The NEDD8 pathway mediated Skp2 degradation in chemoprevention by FKA

项目摘要 在未经选择的人群中进行化学预防在很大程度上是不成功的；因此 通过分子分型对高危人群进行化学预防可能是一种很有前途的未来策略。 Flavokawain A(FKA)是从卡瓦植物中鉴定出的一种天然查尔酮。流行病学研究 已经表明，食用卡瓦可能与#年前列腺癌(PCA)的发病率低有关 卡瓦消费量最高的三个国家(瓦努阿图、斐济和西萨摩亚)。我们已经证明了FKA 与正常细胞相比，选择性地抑制前列腺癌细胞系和RB缺陷细胞的生长。此外, 饲喂FKA可抑制高级别前列腺上皮内瘤变病变的形成和 前列腺癌，减轻了肿瘤负担，完全消除了远处器官转移。 TRAMP转基因小鼠模型没有表现出任何毒性。 FKA的作用机制，特别是FKA的抗癌分子靶点(S)仍然存在 很大程度上是未知的。因此，我们进行了广泛的初步研究。我们发现(1)FKA是5 对过度表达Skp2的PC3细胞的生长抑制作用更强(IC50约为8M) (2)FKA通过蛋白酶体加速Skp2的降解， 泛素化依赖途径；(3)FKA对Skp2的降解不依赖于CDH1的表达，而是依赖于CDH1的表达 (4)FKA体外抑制NEDD8与Uba3和Ubc12的结合 (5)由于FKA含有α，β不饱和的羰基部分，FKA可能与SH发生反应 Uba3和Ubc12中的半胱氨酸基团。FKA类似物和α，β-不饱和羰基的焦点文库 因此，化合物已经准备好进行结构-活性关系研究。基于这些初步的 研究表明，我们假设FKA作为一种新的NEDD8结合抑制因子而导致Skp2 泛素化和降解；FKA通过导致积聚的 Skp2底物(即p27、FOXO1、-TrCP等)导致细胞周期停滞和细胞凋亡。 为了检验假设，我们将确定：(I)FKA抑制FKA过程的机制 NEDD8接合；(Ii)FKA抑制NEDD8接合是否与Skp2有因果关系 降解；(Iii)FKA或FKA代谢物在小鼠前列腺和血浆中的活性浓度 可实现有效抑制NEDD8结合和下调Skp2；(Vi)是否 FKA在其有效浓度下对靶标(即NEDD8和Skp2)的抑制将阻止或推迟 前列腺癌的发生；(V)是否可以通过研究 FKA类似物和α，β-不饱和羰基化合物在DNEDD化反应中的构效关系 Skp2降解。 由于pRb和pten缺失在前列腺癌中很常见，而Skp2已被证明是一种主要的致癌因素 靶标在pRb或pten缺失引发癌症的过程中，这些分子提供了具有吸引力的靶标 化学预防。对上述问题的回答将为预防前列腺癌带来新的影响 确定一种新的靶向肿瘤抑制基因pRb或Pten缺失介导的PCA启动途径的化合物。

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 α, β-unsaturated carbonyl moiety , FKA may react with SH groups of cysteine in the Uba3 and Ubc12. A focused library of FKA analogs and α, β-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 α, β-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.