Enzymes that digest proteins (proteases) are required for normal development of the breast as well as changes in development that occur in pregnancy. Proteases unfortunately can also initiate development of breast cancer and accelerate its progression from a local disease to a metastatic (or disseminated) disease. The digestion of proteins or protease activity is required for these processes. In animal models, protease antagonists or inhibitors have been shown to reduce protease activity and thereby decrease the progression of mammary (breast) cancer. In human breast cancer, proteases would seem to be potential targets for treatment with protease inhibitors. The purpose of this proposal is to identify those proteases that are valid drug targets in breast cancer and to develop systems for assessing inhibition of protease activity. We will use new molecular approaches to catalog the proteases that are expressed during initiation and progression of breast cancer in both mouse model systems and patient biopsies. Proteases that are expressed in both mouse models and human biopsies will be chosen for further study. To ensure the greatest future impact on breast cancer treatment, we will select proteases that change in expression during early progression of breast cancer and/or cause the conversion of premalignant lesions to cancers. We will verify that the selected proteases cause progression and/or conversion by comparing mouse model systems in which the selected protease is present or absent. In parallel, we will develop reagents to measure protease activity by imaging approaches. We will incorporate into the reagents small proteins (peptides) that can be digested by the proteases, producing reagents that measure protease activity. We will produce reagents for two types of imaging: optical and magnetic resonance. Both types of imaging are noninvasive (i.e., can be performed from outside the body). The reagents and technologies that we are developing are novel and innovative and will improve our ability to detect protease activity in vivo. The reagents will be selective for breast cancer-associated proteases and therefore should improve the detection of breast cancer. Ultimately, these reagents and technologies will improve treatment of breast cancer, as they will allow the clinician to determine which proteases are active in a patient¿s tumor and thus what treatment protocols to employ.