Abstract 2326: Loss of prostate-specific antigen slows growth and alters the extracellular proteome of LNCaP human prostate cancer cells

Abstract

Abstract Prostate-specific antigen (PSA) is the most extensively used biomarker in cancer. There continues to be intense debate regarding the use of PSA in screening for prostate cancer (PCa) and how it may be used for optimal benefit. The role PSA plays in reproductive biology and PCa has been an area of ongoing interest. As a serine protease with novel substrate preferences, PSA continues to be secreted at high levels (mg/mL) even by the majority of advanced prostatic carcinomas. This protease has been shown to affect the growth or viability of a variety of cell types, including epithelial cells, osteoclasts, T-cells, and human PCa cell lines. In this study, we stably reduced endogenous PSA gene expression using lentiviral short hairpin loop RNA (shRNA) technology in the LNCaP human PCa cell line. We then investigated any resulting changes to LNCaP growth rates in vitro and ex vivo. Acting on the hypothesis that any PSA-induced changes to LNCaP behavior are the result of the enzyme's extracellular activity, we used a high-throughput shot-gun mass spectrometry-based (MS) approach to analyze the in vitro extracellular LNCaP proteome in control and PSA-targeted shRNA populations. Having demonstrated that our shRNA strategy targeted to PSA caused as much as a 90% reduction in PSA protein production compared to control cells, we next showed that in vitro doubling times are slowed from 2 days to 11 days. Subcutaneous tumors displayed an almost 50% reduction in take rate, with the final weights of those remaining only reaching 10% of controls after 8 weeks of growth. Serum-free media conditioned for 24 hours by control or PSA-targeted LNCaP populations were then subjected to global semi-quantitative proteomics analysis using the high resolution LTQ-Orbitrap mass spectrometer (Thermo Electron, San Jose, CA). In one 200 minute LTQ-Orbitrap analysis, in conjunction with the Mascot Search algorithm (Matrix Science, Boston, MA), we were able to identify 1784 proteins in the LNCaP conditioned media. Of these, 686 were specific to the control populations, 527 were specific to the reduced PSA populations, and 571 were common to both. These results demonstrate the utility of high-throughput proteomic strategies as an unbiased approach for the identification of candidate mechanisms behind novel observations in cancer biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2326.