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DNA methylation of heparanase promoter influences its expression and associated with the progression of human breast cancer.

ABSTRACT: Heparanase promotes tumor invasion and metastasis in several malignancies including breast cancer. However, the roles and regulation mechanisms of heparanase during breast cancer progression are still not fully understood. The aim of this study is to determine the differential regulation of heparanase gene expression in specific stages of breast cancer by DNA methylation. We detected levels of heparanase expression and DNA methylation patterns of its promoter in breast cancer cell lines (MCF-7 and MDA-MB-435) and clinical tissues, respectively. It has been observed that heparanase is highly expressed in the invasive MDA-MB-435 cells with low methylation modification in the heparanase promoter. In contrast, lower expression of heparanase in MCF-7 cells is accompanied by higher methylation in the promoter. Treatment of MCF-7 cells with 5-aza-2'-deoxycytidine (5-aza-dC), a potent demethylating agent, results in induction of heparanase expression and higher invasion potential in vitro and leads to an advantage of tumor formation in vivo. In 54 tissue samples, cancer samples at late stages (stage IV) showed the highest heparanase expression accomplished by little DNA methylation. On the contrary, methylation prevalence is highest in normal tissue and inversely correlated with heparanase expression. A significant correlation between DNA methylation and clinical stage was demonstrated (p = 0.012). Collectively, these results demonstrate that DNA methylation play the regulation role in heparanase gene in different stages of breast cancer and present a direct effect on tumor progression.

SUBMITTER: Jiao F

PROVIDER: S-EPMC3954879 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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DNA methylation of heparanase promoter influences its expression and associated with the progression of human breast cancer.

Jiao Fei F Bai Shi-Yu SY Ma Ying Y Yan Zhong-Hai ZH Yue Zhen Z Yu Yuan Y Wang Xin X Wang Juan J

PloS one 20140314 3

Heparanase promotes tumor invasion and metastasis in several malignancies including breast cancer. However, the roles and regulation mechanisms of heparanase during breast cancer progression are still not fully understood. The aim of this study is to determine the differential regulation of heparanase gene expression in specific stages of breast cancer by DNA methylation. We detected levels of heparanase expression and DNA methylation patterns of its promoter in breast cancer cell lines (MCF-7 a ...[more]

PMID: 24632672

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Project description:BACKGROUND: Inhibitor of DNA binding/Inhibitor of differentiation 4 (ID4) is a critical factor for cell proliferation and differentiation in normal vertebrate development. ID4 has regulative functions for differentiation and growth of the developing brain. The role of ID1, ID2 and ID3 are expected to be oncogenic due to their overexpression in pancreatic cancer and colorectal adenocarcinomas, respectively. Aside from these findings, loss of ID3 expression was demonstrated in ovarian cancer. The aim of the present study was to reveal the factual role of ID4 in carcinogenesis in more detail, since its role for the pathogenesis of human breast cancer has been discussed controversially, assigning both oncogenic and tumour suppressive functions. METHODS: ID4 promoter methylation, ID4 mRNA expression and ID4 protein expression were analysed in primary human breast cancer specimens using methylation-specific PCR (MSP) (n=170), semiquantitative realtime RT-PCR (n=46) and immunhistochemistry (n=3), respectively. In order to demonstrate a functional association of ID4 promoter methylation with its gene silencing, we performed DNA demethylation analysis with four human breast cell lines using MSP and semiquantitative realtime RT-PCR. In addition, we performed correlations of ID4 promoter methylation with ID4 mRNA and ID4 protein expression in matched samples of breast tumour and corresponding normal tissue. We carried out statistical analyses in order to find correlations between ID4 promoter methylation and clinicopathological parameters. RESULTS: Frequent ID4 promoter methylation was observed in primary breast cancer samples (69%, 117/170). We found a tight correlation (P<0.0001) between ID4 promoter methylation and loss of ID4 expression in primary breast cancer 3 specimens. Demethylating treatment with breast cancer cell lines was associated with clear ID4 mRNA re-expression. Tumours with ID4 promoter methylation showed distinct loss of ID4 expression on both transcription and protein level. Interestingly, ID4 promoter methylation was a factor for unfavourable recurrence-free survival (P=0.036) and increased risk for lymph node metastasis (P=0.030). CONCLUSION: ID4 is indeed a novel tumour suppressor gene in normal human breast tissue and is epigenetically silenced during cancer development, indicating increased risk for tumour relapse. Thus, ID4 methylation status could serve as a prognostic biomarker in human breast cancer.

Project description:Epigenetic inactivation of genes by DNA hypermethylation plays an important role in carcinogenesis. An in vitro model of human breast epithelial cell transformation was used to study epigenetic changes induced by estradiol during the neoplastic process. Different stages of tumor initiation and progression are represented in this model being MCF-10F the normal stage; trMCF cells, the transformed stage; bsMCF cells, the invasive stage and, caMCF cells, the tumor stage. Global methylation studies by restriction landmark genomic scanning (RLGS) showed an increased DNA methylation during the in the invasive and tumor stages. Expression studies showed that NRG1 (neuregulin 1), CSS3 (chondroitin sulfate synthase 3) and SNIP (SNAP-25-interacting protein) were downregulated in the invasive and tumor cells. The transformed cells showed low expression of STXBP6 (amysin) compared to the parental cells MCF-10F. The treatment of these cells with the demethylating agent 5-aza-dC alone or in combination with the histone deacetylase inhibitor trichostatin increased the expression of NRG1, STXBP6, CSS3 and SNIP confirming that DNA methylation plays an important role in the regulation of the expression of these genes. The NRG1 exon 1 has a region located between -136 and +79 (considering +1, the translational initiation site) rich in CpG sites that was analyzed by methylation specific PCR (MSP). NRG1 exon 1 showed progressive changes in the methylation pattern associated with the progression of the neoplastic process in this model; NRG1 exon 1 was unmethylated in MCF-10F and trMCF cells, becoming hypermethylated in the invasive (bsMCF) and tumor (caMCF) stages. Studies of human breast tissue samples showed that NRG1 exon 1 was partially methylated in 14 out of 17 (82.4%) invasive carcinomas although it was unmethylated in normal tissues (8 out of 10 normal breast tissue samples). Furthermore, NRG1 exon 1 was partially methylated in 9 out of 14 (64.3%) morphologically normal tissue samples adjacent to invasive carcinomas.

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Project description:BackgroundSex differences are known to impact muscle phenotypes, metabolism, and disease risk. Skeletal muscle stem cells (satellite cells) are important for muscle repair and to maintain functional skeletal muscle. Here we studied, for the first time, effects of sex on DNA methylation and gene expression in primary human myoblasts (activated satellite cells) before and after differentiation into myotubes.MethodWe used an array-based approach to analyse genome-wide DNA methylation and gene expression in myoblasts and myotubes from 13 women and 13 men. The results were followed up with a reporter gene assay.ResultsGenome-wide DNA methylation and gene expression differences between the sexes were detected in both myoblasts and myotubes, on the autosomes as well as the X-chromosome, despite lack of exposure to sex hormones and other factors that differ between sexes. Pathway analysis revealed higher expression of oxidative phosphorylation and other metabolic pathways in myoblasts from women compared to men. Oxidative phosphorylation was also enriched among genes with higher expression in myotubes from women. Forty genes in myoblasts and 9 in myotubes had differences in both DNA methylation and gene expression between the sexes, including LAMP2 and SIRT1 in myoblasts and KDM6A in myotubes. Furthermore, increased DNA methylation of LAMP2 promoter had negative effects on reporter gene expression. Five genes (CREB5, RPS4X, SYAP1, XIST, and ZRSR2) showed differential DNA methylation and gene expression between the sexes in both myoblasts and myotubes. Interestingly, differences in DNA methylation and expression between women and men were also found during differentiation (myoblasts versus myotubes), e.g., in genes involved in energy metabolism. Interestingly, more DNA methylation changes occur in women compared to men on autosomes.ConclusionAll together, we show that epigenetic and transcriptional differences exist in human myoblasts and myotubes as well as during differentiation between women and men. We believe that these intrinsic differences might contribute to sex dependent differences in muscular phenotypes.

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DNA methylation of heparanase promoter influences its expression and associated with the progression of human breast cancer.

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DNA methylation of heparanase promoter influences its expression and associated with the progression of human breast cancer.

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