Abstract
Cullin-RING ubiquitin ligases (CRLs) are the largest family of E3 ligases and require cullin neddylation for their activation. The NEDD8-activating enzyme inhibitor MLN4924 reportedly blocked cullin neddylation and inactivated CRLs, which resulted in apoptosis induction and tumor suppression. However, CRL roles in ovarian cancer cell survival and the ovarian tumor repressing effects of MLN4924 are unknown. We show here that CRL4 components are highly expressed in human epithelial ovarian cancer tissues. MLN4924-induced DNA damage, cell cycle arrest, and apoptosis in ovarian cancer cells in a time- and dose-dependent manner. In addition, MLN4924 sensitized ovarian cancer cells to other chemotherapeutic drug treatments. Depletion of CRL4 components Roc1/2, Cul4a, and DDB1 had inhibitory effects on ovarian cancer cells similar to MLN4924 treatment, which suggested that CRL4 inhibition contributed to the chemotherapeutic effect of MLN4924 in ovarian cancers. We also investigated for key CRL4 substrate adaptors required for ovarian cancer cells. Depleting Vprbp/Dcaf1 did not significantly affect ovarian cancer cell growth, even though it was expressed by ovarian cancer tissues. However, depleting Cdt2/Dcaf2 mimicked the pharmacological effects of MLN4924 and caused the accumulation of its substrate, CDT1, both in vitro and in vivo. MLN4924-induced DNA damage and apoptosis were partially rescued by Cdt1 depletion, suggesting that CRL4(CDT2) repression and CDT1 accumulation were key biochemical events contributing to the genotoxic effects of MLN4924 in ovarian cancer cells. Taken together, these results indicate that CRL4(CDT2) is a potential drug target in ovarian cancers and that MLN4924 may be an effective anticancer agent for targeted ovarian cancer therapy.
Highlights
Cullin-RING ubiquitin ligases are the largest family of E3 ligases, but their roles in ovarian cancers remain uninvestigated
DDB1, VPRBP/DCAF1, CUL4B, and ROC1 were primarily localized in nuclei (Fig. 1B), whereas more CUL4A and CDT2 signals were detected in the cytoplasm of ovarian cancer cells
At the protein level, compared with non-tumor somatic cells, including mouse embryonic fibroblasts (MEFs), granular cells (GCs), and mouse ovarian surface epithelia cells, CRL4 E3 ligase components were highly expressed in ovarian cancer cell lines (Fig. 1C)
Summary
Cullin-RING ubiquitin ligases are the largest family of E3 ligases, but their roles in ovarian cancers remain uninvestigated. A DNA damage response was triggered, cellular apoptosis was induced, and remarkable anticancer effects were observed both in vitro and in vivo (4 – 6) These findings further validated CRL ubiquitin ligases as promising cancer targets and showed MLN4924 to be a novel anticancer agent. As one major target of MLN4924, its inhibition of CRL4CDT2 activity caused the accumulation of its substrate DNA replication licensing factor, CDT1, activation of a DNA damage checkpoint, and cell cycle arrest. By inducing these biochemical effects, MLN4924 could increase the anti-cancer effects of other therapeutic drugs. These results indicate that MLN4924 may be useful as an effective anticancer agent for targeted ovarian cancer therapy
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