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2.
Genes Dev ; 29(7): 732-45, 2015 Apr 01.
Article in English | MEDLINE | ID: mdl-25838542

ABSTRACT

Glioblastoma multiforme (GBM) is a lethal, therapy-resistant brain cancer consisting of numerous tumor cell subpopulations, including stem-like glioma-initiating cells (GICs), which contribute to tumor recurrence following initial response to therapy. Here, we identified miR-182 as a regulator of apoptosis, growth, and differentiation programs whose expression level is correlated with GBM patient survival. Repression of Bcl2-like12 (Bcl2L12), c-Met, and hypoxia-inducible factor 2α (HIF2A) is of central importance to miR-182 anti-tumor activity, as it results in enhanced therapy susceptibility, decreased GIC sphere size, expansion, and stemness in vitro. To evaluate the tumor-suppressive function of miR-182 in vivo, we synthesized miR-182-based spherical nucleic acids (182-SNAs); i.e., gold nanoparticles covalently functionalized with mature miR-182 duplexes. Intravenously administered 182-SNAs penetrated the blood-brain/blood-tumor barriers (BBB/BTB) in orthotopic GBM xenografts and selectively disseminated throughout extravascular glioma parenchyma, causing reduced tumor burden and increased animal survival. Our results indicate that harnessing the anti-tumor activities of miR-182 via safe and robust delivery of 182-SNAs represents a novel strategy for therapeutic intervention in GBM.


Subject(s)
Apoptosis/genetics , Cell Differentiation/genetics , Glioblastoma/genetics , MicroRNAs/metabolism , Animals , Antineoplastic Agents/therapeutic use , Brain Neoplasms/drug therapy , Brain Neoplasms/genetics , Brain Neoplasms/physiopathology , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic , Glioblastoma/drug therapy , Glioblastoma/physiopathology , Humans , Mice , Mice, SCID , MicroRNAs/administration & dosage , MicroRNAs/genetics , Muscle Proteins/genetics , Muscle Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Survival Analysis
3.
Proc Natl Acad Sci U S A ; 114(31): 8366-8371, 2017 08 01.
Article in English | MEDLINE | ID: mdl-28716909

ABSTRACT

CD44 has been postulated as a cell surface coreceptor for augmenting receptor tyrosine kinase (RTK) signaling. However, how exactly CD44 triggers RTK-dependent signaling remained largely unclear. Here we report an unexpected mechanism by which the CD44s splice isoform is internalized into endosomes to attenuate EGFR degradation. We identify a CD44s-interacting small GTPase, Rab7A, and show that CD44s inhibits Rab7A-mediated EGFR trafficking to lysosomes and subsequent degradation. Importantly, CD44s levels correlate with EGFR signature and predict poor prognosis in glioblastomas. Because Rab7A facilitates trafficking of many RTKs to lysosomes, our findings identify CD44s as a Rab7A regulator to attenuate RTK degradation.


Subject(s)
Endosomes/metabolism , ErbB Receptors/metabolism , Glioblastoma/pathology , Hyaluronan Receptors/metabolism , rab GTP-Binding Proteins/metabolism , Cell Line , ErbB Receptors/antagonists & inhibitors , Glioblastoma/genetics , HEK293 Cells , Humans , Hyaluronan Receptors/genetics , Lysosomes/metabolism , Protein Isoforms/genetics , Protein Transport/genetics , Protein Transport/physiology , Signal Transduction/genetics , rab GTP-Binding Proteins/antagonists & inhibitors , rab7 GTP-Binding Proteins
4.
Proc Natl Acad Sci U S A ; 114(16): 4129-4134, 2017 04 18.
Article in English | MEDLINE | ID: mdl-28373576

ABSTRACT

RNA interference (RNAi)-based gene regulation platforms have shown promise as a novel class of therapeutics for the precision treatment of cancer. Techniques in preclinical evaluation of RNAi-based nanoconjugates have yet to allow for optimization of their gene regulatory activity. We have developed spherical nucleic acids (SNAs) as a blood-brain barrier-/blood-tumor barrier-penetrating nanoconjugate to deliver small interfering (si) and micro (mi)RNAs to intracranial glioblastoma (GBM) tumor sites. To identify high-activity SNA conjugates and to determine optimal SNA treatment regimens, we developed a reporter xenograft model to evaluate SNA efficacy in vivo. Engrafted tumors stably coexpress optical reporters for luciferase and a near-infrared (NIR) fluorescent protein (iRFP670), with the latter fused to the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT). Using noninvasive imaging of animal subjects bearing reporter-modified intracranial xenografts, we quantitatively assessed MGMT knockdown by SNAs composed of MGMT-targeting siRNA duplexes (siMGMT-SNAs). We show that systemic administration of siMGMT-SNAs via single tail vein injection is capable of robust intratumoral MGMT protein knockdown in vivo, with persistent and SNA dose-dependent MGMT silencing confirmed by Western blotting of tumor tissue ex vivo. Analyses of SNA biodistribution and pharmacokinetics revealed rapid intratumoral uptake and significant intratumoral retention that increased the antitumor activity of coadministered temozolomide (TMZ). Our study demonstrates that dual noninvasive bioluminescence and NIR fluorescence imaging of cancer xenograft models represents a powerful in vivo strategy to identify RNAi-based nanotherapeutics with potent gene silencing activity and will inform additional preclinical and clinical investigations of these constructs.


Subject(s)
Brain Neoplasms/drug therapy , DNA Modification Methylases/antagonists & inhibitors , DNA Repair Enzymes/antagonists & inhibitors , Glioblastoma/drug therapy , Nanoconjugates/administration & dosage , RNA, Small Interfering/genetics , Tumor Suppressor Proteins/antagonists & inhibitors , Animals , Antineoplastic Agents, Alkylating/pharmacology , Brain Neoplasms/genetics , Brain Neoplasms/pathology , DNA Modification Methylases/genetics , DNA Repair Enzymes/genetics , Dacarbazine/analogs & derivatives , Dacarbazine/pharmacology , Female , Fluorescence , Glioblastoma/genetics , Glioblastoma/pathology , Humans , Mice , Mice, SCID , Nanoconjugates/chemistry , RNA Interference , Temozolomide , Tumor Cells, Cultured , Tumor Suppressor Proteins/genetics , Xenograft Model Antitumor Assays
5.
Proc Natl Acad Sci U S A ; 111(15): 5682-7, 2014 Apr 15.
Article in English | MEDLINE | ID: mdl-24706805

ABSTRACT

Therapy resistance is a major limitation to the successful treatment of cancer. Here, we identify Bcl2-like 13 (Bcl2L13), an atypical member of the Bcl-2 family, as a therapy susceptibility gene with elevated expression in solid and blood cancers, including glioblastoma (GBM). We demonstrate that mitochondria-associated Bcl2L13 inhibits apoptosis induced by a wide spectrum of chemo- and targeted therapies upstream of Bcl2-associated X protein activation and mitochondrial outer membrane permeabilization in vitro and promotes GBM tumor growth in vivo. Mechanistically, Bcl2L13 binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6) via a unique C-terminal 250-aa sequence located between its Bcl-2 homology and membrane anchor domains and blocks homo- and heteromeric CerS2/6 complex formation and activity. Correspondingly, CerS2/6 activity and Bcl2L13 abundance are inversely correlated in GBM tumors. Thus, our genetic and functional studies identify Bcl2L13 as a regulator of therapy susceptibility and point to the Bcl2L13-CerS axis as a promising target to enhance responses of therapy-refractory cancers toward conventional and targeted regimens currently in clinical use.


Subject(s)
Drug Resistance/genetics , Gene Expression Regulation, Enzymologic/physiology , Glioblastoma/enzymology , Oxidoreductases/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/metabolism , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Blotting, Western , Cloning, Molecular , Computational Biology , DNA Primers/genetics , Gene Library , Glioblastoma/drug therapy , Humans , Membrane Proteins/metabolism , Polymerase Chain Reaction , Saccharomyces cerevisiae , Sphingosine N-Acyltransferase/metabolism , Tumor Suppressor Proteins/metabolism , Two-Hybrid System Techniques
6.
Mamm Genome ; 23(5-6): 346-55, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22258617

ABSTRACT

Genome-wide mutagenesis was performed in mice to identify candidate genes for male infertility, for which the predominant causes remain idiopathic. Mice were mutagenized using N-ethyl-N-nitrosourea (ENU), bred, and screened for phenotypes associated with the male urogenital system. Fifteen heritable lines were isolated and chromosomal loci were assigned using low-density genome-wide SNP arrays. Ten of the 15 lines were pursued further using higher-resolution SNP analysis to narrow the candidate gene regions. Exon sequencing of candidate genes identified mutations in mice with cystic kidneys (Bicc1), cryptorchidism (Rxfp2), restricted germ cell deficiency (Plk4), and severe germ cell deficiency (Prdm9). In two other lines with severe hypogonadism, candidate sequencing failed to identify mutations, suggesting defects in genes with previously undocumented roles in gonadal function. These genomic intervals were sequenced in their entirety and a candidate mutation was identified in SnrpE in one of the two lines. The line harboring the SnrpE variant retains substantial spermatogenesis despite small testis size, an unusual phenotype. In addition to the reproductive defects, heritable phenotypes were observed in mice with ataxia (Myo5a), tremors (Pmp22), growth retardation (unknown gene), and hydrocephalus (unknown gene). These results demonstrate that the ENU screen is an effective tool for identifying potential causes of male infertility.


Subject(s)
Ethylnitrosourea/toxicity , Hypogonadism/genetics , Infertility, Male/genetics , Mutagenesis , Animals , Humans , Male , Mice , Mice, Inbred C57BL , Proteins/genetics
7.
Sci Transl Med ; 13(584)2021 03 10.
Article in English | MEDLINE | ID: mdl-33692132

ABSTRACT

Glioblastoma (GBM) is one of the most difficult cancers to effectively treat, in part because of the lack of precision therapies and limited therapeutic access to intracranial tumor sites due to the presence of the blood-brain and blood-tumor barriers. We have developed a precision medicine approach for GBM treatment that involves the use of brain-penetrant RNA interference-based spherical nucleic acids (SNAs), which consist of gold nanoparticle cores covalently conjugated with radially oriented and densely packed small interfering RNA (siRNA) oligonucleotides. On the basis of previous preclinical evaluation, we conducted toxicology and toxicokinetic studies in nonhuman primates and a single-arm, open-label phase 0 first-in-human trial (NCT03020017) to determine safety, pharmacokinetics, intratumoral accumulation and gene-suppressive activity of systemically administered SNAs carrying siRNA specific for the GBM oncogene Bcl2Like12 (Bcl2L12). Patients with recurrent GBM were treated with intravenous administration of siBcl2L12-SNAs (drug moniker: NU-0129), at a dose corresponding to 1/50th of the no-observed-adverse-event level, followed by tumor resection. Safety assessment revealed no grade 4 or 5 treatment-related toxicities. Inductively coupled plasma mass spectrometry, x-ray fluorescence microscopy, and silver staining of resected GBM tissue demonstrated that intravenously administered SNAs reached patient tumors, with gold enrichment observed in the tumor-associated endothelium, macrophages, and tumor cells. NU-0129 uptake into glioma cells correlated with a reduction in tumor-associated Bcl2L12 protein expression, as indicated by comparison of matched primary tumor and NU-0129-treated recurrent tumor. Our results establish SNA nanoconjugates as a potential brain-penetrant precision medicine approach for the systemic treatment of GBM.


Subject(s)
Brain Neoplasms , Glioblastoma , Metal Nanoparticles , Nucleic Acids , Brain Neoplasms/genetics , Brain Neoplasms/therapy , Glioblastoma/genetics , Glioblastoma/therapy , Gold , Humans , Muscle Proteins/metabolism , Neoplasm Recurrence, Local , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA Interference
8.
Sci Adv ; 5(1): eaat0456, 2019 01.
Article in English | MEDLINE | ID: mdl-30613765

ABSTRACT

Mutation or transcriptional up-regulation of isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) promotes cancer progression through metabolic reprogramming and epigenetic deregulation of gene expression. Here, we demonstrate that IDH3α, a subunit of the IDH3 heterotetramer, is elevated in glioblastoma (GBM) patient samples compared to normal brain tissue and promotes GBM progression in orthotopic glioma mouse models. IDH3α loss of function reduces tricarboxylic acid (TCA) cycle turnover and inhibits oxidative phosphorylation. In addition to its impact on mitochondrial energy metabolism, IDH3α binds to cytosolic serine hydroxymethyltransferase (cSHMT). This interaction enhances nucleotide availability during DNA replication, while the absence of IDH3α promotes methionine cycle activity, S-adenosyl methionine generation, and DNA methylation. Thus, the regulation of one-carbon metabolism via an IDH3α-cSHMT signaling axis represents a novel mechanism of metabolic adaptation in GBM.


Subject(s)
Brain Neoplasms/metabolism , Glioblastoma/metabolism , Glycine Hydroxymethyltransferase/metabolism , Isocitrate Dehydrogenase/metabolism , Animals , Brain Neoplasms/genetics , Cell Line, Tumor , Citric Acid Cycle/genetics , Cytosol/metabolism , DNA Methylation/genetics , Female , Glioblastoma/genetics , HEK293 Cells , Heterografts , Humans , Isocitrate Dehydrogenase/genetics , Mice , Mice, SCID , Oxidative Phosphorylation , S Phase Cell Cycle Checkpoints , Transfection
9.
Endocrinology ; 149(12): 6198-206, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18719025

ABSTRACT

The estrogen receptor-alpha (ERalpha) acts through multiple pathways, including estrogen response element (ERE)-dependent (classical) and ERE-independent (nonclassical) mechanisms. We previously created a mouse model harboring a two-amino-acid mutation of the DNA-binding domain (E207A, G208A) that precludes direct binding of ERalpha to an ERE. After crossing heterozygous mutant mice with an ERalpha knockout (ERKO) line, it was possible to assess the degree of physiological rescue by the isolated ERalpha nonclassical allele (-/AA; AA) when compared with ERKO mice (-/-) and to wild type (+/+; WT). In male ERKO mice up to 8 months of age, testosterone levels were high, although LH levels were similar to WT. Testosterone was normal in the AA mice, indicating that the AA allele rescues the enhanced testosterone biosynthesis in ERKO mice. Male ERKO mice exhibited distention of the seminiferous tubules as early as 2-3 months of age as a consequence of decreased water resorption in the efferent ducts. By 3-4 months of age, ERKO mice had impaired spermatogenesis in approximately 40% of their tubules, and sperm counts and motility declined in association with the histological changes. In the AA mice, histological defects were greatly reduced or absent, and sperm counts and motility were rescued. Levels of aquaporins 1 and 9, which contribute to water uptake in the efferent ducts, were reduced in ERKO mice and partially or fully rescued in AA mice, whereas another water transporter, sodium-hydrogen exchanger-3, was decreased in both ERKO and AA mice. We conclude that non-ERE-dependent estrogen pathways are sufficient to rescue the defective spermatogenesis observed in ERKO mice and play a prominent role in ERalpha action in the testis, including pathways that regulate water resorption and androgen biosynthesis.


Subject(s)
Estrogen Receptor alpha/physiology , Estrogens/pharmacology , Response Elements/genetics , Seminiferous Tubules/drug effects , Signal Transduction/drug effects , Animals , Blotting, Western , Estrogen Receptor alpha/genetics , Follicle Stimulating Hormone/blood , Immunohistochemistry , Luteinizing Hormone/blood , Male , Mice , Mice, Knockout , Mutation , Seminiferous Tubules/cytology , Seminiferous Tubules/metabolism , Signal Transduction/genetics , Signal Transduction/physiology , Sperm Count , Sperm Motility , Spermatogenesis/drug effects , Testosterone/blood
10.
J Nutr Biochem ; 18(4): 250-8, 2007 Apr.
Article in English | MEDLINE | ID: mdl-16781858

ABSTRACT

A number of studies have investigated the effects of fish oil on the production of pro-inflammatory cytokines using peripheral blood mononuclear cell models. The majority of these studies have employed heterogeneous blends of long-chain n-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which preclude examination of the individual effects of LC n-3 PUFA. This study investigated the differential effects of pure EPA and DHA on cytokine expression and nuclear factor kappaB (NF-kappaB) activation in human THP-1 monocyte-derived macrophages. Pretreatment with 100 microM EPA and DHA significantly decreased lipopolysaccharide (LPS)-stimulated THP-1 macrophage tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta and IL-6 production (P<.02), compared to control cells. Both EPA and DHA reduced TNF-alpha, IL-1beta and IL-6 mRNA expression. In all cases, the effect of DHA was significantly more potent than that of EPA (P<.01). Furthermore, a low dose (25 microM) of DHA had a greater inhibitory effect than that of EPA on macrophage IL-1beta (P<.01 and P<.04, respectively) and IL-6 (P<.003 and P<.003, respectively) production following 0.01 and 0.1 microg/ml LPS stimulation. Both EPA and DHA down-regulated LPS-induced NF-kappaB/DNA binding in THP-1 macrophages by approximately 13% (P< or =.03). DHA significantly decreased macrophage nuclear p65 expression (P< or =.05) and increased cytoplasmic IkappaBalpha expression (P< or =.05). Although similar trends were observed with EPA, they were not significant. Our findings suggest that DHA may be more effective than EPA in alleviating LPS-induced pro-inflammatory cytokine production in macrophages - an effect that may be partly mediated by NF-kappaB. Further work is required to elucidate additional divergent mechanisms to account for apparent differences between EPA and DHA.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Docosahexaenoic Acids/pharmacology , Eicosapentaenoic Acid/pharmacology , Inflammation/prevention & control , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/physiology , Cell Line , Dose-Response Relationship, Drug , Humans , I-kappa B Proteins/biosynthesis , Interleukin-1beta/biosynthesis , Interleukin-6/biosynthesis , NF-KappaB Inhibitor alpha , NF-kappa B/metabolism , RNA, Messenger/metabolism , Tumor Necrosis Factor-alpha/biosynthesis
11.
Cell Rep ; 19(9): 1858-1873, 2017 05 30.
Article in English | MEDLINE | ID: mdl-28564604

ABSTRACT

Oncogenic mutations in two isocitrate dehydrogenase (IDH)-encoding genes (IDH1 and IDH2) have been identified in acute myelogenous leukemia, low-grade glioma, and secondary glioblastoma (GBM). Our in silico and wet-bench analyses indicate that non-mutated IDH1 mRNA and protein are commonly overexpressed in primary GBMs. We show that genetic and pharmacologic inactivation of IDH1 decreases GBM cell growth, promotes a more differentiated tumor cell state, increases apoptosis in response to targeted therapies, and prolongs the survival of animal subjects bearing patient-derived xenografts (PDXs). On a molecular level, diminished IDH1 activity results in reduced α-ketoglutarate (αKG) and NADPH production, paralleled by deficient carbon flux from glucose or acetate into lipids, exhaustion of reduced glutathione, increased levels of reactive oxygen species (ROS), and enhanced histone methylation and differentiation marker expression. These findings suggest that IDH1 upregulation represents a common metabolic adaptation by GBMs to support macromolecular synthesis, aggressive growth, and therapy resistance.


Subject(s)
Drug Resistance, Neoplasm , Glioblastoma/enzymology , Glioblastoma/pathology , Isocitrate Dehydrogenase/genetics , Molecular Targeted Therapy , Mutation/genetics , Animals , Apoptosis/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Disease Progression , Drug Resistance, Neoplasm/drug effects , Erlotinib Hydrochloride/pharmacology , Erlotinib Hydrochloride/therapeutic use , Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Glioblastoma/drug therapy , Glioblastoma/genetics , Histones/metabolism , Isocitrate Dehydrogenase/metabolism , Ketoglutaric Acids/metabolism , Lipids/biosynthesis , Methylation , Mice , Mice, SCID , NADP/metabolism , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/pathology , Protein Kinase Inhibitors/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism
12.
Sci Transl Med ; 5(209): 209ra152, 2013 Oct 30.
Article in English | MEDLINE | ID: mdl-24174328

ABSTRACT

Glioblastoma multiforme (GBM) is a neurologically debilitating disease that culminates in death 14 to 16 months after diagnosis. An incomplete understanding of how cataloged genetic aberrations promote therapy resistance, combined with ineffective drug delivery to the central nervous system, has rendered GBM incurable. Functional genomics efforts have implicated several oncogenes in GBM pathogenesis but have rarely led to the implementation of targeted therapies. This is partly because many "undruggable" oncogenes cannot be targeted by small molecules or antibodies. We preclinically evaluate an RNA interference (RNAi)-based nanomedicine platform, based on spherical nucleic acid (SNA) nanoparticle conjugates, to neutralize oncogene expression in GBM. SNAs consist of gold nanoparticles covalently functionalized with densely packed, highly oriented small interfering RNA duplexes. In the absence of auxiliary transfection strategies or chemical modifications, SNAs efficiently entered primary and transformed glial cells in vitro. In vivo, the SNAs penetrated the blood-brain barrier and blood-tumor barrier to disseminate throughout xenogeneic glioma explants. SNAs targeting the oncoprotein Bcl2Like12 (Bcl2L12)--an effector caspase and p53 inhibitor overexpressed in GBM relative to normal brain and low-grade astrocytomas--were effective in knocking down endogenous Bcl2L12 mRNA and protein levels, and sensitized glioma cells toward therapy-induced apoptosis by enhancing effector caspase and p53 activity. Further, systemically delivered SNAs reduced Bcl2L12 expression in intracerebral GBM, increased intratumoral apoptosis, and reduced tumor burden and progression in xenografted mice, without adverse side effects. Thus, silencing antiapoptotic signaling using SNAs represents a new approach for systemic RNAi therapy for GBM and possibly other lethal malignancies.


Subject(s)
Brain Neoplasms/therapy , Glioblastoma/therapy , Nanoparticles/chemistry , Nucleic Acids/chemistry , RNA Interference , Animals , Apoptosis , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/pathology , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Line, Tumor , Down-Regulation , Female , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Mice , Mice, SCID , Muscle Proteins/metabolism , Nucleic Acids/administration & dosage , Proto-Oncogene Proteins c-bcl-2/metabolism , Tumor Burden , Xenograft Model Antitumor Assays
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