Flavokawains Degrade S-Phase Kinase-Associated Protein 2 (Skp2) Via Deneddylation Of Cullin-1 And Inhibit Prostate Cancer In The Transgenic Adenocarcinoma Of The Mouse Prostate (Tramp) Model

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The E3 ubiquitin ligase Skp2 was found to be overexpressed in the majority of primary prostate cancer (PCa) and pre-malignant high-grade prostatic intra-epithelial neoplasia (HG-PIN) lesions. In a search for potential Skp2 targeting agents for PCa chemoprevention, we found that flavokawains (including A and B), novel chalcones isolated from the Kava plant, potently decreased protein levels of Skp2 in multiple PCa and other cancer cell lines, including colon, cervical, bladder, melanoma and sarcoma cancer without affecting Skp2 mRNA expression or transcriptional activities. These results indicated that the effects of flavokawains on Skp2 protein expression are not dependent on the genetic backgrounds of these cell lines, suggesting a direct regulation of Skp2 expression by flavokawains. Further studies showed that flavokawains accelerated the degradation of Skp2. Skp2 degradation can be regulated by CDH1 or by autocatalytic degradation. Flavokawains did not increase the expression of CDH1, but decreased cullin-1 neddylation levels in these cell lines. Inhibition of cullin-1 function by expression of a dominant-negative cullin-1 into cells rescued flavokawain B-induced Skp2 degradation, while siRNA knock-down of CSN5 acted synergistically with flavokawain B in degradation of Skp2. In an in vitro neddylation reconstitution assay, flavokawains inhibited the neddylation of both cullin-1 and UBC12, the E2 ligase of neddylation. These results suggest that flavokawains directly inhibit cullin-1 neddylation, resulting in autoubiquition and subsequent degradation of Skp2. To determine the preventive and therapeutic efficacies of flavokawain A, TRAMP mice were fed with flavokawain A supplemented food. In the prevention protocol, TRAMP mice were fed with 3 g flavokawain A /kg food starting at 6 weeks of age and ending at 12 weeks of age, resulting in numbers of HG-PIN and moderately differentiated adenocarcinomas in flavokawain A fed mice which were significantly decreased compared to the mice fed with control food (p<0.01). In the intervention protocol, in which TRAMP mice were fed with 6 g flavokawain A /kg food starting at 12 weeks of age and ending at 24 weeks of age, the average genitourinary weight of flavokawain A fed mice was significantly reduced when compared to control food fed mice (1.45±0.88 gram vs.2.22±0.77 gram, p<0.05, Mann-Whitney U and Kolmogorov-Smirnov test). No toxicity of flavokawain A treatment was observed either in body or organ weight or in histology examination. Western blotting analysis of prostate tissue lystates revealed that the protein levels of Skp2 were markedly reduced in the prostates and tumors of flavokawain A fed mice compared to control. Taken together, Flavokawains are novel Skp2 targeting agents, deserving of further investigation for PCa chemoprevention and therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 857. doi:10.1158/1538-7445.AM2011-857