RESUMEN
BACKGROUND: Pancreatic cancer is one of the most lethal tumors. The aim of this study is to provide an effective therapeutic discovery platform for pancreatic cancer by establishing and characterizing patient-derived organoids (PDOs). METHODS: PDOs were established from pancreatic tumor surgical specimens, and the mutations were examined using a panel sequence. Expression of markers was assessed by PCR, immunoblotting, and immunohistochemistry; tumorigenicity was examined using immunodeficient mice, and drug responses were examined in vitro and in vivo. RESULTS: PDOs were established from eight primary and metastatic tumors, and the characteristic mutations and expression of cancer stem cell markers and CA19-9 were confirmed. Tumorigenicity of the PDOs was confirmed in subcutaneous transplantation and in the peritoneal cavity in the case of PDOs derived from disseminated nodules. Gemcitabine-sensitive/resistant PDOs showed consistent responses in vivo. High throughput screening in PDOs identified a compound effective for inhibiting tumor growth of a gemcitabine-resistant PDO xenograft model. CONCLUSIONS: This PDO-based platform captures important aspects of treatment-resistant pancreatic cancer and its metastatic features, suggesting that this study may serve as a tool for the discovery of personalized therapies.
Asunto(s)
Organoides , Neoplasias Pancreáticas , Animales , Descubrimiento de Drogas , Humanos , Ratones , Organoides/patología , Páncreas/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias PancreáticasRESUMEN
The modification of DNA by 5-methylcytosine (5mC) has essential roles in cell differentiation and development through epigenetic gene regulation. 5mC can be converted to another modified base, 5-hydroxymethylcytosine (5hmC), by the tet methylcytosine dioxygenase (Tet) family of enzymes. Notably, the balance between 5hmC and 5mC in the genome is linked with cell-differentiation processes such as pluripotency and lineage commitment. We have previously reported that the maternal factor PGC7 (also known as Dppa3, Stella) is required for the maintenance of DNA methylation in early embryogenesis, and protects 5mC from conversion to 5hmC in the maternal genome. Here we show that PGC7 protects 5mC from Tet3-mediated conversion to 5hmC by binding to maternal chromatin containing dimethylated histone H3 lysine 9 (H3K9me2) in mice. In addition, imprinted loci that are marked with H3K9me2 in mature sperm are protected by PGC7 binding in early embryogenesis. This type of regulatory mechanism could be involved in DNA modifications in somatic cells as well as in early embryos.
Asunto(s)
5-Metilcitosina/metabolismo , Citosina/análogos & derivados , Embrión de Mamíferos/metabolismo , Histonas/química , Histonas/metabolismo , Proteínas Represoras/metabolismo , Animales , Cromatina/química , Cromatina/metabolismo , Proteínas Cromosómicas no Histona , Citosina/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Dioxigenasas , Embrión de Mamíferos/embriología , Desarrollo Embrionario , Femenino , Impresión Genómica/genética , Lisina/química , Lisina/metabolismo , Masculino , Metilación , Ratones , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , ARN Largo no Codificante , ARN no Traducido/genética , Espermatozoides/metabolismo , ras-GRF1/genéticaRESUMEN
Systemic administration of small molecule toll-like receptor (TLR)-7 agonists leads to potent activation of innate immunity and to the generation of anti-tumor immune responses. However, activation of TLRs with small molecule agonists may lead to the induction of TLR tolerance, defined as a state of hyporesponsiveness to subsequent agonism, which may limit immune activation, the generation of anti-tumor responses and clinical response. Our data reveal that dose scheduling impacts on the efficacy of systemic therapy with the selective TLR7 agonist, 6-amino-2-(butylamino)-9-((6-(2-(dimethylamino)ethoxy)pyridin-3-yl)methyl)-7,9-dihydro-8H-purin-8-one (DSR-6434). In a preclinical model of renal cell cancer, systemic administration of DSR-6434 dosed once weekly resulted in a significant anti-tumor response. However, twice weekly dosing of DSR-6434 led to the induction of TLR tolerance, and no anti-tumor response was observed. We show that TLR7 tolerance was independent of type I interferon (IFN) negative feedback because induction of TLR7 tolerance was also observed in IFN-α/ß receptor knockout mice treated with DSR-6434. Moreover, our data demonstrate that treatment of bone marrow-derived plasmacytoid dendritic cells (BM-pDC) with DSR-6434 led to downregulation of TLR7 expression. From our data, dose scheduling of systemically administered TLR7 agonists can impact on anti-tumor activity through the induction of TLR tolerance. Furthermore, TLR7 expression on pDC may be a useful biomarker of TLR7 tolerance and aid in the optimization of dosing schedules involving systemically administered TLR7 agonists.
Asunto(s)
Adenina/análogos & derivados , Carcinoma de Células Renales/inmunología , Glicoproteínas de Membrana/metabolismo , Receptor Toll-Like 7/metabolismo , Adenina/administración & dosificación , Adenina/farmacología , Animales , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Protocolos Clínicos , Citotoxicidad Inmunológica , Humanos , Tolerancia Inmunológica , Inmunidad Innata , Interferón Tipo I/metabolismo , Glicoproteínas de Membrana/agonistas , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Experimentales , Transducción de Señal , Receptor Toll-Like 7/agonistasRESUMEN
DNA methylation is a key epigenetic regulator in mammals, and the dynamic balance between methylation and demethylation impacts various processes, from development to disease. DNA methylation is erased during replication when DNA methyltransferase 1 (DNMT1) fails to methylate the daughter strand, in a process known as passive DNA demethylation. We found that the enforced expression of Stella (also known as PGC7, Dppa3), a maternal factor required for the maintenance of DNA methylation in early embryos, induced global DNA demethylation in NIH3T3 cells. This demethylation was caused by the binding of Stella to Np95 (also known as Uhrf1, ICBP90) and the subsequent inhibition of DNMT1 recruitment. Considering that impaired DNA methylation profiles are associated with various developmental or disease phenomena, Stella may be a powerful tool with which to study the biological effects of global DNA hypomethylation.
Asunto(s)
Metilación de ADN/fisiología , Proteínas/fisiología , Animales , Proteínas Cromosómicas no Histona , Células HEK293 , Humanos , Ratones , Células 3T3 NIH , Técnicas del Sistema de Dos HíbridosRESUMEN
DNA methylation is an important means of epigenetic gene regulation and must be carefully controlled as a prerequisite for normal early embryogenesis. Although global demethylation occurs soon after fertilization, it is not evenly distributed throughout the genome. Genomic imprinting and epigenetic asymmetry between parental genomes, that is, delayed demethylation of the maternal genome after fertilization, are clear examples of the functional importance of DNA methylation. Here, we show that PGC7/Stella, a maternal factor essential for early development, protects the DNA methylation state of several imprinted loci and epigenetic asymmetry. After determining that PGC7/Stella binds to Ran binding protein 5 (RanBP5; a nuclear transport shuttle protein), mutant versions of the two proteins were used to examine exactly when and where PGC7/Stella functions within the cell. It is likely that PGC7/Stella protects the maternal genome from demethylation only after localizing to the nucleus, where it maintains the methylation of several imprinted genes. These results demonstrate that PGC7/Stella is indispensable for the maintenance of methylation involved in epigenetic reprogramming after fertilization.
Asunto(s)
Metilación de ADN , Desarrollo Embrionario/genética , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Proteínas/fisiología , Animales , Células COS , Línea Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Proteínas Cromosómicas no Histona , Femenino , Fertilización/genética , Humanos , Masculino , Ratones , Proteínas/genética , Proteínas/metabolismo , TransfecciónRESUMEN
TLR7 is an innate immune receptor that recognizes single-stranded RNAs, and its activation leads to anti-tumor immune effects. Although it is the only approved TLR7 agonist in cancer therapy, imiquimod is allowed to be administered with topical formulation. Thus, systemic administrative TLR7 agonist is expected in terms of expanding applicable cancer types. Here, we demonstrated the identification and characterization of DSP-0509 as a novel small-molecule TLR7 agonist. DSP-0509 is designed to have unique physicochemical features that could be administered systemically with a short half-life. DSP-0509 activated bone marrow-derived dendritic cells (BMDCs) and induced inflammatory cytokines including type I interferons. In the LM8 tumor-bearing mouse model, DSP-0509 reduced tumor growth not only in subcutaneous primary lesions but also in lung metastatic lesions. DSP-0509 inhibited tumor growth in several syngeneic tumor-bearing mouse models. We found that the CD8+ T cell infiltration of tumor before treatment tended to be positively correlated with anti-tumor efficacy in several mouse tumor models. The combination of DSP-0509 with anti-PD-1 antibody significantly enhanced the tumor growth inhibition compared to each monotherapy in CT26 model mice. In addition, the effector memory T cells were expanded in both the peripheral blood and tumor, and rejection of tumor re-challenge occurred in the combination group. Moreover, synergistic anti-tumor efficacy and effector memory T cell upregulation were also observed for the combination with anti-CTLA-4 antibody. The analysis of the tumor-immune microenvironment by using the nCounter assay revealed that the combination of DSP-0509 with anti-PD-1 antibody enhanced infiltration by multiple immune cells including cytotoxic T cells. In addition, the T cell function pathway and antigen presentation pathway were activated in the combination group. We confirmed that DSP-0509 enhanced the anti-tumor immune effects of anti-PD-1 antibody by inducing type I interferons via activation of dendritic cells and even CTLs. In conclusion, we expect that DSP-0509, a new TLR7 agonist that synergistically induces anti-tumor effector memory T cells with immune checkpoint blockers (ICBs) and can be administered systemically, will be used in the treatment of multiple cancers.
Asunto(s)
Inhibidores de Puntos de Control Inmunológico , Interferón Tipo I , Neoplasias , Receptor Toll-Like 7 , Animales , Ratones , Adyuvantes Inmunológicos/farmacología , Modelos Animales de Enfermedad , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias/tratamiento farmacológico , Receptor Toll-Like 7/agonistas , Microambiente TumoralRESUMEN
Reprogramming of the epigenetic state from differentiated to pluripotent cells can be attained by cell fusion of differentiated somatic cells with embryonic stem (ES) cells or transfer of the nucleus of a differentiated cell into an enucleated oocyte. Activation of Akt signaling is sufficient to maintain pluripotency of ES cells and promotes derivation of embryonic germ (EG) cells from primordial germ cells (PGCs). Here we analyzed the effects of Akt signaling on somatic cell nuclear reprogramming after cell fusion and nuclear transfer. We found that forced activation of Akt signaling stimulated reprogramming after cell fusion of ES cells with thymocytes or mouse embryonic fibroblasts. These hybrid cells showed ES cell characteristics, including in vitro and in vivo differentiation capacity. In contrast, Akt signaling significantly reduced the efficiency of reprogramming with nuclear transfer. Our results demonstrate that Akt signaling plays important roles on the nuclear reprogramming of somatic cells.
Asunto(s)
Reprogramación Celular , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Animales , Diferenciación Celular , Línea Celular , Activación Enzimática , Ratones , Técnicas de Transferencia Nuclear , Proteínas Proto-Oncogénicas c-akt/genéticaRESUMEN
Embryonic stem (ES) cells are derived from the inner cell mass (ICM) of blastocysts. The use of ES cells as a source of differentiated cells holds great promise for cell transplantation therapy. The efficiency of ES cell derivation is affected by genetic variation in mice; that is, some mouse strains, such as C57BL/6, are amenable to ES cell derivation, whereas others, such as BALB/c, are refractory. Developing an efficient method to establish ES cells from strains of various genetic backgrounds should be valuable for derivation of ES cells in various mammalian species, including human. Although it is well-established that various signaling pathways, including phosphoinositide 3-kinase (PI3K)/Akt and Wnt/beta-catenin, regulate the maintenance of ES cell pluripotency, little is known about the signaling pathways involved in the derivation of ES cells from ICMs. In this study, we demonstrated that inhibition of glycogen synthase kinase-3 (GSK-3), one of the crucial molecules in the regulation of the Wnt/beta-catenin, Hedgehog, and Notch signaling pathways, dramatically augmented ES cell derivation from both C57BL/6 and BALB/c mouse strains. In contrast, Akt signaling activation enhanced the growth of ICM but did not increase the efficiency of ES cell derivation. Our study establishes an efficient means for ES cell derivation by pharmacological inhibition of GSK-3.
Asunto(s)
Células Madre Embrionarias/enzimología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Femenino , Glucógeno Sintasa Quinasa 3/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos ICR , Ratones Transgénicos , Embarazo , Inhibidores de Proteínas Quinasas/farmacología , Especificidad de la EspecieRESUMEN
Strategies to augment anti-cancer immune responses have recently demonstrated therapeutic utility. To date clinical success has been achieved through targeting co-inhibitory checkpoints such as CTLA-4, PD-1, and PD-L1. However, approaches that target co-activatory pathways are also being actively being developed. Here we report that the novel TLR7-selective agonist DSR-29133 is well tolerated in mice and leads to acute immune activation. Administration of DSR-29133 leads to the induction of IFNα/γ, IP-10, TNFα, IL-1Ra and IL-12p70, and to a reduction in tumor burden in syngeneic models of renal cancer (Renca), metastatic osteosarcoma (LM8) and colorectal cancer (CT26). Moreover, we show that the efficacy of DSR-29133 was significantly improved when administered in combination with low-dose fractionated radiotherapy (RT). Effective combination therapy required weekly administration of DSR-29133 commencing on day 1 of a fractionated RT treatment cycle, whereas no enhancement of radiation response was observed when DSR-29133 was administered at the end of the fractionated RT cycle. Combined therapy resulted in curative responses in a high proportion of mice bearing established CT26 tumors which was dependent on the activity of CD8+ T-cells but independent of CD4+ T-cells and NK/NKT cells. Moreover, long-term surviving mice originally treated with DSR-29133 and RT were protected by a tumor-specific memory immune response which could prevent tumor growth upon rechallenge. These results demonstrate that DSR-29133 is a potent selective TLR7 agonist that when administered intravenously can induce anti-tumor immune responses that can be further enhanced through combination with low-dose fractionated RT.
Asunto(s)
Adenina/análogos & derivados , Antineoplásicos/farmacología , Quimioradioterapia/métodos , Neoplasias Experimentales/tratamiento farmacológico , Receptor Toll-Like 7/agonistas , Adenina/farmacología , Administración Intravenosa , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Fraccionamiento de la Dosis de Radiación , Humanos , Activación de Linfocitos/efectos de los fármacos , Ratones , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/radioterapiaRESUMEN
Global DNA hypomethylation and DNA hypermethylation of promoter regions are frequently detected in human cancers. Although many studies have suggested a contribution to carcinogenesis, it is still unclear whether the aberrant DNA hypomethylation observed in tumors is a consequence or a cause of cancer. Here, we show that the enforced expression of Stella (also known as PGC7 and Dppa3) induced not only global DNA demethylation but also transformation of NIH3T3 cells. Furthermore, overexpression of Stella enhanced the metastatic ability of B16 melanoma cells, presumably through the induction of metastasis-related genes. These results provide new insights into the function of global DNA hypomethylation in carcinogenesis.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Metilación de ADN , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Metástasis de la Neoplasia , Regiones Promotoras Genéticas , Proteínas/metabolismo , Animales , Transformación Celular Neoplásica/patología , Proteínas Cromosómicas no Histona , Células Clonales , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Metástasis de la Neoplasia/patología , Trasplante de Neoplasias , Proteínas/antagonistas & inhibidores , Proteínas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Carga TumoralRESUMEN
Primordial germ cells (PGCs) are embryonic germ cell precursors. Although the developmental potency of PGCs is restricted to the germ lineage, PGCs can acquire pluripotency, as verified by the in vitro establishment of embryonic germ (EG) cells and the in vivo production of testicular teratomas. PGC-specific inactivation of PTEN, which is a lipid phosphatase antagonizing phosphoinositide-3 kinase (PI3K), enhances both EG cell production and testicular teratoma formation. Here, we analyzed the effect of the serine/threonine kinase AKT, one of the major downstream effectors of PI3K, on the developmental potency of PGCs. We used transgenic mice that expressed an AKT-MER fusion protein, the kinase activity of which could be regulated by the ligand of modified estrogen receptor (MER), 4-hydroxytamoxifen. We found that hyperactivation of AKT signaling in PGCs at the proliferative phase dramatically augmented the efficiency of EG cell establishment. Furthermore, AKT signaling activation substituted to some extent for the effects of bFGF, an essential growth factor for EG cell establishment. By contrast, AKT activation had no effect on germ cells that were in mitotic arrest or that began meiosis at a later embryonic stage. In the transgenic PGCs, AKT activation induced phosphorylation of GSK3, which inhibits its kinase activity; enhanced the stability and nuclear localization of MDM2; and suppressed p53 phosphorylation, which is required for its activation. The p53 deficiency, but not GSK3 inhibition, recapitulated the effects of AKT hyperactivation on EG cell derivation, suggesting that p53 is one of the crucial downstream targets of the PI3K/AKT signal and that GSK3 is not.
Asunto(s)
Óvulo/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Espermatozoides/fisiología , Animales , Apoptosis , Técnicas de Cultivo de Célula , División Celular , Embrión de Mamíferos/fisiología , Femenino , Citometría de Flujo , Glucógeno Sintasa Quinasa 3/metabolismo , Masculino , Ratones , Ratones Transgénicos , Óvulo/citología , Óvulo/efectos de los fármacos , Fosforilación , Proteínas Proto-Oncogénicas c-akt/genética , Espermatozoides/citología , Espermatozoides/efectos de los fármacos , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologíaRESUMEN
GATA-2 is a zinc finger transcription factor essential for differentiation of immature hematopoietic cells. We analyzed the function of GATA-2 by a combined method of tetracycline-dependent conditional gene expression and in vitro hematopoietic differentiation from mouse embryonic stem (ES) cells using OP9 stroma cells (OP9 system). In the presence of macrophage colony-stimulating factor (M-CSF), the OP9 system induced macrophage differentiation. GATA-2 expression in this system inhibited macrophage differentiation and redirected the fate of hematopoietic differentiation to other hematopoietic lineages. GATA-2 expression commencing at day 5 or day 6 induced megakaryocytic or erythroid differentiation, respectively. Expression levels of PU.1, a hematopoietic transcription factor that interferes with GATA-2, appeared to play a critical role in differentiation to megakaryocytic or erythroid lineages. Transcription of PU.1 was affected by histone acetylation induced by binding of GATA-2 to the PU.1 promoter region. This study demonstrates that the function of GATA-2 is modified in a context-dependent manner by expression of PU.1, which in turn is regulated by GATA-2.
Asunto(s)
Diferenciación Celular/fisiología , Embrión de Mamíferos/fisiología , Factor de Transcripción GATA2/metabolismo , Hematopoyesis/fisiología , Células Progenitoras Mieloides/fisiología , Células Madre/fisiología , Animales , Antibacterianos/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Embrión de Mamíferos/citología , Factor de Transcripción GATA2/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Hematopoyesis/efectos de los fármacos , Macrófagos/citología , Macrófagos/fisiología , Megacariocitos/citología , Megacariocitos/fisiología , Ratones , Células Progenitoras Mieloides/citología , Proteínas Proto-Oncogénicas/metabolismo , Células Madre/citología , Células del Estroma/citología , Células del Estroma/fisiología , Tetraciclina/farmacología , Transactivadores/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genéticaRESUMEN
Primordial germ cells (PGCs) are germ cell precursors that are committed to sperm or oocytes. Dramatic proliferation during PGC development determines the number of founder spermatogonia and oocytes. Although specified to a germ lineage, PGCs produce pluripotent embryonic germ (EG) cells in vitro and testicular teratomas in vivo. Wnt/beta-catenin signaling regulates pluripotency and differentiation in various stem cell systems, and dysregulation of this signaling causes various human cancers. Here, we examined the role of Wnt/beta-catenin signaling in PGC development. In normal PGC development, Wnt/beta-catenin signaling is suppressed by the GSK3beta-mediated active degradation of beta-catenin and the low expression of canonical Wnt molecules. The effects of aberrant activation of Wnt/beta-catenin signaling in PGCs were analyzed using mice carrying a deletion of the exon that encodes the GSK3beta phosphorylation sites in the beta-catenin locus. Despite the potential activity of Wnt/beta-catenin signaling in stem cell maintenance and carcinogenesis in various cell lineages, teratomas were not induced in the mice expressing the nuclear-localized beta-catenin in PGCs. Instead, the mutant mice showed germ cell deficiency caused by the delayed cell cycle progression of the proliferative phase PGCs. Our results show that the suppression of Wnt/beta-catenin signaling is a prerequisite for the normal development of PGCs.