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.
| Sathler, Plinio Cunha; Lourenco, Andre Luiz; Miceli, Leonardo Alves et al. (2014) Structural model of haptoglobin and its complex with the anticoagulant ecotin variants: structure-activity relationship study and analysis of interactions. J Enzyme Inhib Med Chem 29:256-62 |
| LeBeau, Aaron M; Duriseti, Sai; Murphy, Stephanie T et al. (2013) Targeting uPAR with antagonistic recombinant human antibodies in aggressive breast cancer. Cancer Res 73:2070-81 |
| Darragh, Molly R; Schneider, Eric L; Lou, Jianlong et al. (2010) Tumor detection by imaging proteolytic activity. Cancer Res 70:1505-12 |
| Sounni, Nor E; Dehne, Kerstin; van Kempen, Leon et al. (2010) Stromal regulation of vessel stability by MMP14 and TGFbeta. Dis Model Mech 3:317-32 |
| Littlepage, Laurie E; Sternlicht, Mark D; Rougier, Nathalie et al. (2010) Matrix metalloproteinases contribute distinct roles in neuroendocrine prostate carcinogenesis, metastasis, and angiogenesis progression. Cancer Res 70:2224-34 |
| Kessenbrock, Kai; Plaks, Vicki; Werb, Zena (2010) Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 141:52-67 |
| Lederle, Wiltrud; Hartenstein, Bettina; Meides, Alice et al. (2010) MMP13 as a stromal mediator in controlling persistent angiogenesis in skin carcinoma. Carcinogenesis 31:1175-84 |
| Barkan, David T; Hostetter, Daniel R; Mahrus, Sami et al. (2010) Prediction of protease substrates using sequence and structure features. Bioinformatics 26:1714-22 |
| Sun, Cheng; Su, Kai-Hung; Valentine, Jason et al. (2010) Time-resolved single-step protease activity quantification using nanoplasmonic resonator sensors. ACS Nano 4:978-84 |
| Andreu, Pauline; Johansson, Magnus; Affara, Nesrine I et al. (2010) FcRgamma activation regulates inflammation-associated squamous carcinogenesis. Cancer Cell 17:121-34 |
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