Methylation Profile of Several Tumor Suppressor Genes in Non-Small-Cell Lung Cancer

Multiplex methylation-sensitive PCR was employed in studying the methylation of CpG islands in the RB1, p16/CDKN2A, p15/CDKN2B, p14/ARF, CDH1, HIC1, and N33 5 regions in non-small cell lung cancer (51 tumors). Methylation was observed for the two suppressor genes involved in controlling the cell cycle through the Cdk–Rb–E2F signaling pathway, RB1 (10/51, 19%) and p16 (20/51, 39%). The highest methylation frequencies were established for CDH1 (72%) and HIC1 (82%). The CpG islands of p14 and p15 proved to be nonmethylated. At least one gene was methylated in 90% (46/51) tumors and no gene, in 10% (5/51) tumors. In addition, the genes were tested for methylation in peripheral blood lymphocytes of healthy subjects. Methylation frequency significantly differed between tumors and normal cells in the case of RB1, p16, CDH1, HIC1, and N33. Gene methylation frequency was tested for association with histological type of the tumor and stage of tumor progression. Methylation index of a panel of tumor suppressor genes was established for groups of tumors varying in clinical and morphological parameters.     

Magnesium deficiency in pregnant rats alters methylation of specific cytosines in the hepatic hydroxysteroid dehydrogenase-2 promoter of the offspring

Prenatal under-nutrition involves changes in the epigenetic regulation of specific genes. Maternal magnesium (Mg) deficiency affects maternal glucocorticoid metabolism, but the mechanisms underlying changes in glucocorticoid homeostasis of offspring are not well understood. In this study, we investigated the effects of feeding pregnant rats a Mg-deficient diet (0.003% magnesium) on the methylation of cytosine-guanine (CpG) dinucleotides in hepatic glucocorticoid genes of neonatal offspring, compared with controls (0.082% magnesium). Methylation of CpG dinucleotides in the peroxisome proliferator-activated receptor α (Ppara), glucocorticoid receptor (Nr3c1) and 11β-hydroxysteroid dehydrogenase-2 (Hsd11b2) promoters in the liver were measured by pyrosequencing. Quantitative real-time PCR was used to assess hepatic mRNA expression of each gene. Mean methylation of the Hsd11b2 promoter in the Mg-deficient offspring (33.2%) was higher than in controls (10.4%). This was due to a specific increase at CpG dinucleotides 1 (20.0% vs. control 10.1%), 2 (58.8% vs. 17.0%), 3 (29.7% vs. 6.2%) and 4 (38.7% vs. 8.8%) (p < 0.05). Ppara and Nr3c1 methylation status and expression did not differ between the groups. No significant difference was noted between male and female pups, which were equally represented. Therefore, a Mg-deficient diet alters glucocorticoid metabolism, predicting higher hepatic intracellular glucocorticoid concentrations, and is possibly a key mechanism that induces the metabolic complications of Mg deficiency.     

Genome-wide methylation profiling reveals Zinc finger protein 516 (ZNF516) and FK-506-binding protein 6 (FKBP6) promoters frequently methylated in cervical neoplasia,associated with HPV status and ethnicity in a Chilean population

Cervical cancer is a major health concern among women in Latin America due to its high incidence and mortality. Therefore, the discovery of molecular markers for cervical cancer screening and triage is imperative. The aim of this study was to use a genome wide DNA methylation approach to identify novel methylation biomarkers in cervical cancer. DNA from normal cervical mucosa and cervical cancer tissue samples from Chile was enriched with Methylated DNA Immunoprecipitation (MeDIP), hybridized to oligonucleotide methylation microarrays and analyzed with a stringent bioinformatics pipeline to identify differentially methylated regions (DMRs) as candidate biomarkers. Quantitative Methylation Specific PCR (qMSP) was used to study promoter methylation of candidate DMRs in clinical samples from two independent cohorts. HPV detection and genotyping were performed by Reverse Line Blot analysis. Bioinformatics analysis revealed GGTLA4, FKBP6, ZNF516, SAP130, and INTS1 to be differentially methylated in cancer and normal tissues in the Discovery cohort. In the Validation cohort FKBP6 promoter methylation had 73% sensitivity and 80% specificity (AUC = 0.80). ZNF516 promoter methylation was the best biomarker, with both sensitivity and specificity of 90% (AUC = 0.92), results subsequently corroborated in a Prevalence cohort. Together, ZNF516 and FKBP6 exhibited a sensitivity of 84% and specificity of 81%, when considering both cohorts. Our genome wide DNA methylation assessment approach (MeDIP-chip) successfully identified novel biomarkers that differentiate between cervical cancer and normal samples, after adjusting for age and HPV status. These biomarkers need to be further explored in case-control and prospective cohorts to validate them as cervical cancer biomarkers.     

Aberrant Keap1 methylation in breast cancer and association with clinicopathological features

Keap1 (Kelch-like ECH-associated protein 1) is an adaptor protein that mediates the ubiquitination/degradation of genes regulating cell survival and apoptosis under oxidative stress conditions. We determined methylation status of the KEAP1 promoter in 102 primary breast cancers, 14 pre-invasive lesions, 38 paired normal breast tissues and 6 normal breast from reductive mammoplasty by quantitative methylation specific PCR (QMSP). Aberrant promoter methylation was detected in 52 out of the 102 primary breast cancer cases (51%) and 10 out of 14 pre-invasive lesions (71%). No mutations of the KEAP1 gene were identified in the 20 breast cancer cases analyzed by fluorescence based direct sequencing. Methylation was more frequent in the subgroup of patients identified as ER positive-HER2 negative tumors (66.7%) as compared with triple-negative breast cancers (35%) (p = 0.05, Chi-square test). The impact of the interactions between Er, PgR, Her2 expression and KEAP1 methylation on mortality was investigated by RECPAM multivariable statistical analysis, identifying four prognostic classes at different mortality risks. Triple-negative breast cancer patients with KEAP1 methylation had higher mortality risk than patients without triple-negative breast cancer (HR = 14.73, 95%CI: 3.65–59.37). Both univariable and multivariable COX regressions analyses showed that KEAP1 methylation was associated with a better progression free survival in patients treated with epirubicin/cyclophosfamide and docetaxel as sequential chemotherapy (HR = 0.082; 95%CI: 0.007–0.934). These results indicate that aberrant promoter methylation of the KEAP1 gene is involved in breast cancerogenesis. In addition, identifying patients with KEAP1 epigenetic abnormalities may contribute to disease progression prediction in breast cancer patients.     

The 3p14.2 tumour suppressor ADAMTS9 is inactivated by promoter CpG methylation and inhibits tumour cell growth in breast cancer

Chromosome region 3p12‐14 is an important tumour suppressor gene (TSG) locus for multiple cancers. ADAMTS9, a member of the metalloprotease large family, has been identified as a candidate 3p14.2 TSG inactivated by aberrant promoter CpG methylation in several carcinomas, but little known about its expression and function in breast cancer. In this report, ADAMTS9 expression and methylation was analysed in breast cancer cell lines and tissue samples. ADAMTS9 RNA was significantly down‐regulated in breast cancer cell lines (6/8). After treating the cells with demethylation agent Aza and TSA, ADAMTS9 expression was dramatically increased. Bisulphite genomic sequencing and methylation‐specific PCR detected promoter methylation, which was associated with decreased ADAMTS9 expression. Hypermethylation was also detected in 130/219 (59.4%) of primary tumours but only in 4.5% (2/44) of paired surgical margin tissues. Ectopic expression of ADAMTS9 in tumor cells induced significant growth suppression, cell cycle arrest at the G0/G1 phase, enhanced apoptosis and reduced cell migration and invasion. Conditioned culture medium from ADAMTS9‐transfected BT549 cells markedly disrupted tube formation ability of human umbilical vein endothelial cell (HUVEC) in Matrigel. Furthermore, ADAMTS9 inhibited AKT signaling and its downstream targets (MDM2, p53, p21, p27, E‐cadherin, VIM, SNAIL, VEGFA, NFκB‐p65 and MMP2). In addition, we demonstrated, for the first time, that ADAMTS9 inhibits AKT signaling, through suppressing its upstream activators EGFR and TGFβ1/TβR(I/II) in breast cancer cells. Our results suggest that ADAMTS9 is a TSG epigenetically inactivated in breast cancer, which functions through blocking EGFR‐ and TGFβ1/TβR(I/II)‐activated AKT signaling.     

Genome-wide DNA methylation profiling of chronic lymphocytic leukemia allows identification of epigenetically repressed molecular pathways with clinical impact

We performed a genome-wide analysis of aberrant DNA methylation in chronic lymphocytic leukemia (CLL) using methylated CpG island amplification (MCA) coupled with a promoter microarray. We identified 280 potential targets of aberrant DNA methylation in CLL. These genes were located more frequently in chromosomes 19 (16%, p=0.001), 16 (11%, p=0.001), 17 (10%, p=0.02) and 11 (9%, p=0.02) and could be grouped in several functional networks. Methylation status was confirmed for 22 of these genes (SOX11, DLX1, FAM62C, SOX14, RSPO1, ADCY5, HAND2,SPOCK, MLL, ING1, PRIMA1, BCL11B, LTBP2, BNC1, NR2F2, SALL1, GALGT2, LHX1, DLX4, KLK10, TFAP2 and APP) in 78 CLL patients by pyrosequencing. As a proof of principle, we analyzed the expression of 2 genes, PRIMA1 and APP, in primary cells and of GALGT2, TFAP2C and PRIMA1 in leukemia cells. There was an inverse association between methylation and gene expression. This could be reversed by treatment with 5-aza-2’-deoxycytidine in cell lines. Treatment in a clinical trial with 5-azacitidine resulted in decreased methylation of LINE, DLX4 and SALL1 in the peripheral blood B-cells of patients with CLL. IgVH mutational status or ZAP-70 expression were not associated with specific methylation profiles. By multivariate analysis, methylation of LINE and APP was associated with shorter overall survival (p = 0.045 and 0.0035, respectively). This study demonstrates that aberrant DNA methylation is common and has potential prognostic and therapeutic value in CLL.     

Chromosome 5q deletion and epigenetic suppression of the gene encoding alpha-catenin (CTNNA1) in myeloid cell transformation

Interstitial loss of all or part of the long arm of chromosome 5, or del(5q), is a frequent clonal chromosomal abnormality in human myelodysplastic syndrome (MDS, a preleukemic disorder) and acute myeloid leukemia (AML), and is thought to contribute to the pathogenesis of these diseases by deleting one or more tumor-suppressor genes. Although a major commonly deleted region (CDR) has been delineated on chromosome band 5q31.1 (refs. 3-7), attempts to identify tumor suppressors within this band have been unsuccessful. We focused our analysis of gene expression on RNA from primitive leukemia-initiating cells, which harbor 5q deletions, and analyzed 12 genes within the CDR that are expressed by normal hematopoietic stem cells. Here we show that the gene encoding alpha-catenin (CTNNA1) is expressed at a much lower level in leukemia-initiating stem cells from individuals with AML or MDS with a 5q deletion than in individuals with MDS or AML lacking a 5q deletion or in normal hematopoietic stem cells. Analysis of HL-60 cells, a myeloid leukemia line with deletion of the 5q31 region, showed that the CTNNA1 promoter of the retained allele is suppressed by both methylation and histone deacetylation. Restoration of CTNNA1 expression in HL-60 cells resulted in reduced proliferation and apoptotic cell death. Thus, loss of expression of the alpha-catenin tumor suppressor in hematopoietic stem cells may provide a growth advantage that contributes to human MDS or AML with del(5q).     

DNA Methylation Profiles and Their Relationship with Cytogenetic Status in Adult Acute Myeloid Leukemia

Background

Aberrant promoter DNA methylation has been shown to play a role in acute myeloid leukemia (AML) pathophysiology. However, further studies to discuss the prognostic value and the relationship of the epigenetic signatures with defined genomic rearrangements in acute myeloid leukemia are required.

Methodology/Principal Findings

We carried out high-throughput methylation profiling on 116 de novo AML cases and we validated the significant biomarkers in an independent cohort of 244 AML cases. Methylation signatures were associated with the presence of a specific cytogenetic status. In normal karyotype cases, aberrant methylation of the promoter of DBC1 was validated as a predictor of the disease-free and overall survival. Furthermore, DBC1 expression was significantly silenced in the aberrantly methylated samples. Patients with chromosome rearrangements showed distinct methylation signatures. To establish the role of fusion proteins in the epigenetic profiles, 20 additional samples of human hematopoietic stem/progenitor cells (HSPC) transduced with common fusion genes were studied and compared with patient samples carrying the same rearrangements. The presence of MLL rearrangements in HSPC induced the methylation profile observed in the MLL-positive primary samples. In contrast, fusion genes such as AML1/ETO or CBFB/MYH11 failed to reproduce the epigenetic signature observed in the patients.

Conclusions/Significance

Our study provides a comprehensive epigenetic profiling of AML, identifies new clinical markers for cases with a normal karyotype, and reveals relevant biological information related to the role of fusion proteins on the methylation signature.     

Methylation of the Putative Tumor Suppressor Gene RASSF1A in Primary Cervical Tumors

Methylation-sensitive restriction endonuclease analysis (MSRA) followed by polymerase chain reaction (PCR) have been used to estimate the methylation level of 13 CpG dinucleotides in the promoter region of the putative suppressor gene RASSF1A (3p21.31) in squamous cell carcinomas of the uterine cervix (SCCs) carrying human papillomavirus (HPV) types 16, 18, and related types. Methylation of 3 to 13 CpG pairs has been found in 64% (25 out of 39) tumor DNA samples, 22% (2 out of 9) DNA samples from morphologically normal tissues adjacent to the tumor (P = 0.0306), and two out of three DNA samples from peripheral blood leukocytes of carcinoma patients. These CpG pairs are not methylated in the DNA of leukocytes of healthy donors (0 out of 10). The methylation level of the RASSF1A promoter region studied in tumors of the patients with regional lymph node metastases is significantly higher than in tumors of the patient that have no metastases (P = 8.5 × 10^–12). The methylation frequency of gene RASSF1A is two times higher than the frequency of hemi- and homozygous deletions in the chromosome 3 region where the gene is located. The data obtained indicate that methylation is one of the main mechanisms of the RASSF1A gene inactivation in HPV-positive human cervical tumors. The methylation of this gene may be an early event in the genesis of cervical tumors, the methylation level increasing with tumor progression.     

Hypermethylation-mediated partial transcriptional silencing of DAP-kinase gene in bladder cancer

Death-associated protein kinase (DAP-kinase) is a novel serine/threonine kinase whose expression is required for interferon-γ-induced apoptosis. This study evaluated the methylation pattern and its impact on the expression of the DAP-kinase gene in transitional cell carcinoma of the bladder as hypermethylation is one of the earliest and most frequent alterations leading to cancer. The frequency of hypermethylation of the gene promoter was 37.8%. On correlation with clinicopathological features, methylation was seen mostly in superficial tumours in the group aged?>?60 years (42.9 vs 33.3% of those?≤?60 years) and in smokers (48.1 vs 27.4% of non-smokers). The increased risk of bladder cancer was 6.70-fold (95% confidence interval (CI) 2.09–23.87; p?=?0.000) in those carrying methylated DAP-kinase and it was elevated in patients who smoked (odds ratio 7.87; 95% CI 1.50–54.96; p?=?0.007). This study demonstrated that methylation in the gene promoter on its own could significantly decrease the mRNA expression level of DAP-kinase by 27.68%. Interestingly, patients within the group aged?>?60 years and with a smoking habit showed increased downregulation of mRNA compared with non-smokers of this age group (similar pattern of methylation). Hypermethylation can decrease the expression of DAP-kinase and may be one of the reasons for conversion of normal cells to malignant cells, as the frequency of methylation at the early stage (superficial) of tumours was elevated. Methylation of DAP-kinase can be considered as one of the prognosis indicators for progression and development of bladder cancer.     

Colorectal adenoma and cancer detection based on altered methylation pattern of SFRP1, SFRP2, SDC2, and PRIMA1 in plasma samples

Aberrant methylation is one of the most frequent epigenetic alterations that can contribute to tumor formation. Cell-free DNA can originate from tumor tissue; therefore, the evaluation of methylation markers in cell-free DNA can be a promising method for cancer screening. Our aim was to develop a panel of biomarkers with altered methylation along the colorectal adenoma-carcinoma sequence in both colonic tissue and plasma. Methylation of selected CpG sites in healthy colonic (n = 15), adenoma (n = 15), and colorectal cancer (n = 15) tissues was analyzed by pyrosequencing. MethyLight PCR was applied to study the DNA methylation of SFRP1, SFRP2, SDC2, and PRIMA1 gene promoters in 121 plasma and 32 biopsy samples. The effect of altered promoter methylation on protein expression was examined by immunohistochemistry. Significantly higher (P < 0.05) DNA methylation levels were detected in the promoter regions of all 4 markers, both in CRC and adenoma tissues compared with healthy controls. Methylation of SFRP1, SFRP2, SDC2, and PRIMA1 promoter sequences was observed in 85.1%, 72.3%, 89.4%, and 80.9% of plasma samples from patients with CRC and 89.2%, 83.8%, 81.1% and 70.3% from adenoma patients, respectively. When applied as a panel, CRC patients could be distinguished from controls with 91.5% sensitivity and 97.3% specificity [area under the curve (AUC) = 0.978], while adenoma samples could be differentiated with 89.2% sensitivity and 86.5% specificity (AUC = 0.937). Immunohistochemical analysis indicated decreasing protein levels of all 4 markers along the colorectal adenoma-carcinoma sequence. Our findings suggest that this methylation biomarker panel allows non-invasive detection of colorectal adenoma and cancer from plasma samples.     

Promoter methylation of p16 and EDNRB gene in leukemia patients in Taiwan

Both epigenetic and genetic alternations are involved in cancer formation. In this study, we have identified the methylation frequency of p16 and endothelin receptor type B (EDNRB) of 26 leukemia patients and 8 randomly selected normal blood donors in Taiwan. Promoter methylation of p16 was detected in 85% of acute lymphocytic leukemia (ALL), 83% in acute myeloid leukemia (AML) whereas no methylation was detected in chronic myeloid leukemia (CML) in blast crisis. Hypermethylation of EDNRB was observed in 92% of ALL, 75% AML and 100% in CML in blast crisis. No aberrant methylation of p16 and EDNRB was found in 8 normal blood donors. Taken together, aberrant methylation of p16 and EDNRB was highly prevalent in leukemia patients in Taiwan.     

RB1 and CDKN2A Functional Defects Resulting in Retinoblastoma

Multiplex methylation-sensitive PCR was employed in studying the methylation of the RB1 and CDKN2A/p16 promoter regions in 52 retinoblastomas. Aberrant methylation inactivating RB1 was detected in 14 (27%) tumors. Methylation of p16 was for the first time observed in retinoblastoma (9 tumors, 17%). Both promoters proved to be methylated in two tumors. In four tumors, aberrant methylation was combined with structural defects of both RB1 alleles. Aberrant methylation of the p16 promoter was the second mutation event in two tumors and was not accompanied by RB1 defects in one tumor. Complex testing for RB1 mutations, loss of heterozygosity, and functional inactivation of the two genes revealed molecular defects in at least one allele in 51 (98%) tumors.