Project Summary Chemoprevention in unselected populations has been largely unsuccessful; therefore targeting chemoprevention to high-risk populations by molecular subtyping may represent a promising future strategy. Flavokawain A (FKA) is a naturally occurring chalcone identified from the kava plant. Epidemiological studies have suggested that kava consumption may be associated with much lower prostate cancer (PCa) incidence in the three highest kava-drinking countries (Vanuatu, Fiji, and Western Samoa). We have shown that FKA selectively inhibits the growth of PCa cell lines and Rb deficient cells compared to normal cells. In addition, dietary feeding of FKA inhibited the formation of high-grade prostatic intra-epithelial neoplasia lesions and prostate adenocarcinomas, reduced the tumor burden and completely abolished distant organ metastasis.in the TRAMP transgenic mouse model without showing any toxicity. The mechanism of FKA?s action, in particular the anti-cancer molecular target (s) of FKA, remains largely unknown. We have therefore carried out extensive preliminary studies. We found that (1) FKA is five times more effective in inhibiting the growth of PC3 cells with over-expression of Skp2 (the IC50 is about 8 M) than PC3 cells expressing a vector control; (2) FKA accelerates Skp2 degradation via a proteasome and ubiquitination dependent pathway; (3) Skp2 degradation by FKA is not dependent on Cdh1 expression but associated with functional Cullin1; (4) FKA inhibits the conjugation of NEDD8 to Uba3 and Ubc12 in an in vitro assay and in cell culture; and (5) Given FKA has ?, ?-unsaturated carbonyl moiety , FKA may react with SH groups of cysteine in the Uba3 and Ubc12. A focused library of FKA analogs and ?, ?-unsaturated carbonyl compounds has therefore been prepared for a structure-activity relationship study. Based on these preliminary studies, we hypothesize that FKA functions as a novel inhibitor of NEDD8 conjugation and causes Skp2 ubiquitination and degradation; and that FKA exhibits chemopreventive activity by causing the accumulation of Skp2 substrates (i.e. p27, FOXO1, -TrCP, etc.) leading to cell cycle arrest and apoptosis. To test the hypotheses, we will determine: (i) The mechanisms by which FKA inhibits the process of NEDD8 conjugation; (ii) Whether inhibition of NEDD8 conjugation by FKA is causally associated with Skp2 degradation; (iii) The active concentrations of FKA or FKA metabolites in the mouse prostate and plasma that can be achieved for effective inhibition of NEDD8 conjugation and for down-regulation Skp2; (vi) Whether inhibition of the targets (i.e. NEDD8 and Skp2) by FKA at its effective concentrations will prevent or delay prostate carcinogenesis; (v) Whether more potent Skp2 targeting agents can be identified by studying the structure-activity relationship of FKA analogs and ?, ?-unsaturated carbonyl compounds in deNEDDylation and Skp2 degradation. Since pRb and Pten loss are common in PCa and Skp2 has been shown to be a major oncogenic target during the process of the pRb or Pten loss initiated cancer, these molecules present attractive targets for chemoprevention. Answers to the questions described above would bring novel impact on PCa prevention by defining a novel compound targeting the tumor suppressor pRb or Pten loss mediated PCa initiation pathway.