Proteomic identification of extracelluar proteins differentially expressed in prostate cancer reveals novel opportunities for targeting tumor cells

B173 Prostate cancer is the second leading cause of cancer death in American men. The growing number of elderly people across the major markets will expand the overall prostate cancer incident population in the coming years. Identifying novel targets by proteomics may lead to opportunities for the development of targeted therapies or biomarkers. Additionally a significant clinical unmet need for prostate cancer is an effective drug therapy that is able to prevent the disease from progressing to androgen-refractory status or that can prolong the survival of patients with androgen-refractory disease.
 By employing mass spectrometry (MS) as a tool to identify proteins that are over-expressed in prostate tumor relative to normal prostate cells, we aimed to discover new targets that could be utilized in prostate cancer therapy. We developed proteomic methods that allowed us to focus our studies on the discovery of cell surface/secreted proteins, as they represent key antibody therapeutic and biomarker opportunities. Cell-surface and secreted proteins from normal and prostate tumor cell lines were preferentially captured, digested with trypsin and subjected to MS analysis. Peptides were first quantified, and then sequence composition of differentially expressed peptides was resolved by MS analysis. To date, we have identified in excess of 200 proteins over-expressed in prostate cancer cell lines, including known markers such as PSA, PSMA and a panel of proteins not previously associated with prostate cancer. Our analysis also showed 23 of identified proteins were uniquely found in prostate with no overlap with other oncology indications.
 Further analyses identified 37 proteins as over-expressed in androgen-refractory cell lines compared to androgen sensitive cell lines. Independently, a mouse xenograft study of androgen-dependent 22Rv1 prostate cell line was performed to investigate modulation of prostate tumor proteome expression upon androgen withdrawal. Xenografts from pre-castrated or re-grown post-castrated androgen-independent phase of the study were compared. Initial studies identified 21 proteins as over-expressed in 22Rv1 xenografts derived from post-castrated androgen-independent mice compared to pre-castrated mice.
 To validate proteins identified by MS, we performed additional confirmatory studies including immunohistochemistry (IHC), mRNA profiling, Flow Cytometry and functional analyses using RNAi-mediated transfection to evaluate the effect on prostate cell proliferation and/or apoptosis. A subset of targets was selected for validation based on criteria such as druggability of the proteins, novelty, and intensity of tumor over-expression. From this subset, 15 targets confirmed over-expression by IHC in prostate cancer tissues while 16 targets demonstrated RNAi function in prostate cell lines. Examples of such validated targets will be presented.
 These studies highlight that our large scale proteomic mapping capabilities can provide a platform for identification of novel therapeutics and biomarkers. Together with additional functional and expression characterization, this approach represents a unique opportunity for the discovery of targets that may be exploited in the diagnosis and treatment of prostate cancer.