Reversal of the Hypomethylation Status of Urokinase (uPA) Promoter Blocks Breast Cancer Growth and Metastasis

Abstract

Metastasis is a leading cause of mortality and morbidity in cancer. Urokinase (uPA), only expressed by the highly invasive cancer cells, has been implicated in invasion, metastases, and angiogenesis of several malignancies including breast cancer. Because uPA expression is strongly correlated with its hypomethylated state, we utilized the uPA gene in the highly invasive MDA-231 human breast cancer cells as a model system to test the hypothesis that pharmacological reversal of the uPA promoter hypomethylation would result in its silencing and inhibition of metastasis. S-Adenosyl-L-methionine (AdoMet) has previously been shown to cause hypermethylation and inhibit demethylation. Treatment of MDA-231 cells with AdoMet, but not its unmethylated analogue S-adenosylhomocysteine, significantly inhibits uPA expression and tumor cell invasion in vitro and tumor growth and metastasis in vivo. The effects of AdoMet on uPA expression were reversed by the demethylating agent 5-azacytidine, supporting the conclusion that AdoMet effects are caused by hypermethylation. Knockdown of the methyl-binding protein 2 also causes a significant inhibition of uPA expression in vitro and tumor growth and metastasis in vivo. These treatments did not have any effects on estrogen receptor expression, suggesting that inhibition of hypomethylation will not affect genes already silenced by hypermethylation. These data are consistent with the hypothesis that hypomethylation of critical genes like uPA plays a causal role in metastasis. Inhibition of hypomethylation can thus be used as a novel therapeutic approach to silence the pro-metastatic gene uPA and block breast cancer progression into the aggressive and metastatic stages of the disease.Metastasis of tumor cells to different organs is the leading cause of cancer-associated morbidity and mortality in patients with breast cancer (1, 2). One of the key mediators of this process is urokinase (uPA), 1 a member of the serine protease family that catalyzes the conversion of inactive zymogen plasminogen to its active form plasmin (1, 3). When activated, plasmin degrades most components of the ECM, such as laminin, fibronectin, and collagen (3). Produced by tumor cells and tumor surrounding stroma, uPA is involved in the process of tumor progression and has been implicated in the invasion, metastasis, and angiogenesis of several malignancies including breast cancer (1). Breast cancer is a hormone-dependent malignancy that is often initiated as a less aggressive hormone responsive type and gradually progresses to a highly invasive hormone-insensitive phenotype associated with mutations in estrogen receptor (ER) or interference with ER signaling (4). The molecular mechanism regulating this transition is poorly understood.Abnormal patterns of DNA methylation are hallmarks of most malignancies, including breast cancer (5). Widespread global DNA hypomethylation accompanied by region-specific hypermethylation and increased levels of expression and activity of DNMT1 are associated with the malignant phenot...

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