The NEDD8 pathway mediated Skp2 degradation in chemoprevention by FKA

FKA 化学预防中 NEDD8 通路介导的 Skp2 降解

• 批准号: 9176833
• 负责人: Xiaolin Zi
• 金额: $ 35.34万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9176833
• 关键词: Age; Androgen Receptor; Apoptosis; Biochemical; Biochemistry; Biological; Biological Availability; Bone Marrow Cells; Cell Culture Techniques; Cell Cycle Arrest; Cell Proliferation; Cells; Chemoprevention; Chemopreventive Agent; Complex; Consumption; Country; Cullin Proteins; Cysteine; Cytochrome P450; Development; Diet; Distant; Dominant-Negative Mutation; Dose; Down-Regulation; Drug Kinetics; Drug Targeting; Epidemiologic Studies; Epithelial; Exhibits; FOXO1A gene; Fiji; Future; Growth; Hepatic; Human; Immunohistochemistry; Immunoprecipitation; In Vitro; Incidence; Independent State of Samoa; Induction of Apoptosis; Kava; Lesion; Letters; Libraries; Malignant Neoplasms; Malignant neoplasm of prostate; Mediating; Metabolism; Modeling; Molecular Target; Mus; Mutate; Neoplasm Metastasis; Neoplasms; Normal Cell; Nude Mice; Oncogenes; Oncogenic; Oral; Organ; PC3 cell line; PTEN gene; Pathway interactions; Pharmacodynamics; Plants; Plasma; Population; Prevention therapy; Process; Prostate; Prostate Adenocarcinoma; Prostatic Intraepithelial Neoplasias; Proteins; Publications; Publishing; RNA Interference; Research Personnel; Signal Pathway; Skp2 Proteins; Small Interfering RNA; Specificity; Structure-Activity Relationship; Testing; Time; Tissues; Toxic effect; Transcriptional Regulation; Transgenic Mice; Transgenic Organisms; Tumor Burden; Tumor Suppressor Proteins; Ubiquitination; Vanuatu; Western Blotting; Xenograft procedure; analog; base; cancer initiation; carbonyl compound; cell growth; design; drinking; feeding; high risk; in vitro Assay; in vivo; inhibitor/antagonist; intravenous injection; killings; knock-down; molecular subtypes; mouse model; multicatalytic endopeptidase complex; neural precursor cell; novel; overexpression; p19ARF; prevent; promoter; prostate cancer cell; prostate cancer cell line; prostate cancer prevention; prostate carcinogenesis; research study; survivin; targeted agent; tibia; transgene expression; transgenic adenocarcinoma of mouse prostate; tumor; vector control

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.

项目摘要 化学预防在艾滋病人群中基本上是不成功的;因此， 通过分子分型对高危人群进行化学预防可能是一种有前途的未来策略。 Flavokawain A（FKA）是一种天然存在的查耳酮，从卡瓦植物中鉴定出来。流行病学研究 已经表明，卡瓦消费可能与前列腺癌（PCa）发病率低得多相关， 三个卡瓦酒饮用量最高的国家（瓦努阿图、斐济和西萨摩亚）。我们已经证明，FKA 与正常细胞相比，选择性抑制PCa细胞系和Rb缺陷细胞的生长。此外，本发明还提供了一种方法， FKA的饮食喂养抑制了高级别前列腺上皮内瘤病变的形成， 前列腺癌，减少了肿瘤负荷，并完全消除了远处器官metastasis.in TRAMP转基因小鼠模型未显示任何毒性。 FKA的作用机制，特别是FKA的抗癌分子靶点，仍然是未知的。 大部分未知。因此，我们进行了广泛的初步研究。我们发现（1）FKA是5 对Skp 2过表达的PC 3细胞生长的抑制效果提高1倍（IC 50约为8 M） 与表达载体对照的PC 3细胞相比;（2）FKA通过蛋白酶体加速Skp 2降解， （3）FKA对Skp 2的降解不依赖于Cdh 1的表达， 与功能性Cullin 1相关;（4）FKA在体外抑制NEDD 8与Uba 3和Ubc 12的结合。 试验和细胞培养中;和（5）鉴于FKA具有α，β-不饱和羰基部分，FKA可能与SH反应 Uba 3和Ubc 12中的半胱氨酸基团。FKA类似物和α，β-不饱和羰基的聚焦库 因此，制备化合物用于结构-活性关系研究。基于这些初步 研究中，我们假设FKA作为一种新的NEDD 8结合抑制剂发挥作用，并导致Skp 2 泛素化和降解;以及FKA通过引起 Skp 2底物（即p27、FOXO 1、-TrCP等）导致细胞周期停滞和凋亡。 为了检验假设，我们将确定：（i）FKA抑制细胞增殖过程的机制。 NEDD 8偶联;（ii）FKA对NEDD 8偶联的抑制是否与Skp 2有因果关系 （iii）小鼠前列腺和血浆中FKA或FKA代谢物的活性浓度， 可以实现有效抑制NEDD 8缀合和下调Skp 2;（vi）是否 FKA在其有效浓度下对靶点（即NEDD 8和Skp 2）的抑制将阻止或延迟 （v）是否可以通过研究前列腺癌的发生机制来鉴定更有效的Skp 2靶向剂。 FKA类似物和α，β-不饱和羰基化合物在去NEDD化和 Skp 2降解。 由于pRb和Pten缺失在PCa中很常见，Skp 2已被证明是PCa的主要致癌基因。 在pRb或Pten丢失引发的癌症过程中，这些分子是有吸引力的靶点， 化学预防。上述问题的答案将通过以下方式对PCa预防产生新的影响： 定义了靶向肿瘤抑制因子pRb或Pten丢失介导的PCa起始途径的新化合物。