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 methylation-sensitive 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 promoter-luciferase 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.
Highlights
Urokinase-type plasminogen activator 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
Using methylationsensitive PCR, we show that 90% of CpG dinucleotides in the Urokinase-type plasminogen activator (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
Evaluation of uPA mRNA Expression and Its Effect on Tumor Cell Invasion—To test the hypothesis that uPA might play a role in breast cancer cell invasion, we first examined the expression of uPA mRNA in the estrogen receptor (ER)-positive, hormone-sensitive human breast cancer cell line MCF-7 and in highly invasive ER-negative, hormone-insensitive MDA
Summary
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. 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. Whereas hypermethylation and silencing of tumor suppressor genes has attracted much attention recently [13, 15, 16], the molecular mechanisms underlying hypomethylation of tumor progression factors such as ras, myc, hox, and xmrk that are up-regulated during cancer development are poorly described This suggests that hypomethylation may play an important role in regulating gene expression during tumorigenesis similar to hypermethylation [17,18,19,20]. We tested the hypothesis that the expression of uPA, a well defined marker of highly invasive tumor cells and activated at the late stages of breast cancer, can be regulated via the changes in the methylation status of its promoter region. As a follow-up to our previous studies where we demonstrated that the uPA gene is transcriptionally suppressed by DNA methylation [21], we have focused on the examination of the role of DNA demethylation and DNA methylating enzymes such as DNA methyltransferase (DNMT) and demethylase (DMase) in regulating uPA expression during breast cancer progression
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