Flavokawain B targets protein neddylation for enhancing the anti-prostate cancer effect of Bortezomib via Skp2 degradation

Xuesen Li,Xiaolin Zi,Bang H Hoang,Victor Pham,Edward Uchio,Raymond Rendon,Matthew Tippin,Dongjun Fu,Liankun Song

doi:10.1186/s12964-019-0338-2

Xuesen Li, Xiaolin Zi + Show 7 more

Open Access

https://doi.org/10.1186/s12964-019-0338-2

Copy DOI

Abstract

BackgroundFlavokawain B (FKB) has been identified from kava root extracts as a potent apoptosis inducer for inhibiting the growth of various cancer cell lines, including prostate cancer. However, the molecular targets of FKB in prostate cancer cells remain unknown.MethodsAn in vitro NEDD8 Initiation Conjugation Assay was used to evaluate the neddylation inhibitory activity of FKB. Molecular docking and a cellular thermal shift assay were performed to assess the direct interaction between FKB and the NEDD8 activating enzyme (NAE) complex. Protein neddylation, ubiqutination, stability and expression in cells were assessed with immunoprecipitation and Western blotting methods using specific antibodies. Deletion and site specific mutants and siRNAs were used to evaluate deep mechanisms by which FKB induces Skp2 degradation. Cell growth inhibition and apoptosis induction were measured by MTT, ELISA and Western blotting methods.ResultsFKB inhibits NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 neddylation in an in vitro assay. Molecular docking study and a cellular thermal shift assay reveal that FKB interacts with the regulatory subunit (i.e. APP-BP1) of the NAE. In addition, FKB causes Skp2 degradation in an ubiquitin and proteasome dependent manner. Overexpression of dominant-negative cullin1 (1–452), K720R mutant (the neddylation site) Cullin1 or the F-box deleted Skp2 that losses its binding to the Skp1/Cullin1 complex causes the resistance to FKB-induced Skp2 degradation, whereas siRNA knock-down of Cdh1, a known E3 ligase of Skp2 for targeted degradation, didn’t attenuate the effect of FKB on Skp2 degradation. These results suggest that degradation of Skp2 by FKB is involved in a functional Cullin1. Furthermore, proteasome inhibitors Bortezomib and MG132 transcriptionally down-regulate the expression of Skp2, and their combinations with FKB result in enhanced inhibitory effects on the growth of prostate cancer cell lines via synergistic down-regulation of Skp2 and up-regulation of p27/Kip1 and p21/WAF1 protein expression. FKB also selectively inhibits the growth of RB deficient cells with high expression of Skp2.ConclusionThese findings provide a rationale for further investigating combination of FKB and Bortezomib for treatment of RB deficient, castration-resistant prostate cancer.

Highlights

Flavokawain B (FKB) has been identified from kava root extracts as a potent apoptosis inducer for inhibiting the growth of various cancer cell lines, including prostate cancer
The neddylation of Cullin1 occurs via a conjugation cascade-the neddylation pathway, which is initiated by an E1 (i.e. NEDD8 activating enzyme, NAE) enzyme consisting of Amyloid Precursor Protein-binding Protein1 (APP-BP1) and Ubiquitin-Like Modifier Activating Enzyme 3 (UBA3) proteins
The result demonstrates that the level of NEDD8-modified Cullin-1 decreases in both DU145 and PC3 cells when treated with FKB (Fig. 1c)

Summary

Introduction

Flavokawain B (FKB) has been identified from kava root extracts as a potent apoptosis inducer for inhibiting the growth of various cancer cell lines, including prostate cancer. Neural Precursor Cell Expressed, Developmentally Down-Regulated 8 (NEDD8), an ubiquitin-like protein, plays an important role in the modification of Cullin-1 to turn on the Skp1-Cullin-F box protein (SCF) complex for regulation of the stability of its target proteins [1]. The neddylation of Cullin occurs via a conjugation cascade-the neddylation pathway, which is initiated by an E1 (i.e. NEDD8 activating enzyme, NAE) enzyme consisting of Amyloid Precursor Protein-binding Protein (APP-BP1) and Ubiquitin-Like Modifier Activating Enzyme 3 (UBA3) proteins. Many components of the neddylation pathway, such as NEDD8, NAE and DCN1, have been reported to be over-expressed in several cancers [2,3,4]. The neddylation pathway could be targeted for development of novel cancer therapies. There is a need for development of more efficient or less toxic NAE inhibitors or novel combination therapies

Methods

Results

Discussion

Conclusion