Regulation of DNA Methylation in Human Breast Cancer

Abstract

Urokinase-type plasminogen activator (uPA) is a member of the serine protease family and can break down various components of the extracellular matrix to promote growth, invasion, and metastasis of several malignancies including breast cancer. In the current study we examined the role that the DNA methylation machinery might be playing in regulating differential uPA gene expression in breast cancer cell lines. uPA mRNA is expressed in the highly invasive, hormone-insensitive human breast cancer cell line MDA-MB-231 but not in hormone-responsive cell line MCF-7. Using methylationsensitive PCR, we show that 90% of CpG dinucleotides in the uPA promoter are methylated in MCF-7 cells, whereas fully demethylated CpGs were detected in MDA-MB-231 cells. uPA promoter activity, which is directly regulated by the Ets-1 transcription factor, is inhibited by methylation as determined by uPA promoterluciferase reporter assays. We then tested whether the state of expression and methylation of the uPA promoter correlates with the global level of DNA methyltransferase and demethylase activities in these cell lines. We show that maintenance DNA methyltransferase activity is significantly higher in MCF-7 cells than in MDA-MB-231 cells, whereas demethylase activity is higher in MDA-MB-231 cells. We suggest that the combination of increased DNA methyltransferase activity with reduced demethylase activity contributes to the methylation and silencing of uPA expression in MCF-7 cells. The converse is true in MDA-MB-231 cells, which represents a late stage highly invasive breast cancer. The histone deacetylase inhibitor, Trichostatin A, induces the expression of the uPA gene in MDA-MB-231 cells but not in MCF-7 cells. This supports the hypothesis that DNA methylation is the dominant mechanism involved in the silencing of uPA gene expression. Taken together, these results provide insight into the mechanism regulating the transcription of the uPA gene in the complex multistep process of breast cancer progression.

Cigarette smoke induces demethylation of prometastatic oncogene synuclein-γ in lung cancer cells by downregulation of DNMT3B

“…De novo DNA methyltransferase activity assay The DNA methyltransferase assay was performed as described previously (Belinsky et al, 1996;Patra et al, 2006;Guo et al, 2002) with some modification. Twenty micrograms of nuclear extract was incubated at 371C with 1.5 mg of unmethylated poly(dI-dC)-poly(dI-dC) (Sigma) as the template and 2 mCi of 3 H-labeled S-adenosylmethionine (Amersham Biosciences, Piscataway, NJ, USA) in a total volume of 40 ml of reaction buffer (pH 7.4), containing 20 mM Tris-HCl, 25% glycerol (v/v), 10 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride and 20 mM 2-mercaptoethanol.…”

Epigenetic Up-Regulation of Leukemia Inhibitory Factor (LIF) Gene During the Progression to Breast Cancer

“…Accumulating evidence supports the role of such epigenetic silencing mechanisms as methylation and inactive histone modifications in the transcriptional inactivation of tumor suppressor genes during cancer developmental processes (Ballestar and Esteller, 2008;Baylin and Jones, 2007;Robertson, 2005). In contrast, tumor progression may involve the epigenetic activation of oncogene-like genes, as evidenced by the upregulation of galectin-7, CLDN4, uPA, and MMP-2 genes in several cancer cells via DNA hypomethylation in their promoter regions (Chernov et al, 2009;Demers et al, 2009;Guo et al, 2002;Honda et al, 2006;Pakneshan et al, 2004;Shvachko, 2009). This epigenetic activation involves the reversal of inactive epigenetic marks including histone deacetylation by histone acetylation and DNA methylation by DNA hypomethylation.…”

“…Certain cancer cell types methylate the BubR1, 7-dehydrocholesterol reductase (Dhcr7), aquaporin-5 (AQP5), RUNX3, lysophosphatidic acid receptor-1 (lpa1), galectin-3, CDX1, MUC5B, PDZ-LIM domain-containing protein 2 (PDLIM2), LOT1 (PLAGL1/ZAC1), TNFSF7 (CD70) genes, leading to their transcriptional suppression DNA hypomethylation at CpG islands within the promoters (Abdollahi et al, 2003;Ahmed et al, 2007;Kim et al, 2005;Motegi et al, 2005;Park et al, 2005;Perrais et al, 2001;Qu et al, 2010;Suh et al, 2002;Tsujiuchi et al, 2006;Yu et al, 2010). The upregulation of galectin-7, CDX4, and urokinase-type plasminogen activator (uPA), and MMP-2 genes in several cancer types including breast cancer, ovarian cancer, and lymphoma cells is mediated by DNA hypomethylation (Chernov et al, 2009;Demers et al, 2009;Guo et al, 2002;Honda et al, 2006;Pakneshan et al, 2004). For transcription factors such as STAT1, NFAT and Oct-1, methylation at specific CpGs has been shown to directly inhibit protein binding and thus inhibit transcription in colon carcinoma cells and human T cells (McGough et al, 2008;Murayama et al, 2006).…”

Inhibitory effect of HGF on invasiveness of aggressive MDA-MB231 breast carcinoma cells, and role of HDACs

“…First, MDA-MB231 cells showed spontaneous mesenchymal movement, related to the key function of proteolytic enzymes (Guo et al, 2002;Wolf et al, 2008), that decreased after HGF exposure. One explanation might be that HGF reduces ETS1 activity, possibly affecting the expression of target genes such as proteolytic enzymes (Guo et al, 2002;Maroni et al, 2007;Furlan et al, 2008).…”

“…The expression of CXCR4 is lower in lymph node metastases than in primary breast cancer (Shim et al, 2006). In low (MCF-7) and highly (MDA-MB231) invasive breast carcinoma cells (Guo et al, 2002;Yang et al, 2007), HGF respectively raises and lowers the expression of CXCR4 (Matteucci et al, 2005).…”