Abstract 5058: Expansion and validation of the prostate-specific antigen substrate degradome.

Kallikrein-related peptidase 3 (KLK3), better known as prostate-specific antigen (PSA), remains the most important serum biomarker in all of cancer. Its continued expression by tumors throughout the progression of prostate cancer in most patients has led to some speculation as to the possibility of PSA playing an active role in the disease. Although the use of PSA as a biomarker continues to be the subject of great discussion, our understanding of its functions in physiological and pathophysiological contexts is in its infancy. PSA is a secreted serine protease with substrate cleavage preferences similar to chymotrypsin. The principal physiological substrates of PSA in reproduction are semenogelins I and II. Currently, the number of PSA substrates, the so-called substrate degradome, has been identified by a candidate gene approach, and is limited to less than 20 gene products. Our working hypothesis is that the PSA substrate degradome is greatly understated, and that candidate identification by a bioinformatics approach, followed by biochemical validation would serve to dramatically expand the known substrates and help further our understanding of the roles played by this protease in reproduction and prostate cancer biology. Using the Merops database, a series of 32 unique consensus PSA substrate recognition sites was generated, based on reported P4-P1 residues on the N-terminal of known substrate cleavage sites. These peptide sequences were each used to search the PIR (Protein information resource) and IPA (Ingenuity Pathway Analysis) databases for the corresponding genes containing matching sequences. The resulting gene list was consolidated and filtered based on cellular localization. Cell-surface and extracellular gene products were retained for further analysis. Novel, highly active recombinant PSA was expressed, isolated, and used to biochemically validate a subset of the candidate substrates identified in silico. Finally, immunoblotting was used to determine PSA-induced cleavage of candidate substrates. The series of 32 consensus PSA substrate recognition sequences matched almost 10,000 gene products. Of these, approximately 90% were proteins not secreted or found on cell surfaces, and therefore not considered accessible to PSA. The remaining 900 gene products included cytokines, cell surface receptors, proteases, extracellular matrix proteins and immume modulators, many with reported roles in prostate cancer. They were organized by function prior to being subjected to cell-free biochemical validation for time-dependant cleavage. This study has used a combination of bioinformatics-based strategies and biochemistry to dramatically increase the list of potential PSA substrates. The information will be of great use in increasing our understanding of PSA and its functions in reproduction and cancer biology. Citation Format: Sangmi Nam, Niquiche Sangster-Guity, Theodore E. Ewachiw, Samuel R. Denmeade, Simon A. Williams. Expansion and validation of the prostate-specific antigen substrate degradome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5058. doi:10.1158/1538-7445.AM2013-5058