Demethylation of urokinase promoter as a prognostic marker in patients with breast carcinoma

Purpose: Urokinase (uPA) is expressed in a number of highly invasive malignancies including breast cancer. Because production of uPA is associated with breast cancer progression and can serve as a useful prognostic marker, the purpose of this study was to examine the role of uPA promoter methylation as an indicator of uPA production in breast cancer patients. Experimental Design: We examined the methylation status of the uPA promoter and the levels of uPA expression in normal human breast epithelial cells and several human breast cancer cells by bisulfite sequencing analysis and reverse transcription-PCR. We also analyzed the methylation status of the uPA promoter in surgical biopsy samples from patients with breast cancer of different grades, as determined by the Elston-Ellis histological grading system. Results: Expression of uPA mRNA was only detected in the highly invasive estrogen receptor-negative breast cancer cell lines, where the promoter was completely demethylated. In normal and low invasive breast cancer cells, the uPA promoter was methylated, resulting in lack of uPA mRNA expression. Analysis of biopsy samples showed that demethylation of the uPA promoter is associated with malignant transformation. Reverse transcription-PCR analysis revealed that this demethylation of the uPA promoter is directly associated with induction of uPA mRNA expression, which is well known to be associated with poor prognosis in breast cancer patients. Conclusions: This study indicated that uPA expression in breast cancer patients is under epigenetic control via methylation of its promoter. Determination of uPA promoter methylation can therefore serve as an early reliable indicator of uPA production in breast cancer patients.