RESUMEN
Recently, sclerostin (SCL), a circulating glycoprotein, was proposed to be a novel myokine involved in developing metabolic disorders. The association between SCL levels and insulin resistance in skeletal muscle, liver, and adipose tissue was studied in individuals with aggravated glucose tolerance. Thus, we hypothesized that elevated circulating SCL might affect skeletal muscle insulin signaling and hepatic lipid metabolism, and aimed to investigate the effects of SCL on skeletal muscle insulin resistance and hepatic steatosis in obesity using in vitro and in vivo experimental models under hyperlipidemic conditions. In the current study, we found elevated SCL messenger RNA expression levels in myocytes in obese patients. In addition to a higher blood level, SCL was expressed at an elevated level in the skeletal muscle of mice fed a high-fat diet (HFD). Higher SCL release levels and expression were also noticed in palmitate-treated C2C12 myocytes. SCL suppression by in vivo transfection improves skeletal muscle insulin resistance and hepatic steatosis in HFD-fed mice. The treatment of C2C12 myocytes with recombinant SCL aggravated insulin signaling. Furthermore, treatment with SCL augmented lipogenic lipid deposition in human primary hepatocytes. Treatment with SCL upregulated mammalian target of rapamycin (mTOR) phosphorylation and suppressed autophagy markers, thereby causing endoplasmic reticulum (ER) stress. 4-Phenylbutyric acid, a pharmacological ER stress inhibitor, abolished the effects of SCL on insulin signaling in C2C12 myocytes and lipid accumulation in primary hepatocytes. In conclusion, SCL promotes skeletal muscle insulin resistance and hepatic steatosis by upregulating ER stress via the mTOR/autophagy-mediated pathway. The present study suggests that antagonizing SCL might be a novel therapeutic strategy for simultaneously managing insulin resistance and hepatic steatosis in obesity.
Asunto(s)
Hígado Graso , Resistencia a la Insulina , Humanos , Ratones , Animales , Regulación hacia Arriba , Insulina , Serina-Treonina Quinasas TOR , Estrés del Retículo Endoplásmico , Autofagia , Músculo Esquelético , Dieta Alta en Grasa/efectos adversos , Obesidad , Lípidos , Ratones Endogámicos C57BL , MamíferosRESUMEN
Pediatric high-grade glioma (pHGG) is a major contributor to cancer-related death in children. In vitro and in vivo disease models reflecting the intimate connection between developmental context and pathogenesis of pHGG are essential to advance understanding and identify therapeutic vulnerabilities. Here we report establishment of 21 patient-derived pHGG orthotopic xenograft (PDOX) models and eight matched cell lines from diverse groups of pHGG. These models recapitulate histopathology, DNA methylation signatures, mutations and gene expression patterns of the patient tumors from which they were derived, and include rare subgroups not well-represented by existing models. We deploy 16 new and existing cell lines for high-throughput screening (HTS). In vitro HTS results predict variable in vivo response to PI3K/mTOR and MEK pathway inhibitors. These unique new models and an online interactive data portal for exploration of associated detailed molecular characterization and HTS chemical sensitivity data provide a rich resource for pediatric brain tumor research.
Asunto(s)
Heterogeneidad Genética/efectos de los fármacos , Glioma/tratamiento farmacológico , Glioma/genética , Animales , Neoplasias Encefálicas , Línea Celular Tumoral , Proliferación Celular , Niño , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Glioma/patología , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Mutación , Inhibidores de Proteínas Quinasas/uso terapéutico , Serina-Treonina Quinasas TOR , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Regular exercise is the first line of therapy for treating obesity-mediated metabolic disorders, including insulin resistance. It has been reported that developmental endothelial locus-1 (DEL-1) enhances macrophage efferocytosis, resulting in inflammation clearance as well as improves insulin resistance in skeletal muscle. However, the relationship between exercise and DEL-1, and the effects of DEL-1 on insulin signalling in adipocytes have not been fully elucidated to date. Protein expression levels were determined by Western blot analysis. Cells were transfected with small interfering (si) RNA to suppress gene expression. Lipid accumulation levels were detected using Oil red-O staining. Proinflammatory cytokine secretion levels were measured using ELISA. DEL-1 expression levels were induced in the skeletal muscle of people who exercised using microarray analysis. Recombinant DEL-1 augmented AMP-activated protein kinase (AMPK) phosphorylation and haem oxygenase (HO)-1 expression to alleviating inflammation and impairment of insulin signalling in 3T3-L1 adipocytes treated with palmitate. siRNA of AMPK or HO-1 also mitigated the effects of DEL-1 on inflammation and insulin resistance. DEL-1 ameliorates inflammation and insulin resistance in differentiated 3T3-L1 cells via AMPK/HO-1 signalling, suggesting that DEL-1 may be the exercise-mediated therapeutic target for treating insulin resistance and type 2 diabetes.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Adipocitos/metabolismo , Proteínas de Unión al Calcio/genética , Moléculas de Adhesión Celular/genética , Hemo-Oxigenasa 1/metabolismo , Resistencia a la Insulina/genética , Metabolismo de los Lípidos , Transducción de Señal , Células 3T3-L1 , Animales , Proteínas de Unión al Calcio/metabolismo , Moléculas de Adhesión Celular/metabolismo , Ejercicio Físico , Silenciador del Gen , Humanos , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Insulina/metabolismo , Ratones , Músculo Esquelético/metabolismoRESUMEN
Diffuse intrinsic pontine gliomas (DIPGs) are incurable childhood brainstem tumors with frequent histone H3 K27M mutations and recurrent alterations in PDGFRA and TP53. We generated genetically engineered inducible mice and showed that H3.3 K27M enhanced neural stem cell self-renewal while preserving regional identity. Neonatal induction of H3.3 K27M cooperated with activating platelet-derived growth factor receptor α (PDGFRα) mutant and Trp53 loss to accelerate development of diffuse brainstem gliomas that recapitulated human DIPG gene expression signatures and showed global changes in H3K27 posttranslational modifications, but relatively restricted gene expression changes. Genes upregulated in H3.3 K27M tumors were enriched for those associated with neural development where H3K27me3 loss released the poised state of apparently bivalent promoters, whereas downregulated genes were enriched for those encoding homeodomain transcription factors.
Asunto(s)
Neoplasias del Tronco Encefálico/genética , Perfilación de la Expresión Génica/métodos , Glioma/genética , Histonas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Proteína p53 Supresora de Tumor/genética , Animales , Autorrenovación de las Células , Células Cultivadas , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Histonas/metabolismo , Humanos , Ratones , Mutación , Células-Madre Neurales/citología , Rombencéfalo/patología , Análisis de Secuencia de ARN/métodosRESUMEN
Biogenesis of the primary cilium, a cellular organelle mediating various signaling pathways, is generally coordinated with cell cycle exit/re-entry. Although the dynamic cell cycle-associated profile of the primary cilium has been largely accepted, the mechanism governing the link between ciliogenesis and cell cycle progression has been poorly understood. Using a human genome-wide RNAi screen, we identify genes encoding subunits of the spliceosome and proteasome as novel regulators of ciliogenesis. We demonstrate that 1) the mRNA processing-related hits are essential for RNA expression of molecules acting in cilia disassembly, such as AURKA and PLK1, and 2) the ubiquitin-proteasome systems (UPS)-involved hits are necessary for proteolysis of molecules acting in cilia assembly, such as IFT88 and CPAP. In particular, we show that these screen hit-associated mechanisms are crucial for both cilia assembly and cell cycle arrest in response to serum withdrawal. Finally, our data suggest that the mRNA processing mechanism may modulate the UPS-dependent decay of cilia assembly regulators to control ciliary resorption-coupled cell cycle re-entry.
Asunto(s)
Puntos de Control del Ciclo Celular/genética , Ciclo Celular/genética , Cilios/metabolismo , Genoma Humano/genética , Interferencia de ARN , Transcriptoma/genética , Western Blotting , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular , Cilios/fisiología , Análisis por Conglomerados , Medio de Cultivo Libre de Suero/farmacología , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes/genética , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Modelos Genéticos , Morfogénesis/genética , Proteoma/genética , Proteoma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The available evidence suggests that the lethality of glioblastoma is driven by small subpopulations of cells that self-renew and exhibit tumorigenicity. It remains unclear whether tumorigenicity exists as a static property of a few cells or as a dynamically acquired property. We used tumor-sphere and xenograft formation as assays for tumorigenicity and examined subclones isolated from established and primary glioblastoma lines. Our results indicate that glioblastoma tumorigenicity is largely deterministic, yet the property can be acquired spontaneously at low frequencies. Further, these dynamic transitions are governed by epigenetic reprogramming through the lysine-specific demethylase 1 (LSD1). LSD depletion increases trimethylation of histone 3 lysine 4 at the avian myelocytomatosis viral oncogene homolog (MYC) locus, which elevates MYC expression. MYC, in turn, regulates oligodendrocyte lineage transcription factor 2 (OLIG2), SRY (sex determining region Y)-box 2 (SOX2), and POU class 3 homeobox 2 (POU3F2), a core set of transcription factors required for reprogramming glioblastoma cells into stem-like states. Our model suggests epigenetic regulation of key transcription factors governs transitions between tumorigenic states and provides a framework for glioblastoma therapeutic development.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Histona Demetilasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Perfilación de la Expresión Génica , Silenciador del Gen , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Neoplasias/metabolismo , Procesos EstocásticosRESUMEN
Systemic siRNA administration to target and treat glioblastoma, one of the most deadly cancers, requires robust and efficient delivery platform without immunogenicity. Here we report newly emerged multivalent naked RNA nanoparticle (RNP) based on pRNA 3-way-junction (3WJ) from bacteriophage phi29 to target glioblastoma cells with folate (FA) ligand and deliver siRNA for gene silencing. Systemically injected FA-pRNA-3WJ RNPs successfully targeted and delivered siRNA into brain tumor cells in mice, and efficiently reduced luciferase reporter gene expression (4-fold lower than control). The FA-pRNA-3WJ RNP also can target human patient-derived glioblastoma stem cells, thought to be responsible for tumor initiation and deadly recurrence, without accumulation in adjacent normal brain cells, nor other major internal organs. This study provides possible application of pRNA-3WJ RNP for specific delivery of therapeutics such as siRNA, microRNA and/or chemotherapeutic drugs into glioblastoma cells without inflicting collateral damage to healthy tissues.
Asunto(s)
Neoplasias Encefálicas/terapia , Sistemas de Liberación de Medicamentos/métodos , Glioblastoma/terapia , Nanopartículas/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Tratamiento con ARN de Interferencia/métodos , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Femenino , Glioblastoma/genética , Glioblastoma/patología , Humanos , Imagen por Resonancia Magnética , Ratones Desnudos , Microscopía Confocal , Nanopartículas/química , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Carga Tumoral , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
SapC-DOPS is a novel nanotherapeutic that has been shown to target and induce cell death in a variety of cancers, including glioblastoma (GBM). GBM is a primary brain tumor known to frequently demonstrate resistance to apoptosis-inducing therapeutics. Here we explore the mode of action for SapC-DOPS in GBM, a treatment being developed by Bexion Pharmaceuticals for clinical testing in patients. SapC-DOPS treatment was observed to induce lysosomal dysfunction of GBM cells characterized by decreased glycosylation of LAMP1 and altered proteolytic processing of cathepsin D independent of apoptosis and autophagic cell death. We observed that SapC-DOPS induced lysosomal membrane permeability (LMP) as shown by LysoTracker Red and Acridine Orange staining along with an increase of sphingosine, a known inducer of LMP. Additionally, SapC-DOPS displayed strong synergistic interactions with the apoptosis-inducing agent TMZ. Collectively our data suggest that SapC-DOPS induces lysosomal cell death in GBM cells, providing a new approach for treating tumors resistant to traditional apoptosis-inducing agents.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Dacarbazina/análogos & derivados , Glioblastoma/tratamiento farmacológico , Nanoestructuras/administración & dosificación , Fosfatidilserinas/farmacología , Saposinas/farmacología , Animales , Antineoplásicos Alquilantes/administración & dosificación , Antineoplásicos Alquilantes/farmacología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Dacarbazina/administración & dosificación , Dacarbazina/farmacología , Sinergismo Farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Lisosomas/efectos de los fármacos , Ratones , Ratones Desnudos , Distribución Aleatoria , Saposinas/administración & dosificación , Temozolomida , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Neural oncogenesis is currently incurable and invariably lethal. The development of innovative treatments for this devastating cancer will require a deeper molecular understanding of how cancer cells survive, proliferate, and escape from current therapies. In high-grade gliomas (HGGs), glioma stem cells (GSCs) may causally contribute to tumor initiation and propagation, therapeutic resistance, and subsequent recurrence of tumors. Within a tumor mass, GSCs are enriched in a hypoxic niche in which the oxidative stress levels are substantially elevated. Paradoxically, however, recent studies suggest that GSCs appear to generate less reactive oxygen species (ROS), a chemical component responsible for elevation of oxidative stress levels. To date, molecular mechanisms for how GSCs reduce oxidative stress to allow preferential survival in hypoxic areas in tumors remains elusive. This review article summarizes recent studies on the role of ROS-reducing enzymes, including peroxiredoxin 4, in detoxifying oxidative stress preferentially for GSCs in HGGs. In addition, the therapeutic potential of some of the recently identified antioxidant chemotherapeutic agents and avenues for future research in this area are discussed.
Asunto(s)
Glioma/patología , Inactivación Metabólica/fisiología , Células Madre Neoplásicas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Glioma/tratamiento farmacológico , Humanos , Inactivación Metabólica/efectos de los fármacos , Células Madre Neoplásicas/efectos de los fármacos , Peroxirredoxinas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUNDS: Piperlongumine, a natural plant product, kills multiple cancer types with little effect on normal cells. Piperlongumine raises intracellular levels of reactive oxygen species (ROS), a phenomenon that may underlie the cancer-cell killing. Although these findings suggest that piperlongumine could be useful for treating cancers, the mechanism by which the drug selectively kills cancer cells remains unknown. METHODS: We treated multiple high-grade glioma (HGG) sphere cultures with piperlongumine and assessed its effects on ROS and cell-growth levels as well as changes in downstream signaling. We also examined the levels of putative piperlongumine targets and their roles in HGG cell growth. RESULTS: Piperlongumine treatment increased ROS levels and preferentially killed HGG cells with little effect in normal brain cells. Piperlongumine reportedly increases ROS levels after interactions with several redox regulators. We found that HGG cells expressed higher levels of the putative piperlongumine targets than did normal neural stem cells (NSCs). Furthermore, piperlongumine treatment in HGG cells, but not in normal NSCs, increased oxidative inactivation of peroxiredoxin 4 (PRDX4), an ROS-reducing enzyme that is overexpressed in HGGs and facilitates proper protein folding in the endoplasmic reticulum (ER). Moreover, piperlongumine exacerbated intracellular ER stress, an effect that was mimicked by suppressing PRDX4 expression. CONCLUSIONS: Our results reveal that the mechanism by which piperlongumine preferentially kills HGG cells involves PRDX4 inactivation, thereby inducing ER stress. Therefore, piperlongumine treatment could be considered as a novel therapeutic option for HGG treatment.
Asunto(s)
Antineoplásicos/administración & dosificación , Neoplasias Encefálicas/tratamiento farmacológico , Dioxolanos/administración & dosificación , Estrés del Retículo Endoplásmico/efectos de los fármacos , Glioma/tratamiento farmacológico , Peroxirredoxinas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Bases de Datos Factuales , Glioma/metabolismo , Glioma/mortalidad , Humanos , Ratones , Especies Reactivas de Oxígeno/metabolismo , Análisis de Supervivencia , Células Tumorales CultivadasRESUMEN
We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10° steps over 380°. A rectangular region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.
Asunto(s)
Artritis/diagnóstico , Neoplasias Encefálicas/diagnóstico , Colorantes Fluorescentes/administración & dosificación , Nanoestructuras/administración & dosificación , Óptica y Fotónica/métodos , Fosfatidilserinas/administración & dosificación , Saposinas/administración & dosificación , Absorción , Animales , Artritis/metabolismo , Artritis/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Femenino , Colorantes Fluorescentes/farmacocinética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Desnudos , Ratones Transgénicos , Imagen Óptica , Óptica y Fotónica/instrumentación , Imagen de Cuerpo EnteroRESUMEN
PURPOSE: Our goal is to test whether CS1 could be targeted by chimeric antigen receptor (CAR) T cells to treat multiple myeloma (MM). EXPERIMENTAL DESIGN: We generated a retroviral construct of a CS1-specific CAR and engineered primary human T cells expressing the CAR. We then tested the capacity of CS1-CAR T cells to eradicate human MM tumor cells in vitro, ex vivo, and in vivo using orthotopic MM xenograft mouse models. RESULTS: In vitro, compared with mock-transduced T cells, upon recognizing CS1-positive MM cells, CS1-CAR-transduced T cells secreted more IFN-γ as well as interleukin (IL)-2, expressed higher levels of the activation marker CD69, showed higher capacity for degranulation, and displayed enhanced cytotoxicity. Ectopically forced expression of CS1 in MM cells with low CS1 expression enhanced recognition and killing by CAR T cells. Ex vivo, CS1-CAR T cells also showed similarly enhanced activities when responding to primary MM cells. More importantly, in orthotopic MM xenograft mouse models, adoptive transfer of human primary T cells expressing CS1-CAR efficiently suppressed the growth of human MM.1S and IM9 myeloma cells and significantly prolonged mouse survival. CONCLUSIONS: CS1 is a promising antigen that can be targeted by CAR-expressing T cells for treatment of MM.
Asunto(s)
Citotoxicidad Inmunológica/inmunología , Ingeniería Genética , Mieloma Múltiple/prevención & control , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Inmunológicos/genética , Linfocitos T/inmunología , Animales , Western Blotting , Citometría de Flujo , Humanos , Subunidad gamma Común de Receptores de Interleucina , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Receptores de Antígenos de Linfocitos T/genética , Receptores Inmunológicos/inmunología , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Linfocitos T/metabolismo , Linfocitos T/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glioblastoma remains one of the deadliest of human cancers, with most patients succumbing to the disease within two years of diagnosis. The available data suggest that simultaneous inactivation of critical nodes within the glioblastoma molecular circuitry will be required for meaningful clinical efficacy. We conducted parallel genome-wide shRNA screens to identify such nodes and uncovered a number of G-Protein Coupled Receptor (GPCR) neurotransmitter pathways, including the Dopamine Receptor D2 (DRD2) signaling pathway. Supporting the importance of DRD2 in glioblastoma, DRD2 mRNA and protein expression were elevated in clinical glioblastoma specimens relative to matched non-neoplastic cerebrum. Treatment with independent si-/shRNAs against DRD2 or with DRD2 antagonists suppressed the growth of patient-derived glioblastoma lines both in vitro and in vivo. Importantly, glioblastoma lines derived from independent genetically engineered mouse models (GEMMs) were more sensitive to haloperidol, an FDA approved DRD2 antagonist, than the premalignant astrocyte lines by approximately an order of magnitude. The pro-proliferative effect of DRD2 was, in part, mediated through a GNAI2/Rap1/Ras/ERK signaling axis. Combined inhibition of DRD2 and Epidermal Growth Factor Receptor (EGFR) led to synergistic tumoricidal activity as well as ERK suppression in independent in vivo and in vitro glioblastoma models. Our results suggest combined EGFR and DRD2 inhibition as a promising strategy for glioblastoma treatment.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Receptores ErbB/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , ARN Interferente Pequeño/genética , Receptores de Dopamina D2/metabolismo , Animales , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioblastoma/patología , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Fosforilación , ARN Interferente Pequeño/metabolismo , Transducción de Señal , TransfecciónRESUMEN
Glioblastoma is the most common and aggressive histologic subtype of brain cancer with poor outcomes and limited treatment options. Here, we report the selective overexpression of the protein arginine methyltransferase PRMT5 as a novel candidate theranostic target in this disease. PRMT5 silences the transcription of regulatory genes by catalyzing symmetric dimethylation of arginine residues on histone tails. PRMT5 overexpression in patient-derived primary tumors and cell lines correlated with cell line growth rate and inversely with overall patient survival. Genetic attenuation of PRMT5 led to cell-cycle arrest, apoptosis, and loss of cell migratory activity. Cell death was p53-independent but caspase-dependent and enhanced with temozolomide, a chemotherapeutic agent used as a present standard of care. Global gene profiling and chromatin immunoprecipitation identified the tumor suppressor ST7 as a key gene silenced by PRMT5. Diminished ST7 expression was associated with reduced patient survival. PRMT5 attenuation limited PRMT5 recruitment to the ST7 promoter, led to restored expression of ST7 and cell growth inhibition. Finally, PRMT5 attenuation enhanced glioblastoma cell survival in a mouse xenograft model of aggressive glioblastoma. Together, our findings defined PRMT5 as a candidate prognostic factor and therapeutic target in glioblastoma, offering a preclinical justification for targeting PRMT5-driven oncogenic pathways in this deadly disease.
Asunto(s)
Neoplasias Encefálicas/enzimología , Glioblastoma/enzimología , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Supresoras de Tumor/genética , Animales , Apoptosis , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/terapia , Línea Celular Tumoral , Proliferación Celular , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glioblastoma/mortalidad , Glioblastoma/terapia , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Noqueados , Ratones Desnudos , Terapia Molecular Dirigida , Trasplante de Neoplasias , Proteína-Arginina N-Metiltransferasas/metabolismo , ARN Interferente Pequeño/genética , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Over the last decade, cytomegalovirus (CMV) has been suggested to promote the development of glioblastoma multiforme (GBM). Recent evidence demonstrates that CMV contributes to the progression of GBM in the context of oncosuppressor gene mutations. This finding provides further insights into the mechanisms whereby CMV exacerbates the malignancy of GBM.
RESUMEN
To study the controversial role of cytomegalovirus (CMV) in glioblastoma, we assessed the effects of murine CMV (MCMV) perinatal infection in a GFAP-cre; Nf1(loxP/+); Trp53(-/+) genetic mouse model of glioma (Mut3 mice). Early on after infection, MCMV antigen was predominantly localized in CD45+ lymphocytes in the brain with active viral replication and local areas of inflammation, but, by 7 weeks, there was a generalized loss of MCMV in brain, confirmed by bioluminescent imaging. MCMV-infected Mut3 mice exhibited a shorter survival time from their gliomas than control Mut3 mice perinatally infected with mock or with a different neurotropic virus. Animal survival was also significantly shortened when orthotopic gliomas were implanted in mice perinatally infected with MCMV versus controls. MCMV infection increased phosphorylated STAT3 (p-STAT3) levels in neural stem cells (NSC) harvested from Mut3 mice subventricular zone, and, in vivo, there was increased p-STAT3 in NSCs in MCMV-infected compared with control mice. Of relevance, human CMV (HCMV) also increased p-STAT3 and proliferation of patient-derived glioblastoma neurospheres, whereas a STAT3 inhibitor reversed this effect in vitro and in vivo. These findings thus associate CMV infection to a STAT3-dependent modulatory role in glioma formation/progression in the context of tumor suppressor mutations in mice and possibly in humans.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/virología , Infecciones por Citomegalovirus/patología , Citomegalovirus/genética , Glioblastoma/genética , Glioblastoma/virología , Supresión Genética , Animales , Encefalopatías/genética , Encefalopatías/metabolismo , Encefalopatías/patología , Encefalopatías/virología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Citomegalovirus/metabolismo , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/virología , Femenino , Glioblastoma/metabolismo , Glioblastoma/patología , Xenoinjertos , Humanos , Masculino , Ratones , Células 3T3 NIH , Embarazo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Replicación ViralRESUMEN
Saposin C-dioleoylphosphatidylserine (SapC-DOPS) nanovesicles are a nanotherapeutic which effectively target and destroy cancer cells. Here, we explore the systemic use of SapC-DOPS in several models of brain cancer, including glioblastoma multiforme (GBM), and the molecular mechanism behind its tumor-selective targeting specificity. Using two validated spontaneous brain tumor models, we demonstrate the ability of SapC-DOPS to selectively and effectively cross the blood-brain tumor barrier (BBTB) to target brain tumors in vivo and reveal the targeting to be contingent on the exposure of the anionic phospholipid phosphatidylserine (PtdSer). Increased cell surface expression of PtdSer levels was found to correlate with SapC-DOPS-induced killing efficacy, and tumor targeting in vivo was inhibited by blocking PtdSer exposed on cells. Apart from cancer cell killing, SapC-DOPS also exerted a strong antiangiogenic activity in vitro and in vivo. Interestingly, unlike traditional chemotherapy, hypoxic cells were sensitized to SapC-DOPS-mediated killing. This study emphasizes the importance of PtdSer exposure for SapC-DOPS targeting and supports the further development of SapC-DOPS as a novel antitumor and antiangiogenic agent for brain tumors.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Antineoplásicos/administración & dosificación , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Nanopartículas/administración & dosificación , Fosfatidilserinas/química , Saposinas/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Hipoxia de la Célula , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Modelos Animales de Enfermedad , Femenino , Glioblastoma/tratamiento farmacológico , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Masculino , Ratones , Nanopartículas/química , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saposinas/administración & dosificación , Saposinas/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The Polycomb Repressor Complex (PRC) is an epigenetic regulator of transcription whose action is mediated by 2 protein complexes, PRC1 and PRC2. PRC is oncogenic in glioblastoma, where it is involved in cancer stem cell maintenance and radioresistance. METHODS: We used a set of glioblastoma patient samples, glioma stem cells, and neural stem cells from a mouse model of glioblastoma. We characterized gene/protein expression and cellular phenotypes by quantitative PCR/Western blotting and clonogenic, cell-cycle, and DNA damage assays. We performed overexpression/knockdown studies by lentiviral infection and microRNA/small interfering RNA oligonucleotide transfection. RESULTS: We show that microRNA-128 (miR-128) directly targets mRNA of SUZ12, a key component of PRC2, in addition to BMI1, a component of PRC1 that we previously showed as a target as well. This blocks the partially redundant functions of PRC1/PRC2, thereby significantly reducing PRC activity and its associated histone modifications. MiR-128 and SUZ12/BMI1 show opposite expression in human glioblastomas versus normal brain and in glioma stemlike versus neural stem cells. Furthermore, miR-128 renders glioma stemlike cells less radioresistant by preventing the radiation-induced expression of both PRC components. Finally, miR-128 expression is significantly reduced in neural stem cells from the brain of young, presymptomatic mice in our mouse model of glioblastoma. This suggests that loss of miR-128 expression in brain is an early event in gliomagenesis. Moreover, knockdown of miR-128 expression in nonmalignant mouse and human neural stem cells led to elevated expression of PRC components and increased clonogenicity. CONCLUSIONS: MiR-128 is an important suppressor of PRC activity, and its absence is an early event in gliomagenesis.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , MicroARNs/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Animales , Neoplasias Encefálicas/genética , Glioma/genética , Humanos , Ratones , Proteínas de Neoplasias , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Factores de TranscripciónRESUMEN
PURPOSE: Glioblastoma multiforme (GBM) is a devastating disease. Recent studies suggest that the stem cell properties of GBM contribute to the development of therapy resistance. EXPERIMENTAL DESIGN: The expression of Survivin and Ran was evaluated by immunohistochemistry with GBM tissues, and quantitative reverse transcriptase (qRT)-PCR and immunocytochemistry with patient-derived GBM sphere cultures. With a computational structure-based drug design, 11 small-molecule compounds were designed, synthesized, and evaluated as inhibitor candidates for the molecular interaction of Survivin protein. The molecular mechanism of the lead compound, LLP-3, was determined by Western blot, ELISA, in situ proximity ligation assay, and immunocytochemistry. The effects of LLP-3 treatment on GSCs were evaluated both in vitro and in vivo. Quantitative immunohistochemistry was carried out to compare Survivin expression in tissues from 44 newly diagnosed and 31 recurrent post-chemoradiation GBM patients. Lastly, the sensitivities of temozolomide-resistant GBM spheres to LLP-3 were evaluated in vitro. RESULTS: Survivin and Ran were strongly expressed in GBM tissues, particularly in the perivasculature, and also in patient-derived GSC cultures. LLP-3 treatment disrupted the Survivin-Ran protein complex in cancer cells and abolished the growth of patient-derived GBM spheres in vitro and in vivo. This inhibition was dependent on caspase activity and associated with p53 status of cells. Immunohistochemistry showed that Survivin expression is significantly increased in recurrent GBM compared with newly diagnosed tumors, and temozolomide-resistant GBM spheres exhibited high sensitivities to LLP-3 treatment. CONCLUSIONS: Disruption of the Survivin-Ran complex by LLP-3 abolishes survival and growth of GSCs both in vitro and in vivo, indicating an attractive novel therapeutic approach for GBM.
Asunto(s)
Glioblastoma/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Células Madre Neoplásicas/metabolismo , Proteína de Unión al GTP ran/metabolismo , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Resistencia a Antineoplásicos , Glioblastoma/mortalidad , Glioblastoma/cirugía , Humanos , Proteínas Inhibidoras de la Apoptosis/química , Modelos Moleculares , Pronóstico , Unión Proteica/efectos de los fármacos , Conformación Proteica , Multimerización de Proteína , Transducción de Señal , Survivin , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína de Unión al GTP ran/químicaRESUMEN
Cytomegalovirus (CMV) has been detected in several human cancers, but it has not proven to be oncogenic. However, recent studies have suggested mechanisms through which cytomegalovirus may modulate the tumor environment, encouraging its study as a positive modifier of tumorigenesis. In this study, we investigated the effects of cytomegalovirus infection in Trp53 heterozygous mice. Animals were infected with murine cytomegalovirus (MCMV) after birth at 2 days (P2) or 4 weeks of age and then monitored for tumor formation. Mice injected at 2 days of age developed tumors at a high frequency (43%) by 9 months of age. In contrast, only 3% of mock-infected or mice infected at 4 weeks developed tumors. The majority of tumors from P2 MCMV-infected mice were pleomorphic rhabdomyosarcomas (RMS) harboring MCMV DNA, RNA, and protein. An examination of clinical cases revealed that human RMS (embryonal, alveolar, and pleomorphic) harbored human cytomegalovirus IE1 and pp65 protein as well as viral RNA. Taken together, our findings offer support for the hypothesis that cytomegalovirus contributes to the development of pleomorphic RMS in the context of Trp53 mutation, a situation that occurs with high frequency in human RMS.