Promoter Methylation

Colorectal cancers exhibit a high telomerase activity, usually correlated with the hypermethylation of the promoter of its hTERT catalytic subunit. Although telomerase is not expressed in normal tissue, certain proliferative somatic cells such as intestinal crypt cells have demonstrated telomerase activity. The aim of this study was to determine whether a correlation exists between telomerase activity, levels of hTERT methylation and telomere length in tumoral and normal colorectal tissues. Tumor, transitional and normal tissues were obtained from 11 patients with a colorectal cancer. After bisulfite modification of genomic DNA, hTERT promoter methylation was analyzed by methylation-sensitive single-strand conformation analysis (MS-SSCA). Telomerase activity and telomere length were measured by a fluorescent-telomeric repeat amplification protocol assay and by Southern blotting, respectively. A significant increase of hTERT methylation and telomerase activity, and a reduction of the...

Germline mutations in BRCA1/2 predispose individuals to breast cancer (termed germline-mutated BRCA1/2 breast cancer, gBRCA-BC) by impairing homologous recombination (HR) and causing genomic instability. HR also repairs DNA lesions caused by platinum agents and PARP inhibitors. Triple-negative breast cancers (TNBCs) harbor subpopulations with BRCA1/2 mutations, hypothesized to be especially platinum-sensitive. Cancers in putative 'BRCAness' subgroups-tumors with BRCA1 methylation; low levels of BRCA1 mRNA (BRCA1 mRNA-low); or mutational signatures for HR deficiency and those with basal phenotypes-may also be sensitive to platinum. We assessed the efficacy of carboplatin and another mechanistically distinct therapy, docetaxel, in a phase 3 trial in subjects with unselected advanced TNBC. A prespecified protocol enabled biomarker-treatment interaction analyses in gBRCA-BC and BRCAness subgroups. The primary endpoint was objective response rate (ORR). In the unselected populati...

Inhalation exposure to asbestos is associated with lung and pleural diseases in humans and remains a major public health issue worldwide. Human bronchial epithelial cells (16HBE) were exposed to UICC amosite, crocidolite and chrysotile. Cytotoxicity, genotoxicity, global DNA methylation on cytosine residues (using LC-MS/MS) were investigated at different doses (2.5-100 μg/ml). Gene-specific DNA methylation alterations at the whole genome were investigated using a microarray that interrogates >450 thousand CpG sites. Subsequently, gene functional analyses (KEGG pathway, Gene Ontology and functional classification) were performed on genes with differentially methylated gene promoters. At non-cytotoxic doses, global DNA methylation was altered after 24 h exposure to amosite and crocidolite (>2.5 μg/ml). Exposure to amosite and crocidolite (amphibole type asbestos) induced both hypomethylation and hypermethylation at single CpG site and gene promoter levels whereas exposure to chr...

Colorectal cancers exhibit a high telomerase activity, usually correlated with the hypermethylation of the promoter of its hTERT catalytic subunit. Although telomerase is not expressed in normal tissue, certain proliferative somatic cells such as intestinal crypt cells have demonstrated telomerase activity. The aim of this study was to determine whether a correlation exists between telomerase activity, levels of hTERT methylation and telomere length in tumoral and normal colorectal tissues. Tumor, transitional and normal tissues were obtained from 11 patients with a colorectal cancer. After bisulfite modification of genomic DNA, hTERT promoter methylation was analyzed by methylation-sensitive single-strand conformation analysis (MS-SSCA). Telomerase activity and telomere length were measured by a fluorescent-telomeric repeat amplification protocol assay and by Southern blotting, respectively. A significant increase of hTERT methylation and telomerase activity, and a reduction of the...

BackgroundDNA methylation has been shown to be associated with adiposity in adulthood. However, whether similar DNA methylation patterns are associated with childhood and adolescent body mass index (BMI) is largely unknown. More insight into this relationship at younger ages may have implications for future prevention of obesity and its related traits.MethodsWe examined whether DNA methylation in cord blood and whole blood in childhood and adolescence was associated with BMI in the age range from 2 to 18 years using both cross-sectional and longitudinal models. We performed meta-analyses of epigenome-wide association studies including up to 4133 children from 23 studies. We examined the overlap of findings reported in previous studies in children and adults with those in our analyses and calculated enrichment.ResultsDNA methylation at three CpGs (cg05937453, cg25212453, and cg10040131), each in a different age range, was associated with BMI at Bonferroni significance,P < 1.06 × 1...

Silencing of a Nicotiana plumbaginifolia P-l,&glucanase (gnl) transgene in tobacco line T17 occurs in homozygous and in haploid plants with one transgene locus dosage per chromosome ser. We have previously shown that the silent state is manifested by a reduced gnl steady state mRNA leve1 and results from a post-transcriptional process that is under developmental control in homozygous T17 plants. In this study, we show that specific endogenous P-1,S-glucanase genes are cosuppressed with gnl in homozygous T17 plants. We also demonstrate that the developmental timing of cosuppression depends on environmental conditions and that once silencing is established it is stably maintained during plant development. Analyses of additional transgenic tobacco lines revealed that gnl silencing is not restricted to the Ti7 line and showed that silencing can also take place in Ro plants containing independent loci in hemizygous states. Furthermore, silencing can also be obtained in progeny plants in which expressing loci have been combined. Importantly, cosuppression occurs via a post-transcriptional mechanism that does not interfere with the accumulation of transgene nuclear mRNA. These results strongly suggest that the silencing mechanism operates at RNA transpart andlor RNA stability levels.

Recent reports indicate that the alterations in the p16 and pRb pathways can influence tumour progression and poor prognosis in several tumours. The objective of this study was to analyse p16 and pRb expression in161 patients with malignant fibrous histiocytomas (MFH). By immunohistochemistry, p16 and pRb were demonstrated in 25% and 56% of MFH, respectively. Cox regression analysis demonstrated an independent prognostic influence of both genes. Generally, the loss of p16 and pRb expression correlated with poorer prognosis. Promoter methylation of p16 was found in 16/42 of p16 negative MFH and of pRb in 2/42 of pRb-negative MFH. It can be concluded that p16 and pRb alterations play an important role in the progression of soft tissue sarcomas.

BackgroundDNA methylation has been shown to be associated with adiposity in adulthood. However, whether similar DNA methylation patterns are associated with childhood and adolescent body mass index (BMI) is largely unknown. More insight into this relationship at younger ages may have implications for future prevention of obesity and its related traits.MethodsWe examined whether DNA methylation in cord blood and whole blood in childhood and adolescence was associated with BMI in the age range from 2 to 18 years using both cross-sectional and longitudinal models. We performed meta-analyses of epigenome-wide association studies including up to 4133 children from 23 studies. We examined the overlap of findings reported in previous studies in children and adults with those in our analyses and calculated enrichment.ResultsDNA methylation at three CpGs (cg05937453, cg25212453, and cg10040131), each in a different age range, was associated with BMI at Bonferroni significance,P < 1.06 × 1...

Several cross-sectional studies have demonstrated the relevance of DNA methylation of the glucocorticoid receptor exon 1 F region (GR-1 F) for trauma-related psychopathology. We conducted a longitudinal study to examine GR-1 F methylation changes over time in relation to trauma exposure and the development of post-deployment psychopathology. GR-1 F methylation (52 loci) was quantified using pyrosequencing in whole blood of 92 military men 1 month before and 6 months after a 4-month deployment period to Afghanistan. GR-1 F methylation overall (mean methylation and the number of methylated loci) and functional methylation (methylation at loci associated with GR exon 1 F expression) measures were examined. We first investigated the effect of exposure to potentially traumatic events during deployment on these measures. Subsequently, changes in GR-1 F methylation were related to changes in mental health problems (total Symptom Checklist-90 score) and posttraumatic stress disorder (PTSD) symptoms (Self-Report Inventory for PTSD). Trauma exposure during deployment was associated with an increase in all methylation measures, but development of mental health problems 6 months after deployment was only significantly associated with an increased functional methylation. Emergence of post-deployment PTSD symptoms was not related to increased functional methylation over time. Pre-deployment methylation levels did not predict post-deployment psychopathology. To our knowledge, this is the first study to prospectively demonstrate trauma-related increases in GR-1 F methylation, and it shows that only increases at specific functionally relevant sites predispose for post-deployment psychopathology.

Purpose: We sought to identify predictive biomarkers for a novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitor. Experimental Design: We use a NAMPT inhibitor, GNE-617, to evaluate nicotinic acid rescue status in a panel of more than 400 cancer cell lines. Using correlative analysis and RNA interference (RNAi), we identify a specific biomarker for nicotinic acid rescue status. We next determine the mechanism of regulation of expression of the biomarker. Finally, we develop immunohistochemical (IHC) and DNA methylation assays and evaluate cancer tissue for prevalence of the biomarker across indications. Results: Nicotinate phosphoribosyltransferase (NAPRT1) is necessary for nicotinic acid rescue and its expression is the major determinant of rescue status. We demonstrate that NAPRT1 promoter methylation accounts for NAPRT1 deficiency in cancer cells, and NAPRT1 methylation is predictive of rescue status in cancer cell lines. Bisulfite next-generation sequencing mapping of the NAPRT1 promoter identified tumor-specific sites of NAPRT1 DNA methylation and enabled the development of a quantitative methylation-specific PCR (QMSP) assay suitable for use on archival formalin-fixed paraffin-embedded tumor tissue. Conclusions: Tumor-specific promoter hypermethylation of NAPRT1 inactivates one of two NAD salvage pathways, resulting in synthetic lethality with the coadministration of a NAMPT inhibitor. NAPRT1 expression is lost due to promoter hypermethylation in most cancer types evaluated at frequencies ranging from 5% to 65%. NAPRT1-specific immunohistochemical or DNA methylation assays can be used on archival formalin paraffin-embedded cancer tissue to identify patients likely to benefit from coadministration of a Nampt inhibitor and nicotinic acid. Clin Cancer Res; 19(24); 6912-23. Ó2013 AACR.

Purpose: We sought to identify predictive biomarkers for a novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitor.Experimental Design: We use a NAMPT inhibitor, GNE-617, to evaluate nicotinic acid rescue status in a panel of more than 400 cancer cell lines. Using correlative analysis and RNA interference (RNAi), we identify a specific biomarker for nicotinic acid rescue status. We next determine the mechanism of regulation of expression of the biomarker. Finally, we develop immunohistochemical (IHC) and DNA methylation assays and evaluate cancer tissue for prevalence of the biomarker across indications.Results: Nicotinate phosphoribosyltransferase (NAPRT1) is necessary for nicotinic acid rescue and its expression is the major determinant of rescue status. We demonstrate that NAPRT1 promoter methylation accounts for NAPRT1 deficiency in cancer cells, and NAPRT1 methylation is predictive of rescue status in cancer cell lines. Bisulfite next-generation sequencing mapping of the...

Identifying biomarkers in body fluids may improve the noninvasive detection of colorectal cancer. Previously, we identified N-Myc downstream-regulated gene 4 (NDRG4) and GATA binding protein 5 (GATA5) methylation as promising biomarkers for colorectal cancer in stool DNA. Here, we examined the utility of NDRG4, GATA5, and two additional markers [Forkhead box protein E1 (FOXE1) and spectrin repeat containing nuclear envelope 1 (SYNE1)] promoter methylation as biomarkers in plasma DNA. Quantitative methylation-specific PCR was performed on plasma DNA from 220 patients with colorectal cancer and 684 noncancer controls, divided in a training set and a test set. Receiver operating characteristic analysis was performed to measure the area under the curve of GATA5, NDRG4, SYNE1, and FOXE1 methylation. Functional assays were performed in SYNE1 and FOXE1 stably transfected cell lines. The sensitivity of NDRG4, GATA5, FOXE1, and SYNE1 methylation in all stages of colorectal cancer (154 cases,...

Purpose: Epigenetic host cell changes involved in cervical cancer development following a persistent high-risk human papillomavirus (hrHPV) infection, provide promising markers for the management of hrHPV-positive women. In particular, markers based on DNA methylation of tumor suppressor gene promoters are valuable. These markers ideally identify hrHPV-positive women with precancer (CIN2/3) in need of treatment. Here, we set out to identify biologically relevant methylation markers by genomewide methylation analysis of both hrHPV-transformed cell lines and cervical tissue specimens. Experimental Design and Results: Genome-wide discovery by next-generation sequencing (NGS) of methyl-binding domainenriched DNA (MBD-Seq) yielded 20 candidate methylation target genes. Further verification and validation by multiplextargeted bisulfite NGS and (quantitative) methylation-specific PCR (MSP) resulted in 3 genes (GHSR, SST, and ZIC1) that showed a significant increase in methylation with severity of disease in both tissue specimens and cervical scrapes (P < 0.005). The area under the ROC curve for CIN3 or worse varied between 0.86 and 0.89. Within the group of CIN2/3, methylation levels of all 3 genes increased with duration of lesion existence (P < 0.0005), characterized by duration of preceding hrHPV infection, and were significantly higher in the presence of a 3q gain (P < 0.05) in the corresponding tissue biopsy. Conclusions: By unbiased genome-wide DNA methylation profiling and comprehensive stepwise verification and validation studies using in vitro and patient-derived samples, we identified 3 promising methylation markers (GHSR, SST, and ZIC1) associated with a 3q gain for the detection of cervical (pre)cancer.

The role of gene-specific methylation in white blood cells (WBC) as a marker of breast cancer risk is currently unclear. We determined whether promoter hypermethylation in blood DNA of candidate tumor suppressor genes frequently methylated in breast tumors can be used as a surrogate biomarker for breast cancer risk. Promoter methylation of BRCA1, CDH1 and RARβ was analyzed in WBC DNA from a population-based sample of 1,021 breast cancer patients and 1,036 controls by the MethyLight assay. Gene-specific promoter methylation in the DNA of 569 tumor tissue samples was also analyzed to determine the correlation of methylation levels with blood from the same individual. Hypermethylation of BRCA1 (OR: 1.31; 95% CI: 0.98-1.75) in WBC was associated with an increased risk of breast cancer when positive methylation was defined as ≥0.1% methylated. There was lack of concordance between tumor tissue and paired WBC DNA methylation. These results provide limited support that hypermethylation of BRCA1 in WBC DNA may be useful for determination of breast cancer risk. Additional studies with larger numbers of genes are needed to fully understand the relationship between WBC methylation and breast cancer risk.

Background Lung cancer is the major cause of cancer-related deaths worldwide. Differential diagnosis can be difficult, especially when only small samples are available. Epigenetic changes are frequently tissue-specific events in carcinogenesis and hence may serve as diagnostic biomarkers.

Background: Cancer cells evolve and constitute heterogeneous populations that fluctuate in space and time and are subjected to selection generating intratumor heterogeneity. This phenomenon is determined by the acquisition of genetic/epigenetic alterations and their selection over time which has clinical implications on drug resistance. Methods: DNA extracted from different tumor cell populations (breast carcinomas, cancer cell lines and cellular clones) were analyzed by MS-MLPA. Methylation profiles were used to generate a heterogeneity index to quantify the magnitude of epigenetic heterogeneity in these populations. Cellular clones were obtained from single cells derived of MDA-MB 231 cancer cell lines applying serial limiting dilution method and morphology was analyzed by optical microscopy and flow cytometry. Clones characteristics were examined through cellular proliferation, migration capacity and apoptosis. Heterogeneity index was also calculated from beta values derived from methylation profiles of TCGA tumors. Results: The study of methylation profiles of 23 fresh breast carcinomas revealed heterogeneous allele populations in these tumor pieces. With the purpose to measure the magnitude of epigenetic heterogeneity, we developed an heterogeneity index based on methylation information and observed that all tumors present their own heterogeneity level. Applying the index calculation in pure cancer cell populations such as cancer cell lines (MDA-MB 231, MCF-7, T47D, HeLa and K-562), we also observed epigenetic heterogeneity. In addition, we detected that clones obtained from the MDA-MB 231 cancer cell line generated their own new heterogeneity over time. Using TCGA tumors, we determined that the heterogeneity index correlated with prognostic and predictive factors like tumor size (p = 0.0088), number of affected axillary nodes (p = 0.007), estrogen receptor expression (p < 0.0001) and HER2 positivity (p = 0.0007). When we analyzed molecular subtypes we found that they presented different heterogeneity levels. Interestingly, we also observed that all mentioned tumor cell populations shared a similar Heterogeneity index (HI) mean. Conclusions: Our results show that each tumor presents a unique epigenetic heterogeneity level, which is associated with prognostic and predictive factors. We also observe that breast tumor subtypes differ in terms of epigenetic heterogeneity, which could serve as a new contribution to understand the different prognosis of these groups.

Purpose: Conventional chemotherapy has modest efficacy in advanced adenoid cystic carcinomas (ACC). Tumor recurrence is a major challenge in the management of ACC patients. Here, we evaluated the antitumor effect of a novel small-molecule inhibitor of the MDM2-p53 interaction (MI-773) combined with cisplatin in patient-derived xenograft (PDX) ACC tumors. Experimental Design: Therapeutic strategies with MI-773 and/or cisplatin were evaluated in SCID mice harboring PDX ACC tumors (UM-PDX-HACC-5) and in low passage primary human ACC cells (UM-HACC-2A,-2B,-5,-6) in vitro. The effect of therapy on the fraction of cancer stem cells (CSC) was determined by flow cytometry for ALDH activity and CD44 expression. Results: Combined therapy with MI-773 with cisplatin caused p53 activation, induction of apoptosis, and regression of ACC PDX tumors. Western blots revealed induction of MDM2, p53 and downstream p21 expression, and regulation of apoptosis-related proteins PUMA, BAX, Bcl-2, Bcl-x L , and active caspase-9 upon MI-773 treatment. Both single-agent MI-773 and MI-773 combined with cisplatin decreased the fraction of CSCs in PDX ACC tumors. Notably, neoadjuvant MI-773 and surgery eliminated tumor recurrences during a postsurgical follow-up of more than 300 days. In contrast, 62.5% of mice that received vehicle control presented with palpable tumor recurrences within this time period (P ¼ 0.0097). Conclusions: Collectively, these data demonstrate that therapeutic inhibition of MDM2-p53 interaction by MI-773 decreased the CSC fraction, sensitized ACC xenograft tumors to cisplatin, and eliminated tumor recurrence. These results suggest that patients with ACC might benefit from the therapeutic inhibition of the MDM2-p53 interaction. Clin Cancer Res; 23(4); 1036-48. Ó2016 AACR.

Germline mutations in BRCA1/2 predispose individuals to breast cancer (termed germline-mutated BRCA1/2 breast cancer, gBRCA-BC) by impairing homologous recombination (HR) and causing genomic instability. HR also repairs DNA lesions caused by platinum agents and PARP inhibitors. Triple-negative breast cancers (TNBCs) harbor subpopulations with BRCA1/2 mutations, hypothesized to be especially platinum-sensitive. Cancers in putative &#39;BRCAness&#39; subgroups-tumors with BRCA1 methylation; low levels of BRCA1 mRNA (BRCA1 mRNA-low); or mutational signatures for HR deficiency and those with basal phenotypes-may also be sensitive to platinum. We assessed the efficacy of carboplatin and another mechanistically distinct therapy, docetaxel, in a phase 3 trial in subjects with unselected advanced TNBC. A prespecified protocol enabled biomarker-treatment interaction analyses in gBRCA-BC and BRCAness subgroups. The primary endpoint was objective response rate (ORR). In the unselected populati...

Background Lung cancer is the major cause of cancer-related deaths worldwide. Differential diagnosis can be difficult, especially when only small samples are available. Epigenetic changes are frequently tissue-specific events in carcinogenesis and hence may serve as diagnostic biomarkers.

In addition to genetic changes, the occurrence of epigenetic alterations is associated with accumulation of both genetic and epigenetic events that promote the development and progression of human cancer. Previously, we reported a set of candidate genes that comprise part of the emerging ''cancer methylome''. In the present study, we first tested 23 candidate genes for promoter methylation in a small number of primary colon tumor tissues and controls. Based on these results, we then examined the methylation frequency of Oncostatin M receptor-b (OSMR) in a larger number of tissue and stool DNA samples collected from colon cancer patients and controls. We found that OSMR was frequently methylated in primary colon cancer tissues (80%, 80/100), but not in normal tissues (4%, 4/100). Methylation of OSMR was also detected in stool DNA from colorectal cancer patients (38%, 26/69) (cut-off in TaqMan-MSP, 4). Detection of other methylated markers in stool DNA improved sensitivity with little effect on specificity. Promoter methylation mediated silencing of OSMR in cell lines, and CRC cells with low OSMR expression were resistant to growth inhibition by Oncostatin M. Our data provide a biologic rationale for silencing of OSMR in colon cancer progression and highlight a new therapeutic target in this disease. Moreover, detection and quantification of OSMR promoter methylation in fecal DNA is a highly specific diagnostic biomarker for CRC.

Background Glioblastoma is associated with a poor prognosis in the elderly. Survival has been shown to increase among patients 70 years of age or younger when temozolomide chemotherapy is added to standard radiotherapy (60 Gy over a period of 6 weeks). In elderly patients, more convenient shorter courses of radiotherapy are commonly used, but the benefit of adding temozolomide to a shorter course of radiotherapy is unknown. Methods We conducted a trial involving patients 65 years of age or older with newly diagnosed glioblastoma. Patients were randomly assigned to receive either radiotherapy alone (40 Gy in 15 fractions) or radiotherapy with concomitant and adjuvant temozolomide. Results A total of 562 patients underwent randomization, 281 to each group. The median age was 73 years (range, 65 to 90). The median overall survival was longer with radiotherapy plus temozolomide than with radiotherapy alone (9.3 months vs. 7.6 months; hazard ratio for death, 0.67; 95% confidence interval...

N-methyl-D-aspartate receptors (NMDARs) are the predominant excitatory neurotransmitter receptors in the mammalian brain. We found that among the three NMDARs examined (NMDAR1, NMDAR2A, NMDAR2B), only NMDAR2A was silenced in colorectal carcinoma (CRC) cell lines at basal line and reactivated by the demethylating agent, 5-aza-2 0deoxycytidine. NMDAR2A was expressed in normal colon epithelium, while expression was hardly detectable in colon cancer tissues. Promoter methylation of NMDAR2A was confirmed by bisulfite sequencing and combined bisulfite restriction analysis in the CRC cell lines and primary tumors. Quantitative methylation-specific PCR demonstrated NMDAR2A promoter hypermethylation in 82 of 100 primary human CRC, 15 of 100 normal corresponding epithelial tissues and 1 of 11 (9%) normal colon mucosa samples obtained from patients without cancer. Moreover, forced expression of full-length NMDAR2A in CRC cell lines induced apoptosis and almost abolished the ability of the cells to form colonies in culture, while NMDAR2A knockdown increased cell growth. Thus, NMDAR2A is commonly hypermethylated in primary human CRC and possesses tumor-suppressive activity.

The aim of this study was to identity in silico the relationships among microRNAs (miRNAs) and genes encoding transcription factors, ubiquitylation, DNA methylation, and histone modifications in systemic lupus erythematosus (SLE). To identify miRNA dysregulation in SLE, we used miR2Disease and PhenomiR for information about miRNAs exhibiting differential regulation in disease and other biological processes, and HMDD for information about experimentally supported human miRNA-disease association data from genetics, epigenetics, circulating miRNAs, and miRNA-target interactions. This information was incorporated into the miRNA analysis. High-throughput sequencing revealed circulating miRNAs associated with kidney damage in patients with SLE. As the main finding of our in silico analysis of miRNAs differentially expressed in SLE and their interactions with disease-susceptibility genes, post-translational modifications, and transcription factors; we highlight 226 miRNAs associated with genes and processes. Moreover, we highlight that alterations of miRNAs such as hsa-miR-30a-5p, hsa-miR-16-5p, hsa-miR-142-5p, and hsa-miR-324-3p are most commonly associated with post-translational modifications. In addition, altered miRNAs that are most frequently associated with susceptibility-related genes are hsa-miR-16-5p, hsa-miR-374a-5p, hsa-miR-34a-5p, hsa-miR-31-5p, and hsa-miR-1-3p.

A cancer biomarker refers to a substance or process that is indicative of the presence of cancer it may be a molecule secreted by a tumor or a specific response of the body to the presence of cancer. Background: The aim of the present study is to investigate the feasibility and potential of the molecules Cytokeratin 20, Cytokeratin 17, Cytokeratin 10 and Anillin as tumor specific markers to rapidly detect cancer. Materials and Methods: The expression of biomarkers was analyzed by preparing RNA and synthesized cDNA. Specific primers were synthesized for the biomarkers Cytokeratin 10, Cytokeratin 20 Cytokeratin 17 and Anillin and semi quantitative gene expression was carried out using mMLuv reverse transcriptase. Results: All the three biomarkers showed an enhanced expression compared with normal fibroblasts while the expression of cytokeratin 10 did not show much expression in HCT116 while Cytokeratin 20 and Anillin showed better expression in both cell lines. Conclusion: All the bio...

Germline mutations in BRCA1/2 predispose individuals to breast cancer (termed germline-mutated BRCA1/2 breast cancer, gBRCA-BC) by impairing homologous recombination (HR) and causing genomic instability. HR also repairs DNA lesions caused by platinum agents and PARP inhibitors. Triple-negative breast cancers (TNBCs) harbor subpopulations with BRCA1/2 mutations, hypothesized to be especially platinum-sensitive. Cancers in putative &#39;BRCAness&#39; subgroups-tumors with BRCA1 methylation; low levels of BRCA1 mRNA (BRCA1 mRNA-low); or mutational signatures for HR deficiency and those with basal phenotypes-may also be sensitive to platinum. We assessed the efficacy of carboplatin and another mechanistically distinct therapy, docetaxel, in a phase 3 trial in subjects with unselected advanced TNBC. A prespecified protocol enabled biomarker-treatment interaction analyses in gBRCA-BC and BRCAness subgroups. The primary endpoint was objective response rate (ORR). In the unselected populati...

Poly(ADP-ribose) polymerase (PARP) is involved in DNA repair and chromatin regulation. 5-Aza-2′-deoxycytidine (5-aza-dC) inhibits DNA methyltransferases, induces hypomethylation, blocks DNA replication, and causes DNA single strand breaks (SSBs). As the PARP inhibitor is expected to affect both DNA repair and transcriptional regulations, we investigated the effect of combinational use of PARP inhibitors on cytotoxicity of 5-aza-dC in human cancer cell lines. The combinational treatment of 5-aza-dC and PARP inhibitor PJ-34 exhibited a stronger cytotoxicity compared with their treatment alone in blood cancer HL-60, U937, and colon cancer HCT116 and RKO cells. Treatment with 5-aza-dC but not PJ-34 caused SSBs in HCT116 cell lines. Global genome DNA demethylation was observed after treatment with 5-aza-dC but not with PJ-34. Notably, in microarray analysis, combinational treatment with PJ-34 and 5-aza-dC caused dissimilar broad changes in gene expression profiles compared with their sin...

Background Glioblastoma is associated with a poor prognosis in the elderly. Survival has been shown to increase among patients 70 years of age or younger when temozolomide chemotherapy is added to standard radiotherapy (60 Gy over a period of 6 weeks). In elderly patients, more convenient shorter courses of radiotherapy are commonly used, but the benefit of adding temozolomide to a shorter course of radiotherapy is unknown. Methods We conducted a trial involving patients 65 years of age or older with newly diagnosed glioblastoma. Patients were randomly assigned to receive either radiotherapy alone (40 Gy in 15 fractions) or radiotherapy with concomitant and adjuvant temozolomide. Results A total of 562 patients underwent randomization, 281 to each group. The median age was 73 years (range, 65 to 90). The median overall survival was longer with radiotherapy plus temozolomide than with radiotherapy alone (9.3 months vs. 7.6 months; hazard ratio for death, 0.67; 95% confidence interval...

Background: Even though coexistence of multiple phenotypes sharing the same genomic background is interesting, it remains incompletely understood. Epigenomic profiles may represent key factors, with unknown contributions to the development of multiple phenotypes, and social-insect castes are a good model for elucidation of the underlying mechanisms. Nonetheless, previous studies have failed to identify genes associated with aberrant gene expression and methylation profiles because of the lack of suitable methodology that can address this problem properly. Methods: A recently proposed principal component analysis (PCA)-based and tensor decomposition (TD)-based unsupervised feature extraction (FE) can solve this problem because these two approaches can deal with gene expression and methylation profiles even when a small number of samples is available. Results: PCA-based and TD-based unsupervised FE methods were applied to the analysis of gene expression and methylation profiles in the brains of two social insects, Polistes canadensis and Dinoponera quadriceps. Genes associated with differential expression and methylation between castes were identified, and analysis of enrichment of Gene Ontology terms confirmed reliability of the obtained sets of genes from the biological standpoint. Conclusions: Biologically relevant genes, shown to be associated with significant differential gene expression and methylation between castes, were identified here for the first time. The identification of these genes may help understand the mechanisms underlying epigenetic control of development of multiple phenotypes under the same genomic conditions.

Dengue haemorrhagic fever (DHF) sometimes occurs after recovery from the disease caused by Dengue virus (DENV), and is often fatal. However, the mechanism of DHF has not been determined, possibly because no suitable methodologies are available to analyse this disease. Therefore, more innovative methods are required to analyse the gene expression profiles of DENV-infected patients. Principal components analysis (PCA)-based unsupervised feature extraction (FE) was applied to the gene expression profiles of DENV-infected patients, and an integrated analysis of two independent data sets identified 46 genes as critical for DHF progression. PCA using only these 46 genes rendered the two data sets highly consistent. The application of PCA to the 46 genes of an independent third data set successfully predicted the progression of DHF. A fourth in vitro data set confirmed the identification of the 46 genes. These 46 genes included interferon- and heme-biosynthesis-related genes. The former ar...

Background: Transgenerational epigenetics (TGE) are currently considered important in disease, but the mechanisms involved are not yet fully understood. TGE abnormalities expected to cause disease are likely to be initiated during development and to be mediated by aberrant gene expression associated with aberrant promoter methylation that is heritable between generations. However, because methylation is removed and then reestablished during development, it is not easy to identify promoter methylation abnormalities by comparing normal lineages with those expected to exhibit TGE abnormalities. Methods: This study applied the recently proposed principal component analysis (PCA)-based unsupervised feature extraction to previously reported and publically available gene expression/promoter methylation profiles of rat primordial germ cells, between E13 and E16 of the F3 generation vinclozolin lineage that are expected to exhibit TGE abnormalities, to identify multiple genes that exhibited aberrant gene expression/promoter methylation during development. Results: The biological feasibility of the identified genes were tested via enrichment analyses of various biological concepts including pathway analysis, gene ontology terms and protein-protein interactions. All validations suggested superiority of the proposed method over three conventional and popular supervised methods that employed t test, limma and significance analysis of microarrays, respectively. The identified genes were globally related to tumors, the prostate, kidney, testis and the immune system and were previously reported to be related to various diseases caused by TGE. Conclusions: Among the genes reported by PCA-based unsupervised feature extraction, we propose that chemokine signaling pathways and leucine rich repeat proteins are key factors that initiate transgenerational epigenetic-mediated diseases, because multiple genes included in these two categories were identified in this study.

Background: Public domain databases nowadays provide multiple layers of genome-wide data e.g., promoter methylation, mRNA expression, and miRNA expression and should enable integrative modeling of the mechanisms of regulation of gene expression. However, researches along this line were not frequently executed. Results: Here, the public domain dataset of mRNA expression, microRNA (miRNA) expression and promoter methylation patterns in four regions, the frontal cortex, temporal cortex, pons and cerebellum, of human brain were sourced from the National Center for Biotechnology Informations gene expression omnibus, and reanalyzed computationally. A large number of miRNA-mediated regulation of target genes and miRNA-targeting-specific promoter methylation were identified in the six pairwise comparisons among the four brain regions. The miRNA-mediated regulation of target genes was found to be highly correlated with one or both of miRNA-targeting-specific promoter methylation and differential miRNA expression. Genes enriched for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were related to brain function and/or development were found among the target genes of miRNAs whose differential expression patterns were highly correlated with the miRNA-mediated regulation of their target genes. Conclusions: The combinatorial analysis of miRNA-mediated regulation of target genes, miRNA-targeting-specific promoter methylation and differential miRNA expression can help reveal the brain region-specific contributions of miRNAs to brain function and development.

BACKGROUND: There is a strong need to determine the best technique for O 6-methylguanine-DNA-methyltranferase (MGMT) analysis, because MGMT status is currently used in clinical trials and occasionally in routine clinical practice for glioblastoma patients. METHODS: The authors compared analytical performances and predictive values of 5 techniques in a series of 100 glioblastoma patients who received standard of care treatment (Stupp protocol). RESULTS: MGMT promoter was considered methylated in 33%, 33%, 42%, and 60% of patients by methylation-sensitive high-resolution melting, MethyLight, pyrosequencing (with an optimal risk cutoff at 8% for the average percentage of the 5 CpGs tested), and methylation-specific polymerase chain reaction (MS-PCR), respectively. Fifty-nine percent of the samples had <23% (the optimal risk cutoff) of MGMT-positive tumor cells. The best predictive values for overall survival (OS), after adjustment for age and performance status, were obtained by pyrosequencing (hazard ratio [HR], 0.32; P < .0001), MS-PCR (HR, 0.37; P < .0001), and immunohistochemistry (HR, 0.43; P ¼ .0005) as compared with methylation-sensitive high-resolution melting (HR, 0.52 P ¼ .02) and MethyLight (HR, 0.6; P ¼ .05). For progression-free survival (PFS), the best predictive values were obtained with pyrosequencing (HR, 0.35; P < .0001), methylation-sensitive high-resolution melting (HR, 0.46; P ¼ .002), and MS-PCR (HR, 0.49; P ¼ .002). Combining pyrosequencing and immunohistochemistry slightly improved predictive power for OS, but not for PFS. Poor reproducibility and interobserver variability were, however, observed for immunohistochemistry. CONCLUSIONS: Good prediction of survival in addition to high reproducibility and sensitivity made pyrosequencing the best among the 5 techniques tested in this study.

We sought to use PCR followed by high-resolution melting analysis to develop a single closed-tube screening panel to screen for Lynch syndrome. This comprises tests for microsatellite instability (MSI), MLH1 methylation promoter andmutation. For MSI testing, five mononucleotide markers (BAT25, BAT26, BCAT25,,) were developed. In addition, primers were designed to interrogate Region C of the MLH1 promoter for methylation (using bisulphite-modified DNA) and to test for mutations in codon 600 of BRAF. Two separate cohorts from Nottingham (n=99, 46 with MSI, 53 being microsatellite stable (MSS)) and Edinburgh (n=88, 45 MSI, 43 MSS) were tested. All the cases (n=187) were blind tested for MSI and all were correctly characterised by our panel. Thepromoter andwere tested only in the Nottingham cohort. Successful blinded analysis was performed on the MLH1 promoter in 97 cases. All MSS cases showed a pattern of non-methylation while 41/44 cases with MSI showed full methylation. The three cas...

Germline mutations in BRCA1/2 predispose individuals to breast cancer (termed germline-mutated BRCA1/2 breast cancer, gBRCA-BC) by impairing homologous recombination (HR) and causing genomic instability. HR also repairs DNA lesions caused by platinum agents and PARP inhibitors. Triple-negative breast cancers (TNBCs) harbor subpopulations with BRCA1/2 mutations, hypothesized to be especially platinum-sensitive. Cancers in putative &#39;BRCAness&#39; subgroups-tumors with BRCA1 methylation; low levels of BRCA1 mRNA (BRCA1 mRNA-low); or mutational signatures for HR deficiency and those with basal phenotypes-may also be sensitive to platinum. We assessed the efficacy of carboplatin and another mechanistically distinct therapy, docetaxel, in a phase 3 trial in subjects with unselected advanced TNBC. A prespecified protocol enabled biomarker-treatment interaction analyses in gBRCA-BC and BRCAness subgroups. The primary endpoint was objective response rate (ORR). In the unselected populati...

Background: The goal of this prospective multicentric trial was to validate a technique that allowed for MGMT promoter methylation analysis in routine clinical practice. Methods: The MGMT status of 139 glioblastoma patients, whom had received standard first line treatment, was determined using pyrosequencing (PSQ) and a semi-quantitative Methylation-specific PCR (sqMS-PCR) method, using both frozen and formalin-fixed paraffin-embedded FFPE samples. Eight participating centers locally performed the analysis, including external quality controls. Results: There was a strong correlation between results from FFPE and frozen samples. With cutoffs of 12% and 13%, 98% and 91% of samples were identically classified with PSQ and sqMS-PCR respectively. In 12% of cases frozen samples were excluded because they had a low percentage of tumor cells. In 5-6% of cases the analysis was not feasible on FFPE samples. The optimized risk cutoffs were higher in both techniques when using FFPE samples, in comparison to frozen samples. For sqMS-PCR, we validated a cutoff between 13-15% to dichotomize patients. For Oncotarget 2 www.impactjournals.com/oncotarget PSQ, patients with a low level of methylation (<= 8%) had a median progressionfree survival under 9 months, as compared with more than 15.5 months for those with a level above 12%. For intermediate values (9-12%), more discordant results between FFPE and frozen samples were observed and there was not a clear benefit of temozolomide treatment, which indicated a "grey zone". Conclusions: MGMT status can reliably be investigated in local laboratories. PSQ is the ideal choice as proven by strong interlaboratory reproducibility, along with threshold agreements across independent studies.

BACKGROUND: There is a strong need to determine the best technique for O 6-methylguanine-DNA-methyltranferase (MGMT) analysis, because MGMT status is currently used in clinical trials and occasionally in routine clinical practice for glioblastoma patients. METHODS: The authors compared analytical performances and predictive values of 5 techniques in a series of 100 glioblastoma patients who received standard of care treatment (Stupp protocol). RESULTS: MGMT promoter was considered methylated in 33%, 33%, 42%, and 60% of patients by methylation-sensitive high-resolution melting, MethyLight, pyrosequencing (with an optimal risk cutoff at 8% for the average percentage of the 5 CpGs tested), and methylation-specific polymerase chain reaction (MS-PCR), respectively. Fifty-nine percent of the samples had <23% (the optimal risk cutoff) of MGMT-positive tumor cells. The best predictive values for overall survival (OS), after adjustment for age and performance status, were obtained by pyrosequencing (hazard ratio [HR], 0.32; P < .0001), MS-PCR (HR, 0.37; P < .0001), and immunohistochemistry (HR, 0.43; P ¼ .0005) as compared with methylation-sensitive high-resolution melting (HR, 0.52 P ¼ .02) and MethyLight (HR, 0.6; P ¼ .05). For progression-free survival (PFS), the best predictive values were obtained with pyrosequencing (HR, 0.35; P < .0001), methylation-sensitive high-resolution melting (HR, 0.46; P ¼ .002), and MS-PCR (HR, 0.49; P ¼ .002). Combining pyrosequencing and immunohistochemistry slightly improved predictive power for OS, but not for PFS. Poor reproducibility and interobserver variability were, however, observed for immunohistochemistry. CONCLUSIONS: Good prediction of survival in addition to high reproducibility and sensitivity made pyrosequencing the best among the 5 techniques tested in this study.

Iron is an essential nutrient needed for physiological functions, particularly during the developmental period of the early childhood of at-risk populations. The purpose of this study was to investigate, in an experimental colitis, the consequences of daily oral iron ingestion in the early period on the inflammatory response, the spleen T helper (Th) profiles and the associated molecular mechanisms. Juvenile mice orally received microencapsulated ferric iron or water for 6 weeks. On adult mice, we induced a sham or experimental trinitrobenzene sulfonic acid (TNBS) moderate colitis during the last week of the experiment before sacrificing the animals 7 days later. The severity of the gut inflammation was assessed by macroscopic damage scores (MDS) and the myeloperoxidase activity (MPO). Th profiles were evaluated by the examination of the splenic gene expression of key transcription factors of the Th differentiation (Tbet, Gata3, Foxp3 and RORγ) and the methylation of their respectiv...

Background Breast cancer outcome, including response to therapy, risk of metastasis and survival, is difficult to predict using currently available methods, highlighting the urgent need for more informative biomarkers. Androgen receptor (AR) has been implicated in breast carcinogenesis however its potential to be an informative biomarker has yet to be fully explored. In this study, AR protein levels were determined in a cohort of 73 Grade III invasive breast ductal adenocarcinomas. Methods The levels of Androgen receptor protein in a cohort of breast tumour samples was determined by immunohistochemistry and the results were compared with clinical characteristics, including survival. The role of defects in the regulation of Androgen receptor gene expression were examined by mutation and methylation screening of the 5&#39; end of the gene, reporter assays of the 5&#39; and 3&#39; end of the AR gene, and searching for miRNAs that may regulate AR gene expression. Results AR was expresse...

The aquaporins represent a family of transmembrane water channel proteins that play a major role in transcellular and transepithelial water movement. Most tumors have been shown to exhibit high vascular permeability and interstitial fluid pressure, but the transport pathways for water within tumors remain unknown. Here, we tested 10 non-small cell lung cancer cell lines of various origins by reverse transcriptase-polymerase chain reaction and Western blot analysis and identified clear expression of aquaporin 1 (AQP1) in seven cell lines. We next examined the distribution of the AQP1 protein in several types of primary lung tumors (16 squamous cell carcinomas, 21 adenocarcinomas, and 7 bronchoalveolar carcinomas) by immunohistochemical staining. AQP1 was overexpressed in 62% (13 of 21) and 75% (6 of 8) of adenocarcinoma and bronchoalveolar carcinoma, respectively, whereas all cases of squamous cell carcinoma and normal lung tissue were negative. Forced expression of full-length AQP1 cDNA in NIH-3T3 cells induced many phenotypic changes characteristic of transformation, including cell proliferation-enhancing activity by the MTT assay and anchorage-independent growth in soft agar. Although further details on the molecular function of AQP1 related to tumorigen-esis remain to be elucidated, our results suggest a potential role of AQP1 as a novel therapeutic target for the management of lung cancer.

Background Homeobox (HOX) genes deregulation has been largely implicated in the development of human leukemia. Among the HOXB cluster, HOXB1 was silent in a number of analyzed acute myeloid leukemia (AML) primary cells and cell lines, whereas it was expressed in normal terminally differentiated peripheral blood cells. Methods We evaluated the biological effects and the transcriptome changes determined by the retroviral transduction of HOXB1 in the human promyelocytic cell line HL60. Results Our results suggest that the enforced expression of HOXB1 reduces cell growth proliferation, inducing apoptosis and cell differentiation along the monocytic and granulocytic lineages. Accordingly, gene expression analysis showed the HOXB1-dependent down-regulation of some tumor promoting genes, paralleled by the up-regulation of apoptosis- and differentiation-related genes, thus supporting a tumor suppressor role for HOXB1 in AML. Finally, we indicated HOXB1 promoter hypermethylation as a mechani...

Recent reports indicate that the alterations in the p16 and pRb pathways can influence tumour progression and poor prognosis in several tumours. The objective of this study was to analyse p16 and pRb expression in161 patients with malignant fibrous histiocytomas (MFH). By immunohistochemistry, p16 and pRb were demonstrated in 25% and 56% of MFH, respectively. Cox regression analysis demonstrated an independent prognostic influence of both genes. Generally, the loss of p16 and pRb expression correlated with poorer prognosis. Promoter methylation of p16 was found in 16/42 of p16 negative MFH and of pRb in 2/42 of pRb-negative MFH. It can be concluded that p16 and pRb alterations play an important role in the progression of soft tissue sarcomas.

Poly(ADP-ribose) polymerase (PARP) is involved in DNA repair and chromatin regulation. 5-Aza-2′-deoxycytidine (5-aza-dC) inhibits DNA methyltransferases, induces hypomethylation, blocks DNA replication, and causes DNA single strand breaks (SSBs). As the PARP inhibitor is expected to affect both DNA repair and transcriptional regulations, we investigated the effect of combinational use of PARP inhibitors on cytotoxicity of 5-aza-dC in human cancer cell lines. The combinational treatment of 5-aza-dC and PARP inhibitor PJ-34 exhibited a stronger cytotoxicity compared with their treatment alone in blood cancer HL-60, U937, and colon cancer HCT116 and RKO cells. Treatment with 5-aza-dC but not PJ-34 caused SSBs in HCT116 cell lines. Global genome DNA demethylation was observed after treatment with 5-aza-dC but not with PJ-34. Notably, in microarray analysis, combinational treatment with PJ-34 and 5-aza-dC caused dissimilar broad changes in gene expression profiles compared with their sin...

Background DNA methylation has been shown to be associated with adiposity in adulthood. However, whether similar DNA methylation patterns are associated with childhood and adolescent body mass index (BMI) is largely unknown. More insight into this relationship at younger ages may have implications for future prevention of obesity and its related traits. Methods We examined whether DNA methylation in cord blood and whole blood in childhood and adolescence was associated with BMI in the age range from 2 to 18 years using both cross-sectional and longitudinal models. We performed meta-analyses of epigenome-wide association studies including up to 4133 children from 23 studies. We examined the overlap of findings reported in previous studies in children and adults with those in our analyses and calculated enrichment. Results DNA methylation at three CpGs (cg05937453, cg25212453, and cg10040131), each in a different age range, was associated with BMI at Bonferroni significance, P &lt; 1....

TPS619 Background: Triple negative breast cancers (TNBCs) are a biologically diverse and aggressive subgroup lacking targeted therapy. TNBC and germline BRCA (gBRCA) breast cancer share certain phenotypic and molecular similarities, with gBRCA mutations seen in 10% to 20% of TNBC patients. Homologous recombination-deficient tumors, especially those caused by germline or somatic BRCA mutations, are thought to be particularly sensitive to PARP inhibitors. Methods: This is a 3-stage open-label randomized phase II/III trial of neoadjuvant paclitaxel and carboplatin +/- olaparib, followed by clinicians&#39; choice of anthracycline regimen. The aim is to establish whether the addition of olaparib to neoadjuvant platinum-based chemotherapy in the treatment of basal TNBC and/or gBRCA breast cancer is safe and increases efficacy. In stages 1 and 2, all patients receive 4 cycles of 3-weekly carboplatin AUC5/weekly paclitaxel 80mg/m2. They are randomly assigned 1:1:1 to a control arm, or to on...

In addition to genetic changes, the occurrence of epigenetic alterations is associated with accumulation of both genetic and epigenetic events that promote the development and progression of human cancer. Previously, we reported a set of candidate genes that comprise part of the emerging ''cancer methylome''. In the present study, we first tested 23 candidate genes for promoter methylation in a small number of primary colon tumor tissues and controls. Based on these results, we then examined the methylation frequency of Oncostatin M receptor-b (OSMR) in a larger number of tissue and stool DNA samples collected from colon cancer patients and controls. We found that OSMR was frequently methylated in primary colon cancer tissues (80%, 80/100), but not in normal tissues (4%, 4/100). Methylation of OSMR was also detected in stool DNA from colorectal cancer patients (38%, 26/69) (cut-off in TaqMan-MSP, 4). Detection of other methylated markers in stool DNA improved sensitivity with little effect on specificity. Promoter methylation mediated silencing of OSMR in cell lines, and CRC cells with low OSMR expression were resistant to growth inhibition by Oncostatin M. Our data provide a biologic rationale for silencing of OSMR in colon cancer progression and highlight a new therapeutic target in this disease. Moreover, detection and quantification of OSMR promoter methylation in fecal DNA is a highly specific diagnostic biomarker for CRC.

Substance abuse is known as a relapsing and chronic disorder. Drug abuse has a significant role in the dopaminergic system in which dopamine is an important neurotransmitter.  Binding dopamine and dopamine receptors could activate major intracellular signalling through protein kinase A (PKA). Activation of intracellular signalling such CREB affect gene expression of some genes. As dopamine and dopaminergic system have an important regulation role in the inhibition of immune system we analysed the effects of drug addiction on the DNA methylation and expression of cytotoxic T-lymphocyte antigen 4 (CTLA-4). We evaluate promoter methylation and mRNA expression of CTLA-4 in individual with drug addiction. DNA was extracted from blood of 74 patients with drug addiction and 59 healthy controls. Methylation of CTLA-4 promoter was assessed using a methylation-specific PCR technique. In addition the expression level of CTLA-4 was also indicated in 17 individuals with drug addiction and 18 peo...