Higher Promoter Methylation Levels Research Articles

Epigenetic instability is rare in fibrolamellar carcinomas but common in viral-associated hepatocellular carcinomas

Fibrolamellar carcinomas have a unique predilection for younger individuals and arise in livers without recognizable liver disease. In contrast to typical hepatocellular carcinomas, fibrolamellar carcinomas show few chromosomal changes and lack mutation in key genes such as TP53 and CTNNB1. Epigenetic instability, manifesting as methylation of important tumor suppressor gene promoters, has not been investigated in fibrolamellar carcinomas. Thus, the methylation status of 11 tumor suppressor gene promoters was investigated using methylation-specific PCR: RASSF1, CDH1, CDKN2B, HPP1, CDKN2A, GSTP1, P16, RARA, FLJ13081, SOCS1, and TP53. Nine fibrolamellar carcinomas were studied including primary tumors (N=5) and metastatic deposits (N=4) along with control groups of typical hepatocellular carcinoma arising in livers with (N=21) and without cirrhosis (N=10). In fibrolamellar carcinomas, RASSF1A and CDH1 (e-cadherin) were the most commonly methylated genes with 80–100% of tumors methylated. However, overall fibrolamellar carcinomas showed low levels of methylation with no differences between fibrolamellar carcinomas and their paired normal livers. However, fibrolamellar carcinomas showed significantly less methylation than hepatocellular carcinomas that arose in the background of viral cirrhosis. Overall, methylation was most strongly linked to viral cirrhosis. In conclusion, fibrolamellar carcinoma shows low levels of methylation. In contrast, higher levels of promoter methylation are associated with hepatocellular carcinomas that arise in the setting of viral induced cirrhosis.

High Promoter Methylation Levels of APC Predict Poor Prognosis in Sextant Biopsies from Prostate Cancer Patients

Prostate cancer is a highly prevalent malignancy and constitutes a major cause of cancer-related morbidity and mortality. Owing to the limitations of current clinical, serologic, and pathologic parameters in predicting disease progression, we sought to investigate the prognostic value of promoter methylation of a small panel of genes by quantitative methylation-specific PCR (QMSP) in prostate biopsies. Promoter methylation levels of APC, CCND2, GSTP1, RARB2, and RASSF1A were determined by QMSP in a prospective series of 83 prostate cancer patients submitted to sextant biopsy. Clinicopathologic data [age, serum prostate-specific antigen (PSA), stage, and Gleason score] and time to progression and/or death from prostate cancer were correlated with methylation findings. Log-rank test and Cox regression model were used to identify which epigenetic markers were independent predictors of prognosis. At a median follow-up time of 45 months, 15 (18%) patients died from prostate cancer, and 37 (45%) patients had recurrent disease. In univariate analysis, stage and hypermethylation of APC were significantly associated with worse disease-specific survival, whereas stage, Gleason score, high diagnostic serum PSA levels, and hypermethylation of APC, GSTP1, and RASSF1A were significantly associated with poor disease-free survival. However, in the final multivariate analysis, only clinical stage and high methylation of APC were significantly and independently associated with unfavorable prognosis, i.e., decreased disease-free and disease-specific survival. High-level APC promoter methylation is an independent predictor of poor prognosis in prostate biopsy samples and might provide relevant prognostic information for patient management.

Identification of a unique epigenetic sub‐microenvironment in prostate cancer

The glutathione S-transferase P1 (GSTP1) gene promoter is methylated in tumour cells in more than 90% of prostate carcinomas. Recently, GSTP1 promoter methylation was identified in tumour-associated stromal cells in addition to the tumour epithelium. To define the extent and location of stromal methylation, epigenetic mapping using pyrosequencing quantification of GSTP1 promoter methylation and an anatomical three-dimensional reconstruction of an entire human prostate specimen with cancer were performed. Normal epithelium and stroma, tumour epithelium, and tumour-associated stromal cells were laser capture-microdissected from multiple locations throughout the gland. As expected, the GSTP1 promoter in both normal epithelium and normal stromal cells distant from the tumour was not methylated and the tumour epithelium showed consistently high levels of promoter methylation throughout. However, tumour-associated stromal cells were found to be methylated only in a localized and distinct anatomical sub-field of the tumour, revealing the presence of an epigenetically unique microenvironment within the cancer. Morphologically, the sub-field consisted of typical, non-reactive stroma, representing a genomic alteration in cells that appeared otherwise histologically normal. Similar epigenetic anatomical mapping of a control prostate gland without cancer showed low background methylation levels in all cell types throughout the specimen. These data suggest that stromal cell methylation can occur in a distinct sub-region of prostate cancer and may have implications for understanding tumour biology and clinical intervention.

Relationships between DNA Methylation and Expression in Erythrocyte Membrane Protein (Band 3, Protein 4.2, and 3-Spectrin) Genes during Human Erythroid Development and Differentiation

Red cell membrane proteins are sequentially expressed during erythroid development and differentiation. Spectrins have already been synthesized in early erythroid precursors such as pronormoblasts, and band 3 (B3) appears at nearly the same stage. Protein 4.1 appears next, followed by protein 4.2 (P4.2) at the very late erythroblast stage. The methylation states of the promoter 5'-CG-3' sites are known to be linked to the regulation of promoter function by modulating DNA-protein interactions and the structure of chromatin. Hence, the genes for B3, P4.2, and beta-spectrin (beta-SP) appear to be suitable models to study the relationship between methylation of promoter 5'-CG-3' sites and the sequential expression of genes during human erythroid development and differentiation. We have examined methylation profiles in the promoter regions of the genes (ELB42, EPB3, and SPTB) for the human erythroid membrane proteins P4.2, B3, and beta-SP by applying the bisulfite genomic sequencing method. Our results demon strate the following: (1) The promoter regions of EPB3 and ELB42 are extensively methylated in DNA from human peripheral blood mononuclear cells, but the SPTB promoter is totally unmethylated. (2) During erythroid differentiation, DNA methylation patterns change as follows: (a) ELB42 is unmethylated in DNA from erythroid-committed blastic cells, such as the human cell lin UT-7/EPO, but is methylated in erythroblasts from peripheral blood burst-forming unit erythroid (BFU-E) in the second phase of the liquid-culture method. Messenger RNA (mRNA) from ELB42 is first detected in early erythroblasts, and P4.2 is expressed in late erythroblasts. (b) In contrast, EPB3 is consistently methylated in UT-7/EPO cells and in cultured erythroblasts from BFU-E from human peripheral blood. B3 mRNA and protein are already expressed in early erythroblasts. (c) SPTB remains unmethylated in human DNA from UT-7/EPO cells and from cultured erythroblasts. These results document the diversity of the reactions of human promoter sequences to the modulating influence of DNA methylation. Whereas the human SPTB promoter conforms to expectations in that it is unmethylated and fully active throughout erythroid development, high levels of promoter methylation correlate with promoter activity for the EPB3 and ELB42 genes during their sequential activation in erythrocyte differentiation.

Association of breast cancer DNA methylation profiles with hormone receptor status and response to tamoxifen.

We have generated DNA methylation profiles of 148 human breast tumors and found significant differences in hormone receptor (HR) status between clusters of DNA methylation profiles. Of 35 DNA methylation markers analyzed, the ESR1 gene, encoding estrogen receptor alpha, proved to be the best predictor of progesterone receptor status, whereas methylation of the PGR gene, encoding progesterone receptor, was the best predictor of estrogen receptor status. ESR1 methylation outperformed HR status as a predictor of clinical response in patients treated with the antiestrogen tamoxifen, whereas promoter methylation of the CYP1B1 gene, encoding a tamoxifen- and estradiol-metabolizing cytochrome p450, predicted response differentially in tamoxifen-treated and nontamoxifen-treated patients. High levels of promoter methylation of the ARHI gene, encoding a RAS-related small G-protein, were strongly predictive of good survival in patients who had not received tamoxifen therapy. Our results reveal an as yet unrecognized degree of interaction between DNA methylation and HR biology in breast cancer cells and suggest potentially clinically useful novel DNA methylation predictors of response to hormonal and non-hormonal breast cancer therapy.