Demethylation of Urokinase Promoter as a Prognostic Marker in Patients with Breast Carcinoma Pouya Pakneshan, 1 Bernard Te ˆtu, 2 and Shafaat A. Rabbani 1 1 Departments of Medicine and Oncology, McGill University Health Center, Montreal, Canada, and 2 Department of Pathology, Universite ´ Laval, Quebec, Canada ABSTRACT Purpose: Urokinase (uPA) is expressed in a number of highly invasive malignancies including breast cancer. Be- cause 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 pro- moter 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 re- verse 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 deter- mined 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 dem- ethylation of the uPA promoter is associated with malignant transformation. Reverse transcription-PCR analysis re- vealed that this demethylation of the uPA promoter is di- rectly 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 pro- moter methylation can therefore serve as an early reliable indicator of uPA production in breast cancer patients. INTRODUCTION The leading cancer-associated cause of morbidity and mor- tality in patients with breast cancer is metastasis of tumor cells to different organs (1). Tumor metastasis is a multistep process involving local invasion, degradation of the extracellular matrix, angiogenesis, intravasation, survival of malignant cells in the circulation, extravasation, and, finally, establishment of a sec- ondary growth in distant organs (2). One of the key mediators of this process is urokinase (uPA), a member of the serine protease family that catalyzes the conversion of inactive zymogen plas- minogen to its active form, plasmin (3). When activated, plas- min degrades most components of the extracellular matrix, such as laminin, fibronectin, and collagen (3). In addition to its proteolytic activity, uPA is known to exert additional activities including stimulation of cellular proliferation, enhancement of cellular migration, alteration of cellular adhesive properties, and activation of specific growth factors such as vascular endothelial growth factor and hepatocyte growth factor that play an impor- tant role in angiogenesis (3, 4). Increased expression of uPA is directly related to higher tumor growth and metastasis due to the ability of uPA to induce these angiogenic factors (1). Recently, the tumor-promoting effects of uPA have been linked to the ability of uPA to prevent tumor cell apoptosis (5). Such func- tions of uPA have implicated this protease as a major player in promoting the process of tumor growth, invasion, metastasis, and angiogenesis of several malignancies, including breast cancer (6). Over 14 years ago, it was proposed for the first time that breast cancer patients with high levels of uPA in their primary tumors have a lower overall survival rate than patients with low levels of uPA (7). Since then, a large number of studies by different groups have reported the prognostic value of uPA as a marker for breast cancer progression (8 –10). It has been shown that determination of the levels of uPA production in breast cancer patients can serve as a reliable marker that is independent of and stronger than most traditional prognostic markers such as tumor size, tumor grade, patient age, axillary node status, and steroid receptor status (8 –10). These studies have also identified uPA as an excellent therapeutic target for blocking tumor pro- gression (10 –12). Cytosine methylation within the context of CpG dinucle- otides in the genome is a molecular mechanism that causes epigenetic changes in the chromatin structure and leads to transcriptional silencing of genes in many human cancers (13). This epigenetic alteration is heritable but does not alter the nucleotide sequence, making the modification potentially re- versible (14 –16). DNA methylation markers, useful in both epidemiological and clinical studies, have been developed over the years using both targeted genes and genome-wide scanning techniques (17, 18). Hypermethylated CpG islands detected in serum, urine, bronchoalveolar lavage fluid, and lymph nodes derived from patients with various types of malignancies have Received 11/7/03; revised 1/12/04; accepted 1/22/04. Grant support: Grant MOP-12609 from the Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Shafaat A. Rabbani, McGill University Health Centre, 687 Pine Avenue West, Room H4.67, Montreal, Quebec, H3A 1A1 Canada. Phone: (514) 843-1632; Fax: (514) 843-1712; E-mail: shafaat.rabbani@mcgill.ca. 3035 Vol. 10, 3035–3041, May 1, 2004 Clinical Cancer Research Research. on May 29, 2020. © 2004 American Association for Cancer clincancerres.aacrjournals.org Downloaded from