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1.
Invest Ophthalmol Vis Sci ; 60(15): 4991-4999, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31794598

RESUMO

Purpose: Genomic reprogramming and cellular dedifferentiation are critical to the success of de novo tissue regeneration in lower vertebrates such as zebrafish and axolotl. In tissue regeneration following injury or disease, differentiated cells must retain lineage while assuming a progenitor-like identity in order to repopulate the damaged tissue. Understanding the epigenetic regulation of programmed cellular dedifferentiation provides unique insights into the biology of stem cells and cancer and may lead to novel approaches for treating human degenerative conditions. Methods: Using a zebrafish in vivo model of adult muscle regeneration, we utilized chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) to characterize early changes in epigenetic signals, focusing on three well-studied histone modifications-histone H3 trimethylated at lysine 4 (H3K4me3), and histone H3 trimethylated or acetylated at lysine 27 (H3K27me3 and H3K27Ac, respectively). Results: We discovered that zebrafish myocytes undergo a global, rapid, and transient program to drive genomic remodeling. The timing of these epigenetic changes suggests that genomic reprogramming itself represents a distinct sequence of events, with predetermined checkpoints, to generate cells capable of de novo regeneration. Importantly, we uncovered subsets of genes that maintain epigenetic marks paradoxical to changes in expression, underscoring the complexity of epigenetic reprogramming. Conclusions: Within our model, histone modifications previously associated with gene expression act for the most part as expected, with exceptions suggesting that zebrafish chromatin maintains an easily editable state with a number of genes paradoxically marked for transcriptional activity despite downregulation.


Assuntos
Reprogramação Celular/genética , DNA/genética , Epigênese Genética , Músculos Oculomotores/fisiologia , Regeneração/genética , Animais , Imunoprecipitação da Cromatina , Histonas/genética , Modelos Animais , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Peixe-Zebra
2.
Mol Cell ; 76(6): 965-980.e12, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31588023

RESUMO

Development of effective targeted cancer therapies is fundamentally limited by our molecular understanding of disease pathogenesis. Diffuse intrinsic pontine glioma (DIPG) is a fatal malignancy of the childhood pons characterized by a unique substitution to methionine in histone H3 at lysine 27 (H3K27M) that results in globally altered epigenetic marks and oncogenic transcription. Through primary DIPG tumor characterization and isogenic oncohistone expression, we show that the same H3K27M mutation displays distinct modes of oncogenic reprogramming and establishes distinct enhancer architecture depending upon both the variant of histone H3 and the cell context in which the mutation occurs. Compared with non-malignant pediatric pontine tissue, we identify and functionally validate both shared and variant-specific pathophysiology. Altogether, we provide a powerful resource of epigenomic data in 25 primary DIPG samples and 5 rare normal pediatric pontine tissue samples, revealing clinically relevant functional distinctions previously unidentified in DIPG.


Assuntos
/genética , Histonas/genética , Encéfalo/patologia , Neoplasias Encefálicas/genética , Reprogramação Celular/genética , Elementos Facilitadores Genéticos/genética , Epigênese Genética/genética , Epigenômica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Glioma/genética , Glioma/metabolismo , Humanos , Lisina/genética , Mutação/genética , Ponte/metabolismo , Transdução de Sinais , Transcriptoma/fisiologia
3.
PLoS Genet ; 15(10): e1008410, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31584940

RESUMO

Mitochondria have been increasingly recognized as a central regulatory nexus for multiple metabolic pathways, in addition to ATP production via oxidative phosphorylation (OXPHOS). Here we show that inducing mitochondrial DNA (mtDNA) stress in Drosophila using a mitochondrially-targeted Type I restriction endonuclease (mtEcoBI) results in unexpected metabolic reprogramming in adult flies, distinct from effects on OXPHOS. Carbohydrate utilization was repressed, with catabolism shifted towards lipid oxidation, accompanied by elevated serine synthesis. Cleavage and translocation, the two modes of mtEcoBI action, repressed carbohydrate rmetabolism via two different mechanisms. DNA cleavage activity induced a type II diabetes-like phenotype involving deactivation of Akt kinase and inhibition of pyruvate dehydrogenase, whilst translocation decreased post-translational protein acetylation by cytonuclear depletion of acetyl-CoA (AcCoA). The associated decrease in the concentrations of ketogenic amino acids also produced downstream effects on physiology and behavior, attributable to decreased neurotransmitter levels. We thus provide evidence for novel signaling pathways connecting mtDNA to metabolism, distinct from its role in supporting OXPHOS.


Assuntos
Reprogramação Celular/genética , DNA Mitocondrial/genética , Diabetes Mellitus Tipo 2/genética , Mitocôndrias/genética , Trifosfato de Adenosina/genética , Animais , Metabolismo dos Carboidratos/genética , Carboidratos/genética , Enzimas de Restrição do DNA/genética , Diabetes Mellitus Tipo 2/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Redes e Vias Metabólicas/genética , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Estresse Oxidativo/genética
4.
Nat Cell Biol ; 21(10): 1179-1190, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548608

RESUMO

Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors, as is exemplified by reprogramming somatic cells to pluripotent stem cells through the expression of OCT4, KLF4, SOX2 and cMYC. How transcription factors orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a transcription-factor-centric view of chromatin reorganization and its association with three-dimensional enhancer rewiring and transcriptional changes during the reprogramming of mouse embryonic fibroblasts to pluripotent stem cells. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in enhancer connectivity, whereas disruption of individual KLF4 binding sites within pluripotent-stem-cell-specific enhancers was sufficient to impair enhancer-promoter contacts and reduce the expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying transcription factor and offers novel insights into the nature of the molecular events that follow transcription factor binding.


Assuntos
Reprogramação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Elementos Facilitadores Genéticos , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Células-Tronco Pluripotentes/metabolismo
5.
J Biosci ; 44(4)2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31502583

RESUMO

It has been proposed that age reprogramming enables old cells to be rejuvenated without passage through an embryonic stage (Singh and Zacouto in J. Biosci. 35 315-319, 2010). As such, age reprogramming stands apart from the induced pluripotent stem (iPS) and nuclear transfer-embryonic stem (NT-ES) cell therapies where histo-compatible cells are produced only after passage through an embryonic stage. It avoids many of the disadvantages associated with iPS and NT-ES cell therapies. Experimental evidence in support of age reprogramming is burgeoning. Here, we discuss possible new approaches to enhance age reprogramming, which will have considerable benefits for regenerative therapies.


Assuntos
Envelhecimento/genética , Reprogramação Celular/genética , Epigênese Genética/genética , Células-Tronco Embrionárias/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Medicina Regenerativa
6.
Nucleic Acids Res ; 47(19): 10115-10133, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31555818

RESUMO

Pluripotency and cell fates can be modulated through the regulation of super-enhancers; however, the underlying mechanisms are unclear. Here, we showed a novel mechanism in which Ash2l directly binds to super-enhancers of several stemness genes to regulate pluripotency and self-renewal in pluripotent stem cells. Ash2l recruits Oct4/Sox2/Nanog (OSN) to form Ash2l/OSN complex at the super-enhancers of Jarid2, Nanog, Sox2 and Oct4, and further drives enhancer activation, upregulation of stemness genes, and maintains the pluripotent circuitry. Ash2l knockdown abrogates the OSN recruitment to all super-enhancers and further hinders the enhancer activation. In addition, CRISPRi/dCas9-mediated blocking of Ash2l-binding motifs at these super-enhancers also prevents OSN recruitment and enhancer activation, validating that Ash2l directly binds to super-enhancers and initiates the pluripotency network. Transfection of Ash2l with W118A mutation to disrupt Ash2l-Oct4 interaction fails to rescue Ash2l-driven enhancer activation and pluripotent gene upregulation in Ash2l-depleted pluripotent stem cells. Together, our data demonstrated Ash2l formed an enhancer-bound Ash2l/OSN complex that can drive enhancer activation, govern pluripotency network and stemness circuitry.


Assuntos
Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição/genética , Animais , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Autorrenovação Celular/genética , Reprogramação Celular/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Mutação/genética , Proteína Homeobox Nanog/genética , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXB1/genética , Transfecção
7.
Int J Mol Sci ; 20(18)2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31489928

RESUMO

In the last few decades, many pathogenic or likely pathogenic genetic mutations in over hundred different genes have been described for non-ischemic, genetic cardiomyopathies. However, the functional knowledge about most of these mutations is still limited because the generation of adequate animal models is time-consuming and challenging. Therefore, human induced pluripotent stem cells (iPSCs) carrying specific cardiomyopathy-associated mutations are a promising alternative. Since the original discovery that pluripotency can be artificially induced by the expression of different transcription factors, various patient-specific-induced pluripotent stem cell lines have been generated to model non-ischemic, genetic cardiomyopathies in vitro. In this review, we describe the genetic landscape of non-ischemic, genetic cardiomyopathies and give an overview about different human iPSC lines, which have been developed for the disease modeling of inherited cardiomyopathies. We summarize different methods and protocols for the general differentiation of human iPSCs into cardiomyocytes. In addition, we describe methods and technologies to investigate functionally human iPSC-derived cardiomyocytes. Furthermore, we summarize novel genome editing approaches for the genetic manipulation of human iPSCs. This review provides an overview about the genetic landscape of inherited cardiomyopathies with a focus on iPSC technology, which might be of interest for clinicians and basic scientists interested in genetic cardiomyopathies.


Assuntos
Cardiomiopatias/genética , Predisposição Genética para Doença , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Biomarcadores , Cardiomiopatias/diagnóstico , Cardiomiopatias/metabolismo , Diferenciação Celular/genética , Reprogramação Celular/genética , Estudos de Associação Genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mutação , Miócitos Cardíacos/citologia
8.
Mol Cell ; 75(3): 644-660.e5, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31398325

RESUMO

Cell-cell communication via ligand-receptor signaling is a fundamental feature of complex organs. Despite this, the global landscape of intercellular signaling in mammalian liver has not been elucidated. Here we perform single-cell RNA sequencing on non-parenchymal cells isolated from healthy and NASH mouse livers. Secretome gene analysis revealed a highly connected network of intrahepatic signaling and disruption of vascular signaling in NASH. We uncovered the emergence of NASH-associated macrophages (NAMs), which are marked by high expression of triggering receptors expressed on myeloid cells 2 (Trem2), as a feature of mouse and human NASH that is linked to disease severity and highly responsive to pharmacological and dietary interventions. Finally, hepatic stellate cells (HSCs) serve as a hub of intrahepatic signaling via HSC-derived stellakines and their responsiveness to vasoactive hormones. These results provide unprecedented insights into the landscape of intercellular crosstalk and reprogramming of liver cells in health and disease.


Assuntos
Comunicação Celular/genética , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Análise de Sequência de RNA , Animais , Reprogramação Celular/genética , Modelos Animais de Doenças , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Ligantes , Fígado/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Transdução de Sinais/genética , Análise de Célula Única
9.
Genes Cells ; 24(10): 667-673, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31386786

RESUMO

Analysis of gene expression in single cells is required to understand somatic cell reprogramming into human induced pluripotent stem cells (iPSCs). To facilitate this, we established intermediately reprogrammed stem cells (iRSCs), pre-iPSC lines. The iRSC-iPSC conversion system enables the reproducible monitoring of reprogramming events and the analysis of progressive gene expression profiles using single-cell microarray analysis and genome editing. Here, single-cell microarray analysis showed the stage-specific sequential gene activation during the conversion of iRSCs into iPSCs, using OCT4, TDGF1 and E-CADHERIN as marker genes. Out of 75 OCT4-related genes, which were significantly up-regulated after the activation of OCT4, and entry into the mesenchymal-to-epithelial transition (MET), LIN28 (LIN28A) and FOXO1 were selected for applying to gene expression visualization. Multicolored visualization was achieved by the genome editing of LIN28 or FOXO1 with mCherry into OCT4-GFP iRSCs. Fluorescent analysis of gene activity in individual cells showed that OCT4 was dispensable for maintenance, but required for activation, of the LIN28 and FOXO1 expression in reprogramming.


Assuntos
Técnicas de Reprogramação Celular/métodos , Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Caderinas/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Transição Epitelial-Mesenquimal , Proteínas Ligadas por GPI/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Proteínas de Neoplasias/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Análise de Célula Única/métodos , Ativação Transcricional
10.
Nat Commun ; 10(1): 3477, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375664

RESUMO

Oct4, along with Sox2 and Klf4 (SK), can induce pluripotency but structurally similar factors like Oct6 cannot. To decode why Oct4 has this unique ability, we compare Oct4-binding, accessibility patterns and transcriptional waves with Oct6 and an Oct4 mutant defective in the dimerization with Sox2 (Oct4defSox2). We find that initial silencing of the somatic program proceeds indistinguishably with or without Oct4. Oct6 mitigates the mesenchymal-to-epithelial transition and derails reprogramming. These effects are a consequence of differences in genome-wide binding, as the early binding profile of Oct4defSox2 resembles Oct4, whilst Oct6 does not bind pluripotency enhancers. Nevertheless, in the Oct6-SK condition many otherwise Oct4-bound locations become accessible but chromatin opening is compromised when Oct4defSox2 occupies these sites. We find that Sox2 predominantly facilitates chromatin opening, whilst Oct4 serves an accessory role. Formation of Oct4/Sox2 heterodimers is essential for pluripotency establishment; however, reliance on Oct4/Sox2 heterodimers declines during pluripotency maintenance.


Assuntos
Reprogramação Celular/genética , Cromatina/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos , Transição Epitelial-Mesenquimal/genética , Fibroblastos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos Transgênicos , Mutação , Fator 3 de Transcrição de Octâmero/genética , Fator 6 de Transcrição de Octâmero/metabolismo , Cultura Primária de Células , Multimerização Proteica/genética , Fatores de Transcrição SOXB1/genética , Fatores de Tempo
11.
J Exp Clin Cancer Res ; 38(1): 312, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31378204

RESUMO

BACKGROUND: Pancreatic Ductal Adenocarcinoma (PDAC) is an aggressive and lethal disease, lacking effective therapeutic approaches. Available therapies only marginally prolong patient survival and are frequently coupled with severe adverse events. It is therefore pivotal to investigate novel and safe pharmacological approaches. We have recently identified the ABC transporter, ABCC3, whose expression is dependent on mutation of TP53, as a novel target in PDAC. ABCC3-mediated regulation of PDAC cell proliferation and tumour growth in vivo was demonstrated and was shown to be conferred by upregulation of STAT3 signalling and regulation of apoptosis. METHODS: To verify the potential of ABCC3 as a pharmacological target, a small molecule inhibitor of ABCC3, referred to here as MCI-715, was designed. In vitro assays were performed to assess the effects of ABCC3 inhibition on anchorage-dependent and anchorage-independent PDAC cell growth. The impact of ABCC3 inhibition on specific signalling pathways was verified by Western blotting. The potential of targeting ABCC3 with MCI-715 to counteract PDAC progression was additionally tested in several animal models of PDAC, including xenograft mouse models and transgenic mouse model of PDAC. RESULTS: Using both mouse models and human cell lines of PDAC, we show that the pharmacological inhibition of ABCC3 significantly decreased PDAC cell proliferation and clonal expansion in vitro and in vivo, remarkably slowing tumour growth in mice xenografts and patient-derived xenografts and increasing the survival rate in a transgenic mouse model. Furthermore, we show that stromal cells in pancreatic tumours, which actively participate in PDAC progression, are enriched for ABCC3, and that its inhibition may contribute to stroma reprogramming. CONCLUSIONS: Our results indicate that ABCC3 inhibition with MCI-715 demonstrated strong antitumor activity and is well tolerated, which leads us to conclude that ABCC3 inhibition is a novel and promising therapeutic strategy for a considerable cohort of patients with pancreatic cancer.


Assuntos
Antineoplásicos/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Animais , Apoptose , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reprogramação Celular/genética , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/mortalidade , Prognóstico , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Estromais/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Oncogene ; 38(34): 6211-6225, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31289360

RESUMO

One-carbon metabolism plays a central role in a broad array of metabolic processes required for the survival and growth of tumor cells. However, the molecular basis of how one-carbon metabolism may influence RNA methylation and tumorigenesis remains largely unknown. Here we show MTHFD2, a mitochondrial enzyme involved in one-carbon metabolism, contributes to the progression of renal cell carcinoma (RCC) via a novel epitranscriptomic mechanism that involves HIF-2α. We found that expression of MTHFD2 was significantly elevated in human RCC tissues, and MTHFD2 knockdown strongly reduced xenograft tumor growth. Mechanistically, using an unbiased methylated RNA immunoprecipitation sequencing (meRIP-Seq) approach, we found that MTHFD2 plays a critical role in controlling global N6-methyladenosine (m6A) methylation levels, including the m6A methylation of HIF-2α mRNA, which results in enhanced translation of HIF-2α. Enhanced HIF-2α translation, in turn, promotes the aerobic glycolysis, linking one-carbon metabolism to HIF-2α-dependent metabolic reprogramming through RNA methylation. Our findings also suggest that MTHFD2 and HIF-2α form a positive feedforward loop in RCC, promoting metabolic reprograming and tumor growth. Taken together, our results suggest that MTHFD2 links RNA methylation status to the metabolic state of tumor cells in RCC.


Assuntos
Aminoidrolases/fisiologia , Carcinoma de Células Renais/metabolismo , Glicólise/genética , Neoplasias Renais/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/fisiologia , Metiltransferases/metabolismo , Enzimas Multifuncionais/fisiologia , Processamento Pós-Transcricional do RNA/genética , Animais , Metabolismo dos Carboidratos/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Reprogramação Celular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Metilação , Camundongos , Camundongos Nus
13.
Cells ; 8(7)2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31277520

RESUMO

Coronary artery disease is the most common form of cardiovascular diseases, resulting in the loss of cardiomyocytes (CM) at the site of ischemic injury. To compensate for the loss of CMs, cardiac fibroblasts quickly respond to injury and initiate cardiac remodeling in an injured heart. In the remodeling process, cardiac fibroblasts proliferate and differentiate into myofibroblasts, which secrete extracellular matrix to support the intact structure of the heart, and eventually differentiate into matrifibrocytes to form chronic scar tissue. Discovery of direct cardiac reprogramming offers a promising therapeutic strategy to prevent/attenuate this pathologic remodeling and replace the cardiac fibrotic scar with myocardium in situ. Since the first discovery in 2010, many progresses have been made to improve the efficiency and efficacy of reprogramming by understanding the mechanisms and signaling pathways that are activated during direct cardiac reprogramming. Here, we overview the development and recent progresses of direct cardiac reprogramming and discuss future directions in order to translate this promising technology into an effective therapeutic paradigm to reverse cardiac pathological remodeling in an injured heart.


Assuntos
Reprogramação Celular/genética , Doença da Artéria Coronariana/terapia , Miocárdio/patologia , Medicina Regenerativa/métodos , Fatores de Transcrição/genética , Animais , Doença da Artéria Coronariana/patologia , Modelos Animais de Doenças , Matriz Extracelular/patologia , Fibrose , Regulação da Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Injeções Intralesionais , Miocárdio/citologia , Miócitos Cardíacos/fisiologia , Miofibroblastos/fisiologia , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo
14.
BMC Cancer ; 19(1): 711, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324166

RESUMO

BACKGROUND: Differentiated cells that arise from stem cells in early development contain DNA methylation features that provide a memory trace of their fetal cell origin (FCO). The FCO signature was developed to estimate the proportion of cells in a mixture of cell types that are of fetal origin and are reminiscent of embryonic stem cell lineage. Here we implemented the FCO signature estimation method to compare the fraction of cells with the FCO signature in tumor tissues and their corresponding nontumor normal tissues. METHODS: We applied our FCO algorithm to discovery data sets obtained from The Cancer Genome Atlas (TCGA) and replication data sets obtained from the Gene Expression Omnibus (GEO) data repository. Wilcoxon rank sum tests, linear regression models with adjustments for potential confounders and non-parametric randomization-based tests were used to test the association of FCO proportion between tumor tissues and nontumor normal tissues. P-values of < 0.05 were considered statistically significant. RESULTS: Across 20 different tumor types we observed a consistently lower FCO signature in tumor tissues compared with nontumor normal tissues, with 18 observed to have significantly lower FCO fractions in tumor tissue (total n = 6,795 tumor, n = 922 nontumor, P < 0.05). We replicated our findings in 15 tumor types using data from independent subjects in 15 publicly available data sets (total n = 740 tumor, n = 424 nontumor, P < 0.05). CONCLUSIONS: The results suggest that cancer development itself is substantially devoid of recapitulation of normal embryologic processes. Our results emphasize the distinction between DNA methylation in normal tightly regulated stem cell driven differentiation and cancer stem cell reprogramming that involves altered methylation in the service of great cell heterogeneity and plasticity.


Assuntos
Metilação de DNA/genética , Células-Tronco Embrionárias Humanas/metabolismo , Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Adulto , Algoritmos , Plasticidade Celular , Reprogramação Celular/genética , Ilhas de CpG , Epigênese Genética , Feminino , Heterogeneidade Genética , Loci Gênicos , Humanos , Modelos Lineares , Masculino , Neoplasias/patologia , Gravidez , Estatísticas não Paramétricas , Transcriptoma
15.
Oncogene ; 38(34): 6226-6239, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31308488

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) arises through accumulation of multiple genetic alterations. However, cancer cells also acquire and depend on cancer-specific epigenetic changes. To conclusively demonstrate the crucial relevance of the epigenetic programme for the tumourigenicity of the cancer cells, we used cellular reprogramming technology to reverse these epigenetic changes. We reprogrammed human PDAC cultures using three different techniques - (1) lentivirally via induction of Yamanaka Factors (OSKM), (2) the pluripotency-associated gene OCT4 and the microRNA mir-302, or (3) using episomal vectors as a safer alternative without genomic integration. We found that induction with episomal vectors was the most efficient method to reprogram primary human PDAC cultures as well as primary human fibroblasts that served as positive controls. Successful reprogramming was evidenced by immunostaining, alkaline phosphatase staining, and real-time PCR. Intriguingly, reprogramming of primary human PDAC cultures drastically reduced their in vivo tumourigenicity, which appeared to be driven by the cells' enhanced differentiation and loss of stemness upon transplantation. Our study demonstrates that reprogrammed primary PDAC cultures are functionally distinct from parental PDAC cells resulting in drastically reduced tumourigenicity in vitro and in vivo. Thus, epigenetic alterations account at least in part for the tumourigenicity and aggressiveness of pancreatic cancer, supporting the notion that epigenetic modulators could be a suitable approach to improve the dismal outcome of patients with pancreatic cancer.


Assuntos
Carcinogênese/genética , Carcinoma Ductal Pancreático/patologia , Reprogramação Celular/genética , Epigênese Genética/fisiologia , Neoplasias Pancreáticas/patologia , Animais , Carcinogênese/patologia , Carcinoma Ductal Pancreático/genética , Células Cultivadas , Embrião de Mamíferos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Metástase Neoplásica , Neoplasias Pancreáticas/genética , Cultura Primária de Células
16.
Biochim Biophys Acta Gene Regul Mech ; 1862(9): 194407, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31356991

RESUMO

Reprogramming to induced pluripotency through expression of OCT4, SOX2, KLF4, MYC (OSKM) factors is often considered the dedifferentiation of somatic cells. This would suggest that reprogramming represents the reversal of embryonic differentiation. Indeed, molecular events involving the activity of the pluripotency network occur in opposite directions. However, reprogramming and development substantially differ as OSKM bind to accessible regulatory elements in the genome of somatic cells due to their overexpression, including regulatory elements never bound by these factors during normal differentiation. In addition, rewiring the transcriptional network back to pluripotency involves overcoming molecular barriers that protect or stabilize the somatic identity, whereas extrinsic and intrinsic cues will drive differentiation in an energetically favorable landscape in the embryo. This review focuses on how cell fate transitions in reprogramming and development are differentially governed by interactions between transcription factors and chromatin. We also discuss how these interactions shape chromatin architecture and the transcriptional output. Major technological advances have resulted in a better understanding of both differentiation and reprogramming, which is essential to exploit reprogramming regimes for regenerative medicine.


Assuntos
Linhagem da Célula/genética , Reprogramação Celular/genética , Cromatina/genética , Fatores de Transcrição/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Genoma/genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/genética
17.
Int J Mol Sci ; 20(15)2019 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-31357694

RESUMO

Cancer cells have an unusual regulation of hydrogen ion dynamics that are driven by poor vascularity perfusion, regional hypoxia, and increased glycolysis. All these forces synergize/orchestrate together to create extracellular acidity and intracellular alkalinity. Precisely, they lead to extracellular pH (pHe) values as low as 6.2 and intracellular pH values as high as 8. This unique pH gradient (∆pHi to ∆pHe) across the cell membrane increases as the tumor progresses, and is markedly displaced from the electrochemical equilibrium of protons. These unusual pH dynamics influence cancer cell biology, including proliferation, metastasis, and metabolic adaptation. Warburg metabolism with increased glycolysis, even in the presence of Oxygen with the subsequent reduction in Krebs' cycle, is a common feature of most cancers. This metabolic reprogramming confers evolutionary advantages to cancer cells by enhancing their resistance to hypoxia, to chemotherapy or radiotherapy, allowing rapid production of biological building blocks that support cellular proliferation, and shielding against damaging mitochondrial free radicals. In this article, we highlight the interconnected roles of dysregulated pH dynamics in cancer initiation, progression, adaptation, and in determining the programming and re-programming of tumor cell metabolism.


Assuntos
Transformação Celular Neoplásica/genética , Radicais Livres/metabolismo , Neoplasias/genética , Trocador 1 de Sódio-Hidrogênio/genética , Proliferação de Células/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Reprogramação Celular/genética , Glicólise/genética , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação Oxidativa , Microambiente Tumoral/genética
18.
Mol Carcinog ; 58(10): 1876-1885, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31292999

RESUMO

Sirtuin-1 and -3 (SIRT1 and SIRT3) are important nicotinamide adenine dinucleotide (NAD+ )-dependent deacetylases known to regulate a variety of cellular functions. Studies have shown that SIRT1 and SIRT3 were overexpressed in human melanoma cells and tissues and their inhibition resulted in a significant antiproliferative response in human melanoma cells and antitumor response in a mouse xenograft model of melanoma. In this study, we determined the antiproliferative efficacy of a newly identified dual small molecule inhibitor of SIRT1 and SIRT3, 4'-bromo-resveratrol (4'-BR), in human melanoma cell lines (G361, SK-MEL-28, and SK-MEL-2). Our data demonstrate that 4'-BR treatment of melanoma cells resulted in (a) decrease in proliferation and clonogenic survival; (b) induction of apoptosis accompanied by a decrease in procaspase-3, procaspase-8, and increase in the cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP); (c) marked downregulation of proliferating cell nuclear antigen (PCNA); and (d) inhibition of melanoma cell migration. Further, 4'-BR caused a G0/G1 phase arrest of melanoma cells that was accompanied by an increase in WAF-1/P21 and decrease in Cyclin D1/Cyclin-dependent kinase 6 protein levels. Furthermore, we found that 4'-BR causes a decrease in lactate production, glucose uptake, and NAD+ /NADH ratio. These responses were accompanied by downregulation in lactate dehydrogenase A and glucose transporter 1 in melanoma cells. Collectively, our data suggest that dual inhibition of SIRT1 and SIRT3 using 4'-BR imparted antiproliferative effects in melanoma cells through a metabolic reprogramming and affecting the cell cycle and apoptosis signaling. Therefore, concomitant pharmacological inhibition of SIRT1 and SIRT3 needs further investigation for melanoma management.


Assuntos
Melanoma/tratamento farmacológico , Resorcinóis/farmacologia , Sirtuína 1/genética , Sirtuína 3/genética , Estilbenos/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ácido Láctico/metabolismo , Melanoma/genética , Melanoma/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Sirtuína 1/antagonistas & inibidores , Sirtuína 3/antagonistas & inibidores
19.
Genes Dev ; 33(15-16): 1048-1068, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31221665

RESUMO

Fetal hematopoietic stem and progenitor cells (HSPCs) hold promise to cure a wide array of hematological diseases, and we previously found a role for the RNA-binding protein (RBP) Lin28b in respecifying adult HSPCs to resemble their fetal counterparts. Here we show by single-cell RNA sequencing that Lin28b alone was insufficient for complete reprogramming of gene expression from the adult toward the fetal pattern. Using proteomics and in situ analyses, we found that Lin28b (and its closely related paralog, Lin28a) directly interacted with Igf2bp3, another RBP, and their enforced co-expression in adult HSPCs reactivated fetal-like B-cell development in vivo more efficiently than either factor alone. In B-cell progenitors, Lin28b and Igf2bp3 jointly stabilized thousands of mRNAs by binding at the same sites, including those of the B-cell regulators Pax5 and Arid3a as well as Igf2bp3 mRNA itself, forming an autoregulatory loop. Our results suggest that Lin28b and Igf2bp3 are at the center of a gene regulatory network that mediates the fetal-adult hematopoietic switch. A method to efficiently generate induced fetal-like hematopoietic stem cells (ifHSCs) will facilitate basic studies of their biology and possibly pave a path toward their clinical application.


Assuntos
Reprogramação Celular/genética , Proteínas de Ligação a DNA/metabolismo , Redes Reguladoras de Genes , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Ligação a RNA/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Proteínas de Ligação a DNA/genética , Camundongos , MicroRNAs/metabolismo , Modelos Animais , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética
20.
Nat Commun ; 10(1): 2707, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31222004

RESUMO

Bone metastases occur in most advanced breast cancer patients and cause serious skeletal-related complications. The mechanisms by which bone metastasis seeds develop in primary tumors and specifically colonize the bone remain to be elucidated. Here, we show that forkhead box F2 (FOXF2) functions as a master transcription factor for reprogramming cancer cells into an osteomimetic phenotype by pleiotropic transactivation of the BMP4/SMAD1 signaling pathway and bone-related genes that are expressed at early stages of bone differentiation. The epithelial-to-osteomimicry transition regulated by FOXF2 confers a tendency on cancer cells to metastasize to bone which leads to osteolytic bone lesions. The BMP antagonist Noggin significantly inhibits FOXF2-driven osteolytic bone metastasis of breast cancer cells. Thus, targeting the FOXF2-BMP/SMAD axis might be a promising therapeutic strategy to manage bone metastasis. The role of FOXF2 in transactivating bone-related genes implies a biological function of FOXF2 in regulating bone development and remodeling.


Assuntos
Neoplasias Ósseas/patologia , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Animais , Proteína Morfogenética Óssea 4/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/secundário , Neoplasias da Mama/cirurgia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/secundário , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Reprogramação Celular/genética , Feminino , Seguimentos , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Osteoblastos , Transdução de Sinais/genética , Proteína Smad1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
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