PROTEASES IN CANCER--BIOLOGY AND DRUG DEVELOPMENT

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There are three major goals of this proposal: 1) to identify the serine, metallo-, and cysteine proteases expresses by epithelial cancers of the prostate, colon, and skin, 2) to characterize the function of these proteases in transformation, tumor growth, and metastasis, and 3) to develop new chemotherapeutic leads based on inhibitions of key proteases identified in 1) and 2). We hope to exploit differential location of proteases in cancer versus normal issue as well as active site specificity for inhibitor design. Predominant proteases and the cell of origin present in the targeted human tumors will be identified by immunohistochemistry, biotinylated active site probes, and enzyme histochemistry. RT-PCR amplification of the proteases will be used to confirm sequence identity, and to subclone into expression vectors for production of reagent quantities (Core B/Project 1). Putative novel small molecule protease inhibitors will be identified by database screening procedures using molecular modeling and UCSF software. In addition, small molecule protease inhibitors will be obtained from corporate and/or academic sources including our own synthetic inhibitor, library, for testing (Project 1). A second approach will be to use protein engineering techniques to modulate macromolecular inhibitor activity and increase specificity. Phage display will be used incorporating random and directed engineering approaches. Specific macromolecules which will be investigated at the outset for inhibitory activity against proteases include Ecotin. Tissue Inhibitor of Metalloprotease, and Cystatin (Project 1). Inhibitors identified in Project 1 and by our collaborators which are potent against the enzymes identified in the targeted tumors will be tested in in vivo cellular and transgenic and immunodeficient mouse models (Project 2). Core C will develop transgenic mice which either overexpress 1) proteases in targeted tumor tissue to determine the role of these proteases or 2) inhibitors of these proteases. By inhibiting proteases either by administration of exogenous proteins or small molecules, or endogenous transgenic expression of the inhibitors in the tumor cells, we will be able to determine the role of these proteases in tumor progression. In Core D, the metabolism and elimination of inhibitors from Project 1 and our collaborators will be studied in order to improve their delivery to sites of action, and their in vivo pharmacokinetics. In Project 3, the temporal and spatial expression of proteases in transformation of squamous epithelium of the skin will be determined. In the K14-HPV16 transgenic mouse model of epithelial carcinogenesis that undergoes multistage neoplastic progression to invasive malignancy. This model will be used to identify and characterize which proteases are expressed at various stages of neoplasia as well as the biological significance of altered ECM- degrading proteases and/or inhibitor expression on neoplastic progression by genetic complementation. By taking this multifaceted, multidisciplinary coordinated approach, we are optimistic that we will 1) obtain important insights into the role of proteases in transformation as well as tumor growth and metastasis, and 2) be successful in obtaining novel therapies for treating cancer.