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1.
J Cell Physiol ; 237(11): 4226-4237, 2022 11.
Article in English | MEDLINE | ID: mdl-36087347

ABSTRACT

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.


Subject(s)
Fatty Liver , Insulin Resistance , Humans , Mice , Animals , Up-Regulation , Insulin , TOR Serine-Threonine Kinases , Endoplasmic Reticulum Stress , Autophagy , Muscle, Skeletal , Diet, High-Fat/adverse effects , Obesity , Lipids , Mice, Inbred C57BL , Mammals
2.
Nat Commun ; 12(1): 4089, 2021 07 02.
Article in English | MEDLINE | ID: mdl-34215733

ABSTRACT

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.


Subject(s)
Genetic Heterogeneity/drug effects , Glioma/drug therapy , Glioma/genetics , Animals , Brain Neoplasms , Cell Line, Tumor , Cell Proliferation , Child , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Glioma/pathology , High-Throughput Screening Assays , Humans , Mice , Mutation , Protein Kinase Inhibitors/therapeutic use , TOR Serine-Threonine Kinases , Xenograft Model Antitumor Assays
3.
Adipocyte ; 9(1): 576-586, 2020 12.
Article in English | MEDLINE | ID: mdl-32954935

ABSTRACT

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.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Adipocytes/metabolism , Calcium-Binding Proteins/genetics , Cell Adhesion Molecules/genetics , Heme Oxygenase-1/metabolism , Insulin Resistance/genetics , Lipid Metabolism , Signal Transduction , 3T3-L1 Cells , Animals , Calcium-Binding Proteins/metabolism , Cell Adhesion Molecules/metabolism , Exercise , Gene Silencing , Humans , Inflammation/etiology , Inflammation/metabolism , Inflammation/pathology , Insulin/metabolism , Mice , Muscle, Skeletal/metabolism
4.
Cancer Cell ; 35(1): 140-155.e7, 2019 01 14.
Article in English | MEDLINE | ID: mdl-30595505

ABSTRACT

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.


Subject(s)
Brain Stem Neoplasms/genetics , Gene Expression Profiling/methods , Glioma/genetics , Histones/genetics , Receptor, Platelet-Derived Growth Factor alpha/genetics , Tumor Suppressor Protein p53/genetics , Animals , Cell Self Renewal , Cells, Cultured , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Histones/metabolism , Humans , Mice , Mutation , Neural Stem Cells/cytology , Rhombencephalon/pathology , Sequence Analysis, RNA/methods
5.
Biochim Biophys Acta ; 1863(6 Pt A): 1307-18, 2016 Jun.
Article in English | MEDLINE | ID: mdl-27033521

ABSTRACT

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.


Subject(s)
Cell Cycle Checkpoints/genetics , Cell Cycle/genetics , Cilia/metabolism , Genome, Human/genetics , RNA Interference , Transcriptome/genetics , Blotting, Western , Cell Cycle Checkpoints/drug effects , Cell Line , Cilia/physiology , Cluster Analysis , Culture Media, Serum-Free/pharmacology , Gene Expression Profiling/methods , Gene Regulatory Networks/genetics , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Confocal , Models, Genetic , Morphogenesis/genetics , Proteome/genetics , Proteome/metabolism , Reverse Transcriptase Polymerase Chain Reaction
6.
Proc Natl Acad Sci U S A ; 112(30): E4055-64, 2015 Jul 28.
Article in English | MEDLINE | ID: mdl-26159421

ABSTRACT

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.


Subject(s)
Brain Neoplasms/metabolism , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Glioblastoma/metabolism , Histone Demethylases/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Animals , Cell Line, Tumor , Cell Transformation, Neoplastic , Gene Expression Profiling , Gene Silencing , Humans , Male , Mice , Mice, Inbred C57BL , Neoplasm Transplantation , Neoplasms/metabolism , Stochastic Processes
7.
Oncotarget ; 6(17): 14766-76, 2015 Jun 20.
Article in English | MEDLINE | ID: mdl-25885522

ABSTRACT

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.


Subject(s)
Brain Neoplasms/therapy , Drug Delivery Systems/methods , Glioblastoma/therapy , Nanoparticles/administration & dosage , RNA, Small Interfering/administration & dosage , RNAi Therapeutics/methods , Animals , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cell Line, Tumor , Female , Glioblastoma/genetics , Glioblastoma/pathology , Humans , Magnetic Resonance Imaging , Mice, Nude , Microscopy, Confocal , Nanoparticles/chemistry , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , Tumor Burden , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
8.
Oncotarget ; 5(20): 9703-9, 2014 Oct 30.
Article in English | MEDLINE | ID: mdl-25210852

ABSTRACT

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.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Brain Neoplasms/drug therapy , Dacarbazine/analogs & derivatives , Glioblastoma/drug therapy , Nanostructures/administration & dosage , Phosphatidylserines/pharmacology , Saposins/pharmacology , Animals , Antineoplastic Agents, Alkylating/administration & dosage , Antineoplastic Agents, Alkylating/pharmacology , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Death/drug effects , Cell Line, Tumor , Dacarbazine/administration & dosage , Dacarbazine/pharmacology , Drug Synergism , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Lysosomes/drug effects , Mice , Mice, Nude , Random Allocation , Saposins/administration & dosage , Temozolomide , Xenograft Model Antitumor Assays
9.
J Neurosci Res ; 92(11): 1419-24, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25043479

ABSTRACT

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.


Subject(s)
Glioma/pathology , Inactivation, Metabolic/physiology , Neoplastic Stem Cells/metabolism , Oxidative Stress/drug effects , Animals , Antioxidants/pharmacology , Antioxidants/therapeutic use , Glioma/drug therapy , Humans , Inactivation, Metabolic/drug effects , Neoplastic Stem Cells/drug effects , Peroxiredoxins/metabolism , Reactive Oxygen Species/metabolism
10.
Neuro Oncol ; 16(10): 1354-64, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24879047

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/administration & dosage , Brain Neoplasms/drug therapy , Dioxolanes/administration & dosage , Endoplasmic Reticulum Stress/drug effects , Glioma/drug therapy , Peroxiredoxins/metabolism , Animals , Apoptosis/drug effects , Brain Neoplasms/metabolism , Brain Neoplasms/mortality , Databases, Factual , Glioma/metabolism , Glioma/mortality , Humans , Mice , Reactive Oxygen Species/metabolism , Survival Analysis , Tumor Cells, Cultured
11.
J Vis Exp ; (87)2014 May 02.
Article in English | MEDLINE | ID: mdl-24837630

ABSTRACT

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.


Subject(s)
Arthritis/diagnosis , Brain Neoplasms/diagnosis , Fluorescent Dyes/administration & dosage , Nanostructures/administration & dosage , Optics and Photonics/methods , Phosphatidylserines/administration & dosage , Saposins/administration & dosage , Absorption , Animals , Arthritis/metabolism , Arthritis/pathology , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Female , Fluorescent Dyes/pharmacokinetics , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Nude , Mice, Transgenic , Optical Imaging , Optics and Photonics/instrumentation , Whole Body Imaging
12.
Oncotarget ; 5(4): 882-93, 2014 Feb 28.
Article in English | MEDLINE | ID: mdl-24658464

ABSTRACT

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.


Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/metabolism , ErbB Receptors/metabolism , Glioblastoma/genetics , Glioblastoma/metabolism , RNA, Small Interfering/genetics , Receptors, Dopamine D2/metabolism , Animals , Brain Neoplasms/pathology , Cell Line, Tumor , Glioblastoma/pathology , Heterografts , Humans , Mice , Mice, Nude , Phosphorylation , RNA, Small Interfering/metabolism , Signal Transduction , Transfection
13.
Clin Cancer Res ; 20(15): 3989-4000, 2014 Aug 01.
Article in English | MEDLINE | ID: mdl-24677374

ABSTRACT

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.


Subject(s)
Cytotoxicity, Immunologic/immunology , Genetic Engineering , Multiple Myeloma/prevention & control , Receptors, Antigen, T-Cell/immunology , Receptors, Immunologic/genetics , T-Lymphocytes/immunology , Animals , Blotting, Western , Flow Cytometry , Humans , Interleukin Receptor Common gamma Subunit , Male , Mice , Mice, Inbred NOD , Mice, SCID , Multiple Myeloma/immunology , Multiple Myeloma/pathology , Receptors, Antigen, T-Cell/genetics , Receptors, Immunologic/immunology , Signaling Lymphocytic Activation Molecule Family , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
14.
Cancer Res ; 74(6): 1752-65, 2014 Mar 15.
Article in English | MEDLINE | ID: mdl-24453002

ABSTRACT

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.


Subject(s)
Brain Neoplasms/enzymology , Glioblastoma/enzymology , Protein-Arginine N-Methyltransferases/genetics , Tumor Suppressor Proteins/genetics , Animals , Apoptosis , Brain Neoplasms/mortality , Brain Neoplasms/therapy , Cell Line, Tumor , Cell Proliferation , Gene Expression , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Glioblastoma/mortality , Glioblastoma/therapy , Humans , Kaplan-Meier Estimate , Mice , Mice, Knockout , Mice, Nude , Molecular Targeted Therapy , Neoplasm Transplantation , Protein-Arginine N-Methyltransferases/metabolism , RNA, Small Interfering/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Proteins/metabolism
15.
Oncoimmunology ; 2(9): e25620, 2013 Sep 01.
Article in English | MEDLINE | ID: mdl-24319635

ABSTRACT

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.

16.
Cancer Res ; 73(11): 3441-50, 2013 Jun 01.
Article in English | MEDLINE | ID: mdl-23729642

ABSTRACT

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.


Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/virology , Cytomegalovirus Infections/pathology , Cytomegalovirus/genetics , Glioblastoma/genetics , Glioblastoma/virology , Suppression, Genetic , Animals , Brain Diseases/genetics , Brain Diseases/metabolism , Brain Diseases/pathology , Brain Diseases/virology , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cytomegalovirus/metabolism , Cytomegalovirus/physiology , Cytomegalovirus Infections/genetics , Cytomegalovirus Infections/virology , Female , Glioblastoma/metabolism , Glioblastoma/pathology , Heterografts , Humans , Male , Mice , NIH 3T3 Cells , Pregnancy , STAT3 Transcription Factor/metabolism , Signal Transduction , Virus Replication
17.
Mol Ther ; 21(8): 1517-25, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23732993

ABSTRACT

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.


Subject(s)
Angiogenesis Inhibitors/administration & dosage , Antineoplastic Agents/administration & dosage , Brain Neoplasms/metabolism , Glioblastoma/metabolism , Nanoparticles/administration & dosage , Phosphatidylserines/chemistry , Saposins/metabolism , Animals , Blood-Brain Barrier/metabolism , Brain Neoplasms/drug therapy , Brain Neoplasms/mortality , Brain Neoplasms/pathology , Cell Hypoxia , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/metabolism , Disease Models, Animal , Female , Glioblastoma/drug therapy , Glioblastoma/mortality , Glioblastoma/pathology , Humans , Male , Mice , Nanoparticles/chemistry , Neovascularization, Physiologic/drug effects , Recombinant Proteins/administration & dosage , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saposins/administration & dosage , Saposins/chemistry , Xenograft Model Antitumor Assays
18.
Neuro Oncol ; 15(9): 1212-24, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23733246

ABSTRACT

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.


Subject(s)
Brain Neoplasms/metabolism , Glioma/metabolism , MicroRNAs/metabolism , Neoplastic Stem Cells/metabolism , Polycomb-Group Proteins/metabolism , Animals , Brain Neoplasms/genetics , Glioma/genetics , Humans , Mice , Neoplasm Proteins , Polycomb Repressive Complex 1/metabolism , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism , Transcription Factors
19.
Clin Cancer Res ; 19(3): 631-42, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23251006

ABSTRACT

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.


Subject(s)
Glioblastoma/metabolism , Inhibitor of Apoptosis Proteins/metabolism , Neoplastic Stem Cells/metabolism , ran GTP-Binding Protein/metabolism , Apoptosis , Cell Line, Tumor , Cell Proliferation , Cell Survival , Drug Resistance, Neoplasm , Glioblastoma/mortality , Glioblastoma/surgery , Humans , Inhibitor of Apoptosis Proteins/chemistry , Models, Molecular , Prognosis , Protein Binding/drug effects , Protein Conformation , Protein Multimerization , Signal Transduction , Survivin , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor Assays , ran GTP-Binding Protein/chemistry
20.
Cancer Res ; 72(22): 5669-74, 2012 Nov 15.
Article in English | MEDLINE | ID: mdl-23002204

ABSTRACT

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.


Subject(s)
Cytomegalovirus Infections/genetics , Cytomegalovirus Infections/pathology , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma/virology , Tumor Suppressor Protein p53/genetics , Animals , Antigens, Viral/biosynthesis , Cytomegalovirus/genetics , Cytomegalovirus/immunology , Cytomegalovirus/isolation & purification , Cytomegalovirus Infections/metabolism , DNA, Viral/analysis , Gene Expression , Mice , Mice, Transgenic , Muscle Neoplasms/genetics , Muscle Neoplasms/metabolism , Muscle Neoplasms/virology , Neoplasms, Connective Tissue/genetics , Neoplasms, Connective Tissue/metabolism , Neoplasms, Connective Tissue/virology , Rhabdomyosarcoma/metabolism , Tumor Suppressor Protein p53/biosynthesis
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