Methylation Markers of Early-Stage Non-Small Cell Lung Cancer

Despite of intense research in early cancer detection, there is a lack of biomarkers for the reliable detection of malignant tumors, including non-small cell lung cancer (NSCLC). DNA methylation changes are common and relatively stable in various types of cancers, and may be used as diagnostic or prognostic biomarkers.We performed DNA methylation profiling of samples from 48 patients with stage I NSCLC and 18 matching cancer-free lung samples using microarrays that cover the promoter regions of more than 14,500 genes. We correlated DNA methylation changes with gene expression levels and performed survival analysis.We observed hypermethylation of 496 CpGs in 379 genes and hypomethylation of 373 CpGs in 335 genes in NSCLC. Compared to adenocarcinoma samples, squamous cell carcinoma samples had 263 CpGs in 223 hypermethylated genes and 513 CpGs in 436 hypomethylated genes. 378 of 869 (43.5%) CpG sites discriminating the NSCLC and control samples showed an inverse correlation between CpG site methylation and gene expression levels. As a result of a survival analysis, we found 10 CpGs in 10 genes, in which the methylation level differs in different survival groups.We have identified a set of genes with altered methylation in NSCLC and found that a minority of them showed an inverse correlation with gene expression levels. We also found a set of genes that associated with the survival of the patients. These newly-identified marker candidates for the molecular screening of NSCLC will need further analysis in order to determine their clinical utility

Genome-wide methylation and transcriptome analysis in penile carcinoma: uncovering new molecular markers

Background: Despite penile carcinoma (PeCa) being a relatively rare neoplasm, it remains an important public health issue for poor and developing countries. Contrary to most tumors, limited data are available for markers that are capable of assisting in diagnosis, prognosis, and treatment of PeCa. We aimed to identify molecular markers for PeCa by evaluating their epigenomic and transcriptome profiles and comparing them with surrounding non-malignant tissue (SNT) and normal glans (NG).Results: Genome-wide methylation analysis revealed 171 hypermethylated probes in PeCa. Transcriptome profiling presented 2,883 underexpressed and 1,378 overexpressed genes. Integrative analysis revealed a panel of 54 genes with an inverse correlation between methylation and gene expression levels. Distinct methylome and transcriptome patterns were found for human papillomavirus (HPV)-positive (38.6%) and negative tumors. Interestingly, grade 3 tumors showed a distinct methylation profile when compared to grade 1. In addition, univariate analysis revealed that low BDNF methylation was associated with lymph node metastasis and shorter disease-free survival. CpG hypermethylation and gene underexpression were confirmed for a panel of genes, including TWIST1, RSOP2, SOX3, SOX17, PROM1, OTX2, HOXA3, and MEIS1.Conclusions: A unique methylome signature was found for PeCa compared to SNT, with aberrant DNA methylation appearing to modulate the expression of specific genes. This study describes new pathways with the potential to regulate penile carcinogenesis, including stem cell regulatory pathways and markers associated to a worse prognosis. These findings may be instrumental in the discovery and application of new genetic and epigenetic biomarkers in PeCa.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Hypermethylation of Tumor Suppressor Genes Involved in Critical Regulatory Pathways for Developing a Blood-Based Test in Breast Cancer

Aberrant DNA methylation patterns might be used as a biomarker for diagnosis and management of cancer patients

Multilayer OMIC data in medullary thyroid carcinoma identifies the STAT3 pathway as a potential therapeutic target in RETM918T tumors

Purpose: Medullary thyroid carcinoma (MTC) is a rare disease with few genetic drivers, and the etiology specific to each known susceptibility mutation remains unknown. Exploiting multilayer genomic data, we focused our interest on the role of aberrant DNA methylation in MTC development.Experimental Design: We performed genome-wide DNA methylation profiling assessing more than 27,000 CpGs in the largest MTC series reported to date, comprising 48 molecularly characterized tumors. mRNA and miRNA expression data were available for 33 and 31 tumors, respectively. Two human MTC cell lines and 101 paraffin-embedded MTCs were used for validation.Results: The most distinctive methylome was observed for RETM918T-related tumors. Integration of methylation data with mRNA and miRNA expression data identified genes negatively regulated by promoter methylation. These in silico findings were confirmed in vitro for PLCB2, DKK4, MMP20, and miR-10a, -30a, and -200c. The mutation-specific aberrant methylation of PLCB2, DKK4, and MMP20 was validated in 25 independent MTCs by bisulfite pyrosequencing. The methylome and transcriptome data underscored JAK/Stat pathway involvement in RETM918T MTCs. Immunostaining [immunohistochemistry (IHC)] for the active form of signaling effector STAT3 was performed in a series of 101 MTCs. As expected, positive IHC was associated with RETM918T-bearing tumors (P < 0.02). Pharmacologic inhibition of STAT3 activity increased the sensitivity to vandetanib of the RETM918T-positive MTC cell line, MZ-CRC-1.Conclusions: Multilayer OMIC data analysis uncovered methylation hallmarks in genetically defined MTCs and revealed JAK/Stat signaling effector STAT3 as a potential therapeutic target for the treatment of RETM918T MTCs.This work was supported by the Fondo de Investigaciones Sanitarias (FIS) project PI14/00240 and the Comunidad de Madrid (Grant S2011/BMD-2328 TIRONET) to MR. LI-P is supported by the Centro de Investigacion Biomédica en Red de Enfermedades Raras (CIBERER). VM was supported by a predoctoral fellowship from the "la Caixa"/CNIO international PhD programme. CM-C is supported by a postdoctoral fellowship from the Fundación AECC

Epigenetic Events Suggest A Distinct Molecular Pathogenesis In Braf-Associated Papillary Thyroid Cancer

EPIGENETIC EVENTS SUGGEST A DISTINCT MOLECULAR PATHOGENESIS IN BRAF-ASSOCIATED PAPILLARY THYROID CANCER. Ogechukwu Eze, Lee Starker, William Long, Robert Udelsman, Peyman Björklund, and Tobias Carling. Department of Surgery, Yale University School of Medicine, New Haven, CT, USA. Papillary thyroid cancer (PTC) associated with a somatic V-raf murine sarcoma viral oncogene homolog B1 (BRAF) V600E mutation has been associated with a more aggressive phenotype (i.e. extrathyroidal extension, lymph node metastasis, high TNM stage and recurrence) than those with wildtype (WT) BRAF. The underlying molecular mechanism for this association is incompletely clarified. Epigenetic alterations, such as DNA methylation participate with genetic abnormalities to cause altered patterns of gene expression and/or function leading to oncogenesis. Using a quantitative, genome-wide approach to evaluate the PTC DNA methylome, we identified a distinct DNA methylation profile in BRAF-associated PTC. This represents the first, unbiased, systematic, quantitative genome-wide evaluation of DNA methylation alterations in PTC. A distinct DNA methylation profile was noted in BRAF V600E PTC versus BRAF WT PTC. Twenty-four genes were significantly hypermethylated in BRAF V600E PTC versus BRAF WT (p\u3c0.005 for all). Genes frequently and significantly hypermethylated in BRAF V600E PTC included those involved in transcriptional regulation and cell cycle control such as CDKN2B/p15, RASSF1A and CD6. 25 BRAF V600E PTC and 25 BRAF WT PTC were used for validation of the methylation profile of RASSF1 and analysis of its gene expression. One of two CpG islands in the RASSF1A promoter was differentially methylated with an average of 73% and 8% methylation in BRAF V600E PTC and BRAF WT PTC respectively. In addition, 92% (n=23) of the BRAF V600E tumors were hypermethylated at this CpG island, compared to 12% (n=3) of the BRAF WT tumors. Expression of RASSF1A was decreased in BRAF V600E PTC relative to expression in BRAF WT PTC. The DNA methylation profile of PTC correlates with BRAF mutational status, underscoring its importance in PTC development. The molecular mechanism of the more aggressive phenotype of BRAF V600E PTC may be related to aberrant DNA methylation of genes involved in transcriptional and cell cycle control. Functional studies assessing the effect of azanucleoside, a demethylating agent on RASSF1A gene expression may further determine a cause-effect relationship. Furthermore, conducting such functional experiments in BRAF V600E PTC and BRAF WT PTC cell cultures would be critical in assessing the possibility of clinically applying a demethylating agent to treatment of aggressive, BRAF V600E PTC

Discovery of DNA methylation markers in cervical cancer using relaxation ranking

<p>Abstract</p> <p>Background</p> <p>To discover cancer specific DNA methylation markers, large-scale screening methods are widely used. The pharmacological unmasking expression microarray approach is an elegant method to enrich for genes that are silenced and re-expressed during functional reversal of DNA methylation upon treatment with demethylation agents. However, such experiments are performed in <it>in vitro </it>(cancer) cell lines, mostly with poor relevance when extrapolating to primary cancers. To overcome this problem, we incorporated data from primary cancer samples in the experimental design. A strategy to combine and rank data from these different data sources is essential to minimize the experimental work in the validation steps.</p> <p>Aim</p> <p>To apply a new relaxation ranking algorithm to enrich DNA methylation markers in cervical cancer.</p> <p>Results</p> <p>The application of a new sorting methodology allowed us to sort high-throughput microarray data from both cervical cancer cell lines and primary cervical cancer samples. The performance of the sorting was analyzed <it>in silico</it>. Pathway and gene ontology analysis was performed on the top-selection and gives a strong indication that the ranking methodology is able to enrich towards genes that might be methylated. Terms like regulation of progression through cell cycle, positive regulation of programmed cell death as well as organ development and embryonic development are overrepresented. Combined with the highly enriched number of imprinted and X-chromosome located genes, and increased prevalence of known methylation markers selected from cervical (the highest-ranking known gene is <it>CCNA1</it>) as well as from other cancer types, the use of the ranking algorithm seems to be powerful in enriching towards methylated genes.</p> <p>Verification of the DNA methylation state of the 10 highest-ranking genes revealed that 7/9 (78%) gene promoters showed DNA methylation in cervical carcinomas. Of these 7 genes, 3 (<it>SST</it>, <it>HTRA3 </it>and <it>NPTX1</it>) are not methylated in normal cervix tissue.</p> <p>Conclusion</p> <p>The application of this new relaxation ranking methodology allowed us to significantly enrich towards methylation genes in cancer. This enrichment is both shown <it>in silico </it>and by experimental validation, and revealed novel methylation markers as proof-of-concept that might be useful in early cancer detection in cervical scrapings.</p

Integrated Epigenetics of Human Breast Cancer: Synoptic Investigation of Targeted Genes, MicroRNAs and Proteins upon Demethylation Treatment

The contribution of aberrant DNA methylation in silencing of tumor suppressor genes (TSGs) and microRNAs has been investigated. Since these epigenetic alterations are reversible, it became of interest to determine the effects of the 5-aza-2'-deoxycytidine (DAC) demethylation therapy in breast cancer at different molecular levels

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type–independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors