trichostatin-a and Adenoma

Primary hyperparathyroidism (PHPT) results from the hypersecretion of parathyroid hormone from parathyroid tumors. A transcription factor, namely Paired box1 (PAX1), is active in parathyroid gland development.. We aimed to study potential epigenetic-mediated mechanism of PAX1 gene in sporadic parathyroid adenomas.. In parathyroid adenomas tissues, we analyzed the DNA methylation via bisulfite-specific polymerase chain reaction (BSP) and histone modifications via chromatin immunoprecipitation in regulating the differential expression of PAX1.. The results showed that mRNA and protein expression of PAX1 was significantly reduced in parathyroid adenomas. Bisulfite sequencing demonstrated hypermethylation in the promoter region of PAX1 (35%; 14/40) and lower levels of histone 3 lysine 9 acetylation (H3K9ac) were observed on the promoter region of PAX1 (6-fold; P < .004) in parathyroid adenomas. Furthermore, upon treatment with a pharmacologic inhibitor, namely 5'aza-2 deoxycytidine, in rat parathyroid continuous cells, we found re-expression of PAX1 gene.. Our study not only reveals expression of PAX1 is epigenetically deregulated but also paves a way for clinical and therapeutic implications in patients with PHPT.

Topics: Acetylation; Adenoma; Adolescent; Adult; Aged; Animals; Carcinogenesis; Case-Control Studies; Cell Line, Tumor; Decitabine; DNA Methylation; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Healthy Volunteers; Histone Code; Humans; Hydroxamic Acids; Male; Middle Aged; Paired Box Transcription Factors; Parathyroid Glands; Parathyroid Neoplasms; Parathyroidectomy; Promoter Regions, Genetic; Rats; Young Adult

Transgenic mice overexpressing the high mobility group A (HMGA) genes, Hmga1 or Hmga2 develop pituitary tumours and their overexpression is also a frequent finding in human pituitary adenomas. In some cases, increased expression of HMGA2 but not that of HMGA1 is consequent to genetic perturbations. However, recent studies show that down-regulation of microRNA (miRNA), that contemporaneously target the HMGA1 and HMGA2 transcripts, are associated with their overexpression.. In a cohort of primary pituitary adenoma we determine the impact of epigenetic modifications on the expression of HMGA-targeting miRNA. For these miRNAs, chromatin immunoprecipitations showed that transcript down-regulation is correlated with histone tail modifications associated with condensed silenced genes. The functional impact of epigenetic modification on miRNA expression was determined in the rodent pituitary cell line, GH3. In these cells, histone tail, miRNA-associated, modifications were similar to those apparent in human adenoma and likely account for their repression. Indeed, challenge of GH3 cells with the epidrugs, zebularine and TSA, led to enrichment of the histone modification, H3K9Ac, associated with active genes, and depletion of the modification, H3K27me3, associated with silent genes and re-expression of HMGA-targeting miRNA. Moreover, epidrugs challenges were also associated with a concomitant decrease in hmga1 transcript and protein levels and concurrent increase in bmp-4 expression.. These findings show that the inverse relationship between HMGA expression and targeting miRNA is reversible through epidrug interventions. In addition to showing a mechanistic link between epigenetic modifications and miRNA expression these findings underscore their potential as therapeutic targets in this and other diseases.

Topics: Adenoma; Animals; Antineoplastic Agents; Cell Line; Chromatin Assembly and Disassembly; CpG Islands; Cytidine; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; HMGA Proteins; HMGA2 Protein; Humans; Hydroxamic Acids; MicroRNAs; Pituitary Gland; Pituitary Neoplasms; Rats; Rats, Sprague-Dawley; Up-Regulation

Retinoic acid (RA)-induced expression of bone morphogenetic protein-4 (BMP-4) inhibits in vitro and in vivo cell proliferation and ACTH synthesis in corticotroph-derived tumor cells. Reduced expression of BMP-4 in this adenoma subtype is associated with epigenomic silencing, and similar silencing mechanisms are also associated with the RA-responsive dopamine D2 receptor (D2R) in somatolactotroph cells. We now show that preincubation with the epidrugs zebularine and trichostatin A is obligate and permissive for RA-induced expression of the BMP-4 and the D2R genes in pituitary tumor cells. Combined epidrug challenges are associated with marginal reduction in CpG island methylation. However, significant change to histone tail modifications toward those associated with expression-competent genes is apparent, whereas RA challenge alone or in combined incubations does not have an impact on these modifications. Epidrug-mediated and RA-augmented expression of endogenous BMP-4 increased or decreased cell proliferation and colony-forming efficiency in GH3 and AtT-20 pituitary tumor cells, respectively, recapitulating recent reports of challenges of these cells with exogenous ligand. The specificity of the BMP-4-mediated effects was further supported by knock-down experiments of the BMP-4 antagonist noggin (small interfering RNA [siRNA]). Knock-down of noggin, in the absence and the presence of epidrugs, induced and augmented BMP-4 expression, respectively. In cell proliferation assays, challenge with either epidrugs or siRNA led to significant increase in cell numbers at the 72-hour time point; however, in siRNA-treated cells coincubated with epidrugs, a significant increase was apparent at the 48-hour time point. These studies show the potential of combined drug challenges as a treatment option, where epidrug renders silenced genes responsive to conventional therapeutic options.

Topics: Adenoma; Animals; Bone Morphogenetic Protein 4; Cell Culture Techniques; Cytidine; Drug Synergism; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mice; Pituitary Neoplasms; Prodrugs; Receptors, Dopamine D2; Time Factors; Tretinoin; Tumor Cells, Cultured

Nucleophosmin (NPM) is a multifunctional nucleolar protein that, depending on the context, can act as oncogene or tumour suppressor. Mutations of the NPM1 gene induce delocalization of NPM in acute myeloid leukaemia. Differently, in solid tumours, only NPM overexpression, but not delocalization, has been so far reported. Here, NPM localization in thyroid tumours was investigated. By using immunohistochemistry, we show increase of NPM cytoplasmic localization in follicular adenomas and papillary carcinomas compared to normal thyroid tissue (p = 0.0125 and <0.0001, respectively). NPM1 mutations commonly found in human leukaemia are not present in thyroid tumours. Immunofluorescence in cultured cell lines was utilized to discriminate between nucleolar and nuclear localization. We show that in thyroid cancer cell lines NPM localizes both in the nucleolus and in nucleus, while in non-tumorigenic thyroid cell lines localizes only in nucleolus. Either presence of the histone deacetylase inhibitor trichostatin A or absence of thyroid-stimulating hormone induces NPM nuclear localization in non-tumorigenic thyroid cell lines.

Topics: Adenocarcinoma, Follicular; Adenoma; Biomarkers, Tumor; Carcinoma; Carcinoma, Papillary; Cell Line, Tumor; Cell Nucleolus; Cell Nucleus; DNA Mutational Analysis; DNA, Neoplasm; Humans; Hydroxamic Acids; Nuclear Proteins; Nucleophosmin; Thyroid Neoplasms; Thyrotropin

Ca2+ is a chemopreventive agent for colon cancer. Ion transport systems are often altered in human cancer. The aim of this study was to clarify the alterations of calcium-sensing receptor (CASR), a member of the G protein-coupled receptor family, in colorectal carcinogenesis. We analyzed the expression of CASR in colorectal cancer cell lines and in cancer and adenoma tissues by RT-PCR and immunostaining. In addition, we analyzed methylation of the CASR promoter by using bisulfite sequence analysis and methylation-specific PCR. CASR mRNA and protein expression was significantly downregulated in most of the cancer cell lines. CpG islands were densely methylated in cancer cell lines with reduced CASR mRNA expression. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine, and/or a histone deacetylase inhibitor, trichostatin A, restored CASR expression in the cancer cell lines. Disruption of CASR expression in CASR-unmethylated HCT-8 cells blocked the enhancing effect of Ca2+ on the cytotoxic response to 5-fluorouracil. CASR expression was observed in normal colonic epithelial cells and was retained in most adenoma tissues. CASR mRNA and protein expression was significantly downregulated in cancer tissues. There was an inverse relationship between CASR expression and degree of differentiation. Immunohistochemical CASR staining was reduced more predominantly in less-differentiated cancer tissues and/or in cancer cells at the invasive front, where nuclear/cytoplasmic β-catenin was often localized. CASR methylation was detected in 69% of colorectal cancer tissues and 90% of lymph node metastatic tissues and was significantly correlated with reduced CASR expression. CASR methylation was also detected in 32% of advanced adenoma tissues but was detected in only 9% of adenoma tissues and was not detected in hyperplastic polyp tissues. CASR methylation seems to occur at an early stage and progress in colorectal carcinogenesis. The results suggest that epigenetic inactivation of CASR has an important role in colorectal carcinogenesis.

Topics: Adenocarcinoma; Adenoma; Antineoplastic Agents, Alkylating; Azacitidine; Caco-2 Cells; Cell Survival; Colonic Neoplasms; CpG Islands; Decitabine; DNA Methylation; DNA, Neoplasm; Gene Expression; Gene Silencing; Humans; Hydroxamic Acids; Receptors, Calcium-Sensing; RNA, Messenger; Tissue Array Analysis

Although growing evidence demonstrates that TWIST1 is an interesting tumor biomarker, little is known about the clinical significance of TWIST1 expression and TWIST1 methylation in human primary colorectal cancer. In this study, we examined the association of TWIST1 expression and TWIST1 methylation with clinicopathologic features in human primary colorectal tumors. Primary colorectal cancer (CRC) specimens from 319 patients, corresponding normal colorectal nontumorous mucosa from 251 patients with cancer, and colorectal adenomas from 189 patients were used. Methylation and expression levels of TWIST1 were compared with clinicopathologic features. The TWIST1 methylation level was higher in colorectal adenoma and cancer than in normal colorectal mucosa. Elevated TWIST1 mRNA expression in normal colorectal mucosa in patients with CRC as well as in primary CRC specimens was associated with unfavorable outcomes. There was no correlation between TWIST1 methylation and TWIST1 expression. Our results suggest that TWIST1 methylation may be a useful biomarker for screening colorectal tumors. In addition, TWIST1 mRNA expression is a possible molecular marker for predicting the outcome in patients with CRC. Confirmatory studies using independent data sets are needed to confirm our findings.

Topics: Adenoma; Adult; Aged; Aged, 80 and over; Azacitidine; Case-Control Studies; Cell Line, Tumor; Chromosome Mapping; Colorectal Neoplasms; Decitabine; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Intestinal Mucosa; Linear Models; Male; Middle Aged; Nuclear Proteins; Polymerase Chain Reaction; Prognosis; Sequence Analysis, DNA; Statistics, Nonparametric; Twist-Related Protein 1

To investigate the functions of promoter hypermethylation of secreted Wnt-antagonist genes in colorectal tumorigenesis and progression.. Two colorectal cancer cell lines, HCT116 and SW480, were treated by 5-aza-2'-deoxycytidine (DAC) and trichostatin A (TSA) for demethylation. The promoter hypermethylation and expression of sFRP and WIF-1 genes in different stages of colorectal tumor and colorectal cancer cell lines were detected by methylation-specific PCR and reverse transcription PCR, respectively.. None of the normal colorectal mucosa samples showed methylated bands of any sFRP and WIF-1genes. Hypermethylation of sFRP1, 2, 4, 5 and WIF-1 was detected in 93.1% (67/72), 83.3% (60/72), 36.1% (26/72), 52.8% (38/72) and 84.7% (61/72) of adenocarcinomas, 87.9% (29/33), 81.8% (27/33), 24.2% (8/33), 57.6% (19/33) and 72.7% (24/33) of adenomas, 52.6%, 28.9%, 2.6%, 18.4%, 23.7% of the adjacent normal mucosa. Methylation was more frequently found in colorectal tumors than in normal mucosa and adjacent normal mucosa from patients with tumor (P < 0.05). No significant association between Wnt-antagonist genes hypermethylation and clinicopathological characteristics was found (P > 0.05). SFRP1, 2, 4, 5 and WIF-1 genes were methylated in HCT116 cell line. SFRP1, 2 and WIF-1 were methylated in SW480 cell line. The mRNA expression of sFRPs and WIF-1 genes was absent or significantly downregulated (P < 0.01) when they were methylated in two colorectal cancer cell lines. SFRP3 was expressed in two colorectal carcinoma cell lines. DAC/TSA combination treatment re-expressed the silenced sFRPs and WIF-1 genes mRNA expressions effectively. A single application of TSA could not re-express sFRPs and WIF-1 genes mRNA expressions. The influence of demethylation treatment on sFRP3 expression was minimal.. Hypermethylation of Wnt-antagonist genes is a common early event in the evolution of colorectal tumor. Methylation of sFRP1, 2, 5 and WIF-1 genes might serve as biomarkers for the early detection of colorectal tumor.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Adenoma; Adult; Aged; Azacitidine; Cell Line, Tumor; Colorectal Neoplasms; Decitabine; DNA Methylation; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; HCT116 Cells; Humans; Hydroxamic Acids; Intestinal Mucosa; Intracellular Signaling Peptides and Proteins; Male; Middle Aged; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

To investigate the functions of promoter hypermethylation of secreted frizzled-related protein (sFRP) genes in colorectal tumorigenesis and progression.. Three colorectal cancer cell lines, RKO, HCTll6 and SW480, were treated hy 5-aza-2'-deoxycytidine and trichostatin A for demethylation. The promoter hypermethylation and expression of sFRP genes in colorectal tumor tissue and colorectal cancer cell lines were detected hy methylation-specific PCR and reverse transcription PCR, respectively.. None of the normal colorectal mucosa tissues showed methylation of sFRP genes. sFRP1, 2, 4 and 5 were frequently methylated in colorectal adenocarcinoma, adenoma and aberrant crypt foci (ACF) (sFRP1 > 85%, sFRP2 > 75%, sFRP5 > 50%), the differences between any two of them were not significant (P >0.05). Methylation was more frequent in colorectal tumors than in normal mucosa and adjacent normal mucosa from patients with tumor. Hypermethylation of sFRP genes was present in three colorectal cancer cell lines. When sFRP genes were methylated, their corresponding mRNA expression was absent. After cells were treated by DAC/TSA combination, the silenced sFRP expression could be effectively re-expressed.. Hypermethylation of sFRP genes is a common early event in the evolution of colorectal tumors that occurs frequently in ACF. Methylation of sFRP1, 2 and 5 genes might serve as biomarkers for the early detection of colorectal tumors. Demethylation can effectively reverse gene expression that appears possibly to be an effective way for tumor therapy.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Adenoma; Adult; Aged; Azacitidine; Biomarkers, Tumor; Colonic Neoplasms; Colorectal Neoplasms; Decitabine; DNA Methylation; DNA Modification Methylases; Eye Proteins; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; HCT116 Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Male; Membrane Proteins; Middle Aged; RNA, Messenger

CDX2 is a caudal-related homeobox transcription factor whose expression in the adult is normally restricted to intestinal epithelium. Mice heterozygous for germ line Cdx2 inactivation develop intestinal polyps, and the lesions lack Cdx2 expression. Prior studies indicate some human colon carcinomas also lack CDX2 expression. To address the role of CDX2 defects in colon cancer development, we analyzed CDX2 expression in 45 primary colorectal carcinomas. Four carcinomas lacked CDX2 expression, and three others showed aberrant cytoplasmic localization of CDX2, although no significant CDX2 gene defects were seen in the seven tumors. Marked reductions in CDX2 transcript and protein levels were seen in five of 13 colorectal cell lines, and nuclear run-off data indicated reduced transcription was a major factor in CDX2 silencing. Treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine and/or the histone deacetylase inhibitor trichostatin A did not restore CDX2 expression in CDX2-negative lines. However, consistent with a role for dominant repression mechanisms in CDX2 silencing, all somatic cell hybrids resulting from pairwise fusions between colon cancer lines with intact CDX2 expression and lines lacking CDX2 had reduced CDX2 transcripts and protein. A roughly 9.5-kb 5'-flanking region from the human CDX2 gene contained key cis elements for regulating transcription in colon cancer cells. Restoration of CDX2 expression suppressed proliferation and soft agar growth in the CDX2-negative HT-29 colon cancer cell line. Our findings suggest CDX2 inactivation in colon cancer results from defects in trans-acting pathways regulating CDX2 transcription, and CDX2 silencing contributes to the altered phenotype of some colorectal cancers.

Topics: Adenoma; Azacitidine; Blotting, Western; CDX2 Transcription Factor; Cell Division; Colonic Neoplasms; Cytoplasm; Decitabine; DNA Methylation; Enzyme Inhibitors; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Homeodomain Proteins; Humans; Hydroxamic Acids; Immunohistochemistry; Methyltransferases; Mutation; Mutation, Missense; Phenotype; Phosphatidylinositol 3-Kinases; Rectal Neoplasms; Trans-Activators; Transcription, Genetic

Ectopic secretion of ACTH, from sites such as small cell lung cancer (SCLC), results in severe Cushing's syndrome. ACTH is cleaved from POMC. The syndrome may occur when the highly tissue-specific promoter of the human POMC gene (POMC) is activated. The mechanism of activation is not fully understood. This promoter is embedded within a defined CpG island, and CpG islands are usually considered to be unmethylated in all tissues. We demonstrate that much of this CpG island is methylated in normal nonexpressing tissues, in contrast to somatically expressed CpG island promoters reported to date, and is specifically unmethylated in expressing tissues, tumors, and the POMC-expressing DMS-79 SCLC cell line. A narrow 100-bp region is free of methylation in all tissues. E2F factors binding to the upstream domain IV region of the promoter have been shown to be involved in the expression of POMC in SCLC. We show that these sites are methylated in normal nonexpressing tissues, which will prevent binding of E2F, but are unmethylated in expressing tissue. Methylation in vitro is sufficient for silencing of expression, which is not reversed by treatment with Trichostatin A, suggesting that inhibition of expression may be mediated by means other than recruitment of histone deacetylase activity. The DMS-79 cells lack POMC demethylating activity, implying that the methylation and expression patterns are likely to be set early or before neoplastic transformation, and that targeted de novo methylation might be a potential therapeutic strategy.

Topics: Adenoma; Adrenocorticotropic Hormone; Carcinoma, Small Cell; Carrier Proteins; Cell Cycle Proteins; Cell Line; CpG Islands; DNA Methylation; DNA-Binding Proteins; E2F Transcription Factors; Enzyme Inhibitors; Gene Expression; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; Neoplasms; Organ Specificity; Pituitary Neoplasms; Plasmids; Pro-Opiomelanocortin; Promoter Regions, Genetic; Retinoblastoma-Binding Protein 1; Sequence Analysis, DNA; Transcription Factor DP1; Transcription Factors; Transcription, Genetic; Transfection