RESUMEN
Aberrant methylation of multiple promoter CpG islands could be related to the biology of ovarian tumors and its determination could help to improve treatment strategies. DNA methylation profiling was performed using the Methylation Ligation-dependent Macroarray (MLM), an array-based analysis. Promoter regions of 41 genes were analyzed in 102 ovarian tumors and 17 normal ovarian samples. An average of 29% of hypermethylated promoter genes was observed in normal ovarian tissues. This percentage increased slightly in serous, endometrioid, and mucinous carcinomas (32%, 34%, and 45%, respectively), but decreased in germ cell tumors (20%). Ovarian tumors had methylation profiles that were more heterogeneous than other epithelial cancers. Unsupervised hierarchical clustering identified four groups that are very close to the histological subtypes of ovarian tumors. Aberrant methylation of three genes (BRCA1, MGMT, and MLH1), playing important roles in the different DNA repair mechanisms, were dependent on the tumor subtype and represent powerful biomarkers for precision therapy. Furthermore, a promising relationship between hypermethylation of MGMT, OSMR, ESR1, and FOXL2 and overall survival was observed. Our study of DNA methylation profiling indicates that the different histotypes of ovarian cancer should be treated as separate diseases both clinically and in research for the development of targeted therapies.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Metilación de ADN/genética , Neoplasias Ováricas/genética , Análisis por Conglomerados , Femenino , Humanos , Estimación de Kaplan-Meier , Regiones Promotoras GenéticasRESUMEN
Most types of cancer cells are characterized by aberrant methylation of promoter genes. In this study, we described a rapid, reproducible, and relatively inexpensive approach allowing the detection of multiple human methylated promoter genes from many tissue samples, without the need of bisulfite conversion. The Methylation Ligation-dependent Macroarray (MLM), an array-based analysis, was designed in order to measure methylation levels of 58 genes previously described as putative biomarkers of cancer. The performance of the design was proven by screening the methylation profile of DNA from esophageal cell lines, as well as microdissected formalin-fixed and paraffin-embedded (FFPE) tissues from esophageal adenocarcinoma (EAC). Using the MLM approach, we identified 32 (55%) hypermethylated promoters in EAC, and not or rarely methylated in normal tissues. Among them, 21promoters were found aberrantly methylated in more than half of tumors. Moreover, seven of them (ADAMTS18, APC, DKK2, FOXL2, GPX3, TIMP3 and WIF1) were found aberrantly methylated in all or almost all the tumor samples, suggesting an important role for these genes in EAC. In addition, dysregulation of the Wnt pathway with hypermethylation of several Wnt antagonist genes was frequently observed. MLM revealed a homogeneous pattern of methylation for a majority of tumors which were associated with an advanced stage at presentation and a poor prognosis. Interestingly, the few tumors presenting less methylation changes had a lower pathological stage. In conclusion, this study demonstrated the feasibility and accuracy of MLM for DNA methylation profiling of FFPE tissue samples.
Asunto(s)
Adenocarcinoma/genética , Metilación de ADN , Neoplasias Esofágicas/genética , Regiones Promotoras Genéticas/genética , Adenocarcinoma/patología , Biomarcadores de Tumor/genética , Línea Celular Tumoral , ADN de Neoplasias/química , ADN de Neoplasias/genética , Neoplasias Esofágicas/patología , Estudios de Factibilidad , Fijadores/química , Formaldehído/química , Humanos , Análisis por Micromatrices/métodos , Adhesión en Parafina , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN/métodos , Fijación del Tejido , Vía de Señalización Wnt/genéticaRESUMEN
Telomerase activity, not detectable in somatic cells but frequently activated during carcinogenesis, confers immortality to tumors. Mechanisms governing expression of the catalytic subunit hTERT, the limiting factor for telomerase activity, still remain unclear. We previously proposed a model in which the binding of the transcription factor CTCF to the two first exons of hTERT results in transcriptional inhibition in normal cells. This inhibition is abrogated, however, by methylation of CTCF binding sites in 85% of tumors. Here, we showed that hTERT was unmethylated in testicular and ovarian tumors and in derivative cell lines. We demonstrated that CTCF and its paralogue, BORIS/CTCFL, were both present in the nucleus of the same cancer cells and bound to the first exon of hTERT in vivo. Moreover, exogenous BORIS expression in normal BORIS-negative cells was sufficient to activate hTERT transcription with an increasing number of cell passages. Thus, expression of BORIS was sufficient to allow hTERT transcription in normal cells and to counteract the inhibitory effect of CTCF in testicular and ovarian tumor cells. These results define an important contribution of BORIS to immortalization during tumorigenesis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Ováricas/genética , Telomerasa/genética , Neoplasias Testiculares/genética , Sitios de Unión , Línea Celular Tumoral , Supervivencia Celular , Metilación de ADN , Exones , Femenino , Regulación Enzimológica de la Expresión Génica , Células HeLa , Humanos , Masculino , Neoplasias Ováricas/metabolismo , Neoplasias Testiculares/metabolismoRESUMEN
TBX15 is a gene involved in the development of mesodermal derivatives. As the ovaries and the female reproductive system are of mesodermal origin, the aim of the present study was to determine the methylation status of the TBX15 gene promoter and the expression levels of TBX15 in ovarian carcinoma, which is the most lethal and aggressive type of gynecological tumor, in order to determine the role of TBX15 in the pathogenesis of ovarian carcinoma. This alteration could be used to predict tumor development, progression, recurrence and therapeutic effects. The study was conducted on 80 epithelial ovarian carcinoma and 17 control cases (normal ovarian and tubal tissues). TBX15 promoter methylation was first determined by pyrosequencing following bisulfite modification, then by cloning and sequencing, in order to obtain information about the epigenetic haplotype. Immunohistochemical analysis was performed to evaluate the correlation between the methylation and protein expression levels. Data revealed a statistically significant increase of the TBX15 promoter region methylation in 82% of the tumor samples and in various histological subtypes. Immunohistochemistry showed an inverse correlation between methylation levels and the expression of the TBX15 protein. Furthermore, numerous tumor samples displayed varying degrees of intratumor heterogeneity. Thus, the present study determined that ovarian carcinoma typically expresses low levels of TBX15 protein, predominantly due to an epigenetic mechanism. This may have a role in the pathogenesis of ovarian carcinoma independent of the histological subtype.
RESUMEN
Cancer stem cells are cancer cells characterized by stem cell properties and represent a small population of tumor cells that drives tumor development, progression, metastasis and drug resistance. To date, the molecular mechanisms that generate and regulate cancer stem cells are not well defined. BORIS (Brother of Regulator of Imprinted Sites) or CTCFL (CTCF-like) is a DNA-binding protein that is expressed in normal tissues only in germ cells and is re-activated in tumors. Recent evidences have highlighted the correlation of BORIS/CTCFL expression with poor overall survival of different cancer patients. We have previously shown an association of BORIS-expressing cells with stemness gene expression in embryonic cancer cells. Here, we studied the role of BORIS in epithelial tumor cells. Using BORIS-molecular beacon that was already validated, we were able to show the presence of BORIS mRNA in cancer stem cell-enriched populations (side population and spheres) of cervical, colon and breast tumor cells. BORIS silencing studies showed a decrease of sphere formation capacity in breast and colon tumor cells. Importantly, BORIS-silencing led to down-regulation of hTERT, stem cell (NANOG, OCT4, SOX2 and BMI1) and cancer stem cell markers (ABCG2, CD44 and ALDH1) genes. Conversely, BORIS-induction led to up-regulation of the same genes. These phenotypes were observed in cervical, colon and invasive breast tumor cells. However, a completely different behavior was observed in the non-invasive breast tumor cells (MCF7). Indeed, these cells acquired an epithelial mesenchymal transition phenotype after BORIS silencing. Our results demonstrate that BORIS is associated with cancer stem cell-enriched populations of several epithelial tumor cells and the different phenotypes depend on the origin of tumor cells.
Asunto(s)
Biomarcadores de Tumor/genética , Proteínas de Unión al ADN/genética , Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Células Madre Neoplásicas/metabolismo , Esferoides Celulares/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/patología , Transición Epitelial-Mesenquimal/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Proteína Homeótica Nanog , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/patología , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Especificidad de Órganos , Fenotipo , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Retinal-Deshidrogenasa/genética , Retinal-Deshidrogenasa/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal , Esferoides Celulares/patología , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
BORIS/CTCFL is a member of cancer testis antigen family normally expressed in germ cells. In tumors, it is aberrantly expressed although its functions are not completely well-defined. To better understand the functions of BORIS in cancer, we selected the embryonic cancer cells as a model. Using a molecular beacon, which specifically targets BORIS mRNA, we demonstrated that BORIS positive cells are a small subpopulation of tumor cells (3-5% of total). The BORIS-positive cells isolated using BORIS-molecular beacon, expressed higher telomerase hTERT, stem cell (NANOG, OCT4, SOX2) and cancer stem cell marker genes (CD44 and ALDH1) compared to the BORIS-negative tumor cells. In order to define the functional role of BORIS, stable BORIS-depleted embryonic cancer cells were generated. BORIS silencing strongly down-regulated the expression of hTERT, stem cell and cancer stem cell marker genes. Moreover, the BORIS knockdown increased cellular senescence in embryonic cancer cells, revealing a putative role of BORIS in the senescence biological program. Our data indicate an association of BORIS expressing cells subpopulation with the expression of stemness genes, highlighting the critical role played by BORIS in embryonic neoplastic disease.