This proposal is responsive to an RFA designed to fund exploratory/ developmental projects between a basic scientist and a metastasis biologist in the area of molecular and cellular biology of metastatic tumor cells. They present here a project which creates such a synergistic collaboration and which sets out to characterize a novel serine protease and its inhibitor in human breast cancer. Local invasion and metastasis of breast cancer have been proposed to require imbalanced or unregulated presentation of proteases at invading edges of carcinoma cells. To date, most of the proteases found associated with the disease (metalloproteases, cathepsins, and urokinase) have been observed to be synthesized in the tumor stroma, as a host reaction to the disease. Some of these proteases are thought to bind to receptors on tumor cell surfaces or to act through indirect mechanisms for promotion of malignaant processes. In contrast, they have now characterized a novel, tyrpsin-related serine protease whose synthesis is up-regulated directly in human carcinomas of various sites, including breast, ovary, endometrium, and colon. Expression of the protease is negligible in ovarian tumors of stromal origins and in human sarcomas with various histological grades and origins. A specific inhibitor of the protease, a Kunitz-type serine protease inhibitor (KSPI), has also been identified. This inhibitor is specifically localized in normal mammary gland epithelial cells and breast cancer cells in non-invasive lesions. However, expression of the inhibitor is down regulated in invasive and metastatic lesions and this protein is also prevented by an unknown mechanism from binding to the protease in disease progression. They now propose to characterize in detail the substrate specificity of the protease, focussing on its possible role to activate pro-uPA, plasminogen, and hepatocyte growth factor. Second, they propose, to examine in detail the biochemical mechanism of the interaction of the KSPI with the protease and its disruption in breast carcinogenesis. Finally, they will examine in a pilot study expression of the protease and the inhibitor in primary human breast tumors and overexpress the inhibitor in a metastatic cell line to assess the effects of the inhibitor in a xenograft sitting in vivo. These studies could lead to a new perspective on invasive mechanisms in the disease and provide new avenues for its therapy. In addition, they will focus the research of a protease biochemist early in his career on the problem of metastasis.
