RESUMO
Diagnosis of malignant pleural effusion (MPE) is made by cytological examination of pleural fluid or histological examination of pleural tissue from biopsy. Unfortunately, detection of malignancy using cytology has an overall sensitivity of 50%, and is dependent upon tumor load, volume of fluid assessed, and cytopathologist experience. The diagnostic yield of pleural fluid cytology is also compromised by low abundance of tumor cells or when morphology is obscured by inflammation or reactive mesothelial cells. A reliable molecular marker that may complement fluid cytology for the diagnosis of malignant pleural effusion is needed. The purpose of this study was to establish a molecular diagnostic approach based on pleural effusion cell-free DNA methylation analysis for the differential diagnosis of malignant pleural effusion and benign pleural effusion. This was a blind, prospective case-control biomarker study. We recruited 104 patients with pleural effusion for the study. We collected pleural fluid from patients with: MPE (n = 48), indeterminate pleural effusion in subjects with known malignancy or IPE (n = 28), and benign PE (n = 28), and performed the Sentinel-MPE liquid biopsy assay. The methylation level of Sentinel-MPE was markedly higher in the MPE samples compared to BPE control samples (p < 0.0001) and the same tendency was observed relative to IPE (p = 0.004). We also noted that the methylation signal was significantly higher in IPE relative to BPE (p < 0.001). We also assessed the diagnostic efficiency of the Sentinel-MPE test by performing receiver operating characteristic analysis (ROC). For the ROC analysis we combined the malignant and indeterminate pleural effusion groups (n = 76) and compared against the benign group (n = 28). The detection sensitivity and specificity of the Sentinel-MPE test was high (AUC = 0.912). The Sentinel-MPE appears to have better performance characteristics than cytology analysis. However, combining Sentinel-MPE with cytology analysis could be an even more effective approach for the diagnosis of MPE. The Sentinel-MPE test can discriminate between BPE and MPE. The Sentinel-MPE liquid biopsy test can detect aberrant DNA in several different tumor types. The Sentinel-MPE test can be a complementary tool to cytology in the diagnosis of MPE.
Assuntos
Ácidos Nucleicos Livres , Derrame Pleural Maligno , Derrame Pleural , Humanos , Derrame Pleural Maligno/diagnóstico , Derrame Pleural Maligno/genética , Derrame Pleural Maligno/patologia , Metilação de DNA , Biomarcadores Tumorais/metabolismo , Derrame Pleural/diagnóstico , Derrame Pleural/patologiaRESUMO
The nucleotide 5-methylcytosine is involved in processes crucial in mammalian development, such as X-chromosome inactivation and gene imprinting. In addition, cytosine methylation has long been speculated to be involved in the establishment and maintenance of cell type specific expression of developmentally regulated genes; however, it has been difficult to identify clear examples of such genes, particularly in humans. Here we provide evidence that cytosine methylation of the maspin gene (SERPINB5) promoter controls, in part, normal cell type specific SERPINB5 expression. In normal cells expressing SERPINB5, the SERPINB5 promoter is unmethylated and the promoter region has acetylated histones and an accessible chromatin structure. By contrast, normal cells that do not express SERPINB5 have a completely methylated SERPINB5 promoter with hypoacetylated histones, an inaccessible chromatin structure and a transcriptional repression that is relieved by inhibition of DNA methylation. These findings indicate that cytosine methylation is important in the establishment and maintenance of cell type restricted gene expression.
Assuntos
Metilação de DNA , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas/genética , Serpinas/genética , Linhagem Celular , Citosina/fisiologia , Genes Supressores de Tumor , Humanos , Especificidade de Órgãos/genética , Especificidade de Órgãos/fisiologia , Biossíntese de Proteínas , Serpinas/biossínteseRESUMO
Identification of cancer-specific methylation of DNA released by tumours can be used for non-invasive diagnostics and monitoring. We previously reported in silico identification of DNA methylation loci specifically hypermethylated in common human cancers that could be used as epigenetic biomarkers. Using DNA methylation specific qPCR we now clinically tested a group of these cancer-specific loci on cell-free DNA (cfDNA) extracted from the plasma fraction of blood samples from healthy controls and non-small cell lung cancer (NSCLC) patients. These DNA methylation biomarkers distinguish lung cancer cases from controls with high sensitivity and specificity (AUC = 0.956), and furthermore, the signal from the markers correlates with tumour size and decreases after surgical resection of lung tumours. Presented observations suggest the clinical value of these DNA methylation biomarkers for NSCLC diagnostics and monitoring. Since we successfully validated the biomarkers using independent DNA methylation data from multiple additional common carcinoma cohorts (bladder, breast, colorectal, oesophageal, head and neck, pancreatic or prostate cancer) we predict that these DNA methylation biomarkers will detect additional carcinoma types from plasma samples as well.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA , Neoplasias Pulmonares/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/normas , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/patologia , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Ácidos Nucleicos Livres/normas , Feminino , Humanos , Biópsia Líquida , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Sensibilidade e EspecificidadeRESUMO
Using an integrated approach of epigenomic scanning and gene expression profiling, we found aberrant methylation and epigenetic silencing of a small neighborhood of contiguous genes-the HOXA gene cluster in human breast cancer. The observed transcriptional repression was localized to approximately 100 kb of the HOXA gene cluster and did not extend to genes located upstream or downstream of the cluster. Bisulfite sequencing, chromatin immunoprecipitation, and quantitative reverse transcription-PCR analysis confirmed that the loss of expression of the HOXA gene cluster in human breast cancer is closely linked to aberrant DNA methylation and loss of permissive histone modifications in the region. Pharmacologic manipulations showed the importance of these aberrant epigenetic changes in gene silencing and support the hypothesis that aberrant DNA methylation is dominant to histone hypoacetylation. Overall, these data suggest that inactivation of the HOXA gene cluster in breast cancer may represent a new type of genomic lesion-epigenetic microdeletion. We predict that epigenetic microdeletions are common in human cancer and that they functionally resemble genetic microdeletions but are defined by epigenetic inactivation and transcriptional silencing of a relatively small set of contiguous genes along a chromosome, and that this type of genomic lesion is metastable and reversible in a classic epigenetic fashion.
Assuntos
Neoplasias da Mama/genética , Proteínas de Homeodomínio/genética , Família Multigênica , Linhagem Celular Tumoral , Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , HumanosRESUMO
p53 is an important transcriptional regulator that is frequently mutated in cancer. Gene-profiling experiments of breast cancer cells infected with wt p53 revealed both MASPIN and desmocollin 3 (DSC3) to be p53-target genes, even though both genes are silenced in association with aberrant cytosine methylation of their promoters. Despite the transcriptional repression of these genes by aberrant DNA methylation, restoration of p53 resulted in the partial reactivation of both genes. This reactivation is a result of wt p53 binding to its consensus DNA-binding sites within the MASPIN and DSC3 promoters, stimulating histone acetylation, and enhancing chromatin accessibility of their promoters. Interestingly, wt p53 alone did not affect the methylation status of either promoter, suggesting that p53 itself can partially overcome the repressive barrier of DNA methylation. Pharmacologic inhibition of DNA methylation with 5-aza-2'-deoxycytidine in combination with restoration of wt p53 status resulted in a synergistic reactivation of these genes to near-normal levels. These results suggest that cancer treatments that target both genetic and epigenetic facets of gene regulation may be a useful strategy towards the therapeutic transcriptional reprogramming of cancer cells.
Assuntos
Neoplasias da Mama/genética , Citosina/metabolismo , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Mutação , Proteína Supressora de Tumor p53/genética , Azacitidina/farmacologia , Neoplasias da Mama/tratamento farmacológico , Cromatina/metabolismo , Cromatina/ultraestrutura , Metilação de DNA/efeitos dos fármacos , DNA-Citosina Metilases/metabolismo , Desmocolinas , Feminino , Inativação Gênica , Genes Supressores de Tumor , Histonas/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Regiões Promotoras Genéticas , Proteínas/genética , Serpinas/genética , Células Tumorais CultivadasRESUMO
Epigenetic control participates in processes crucial in mammalian development, such as X-chromosome inactivation, gene imprinting, and cell type-specific gene expression. We provide evidence that the p53-inducible gene 14-3-3sigma is a new example of a gene important to human cancer, where epigenetic mechanisms participate in the control of normal cell type-specific expression, as well as aberrant gene silencing in cancer cells. Like a previously identified cell type-specific gene maspin, 14-3-3sigma is a p53-inducible gene; however, it participates in G2/M arrest in response to DNA-damaging agents. 14-3-3Sigma expression is restricted to certain epithelial cell types, including breast and prostate, whereas expression is absent in nonepithelial tissues such as fibroblasts and lymphocytes. In this report, we show that in normal cells expressing 14-3-3sigma, the 14-3-3sigma CpG island is unmethylated; associated with acetylated histones, unmethylated histone H3 lysine 9; and an accessible chromatin structure. By contrast, normal cells that do not express 14-3-3sigma have a methylated 14-3-3sigma CpG island with hypoacetylated histones, methylated histone H3 lysine 9, and an inaccessible chromatin structure. These findings extend the spectrum of cell type-specific genes controlled, partly, by normal epigenetic mechanisms, and suggest that this subset of genes may represent important targets of epigenetic dysregulation in human cancer.
Assuntos
Biomarcadores Tumorais/genética , Epigênese Genética , Exonucleases/genética , Regulação da Expressão Gênica , Proteínas de Neoplasias/genética , Proteínas 14-3-3 , Acetilação , Biomarcadores Tumorais/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Cromatina/ultraestrutura , Ilhas de CpG , Metilação de DNA , Exonucleases/metabolismo , Exorribonucleases , Código das Histonas , Histonas/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
INTRODUCTION: Desmocollin 3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and a principle component of desmosomes. Desmosomal proteins such as DSC3 are integral to the maintenance of tissue architecture and the loss of these components leads to a lack of adhesion and a gain of cellular mobility. DSC3 expression is down-regulated in breast cancer cell lines and primary breast tumors; however, the loss of DSC3 is not due to gene deletion or gross rearrangement of the gene. In this study, we examined the prevalence of epigenetic silencing of DSC3 gene expression in primary breast tumor specimens. METHODS: We used bisulfite genomic sequencing to analyze the methylation state of the DSC3 promoter region from 32 primary breast tumor specimens. We also used a quantitative real-time RT-PCR approach, and analyzed all breast tumor specimens for DSC3 expression. Finally, in addition to bisulfite sequencing and RT-PCR, we used an in vivo nuclease accessibility assay to determine the chromatin architecture of the CpG island region from DSC3-negative breast cancer cells lines. RESULTS: DSC3 expression was downregulated in 23 of 32 (72%) breast cancer specimens comprising: 22 invasive ductal carcinomas, 7 invasive lobular breast carcinomas, 2 invasive ductal carcinomas that metastasized to the lymph node, and a mucoid ductal carcinoma. Of the 23 specimens showing a loss of DSC3 expression, 13 (56%) were associated with cytosine hypermethylation of the promoter region. Furthermore, DSC3 expression is limited to cells of epithelial origin and its expression of mRNA and protein is lost in a high proportion of breast tumor cell lines (79%). Lastly, DNA hypermethylation of the DSC3 promoter is highly correlated with a closed chromatin structure. CONCLUSION: These results indicate that the loss of DSC3 expression is a common event in primary breast tumor specimens, and that DSC3 gene silencing in breast tumors is frequently linked to aberrant cytosine methylation and concomitant changes in chromatin structure.
Assuntos
Neoplasias da Mama/genética , Inativação Gênica , Glicoproteínas de Membrana/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Metilação de DNA , Primers do DNA , Desmocolinas , Células Epiteliais/enzimologia , Feminino , Humanos , Masculino , Mastectomia , Mastectomia Segmentar , Regiões Promotoras Genéticas , Próstata/enzimologia , RNA Neoplásico/genéticaRESUMO
p300/CBP-associated factor (PCAF) is a coactivator of the tumor suppressor, p53. PCAF participates in p53's transactivation of target genes through acetylation of both bound p53 and histones within p53 target promoters. Using microarrays, we discovered that PCAF itself is induced by p53 in a panel of breast tumor cell lines. Two p53 mutant breast tumor cell lines, BT-549 and UACC-1179, were chosen for further study of PCAF induction by wild-type p53. PCAF induction following adenoviral transduction of p53 expression was confirmed with real-time polymerase chain reaction in a time course experiment. Chromatin immunoprecipitation experiments then showed that PCAF induction was associated with increased p53 binding to the PCAF promoter, which contains p53 consensus-binding sites. PCAF induction by p53 activity was further demonstrated in wild-type p53 MCF10A cells when PCAF expression was induced following activation of endogenous wild-type p53 with doxorubicin in a dose- and time-dependent manner. Furthermore, the doxorubicin-induced increase in PCAF expression was blocked by pretreatment of the MCF10A cells with siRNA (small interfering RNA) targeted against p53 mRNA. Taken together, the results show that PCAF expression can be induced by wild-type p53.
Assuntos
Acetiltransferases/genética , Acetiltransferases/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína Supressora de Tumor p53/metabolismo , Adenoviridae/genética , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Vetores Genéticos/genética , Histona Acetiltransferases , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Interferente Pequeno/genética , Fatores de Transcrição , Transfecção , Fatores de Transcrição de p300-CBPRESUMO
Aberrations of p53 occur in most, if not all, human cancers. In breast cancer, p53 mutation is the most common genetic defect related to a single gene. Immortalized human mammary epithelial cells resemble the earliest forms of aberrant breast tissue growth but do not express many malignancy-associated phenotypes. We created a model of human mammary epithelial tumorigenesis by infecting hTERT-HME1 immortalized human mammary epithelial cells expressing wild-type p53 with four different mutant p53 constructs to determine the role of p53 mutation on the evolution of tumor phenotypes. We demonstrate that different mutant/wild-type p53 heterozygous models generate loss of function, dominant negative activity, and a spectrum of gain of function activities that induce varying degrees of invasive potential. We suggest that this model can be used to elucidate changes that occur in early stages of human mammary epithelial tumorigenesis. These changes may constitute novel biomarkers or reveal novel treatment modalities that could inhibit progression from primary to metastatic breast disease.
Assuntos
Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica , Mutação/genética , Proteína Supressora de Tumor p53/fisiologia , Adenoviridae/genética , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Imunoprecipitação da Cromatina , DNA/genética , DNA/metabolismo , Progressão da Doença , Citometria de Fluxo , Genes Dominantes , Humanos , Imunoprecipitação , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares , Telomerase/metabolismo , Ensaio Tumoral de Célula-TroncoRESUMO
Changes in DNA methylation patterns are a common characteristic of cancer cells. Recent studies suggest that DNA methylation affects not only discrete genes, but it can also affect large chromosomal regions, potentially leading to LRES. It is unclear whether such long-range epigenetic events are relatively rare or frequent occurrences in cancer. Here, we use a high-resolution promoter tiling array approach to analyze DNA methylation in breast cancer specimens and normal breast tissue to address this question. We identified 3,506 cancer-specific differentially methylated regions (DMR) in human breast cancer with 2,033 being hypermethylation events and 1,473 hypomethylation events. Most of these DMRs are recurrent in breast cancer; 90% of the identified DMRs occurred in at least 33% of the samples. Interestingly, we found a nonrandom spatial distribution of aberrantly methylated regions across the genome that showed a tendency to concentrate in relatively small genomic regions. Such agglomerates of hypermethylated and hypomethylated DMRs spanned up to several hundred kilobases and were frequently found at gene family clusters. The hypermethylation events usually occurred in the proximity of the transcription start site in CpG island promoters, whereas hypomethylation events were frequently found in regions of segmental duplication. One example of a newly discovered agglomerate of hypermethylated DMRs associated with gene silencing in breast cancer that we examined in greater detail involved the protocadherin gene family clusters on chromosome 5 (PCDHA, PCDHB, and PCDHG). Taken together, our results suggest that agglomerative epigenetic aberrations are frequent events in human breast cancer.