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1.
Methods Mol Biol ; 2318: 69-85, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019287

RESUMO

Detection of post-translational modifications in c-Myc is an invaluable tool in assessing Myc status, particularly in cancer. However, it can be challenging to detect these modifications. The evaluation of phosphorylation status of c-Myc can also be challenging with the current commercially available phosphorylation sensitive antibodies. Here, we describe protocols for the immunoprecipitation of endogenous c-Myc to probe for phosphorylation status, as well as the detection of ubiquitination and SUMOylation on c-Myc. We will also discuss the challenges of detecting phosphorylated c-Myc in formalin-fixed paraffin-embedded tissues by immunofluorescence and describe a protocol using a new rat monoclonal antibody we have generated suitable for this purpose.


Assuntos
Imunoprecipitação/métodos , Processamento de Proteína Pós-Traducional/genética , Proteínas Proto-Oncogênicas c-myc/genética , Imunofluorescência , Genes myc/genética , Genes myc/fisiologia , Humanos , Fosforilação , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sumoilação , Ubiquitinação
2.
Methods Mol Biol ; 2318: 231-239, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019293

RESUMO

The MYC gene regulates normal cell growth and is deregulated in many human cancers, contributing to tumor growth and progression. The MYC transcription factor activates RNA polymerases I, II, and III target genes that are considered housekeeping genes. These target genes are largely involved in ribosome biogenesis, fatty acid, protein and nucleotide synthesis, nutrient influx or metabolic waste efflux, glycolysis, and glutamine metabolism. MYC's function as a driver of cell growth has been revealed through RNA sequencing, genome-wide chromatin immunoprecipitation, proteomics, and importantly metabolomics, which is highlighted in this chapter.


Assuntos
Carcinogênese/metabolismo , Metabolômica/métodos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Carcinogênese/genética , Proliferação de Células , Transformação Celular Neoplásica/genética , DNA/genética , Genes myc/genética , Genes myc/fisiologia , Glucose/metabolismo , Glicólise , Humanos , Neoplasias/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase I/metabolismo , Ribossomos/metabolismo
3.
Methods Mol Biol ; 2318: 21-43, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019285

RESUMO

The C-terminal region of the c-MYC transcription factor consists of approximately 100 amino acids that in its native state does not adopt a stable structure. When this region binds to the obligatory partner MAX via a coupled folding-and-binding mechanism, it forms a basic-helix-loop-helix-leucine zipper (bHLHZip) heterodimeric complex. The C-terminal region of MYC is the target for numerous drug discovery programs for direct MYC inhibition via blocking the dimerization event and/or binding to DNA, and a proper understanding of the partially folded, dynamic nature of the heterodimeric complex is essential to these efforts. The bHLHZip motif also drives protein-protein interactions with cofactors that are crucial for both transcriptional repression and activation of MYC target genes. Targeting these interactions could potentially provide a means of developing alternative approaches to halt MYC functions; however, the molecular mechanism of these regulatory interactions is poorly understood. Herein we provide methods to produce high-quality human c-MYC C-terminal by itself and in complex MAX, and how to study them using Nuclear Magnetic Resonance spectroscopy and X-ray crystallography. Our protein expression and purification protocols have already been used to study interactions with cofactors.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/isolamento & purificação , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/isolamento & purificação , Sequência de Aminoácidos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Sítios de Ligação , Cristalografia por Raios X/métodos , DNA/química , DNA/genética , Dimerização , Genes myc/genética , Genes myc/fisiologia , Sequências Hélice-Alça-Hélice/genética , Sequências Hélice-Alça-Hélice/fisiologia , Humanos , Zíper de Leucina/genética , Zíper de Leucina/fisiologia , Espectroscopia de Ressonância Magnética/métodos , Ligação Proteica , Domínios Proteicos/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/metabolismo
4.
Methods Mol Biol ; 2318: 45-67, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019286

RESUMO

By identifying MYC protein-protein interactors, we aim to gain a deeper mechanistic understanding of MYC as a regulator of gene transcription and potent oncoprotein. This information can then be used to devise strategies for disrupting critical MYC protein-protein interactions to inhibit MYC-driven tumorigenesis. In this chapter, we discuss four techniques to identify and validate MYC-interacting partners. First, we highlight BioID, a powerful discovery method used to identify high-confidence proximal interactors in living cells. We also discuss bioinformatic prioritization strategies for the BioID-derived MYC-proximal complexes. Next, we discuss how protein interactions can be validated using techniques such as in vivo-in vitro pull-down assays and the proximity ligation assay (PLA). We conclude with an overview of biolayer interferometry (BLI), a quantitative method used to characterize direct interactions between two proteins in vitro. Overall, we highlight the principles of each assay and provide methodology necessary to conduct these experiments and adapt them to the study of interactors of additional proteins of interest.


Assuntos
Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/isolamento & purificação , Sequência de Aminoácidos/genética , Sítios de Ligação , Biologia Computacional/métodos , DNA/química , DNA/genética , Dimerização , Genes myc/genética , Genes myc/fisiologia , Humanos , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas/métodos , Proteínas Proto-Oncogênicas c-myc/metabolismo
5.
Methods Mol Biol ; 2318: 87-117, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019288

RESUMO

The MYC oncogene was originally identified as a transduced allele (v-myc) in the genome of the highly oncogenic avian retrovirus MC29. The protein product (MYC) of the cellular MYC (c-myc) protooncogene represents the key component of a transcription factor network controlling the expression of a large fraction of all human genes. MYC regulates fundamental cellular processes like growth control, metabolism, proliferation, differentiation, and apoptosis. Mutational deregulation of MYC, leading to increased levels of the MYC protein, is a frequent event in the etiology of human cancers. In this chapter, we describe cell systems and experimental strategies to quantify the oncogenic potential of MYC alleles, to test MYC inhibitors, and to monitor MYC-specific protein-protein interactions that are relevant for the cell transformation process. We also describe experimental procedures to study the evolutionary origin of MYC and to analyze structure, function, and regulation of the ancestral MYC proto-oncogenes.


Assuntos
Neoplasias/genética , Proteínas Proto-Oncogênicas c-myc/genética , Alelos , Sequência de Aminoácidos , Animais , Carcinogênese , Transformação Celular Neoplásica/genética , Evolução Molecular , Genes myc/genética , Genes myc/fisiologia , Humanos , Mutação , Neoplasias/patologia , Oncogenes , Mapeamento de Interação de Proteínas/métodos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/genética
6.
Methods Mol Biol ; 2318: 187-208, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019291

RESUMO

MYC is a transcription factor playing multiple functions both in physiological and pathological settings. Biochemical characterizations, combined with the analyses of MYC chromatin binding, have shown that its pleiotropic activity depends on the chromatin context and its protein-protein interactions with different cofactors. In order to determine the contribution of MYC in a certain biological condition, it would be relevant to analyze the concomitant binding of MYC and its associated proteins, in relationship to the chromatin environment. To this end, we here provide a simple method to parallel map the genome-wide binding of MYC-associated proteins, together with the chromatin profiling of multiple histone modifications. We detail the procedure to perform high-throughput ChIP-seq (HT-ChIP-seq) with a variety of biological samples. In addition, we describe simple bioinformatic steps to determine the distribution of MYC binding with respect to the chromatin context and the association of its cofactors. The described approach will permit the reproducible characterization of MYC activity in different biological contexts.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Epigenômica/métodos , Proteínas Proto-Oncogênicas c-myc/genética , Cromatina/genética , Imunoprecipitação da Cromatina/métodos , Biologia Computacional/métodos , DNA/genética , Epigênese Genética/genética , Genes myc/genética , Genes myc/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Código das Histonas/genética , Histonas/metabolismo , Humanos , Processamento de Proteína Pós-Traducional/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Análise de Sequência de DNA/métodos , Fatores de Transcrição/metabolismo
7.
Methods Mol Biol ; 2318: 241-254, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019294

RESUMO

Cellular senescence plays a role in several physiological processes including aging, embryonic development, tissue remodeling, and wound healing and is considered one of the main barriers against tumor development. Studies of normal and tumor cells both in culture and in vivo suggest that MYC plays an important role in regulating senescence, thereby contributing to tumor development. We have previously described different common methods to measure senescence in cell cultures and in tissues. Unfortunately, there is no unique marker that unambiguously defines a senescent state, and it is therefore necessary to combine measurements of several different markers in order to assure the correct identification of senescent cells. Here we describe protocols for simultaneous detection of multiple senescence markers in situ, a quantitative fluorogenic method to measure senescence-associated ß-galactosidase activity (SA-ß-gal), and a new method to detect senescent cells based on the Sudan Black B (SBB) analogue GL13, which is applicable to formalin-fixed paraffin-embedded tissues. The application of these methods in various systems will hopefully shed further light on the role of MYC in regulation of senescence, and how that impacts normal physiological processes as well as diseases and in particular cancer development.


Assuntos
Senescência Celular/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Coloração e Rotulagem/métodos , Envelhecimento , Compostos Azo/química , Biomarcadores , Células Cultivadas , Senescência Celular/genética , DNA/genética , Corantes Fluorescentes/química , Genes myc/genética , Genes myc/fisiologia , Humanos , Naftalenos/química , Proteínas Proto-Oncogênicas c-myc/genética , beta-Galactosidase/análise , beta-Galactosidase/metabolismo
8.
Methods Mol Biol ; 2318: 255-266, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019295

RESUMO

A central component of Myc's role as a master coordinator of energy metabolism and biomass accumulation is its ability to increase the rate of protein synthesis, driving cell cycle progression, and proliferation. Importantly, Myc-induced alterations in both global and specific mRNA translation is a key determinant of Myc's oncogenic function. Herein, we provide five assays to enable researchers to measure global protein synthesis changes, to identify the translatome uniquely regulated by Myc and to investigate the mechanisms generating the tailored Myc translation network. Metabolic labeling of cells with 35S-containing methionine and cysteine in culture and O-propargyl-puromycin (OP-Puro) incorporation in vivo are presented as methods to measure the overall rate of global protein synthesis. Isolation of polysome-associated mRNAs followed by quantitative real-time PCR (qRT-PCR) and the toeprint assay enable the detection of altered translation of specific mRNAs and isoforms, and visualization of differential ribosomal engagement at start codons uniquely mediated by Myc activation, respectively. Finally, the translation initiation reporter assay is utilized to uncover the molecular mechanism mediating altered translation initiation of a specific mRNA. Together, the protocols detailed in this chapter can be used to illuminate how and to what degree Myc-dependent regulation of translation influences homeostatic cellular functions as well as tumorigenesis.


Assuntos
Metabolômica/métodos , Biossíntese de Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transformação Celular Neoplásica/metabolismo , DNA/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes myc/genética , Genes myc/fisiologia , Homeostase/genética , Homeostase/fisiologia , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Biossíntese de Proteínas/genética , Proteínas/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo
9.
Methods Mol Biol ; 2318: 267-279, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019296

RESUMO

Cellular reprogramming is a process by which adult differentiated cells lose their identity and are converted into pluripotent stem cells, known as induced pluripotent stem (iPS) cells. This process can be achieved in vitro and in vivo and is relevant for many fields including regenerative medicine and cancer. Cellular reprogramming is commonly induced by the ectopic expression of a transcription factor cocktail composed by Oct4, Sox2, Klf4, and Myc (abbreviated as OSKM), and its efficiency and kinetics are strongly dependent on the presence of Myc. Here, we describe a versatile method to study reprogramming in vivo based on the use of adeno-associated viral (AAV) vectors, which allows the targeting of specific organs and cell types. This method can be used to test Myc mutations or genes that may replace Myc, or be combined with different Myc regulators. In vivo reprogramming can be scored by the presence of teratomas and the isolation of in vivo iPS, thereby providing a simple surrogate for the function of Myc in dedifferentiation and stemness. Our protocol can be divided into five steps: (1) intravenous inoculation of AAV vectors; (2) monitoring the animals until the appearance of teratomas; (3) analysis of teratomas; (4) histopathological analysis of mouse organs; and (5) isolation of in vivo-generated iPS cells from teratomas, blood, and bone marrow. The information obtained by this in vivo testing platform may provide relevant information on the role of Myc in tissue regeneration, stemness, and cancer.


Assuntos
Técnicas de Reprogramação Celular/métodos , Reprogramação Celular/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Reprogramação Celular/fisiologia , DNA/genética , Dependovirus/genética , Fibroblastos/citologia , Genes myc/genética , Genes myc/fisiologia , Engenharia Genética/métodos , Vetores Genéticos/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/metabolismo , Transdução Genética
10.
Methods Mol Biol ; 2318: 281-295, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019297

RESUMO

Myc is deregulated in most-if not all-cancers, and it not only promotes tumor progression by inducing cell proliferation but is also responsible for tumor immune evasion. In a nutshell, MYC promotes the development of tumor-associated macrophages, impairs the cellular response to interferons, induces the expression of immunosuppressive molecules, and excludes tumor infiltrating lymphocytes (TILs) from the tumor site. Based on the insights into the role of MYC in promoting and regulating immune evasion by cancer cells, it is of special interest to study the different immune cell populations infiltrating the tumors. MYC inhibition has emerged as a potential new strategy for the treatment of cancer, directly inhibiting tumor progression while also counteracting the immunosuppressive tumor microenvironment, allowing an optimal anti-tumor immune response. Hence, this chapter describes a flow cytometry-based method to study the different immune cell subsets infiltrating the tumor by combining surface, cytoplasmic, and nuclear multicolor protein stainings.


Assuntos
Citometria de Fluxo/métodos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Microambiente Tumoral/imunologia , Proliferação de Células , DNA/genética , Genes myc/genética , Genes myc/fisiologia , Humanos , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias/genética , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/imunologia , Proteínas Proto-Oncogênicas c-myc/genética , Microambiente Tumoral/fisiologia
11.
Methods Mol Biol ; 2318: 297-312, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019298

RESUMO

The tetracycline regulatory system provides a tractable strategy to interrogate the role of oncogenes in the initiation, maintenance, and regression of tumors through both spatial and temporal control of expression. This approach has several potential advantages over conventional methods to generate genetically engineered mouse models. First, continuous constitutive overexpression of an oncogene can be lethal to the host impeding further study. Second, constitutive overexpression fails to model adult onset of disease. Third, constitutive deletion does not permit, whereas conditional overexpression of an oncogene enables the study of the consequences of restoring expression of an oncogene back to endogenous levels. Fourth, the conditional activation of oncogenes enables examination of specific and/or developmental state-specific consequences.Hence, by allowing precise control of when and where a gene is expressed, the tetracycline regulatory system provides an ideal approach for the study of putative oncogenes in the initiation as well as the maintenance of tumorigenesis and the examination of the mechanisms of oncogene addiction. In this protocol, we describe the methods involved in the development of a conditional mouse model of MYC-induced T-cell acute lymphoblastic leukemia.


Assuntos
Engenharia Genética/métodos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Animais Geneticamente Modificados/genética , Apoptose , Carcinogênese/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , DNA/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Genes myc/genética , Genes myc/fisiologia , Humanos , Camundongos , Oncogenes , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Inibidores da Síntese de Proteínas , Proteínas Proto-Oncogênicas c-myc/genética , Linfócitos T/metabolismo , Tetraciclina/farmacologia
12.
Methods Mol Biol ; 2318: 313-320, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019299

RESUMO

In situ hybridization (ISH) allows evaluation of genetic abnormalities, such as changes in chromosome number, chromosome translocations, or gene amplifications, by hybridization of tagged DNA (or RNA) probes with complementary DNA (or RNA) sequences in interphase nuclei of target tissue. However, chromogenic in situ hybridization (CISH) is also applicable to formalin-fixed, paraffin-embedded (FFPE ) tissues, besides metaphase chromosome spreads. CISH is similar to fluorescent in situ hybridization (FISH) regarding pretreatments and hybridization protocols but differs in the way of visualization. Indeed, CISH signal detection is similar to that used in immunohistochemistry, making use of a peroxidase-based chromogenic reaction instead of fluorescent dyes. In particular, tagged DNA probes are indirectly detected using an enzyme-conjugated antibody targeting the tags. The enzymatic reaction of the chromogenic substrate leads to the formation of strong permanent brown signals that can be visualized by bright-field microscopy at 40× magnification. The advantage of CISH is that it allows the simultaneous observation of gene amplification and tissue morphology, and the slides can be stored for a long time.


Assuntos
Hibridização in Situ Fluorescente/métodos , Hibridização In Situ/métodos , Proteínas Proto-Oncogênicas c-myc/imunologia , Compostos Cromogênicos/química , DNA/genética , Sondas de DNA , Amplificação de Genes , Genes myc/genética , Genes myc/fisiologia , Humanos , Imuno-Histoquímica/métodos , Neoplasias , Inclusão em Parafina/métodos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Translocação Genética
13.
Methods Mol Biol ; 2318: 321-336, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019300

RESUMO

Clinical tumor specimens are routinely formalin-fixed and paraffin-embedded (FFPE) in Pathology departments worldwide. FFPE blocks are convenient, long-term stable, and easy to archive and manipulate. However, nucleic acids extracted from FFPE tissues generally show a high degree of fragmentation as well as chemical modifications, mainly due to the fixation process. Methods to determine copy number alterations (CNAs) from FFPE clinical samples have proven challenging, in the fact that they are low-plex, only able to profile single genes or gene clusters (such as in situ hybridization-based methods), and/or show a low degree of robustness with partially degraded samples (array-based, NGS-based) as well as being time-consuming, costly, and with limitations in resolution. The NanoString nCounter® System is a medium-plex, extremely FFPE-robust system, that overcomes several of the frequent issues when dealing with clinical samples. The technique is based on hybridization of molecular barcoded probes directly to FFPE-derived DNA, followed by single molecule imaging to detect hundreds of unique molecules in a single reaction without any amplification steps that might introduce undesired biases. Here we describe nCounter v2 Cancer Copy Number Assay, a robust and highly reproducible method for detecting the copy number status of 87 genes commonly amplified or deleted in cancer, including the MYC proto-oncogene.


Assuntos
Variações do Número de Cópias de DNA , Hibridização de Ácido Nucleico/métodos , Proteínas Proto-Oncogênicas c-myc/genética , DNA/genética , Sondas de DNA , Formaldeído/química , Genes myc/genética , Genes myc/fisiologia , Humanos , Hibridização in Situ Fluorescente/métodos , Neoplasias/genética , Inclusão em Parafina/métodos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fixação de Tecidos/métodos
14.
Methods Mol Biol ; 2318: 337-346, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019301

RESUMO

Oncoproteins encoded by dominant oncogenes have long been considered as targets for chemotherapeutic intervention. However, oncogenic transcription factors have often been dismissed as "undruggable." Members of the Myc family of transcription factors have been identified as promising targets for cancer chemotherapy in multiple publications reporting the requirement of Myc proteins for maintenance of almost every type of tumor. Here, we describe cell-based approaches to identify c-Myc small molecule inhibitors by screening complex libraries of diverse small molecules based on Myc functionality and specificity.


Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Genes myc/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Linhagem Celular Tumoral , Genes myc/genética , Genes myc/fisiologia , Humanos , Proteínas Oncogênicas/efeitos dos fármacos , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo
15.
Life Sci Alliance ; 4(5)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33653688

RESUMO

The huge cadre of genes regulated by Myc has obstructed the identification of critical effectors that are essential for Myc-driven tumorigenesis. Here, we describe how only the lack of the receptor Fzd9, previously identified as a Myc transcriptional target, impairs sustained tumor expansion and ß-cell dedifferentiation in a mouse model of Myc-driven insulinoma, allows pancreatic islets to maintain their physiological structure and affects Myc-related global gene expression. Importantly, Wnt signaling inhibition in Fzd9-competent mice largely recapitulates the suppression of proliferation caused by Fzd9 deficiency upon Myc activation. Together, our results indicate that the Wnt signaling receptor Fzd9 is essential for Myc-induced tumorigenesis in pancreatic islets.


Assuntos
Adenoma de Células das Ilhotas Pancreáticas/fisiopatologia , Carcinogênese/metabolismo , Receptores Frizzled/metabolismo , Adenoma de Células das Ilhotas Pancreáticas/metabolismo , Animais , Movimento Celular , Proliferação de Células , Feminino , Receptores Frizzled/genética , Receptores Frizzled/fisiologia , Genes myc/genética , Genes myc/fisiologia , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo
16.
Cancer Res ; 81(8): 1925-1936, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33472888

RESUMO

MYC is embedded in the transcriptional oasis of the 8q24 gene desert. A plethora of genomic elements has roles in MYC aberrant expression in cancer development by interacting with transcription factors and epigenetics regulators as well as altering the structure of chromatin at the MYC locus and tissue-specific long-range enhancer-promoter contacts. Furthermore, MYC is a master regulator of several human cancers by modulating the transcription of numerous cancer-related genes through epigenetic mechanisms. This review provides a comprehensive overview of the three-dimensional genomic organization around MYC and the role of epigenetic machinery in transcription and function of MYC as well as discusses various epigenetic-targeted therapeutic strategies in MYC-driven cancers.


Assuntos
Cromatina/ultraestrutura , Epigênese Genética/fisiologia , Regulação Neoplásica da Expressão Gênica , Genes myc/fisiologia , Neoplasias/genética , Fatores de Transcrição/fisiologia , Fator de Ligação a CCCTC , Cromossomos Humanos Par 8/genética , Progressão da Doença , Genes Neoplásicos , Sequências Hélice-Alça-Hélice , Humanos , MicroRNAs/fisiologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/fisiologia , Elementos Reguladores de Transcrição
17.
Cancer Res ; 80(18): 3799-3802, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32732221

RESUMO

Myc and Ras are two of the most commonly activated oncogenes in tumorigenesis. Together and independently they regulate many cancer hallmarks including proliferation, apoptosis, and self-renewal. Recently, they were shown to cooperate to regulate host tumor microenvironment programs including host immune responses. But, is their partnership always cooperative or do they have distinguishable functions? Here, we provide one perspective that Myc and Ras cooperation depends on the genetic evolution of a particular cancer. This in turn, dictates when they cooperate via overlapping and identifiably distinct cellular- and host immune-dependent mechanisms that are cancer type specific.


Assuntos
Neoplasias da Mama/genética , Genes myc/fisiologia , Genes ras/fisiologia , Neoplasias Hematológicas/genética , Neoplasias Pulmonares/genética , Microambiente Tumoral/genética , Adenocarcinoma/genética , Carcinogênese/genética , Carcinogênese/imunologia , Fenômenos Fisiológicos Celulares/genética , Feminino , Humanos , Ativação Transcricional
18.
Cell Death Dis ; 11(3): 167, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32127525

RESUMO

Pseudogenes have long been considered as nonfunctional genomic sequences. Recent studies have shown that they can potentially regulate the expression of protein-coding genes and are dysregulated in diseases including cancer. However, the potential roles of pseudogenes in ovarian cancer have not been well studied. Here we characterized the pseudogene expression profile in HGSOC (high-grade serous ovarian carcinoma) by microarray. We identified 577 dysregulated pseudogenes and most of them were up-regulated (538 of 577). HMGA1P6 (High mobility group AT-hook 1 pseudogene 6) was one of the overexpressed pseudogenes and its expression was inversely correlated with patient survival. Mechanistically, HMGA1P6 promoted ovarian cancer cell malignancy by acting as a ceRNA (competitive endogenous RNA) that led to enhanced HMGA1 and HMGA2 expression. Importantly, HMGA1P6 was transcriptionally activated by oncogene MYC in ovarian cancer. Our findings reveal that MYC may contribute to oncogenesis through transcriptional regulation of pseudogene HMGA1P6 in ovarian cancer.


Assuntos
Proteína HMGA1a/genética , Proteína HMGA2/genética , Neoplasias Ovarianas/genética , Pseudogenes/genética , Carcinogênese/genética , Carcinoma Epitelial do Ovário/genética , Cistadenocarcinoma Seroso/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Genes myc/genética , Genes myc/fisiologia , Proteína HMGA1a/metabolismo , Proteína HMGA2/metabolismo , Humanos , Oncogenes/genética , Pseudogenes/fisiologia
19.
Hepatology ; 71(1): 148-163, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31155734

RESUMO

The oncogene c-Myc is aberrantly expressed and plays a key role in malignant transformation and progression of hepatocellular carcinoma (HCC). Here, we report that c-Myc is significantly up-regulated by tumor necrosis factor receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase, in hepatocarcinogenesis. High TRAF6 expression in clinical HCC samples correlates with poor prognosis, and the loss of one copy of the Traf6 gene in Traf6+/- mice significantly impairs liver tumorigenesis. Mechanistically, TRAF6 first interacts with and ubiquitinates histone deacetylase 3 (HDAC3) with K63-linked ubiquitin chains, which leads to the dissociation of HDAC3 from the c-Myc promoter and subsequent acetylation of histone H3 at K9, thereby epigenetically enhancing the mRNA expression of c-Myc. Second, the K63-linked ubiquitination of HDAC3 impairs the HDAC3 interaction with c-Myc and promotes c-Myc protein acetylation, which thereby enhances c-Myc protein stability by inhibiting carboxyl terminus of heat shock cognate 70-kDa-interacting protein-mediated c-Myc ubiquitination and degradation. Importantly, TRAF6/HDAC3/c-Myc signaling is also primed in hepatitis B virus-transgenic mice, unveiling a critical role for a mechanism in inflammation-cancer transition. In clinical specimens, TRAF6 positively correlates with c-Myc at both the mRNA and protein levels, and high TRAF6 and c-Myc expression is associated with an unfavorable prognosis, suggesting that TRAF6 collaborates with c-Myc to promote human hepatocarcinogenesis. Consistently, curbing c-Myc expression by inhibition of TRAF6 activity with a TRAF6 inhibitor peptide or the silencing of c-Myc by small interfering RNA significantly suppressed tumor growth in mice. Conclusion: These findings demonstrate the oncogenic potential of TRAF6 during hepatocarcinogenesis by modulating TRAF6/HDAC3/c-Myc signaling, with potential implications for HCC therapy.


Assuntos
Carcinogênese , Carcinoma Hepatocelular/genética , Genes myc/fisiologia , Histona Desacetilases/fisiologia , Neoplasias Hepáticas/genética , Fator 6 Associado a Receptor de TNF/fisiologia , Animais , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Estabilidade Proteica , Células Tumorais Cultivadas
20.
Med Sci Monit ; 25: 8637-8644, 2019 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-31733054

RESUMO

BACKGROUND Although the promoting roles of Frizzled-7 (Fzd7) have been shown before, its effects in gastric cancer (GC) cell stemness are still unclear. The present study assessed the effects of Fzd7 on GC cell stemness and chemoresistance. MATERIAL AND METHODS Clinical samples were used to detect Fzd7 expression and online datasets were used to analyze the correlation between Fzd7 expression and GC patient prognosis. Quantitative real-time PCR (qPCR), Western blot, and spheroid formation were used to detect the stemness of cells and Fzd7-mediated effects on GC cell stemness. Cell viability was assessed to evaluate the role of Fzd7 in chemoresistance of GC cells. RESULTS We found that the expression of Frizzled-7 (Fzd7), a Wnt receptor, was increased in gastric cancer (GC) cells and tissues. Additionally, Fzd7 expression was correlated with shorter overall survival of GC patients. Knockdown of Fzd7 or using inhibitors of Wnt/Fzd (OMP-18R5/Vantictumad) decreased GC cell stemness, characterized as a decrease of spheroid formation ability and expression of stemness regulators. Notably, Fzd7 knockdown or inhibitors of Wnt/Fzd attenuated the chemoresistance of GC cells. Furthermore, elevation of Myc expression rescued the effects of Fzd7 inhibition on GC cell stemness and chemoresistance. CONCLUSIONS Our results suggest that inhibition of Fzd7 decreases the stemness and chemotherapeutic resistance of GC cells.


Assuntos
Receptores Frizzled/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Gástricas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , China , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Receptores Frizzled/genética , Receptores Frizzled/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Genes myc/genética , Genes myc/fisiologia , Humanos , Estimativa de Kaplan-Meier , Neoplasias Gástricas/genética , Via de Sinalização Wnt/genética , beta Catenina/metabolismo
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