RESUMEN
Lymphoid tissue inducer (LTi) cells are regarded as a subset of innate lymphoid cells (ILCs). However, these cells are not derived from the ILC common progenitor, which generates other ILC subsets and is defined by the expression of the transcription factor PLZF. Here, we examined transcription factor(s) determining the fate of LTi progenitors versus non-LTi ILC progenitors. Conditional deletion of Gata3 resulted in the loss of PLZF+ non-LTi progenitors but not the LTi progenitors that expressed the transcription factor RORγt. Consistently, PLZF+ non-LTi progenitors expressed high amounts of GATA3, whereas GATA3 expression was low in RORγt+ LTi progenitors. The generation of both progenitors required the transcriptional regulator Id2, which defines the common helper-like innate lymphoid progenitor (ChILP), but not cytokine signaling. Nevertheless, low GATA3 expression was necessary for the generation of functionally mature LTi cells. Thus, differential expression of GATA3 determines the fates and functions of distinct ILC progenitors.
Asunto(s)
Factor de Transcripción GATA3/biosíntesis , Células Madre/citología , Subgrupos de Linfocitos T/citología , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Linaje de la Célula/inmunología , Células Cultivadas , Factor de Transcripción GATA3/genética , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/biosíntesis , Receptor de Muerte Celular Programada 1/biosíntesis , Proteína de la Leucemia Promielocítica con Dedos de Zinc/biosíntesis , Células Madre/inmunología , Subgrupos de Linfocitos T/inmunologíaRESUMEN
The poor clinical outcome in pancreatic ductal adenocarcinoma (PDA) is attributed to intrinsic chemoresistance and a growth-permissive tumor microenvironment. Conversion of quiescent to activated pancreatic stellate cells (PSCs) drives the severe stromal reaction that characterizes PDA. Here, we reveal that the vitamin D receptor (VDR) is expressed in stroma from human pancreatic tumors and that treatment with the VDR ligand calcipotriol markedly reduced markers of inflammation and fibrosis in pancreatitis and human tumor stroma. We show that VDR acts as a master transcriptional regulator of PSCs to reprise the quiescent state, resulting in induced stromal remodeling, increased intratumoral gemcitabine, reduced tumor volume, and a 57% increase in survival compared to chemotherapy alone. This work describes a molecular strategy through which transcriptional reprogramming of tumor stroma enables chemotherapeutic response and suggests vitamin D priming as an adjunct in PDA therapy. PAPERFLICK:
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antineoplásicos/farmacología , Calcitriol/análogos & derivados , Carcinoma Ductal Pancreático/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Receptores de Calcitriol/metabolismo , Adenocarcinoma/patología , Animales , Calcitriol/farmacología , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Neoplasias Pancreáticas/patología , Pancreatitis/tratamiento farmacológico , Pancreatitis/prevención & control , Transducción de Señal , Células del Estroma/patologíaRESUMEN
Mutations in LMNA, the gene encoding A-type lamins, cause laminopathies-diseases of striated muscle and other tissues. The aetiology of laminopathies has been attributed to perturbation of chromatin organization or structural weakening of the nuclear envelope (NE) such that the nucleus becomes more prone to mechanical damage. The latter model requires a conduit for force transmission to the nucleus. NE-associated Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes are one such pathway. Using clustered regularly interspaced short palindromic repeats to disrupt the Nesprin-1 KASH (Klarsicht, ANC-1, Syne Homology) domain, we identified this LINC complex protein as the predominant NE anchor for microtubule cytoskeleton components, including nucleation activities and motor complexes, in mouse cardiomyocytes. Loss of Nesprin-1 LINC complexes resulted in loss of microtubule cytoskeleton proteins at the nucleus and changes in nuclear morphology and positioning in striated muscle cells, but with no overt physiological defects. Disrupting the KASH domain of Nesprin-1 suppresses Lmna-linked cardiac pathology, likely by reducing microtubule cytoskeleton activities at the nucleus. Nesprin-1 LINC complexes thus represent a potential therapeutic target for striated muscle laminopathies.
Asunto(s)
Laminopatías , Músculo Estriado , Animales , Ratones , Proteínas de Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de la Membrana/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Matriz Nuclear/genética , Microtúbulos/metabolismo , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Músculo Estriado/metabolismo , Laminopatías/metabolismoRESUMEN
ADGRL1 (latrophilin 1), a well-characterized adhesion G protein-coupled receptor, has been implicated in synaptic development, maturation, and activity. However, the role of ADGRL1 in human disease has been elusive. Here, we describe ten individuals with variable neurodevelopmental features including developmental delay, intellectual disability, attention deficit hyperactivity and autism spectrum disorders, and epilepsy, all heterozygous for variants in ADGRL1. In vitro, human ADGRL1 variants expressed in neuroblastoma cells showed faulty ligand-induced regulation of intracellular Ca2+ influx, consistent with haploinsufficiency. In vivo, Adgrl1 was knocked out in mice and studied on two genetic backgrounds. On a non-permissive background, mice carrying a heterozygous Adgrl1 null allele exhibited neurological and developmental abnormalities, while homozygous mice were non-viable. On a permissive background, knockout animals were also born at sub-Mendelian ratios, but many Adgrl1 null mice survived gestation and reached adulthood. Adgrl1-/- mice demonstrated stereotypic behaviors, sexual dysfunction, bimodal extremes of locomotion, augmented startle reflex, and attenuated pre-pulse inhibition, which responded to risperidone. Ex vivo synaptic preparations displayed increased spontaneous exocytosis of dopamine, acetylcholine, and glutamate, but Adgrl1-/- neurons formed synapses in vitro poorly. Overall, our findings demonstrate that ADGRL1 haploinsufficiency leads to consistent developmental, neurological, and behavioral abnormalities in mice and humans.
Asunto(s)
Trastorno del Espectro Autista , Discapacidad Intelectual , Trastornos del Neurodesarrollo , Receptores Acoplados a Proteínas G , Receptores de Péptidos , Adulto , Animales , Trastorno del Espectro Autista/genética , Modelos Animales de Enfermedad , Haploinsuficiencia/genética , Humanos , Discapacidad Intelectual/genética , Ratones , Ratones Noqueados , Trastornos del Neurodesarrollo/genéticaRESUMEN
Glioblastoma (GBM), the most common brain malignancy, remains fatal with no effective treatment. Analyses of common aberrations in GBM suggest major regulatory pathways associated with disease etiology. However, 90% of GBMs are diagnosed at an advanced stage (primary GBMs), providing no access to early disease stages for assessing disease progression events. As such, both understanding of disease mechanisms and the development of biomarkers and therapeutics for effective disease management are limited. Here, we describe an adult-inducible astrocyte-specific system in genetically engineered mice that queries causation in disease evolution of regulatory networks perturbed in human GBM. Events yielding disease, both engineered and spontaneous, indicate ordered grade-specific perturbations that yield high-grade astrocytomas (anaplastic astrocytomas and GBMs). Impaired retinoblastoma protein RB tumor suppression yields grade II histopathology. Additional activation of v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) network drives progression to grade III disease, and further inactivation of phosphatase and tensin homolog (PTEN) yields GBM. Spontaneous missense mutation of tumor suppressor Trp53 arises subsequent to KRAS activation, but before grade III progression. The stochastic appearance of mutations identical to those observed in humans, particularly the same spectrum of p53 amino acid changes, supports the validity of engineered lesions and the ensuing interpretations of etiology. Absence of isocitrate dehydrogenase 1 (IDH1) mutation, asymptomatic low grade disease, and rapid emergence of GBM combined with a mesenchymal transcriptome signature reflect characteristics of primary GBM and provide insight into causal relationships.
Asunto(s)
Astrocitoma/etiología , Evolución Biológica , Modelos Animales de Enfermedad , Ingeniería Genética/métodos , Glioblastoma/etiología , Animales , Secuencia de Bases , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Redes Reguladoras de Genes/genética , Ratones , Ratones Transgénicos , Análisis por Micromatrices , Datos de Secuencia Molecular , Mutación Missense/genética , Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Análisis de Secuencia de ADN , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The nuclear lamina, comprised of the A and B-type lamins, is important in maintaining nuclear shape and in regulating key nuclear functions such as chromatin organization and transcription. Deletion of the A-type lamins results in genome instability and many cancers show altered levels of A-type lamin expression. Loss of function mutations in the mouse Lmna gene result in early postnatal lethality, usually within 3-5 weeks of birth making an analysis of the role of lamins in carcinogenesis difficult. To circumvent early lethality, and determine the role of the A-type lamins in specific tissues in older mice we derived a conditional allele of Lmna(FL/FL) (floxed). Lmna(FL/FL) was specifically deleted in the gastrointestinal (GI) epithelium by crossing the Lmna(FL/FL) mice with Villin-Cre mice. Mice lacking Lmna in the GI are overtly normal with no effects on overall growth, longevity or GI morphology. On a GI specific sensitized (Apc(Min/+)) background, polyp numbers are unchanged, but polyp size is slightly increased, and only in the duodenum. Our findings reveal that although A-type lamins are dispensable in the postnatal GI epithelium, loss of Lmna under malignant conditions may, to a limited extent, enhance polyp size indicating that A-type lamins may regulate cell proliferation in the transformed GI epithelium.
Asunto(s)
Transformación Celular Neoplásica/genética , Inestabilidad Genómica , Pólipos Intestinales/genética , Lamina Tipo A/genética , Animales , Proliferación Celular/genética , Epitelio/crecimiento & desarrollo , Epitelio/patología , Tracto Gastrointestinal/crecimiento & desarrollo , Tracto Gastrointestinal/patología , Pólipos Intestinales/patología , Lamina Tipo A/metabolismo , Ratones , Especificidad de ÓrganosRESUMEN
Ectodermal appendages, such as the mammary gland (MG), are thought to have evolved from hair-associated apocrine glands to serve the function of milk secretion. Through the directed differentiation of mouse embryonic stem cells (mESCs), here, we report the generation of multilineage ESC-derived mammary organoids (MEMOs). We adapted the skin organoid model, inducing the dermal mesenchyme to transform into mammary-specific mesenchyme via the sequential activation of Bone Morphogenetic Protein 4 (BMP4) and Parathyroid Hormone-related Protein (PTHrP) and inhibition of hedgehog (HH) signaling. Using single-cell RNA sequencing, we identified gene expression profiles that demonstrate the presence of mammary-specific epithelial cells, fibroblasts, and adipocytes. MEMOs undergo ductal morphogenesis in Matrigel and can reconstitute the MG in vivo. Further, we demonstrate that the loss of function in placode regulators LEF1 and TBX3 in mESCs results in impaired skin and MEMO generation. In summary, our MEMO model is a robust tool for studying the development of ectodermal appendages, and it provides a foundation for regenerative medicine and disease modeling.
Asunto(s)
Proteínas Hedgehog , Células Madre Embrionarias de Ratones , Ratones , Animales , Proteínas Hedgehog/metabolismo , Glándulas Mamarias Animales , Células Epiteliales , Diferenciación Celular , OrganoidesRESUMEN
Pancreatic adenocarcinoma (PDAC) is one the most intractable cancers, in part due to its highly inflammatory microenvironment and paucity of infiltrating dendritic cells (DCs). Here, we find that genetic ablation or antibody blockade of tumor necrosis factor receptor 1 (TNFR1) enhanced intratumor T cell activation and slowed PDAC growth. While anti-PD-1 checkpoint inhibition alone had little effect, it further enhanced intratumor T cell activation in combination with anti-TNFR1. The major cellular alteration in the tumor microenvironment in the absence of TNFR1 signaling was a large increase in DC number and immunostimulatory phenotype. This may reflect a direct effect on DCs, because TNF induced TNFR1-dependent apoptosis of bone-marrow-derived DCs. The therapeutic response to anti-TNFR1 alone was superior to the combination of DC-activating agonistic anti-CD40 and Flt3 ligand (Flt3L). These observations suggest that targeting TNFR1, perhaps in concert with other strategies that promote DC generation and mobilization, may have therapeutic benefits.
RESUMEN
All-trans-retinoic acid (ATRA), the retinoic acid receptors (RARs) agonist, regulates cell growth, differentiation, immunity, and survival. We report that ATRA-treatment repressed cancer growth in syngeneic immunocompetent, but not immunodeficient mice. The tumor microenvironment was implicated: CD8+ T cell depletion antagonized ATRA's anti-tumorigenic effects in syngeneic mice. ATRA-treatment with checkpoint blockade did not cooperatively inhibit murine lung cancer growth. To augment ATRA's anti-tumorigenicity without promoting its pro-tumorigenic potential, an RARγ agonist (IRX4647) was used since it regulates T cell biology. Treating with IRX4647 in combination with an immune checkpoint (anti-PD-L1) inhibitor resulted in a statistically significant suppression of syngeneic 344SQ lung cancers in mice-a model known for its resistance to checkpoints and characterized by low basal T cell and PD-L1 expression. This combined treatment notably elevated CD4+ T-cell presence within the tumor microenvironment and increased IL-5 and IL-13 tumor levels, while simultaneously decreasing CD38 in the tumor stroma. IL-5 and/or IL-13 treatments increased CD4+ more than CD8+ T-cells in mice. IRX4647-treatment did not appreciably affect in vitro lung cancer growth, despite RARγ expression. Pharmacokinetic analysis found IRX4647 plasma half-life was 6 h in mice. Yet, RARα antagonist (IRX6696)-treatment with anti-PD-L1 did not repress syngeneic lung cancer growth. Together, these findings provide a rationale for a clinical trial investigating an RARγ agonist to augment check point blockade response in cancers.
Asunto(s)
Linfocitos T CD8-positivos , Neoplasias Pulmonares , Animales , Ratones , Interleucina-13 , Interleucina-5 , Microambiente Tumoral , Receptores de Ácido Retinoico , Neoplasias Pulmonares/tratamiento farmacológico , Tretinoina , CarcinogénesisRESUMEN
During the initial stage of neuromuscular junction (NMJ) formation, nerve-derived agrin cooperates with muscle-autonomous mechanisms in the organization and stabilization of a plaque-like postsynaptic specialization at the site of nerve-muscle contact. Subsequent NMJ maturation to the characteristic pretzel-like appearance requires extensive structural reorganization. We found that the progress of plaque-to-pretzel maturation is regulated by agrin. Excessive cleavage of agrin via transgenic overexpression of an agrin-cleaving protease, neurotrypsin, in motoneurons resulted in excessive reorganizational activity of the NMJs, leading to rapid dispersal of the synaptic specialization. By contrast, expression of cleavage-resistant agrin in motoneurons slowed down NMJ remodeling and delayed NMJ maturation. Neurotrypsin, which is the sole agrin-cleaving protease in the CNS, was excluded as the physiological agrin-cleaving protease at the NMJ, because NMJ maturation was normal in neurotrypsin-deficient mice. Together, our analyses characterize agrin cleavage at its proteolytic α- and ß-sites by an as-yet-unspecified protease as a regulatory access for relieving the agrin-dependent constraint on endplate reorganization during NMJ maturation.
Asunto(s)
Agrina/metabolismo , Unión Neuromuscular/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Línea Celular , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Transgénicos , Neuronas Motoras/metabolismo , Fibras Nerviosas/metabolismo , Serina Endopeptidasas/biosíntesis , Médula Espinal/citología , Transmisión Sináptica/fisiologíaRESUMEN
Nucleocytoplasmic coupling is mediated by outer nuclear membrane (ONM) nesprin proteins and inner nuclear membrane Sun proteins. Interactions spanning the perinuclear space create nesprin-Sun complexes connecting the cytoskeleton to nuclear components. A search for proteins displaying a conserved C-terminal sequence present in nesprins 1-3 identified nesprin 4 (Nesp4), a new member of this family. Nesp4 is a kinesin-1-binding protein that displays Sun-dependent localization to the ONM. Expression of Nesp4 is associated with dramatic changes in cellular organization involving relocation of the centrosome and Golgi apparatus relative to the nucleus. These effects can be accounted for entirely by Nesp4's kinesin-binding function. The implication is that Nesp4 may contribute to microtubule-dependent nuclear positioning.
Asunto(s)
Cinesinas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Proteínas Nucleares/fisiología , Secuencia de Aminoácidos , Animales , Centrosoma/metabolismo , Proteínas Fluorescentes Verdes/química , Células HeLa , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Microfilamentos/química , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Membrana Nuclear/metabolismo , Proteínas Nucleares/química , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Antibody-based therapies designed for human use frequently fail to cross-react with the murine isoform of their target. Because of this problem, preclinical studies of antibody-based mesothelin (Msl)-targeted therapeutics in immunocompetent systems have been limited by the lack of suitable mouse models. Here, we describe two immunocompetent humanized mesothelin transgenic mouse lines that can act as tolerant hosts for C57Bl/6-syngeneic cell lines expressing the human isoform of mesothelin. Thyroid peroxidase (TPO) mice have thyroid-restricted human mesothelin expression. Mesothelin (Msl) mice express human mesothelin in the typical serosal membrane distribution and can additionally be utilized to assess on-target, off-tumor toxicity of human mesothelin-targeted therapeutics. Both transgenic strains shed human mesothelin into the serum like human mesothelioma and patients with ovarian cancer, and serum human mesothelin can be used as a blood-based surrogate of tumor burden. Using these models, we examined the on-target toxicity and antitumor activity of human mesothelin-targeted recombinant immunotoxins. We found that immunotoxin treatment causes acute and chronic histologic changes to serosal membranes in Msl mice, while human mesothelin-expressing thyroid follicular cells in TPO mice are resistant to immunotoxin despite excellent drug delivery. Furthermore, poor delivery of immunotoxin to syngeneic orthotopic human mesothelin-expressing pancreatic adenocarcinoma limits antitumor activity both alone and in combination with immune checkpoint inhibition. In summary, we have developed two high-fidelity, immunocompetent murine models for human cancer that allow for rigorous preclinical evaluation of human mesothelin-targeted therapeutics.
Asunto(s)
Adenocarcinoma/terapia , Mesotelina/administración & dosificación , Mesotelioma/terapia , Neoplasias Pancreáticas/terapia , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Apoptosis , Proliferación Celular , Femenino , Ingeniería Genética , Humanos , Masculino , Mesotelina/genética , Mesotelina/metabolismo , Mesotelioma/genética , Mesotelioma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The desmoplastic stroma of pancreatic cancers forms a physical barrier that impedes intratumoral drug delivery. Attempts to modulate the desmoplastic stroma to increase delivery of administered chemotherapy have not shown positive clinical results thus far, and preclinical reports in which chemotherapeutic drugs were coadministered with antistromal therapies did not universally demonstrate increased genotoxicity despite increased intratumoral drug levels. In this study, we tested whether TGFß antagonism can break the stromal barrier, enhance perfusion and tumoral drug delivery, and interrogated cellular and molecular mechanisms by which the tumor prevents synergism with coadministered gemcitabine. TGFß inhibition in genetically engineered murine models (GEMM) of pancreas cancer enhanced tumoral perfusion and increased intratumoral gemcitabine levels. However, tumors rapidly adapted to TGFß-dependent stromal modulation, and intratumoral perfusion returned to pre-treatment levels upon extended TGFß inhibition. Perfusion was governed by the phenotypic identity and distribution of cancer-associated fibroblasts (CAF) with the myelofibroblastic phenotype (myCAFs), and myCAFs which harbored unique genomic signatures rapidly escaped the restricting effects of TGFß inhibition. Despite the reformation of the stromal barrier and reversal of initially increased intratumoral exposure levels, TGFß inhibition in cooperation with gemcitabine effectively suppressed tumor growth via cooperative reprogramming of T regulatory cells and stimulation of CD8 T cell-mediated antitumor activity. The antitumor activity was further improved by the addition of anti-PD-L1 immune checkpoint blockade to offset adaptive PD-L1 upregulation induced by TGFß inhibition. These findings support the development of combined antistroma anticancer therapies capable of impacting the tumor beyond the disruption of the desmoplastic stroma as a physical barrier to improve drug delivery.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Carcinoma Ductal Pancreático/inmunología , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/inmunología , Receptor Tipo I de Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Células del Estroma/inmunología , Microambiente Tumoral , Animales , Antimetabolitos Antineoplásicos/farmacología , Apoptosis , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Proliferación Celular , Terapia Combinada , Desoxicitidina/farmacología , Humanos , Ratones , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Myelin, the insulating layers of membrane wrapped around axons by oligodendrocytes, is essential for normal impulse conduction. It forms during late stages of fetal development but continues into early adult life. Myelination correlates with cognitive development and can be regulated by impulse activity through unknown molecular mechanisms. Astrocytes do not form myelin, but these nonneuronal cells can promote myelination in ways that are not understood. Here, we identify a link between myelination, astrocytes, and electrical impulse activity in axons that is mediated by the cytokine leukemia inhibitory factor (LIF). These findings show that LIF is released by astrocytes in response to ATP liberated from axons firing action potentials, and LIF promotes myelination by mature oligodendrocytes. This activity-dependent mechanism promoting myelination could regulate myelination according to functional activity or environmental experience and may offer new approaches to treating demyelinating diseases.
Asunto(s)
Astrocitos/efectos de la radiación , Comunicación Celular/fisiología , Estimulación Eléctrica/métodos , Proteínas de la Mielina/metabolismo , Oligodendroglía/fisiología , Potenciales de Acción/fisiología , Potenciales de Acción/efectos de la radiación , Adenosina/farmacología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Adenosina-5'-(N-etilcarboxamida)/farmacología , Anestésicos Locales/farmacología , Animales , Anticuerpos/farmacología , Astrocitos/fisiología , Axones/efectos de los fármacos , Axones/metabolismo , Axones/efectos de la radiación , Compuestos Azo , Western Blotting/métodos , Comunicación Celular/efectos de los fármacos , Comunicación Celular/efectos de la radiación , Recuento de Células/métodos , Células Cultivadas , Corteza Cerebral/citología , Técnicas de Cocultivo/métodos , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Proteínas de Drosophila/metabolismo , Interacciones Farmacológicas , Embrión de Mamíferos , Ensayo de Inmunoadsorción Enzimática/métodos , Ganglios Espinales/citología , Expresión Génica/efectos de los fármacos , Inmunohistoquímica/métodos , Interleucina-6/inmunología , Interleucina-6/metabolismo , Factor Inhibidor de Leucemia , Ratones , Modelos Biológicos , Proteína Básica de Mielina/metabolismo , Glicoproteína Asociada a Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito , Naftalenos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Antígenos O/metabolismo , ARN Mensajero/biosíntesis , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Células Madre , Tetrodotoxina/farmacología , Tionucleótidos/farmacologíaRESUMEN
Emery-Dreifuss muscular dystrophy can be caused by mutations in the nuclear envelope proteins lamin A/C and emerin. We recently demonstrated that A-type lamin-deficient cells have impaired nuclear mechanics and altered mechanotransduction, suggesting two potential disease mechanisms (Lammerding, J., P.C. Schulze, T. Takahashi, S. Kozlov, T. Sullivan, R.D. Kamm, C.L. Stewart, and R.T. Lee. 2004. J. Clin. Invest. 113:370-378). Here, we examined the function of emerin on nuclear mechanics and strain-induced signaling. Emerin-deficient mouse embryo fibroblasts have abnormal nuclear shape, but in contrast to A-type lamin-deficient cells, exhibit nuclear deformations comparable to wild-type cells in cellular strain experiments, and the integrity of emerin-deficient nuclear envelopes appeared normal in a nuclear microinjection assay. Interestingly, expression of mechanosensitive genes in response to mechanical strain was impaired in emerin-deficient cells, and prolonged mechanical stimulation increased apoptosis in emerin-deficient cells. Thus, emerin-deficient mouse embryo fibroblasts have apparently normal nuclear mechanics but impaired expression of mechanosensitive genes in response to strain, suggesting that emerin mutations may act through altered transcriptional regulation and not by increasing nuclear fragility.
Asunto(s)
Núcleo Celular , Fibroblastos , Mecanotransducción Celular/fisiología , Proteínas de la Membrana/metabolismo , Transducción de Señal/fisiología , Timopoyetinas/metabolismo , Animales , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/metabolismo , FN-kappa B/metabolismo , Proteínas Nucleares , Estrés Mecánico , Timopoyetinas/genéticaRESUMEN
Pancreatic cancer is a disease with limited therapeutic options. Resistance to chemotherapies poses a significant clinical challenge for patients with pancreatic cancer and contributes to a high rate of recurrence. Oncogenic KRAS, a critical driver of pancreatic cancer, promotes metabolic reprogramming and upregulates NRF2, a master regulator of the antioxidant network. Here, we show that NRF2 contributed to chemoresistance and was associated with a poor prognosis in patients with pancreatic cancer. NRF2 activation metabolically rewired and elevated pathways involved in glutamine metabolism. This curbed chemoresistance in KRAS-mutant pancreatic cancers. In addition, manipulating glutamine metabolism restrained the assembly of stress granules, an indicator of chemoresistance. Glutaminase inhibitors sensitized chemoresistant pancreatic cancer cells to gemcitabine, thereby improving the effectiveness of chemotherapy. This therapeutic approach holds promise as a novel therapy for patients with pancreatic cancer harboring KRAS mutation. SIGNIFICANCE: These findings illuminate the mechanistic features of KRAS-mediated chemoresistance and provide a rationale for exploiting metabolic reprogramming in pancreatic cancer cells to confer therapeutic opportunities that could be translated into clinical trials. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/8/1630/F1.large.jpg.
Asunto(s)
Resistencia a Antineoplásicos/fisiología , Glutamina/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Antimetabolitos Antineoplásicos/farmacología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/mortalidad , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Glutaminasa/antagonistas & inhibidores , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Mutación , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidad , Pronóstico , Distribución Aleatoria , Análisis de Matrices Tisulares , Regulación hacia Arriba , GemcitabinaRESUMEN
Synthetic host defense peptides (HDP) are a new class of promising therapeutic agents with potential application in a variety of diseases. RP-182 is a 10mer synthetic HDP design, which selectively reduces M2-like tumor associated macrophages via engagement with the cell surface lectin receptor MRC1/CD206 and is currently being developed as an innate immune defense regulator to improve anti-tumor immunity in immunologically cold tumors. Herein, we describe a sensitive and specific liquid chromatography (LC) coupled to quadrupole electron spray tandem mass spectrometry method to measure positively charged HDPs and HDP peptide fragments in complex biological matrices. Carboxylic acid magnetic beads were used as an affinity-capturing agent to extract the positively charged RP-182 from both mouse plasma and tissue homogenates. Beads were eluted with 0.1% (v/v) formic acid and chromatographic separation was achieved on a Waters 2.1â¯×â¯100â¯mm, 3.5⯵m XSelect Peptide CSH C18 column with a Vanguard pre-column of the same phase. MS/MS was performed on a Thermo TSQ Quantiva triple quadrupole mass spectrometer operating in Selected Reaction Monitoring (SRM) mode fragmenting the plus three parent ion 458.9+3 and monitoring ions 624.0+2, 550.5+2, and 597.3+1 for RP-182 and 462.4+3 > 629.1+2, 555.5+2, and 607.3+1 for isotopic RP-182 standard. The assay had good linearity ranging from 1â¯ng to 1000â¯ng in mouse plasma with the lower limit of detection for RP-182 at 1â¯ng in mouse plasma with good intra- and inter-sample precision and accuracy. Recovery ranged from 66% to 77% with minimum matrix effects. The method was successfully applied to an abbreviated pharmacokinetic study in mice after single IP injection of RP-182. The method was successfully tested on a second HDP, the 17mer D4E1, and the cationic human peptide hormone ghrelin suggesting that it might be a general sensitive method applicable to quantifying HDP peptides that are difficult to extract.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Animales , Péptidos Catiónicos Antimicrobianos/sangre , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacocinética , Ácidos Carboxílicos/química , Cromatografía de Afinidad/métodos , Cromatografía Líquida de Alta Presión/métodos , Evaluación Preclínica de Medicamentos/métodos , Ghrelina/sangre , Ghrelina/química , Ghrelina/aislamiento & purificación , Límite de Detección , Fenómenos Magnéticos , Ratones , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodosRESUMEN
Solid tumors elicit a detectable immune response including the infiltration of tumor-associated macrophages (TAMs). Unfortunately, this immune response is co-opted into contributing toward tumor growth instead of preventing its progression. We seek to reestablish an antitumor immune response by selectively targeting surface receptors and endogenous signaling processes of the macrophage subtypes driving cancer progression. RP-182 is a synthetic 10-mer amphipathic analog of host defense peptides that selectively induces a conformational switch of the mannose receptor CD206 expressed on TAMs displaying an M2-like phenotype. RP-182-mediated activation of this receptor in human and murine M2-like macrophages elicits a program of endocytosis, phagosome-lysosome formation, and autophagy and reprograms M2-like TAMs to an antitumor M1-like phenotype. In syngeneic and autochthonous murine cancer models, RP-182 suppressed tumor growth, extended survival, and was an effective combination partner with chemo- or immune checkpoint therapy. Antitumor activity of RP-182 was also observed in CD206high patient-derived xenotransplantation models. Mechanistically, via selective reduction of immunosuppressive M2-like TAMs, RP-182 improved adaptive and innate antitumor immune responses, including increased cancer cell phagocytosis by reprogrammed TAMs.
Asunto(s)
Lectinas de Unión a Manosa , Macrófagos Asociados a Tumores , Animales , Línea Celular Tumoral , Humanos , Inmunidad Innata , Lectinas Tipo C , Receptor de Manosa , Ratones , Receptores de Superficie CelularRESUMEN
The human nuclear envelope proteins emerin and lamina-associated polypeptide 2alpha (LAP2alpha) have been proposed to aid in the early replication steps of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). However, whether these factors are essential for HIV-1 or MLV infection has been questioned. Prior studies in which conflicting results were obtained were highly dependent on RNA interference-mediated gene silencing. To shed light on these contradictory results, we examined whether HIV-1 or MLV could infect primary cells from mice deficient for emerin, LAP2alpha, or both emerin and LAP2alpha. We observed HIV-1 and MLV infectivity in mouse embryonic fibroblasts (MEFs) from emerin knockout, LAP2alpha knockout, or emerin and LAP2alpha double knockout mice to be comparable in infectivity to wild-type littermate-derived MEFs, indicating that both emerin and LAP2alpha were dispensable for HIV-1 and MLV infection of dividing, primary mouse cells. Because emerin has been suggested to be important for infection of human macrophages by HIV-1, we also examined HIV-1 transduction of macrophages from wild-type mice or knockout mice, but again we did not observe a difference in susceptibility. These findings prompted us to reexamine the role of human emerin in supporting HIV-1 and MLV infection. Notably, both viruses efficiently infected human cells expressing high levels of dominant-negative emerin. We thus conclude that emerin and LAP2alpha are not required for the early replication of HIV-1 and MLV in mouse or human cells.
Asunto(s)
Proteínas de Unión al ADN/genética , VIH-1/fisiología , Proteínas de la Membrana/genética , Proteínas Nucleares/genética , Infecciones por Retroviridae/metabolismo , Animales , Línea Celular , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Humanos , Riñón/citología , Virus de la Leucemia Murina/patogenicidad , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Células 3T3 NIH , Proteínas Nucleares/metabolismo , Estructura Terciaria de ProteínaRESUMEN
The synaptic serine protease neurotrypsin is considered to be essential for the establishment and maintenance of cognitive brain functions, because humans lacking functional neurotrypsin suffer from severe mental retardation. Neurotrypsin cleaves agrin at two homologous sites, liberating a 90-kDa and a C-terminal 22-kDa fragment from the N-terminal moiety of agrin. Morphological analyses indicate that neurotrypsin is contained in presynaptic terminals and externalized in association with synaptic activity, while agrin is localized to the extracellular space at or in the vicinity of the synapse. Here, we present a detailed biochemical analysis of neurotrypsin-mediated agrin cleavage in the murine brain. In brain homogenates, we found that neurotrypsin exclusively cleaves glycanated variants of agrin. Studies with isolated synaptosomes obtained by subcellular fractionation from brains of wild-type and neurotrypsin-overexpressing mice revealed that neurotrypsin-dependent cleavage of agrin was concentrated at synapses, where the most heavily glycanated variants of agrin predominate. Because agrin has been shown to play an important role in the formation and the maintenance of excitatory synapses in the central nervous system, its local cleavage at the synapse implicates the neurotrypsin/agrin system in the regulation of adaptive reorganizations of the synaptic circuitry in the context of cognitive functions, such as learning and memory.