-SC-1) and a naturally-occurring CovS-inactivated strain (M4-LC-1) that contains an 11bp covS insertion. M4-LC-1 was significantly more virulent in a mouse bacteremia model but caused smaller lesions in a subcutaneous mouse model. Over 10% of the genome showed significantly different transcript levels in M4-LC-1 vs. M4-SC-1 strain. Notably, the Mga regulon and multiple cell surface protein-encoding genes were strongly upregulated-a finding not observed for CovS-inactivated, encapsulated M1 or M3 GAS strains. Consistent with the transcriptomic data, transmission electron microscopy revealed markedly altered cell surface morphology of M4-LC-1 compared to M4-SC-1. Insertional inactivation of covS in M4-SC-1 recapitulated the transcriptome and cell surface morphology. Analysis of the cell surface following CovS-inactivation revealed that the upregulated proteins were part of the Mga regulon. Inactivation of mga in M4-LC-1 reduced transcript levels of multiple cell surface proteins and reversed the cell surface alterations consistent with the effect of CovS inactivation on cell surface composition being mediated by Mga. CovRS-inactivating mutations were detected in 20% of current invasive serotype M4 strains in the United States. Thus, we discovered that hypervirulent M4 GAS strains with covRS mutations can arise in an acapsular background and that such hypervirulence is associated with profound alteration of the cell surface.
Asunto(s)
Streptococcus pyogenes/patogenicidad , Animales , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/ultraestructura , Proteínas Bacterianas/genética , Membrana Celular/genética , Membrana Celular/ultraestructura , Femenino , Genes Bacterianos , Histidina Quinasa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Microscopía Electrónica de Transmisión , Mutación , Regulón , Proteínas Represoras/genética , Serogrupo , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/genética , Streptococcus pyogenes/ultraestructura , Virulencia/genética , Secuenciación Completa del GenomaRESUMEN
Long noncoding RNA (lncRNA) is yet to be linked to cancer metabolism. Here, we report that upregulation of the lncRNA LINC00538 (YIYA) promotes glycolysis, cell proliferation, and tumor growth in breast cancer. YIYA is associated with the cytosolic cyclin-dependent kinase CDK6 and regulated CDK6-dependent phosphorylation of the fructose bisphosphatase PFK2 (PFKFB3) in a cell-cycle-independent manner. In breast cancer cells, these events promoted catalysis of glucose 6-phosphate to fructose-2,6-bisphosphate/fructose-1,6-bisphosphate. CRISPR/Cas9-mediated deletion of YIYA or CDK6 silencing impaired glycolysis and tumor growth in vivo In clinical specimens of breast cancer, YIYA was expressed in approximately 40% of cases where it correlated with CDK6 expression and unfavorable survival outcomes. Our results define a functional role for lncRNA in metabolic reprogramming in cancer, with potential clinical implications for its therapeutic targeting.Significance: These findings offer a first glimpse into how a long-coding RNA influences cancer metabolism to drive tumor growth. Cancer Res; 78(16); 4524-32. ©2018 AACR.
Asunto(s)
Neoplasias de la Mama/genética , Proliferación Celular/genética , ARN Largo no Codificante/genética , Neoplasias de la Mama/patología , Sistemas CRISPR-Cas , Línea Celular Tumoral , Quinasa 6 Dependiente de la Ciclina/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Glucosa/metabolismo , Glucólisis/genética , Humanos , FosforilaciónRESUMEN
Adenosine (A) to inosine (I) RNA editing introduces many nucleotide changes in cancer transcriptomes. However, due to the complexity of post-transcriptional regulation, the contribution of RNA editing to proteomic diversity in human cancers remains unclear. Here, we performed an integrated analysis of TCGA genomic data and CPTAC proteomic data. Despite limited site diversity, we demonstrate that A-to-I RNA editing contributes to proteomic diversity in breast cancer through changes in amino acid sequences. We validate the presence of editing events at both RNA and protein levels. The edited COPA protein increases proliferation, migration, and invasion of cancer cells in vitro. Our study suggests an important contribution of A-to-I RNA editing to protein diversity in cancer and highlights its translational potential.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Proteómica/métodos , Edición de ARN , Adenosina/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Bases de Datos Genéticas , Regulación Neoplásica de la Expresión Génica , Humanos , Inosina/genética , Análisis de Secuencia de ARN , Espectrometría de Masas en TándemRESUMEN
Despite remarkable progresses in vaccinology, therapeutic cancer vaccines have not achieved their full potential. We previously showed that an excessively long duration of Ag presentation critically reduced the quantity and quality of vaccination-induced T cell responses and subsequent antitumor efficacy. In this study, using a murine model and tumor cell lines, we studied l-tyrosine amino acid-based microparticles as a peptide vaccine adjuvant with a short-term Ag depot function for the induction of tumor-specific T cells. l-Tyrosine microparticles did not induce dendritic cell maturation, and their adjuvant activity was not mediated by inflammasome activation. Instead, prolonged Ag presentation in vivo translated into increased numbers and antitumor activity of vaccination-induced CD8+ T cells. Indeed, prolonging Ag presentation by repeated injection of peptide in saline resulted in an increase in T cell numbers similar to that observed after vaccination with peptide/l-tyrosine microparticles. Our results show that the duration of Ag presentation is critical for optimal induction of antitumor T cells, and can be manipulated through vaccine formulation.
Asunto(s)
Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Péptidos/inmunología , Adyuvantes Inmunológicos , Animales , Línea Celular Tumoral , Células Dendríticas/inmunología , Inflamasomas/inmunología , Ratones , Ratones Endogámicos C57BL , Tirosina/inmunología , Vacunación/métodos , Vacunas de Subunidad/inmunologíaRESUMEN
In Aedes aegypti females, the ammonia released during blood meal digestion is partially metabolized to facilitate the disposal of excess nitrogen. In this study, we used low- and high-resolution liquid chromatography-mass spectrometry (LC/MS) techniques to investigate the role of glucose during ammonia detoxification. Mosquitoes were fed a blood meal supplemented with [1,2-13C2]glucose, and downstream metabolites were measured for 24 h. Quantification of [13C] amino acids in the entire mosquito body was conducted without sample derivatization using selected reaction monitoring of mass transitions that are indicative of the structural position of [13C] atom incorporation. Identification of unlabeled and [13C] isotopologs of 43 compounds, including amino acids, amino acid derivatives, and organic acids, was performed by high-resolution LC/MS techniques. Blood-fed mosquitoes synthesized [13C] metabolites in mainly 2 carbon positions from [1,2-13C2]glucose. [13C2]Ala and [13C2]Pro were the most abundant and rapidly labeled amino acids synthesized. Additional [13C] amino acids, [13C] amino acid derivatives, and [13C] organic acids in 1 or 2 carbon positions were also identified. Two kinetic routes were proposed based on the incorporation of a [13C] atom at position 1 in specific amino acids. Our findings provide evidence that glucose is used for ammonia detoxification and [13C] uric acid synthesis through multiple metabolic pathways, uncovering a metabolic link at the carbon atomic level in ammonia metabolism of A. aegypti-Horvath, T. D., Dagan, S., Lorenzi, P. L., Hawke, D. H., Scaraffia, P. Y. Positional stable isotope tracer analysis reveals carbon routes during ammonia metabolism of Aedes aegypti mosquitoes.
Asunto(s)
Aedes/metabolismo , Amoníaco/metabolismo , Carbono/metabolismo , Aminoácidos/metabolismo , Animales , Isótopos de Carbono/metabolismo , Cromatografía Liquida , Femenino , Glucosa/metabolismo , Isótopos , Espectrometría de Masas , Redes y Vías Metabólicas , Metabolómica , Modelos Biológicos , Nitrógeno/metabolismoRESUMEN
Increasing evidence has demonstrated that small nucleolar RNAs (snoRNAs) play important roles in tumorigenesis. We systematically investigated the expression landscape and clinical relevance of snoRNAs in >10,000 samples across 31 cancer types from The Cancer Genome Atlas. We observed overall elevated expression of snoRNAs and their ribonucleoproteins in multiple cancer types. We showed complex regulation of snoRNA expression by their host genes, copy number variation, and DNA methylation. Unsupervised clustering revealed that the snoRNA expression subtype is highly concordant with other molecular/clinical subtypes. We further identified 46 clinically relevant snoRNAs and experimentally demonstrated functional roles of SNORD46 in promoting cell proliferation, migration, and invasion. We developed a user-friendly data portal, SNORic, to benefit the research community. Our study highlights the significant roles of snoRNAs in the development and implementation of biomarkers or therapeutic targets for cancer and provides a valuable resource for cancer research.
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Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , ARN Nucleolar Pequeño/genética , Biomarcadores de Tumor/metabolismo , Metilación de ADN , Dosificación de Gen , Humanos , Neoplasias/metabolismo , Neoplasias/patología , ARN Nucleolar Pequeño/metabolismo , Programas InformáticosRESUMEN
Translation is principally regulated at the initiation stage. The development of the translation initiation (TI) sequencing (TI-seq) technique has enabled the global mapping of TIs and revealed unanticipated complex translational landscapes in metazoans. Despite the wide adoption of TI-seq, there is no computational tool currently available for analyzing TI-seq data. To fill this gap, we develop a comprehensive toolkit named Ribo-TISH, which allows for detecting and quantitatively comparing TIs across conditions from TI-seq data. Ribo-TISH can also predict novel open reading frames (ORFs) from regular ribosome profiling (rRibo-seq) data and outperform several established methods in both computational efficiency and prediction accuracy. Applied to published TI-seq/rRibo-seq data sets, Ribo-TISH uncovers a novel signature of elevated mitochondrial translation during amino-acid deprivation and predicts novel ORFs in 5'UTRs, long noncoding RNAs, and introns. These successful applications demonstrate the power of Ribo-TISH in extracting biological insights from TI-seq/rRibo-seq data.
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Regiones no Traducidas 5'/genética , Iniciación de la Cadena Peptídica Traduccional/genética , Biología Computacional , Biblioteca de Genes , Genoma Humano , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Modelos Estadísticos , Sistemas de Lectura Abierta , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Análisis de Secuencia de ARNRESUMEN
Conventional therapies for breast cancer brain metastases (BCBMs) have been largely ineffective because of chemoresistance and impermeability of the blood-brain barrier. A comprehensive understanding of the underlying mechanism that allows breast cancer cells to infiltrate the brain is necessary to circumvent treatment resistance of BCBMs. Here, we determined that expression of a long noncoding RNA (lncRNA) that we have named lncRNA associated with BCBM (Lnc-BM) is prognostic of the progression of brain metastasis in breast cancer patients. In preclinical murine models, elevated Lnc-BM expression drove BCBM, while depletion of Lnc-BM with nanoparticle-encapsulated siRNAs effectively treated BCBM. Lnc-BM increased JAK2 kinase activity to mediate oncostatin M- and IL-6-triggered STAT3 phosphorylation. In breast cancer cells, Lnc-BM promoted STAT3-dependent expression of ICAM1 and CCL2, which mediated vascular co-option and recruitment of macrophages in the brain, respectively. Recruited macrophages in turn produced oncostatin M and IL-6, thereby further activating the Lnc-BM/JAK2/STAT3 pathway and enhancing BCBM. Collectively, our results show that Lnc-BM and JAK2 promote BCBMs by mediating communication between breast cancer cells and the brain microenvironment. Moreover, these results suggest targeting Lnc-BM as a potential strategy for fighting this difficult disease.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Neoplasias de la Mama/metabolismo , Janus Quinasa 2/metabolismo , Proteínas de Neoplasias/metabolismo , ARN Largo no Codificante/metabolismo , ARN Neoplásico/metabolismo , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Janus Quinasa 2/genética , Células MCF-7 , Ratones , Ratones Desnudos , Células 3T3 NIH , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , ARN Largo no Codificante/genética , ARN Neoplásico/genética , Transducción de Señal/genética , Microambiente Tumoral/genética , Células U937RESUMEN
Phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3 or PIP3) mediates signalling pathways as a second messenger in response to extracellular signals. Although primordial functions of phospholipids and RNAs have been hypothesized in the 'RNA world', physiological RNA-phospholipid interactions and their involvement in essential cellular processes have remained a mystery. We explicate the contribution of lipid-binding long non-coding RNAs (lncRNAs) in cancer cells. Among them, long intergenic non-coding RNA for kinase activation (LINK-A) directly interacts with the AKT pleckstrin homology domain and PIP3 at the single-nucleotide level, facilitating AKT-PIP3 interaction and consequent enzymatic activation. LINK-A-dependent AKT hyperactivation leads to tumorigenesis and resistance to AKT inhibitors. Genomic deletions of the LINK-A PIP3-binding motif dramatically sensitized breast cancer cells to AKT inhibitors. Furthermore, meta-analysis showed the correlation between LINK-A expression and incidence of a single nucleotide polymorphism (rs12095274: A > G), AKT phosphorylation status, and poor outcomes for breast and lung cancer patients. PIP3-binding lncRNA modulates AKT activation with broad clinical implications.