RESUMO
There is a need to develop effective therapies for pancreatic ductal adenocarcinoma (PDA), a highly lethal malignancy with increasing incidence1 and poor prognosis2. Although targeting tumour metabolism has been the focus of intense investigation for more than a decade, tumour metabolic plasticity and high risk of toxicity have limited this anticancer strategy3,4. Here we use genetic and pharmacological approaches in human and mouse in vitro and in vivo models to show that PDA has a distinct dependence on de novo ornithine synthesis from glutamine. We find that this process, which is mediated through ornithine aminotransferase (OAT), supports polyamine synthesis and is required for tumour growth. This directional OAT activity is usually largely restricted to infancy and contrasts with the reliance of most adult normal tissues and other cancer types on arginine-derived ornithine for polyamine synthesis5,6. This dependency associates with arginine depletion in the PDA tumour microenvironment and is driven by mutant KRAS. Activated KRAS induces the expression of OAT and polyamine synthesis enzymes, leading to alterations in the transcriptome and open chromatin landscape in PDA tumour cells. The distinct dependence of PDA, but not normal tissue, on OAT-mediated de novo ornithine synthesis provides an attractive therapeutic window for treating patients with pancreatic cancer with minimal toxicity.
Assuntos
Ornitina-Oxo-Ácido Transaminase , Neoplasias Pancreáticas , Poliaminas , Animais , Humanos , Camundongos , Arginina/deficiência , Arginina/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Ornitina/biossíntese , Ornitina/metabolismo , Ornitina-Oxo-Ácido Transaminase/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Poliaminas/metabolismo , Microambiente TumoralRESUMO
Genetic mutations or regulatory failures underlie cellular malfunction in many diseases, including colorectal cancer and inflammatory bowel diseases. However, mutational defects alone fail to explain the complexity of such disorders. Epigenetic regulation-control of gene action through chemical and structural changes of chromatin-provides a platform to integrate multiple extracellular inputs and prepares the cellular genome for appropriate gene expression responses. Coregulation by polycomb repressive complex 2-mediated trimethylation of lysine 27 on histone 3 and DNA methylation has emerged as one of the most influential epigenetic controls in colorectal cancer and many other diseases, but molecular details remain inadequate. Here we review the molecular interplay of these epigenetic features in relation to gastrointestinal development, homeostasis, and disease biology. We discuss other epigenetic mechanisms pertinent to the balance of trimethylation of lysine 27 on histone 3 and DNA methylation and their actions in gastrointestinal cancers. We also review the current molecular understanding of chromatin control in the pathogenesis of inflammatory bowel diseases.
Assuntos
Metilação de DNA/genética , Epigênese Genética/genética , Neoplasias Gastrointestinais/genética , Histonas/genética , Células-Tronco/fisiologia , Cromatina/genética , Humanos , Doenças Inflamatórias Intestinais/genética , Lisina/genética , Complexo Repressor Polycomb 2/genética , Processamento de Proteína Pós-TraducionalRESUMO
Hematopoietic clones with leukemogenic mutations arise in healthy people as they age, but progression to acute myeloid leukemia (AML) is rare. Recent evidence suggests that the microenvironment may play an important role in modulating human AML population dynamics. To investigate this concept further, we examined the combined and separate effects of an oncogene (c-MYC) and exposure to interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) on the experimental genesis of a human AML in xenografted immunodeficient mice. Initial experiments showed that normal human CD34+ blood cells transduced with a lentiviral MYC vector and then transplanted into immunodeficient mice produced a hierarchically organized, rapidly fatal, and serially transplantable blast population, phenotypically and transcriptionally similar to human AML cells, but only in mice producing IL-3, GM-CSF, and SCF transgenically or in regular mice in which the cells were exposed to IL-3 or GM-CSF delivered using a cotransduction strategy. In their absence, the MYC+ human cells produced a normal repertoire of lymphoid and myeloid progeny in transplanted mice for many months, but, on transfer to secondary mice producing the human cytokines, the MYC+ cells rapidly generated AML. Indistinguishable diseases were also obtained efficiently from both primitive (CD34+CD38-) and late granulocyte-macrophage progenitor (GMP) cells. These findings underscore the critical role that these cytokines can play in activating a malignant state in normally differentiating human hematopoietic cells in which MYC expression has been deregulated. They also introduce a robust experimental model of human leukemogenesis to further elucidate key mechanisms involved and test strategies to suppress them.
Assuntos
Regulação Leucêmica da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-3/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Transplante de NeoplasiasRESUMO
In eggs, the metabolic activities of the developing embryo produce heat (H) that is dissipated in various forms, including radiation. Given that much of the total heat radiated by an egg (Htot) is heat acquired passively, we asked whether it was possible to detect the fraction produced metabolically (Hmetab) and the extent of its correlation with the embryo's metabolic rate. In chicken and duck eggs at various incubation ages, under standardized experimental conditions of heat conduction and convection, Hmetab was measured by thermography as the difference in Htot between fertile and sterile eggs. Then, Hmetab was correlated to the embryo's oxygen consumption ( [Formula: see text] ), measured by an open-circuit methodology. Heat loss by water evaporation was found to be less than 3% of the total. During the first half of incubation Hmetab was too small to be significantly separated from Htot. In the second half of incubation Hmetab was significant, represented 30-50% of the total energy consumed and correlated linearly with [Formula: see text] for a good fraction of incubation. We conclude that under standardized conditions of heat conduction and convection, in the second half of incubation thermography offers a simple tool not only to verify the progression of the embryo's incubation but also to estimate its metabolic rate.
Assuntos
Galinhas/fisiologia , Patos/fisiologia , Ovos , Embrião não Mamífero/fisiologia , Consumo de Oxigênio , Animais , TemperaturaRESUMO
Precise spatiotemporal and cell type-specific gene expression is essential for proper tissue development and function. Transcription factors (TFs) guide this process by binding to developmental stage-specific targets and establishing an appropriate enhancer landscape. In turn, DNA and chromatin modifications direct the genomic binding of TFs. However, how TFs navigate various chromatin features and selectively bind a small portion of the millions of possible genomic target loci is still not well understood. Here we show that Cdx2 - a pioneer TF that binds distinct targets in developing versus adult intestinal epithelial cells - has a preferential affinity for a non-canonical CpG-containing motif in vivo. A higher frequency of this motif at embryonic and fetal Cdx2 target loci and the specifically methylated state of the CpG during development allows selective Cdx2 binding and activation of developmental enhancers and linked genes. Conversely, demethylation at these enhancers prohibits ectopic Cdx2 binding in adult cells, where Cdx2 binds its canonical motif without a CpG. This differential Cdx2 binding allows for corecruitment of Ctcf and Hnf4, facilitating the establishment of intestinal superenhancers during development and enhancers mediating adult homeostatic functions, respectively. Induced gain of DNA methylation in the adult mouse epithelium or cultured cells causes ectopic recruitment of Cdx2 to the developmental target loci and facilitates cobinding of the partner TFs. Together, our results demonstrate that the differential CpG motif requirements for Cdx2 binding to developmental versus adult target sites allow it to navigate different DNA methylation profiles and activate cell type-specific genes at appropriate times.
RESUMO
MicroRNAs (miRNAs) are commonly deregulated in acute myeloid leukemia (AML), affecting critical genes not only through direct targeting, but also through modulation of downstream effectors. Homeobox (Hox) genes balance self-renewal, proliferation, cell death, and differentiation in many tissues and aberrant Hox gene expression can create a predisposition to leukemogenesis in hematopoietic cells. However, possible linkages between the regulatory pathways of Hox genes and miRNAs are not yet fully resolved. We identified miR-708 to be upregulated in Hoxa9/Meis1 AML inducing cell lines as well as in AML patients. We further showed Meis1 directly targeting miR-708 and modulating its expression through epigenetic transcriptional regulation. CRISPR/Cas9 mediated knockout of miR-708 in Hoxa9/Meis1 cells delayed disease onset in vivo, demonstrating for the first time a pro-leukemic contribution of miR-708 in this context. Overexpression of miR-708 however strongly impeded Hoxa9 mediated transformation and homing capacity in vivo through modulation of adhesion factors and induction of myeloid differentiation. Taken together, we reveal miR-708, a putative tumor suppressor miRNA and direct target of Meis1, as a potent antagonist of the Hoxa9 phenotype but an effector of transformation in Hoxa9/Meis1. This unexpected finding highlights the yet unexplored role of miRNAs as indirect regulators of the Hox program during normal and aberrant hematopoiesis.
Assuntos
Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide Aguda/patologia , MicroRNAs/genética , Células Mieloides/patologia , Proteína Meis1/metabolismo , Animais , Apoptose , Sistemas CRISPR-Cas , Diferenciação Celular , Proliferação de Células , Feminino , Hematopoese , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Células Mieloides/metabolismo , Proteína Meis1/genética , Células Tumorais CultivadasRESUMO
Autophagy is a cell survival process essential for the regulation of immune responses to infections. However, the role of T cell autophagy in anti-tumor immunity is less clear. Here, we demonstrate a cell-autonomous role for autophagy in the regulation of CD8+ T-cell-mediated control of tumors. Mice deficient for the essential autophagy genes Atg5, Atg14, or Atg16L1 display a dramatic impairment in the growth of syngeneic tumors. Moreover, T cells lacking Atg5 have a profound shift to an effector memory phenotype and produce greater amounts of interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α). Mechanistically, Atg5-/- CD8+ T cells exhibit enhanced glucose metabolism that results in alterations in histone methylation, increases in H3K4me3 density, and transcriptional upregulation of both metabolic and effector target genes. Nonetheless, glucose restriction is sufficient to suppress Atg5-dependent increases in effector function. Thus, autophagy-dependent changes in CD8+ T cell metabolism directly regulate anti-tumor immunity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Neoplasias/imunologia , Animais , Autofagia , Humanos , CamundongosRESUMO
Mechanistic studies in human cancer have relied heavily on cell lines and mouse models, but are limited by in vitro adaptation and species context issues, respectively. More recent efforts have utilized patient-derived xenografts; however, these are hampered by variable genetic background, inability to study early events, and practical issues with availability/reproducibility. We report here an efficient, reproducible model of T-cell leukemia in which lentiviral transduction of normal human cord blood yields aggressive leukemia that appears indistinguishable from natural disease. We utilize this synthetic model to uncover a role for oncogene-induced HOXB activation which is operative in leukemia cells-of-origin and persists in established tumors where it defines a novel subset of patients distinct from other known genetic subtypes and with poor clinical outcome. We show further that anterior HOXB genes are specifically activated in human T-ALL by an epigenetic mechanism and confer growth advantage in both pre-leukemia cells and established clones.
Assuntos
Proteínas de Homeodomínio/metabolismo , Leucemia/metabolismo , Família Multigênica , Animais , Proliferação de Células , Epigênese Genética , Feminino , Xenoenxertos , Proteínas de Homeodomínio/genética , Humanos , Leucemia/genética , Leucemia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Modelos Genéticos , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismoRESUMO
Acute leukemias are aggressive malignancies of developmentally arrested hematopoietic progenitors. We sought here to explore the possibility that changes in hematopoietic stem/progenitor cells during development might alter the biology of leukemias arising from this tissue compartment. Using a mouse model of acute T cell leukemia, we found that leukemias generated from fetal liver (FL) and adult bone marrow (BM) differed dramatically in their leukemia stem cell activity with FL leukemias showing markedly reduced serial transplantability as compared to BM leukemias. We present evidence that this difference is due to NOTCH1-driven autocrine IGF1 signaling, which is active in FL cells but restrained in BM cells by EZH2-dependent H3K27 trimethylation. Further, we confirmed this mechanism is operative in human disease and show that enforced IGF1 signaling effectively limits leukemia stem cell activity. These findings demonstrate that resurrecting dormant fetal programs in adult cells may represent an alternate therapeutic approach in human cancer.
Assuntos
Células da Medula Óssea/metabolismo , Epigênese Genética/genética , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Células-Tronco Neoplásicas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Transdução de Sinais/genética , Animais , Células da Medula Óssea/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Células-Tronco Neoplásicas/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologiaRESUMO
We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.
Assuntos
Imunoprecipitação da Cromatina/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Código das Histonas/genética , Nucleossomos/genéticaRESUMO
Conventional cytotoxic therapies for synovial sarcoma provide limited benefit, and no drugs specifically targeting the causative SS18-SSX fusion oncoprotein are currently available. Histone deacetylase (HDAC) inhibition has been shown in previous studies to disrupt the synovial sarcoma oncoprotein complex, resulting in apoptosis. To understand the molecular effects of HDAC inhibition, RNA-seq transcriptome analysis was undertaken in six human synovial sarcoma cell lines. HDAC inhibition induced pathways of cell-cycle arrest, neuronal differentiation, and response to oxygen-containing species, effects also observed in other cancers treated with this class of drugs. More specific to synovial sarcoma, polycomb group targets were reactivated, including tumor suppressor CDKN2A, and proapoptotic transcriptional patterns were induced. Functional analyses revealed that ROS-mediated FOXO activation and proapoptotic factors BIK, BIM, and BMF were important to apoptosis induction following HDAC inhibition in synovial sarcoma. HDAC inhibitor pathway activation results in apoptosis and decreased tumor burden following a 7-day quisinostat treatment in the Ptenfl/fl;hSS2 mouse model of synovial sarcoma. This study provides mechanistic support for a particular susceptibility of synovial sarcoma to HDAC inhibition as a means of clinical treatment. Mol Cancer Ther; 16(12); 2656-67. ©2017 AACR.
Assuntos
Inibidores de Histona Desacetilases/uso terapêutico , Sarcoma Sinovial/metabolismo , Animais , Morte Celular , Linhagem Celular Tumoral , Proliferação de Células , Inibidores de Histona Desacetilases/farmacologia , Humanos , CamundongosRESUMO
Mutations in human Atrophin1, a transcriptional corepressor, cause dentatorubral-pallidoluysian atrophy, a neurodegenerative disease. Drosophila Atrophin (Atro) mutants display many phenotypes, including neurodegeneration, segmentation, patterning and planar polarity defects. Despite Atro's critical role in development and disease, relatively little is known about Atro's binding partners and downstream targets. We present the first genomic analysis of Atro using ChIP-seq against endogenous Atro. ChIP-seq identified 1300 potential direct targets of Atro including engrailed, and components of the Dpp and Notch signaling pathways. We show that Atro regulates Dpp and Notch signaling in larval imaginal discs, at least partially via regulation of thickveins and fringe. In addition, bioinformatics analyses, sequential ChIP and coimmunoprecipitation experiments reveal that Atro interacts with the Drosophila GAGA Factor, Trithorax-like (Trl), and they bind to the same loci simultaneously. Phenotypic analyses of Trl and Atro clones suggest that Atro is required to modulate the transcription activation by Trl in larval imaginal discs. Taken together, these data indicate that Atro is a major Trl cofactor that functions to moderate developmental gene transcription.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Imunoprecipitação da Cromatina , Mapeamento de Interação de Proteínas , Análise de Sequência de DNARESUMO
Nucleosome position, density, and post-translational modification are widely accepted components of mechanisms regulating DNA transcription but still incompletely understood. We present a modified native ChIP-seq method combined with an analytical framework that allows MNase accessibility to be integrated with histone modification profiles. Application of this methodology to the primitive (CD34+) subset of normal human cord blood cells enabled genomic regions enriched in one versus two nucleosomes marked by histone 3 lysine 4 trimethylation (H3K4me3) and/or histone 3 lysine 27 trimethylation (H3K27me3) to be associated with their transcriptional and DNA methylation states. From this analysis, we defined four classes of promoter-specific profiles and demonstrated that a majority of bivalent marked promoters are heterogeneously marked at a single-cell level in this primitive cell type. Interestingly, extension of this approach to human embryonic stem cells revealed an altered relationship between chromatin modification state and nucleosome content at promoters, suggesting developmental stage-specific organization of histone methylation states.
Assuntos
Imunoprecipitação da Cromatina , Nucleossomos/metabolismo , Análise de Sequência de RNA , Antígenos CD34/metabolismo , Ilhas de CpG/genética , DNA/metabolismo , Metilação de DNA/genética , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Histonas/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Nuclease do Micrococo/metabolismo , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , RNA/genética , RNA/metabolismoRESUMO
Transcriptional regulation involves complex and interdependent interactions of noncoding and coding regions of the genome with proteins that interact and modify them. Genetic variation/mutation in coding and noncoding regions of the genome can drive aberrant transcription and disease. In spite of accounting for nearly 98% of the genome comparatively little is known about the contribution of noncoding DNA elements to disease. Genome-wide association studies of complex human diseases including cancer have revealed enrichment for variants in the noncoding genome. A striking finding of recent cancer genome re-sequencing efforts has been the previously underappreciated frequency of mutations in epigenetic modifiers across a wide range of cancer types. Taken together these results point to the importance of dysregulation in transcriptional regulatory control in genesis of cancer. Powered by recent technological advancements in functional genomic profiling, exploration of normal and transformed regulatory networks will provide novel insight into the initiation and progression of cancer and open new windows to future prognostic and diagnostic tools.