RESUMO
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.
Assuntos
Apoptose/genética , Diferenciação Celular/genética , Glioblastoma/genética , MicroRNAs/metabolismo , Animais , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/fisiopatologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/fisiopatologia , Humanos , Camundongos , Camundongos SCID , MicroRNAs/administração & dosagem , MicroRNAs/genética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Análise de SobrevidaRESUMO
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.
Assuntos
Endossomos/metabolismo , Receptores ErbB/metabolismo , Glioblastoma/patologia , Receptores de Hialuronatos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Linhagem Celular , Receptores ErbB/antagonistas & inibidores , Glioblastoma/genética , Células HEK293 , Humanos , Receptores de Hialuronatos/genética , Lisossomos/metabolismo , Isoformas de Proteínas/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , Transdução de Sinais/genética , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , proteínas de unión al GTP Rab7RESUMO
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.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Metilases de Modificação do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/antagonistas & inibidores , Glioblastoma/tratamento farmacológico , Nanoconjugados/administração & dosagem , RNA Interferente Pequeno/genética , Proteínas Supressoras de Tumor/antagonistas & inibidores , Animais , Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Feminino , Fluorescência , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos , Camundongos SCID , Nanoconjugados/química , Interferência de RNA , Temozolomida , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Resistência a Medicamentos/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Glioblastoma/enzimologia , Oxirredutases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Clonagem Molecular , Biologia Computacional , Primers do DNA/genética , Biblioteca Gênica , Glioblastoma/tratamento farmacológico , Humanos , Proteínas de Membrana/metabolismo , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae , Esfingosina N-Aciltransferase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Despite being the leading cause of childhood mortality, pediatric gliomas have been relatively understudied, and the repurposing of immunotherapies has not been successful. Whole transcriptome sequencing, single-cell sequencing, and sequential multiplex immunofluorescence were used to identify an immunotherapy strategy evaluated in multiple preclinical glioma models. MAPK-driven pediatric gliomas have a higher interferon signature relative to other molecular subgroups. Single-cell sequencing identified an activated and cytotoxic microglia population designated MG-Act in BRAF-fused MAPK-activated pilocytic astrocytoma (PA), but not in high-grade gliomas or normal brain. TIM3 is expressed on MG-Act and on the myeloid cells lining the tumor vasculature but not normal brain. TIM3 expression becomes upregulated on immune cells in the PA microenvironment and anti-TIM3 reprograms ex vivo immune cells from human PAs to a pro-inflammatory cytotoxic phenotype. In a genetically engineered murine model of MAPK-driven low-grade gliomas, anti-TIM3 treatment increased median survival over IgG and anti-PD1 treated mice. ScRNA sequencing data during the therapeutic window of anti-TIM3 demonstrates enrichment of the MG-Act population. The therapeutic activity of anti-TIM3 is abrogated in the CX3CR1 microglia knockout background. These data support the use of anti-TIM3 in clinical trials of pediatric low-grade MAPK-driven gliomas.
RESUMO
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.
Assuntos
Etilnitrosoureia/toxicidade , Hipogonadismo/genética , Infertilidade Masculina/genética , Mutagênese , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas/genéticaRESUMO
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.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Nanopartículas Metálicas , Ácidos Nucleicos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Glioblastoma/genética , Glioblastoma/terapia , Ouro , Humanos , Proteínas Musculares/metabolismo , Recidiva Local de Neoplasia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNARESUMO
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.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Glicina Hidroximetiltransferase/metabolismo , Isocitrato Desidrogenase/metabolismo , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico/genética , Citosol/metabolismo , Metilação de DNA/genética , Feminino , Glioblastoma/genética , Células HEK293 , Xenoenxertos , Humanos , Isocitrato Desidrogenase/genética , Camundongos , Camundongos SCID , Fosforilação Oxidativa , Pontos de Checagem da Fase S do Ciclo Celular , TransfecçãoRESUMO
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.
Assuntos
Receptor alfa de Estrogênio/fisiologia , Estrogênios/farmacologia , Elementos de Resposta/genética , Túbulos Seminíferos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Western Blotting , Receptor alfa de Estrogênio/genética , Hormônio Foliculoestimulante/sangue , Imuno-Histoquímica , Hormônio Luteinizante/sangue , Masculino , Camundongos , Camundongos Knockout , Mutação , Túbulos Seminíferos/citologia , Túbulos Seminíferos/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Contagem de Espermatozoides , Motilidade dos Espermatozoides , Espermatogênese/efeitos dos fármacos , Testosterona/sangueRESUMO
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.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Inflamação/prevenção & controle , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Proteínas I-kappa B/biossíntese , Interleucina-1beta/biossíntese , Interleucina-6/biossíntese , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
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.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Glioblastoma/enzimologia , Glioblastoma/patologia , Isocitrato Desidrogenase/genética , Terapia de Alvo Molecular , Mutação/genética , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Cloridrato de Erlotinib/farmacologia , Cloridrato de Erlotinib/uso terapêutico , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Histonas/metabolismo , Isocitrato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Lipídeos/biossíntese , Metilação , Camundongos , Camundongos SCID , NADP/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.