PROJECT SUMMARY: FULL PROJECT 2 Even though the growth of prostate cancer (PCa) is largely driven by androgens, a subset usually develops that is refractory to androgen ablation (also known as castration resistant PCa; CRPC) with potential for metastasis. Preliminary data from our laboratory has implicated fetuin-A, also known as alpha 2-Heremans-Schmid glycoprotein (AHSG), in the growth of PCa cells and in the production of “uptake-competent” exosomes. The objective of the proposed studies is to define the role and significance of fetuin-A in prostate cancer progression. We hypothesize that PCa cells express ectopic fetuin-A which is secreted and taken up by the cells via TLR4 to mediate the biogenesis of `uptake-competent' exosomes that promote PCa growth via activation of pAKT/pERK; moreover, we postulate that elevated fetuin-A expression serves as a prognostic biomarker for PCa. Three specific aims are proposed: Aim 1. To determine if fetuin-A expression is higher in AA PCa tissues relative to Caucasian American (CA) PCa tissues and whether high fetuin-A expression is associated with high Gleason Scores (>6) and enhanced pAKT and pERK. We will analyze mRNA expression of fetuin-A using NanoString as well as pAKT/pERK protein levels using immunohistochemistry (IHC) analysis of human PCa tissues. Multivariable linear regression analysis will be used to determine the correlation between fetuin-A, pAKT, pERK and Gleason scores in PCa tissues of AA and CA patients, as well as other progression parameters such as positive margins and spread of PCa. It is expected that fetuin-A, pAKT and pERK will be expressed at high levels in PCa tissues of AA patients particularly those with high Gleason scores (>6). Aim 2. To determine the role of ectopic fetuin-A in exosome biogenesis, promotion of 2-D and 3-D growth, motility and invasive capacity of PCa cells. In this aim, we will overexpress and knockout fetuin-A in two PCa cell lines to determine whether fetuin-A plays a causal role in the biogenesis of `uptake competent' exosomes that transmit growth signals in recipient cells. We expect to demonstrate that exosomes from fetuin-A overexpressing cells will promote 2-D, 3-D growth and motility and invasion of PCa cells while exosomes from fetuin-A null cells will not. Aim 3. To investigate the efficacy of targeting fetuin-A mediated signaling on the suppression of prostate tumor initiation and growth in mice. In this aim, we will utilize the Pten-null mouse model for PCa to determine whether Pten loss requires intact fetuin-A gene to mediate its tumorigenic role and whether loss of fetuin-A in Pten-/-/fetuin-A-/- double mutant mice attenuates the tumorigenic role of Pten-null. We expect reduced tumor growth in the double mutant mice compared to Pten-null fetuin-A+/+ mice. Significance: There is an urgent need to identify biomarkers that can differentiate CRPC from indolent PCa and this proposal addresses that need and evaluates the process by which fetuin-A e...