Inhibition of NEDD8-Activating Enzyme Induces Rereplication and Apoptosis in Human Tumor Cells Consistent with Deregulating CDT1 Turnover

Michael A Milhollen,Usha Narayanan,Peter G Smith,Petter O Veiby,Benjamin Amidon,Teresa A Soucy

doi:10.1158/0008-5472.can-10-2122

Michael A Milhollen, Usha Narayanan + Show 4 more

Open Access

https://doi.org/10.1158/0008-5472.can-10-2122

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Abstract

Loss of NEDD8-activating enzyme (NAE) function by siRNA knockdown or inhibition by the small molecule NAE inhibitor MLN4924 leads to increased steady-state levels of direct Cullin-RING ligase (CRL) substrates by preventing their ubiquitination and proteasome-dependent degradation. Many of these CRL substrates are involved in cell cycle progression, including a critical DNA replication licensing factor CDT1. Cell cycle analysis of asynchronous and synchronous cultures after NAE inhibition revealed effects on cell cycle distribution and activation of DNA break repair signaling pathways similar to that reported for CDT1 overexpression. The siRNA knockdown of cullins critical for the turnover of CDT1 recapitulated the aberrant rereplication phenotype while CDT1 knockdown was suppressing. Although NAE inhibition leads to deregulation of many CRL substrates, these data demonstrate that CDT1 accumulation mediates the DNA rereplication phenotype resulting from loss of NAE function. DNA rereplication is an unrecoverable cellular insult and the small molecule inhibitor MLN4924, currently in phase I trials, represents an unprecedented opportunity to explore this mechanism of cytotoxicity for the treatment of cancer.

Highlights

Covalent attachment of ubiquitin to cellular proteins regulates many diverse cellular processes [1, 2]
The best characterized substrates modified by NEDD8 are the cullin family of proteins (CUL1, 2, 3, 4A, 4B, 5, and 7) that are components of a family of multisubunit E3 ligases known as cullin-RING ligases (CRL; refs. 9, 10)
HCT-116 colon carcinoma cells were treated with 1 mmol/L MLN4924, resulting in rapid inhibition of NEDD8-cullin conjugation and diminished CRL activity, which coincided with a reciprocal increase in cellular protein levels of various cell cycle regulators (Fig. 1A)

Summary

Introduction

Covalent attachment of ubiquitin to cellular proteins regulates many diverse cellular processes [1, 2]. The effects of tagging proteins with other ubiquitin-like proteins (Ubl) such as NEDD8 and SUMO have been described [3, 4] and include modulation of protein activity, intermolecular interactions, and subcellular localization [5]. Ubls are conjugated to their substrates via 3-step enzymatic cascades exemplified by the well-described ubiquitination cascade [6]. The best characterized substrates modified by NEDD8 are the cullin family of proteins (CUL1, 2, 3, 4A, 4B, 5, and 7) that are components of a family of multisubunit E3 ligases known as cullin-RING ligases The CRL family mediates substrate ubiquitination by recruiting a targeted protein via substrate recognition modules at the cullin N-terminus and the ubiquitin-charged E2 at the cullin C-terminus [11].

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