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1.
Proc Natl Acad Sci U S A ; 110(29): 11994-9, 2013 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-23818607

RESUMO

The ten-eleven translocation 1 (TET1) gene is the founding member of the TET family of enzymes (TET1/2/3) that convert 5-methylcytosine to 5-hydroxymethylcytosine. Although TET1 was first identified as a fusion partner of the mixed lineage leukemia (MLL) gene in acute myeloid leukemia carrying t(10,11), its definitive role in leukemia is unclear. In contrast to the frequent down-regulation (or loss-of-function mutations) and critical tumor-suppressor roles of the three TET genes observed in various types of cancers, here we show that TET1 is a direct target of MLL-fusion proteins and is significantly up-regulated in MLL-rearranged leukemia, leading to a global increase of 5-hydroxymethylcytosine level. Furthermore, our both in vitro and in vivo functional studies demonstrate that Tet1 plays an indispensable oncogenic role in the development of MLL-rearranged leukemia, through coordination with MLL-fusion proteins in regulating their critical cotargets, including homeobox A9 (Hoxa9)/myeloid ecotropic viral integration 1 (Meis1)/pre-B-cell leukemia homeobox 3 (Pbx3) genes. Collectively, our data delineate an MLL-fusion/Tet1/Hoxa9/Meis1/Pbx3 signaling axis in MLL-rearranged leukemia and highlight TET1 as a potential therapeutic target in treating this presently therapy-resistant disease.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Leucemia Mieloide Aguda/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , 5-Metilcitosina/análogos & derivados , Imunoprecipitação da Cromatina , Cromatografia Líquida , Citosina/análogos & derivados , Citosina/metabolismo , Perfilação da Expressão Gênica , Histona-Lisina N-Metiltransferase , Proteínas de Homeodomínio/metabolismo , Humanos , Immunoblotting , Análise em Microsséries , Oxigenases de Função Mista , Proteína Meis1 , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/genética , Espectrometria de Massas em Tandem
2.
Proc Natl Acad Sci U S A ; 110(28): 11511-6, 2013 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-23798388

RESUMO

MicroRNAs (miRNAs), small noncoding RNAs that regulate target gene mRNAs, are known to contribute to pathogenesis of cancers. Acute myeloid leukemia (AML) is a group of heterogeneous hematopoietic malignancies with various chromosomal and/or molecular abnormalities. AML with chromosomal translocations involving the mixed lineage leukemia (MLL) gene are usually associated with poor survival. In the present study, through a large-scale, genomewide miRNA expression assay, we show that microRNA-9 (miR-9) is the most specifically up-regulated miRNA in MLL-rearranged AML compared with both normal control and non-MLL-rearranged AML. We demonstrate that miR-9 is a direct target of MLL fusion proteins and can be significantly up-regulated in expression by the latter in human and mouse hematopoietic stem/progenitor cells. Depletion of endogenous miR-9 expression by an appropriate antagomiR can significantly inhibit cell growth/viability and promote apoptosis in human MLL-rearranged AML cells, and the opposite is true when expression of miR-9 is forced. Blocking endogenous miR-9 function by anti-miRNA sponge can significantly inhibit, whereas forced expression of miR-9 can significantly promote, MLL fusion-induced immortalization/transformation of normal mouse bone marrow progenitor cells in vitro. Furthermore, forced expression of miR-9 can significantly promote MLL fusion-mediated leukemogenesis in vivo. In addition, a group of putative target genes of miR-9 exhibited a significant inverse correlation of expression with miR-9 in a series of leukemia sample sets, suggesting that they are potential targets of miR-9 in MLL-rearranged AML. Collectively, our data demonstrate that miR-9 is a critical oncomiR in MLL-rearranged AML and can serve as a potential therapeutic target to treat this dismal disease.


Assuntos
Leucemia Mieloide Aguda/genética , MicroRNAs/fisiologia , Proteína de Leucina Linfoide-Mieloide/genética , Apoptose/genética , Sobrevivência Celular/genética , Proteínas de Ligação a DNA/fisiologia , Humanos , Leucemia Mieloide Aguda/patologia , Proteína do Locus do Complexo MDS1 e EVI1 , MicroRNAs/genética , Proto-Oncogenes/fisiologia , Fatores de Transcrição/fisiologia
3.
Blood ; 121(8): 1422-31, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23264595

RESUMO

Although PBX proteins are known to increase DNA-binding/transcriptional activity of HOX proteins through their direct binding, the functional importance of their interaction in leukemogenesis is unclear.We recently reported that overexpression of a 4-homeobox-gene signature (ie, PBX3/HOXA7/HOXA9/HOXA11) is an independent predictor of poor survival in patients with cytogenetically abnormal acute myeloid leukemia (CA-AML). Here we show that it is PBX3, but not PBX1 or PBX2, that is consistently coexpressed with HOXA9 in various subtypes of CA-AML, particularly MLL-rearranged AML, and thus appears as a potential pathologic cofactor of HOXA9 in CA-AML. We then show that depletion of endogenous Pbx3 expression by shRNA significantly inhibits MLL-fusion-mediated cell transformation, and coexpressed PBX3 exhibits a significantly synergistic effect with HOXA9 in promoting cell transformation in vitro and leukemogenesis in vivo. Furthermore, as a proof of concept, we show that a small peptide, namely HXR9, which was developed to specifically disrupt the interactions between HOX and PBX proteins, can selectively kill leukemic cells with overexpression of HOXA/PBX3 genes. Collectively, our data suggest that PBX3 is a critical cofactor of HOXA9 in leukemogenesis, and targeting their interaction is a feasible strategy to treat presently therapy resistant CA-AML (eg, MLL-rearranged leukemia) in which HOXA/PBX3 genes are overexpressed.


Assuntos
Regulação Leucêmica da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Animais , Células da Medula Óssea/fisiologia , Transplante de Medula Óssea , Linhagem Celular Transformada , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Rearranjo Gênico/genética , Células HEK293 , Histona-Lisina N-Metiltransferase , Proteínas de Homeodomínio/antagonistas & inibidores , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteína de Leucina Linfoide-Mieloide/genética , Peptídeos/farmacologia , Fator de Transcrição 1 de Leucemia de Células Pré-B , Proteínas Proto-Oncogênicas/antagonistas & inibidores , RNA Interferente Pequeno/genética , Ratos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Proc Natl Acad Sci U S A ; 109(47): 19397-402, 2012 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-23132946

RESUMO

Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic malignancies with variable response to treatment. AMLs bearing MLL (mixed lineage leukemia) rearrangements are associated with intermediate or poor survival. MicroRNAs (miRNAs), a class of small noncoding RNAs, have been postulated to be important gene expression regulators virtually in all biological processes, including leukemogenesis. Through a large-scale, genome-wide miRNA expression profiling assay of 85 human AML and 15 normal control samples, we show that among 48 miRNAs that are significantly differentially expressed between MLL- and non-MLL-rearranged AML samples, only one (miR-495) is expressed at a lower level in MLL-rearranged AML than in non-MLL-rearranged AML; meanwhile, miR-495 is also significantly down-regulated in MLL-rearranged AML samples compared with normal control samples. Through in vitro colony-forming/replating assays and in vivo bone marrow transplantation studies, we show that forced expression of miR-495 significantly inhibits MLL-fusion-mediated cell transformation in vitro and leukemogenesis in vivo. In human leukemic cells carrying MLL rearrangements, ectopic expression of miR-495 greatly inhibits cell viability and increases cell apoptosis. Furthermore, our studies demonstrate that PBX3 and MEIS1 are two direct target genes of miR-495, and forced expression of either of them can reverse the effects of miR-495 overexpression on inhibiting cell viability and promoting apoptosis of human MLL-rearranged leukemic cells. Thus, our data indicate that miR-495 likely functions as a tumor suppressor in AML with MLL rearrangements by targeting essential leukemia-related genes.


Assuntos
Regulação para Baixo/genética , Rearranjo Gênico/genética , Leucemia Mieloide Aguda/genética , MicroRNAs/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Animais , Sequência de Bases , Estudos de Casos e Controles , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Genes Neoplásicos/genética , Estudos de Associação Genética , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , MicroRNAs/genética , Dados de Sequência Molecular , Proteína Meis1 , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas/metabolismo
5.
Toxicol Appl Pharmacol ; 260(3): 250-9, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22426358

RESUMO

Arsenic is a toxicant commonly found in water systems and chronic exposure can result in adverse developmental effects including increased neonatal death, stillbirths, and miscarriages, low birth weight, and altered locomotor activity. Previous studies indicate that 20 nM sodium arsenite exposure to C2C12 mouse myocyte cells delayed myoblast differentiation due to reduced myogenin expression, the transcription factor that differentiates myoblasts into myotubes. In this study, several mechanisms by which arsenic could alter myogenin expression were examined. Exposing differentiating C2C12 cells to 20 nM arsenic increased H3K9 dimethylation (H3K9me2) and H3K9 trimethylation (H3K9me3) by 3-fold near the transcription start site of myogenin, which is indicative of increased repressive marks, and reduced H3K9 acetylation (H3K9Ac) by 0.5-fold, indicative of reduced permissive marks. Protein expression of Glp or Ehmt1, a H3-K9 methyltransferase, was also increased by 1.6-fold in arsenic-exposed cells. In addition to the altered histone remodeling status on the myogenin promoter, protein and mRNA levels of Igf-1, a myogenic growth factor, were significantly repressed by arsenic exposure. Moreover, a 2-fold induction of Ezh2 expression, and an increased recruitment of Ezh2 (3.3-fold) and Dnmt3a (~2-fold) to the myogenin promoter at the transcription start site (-40 to +42), were detected in the arsenic-treated cells. Together, we conclude that the repressed myogenin expression in arsenic-exposed C2C12 cells was likely due to a combination of reduced expression of Igf-1, enhanced nuclear expression and promoter recruitment of Ezh2, and altered histone remodeling status on myogenin promoter (-40 to +42).


Assuntos
Arsenitos/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/efeitos dos fármacos , Miogenina/efeitos dos fármacos , Compostos de Sódio/toxicidade , Animais , Linhagem Celular , Proteína Potenciadora do Homólogo 2 de Zeste , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Fator de Crescimento Insulin-Like I/genética , Camundongos , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Miogenina/genética , Complexo Repressor Polycomb 2 , Regiões Promotoras Genéticas
6.
Toxicol Appl Pharmacol ; 250(2): 154-61, 2011 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20965206

RESUMO

Epidemiological studies have correlated arsenic exposure with cancer, skin diseases, and adverse developmental outcomes such as spontaneous abortions, neonatal mortality, low birth weight, and delays in the use of musculature. The current study used C2C12 mouse myoblast cells to examine whether low concentrations of arsenic could alter their differentiation into myotubes, indicating that arsenic can act as a developmental toxicant. Myoblast cells were exposed to 20 nM sodium arsenite, allowed to differentiate into myotubes, and expression of the muscle-specific transcription factor myogenin, along with the expression of tropomyosin, suppressor of cytokine signaling 3 (Socs3), prostaglandin I2 synthesis (Ptgis), and myocyte enhancer 2 (Mef2), was investigated using QPCR and immunofluorescence. Exposing C2C12 cells to 20 nM sodium arsenite delayed the differentiation process, as evidenced by a significant reduction in the number of multinucleated myotubes, a decrease in myogenin mRNA expression, and a decrease in the total number of nuclei expressing myogenin protein. The expression of mRNA involved in myotube formation, such as Ptgis and Mef2 mRNA, was also significantly reduced by 1.6-fold and 4-fold during differentiation. This was confirmed by immunofluorescence for Mef2, which showed a 2.6-fold reduction in nuclear translocation. Changes in methylation patterns in the promoter region of myogenin (-473 to +90) were examined by methylation-specific PCR and bisulfite genomic sequencing. Hypermethylated CpGs were found at -236 and -126 bp, whereas hypomethylated CpGs were found at -207 bp in arsenic-exposed cells. This study indicates that 20 nM sodium arsenite can alter myoblast differentiation by reducing the expression of the transcription factors myogenin and Mef2c, which is likely due to changes in promoter methylation patterns. The delay in muscle differentiation may lead to developmental abnormalities.


Assuntos
Arsenitos/toxicidade , Diferenciação Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , Miogenina/efeitos dos fármacos , Compostos de Sódio/toxicidade , Animais , Linhagem Celular , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores de Transcrição MEF2 , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Fatores de Regulação Miogênica/efeitos dos fármacos , Fatores de Regulação Miogênica/genética , Miogenina/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/metabolismo
7.
Cancer Cell ; 31(1): 127-141, 2017 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-28017614

RESUMO

N6-Methyladenosine (m6A) represents the most prevalent internal modification in mammalian mRNAs. Despite its functional importance in various fundamental bioprocesses, the studies of m6A in cancer have been limited. Here we show that FTO, as an m6A demethylase, plays a critical oncogenic role in acute myeloid leukemia (AML). FTO is highly expressed in AMLs with t(11q23)/MLL rearrangements, t(15;17)/PML-RARA, FLT3-ITD, and/or NPM1 mutations. FTO enhances leukemic oncogene-mediated cell transformation and leukemogenesis, and inhibits all-trans-retinoic acid (ATRA)-induced AML cell differentiation, through regulating expression of targets such as ASB2 and RARA by reducing m6A levels in these mRNA transcripts. Collectively, our study demonstrates the functional importance of the m6A methylation and the corresponding proteins in cancer, and provides profound insights into leukemogenesis and drug response.


Assuntos
Adenosina/análogos & derivados , Dioxigenase FTO Dependente de alfa-Cetoglutarato/fisiologia , Leucemia Mieloide Aguda/etiologia , Adenosina/metabolismo , Apoptose , Proliferação de Células , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/patologia , Metilação , Nucleofosmina , Receptor alfa de Ácido Retinoico/fisiologia , Proteínas Supressoras da Sinalização de Citocina/fisiologia , Transcriptoma , Tretinoína/farmacologia
8.
Toxicol Sci ; 129(1): 146-56, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22641621

RESUMO

Epidemiological studies have correlated embryonic arsenic exposure with adverse developmental outcomes such as stillbirths, neonatal mortality, and low birth weight. Additionally, arsenic exposure reduces neuronal cell migration and maturation, and reduces skeletal muscle cell formation, alters muscle fiber subtype, and changes locomotor activity. This study used P19 mouse embryonic stem cells to examine whether arsenic exposure could alter their differentiation into skeletal muscles and neurons. When P19 cells were exposed to 0.1, 0.5, or 1.0 µM sodium arsenite, embryoid body (EB) formation was not altered. However, arsenic suppressed their differentiation into muscles and neurons, as evidenced by morphological changes accompanied by a significant reduction in myosin heavy chain and Tuj1 expression. Real-time PCR, immunofluorescence, and immunoblotting were used to confirm that the altered differentiation was due to the repression of muscle- and neuron-specific transcription factors such as Pax3, Myf5, MyoD, myogenin, neurogenin 1, neurogenin 2, and NeuroD in the arsenite-exposed cells. The reductions in transcription factors expression appear to be caused by repressed Wnt/ß-catenin signaling pathways in early embryogenesis, as evidenced by decreased ß-catenin expression in the arsenic-exposed EBs on differentiation days 2 and 5. Interestingly, the expression of Nanog, a transcription factor that maintains the pluripotency of stem cells, was increased after arsenite exposure, indicating that arsenite inhibits their differentiation but not proliferation. This study demonstrates that arsenic can perturb the embryonic differentiation process by repressing the Wnt/ß-catenin signaling pathway. More importantly, this study may provide insight into how arsenic exposure affects skeletal and neuronal differentiation during embryogenesis.


Assuntos
Arsênio/toxicidade , Células-Tronco Embrionárias/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , beta Catenina/metabolismo , Animais , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Primers do DNA , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Imuno-Histoquímica , Camundongos , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/metabolismo
9.
Cancer Cell ; 22(4): 524-35, 2012 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-23079661

RESUMO

Expression of microRNAs (miRNAs) is under stringent regulation at both transcriptional and posttranscriptional levels. Disturbance at either level could cause dysregulation of miRNAs. Here, we show that MLL fusion proteins negatively regulate production of miR-150, an miRNA widely repressed in acute leukemia, by blocking miR-150 precursors from being processed to mature miRNAs through MYC/LIN28 functional axis. Forced expression of miR-150 dramatically inhibited leukemic cell growth and delayed MLL-fusion-mediated leukemogenesis, likely through targeting FLT3 and MYB and thereby interfering with the HOXA9/MEIS1/FLT3/MYB signaling network, which in turn caused downregulation of MYC/LIN28. Collectively, we revealed a MLL-fusion/MYC/LIN28⊣miR-150⊣FLT3/MYB/HOXA9/MEIS1 signaling circuit underlying the pathogenesis of leukemia, where miR-150 functions as a pivotal gatekeeper and its repression is required for leukemogenesis.


Assuntos
Leucemia/etiologia , MicroRNAs/fisiologia , Proteína de Leucina Linfoide-Mieloide/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteínas de Ligação a RNA/fisiologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Metilação de DNA , Regulação para Baixo , Dosagem de Genes , Regulação Leucêmica da Expressão Gênica , Histona-Lisina N-Metiltransferase , Proteínas de Homeodomínio/fisiologia , Humanos , Camundongos , MicroRNAs/análise , MicroRNAs/antagonistas & inibidores , Mutação , Proteína Meis1 , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/genética , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms/genética
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