RESUMO
Evidence for active DNA demethylation in vertebrates is accumulating, but the mechanisms and enzymes remain unclear. Using zebrafish embryos we provide evidence for 5-methylcytosine (5-meC) removal in vivo via the coupling of a 5-meC deaminase (AID, which converts 5-meC to thymine) and a G:T mismatch-specific thymine glycosylase (Mbd4). The injection of methylated DNA into embryos induced a potent DNA demethylation activity, which was attenuated by depletion of AID or the non enzymatic factor Gadd45. Remarkably, overexpression of the deaminase/glycosylase pair AID/Mbd4 in vivo caused demethylation of the bulk genome and injected methylated DNA fragments, likely involving a G:T intermediate. Furthermore, AID or Mbd4 knockdown caused the remethylation of a set of common genes. Finally, Gadd45 promoted demethylation and enhanced functional interactions between deaminase/glycosylase pairs. Our results provide evidence for a coupled mechanism of 5-meC demethylation, whereby AID deaminates 5-meC, followed by thymine base excision by Mbd4, promoted by Gadd45.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Glicosilases/metabolismo , Metilação de DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Timina DNA Glicosilase/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Linhagem Celular , Citidina Desaminase/metabolismo , Embrião não Mamífero/metabolismo , Humanos , Neuropeptídeos/metabolismo , Regulação para Cima , Proteínas GADD45RESUMO
Although DNA methylation is critical for proper embryonic and tissue-specific development, how different DNA methyltransferases affect tissue-specific development and their targets remains unknown. We address this issue in zebrafish through antisense-based morpholino knockdown of Dnmt3 and Dnmt1. Our data reveal that Dnmt3 is required for proper neurogenesis, and its absence results in profound defects in brain and retina. Interestingly, other organs such as intestine remain unaffected suggesting tissue-specific requirements of Dnmt3. Further, comparison of Dnmt1 knockdown phenotypes with those of Dnmt3 suggested that these two families have distinct functions. Consistent with this idea, Dnmt1 failed to complement Dnmt3 deficiency, and Dnmt3 failed to complement Dnmt1 deficiency. Downstream of Dnmt3 we identify a neurogenesis regulator, lef1, as a Dnmt3-specific target gene that is demethylated and up-regulated in dnmt3 morphants. Knockdown of lef1 rescued neurogenesis defects resulting from Dnmt3 absence. Mechanistically, we show cooperation between Dnmt3 and an H3K9 methyltransferase G9a in regulating lef1. Further, like Dnmt1-Suv39h1 cooperativity, Dnmt3 and G9a seemed to function together for tissue-specific development. G9a knockdown, but not Suv39h1 loss, phenocopied dnmt3 morphants and G9a overexpression provided a striking rescue of dnmt3 morphant phenotypes, whereas Suv39h1 overexpression failed, supporting the notion of specific DNMT-histone methyltransferase networks. Consistent with this model, H3K9me3 levels on the lef1 promoter were reduced in both dnmt3 and g9a morphants, and its knockdown rescued neurogenesis defects in g9a morphants. We propose a model wherein specific DNMT-histone methyltransferase networks are utilized to silence critical regulators of cell fate in a tissue-specific manner.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Histona-Lisina N-Metiltransferase/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/metabolismo , Imunoprecipitação da Cromatina , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Histona-Lisina N-Metiltransferase/metabolismo , Hibridização In Situ , Modelos Biológicos , Neurogênese/genética , Retina/citologia , Retina/embriologia , Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Breast cancer, the most common malignancy in women, emerges through a multistep process, encompassing the progressive sequential evolution of morphologically distinct stages from a normal cell to hyperplasia (with and without atypia), carcinoma in situ, invasive carcinoma and metastasis. The success of treatment of breast cancer could be greatly improved by the detection at early stages of cancer. In the present study, we investigated the underlying molecular mechanisms involved in breast carcinogenesis in Augustus and Copenhagen-Irish female rats, a cross between the ACI strains, induced by continuous exposure to 17beta-estradiol. The results of our study demonstrate that early stages of estrogen-induced breast carcinogenesis are characterized by altered global DNA methylation, aberrant expression of proteins responsible for the proper maintenance of DNA methylation pattern and epigenetic silencing of the critical Rassf1a (Ras-association domain family 1, isoform A) tumor suppressor gene. Interestingly, transcriptional repression of the Rassf1a gene in mammary glands during early stages of breast carcinogenesis was associated with an increase in trimethylation of histones H3 lysine 9 and H3 lysine 27 and de novo CpG island methylation and at the Rassf1a promoter and first exon. In conclusion, we demonstrate that epigenetic alterations precede formation of preneoplastic lesions indicating the significance of epigenetic events in induction of oncogenic pathways in early stages of carcinogenesis.
Assuntos
Transformação Celular Neoplásica/genética , Estradiol/toxicidade , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes Supressores de Tumor , Neoplasias Mamárias Experimentais/genética , Animais , Ilhas de CpG , Metilação de DNA , Éxons/genética , Feminino , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Hiperplasia , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/induzido quimicamente , Proteínas de Neoplasias/metabolismo , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Regiões Promotoras Genéticas/genética , Distribuição Aleatória , Ratos , Ratos Endogâmicos ACI , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genéticaRESUMO
Melanoma antigen gene protein-A11 (MAGE-11) of the MAGE family of cancer germ-line antigens increases androgen receptor (AR) transcriptional activity through its interaction with the AR NH(2)-terminal FXXLF motif. The present study investigated the regulatory mechanisms that control MAGE-11 expression during androgen deprivation therapy and prostate cancer progression. Studies include the CWR22 xenograft model of human prostate cancer, clinical specimens of benign and malignant prostate, and prostate cancer cell lines. MAGE-11 mRNA levels increased 100- to 1,500-fold during androgen deprivation therapy and prostate cancer progression, with highest levels in the castration-recurrent CWR22 xenograft and clinical specimens of castration-recurrent prostate cancer. Pyrosequencing of genomic DNA from prostate cancer specimens and cell lines indicated the increase in MAGE-11 resulted from DNA hypomethylation of a CpG island in the 5' promoter of the MAGE-11 gene. Sodium bisulfite sequencing of genomic DNA from benign and malignant prostate tumors and prostate cancer cell lines revealed DNA hypomethylation at individual CpG sites at the transcription start site were most critical for MAGE-11 expression. Cyclic AMP (cAMP) also increased MAGE-11 expression and AR transcriptional activity in prostate cancer cell lines. However, cAMP did not alter DNA methylation of the promoter and its effects were inhibited by extensive DNA methylation in the MAGE-11 promoter region. Increased expression of the AR coregulator MAGE-11 through promoter DNA hypomethylation and cAMP provides a novel mechanism for increased AR signaling in castration-recurrent prostate cancer.
Assuntos
Antígenos de Neoplasias/genética , AMP Cíclico/farmacologia , Metilação de DNA , Regulação da Expressão Gênica/fisiologia , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Adulto , Idoso , Androgênios/farmacologia , Animais , Castração , Ilhas de CpG/genética , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Regiões Promotoras Genéticas , Prostatectomia , Hiperplasia Prostática/genética , Hiperplasia Prostática/patologia , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Androgênicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Transplante HeterólogoRESUMO
Expression of the cancer-germline (CG) (or cancer-testis) antigen gene BORIS/CTCFL has been proposed to mediate activation of CG antigen genes in cancer. Consistent with this idea, we have observed that BORIS is frequently expressed in ovarian cancer, often in conjunction with other CG genes. Here we assessed the role of BORIS in CG antigen gene regulation and DNA methylation using normal and cancerous ovarian cell lines, and the CG genes MAGE-A1, NY-ESO-1, and XAGE-1 as models. Adenoviral vectored BORIS was expressed at robust levels and exhibited predominant nuclear localization in ovarian cells. However, BORIS expression in immortalized ovarian surface epithelial cells or ovarian cancer cell lines did not induce CG antigen gene expression or lead to CG antigen promoter DNA hypomethylation. BORIS overexpression also did not alter global DNA methylation, as assessed by genomic 5-methyl-deoxycytidine levels and LINE-1 methylation. We used decitabine to further assess the role of BORIS in CG gene activation and found that decitabine treatment induced BORIS and other CG genes with similar kinetics, suggesting that BORIS induction does not account for the induction of other CG genes by decitabine in ovarian cancer cells. In agreement, siRNA knockdown of BORIS did not block decitabine-mediated induction of CG genes or DNA hypomethylation in ovarian cancer cells treated with this agent. We conclude that BORIS is insufficient for CG antigen gene expression and DNA hypomethylation in ovarian cell lines, and that additional factors are likely required for CG antigen expression in ovarian cancer.
Assuntos
Antígenos de Neoplasias/biossíntese , Metilação de DNA , Proteínas de Ligação a DNA/biossíntese , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Decitabina , Epigênese Genética , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Especificidade de Órgãos , Regiões Promotoras Genéticas , Sítio de Iniciação de TranscriçãoAssuntos
Crise Blástica/tratamento farmacológico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , beta Catenina/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Crise Blástica/metabolismo , Crise Blástica/patologia , Estudos de Casos e Controles , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Cariotipagem , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Global DNA hypomethylation may result in chromosomal instability and oncogene activation, and as a surrogate of systemic methylation activity, may be associated with breast cancer risk. METHODS: Samples and data were obtained from women with incident early-stage breast cancer (I-IIIa) and women who were cancer free, frequency matched on age and race. In preliminary analyses, genomic methylation of leukocyte DNA was determined by measuring 5-methyldeoxycytosine (5-mdC), as well as methylation analysis of the LINE-1-repetitive DNA element. Further analyses used only 5-mdC levels. Logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for risk of breast cancer in relation to amounts of methylation. RESULTS: In a subset of samples tested (n = 37), 5-mdC level was not correlated with LINE-1 methylation. 5-mdC level in leukocyte DNA was significantly lower in breast cancer cases than healthy controls (P = 0.001), but no significant case-control differences were observed with LINE-1 methylation (P = 0.176). In the entire data set, we noted significant differences in 5-mdC levels in leukocytes between cases (n = 176) and controls (n = 173); P value < 0.001. Compared with women in the highest 5-mdC tertile (T3), women in the second (T2; OR = 1.49, 95% CI = 0.84-2.65) and lowest tertile (T1; OR = 2.86, 95% CI = 1.65-4.94) had higher risk of breast cancer (P for trend < or = 0.001). Among controls only and cases and controls combined, only alcohol intake was found to be inversely associated with methylation levels. CONCLUSION: These findings suggest that leukocyte DNA hypomethylation is independently associated with development of breast cancer.
Assuntos
Neoplasias da Mama/genética , Metilação de DNA , 5-Metilcitosina/análogos & derivados , Adulto , Idoso , Consumo de Bebidas Alcoólicas , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Intervalos de Confiança , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Suplementos Nutricionais , Feminino , Humanos , Leucócitos/metabolismo , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Razão de Chances , Sequências Repetitivas de Ácido Nucleico , Risco , Fatores de Risco , FumarRESUMO
We analyzed DNA methyltransferase (Dnmt) protein expression and DNA methylation patterns during four progressive stages of prostate cancer in the transgenic adenocarcinoma of mouse prostate (TRAMP) model, including prostatic intraepithelial neoplasia, well-differentiated tumors, early poorly differentiated tumors, and late poorly differentiated tumors. Dnmt1, Dnmt3a, and Dnmt3b protein expression were increased in all stages; however, after normalization to cyclin A to account for cell cycle regulation, Dnmt proteins remained overexpressed in prostatic intraepithelial neoplasia and well-differentiated tumors, but not in poorly differentiated tumors. Restriction landmark genomic scanning analysis of locus-specific methylation revealed a high incidence of hypermethylation only in poorly differentiated (early and late) tumors. Several genes identified by restriction landmark genomic scanning showed hypermethylation of downstream regions correlating with mRNA overexpression, including p16INK4a, p19ARF, and Cacna1a. Parallel gene expression and DNA methylation analyses suggests that gene overexpression precedes downstream hypermethylation during prostate tumor progression. In contrast to gene hypermethylation, genomic DNA hypomethylation, including hypomethylation of repetitive elements and loss of genomic 5-methyldeoxycytidine, occurred in both early and late stages of prostate cancer. DNA hypermethylation and DNA hypomethylation did not correlate in TRAMP, and Dnmt protein expression did not correlate with either variable, with the exception of a borderline significant association between Dnmt1 expression and DNA hypermethylation. In summary, our data reveal the relative timing of and relationship between key alterations of the DNA methylation pathway occurring during prostate tumor progression in an in vivo model system.
Assuntos
Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Modelos Animais de Doenças , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Adenocarcinoma/genética , Animais , Canais de Cálcio Tipo N , Canais de Cálcio Tipo P/genética , Canais de Cálcio Tipo P/metabolismo , Canais de Cálcio Tipo Q/genética , Canais de Cálcio Tipo Q/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Genoma/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estadiamento de Neoplasias , Neoplasias da Próstata/genéticaRESUMO
DNA methylation and histone methylation are two key epigenetic modifications that help govern heterochromatin dynamics. The roles for these chromatin-modifying activities in directing tissue-specific development remain largely unknown. To address this issue, we examined the roles of DNA methyltransferase 1 (Dnmt1) and the H3K9 histone methyltransferase Suv39h1 in zebra fish development. Knockdown of Dnmt1 in zebra fish embryos caused defects in terminal differentiation of the intestine, exocrine pancreas, and retina. Interestingly, not all tissues required Dnmt1, as differentiation of the liver and endocrine pancreas appeared normal. Proper differentiation depended on Dnmt1 catalytic activity, as Dnmt1 morphants could be rescued by active zebra fish or human DNMT1 but not by catalytically inactive derivatives. Dnmt1 morphants exhibited dramatic reductions of both genomic cytosine methylation and genome-wide H3K9 trimethyl levels, leading us to investigate the overlap of in vivo functions of Dnmt1 and Suv39h1. Embryos lacking Suv39h1 had organ-specific terminal differentiation defects that produced largely phenocopies of Dnmt1 morphants but retained wild-type levels of DNA methylation. Remarkably, suv39h1 overexpression rescued markers of terminal differentiation in Dnmt1 morphants. Our results suggest that Dnmt1 activity helps direct histone methylation by Suv39h1 and that, together, Dnmt1 and Suv39h1 help guide the terminal differentiation of particular tissues.
Assuntos
Diferenciação Celular/fisiologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Embrião não Mamífero/citologia , Metiltransferases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , Desenvolvimento Embrionário/genética , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Humanos , Intestinos/anormalidades , Intestinos/patologia , Metiltransferases/genética , Especificidade de Órgãos , Pâncreas/anormalidades , Pâncreas/patologia , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retina/anormalidades , Retina/patologia , Proteínas de Peixe-Zebra/genéticaRESUMO
The POTE gene family consists of 14 homologous genes localized to autosomal pericentromeres, and a sub-set of POTEs are cancer-testis antigen (CTA) genes. POTEs are over-expressed in epithelial ovarian cancer (EOC), including the high-grade serous subtype (HGSC), and expression of individual POTEs correlates with chemoresistance and reduced survival in HGSC. The mechanisms driving POTE overexpression in EOC and other cancers is unknown. Here, we investigated the role of epigenetics in regulating POTE expression, with a focus on DNA hypomethylation. Consistent with their pericentromeric localization, Pan-POTE expression in EOC correlated with expression of the pericentromeric repeat NBL2, which was not the case for non-pericentromeric CTAs. POTE genomic regions contain LINE-1 (L1) sequences, and Pan-POTE expression correlated with both global and POTE-specific L1 hypomethylation in EOC. Analysis of individual POTEs using RNA-seq and DNA methylome data from fallopian tube epithelia (FTE) and HGSC revealed that POTEs C, E, and F have increased expression in HGSC in conjunction with DNA hypomethylation at 5' promoter or enhancer regions. Moreover, POTEs C/E/F showed additional increased expression in recurrent HGSC in conjunction with 5' hypomethylation, using patient-matched samples. Experiments using decitabine treatment and DNMT knockout cell lines verified a functional contribution of DNA methylation to POTE repression, and epigenetic drug combinations targeting histone deacetylases (HDACs) and histone methyltransferases (HMTs) in combination with decitabine further increased POTE expression. In summary, several alterations of the cancer epigenome, including pericentromeric activation, global and locus-specific L1 hypomethylation, and locus-specific 5' CpG hypomethylation, converge to promote POTE expression in ovarian cancer.
Assuntos
Carcinoma Epitelial do Ovário/genética , Metilação de DNA , Neoplasias Ovarianas/genética , Proteínas/genética , Regulação para Cima , Linhagem Celular Tumoral , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Elementos Nucleotídeos Longos e Dispersos , Regiões Promotoras GenéticasRESUMO
Genotoxic carcinogens, including 2-acetylaminofluorene (2-AAF), in addition to exerting their genotoxic effects, often cause a variety of non-genotoxic alterations in cells. It is believed that these non-genotoxic effects may be indispensable events in tumorigenesis; however, there is insufficient knowledge to clarify the role of carcinogens in both the genetic and epigenetic changes in premalignant tissues and a lack of conclusive information on the link between epigenetic alterations and carcinogenic exposure. In the current study, we investigated whether or not the mechanism of 2-AAF-induced hepatocarcinogenesis consists of both genotoxic (genetic) and non-genotoxic (epigenetic) alterations. Male and female Sprague-Dawley rats were fed NIH-31 diet containing 0.02% of 2-AAF for 6, 12, 18 or 24 weeks. The levels of DNA adducts obtained from 2-AAF in liver and kidney tissues were assessed by high-performance liquid chromatography combined with electrospray tandem mass spectrometry (HPLC-ES-MS/MS). N-(Deoxyguanosine-8-yl)-2-aminofluorene was the major adduct detected at all time points in both tissues. Global DNA methylation in the livers and kidneys, as determined by an HpaII-based cytosine extension assay and by HPLC-ES-MS/MS, did not change over the 24-week period. In the livers of male rats, there was a progressive decrease of global and long interspersed nucleotide element-1-associated histone H4 lysine 20 trimethylation, as well as hypermethylation of the p16(INK4A) gene. These epigenetic changes were not observed in the livers of female rats or the kidneys of both sexes. Importantly, morphological evidence of formation and progression of neoplastic process was observed in the liver of male rats only. In conclusion, we have demonstrated that exposure of rats to genotoxic hepatocarcinogen 2-AAF, in addition to formation of 2-AAF-specific DNA lesions, resulted in substantial alterations in cellular epigenetic status.
Assuntos
2-Acetilaminofluoreno/toxicidade , Carcinógenos/toxicidade , Epigênese Genética , Neoplasias Hepáticas Experimentais/genética , Lesões Pré-Cancerosas/genética , Animais , Cromatografia Líquida de Alta Pressão , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Adutos de DNA/metabolismo , Feminino , Imuno-Histoquímica , Rim/metabolismo , Fígado/metabolismo , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Reação em Cadeia da Polimerase , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em TandemRESUMO
The maintenance of the cellular epigenomic landscape, which depends on the status of the one-carbon metabolic pathway, is essential for normal central nervous system development and function. In the present study, we examined the epigenetic alterations in the brains of Fisher 344 rats induced by the long-term administration of a diet lacking of essential one-carbon nutrients, methionine, choline, and folic acid. The results demonstrated that feeding a folate/methyl-deficient diet causes global DNA hypermethylation as indicated by an increase of genomic 5-methyl-2'-deoxycytidine (5mdC) content and more importantly, by an increase of methylation within unmethylated CpG-rich DNA domains. Interestingly, these epigenetic changes were opposite to those observed in the livers of the same folate/methyl-deficient rats. The hypermethylation changes were associated with an increased protein expression of de novo DNA methyltransferase DNMT3a and methyl-CpG-binding protein 2. Additionally, the gene expression profiling identified 33 significantly up- or down-regulated genes (fold change > or =1.5 and p< or =0.05) in the brains of rats fed a folate/methyl-deficient diet for 36 weeks. Interestingly, we detected an up-regulation of regulatory factor X, 3 (Rfx3) gene, a sequence-specific DNA-binding protein, that mediates the transcriptional activation of silenced by methylation genes, which may be an adaptive protective brain response to hypermethylation. Together, these data suggest that the proper maintenance of the epigenomic landscape in normal brain depends on the adequate supply of essential nutrients involved in the metabolism of methyl groups.
Assuntos
Encéfalo/metabolismo , Dieta Redutora/efeitos adversos , Epigênese Genética/fisiologia , Deficiência de Ácido Fólico/etiologia , Deficiência de Ácido Fólico/patologia , Animais , Metilases de Modificação do DNA/classificação , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Homocisteína/metabolismo , Masculino , Metionina/deficiência , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Endogâmicos F344 , Fatores de Transcrição de Fator Regulador X , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
We examined the DNA methylation pathway in an autochthonous murine prostate cancer model, transgenic adenocarcinoma of mouse prostate (TRAMP). We observed that, compared with strain-matched normal prostates, primary and metastatic TRAMP tumors display increased cytosine DNA methyltransferase (Dnmt) activity, Dnmt1 and Dnmt3b protein expression, and Dnmt1, Dnmt3a, and Dnmt3b mRNA expression. Increased expression of Dnmt genes correlates with increased expression of cyclin A and E2F target genes, implicating increased cell proliferation and Rb inactivation in Dnmt overexpression. We analyzed DNA methylation in TRAMP and found that global levels of 5-methyl-2'-deoxycytidine are unaltered, whereas specific tumors display centromeric repeat hypomethylation. To interrogate locus-specific methylation, we did restriction landmark genomic scanning (RLGS) on normal prostates and primary tumors. In primary tumors, 2.3% of approximately 1,200 analyzed loci display aberrant DNA hypermethylation, whereas a considerably smaller number of events show hypomethylation. The pattern of RLGS changes was nonrandom, indicating a coordinated methylation defect. Two specific genes identified by RLGS were studied in detail. Surprisingly, methylation of a downstream exon of p16(INK4a) (p16) was the highest frequency hypermethylation event identified in TRAMP, where it is associated with increased p16 mRNA and protein expression. In contrast, hypermethylation of the 5' CpG island region of the homeobox gene Irx3 in TRAMP is associated with reduced gene expression. In summary, our data reveal a systemic DNA methylation pathway defect in TRAMP reminiscent of human prostate cancer, supporting the use of this model to investigate the functional role of DNA methylation pathway alterations in prostate cancer development.
Assuntos
Metilação de DNA , Neoplasias da Próstata/genética , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Animais , Sequência de Bases , Clonagem Molecular , DNA (Citosina-5-)-Metiltransferases/genética , Primers do DNA , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/genética , Neoplasias da Próstata/enzimologia , Mapeamento por Restrição , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The POTE family includes 14 genes in three phylogenetic groups. We determined POTE mRNA expression in normal tissues, epithelial ovarian and high-grade serous ovarian cancer (EOC, HGSC), and pan-cancer, and determined the relationship of POTE expression to ovarian cancer clinicopathology. Groups 1 & 2 POTEs showed testis-specific expression in normal tissues, consistent with assignment as cancer-testis antigens (CTAs), while Group 3 POTEs were expressed in several normal tissues, indicating they are not CTAs. Pan-POTE and individual POTEs showed significantly elevated expression in EOC and HGSC compared to normal controls. Pan-POTE correlated with increased stage, grade, and the HGSC subtype. Select individual POTEs showed increased expression in recurrent HGSC, and POTEE specifically associated with reduced HGSC OS. Consistent with tumors, EOC cell lines had significantly elevated Pan-POTE compared to OSE and FTE cells. Notably, Group 1 & 2 POTEs (POTEs A/B/B2/C/D), Group 3 POTE-actin genes (POTEs E/F/I/J/KP), and other Group 3 POTEs (POTEs G/H/M) show within-group correlated expression, and pan-cancer analyses of tumors and cell lines confirmed this relationship. Based on their restricted expression in normal tissues and increased expression and association with poor prognosis in ovarian cancer, POTEs are potential oncogenes and therapeutic targets in this malignancy.
Assuntos
Antígenos de Neoplasias/genética , Neoplasias Ovarianas/genética , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Família Multigênica , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Análise de SobrevidaRESUMO
Brother of the Regulator of Imprinted Sites (BORIS/CTCFL) is an autosomal cancer germline (CG) or cancer-testis antigen gene and paralog of CTCF that has been proposed to function as an oncogene in human cancer via dysregulation of the cancer epigenome. Here we show that genetic disruption of DNA methylation in human cancer cells induces BORIS expression, coincident with DNA hypomethylation and an altered histone H3 modification pattern at the BORIS promoter. Rapid amplification of cDNA ends (RACE) mapping revealed that the transcriptional start site of BORIS in human testis, DNMT deficient human cancer cells, and human epithelial ovarian cancer (EOC) tissues, is similar and lies within the 5' CpG island. The BORIS promoter is repressed by CpG methylation in a dose-dependent fashion, indicating a direct role for DNA methylation in BORIS transcriptional regulation. In human ovarian cancer cell lines, 5-aza-2'-deoxycytidine treatment activates BORIS expression and reduces BORIS promoter DNA methylation. We quantitatively measured BORIS mRNA expression and promoter DNA methylation in normal ovary (NO; n = 10) and epithelial ovarian cancer (EOC; n = 77) and found that, compared to NO, EOC tumors show increased BORIS expression and decreased BORIS methylation. Importantly, BORIS promoter DNA methylation shows a significant inverse correlation with BORIS mRNA expression in EOC (Kendall's Tau = -0.235, P = 0.007, n = 63). These data establish promoter DNA hypomethylation as a mechanism leading to BORIS expression in human ovarian cancer.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/genética , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferases/deficiência , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Feminino , Humanos , Especificidade de Órgãos , Regiões Promotoras Genéticas , Sítio de Iniciação de TranscriçãoRESUMO
Cancer testis antigens (CTAs) are promising cancer associated antigens in solid tumors, but in acute myeloid leukemia, dense promoter methylation silences their expression. Leukemia cell lines exposed to HMAs induce expression of CTAs. We hypothesized that AML patients treated with standard of care decitabine (20mg/m2 per day for 10 days) would demonstrate induced expression of CTAs. Peripheral blood blasts serially isolated from AML patients treated with decitabine were evaluated for CTA gene expression and demethylation. Induction of NY-ESO-1 and MAGEA3/A6, were observed following decitabine. Re-expression of NY-ESO-1 and MAGEA3/A6 was associated with both promoter specific and global (LINE-1) hypomethylation. NY-ESO-1 and MAGEA3/A6 mRNA levels were increased irrespective of clinical response, suggesting that these antigens might be applicable even in patients who are not responsive to HMA therapy. Circulating blasts harvested after decitabine demonstrate induced NY-ESO-1 expression sufficient to activate NY-ESO-1 specific CD8+ T-cells. Induction of CTA expression sufficient for recognition by T-cells occurs in AML patients receiving decitabine. Vaccination against NY-ESO-1 in this patient population is feasible.
Assuntos
Antígenos de Neoplasias/biossíntese , Antineoplásicos/uso terapêutico , Azacitidina/análogos & derivados , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas de Membrana/biossíntese , Idoso , Idoso de 80 Anos ou mais , Azacitidina/uso terapêutico , Decitabina , Feminino , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
We aimed to determine the effect of SGI-110 on methylation and expression of the cancer testis antigens (CTAs) NY-ESO-1 and MAGE-A in epithelial ovarian cancer (EOC) cells in vitro and in vivo and to establish the impact of SGI-110 on expression of major histocompatibility (MHC) class I and Intracellular Adhesion Molecule 1 (ICAM-1) on EOC cells, and on recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. We also tested the impact of combined SGI-110 and NY-ESO-1-specific CD8+ T-cells on tumor growth and/or murine survival in a xenograft setting. EOC cells were treated with SGI-110 in vitro at various concentrations and as tumor xenografts with 3 distinct dose schedules. Effects on global methylation (using LINE-1), NY-ESO-1 and MAGE-A methylation, mRNA, and protein expression were determined and compared to controls. SGI-110 treated EOC cells were evaluated for expression of immune-modulatory genes using flow cytometry, and were co-cultured with NY-ESO-1 specific T-cell clones to determine immune recognition. In vivo administration of SGI-110 and CD8+ T-cells was performed to determine anti-tumor effects on EOC xenografts. SGI-110 treatment induced hypomethylation and CTA gene expression in a dose dependent manner both in vitro and in vivo, at levels generally superior to azacitidine or decitabine. SGI-110 enhanced the expression of MHC I and ICAM-1, and enhanced recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. Sequential SGI-110 and antigen-specific CD8+ cell treatment restricted EOC tumor growth and enhanced survival in a xenograft setting. SGI-110 is an effective hypomethylating agent and immune modulator and, thus, an attractive candidate for combination with CTA-directed vaccines in EOC.
Assuntos
Azacitidina/análogos & derivados , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Metiltransferases/antagonistas & inibidores , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Animais , Antígenos de Neoplasias/genética , Azacitidina/farmacologia , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Camundongos SCID , Transplante de Neoplasias , Linfócitos TRESUMO
The mechanism of clinical action for the FDA approved hypomethylating drugs azacitidine and decitabine remains unresolved and in this context the potential immunomodulatory effect of these agents on leukemic cells is an area of active investigation. Induced expression of methylated Cancer Testis Antigen (CTA) genes has been demonstrated in leukemic cell lines following exposure to hypomethylating drugs in vitro. SGI-110 is a novel hypomethylating dinucleotide with prolonged in vivo exposure and clinical activity in patients with MDS and AML. We demonstrate that this agent, like decitabine, produces robust re-expression of the CTAs NY-ESO-1 and MAGE-A, both in vitro and in leukemia-bearing AML xenografts. Upregulation of these genes in vitro was sufficient to induce cytotoxicity by HLA-compatible CD8+ T-cells specific for NY-ESO-1, a well-recognized and immunogenic CTA. Additionally, exposure to SGI-110 enhances MHC class I and co-stimulatory molecule expression, potentially contributing to recognition of CTAs. SGI-110, like the parent compound decitabine, induces expression of CTAs and might modulate immune recognition of myeloid malignancy.
Assuntos
Azacitidina/análogos & derivados , Fatores Imunológicos/farmacologia , Leucemia Mieloide Aguda/patologia , Animais , Azacitidina/farmacologia , Linhagem Celular Tumoral , Metilação de DNA , Feminino , Humanos , Camundongos , Camundongos SCID , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The cancer-testis/cancer-germline antigen NY-ESO-1 is a vaccine target in epithelial ovarian cancer (EOC), but its limited expression is a barrier to vaccine efficacy. As NY-ESO-1 is regulated by DNA methylation, we hypothesized that DNA methyltransferase (DNMT) inhibitors may augment NY-ESO-1 vaccine therapy. In agreement, global DNA hypomethylation in EOC was associated with the presence of circulating antibodies to NY-ESO-1. Pre-clinical studies using EOC cell lines showed that decitabine treatment enhanced both NY-ESO-1 expression and NY-ESO-1-specific CTL-mediated responses. Based on these observations, we performed a phase I dose-escalation trial of decitabine, as an addition to NY-ESO-1 vaccine and doxorubicin liposome (doxorubicin) chemotherapy, in 12 patients with relapsed EOC. The regimen was safe, with limited and clinically manageable toxicities. Both global and promoter-specific DNA hypomethylation occurred in blood and circulating DNAs, the latter of which may reflect tumor cell responses. Increased NY-ESO-1 serum antibodies and T cell responses were observed in the majority of patients, and antibody spreading to additional tumor antigens was also observed. Finally, disease stabilization or partial clinical response occurred in 6/10 evaluable patients. Based on these encouraging results, evaluation of similar combinatorial chemo-immunotherapy regimens in EOC and other tumor types is warranted.
Assuntos
Antígenos de Neoplasias/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Vacinas Anticâncer/imunologia , Epigênese Genética/efeitos dos fármacos , Proteínas de Membrana/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/terapia , Adulto , Idoso , Antígenos de Neoplasias/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Azacitidina/administração & dosagem , Azacitidina/análogos & derivados , Vacinas Anticâncer/genética , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Metilação de DNA , Decitabina , Doxorrubicina/administração & dosagem , Doxorrubicina/análogos & derivados , Feminino , Expressão Gênica , Humanos , Imunidade Humoral , Imunoterapia Ativa , Elementos Nucleotídeos Longos e Dispersos , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/imunologia , Neoplasias Epiteliais e Glandulares/terapia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Polietilenoglicóis/administração & dosagem , Subpopulações de Linfócitos T/imunologia , Resultado do TratamentoRESUMO
MAGEA11 is a cancer germline (CG) antigen and androgen receptor co-activator. Its expression in cancers other than prostate, and its mechanism of activation, has not been reported. In silico analyses reveal that MAGEA11 is frequently expressed in human cancers, is increased during tumor progression, and correlates with poor prognosis and survival. In prostate and epithelial ovarian cancers (EOC), MAGEA11 expression was associated with promoter and global DNA hypomethylation, and with activation of other CG genes. Pharmacological or genetic inhibition of DNA methyltransferases (DNMTs) and/or histone deacetylases (HDACs) activated MAGEA11 in a cell line specific manner. MAGEA11 promoter activity was directly repressed by DNA methylation, and partially depended on Sp1, as pharmacological or genetic targeting of Sp1 reduced MAGEA11 promoter activity and endogenous gene expression. Importantly, DNA methylation regulated nucleosome occupancy specifically at the -1 positioned nucleosome of MAGEA11. Methylation of a single Ets site near the transcriptional start site (TSS) correlated with -1 nucleosome occupancy and, by itself, strongly repressed MAGEA11 promoter activity. Thus, DNA methylation regulates nucleosome occupancy at MAGEA11, and this appears to function cooperatively with sequence-specific transcription factors to regulate gene expression. MAGEA11 regulation is highly instructive for understanding mechanisms regulating CG antigen genes in human cancer.