RESUMEN
BACKGROUND: Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) is involved in DNA demethylation and transcriptional regulation, plays a key role in the maintenance of stem cell pluripotency, and is dysregulated in malignant cells. The identification of cancer stem cells (CSCs) driving tumor growth and metastasis is the primary objective of biomarker discovery in aggressive prostate cancer (PCa). In this context, we analyzed TET1 expression in PCa. METHODS: A large-scale immunohistochemical analysis of TET1 was performed in normal prostate (NOR) and PCa using conventional slides (50 PCa specimens) and tissue microarrays (669 NOR and 1371 PCa tissue cores from 371 PCa specimens). Western blotting, RT-qPCR, and 450 K methylation array analyses were performed on PCa cell lines. Genome-wide correlation, gene regulatory network, and functional genomics studies were performed using publicly available data sources and bioinformatics tools. RESULTS: In NOR, TET1 was exclusively expressed in normal cytokeratin 903 (CK903)-positive basal cells. In PCa, TET1 was frequently detected in alpha-methylacyl-CoA racemase (AMACR)-positive tumor cell clusters and was detectable at all tumor stages and Gleason scores. Pearson's correlation analyses of PCa revealed 626 TET1-coactivated genes (r > 0.5) primarily encoding chromatin remodeling and mitotic factors. Moreover, signaling pathways regulating antiviral processes (62 zinc finger, ZNF, antiviral proteins) and the pluripotency of stem cells were activated. A significant proportion of detected genes exhibited TET1-correlated promoter hypomethylation. There were 161 genes encoding transcription factors (TFs), of which 133 were ZNF-TFs with promoter binding sites in TET1 and in the vast majority of TET1-coactivated genes. CONCLUSIONS: TET1-expressing cells are an integral part of PCa and may represent CSCs with oncogenic potential.
Asunto(s)
Oxigenasas de Función Mixta/análisis , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas/análisis , Anciano , Metilación de ADN/genética , Expresión Génica/genética , Expresión Génica/inmunología , Humanos , Masculino , Persona de Mediana Edad , Oxigenasas de Función Mixta/sangre , Oxigenasas de Función Mixta/genética , Proteínas Proto-Oncogénicas/sangre , Proteínas Proto-Oncogénicas/genéticaRESUMEN
AIM: The prostatitis syndrome is a frequent disease affecting men in their reproductive age. The prostatitis syndrome is classified according to the National Institutes of Health (NIH) definition. Andrological implications of the prostatitis syndrome might encompass fertility issues, sexual dysfunctions and endocrinological alterations and influences. METHODS: A medline query using the terms prostatitis AND andrological implication, fertility, sexual dysfunction or endocrinology was performed. RESULTS: Acute bacterial prostatitis and andrological implications have not been adequately addressed. Patients with chronic bacterial prostatitis and chronic pelvic pain syndrome have been investigated evaluating sperm parameters. Some studies showed impaired sperm parameters. In chronic bacterial prostatitis, half of the patients reveal significant bacteriospermia with still debatable deleterious effects on sperm quality. Few interventional studies have addressed fertility issues in those patients. Anti-inflammatory treatment perhaps could have a positive impact on sperm parameters. Sexual dysfunction can be described by different components such as erectile, ejaculatory, orgasmic and sexual desire dysfunctions. Sexual dysfunction in chronic prostatitis adds to the number of positive symptom phenotypes and correlates therefore with increasing symptom scores in patients with chronic prostatitis syndromes. However, prospective interventional studies on the role of sexual dysfunctions are missing. Hormones have been found to modulate the inflammatory response via different receptors, particularly via estrogen receptor alpha. This evidence, however, is mainly limited to pre-clinical studies currently. CONCLUSION: Andrological implications are heterogenous and frequently described in patients with chronic prostatitis syndrome. Nonetheless, andrological factors have not been routinely addressed as primary variables in the different studies, which makes further research necessary.
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Prostatitis/complicaciones , Enfermedad Aguda , Infecciones Bacterianas/complicaciones , Enfermedades del Sistema Endocrino/etiología , Humanos , Infertilidad Masculina/etiología , Masculino , Prostatitis/microbiología , Disfunciones Sexuales Fisiológicas/etiologíaRESUMEN
To investigate intra-tumoural coexistence and heterogeneity of aberrant promoter hypermethylation of different tumour suppressor genes in melanoma, we analyzed the intra-tumoural distribution of promoter methylation of RASSF1A, p16, DAPK, MGMT, and Rb in 339 assays of 34 tumours (15 melanoma primaries, 19 metastases) by methylation-specific PCR, correlation to histopathology and RASSF1A expression. We detected promoter hypermethylation of at least one gene in 74% of tumours (30%, 52%, 33%, 20%, and 40% for RASSF1A, p16, DAPK, MGMT and Rb, respectively). 70% of the cases exhibited an inhomogeneous methylation pattern (17%, 45%, 33%, 20%, and 40% for RASSF1A, p16, DAPK, MGMT and Rb, respectively). Samples from the core of the tumours represented the methylation state of the whole tumours more accurately than the periphery. Local intra-tumoural correlation was found between the promoter hypermethylation state of p16 and Rb or p16 and DAPK, or epitheloid tumour cell type and RASSF1A or p16 methylation. Mitosis rate and sex was correlated with methylation of RASSF1A. Histological results confirmed that promoter hypermethylation of RASSF1A led to aberrant expression patterns. We conclude that intra-tumoural inhomogeneity of promoter hypermethylation is frequent in melanoma and this supports the hypothesis of clonal instability during progression of melanomas. In prognosis studies, missing the intra-tumoural sample representativeness may result in a reduction of the sensitivities or specificities.
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Metilación de ADN , Genes p16 , Heterogeneidad Genética , Melanoma/genética , Melanoma/metabolismo , Regiones Promotoras Genéticas , Adulto , Anciano , Anciano de 80 o más Años , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , ADN/genética , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Proteínas Quinasas Asociadas a Muerte Celular , Femenino , Genes Supresores de Tumor , Humanos , Inmunohistoquímica , Masculino , Melanoma/patología , Persona de Mediana Edad , Metástasis de la Neoplasia , Reacción en Cadena de la Polimerasa , Proteína de Retinoblastoma/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Cystatin M is a potent endogenous inhibitor of lysosomal cysteine proteases. In breast carcinoma, cystatin M expression is frequently downregulated. It has been shown that cystatin M expression suppressed growth and migration of breast cancer cells. We examined the methylation status of the CpG island promoter of cystatin M in four breast cancer cell lines (MDAMB231, ZR75-1, MCF7 and T47D), in 40 primary breast carcinoma and in corresponding normal tissue probes by combined bisulphite restriction analysis. To investigate the effects of cystatin M expression on the growth of breast carcinoma, cystatin M was transfected in T47D. The cystatin M promoter was highly methylated in all four-breast cancer cell lines. Primary breast tumours were significantly more frequently methylated compared to normal tissue samples (60 vs 25%; P=0.006 Fisher's exact test). Treatment of breast cancer cells with 5-aza-2'-deoxycytidine (5-Aza-CdR), reactivated the transcription of cystatin M. Transfection of breast carcinoma cells with cystatin M caused a 30% decrease in colony formation compared to control transfection (P=0.002). Our results show that cystatin M is frequently epigenetically inactivated during breast carcinogenesis and cystatin M expression suppresses the growth of breast carcinoma. These data suggest that cystatin M may encode a novel epigenetically inactivated candidate tumour suppressor gene.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Cistatinas/antagonistas & inhibidores , Cistatinas/genética , Epigénesis Genética , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Línea Celular Tumoral , Islas de CpG/genética , Cistatina M , Cistatinas/metabolismo , Metilación de ADN , Femenino , Silenciador del Gen , Humanos , Proteínas Supresoras de Tumor/metabolismoRESUMEN
AIMS: About 10-30% of primary liver cancers represent intrahepatic cholangiocarcinomas (IHCC). Since chromosomal losses of 3p are detectable in about 40% of cholangiocarcinomas our study aimed at the identification of mechanisms leading to functional deletion of tumor suppressor genes in this region. Our efforts focussed on genomic losses and epigenetic inactivation of two tumor suppressor genes, the fragile histidine triad (FHIT) and the ras association domain family 1 (RASSF1A) genes, both located on the short arm of chromosome 3. METHODS: Methylation-specific PCR (MSP) and combined bisulfite-dependent restriction analysis (COBRA) were applied to detect epigenetic silencing of gene promoters. Genomic duplex PCR was used to identify exon losses of the FHIT gene. Nineteen paraffin-embedded samples of intrahepatic cholangiocarcinomas were studied. RESULTS: Here we report for the first time that in addition to frequent losses of the exons 5 and 6, hypermethylation of the FHIT promoter occured in a significant portion of IHCC. Methylation specific PCR (MSP) detected epigenetic inactivation of the FHIT/FRA3B locus in 8 of 19 (42%) cases. Combined bisulfite restriction analysis (COBRA) revealed that high levels of methylated FHIT promoter sequences were present in 6 of the 8 methylation positive samples. In agreement with previous reports MSP identified hypermethylation of the RASSF1A gene in 13 of 19 (68%) IHCC specimens examined. CONCLUSIONS: Epigenetic silencing of the FHIT tumor suppressor gene is a novel inactivation mechanism to be considered in the development of intrahepatic cholangiocarcinomas. However, a statistically significant inverse correlation between K-Ras activation and RASSF1A inactivation was not found.
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Ácido Anhídrido Hidrolasas/genética , Neoplasias de los Conductos Biliares/genética , Conductos Biliares Intrahepáticos , Colangiocarcinoma/genética , Metilación de ADN , Epigénesis Genética/genética , Exones/genética , Genes Supresores de Tumor , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas/genética , Silenciador del Gen , Genes ras , Humanos , Pérdida de Heterocigocidad , Reacción en Cadena de la Polimerasa , Proteínas Supresoras de Tumor/genéticaRESUMEN
Loss of heterozygosity of the small arm of chromosome 3 is one of the most common alterations in human cancer. Most notably, a segment in 3p21.3 is frequently lost in lung cancer and several other carcinomas. We and others have identified a novel Ras effector at this segment, which was termed Ras Association Domain family 1 (RASSF1A) gene. RASSF1 consists of two main variants (RASSF1A and RASSF1C), which are transcribed from distinct CpG island promoters. Aberrant methylation of the RASSF1A promoter region is one of the most frequent epigenetic inactivation events detected in human cancer and leads to silencing of RASSF1A. Hypermethylation of RASSF1A was commonly observed in primary tumors including lung, breast, pancreas, kidney, liver, cervix, nasopharyngeal, prostate, thyroid and other cancers. Moreover, RASSF1A methylation was frequently detected in body fluids including blood, urine, nipple aspirates, sputum and bronchial alveolar lavages. Inactivation of RASSF1A was associated with an advanced tumor stage (e.g. bladder, brain, prostate, gastric tumors) and poor prognosis (e.g. lung, sarcoma and breast cancer). Detection of aberrant RASSF1A methylation may serve as a diagnostic and prognostic marker. The functional analyses of RASSF1A reveal an involvement in apoptotic signaling, microtubule stabilization and mitotic progression. The tumor suppressor RASSF1A may act as a negative Ras effector inhibiting cell growth and inducing cell death. Thus, RASSF1A may represent an epigenetically inactivated bona fide tumor suppressor in human carcinogenesis.
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Genes Supresores de Tumor , Neoplasias/genética , Proteínas Supresoras de Tumor/genética , Cromosomas Humanos Par 3/genética , Metilación de ADN , ADN de Neoplasias/química , ADN de Neoplasias/genética , Epigénesis Genética , Femenino , Humanos , Pérdida de Heterocigocidad , Masculino , Neoplasias/química , Neoplasias/patología , Pronóstico , Regiones Promotoras Genéticas , Eliminación de SecuenciaRESUMEN
Hypermethylation of CpG island promoters is associated with transcriptional inactivation of tumor suppressor genes in neoplasia. Inactivation of p16 and Pten was related to the development of pheochromocytomas. In this report, we investigated the methylation status of the p16INK4a cell cycle inhibitor gene and other prominent tumor-related genes ( PTEN, RASSF1 A, CDH1, MSH2, MLH1, VHL, and TIMP3) in sporadic and multiple endocrine neoplasia type 2 (MEN2) pheochromocytomas by methylation-specific PCR. Hypermethylation was detected in 48 % of pheochromocytomas for RASSF1 A, 24 % for p16, 36 % for MSH2, 16 % for CDH1, and 8 % for PTEN. No VHL, MLH1, and TIMP3 methylation was observed. Interestingly, the frequency of p16 inactivation in familial tumors was higher (5 out of 12, 42 %) than in sporadic tumors (1 out of 13, 8 %; p = 0.047) and RASSF1 A inactivation was more common in the hereditary tumors (58 %) compared to the sporadic tumors (38 %). Combined methylation of RASSF1 A and p16 was found only in MEN2-related pheochromocytomas. Thus, a subset of hereditary pheochromocytomas displays preferential methylation of p16 and RASSF1 A.
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Neoplasias de las Glándulas Suprarrenales/genética , Metilación de ADN , Genes Supresores de Tumor , Neoplasia Endocrina Múltiple Tipo 2a/genética , Feocromocitoma/genética , Regiones Promotoras Genéticas , Adulto , Anciano , Femenino , Genes p16 , Humanos , Masculino , Persona de Mediana Edad , Proteínas Supresoras de Tumor/genéticaRESUMEN
The Ras GTPases are a superfamily of molecular switches that regulate cellular proliferation and apoptosis in response to extra-cellular signals. The regulation of these pathways depends on the interaction of the GTPases with specific effectors. Recently, we have cloned and characterized a novel gene encoding a putative Ras effector: the Ras-association domain family 1 (RASSF1) gene. The RASSF1 gene is located in the chromosomal segment of 3p21.3. The high allelic loss in a variety of cancers suggested a crucial role of this region in tumorigenesis. At least two forms of RASSF1 are present in normal human cells. The RASSF1A isoform is highly epigenetically inactivated in lung, breast, ovarian, kidney, prostate, thyroid and several other carcinomas. Re-expression of RASSF1A reduced the growth of human cancer cells supporting a role for RASSF1 as a tumor suppressor gene. RASSF1A inactivation and K-ras activation are mutually exclusive events in the development of certain carcinomas. This observation could further pinpoint the function of RASSF1A as a negative effector of Ras in a pro-apoptotic signaling pathway. In malignant mesothelioma and gastric cancer RASSF1A methylation is associated with virus infection of SV40 and EBV, respectively, and suggests a causal relationship between viral infection and progressive RASSF1A methylation in carcinogenesis. Furthermore, a significant correlation between RASSF1A methylation and impaired lung cancer patient survival was reported, and RASSF1A silencing was correlated with several parameters of poor prognosis and advanced tumor stage (e.g. poor differentiation, aggressiveness, and invasion). Thus, RASSF1A methylation could serve as a useful marker for the prognosis of cancer patients and could become important in early detection of cancer.
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Silenciador del Gen , Proteínas de Neoplasias/genética , Neoplasias/genética , Proteínas Supresoras de Tumor , Animales , Islas de CpG/genética , Análisis Mutacional de ADN , Genes Supresores de Tumor , Humanos , Regiones Promotoras Genéticas/genéticaRESUMEN
In order to investigate genomic imbalances, comparative genomic hybridization was applied to 20 malignant fibrous histiocytomas. Deletions were rare and found mainly in chromosomes 2q33-35, 4q32-qter, 8p, 9p21-pter, 12p and 19p, whereas, over-representations frequently affected chromosomes 3, 4q31, 5p, 6, 7, 14q22-ter, 18p, as well as, five distinct amplifications within the regions 12q12-15 and 15q24-qter. The total number of genetic imbalances per tumor was slightly increased in primary tumors when compared to relapses. No relationship was found between the patterns of gain and loss when compared to the histological subtype, tumor grading, the clinical outcome and the p53 mutation status.