Targeting Neddylation pathways to inactivate cullin-RING ligases for anticancer therapy

Abstract

Significance: Protein neddylation is catalyzed by an E1 NEDD8-activating enzyme (NAE), an E2 NEDD8-conjugating enzyme, and an E3 NEDD8 ligase. Known physiological substrates of neddylation are cullin family members. Cullin neddylation leads to activation of cullin-RING ligases (CRLs), the largest family of E3 ubiquitin ligases responsible for ubiquitylation and degradation of many key signaling/regulatory proteins. Thus, through modulating CRLs, neddylation regulates many biological processes, including cell cycle progression, signal transduction, and tumorigenesis. Given that NEDD8 is overexpressed and CRLs are abnormally activated in many human cancers, targeting protein neddylation, in general, and cullin neddylation, in particular, appears to be an attractive anticancer approach.

Recent advances: MLN4924, a small molecule inhibitor of NAE, was discovered that inactivates CRLs and causes accumulation of CRL substrates to suppress tumor cell growth both in vitro and in vivo. Promising preclinical results advanced MLN4924 to several clinical trials for anticancer therapy.

Critical issues: In preclinical settings, MLN4924 effectively suppresses tumor cell growth by inducing apoptosis, senescence, and autophagy, and causes sensitization to chemoradiation therapies in a cellular context-dependent manner. Signal molecules that determine the cell fate upon MLN4924 treatment, however, remain elusive. Cancer cells develop MLN4924 resistance by selecting target mutations.

Future directions: In the clinical side, several Phase 1b trials are under way to determine the safety and efficacy of MLN4924, acting alone or in combination with conventional chemotherapy, against human solid tumors. In the preclinical side, the efforts are being made to develop additional neddylation inhibitors by targeting NEDD8 E2s and E3s.

FIG. 1.

The enzymatic cascades for protein neddylation and deneddylation. Schematic representation of each step of the NEDD8 conjugation pathway, including NEDD8 precursor processing, NEDD8 activation by NAE, E2 loading, conjugation to a substrate by an E3 and recycling of NEDD8 by a NEDD8 isopeptidase. The involving enzymes in each step are listed. NAE, NEDD8-activating enzyme. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

Dynamic regulation of CRL activity by neddylation and deneddylation. The binding of unmodified cullin to CAND1 inhibits the cullin binding to the substrate receptor–adaptor module at its N-terminus. Neddylation of cullin disrupts the inhibitory binding by CAND1 and retains the CRL in an active conformation to promote substrate ubiquitylation. After dissociation of polyubiquitylated substrate from the CRL complex, CSN binds to the neddylation site of cullin and removes NEDD8 from cullin for recycling. CAND1 then binds to cullin and inactivates CRL. CAND1, cullin-associated and neddylation-dissociated-1; CRL, cullin-RING ligase; CSN, COP9 signalosome complex; RING, really interesting new gene. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 3.

Redox regulation of NRF2 via CRL E3s: Under normal physiological conditions, the NRF2 level is kept low as a result of targeted degradation by (i) CRL3 upon Keap1 binding and (ii) CRL1 upon β-TrCP binding, following GSK3-mediated NRF2 phosphorylation. Under oxidative stressed conditions, ROS on one hand inhibits cullin neddylation to inactivate CRLs and on the other hand activates AKT to block GSK3-mediated NRF2 phosphorylation, leading to suppression of NRF2 degradation. Accumulated NRF2 then translocates into the nucleus, where it complexes with MAF to bind to the ARE and transactivates the expression of antioxidant enzymes to scavenge ROS. ARE, antioxidant response element; GSK3, glycongen synthase kinase 3; Keap1, Kelch-like ECH-associated protein 1; NRF2, nuclear factor erythroid 2-related factor 2; ROS, reactive oxygen species. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

MLN4924 as an inhibitor of NAE. (A) Chemical structure of MLN4924. (B) MLN4924 blocks neddylation of all cullins tested. SK-BR3 cells were treated with 1 μM MLN4924 or DMSO vehicle control for the indicated time periods, followed by western blotting with the indicated antibodies.

FIG. 5.

MLN4924 suppresses tumor cell growth via inducing apoptosis, autophagy, and senescence. (A) Induction of apoptosis: MCF7 cells were treated with 1 μM MLN4924 for the indicated time periods, followed by western blotting with the indicated antibodies. (B–D) Induction of autophagy: MDA-MB231 cells stably expressing EGFP-LC3 were treated with 1 μM MLN4924 or DMSO vehicle control for 24 h and 48 h before photography under a fluorescent microscope (B). Detection of autophagosomes by electron microscopy (EM). SK-BR3 cells were treated for 24 h with MLN4924 (1 μM), along with DMSO vehicle control, followed by the EM analysis. Autophagosomes (arrows) are indicated in MLN4924-treated cells (C). SK-BR3 cells were treated with MLN4924 (1 μM), along with DMSO vehicle control for the indicated time periods, followed by western blotting using the indicated antibodies (D). (E, F) Induction of senescence: SK-BR3 cells were treated with MLN4924 (1 μM) for 8 h and stained with β-Gal after the drug washout for 72 h (E). SK-BR3 cells were treated with 1 μM MLN4924 for the indicated time periods, followed by western blotting with the indicated antibodies (F). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

A model for suppression of cancer cell growth and sensitization of chemo- and radiation therapies by MLN4924 (see text for description). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars