Determining Gene Expression, Substrate Specificity and Natural
Substrates for Prostate-Associated Proteases

    In spite of the myriad advances in prostate cancer diagnosis and treatment strategies, it was estimated that in 1997, more than 41,000 men died as a result of this disease (Parker SL, et al, Cancer J Clin. 1997;47:5-27). Recent progress in experimental and theoretical biochemistry presents new approaches to fighting this disease at the molecular level. A class of enzymes known as proteases have been implicated in all stages of cancer progression, including the growth and metastasis of cancer cells. This ability of the malignant cancer cells to invade the surrounding tissue and spread to other sites of the body (metastasis) greatly complicates the treatment of cancer in humans. Inhibition of metastasis through the inhibition of proteases offers a form of treatment that redirects cancer therapy from general cytotoxicity to specific points of molecular intervention and offers hope of new treatment modalities.

    We have discovered two novel proteases expressed in prostate cancers that are implicated in the growth and metastasis of a prostate cancer cell line in animal model studies performed in collaboration with Dr. Marc Shuman at UCSF. Since proteases are enzymes that work by cutting other proteins and peptides, we propose to understand the function of these prostate-derived proteases by examining what amino acids are preferred for protein and peptide cleavage. As a first step, we will screen a chemically synthesized library of peptides to find the composition of amino acids that results in preferred cleavage by the proteases. Additional cleavage information will be derived from a second combinatorial library known as a substrate phage display library that allows selection of a full peptide sequence rather than a truncated, chemically synthesized sequence. This data will assist in the design and synthesis of highly selective and potent protease inhibitors that can be tested in later prostate cancer studies. In addition, knowledge of the ideal composition for cleaved peptides allows the prediction of natural substrates (cleaved proteins) that contain these preferred amino acids for cleavage.

    These predicted protein cleavages can be directly tested in systems where the protein can be synthesized from DNA sequences in small amounts in a test tube. This system will be expanded to screen for novel substrates using small pools of synthesized proteins. If a protein within the pool is cleaved, then the pool can be split until the cleaved protein is identified. The next proposed step is to characterize the quantity and location of identified proteases and substrates within prostate cancer tumors. This analysis can be performed first by examining the levels of messenger RNA (mRNA) that is synthesized within the tumor. Since mRNA is translated into proteins, more mRNA generally leads to more produced proteins. Overproduced proteases such as Prostate Specific Antigen (PSA) may result in the development of more diagnostic markers for prostate cancer, and may give more specific information such as the stage of prostate cancer present.

    In addition to the overall production of the proteases and substrates, we propose to identify the specific location of these proteins within a tumor. These studies can be performed using antibodies that will bind specifically to the target proteins. Tracking of the antibodies reveals the location of the target proteins. These studies are essential for understanding the cancer biology of the proteases and their substrates. Finally, we hope to categorize expressed proteases and discover other novel protease targets by using laser microdissection of prostate tumors, in collaboration with Dr. Shuman's laboratory. We plan to dissect the most aggressive, cancerous cells and quantify the proteases that are present and may be contributing to the aggressive characteristics. In summary, the identification of substrates and characterization of protease and substrate levels will help elucidate the function of these proteases and help evaluate the possible therapeutic potential for the treatment of prostate and/or other cancers.