RESUMO
NF-κB is a key regulator of inflammation and cancer progression, with an important role in leukemogenesis. Despite its therapeutic potential, targeting NF-κB using pharmacologic inhibitors has proven challenging. Here, we describe a myeloid cell-selective NF-κB inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Toll-like receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a, C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6), thereby blocking activation of NF-κB in target cells. IV injections of C-miR146a mimic to miR-146a-deficient mice prevented excessive NF-κB activation in myeloid cells, and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly, C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell-induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions, miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set, we found an inverse correlation of miR-146a levels with NF-κB-related genes and with patient survival. Correspondingly, C-miR146a induced cytotoxic effects in human MDSL, HL-60, and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-κB target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell-targeted miR-146a mimics for the treatment of inflammatory and myeloproliferative disorders.
Assuntos
Síndrome da Liberação de Citocina/prevenção & controle , Inflamação/prevenção & controle , Leucemia Mieloide Aguda/prevenção & controle , MicroRNAs/genética , Células Progenitoras Mieloides/patologia , NF-kappa B/metabolismo , Animais , Apoptose , Proliferação de Células , Síndrome da Liberação de Citocina/genética , Síndrome da Liberação de Citocina/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Células Progenitoras Mieloides/metabolismo , NF-kappa B/genética , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RNA-based therapeutics, i.e. the utilization of synthetic RNA molecules to alter cellular functions, have the potential to address targets which are currently out of scope for traditional drug design pipelines. This potential however hinges on the ability to selectively deliver and internalize therapeutic RNAs into cells of interest. Cell internalizing RNA aptamers selected against surface receptors and discriminatively expressed on target cells hold particular promise as suitable candidates for such delivery agents. Specifically, these aptamers can be combined with a therapeutic cargo and facilitate internalization of the cargo into the cell of interest. A recently proposed method to obtain such aptamer-cargo constructs employs a double-stranded "sticky bridge" where the complementary strands constituting the bridge are conjugated with the aptamer and the cargo respectively. The design of appropriate sticky bridge sequences however has proven highly challenging given the structural and functional constraints imposed on them during synthesis and administration. These include, but are not limited to, guaranteed formation and stability of the complex, non-interference with the aptamer or the cargo, as well as the prevention of spurious aggregation of the molecules during incubation. In order to address these issues, we have developed AptaBlocks - a computational method to design RNA complexes that hybridize via sticky bridges. The effectiveness of our approach has been verified computationally, and experimentally in the context of drug delivery to pancreatic cancer cells. Importantly, AptaBlocks is a general method for the assembly of nucleic acid systems that, in addition to designing of RNA-based drug delivery systems, can be used in other applications of RNA nanotechnology. AptaBlocks is available at https://github.com/wyjhxq/AptaBlocks.
Assuntos
Algoritmos , Aptâmeros de Nucleotídeos/metabolismo , Biologia Computacional/métodos , Sistemas de Liberação de Medicamentos/métodos , Preparações Farmacêuticas/administração & dosagem , RNA/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Linhagem Celular Tumoral , Humanos , Internet , Nanotecnologia/métodos , Neoplasias/genética , Neoplasias/metabolismo , Preparações Farmacêuticas/química , RNA/química , RNA/genética , Reprodutibilidade dos TestesRESUMO
Growing evidence links the aggressiveness of non-Hodgkin's lymphoma, especially the activated B cell-like type diffuse large B cell lymphomas (ABC-DLBCLs) to Toll-like receptor 9 (TLR9)/MyD88 and STAT3 transcription factor signaling. Here, we describe a dual-function molecule consisting of a clinically relevant TLR9 agonist (CpG7909) and a STAT3 inhibitor in the form of a high-affinity decoy oligodeoxynucleotide (dODN). The CpG-STAT3dODN blocked STAT3 DNA binding and activity, thus reducing expression of downstream target genes, such as MYC and BCL2L1, in human and mouse lymphoma cells. We further demonstrated that injections (i.v.) of CpG-STAT3dODN inhibited growth of human OCI-Ly3 lymphoma in immunodeficient mice. Moreover, systemic CpG-STAT3dODN administration induced complete regression of the syngeneic A20 lymphoma, resulting in long-term survival of immunocompetent mice. Both TLR9 stimulation and concurrent STAT3 inhibition were critical for immune-mediated therapeutic effects, since neither CpG7909 alone nor CpG7909 co-injected with unconjugated STAT3dODN extended mouse survival. The CpG-STAT3dODN induced expression of genes critical to antigen-processing/presentation and Th1 cell activation while suppressing survival signaling. These effects resulted in the generation of lymphoma cell-specific CD8/CD4-dependent T cell immunity protecting mice from tumor rechallenge. Our results suggest that CpG-STAT3dODN as a systemic/local monotherapy or in combination with PD1 blockade can provide an opportunity for treating patients with B cell NHL.
Assuntos
Antineoplásicos/farmacologia , Linfoma de Células B/imunologia , Linfoma de Células B/metabolismo , Oligonucleotídeos/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Receptor Toll-Like 9/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Imunoterapia , Linfoma de Células B/patologia , Linfoma de Células B/terapia , Camundongos , Terapia de Alvo Molecular , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Transcrição Gênica , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The promising results of clinical trials using immune checkpoint inhibitors revived interests in cancer immunotherapy. However, it also became apparent that efficacy of immune checkpoint blockade can benefit from combining it with immunostimulatory strategies. Here, we review prior and re-emerging approaches using Toll-like Receptor 9 (TLR9) agonists, CpG oligodeoxynucleotides (ODNs), focused on the generation of antitumor immune responses in cancer patients. While numerous early clinical trials using TLR9 ligands in monotherapies provided evidence of CpG ODNs tolerability and safety, they failed to demonstrate sufficient antitumor efficacy. Recent studies unraveled multiple levels of negative regulation of immunostimulatory TLR9 signaling in immune cells by the tumor microenvironment that can stifle immune activity in cancer patients. Therefore, CpG ODNs-based strategies can greatly benefit from combination with strategies targeting immune checkpoint regulation. The most recent clinical trials of CpG ODNs together with immune checkpoint inhibitors have a chance to generate novel, more effective and safer cancer immunotherapies.
RESUMO
Cervical cancer (CC) is the fourth most common malignant neoplasm among women. Late diagnosis is directly associated with the incidence of metastatic disease and remarkably limits the effectiveness of conventional anticancer therapies at the advanced tumor stage. In this study, we investigated the role of 5'AMP-activated kinase (AMPK) in the metastatic progression of cervical cancer. Since the epithelial mesenchymal transition (EMT) is known as major mechanism enabling cancer cell metastasis, cell lines, which accurately represent this process, have been used as a research model. We used C-4I and HTB-35 cervical cancer cell lines representing distant stages of the disease, in which we genetically modified the expression of the AMPK catalytic subunit α. We have shown that tumor progression leads to metabolic deregulation which results in reduced expression and activity of AMPK. We also demonstrated that AMPK is related to the ability of cells to acquire invasive phenotype and potential for in vivo metastases, and its activity may inhibit these processes. Our findings support the hypothesis that AMPK is a promising therapeutic target and modulation of its expression and activity may improve the efficacy of cervical cancer treatment.
Assuntos
Proteínas Quinases Ativadas por AMP , Neoplasias do Colo do Útero , Humanos , Feminino , Proteínas Quinases Ativadas por AMP/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo do Útero/patologia , Transição Epitelial-Mesenquimal/genética , Metástase NeoplásicaRESUMO
Tropism of neural stem cells (NSCs) to hypoxic tumor areas provides an opportunity for the drug delivery. Here, we demonstrate that NSCs effectively transport antisense oligonucleotides (ASOs) targeting oncogenic and tolerogenic signal transducer and activator of transcription 3 (STAT3) protein into glioma microenvironment. To enable spontaneous, scavenger receptor-mediated endocytosis by NSCs, we used previously described CpG-STAT3ASO conjugates. Following uptake and endosomal escape, CpG-STAT3ASO colocalized with CD63+ vesicles and later with CD63+CD81+ exosomes. Over 3 days, NSCs secreted exosomes loaded up to 80% with CpG-STAT3ASO. Compared to native NSC exosomes, the CpG-STAT3ASO-loaded exosomes potently stimulated immune activity of human dendritic cells or mouse macrophages, inducing nuclear factor κB (NF-κB) signaling and interleukin-12 (IL-12) production. Using orthotopic GL261 tumors, we confirmed that NSC-mediated delivery improved oligonucleotide transfer from a distant injection site into the glioma microenvironment versus naked oligonucleotides. Correspondingly, the NSC-delivered CpG-STAT3ASO enhanced activation of glioma-associated microglia. Finally, we demonstrated that NSC-mediated CpG-STAT3ASO delivery resulted in enhanced antitumor effects against GL261 glioma in mice. Peritumoral injections of 5 × 105 NSCs loaded ex vivo with CpG-STAT3ASO inhibited subcutaneous tumor growth more effectively than the equivalent amount of oligonucleotide alone. Based on these results, we anticipate that NSCs and NSC-derived exosomes will provide a clinically relevant strategy to improve delivery and safety of oligonucleotide therapeutics for glioma treatment.
RESUMO
Both the extracellular matrix (ECM) and DNA epigenetic regulation are critical for maintaining stem cell phenotype and cancer progression. Whether and how ECM regulates epigenetic alterations to influence cancer stem cells (CSCs) remain to be explored. Here we report that ECM through laminin-integrin α6 upregulates ten-eleven translocation enzyme 3 (TET3) dioxygenase. TET3 in turn mediates DNA cytosine 5'-hydroxymethylation (5hmC) and upregulates genes critical for maintenance of glioma stem cells (GSCs). Activating integrin α6-FAK pathway increases STAT3 activity, TET3 expression and 5hmC levels in GSCs. Moreover, targeting STAT3 disrupts integrin α6-FAK signaling and inhibits TET3+ GSC maturation in vivo. STAT3 directly regulates TET3 expression and the two proteins are co-localized with 5hmC in GSC clusters. 5hmC is upregulated by STAT3 at the promoters of several tumorigenic genes, including c-Myc, known to be critical for GSCs. In vivo silencing of TET3 in GSC-enriched tumors reduces 5hmC accumulation and expression of the GSC critical genes, leading to tumor growth inhibition. TET3 expression and 5hmC accumulation also co-segregate with integrin α6 in patient malignant glioma. Thus, ECM- integrin α6-STAT3-TET3 axis regulates hydroxymethylation of genes important for GSCs, thereby increasing GSC tumorigenicity and resistance to therapies.
Assuntos
Metilação de DNA , Dioxigenases/metabolismo , Epigênese Genética , Glioma/genética , Integrina alfa6/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fator de Transcrição STAT3/metabolismo , 5-Metilcitosina , Animais , Linhagem Celular , Dioxigenases/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Glioma/enzimologia , Glioma/metabolismo , Humanos , Masculino , Camundongos Nus , Células-Tronco Neoplásicas/enzimologia , Transdução de SinaisRESUMO
BACKGROUND: In depression, excessive glucocorticoid action may cause maladaptive brain changes, including in the pathways controlling energy metabolism. Insulin and glucagon-like peptide-1 (GLP-1), besides regulation of glucose homeostasis, also possess neurotrophic properties. Current study was aimed at investigating the influence of prenatal stress (PS) on insulin, GLP-1 and their receptor (IR and GLP-1R) levels in the hypothalamus. GLP-1 and GLP-1R were assayed also in the hippocampus and frontal cortex - brain regions mainly affected in depression. The second objective was to determine the influence of exendin-4 and insulin on CRH promoter gene activity in in vitro conditions. METHODS: Adult male PS rats were subjected to acute stress and/or received orally glucose. Levels of hormones and their receptors were assayed with ELISA method. In vitro studies were performed on mHypoA-2/12 hypothalamic cell line, stably transfected with CRH promoter coupled with luciferase. RESULTS: PS has reduced GLP-1 and GLP-1R levels, attenuated glucose-induced increase in insulin concentration and increased the amount of phosphorylated IR in the hypothalamus of animals subjected to additional stress stimuli, and also decreased the GLP-1R level in the hippocampus. In vitro studies demonstrated that insulin is capable of increasing CRH promoter activity in the condition of stimulation of the cAMP/PKA pathway in the applied cellular model. CONCLUSION: Prenatal stress may act as a preconditioning factor, affecting the concentrations of hormones such as insulin and GLP-1 in the hypothalamus in response to adverse stimuli. The decreased GLP-1R level in the hippocampus could be linked with the disturbances in neuronal plasticity.
Assuntos
Depressão/fisiopatologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Hipotálamo/metabolismo , Insulina/metabolismo , Animais , Linhagem Celular , Hormônio Liberador da Corticotropina/genética , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Exenatida/metabolismo , Feminino , Glucose/metabolismo , Masculino , Camundongos , Plasticidade Neuronal/fisiologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Regiões Promotoras Genéticas/genética , Ratos , Ratos Sprague-Dawley , Estresse Psicológico/fisiopatologiaRESUMO
Rhabdomyosarcoma (RMS) is a mesenchymal tumor of soft tissue in children that originates from a myogenic differentiation defect. Expression of SNAIL transcription factor is elevated in the alveolar subtype of RMS (ARMS), characterized by a low myogenic differentiation status and high aggressiveness. In RMS patients SNAIL level increases with higher stage. Moreover, SNAIL level negatively correlates with MYF5 expression. The differentiation of human ARMS cells diminishes SNAIL level. SNAIL silencing in ARMS cells inhibits proliferation and induces differentiation in vitro, and thereby completely abolishes the growth of human ARMS xenotransplants in vivo. SNAIL silencing induces myogenic differentiation by upregulation of myogenic factors and muscle-specific microRNAs, such as miR-206. SNAIL binds to the MYF5 promoter suppressing its expression. SNAIL displaces MYOD from E-box sequences (CANNTG) that are associated with genes expressed during differentiation and G/C rich in their central dinucleotides. SNAIL silencing allows the re-expression of MYF5 and canonical MYOD binding, promoting ARMS cell myogenic differentiation. In differentiating ARMS cells SNAIL forms repressive complex with histone deacetylates 1 and 2 (HDAC1/2) and regulates their expression. Accordingly, in human myoblasts SNAIL silencing induces differentiation by upregulation of myogenic factors. Our data clearly point to SNAIL as a key regulator of myogenic differentiation and a new promising target for future ARMS therapies.
Assuntos
Diferenciação Celular , Proteína MyoD/metabolismo , Fator Regulador Miogênico 5/metabolismo , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/patologia , Fatores de Transcrição da Família Snail/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , DNA de Neoplasias/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Histona Desacetilases/metabolismo , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , MicroRNAs/metabolismo , Desenvolvimento Muscular , Músculos/metabolismo , Músculos/patologia , Fenótipo , Regulação para Cima/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Prostate cancers show remarkable resistance to emerging immunotherapies, partly due to tolerogenic STAT3 signaling in tumor-associated myeloid cells. Here, we describe a novel strategy combining STAT3 inhibition with Toll-like Receptor 9 (TLR9) stimulation to unleash immune response against prostate cancers regardless of the genetic background. EXPERIMENTAL DESIGN: We developed and validated a conjugate of the STAT3 antisense oligonucleotide (ASO) tethered to immunostimulatory TLR9 agonist (CpG oligonucleotide) to improve targeting of human and mouse prostate cancer and myeloid immune cells, such as myeloid-derived suppressor cells (MDSC). RESULTS: CpG-STAT3ASO conjugates showed improved biodistribution and potency of STAT3 knockdown in target cells in vitro and in vivo. Systemic administration of CpG-STAT3ASO (5 mg/kg) eradicated bone-localized, Ras/Myc-driven, and Ptenpc -/- Smad4pc -/- Trp53c -/- prostate tumors in the majority of treated mice. These antitumor effects were primarily immune-mediated and correlated with an increased ratio of CD8+ to regulatory T cells and reduced pSTAT3+/PD-L1+ MDSCs. Both innate and adaptive immunity contributed to systemic antitumor responses as verified by the depletion of Gr1+ myeloid cells and CD8+ and CD4+ T cells, respectively. Importantly, only the bifunctional CpG-STAT3ASO, but not control CpG oligonucleotides, STAT3ASO alone, or the coinjection of both oligonucleotides, succeeded in recruiting neutrophils and CD8+ T cells into tumors. Thus, the concurrence of TLR9 activation with STAT3 inhibition in the same cellular compartment is indispensable for overcoming tumor immune tolerance and effective antitumor immunity against prostate cancer. CONCLUSIONS: The bifunctional, immunostimulatory, and tolerance-breaking design of CpG-STAT3ASO offers a blueprint for the development of effective and safer oligonucleotide strategies for treatment of immunologically "cold" human cancers.
Assuntos
Heterogeneidade Genética , Imunomodulação/efeitos dos fármacos , Oligodesoxirribonucleotídeos/administração & dosagem , Oligonucleotídeos Antissenso/administração & dosagem , Neoplasias de Próstata Resistentes à Castração/etiologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Fator de Transcrição STAT3/genética , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Tolerância Imunológica , Imunofenotipagem , Masculino , Camundongos , Camundongos Transgênicos , Oligodesoxirribonucleotídeos/imunologia , Oligonucleotídeos Antissenso/imunologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia , Fator de Transcrição STAT3/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Receptor Toll-Like 9/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Rhabdomyosarcoma (RMS) is a soft tissue sarcoma, which may originate from impaired differentiation of mesenchymal stem cells (MSC). Expression of MET receptor is elevated in alveolar RMS subtype (ARMS) which is associated with worse prognosis, compared to embryonal RMS (ERMS). Forced differentiation of ARMS cells diminishes MET level and, as shown previously, MET silencing induces differentiation of ARMS. In ERMS cells introduction of TPR-MET oncogene leads to an uncontrolled overstimulation of the MET receptor downstream signaling pathways. In vivo, tumors formed by those cells in NOD-SCID mice display inhibited differentiation, enhanced proliferation, diminished apoptosis and increased infiltration of neutrophils. Consequently, tumors grow significantly faster and they display enhanced ability to metastasize to lungs and to vascularize due to elevated VEGF, MMP9 and miR-378 expression. In vitro, TPR-MET ERMS cells display enhanced migration, chemotaxis and invasion toward HGF and SDF-1. Introduction of TPR-MET into MSC increases survival and may induce expression of early myogenic factors depending on the genetic background, and it blocks terminal differentiation of skeletal myoblasts. To conclude, our results suggest that activation of MET signaling may cause defects in myogenic differentiation leading to rhabdomyosarcoma development and progression.
Assuntos
Diferenciação Celular , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/patologia , Neovascularização Patológica , Proteínas Proto-Oncogênicas c-met/metabolismo , Rabdomiossarcoma/patologia , Animais , Apoptose , Western Blotting , Células Cultivadas , Citometria de Fluxo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mioblastos/citologia , Mioblastos/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rabdomiossarcoma/genética , Rabdomiossarcoma/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The main goal in gene therapy and biomedical research is an efficient transcription factors (TFs) delivery system. SNAIL, a zinc finger transcription factor, is strongly involved in tumor, what makes its signaling pathways an interesting research subject. The necessity of tracking activation of intracellular pathways has prompted fluorescent proteins usage as localization markers. Advanced molecular cloning techniques allow to generate fusion proteins from fluorescent markers and transcription factors. Depending on fusion strategy, the protein expression levels and nuclear transport ability are significantly different. The P2A self-cleavage motif through its cleavage ability allows two single proteins to be simultaneously expressed. The aim of this study was to compare two strategies for introducing a pair of genes using expression vector system. We have examined GFP and SNAI1 gene fusions by comprising common nucleotide polylinker (multiple cloning site) or P2A motif in between them, resulting in one fusion or two independent protein expressions respectively. In each case transgene expression levels and translation efficiency as well as nuclear localization of expressed protein have been analyzed. Our data showed that usage of P2A motif provides more effective nuclear transport of SNAIL transcription factor than conventional genes linker. At the same time the fluorescent marker spreads evenly in subcellular space.