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Role of Coagulant Proteases in Breast Cancer Cell Invasion
Background: Coagulant proteases such as thrombin are generated in the tumor microenvironment. We recently found that thrombin directly stimulates human breast carcinoma cell invasion via activation of protease-activated receptor-1 (PAR1). PAR1 expression is increased in malignant breast cancers and highly invasive cancer cell lines. In contrast, PAR1 expression is minimal or absent in benign hyperplasias, and normal breast epithelial tissue and non-invasive cell lines. Inhibition of PAR1 expression completely ablated thrombin-induced mitogen-activated protein kinase (MAPK) signaling and cellular invasion. Surprisingly, inhibition of epidermal growth factor receptor (EGFR) abolished thrombin-triggered MAPK activation and cellular invasion supporting our hypothesis that transactivation of the EGFR/ErbB pathway by thrombin is critical for promotion of cellular invasion. The mechanisms by which thrombin and upstream coagulant proteases contribute to cellular invasion is not known. Objective/Hypothesis: The objective of this proposal is to determine the contribution of coagulant proteases in breast carcinoma cell invasion and to delineate the mechanism by which coagulant proteases contribute to cellular invasion. The aims are to (1) define the contribution of coagulant proteases in promoting cellular invasion, (2) delineate the role of EGFR/ErbB members in coagulant protease-induced cellular invasion, and (3) define the molecular mechanism by which coagulant proteases transactivate EGFR/ErbB. Study Design: First, we will use purified active and inactive coagulant proteases to determine which coagulant proteases are capable of promoting cancer cell invasion. Second, we will assess the role of EGFR/ErbB transactivation in coagulant-protease cancer cell invasion using a variety of small molecule inhibitors. Third, studies will examine how coagulant proteases transactivate EGFR/ErbB using broad based-approaches. Potential Outcomes/Research Benefits: Hyperactivation of the ErbB pathway is associated with metastatic breast cancer. The mechanisms that lead to sustained ErbB activation are varied and poorly understood. The goal of our study will be to determine whether coagulant proteases contribute to cellular invasion by transactivation of the ErbB pathway. Thus understanding how coagulant proteases contribute to cancer cell invasion may provide new strategies for the prevention and treatment of metastatic breast cancer disease.
Breast cancer is a genetic disease in which normal genes are overexpressed or mutated leading to a loss of normal growth proliferation and survival of cells. ErbB2 (HER2) is overexpressed and consequently hyperactivated in 20-30% of human breast tumors and is correlated with poor prognosis. Therapeutics directed against ErbB2/HER2 reduce the rate of tumor growth and, when used together with chemotherapy, improve metastatic breast cancer patient responses. Thus hyperactivation of the ErbB pathway is critical for breast cancer progression. In addition to HER2 overexpression, hyperactivation of this pathway can be achieved by other mechanisms including overproduction of ligands and/or mutations or defects in other signaling components that impinge upon this pathway. These alternative mechanisms of activating the ErbB pathway remain poorly understood. One possible mediator of activation of the ErbB pathway in breast carcinoma cells may involve transactivation by protease-activated receptors (PARs). PARs are G protein-coupled receptors for coagulant proteases including the main effector thrombin. Several studies determined that active coagulant proteases are present in the tumor microenvironment and that expression of PARs is increased in malignant breast tissue specimens and in highy invasive cell lines. We recently found that the addition of thrombin increases breast carcinoma cellular invasion through the activation of PAR1. More importantly the ability of thrombin and PAR1 to promote cellular invasion involves transactivation of the ErbB pathway. Our studies propose to examine the contribution of coagulant proteases and PARs towards the activation of ErbB pathway and how this affects the ability of coagulant proteases to stimulate breast carcinoma cellular invasion. Thus an understanding of how coagulant proteases and their receptors (PARs) contribute to breast carcinoma cellular invasion may establish PARs as important diagnostic markers or targets for drug discovery for treatment of metastatic breast cancer disease.