Chemotherapy Induces NEDP1-Mediated Destabilization of MDM2

Home , Cullin, Mdm2, NEDD8

Oncogene (2010) 29, 297–304 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc SHORT COMMUNICATION Chemotherapy induces NEDP1-mediated destabilization of MDM2

IR Watson1,2,BKLi1,2, O Roche1, A Blanch2, M Ohh1 and MS Irwin1,2,3

1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; 2Cell Biology Program, Hospital for Sick Children, Toronto, Ontario, Canada and 3Department of Paediatrics and Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada

MDM2 is an E3 ligase that promotes ubiquitin-mediated In response to DNA damage, p53 becomes phos- destruction of p53. Cellular stresses such as DNA damage phorylated by several kinases within the MDM2- can lead to p53 activation due in part to MDM2 binding domain, which prevents MDM2–p53 interac- destabilization. Here, we show that the stability of tion (Bode and Dong, 2004). The stabilization of p53 MDM2 is regulated by an ubiquitin-like NEDD8 pathway then leads to DNA repair, cell cycle arrest, senescence or and identify NEDP1 as a chemotherapy-induced isopepti- apoptosis. Recent studies have shown that MDM2 is dase that deneddylates MDM2, resulting in MDM2 destabilized in response to DNA damage, which promotes destabilization concomitant with p53 activation. Concor- p53 activation (Stommel and Wahl, 2004; Meulmeester dantly, RNAi-mediated knockdown of endogenous et al., 2005). NEDD8 is a ubiquitin-like protein that NEDP1 blocked diminution of MDM2 levels and regulates protein function through covalent modification increased chemoresistance of tumor cells. These findings of substrates such as Cullins, BCA3, EGFR, ribosomal unveil the regulation of MDM2 stability through NEDP1 L11 protein, VHL, p73 and p53 (Xirodimas, 2008). For as a common molecular determinant governing che- example, NEDD8 modification of Cullins not only motherapy-induced p53-dependent cell death. regulates the enzymatic activity of Cullin-based E3 Oncogene (2010) 29, 297–304; doi:10.1038/onc.2009.314; ubiquitin ligases (Pan et al., 2004), but also influences published online 28 September 2009 Cullin protein stability (Morimoto et al., 2003; Wu et al., 2005). Neddylation of p53 mediated by MDM2 was Keywords: chemotherapy; MDM2; NEDD8; NEDP1; p53 shown to inhibit p53-transcriptional activity (Xirodimas et al., 2004). Recent evidence suggests that MDM2 itself is subjected to NEDD8 modification. However, the molecular mechanisms governing MDM2 neddylation and its Introduction biological significance remain unclear. Here, we examined the role of NEDD8 pathway in Deregulation of the p53 pathway is observed in most the regulation of MDM2. We identify for the first time a human tumors. Approximately 50% of tumors harbor biological stimulus that affects the expression of a inactivating mutations in the p53 gene, whereas the NEDD8-specific isopeptidase NEDP1, and show that remaining tumors show suppressed, albeit wild-type modulation of NEDP1 upon various DNA-damaging p53 activity owing to a variety of mechanisms including chemotherapeutic drugs regulates p53 activity through overexpression of MDM2, which represents an alter- regulation of MDM2 protein stability. Furthermore, native and direct mechanism of p53 inactivation (Toledo RNAi-mediated suppression of NEDP1 increased MDM2 and Wahl, 2006). MDM2 is also a critical regulator of levels and chemoresistance of tumor cells. These findings p53 during development, shown by the embryonic uncover a novel role of NEDP1 in p53 activation in lethality phenotype of MDM2-null mice that is rescued response to chemotherapy. by concomitant loss of p53 (Jones et al., 1995; Montes de Oca Luna et al., 1995). In addition, an E3 ligase- defective MDM2 mutant was unable to rescue MDM2- Results and discussion null mice phenotype (Itahana et al., 2007). These observations establish the biological importance of Neddylation stabilizes MDM2 MDM2 E3 activity in the negative regulation of p53. Ectopic expression of T7-NEDD8, but not T7- NEDD8DGG mutant that lacks the essential C-terminal glycine–glycine motif required for covalent conjugation Correspondence: Dr MS Irwin, or Dr M Ohh, Department of of substrates, promoted significant MDM2 neddylation Paediatrics and Cell Biology, The Hospital for Sick Children, 555 (Figure 1a). Although this observation is consistent with University Avenue, Black Wing-9th floor, Toronto, Ontario, Canada a previous report showing MDM2 as a substrate of M5G 1X8. E-mails: [email protected] or [email protected] NEDD8 modification (Xirodimas et al., 2004), we noted Received 13 April 2009; revised 27 August 2009; accepted 1 September a dramatic dose-dependent increase in MDM2 levels 2009; published online 28 September 2009 with increasing amounts of T7-NEDD8 (Figure 1b), NEDP1 promotes MDM2 destabilization IR Watson et al 298

Figure 1 Neddylation of MDM2 increases its protein stability. (a) HEK293 cells transfected with plasmids encoding human MDM2, T7-NEDD8 or conjugation-defective T7-NEDD8DGG, were lysed under denaturing conditions, resolved by SDS–PAGE and immunoblotted with the indicated antibodies (left panel) as previously described (Watson et al., 2006). Cells transfected with empty plasmids (MOCK) or plasmids encoding T7-NEDD8 or T7-NEDD8DGG were lysed, equal amounts of whole cell extracts resolved by SDS–PAGE and immunoblotted with an anti-T7 antibody to validate the conjugation capability of the wild-type T7-NEDD8 versus T7-NEDD8DGG (right panel). (b) Cells were transfected with plasmids encoding MDM2 (left panel) or human HA-MDMX (right panel) (kindly provided by Dr Aart Jochemsen) with increasing amounts of T7-NEDD8 or T7-NEDDDGG. Cells were lysed under denaturing conditions, equal amounts of whole cell extracts resolved by SDS–PAGE and immunoblotted with either anti-MDM2 or anti-MDMX antibodies. (c) Cells transfected with plasmids encoding MDM2 with increasing amounts of His–Ubc12 or dominant- negative Ubc12(C111S) (DN-Ubc12) (kindly provided by Dr Edward Yeh) were lysed, resolved by SDS–PAGE and immunoblotted with the indicated antibodies. (d) Cells were transfected with empty plasmids (MOCK) or plasmids encoding T7-NEDD8 or T7-NEDD8DGG. Forty-eight hours post-transfection, cells were treated with 40 mg/ml cycloheximide (CHX) (Sigma, St Louis, MO, USA) for the indicated time periods, lysed, equal amounts of lysates resolved by SDS–PAGE and immunoblotted with the indicated antibodies (left panel). Protein levels of endogenous MDM2 were quantiﬁed by densitometry using Image J (http://rsb.info.nih.gov/ij) software and plotted as a percentage of the no treatment baseline. A trendline was derived to determine the half-life of MDM2 (right panel) as previously described (Lau et al., 2007). IB: immunoblot.

Oncogene NEDP1 promotes MDM2 destabilization IR Watson et al 299 which was not attributable to changes in MDM2 cleaving neddylated Cul1 than COP9 signalosome transcript levels as determined by semi-quantitative (Yamoah et al., 2005). Consistent with this observation, RT–PCR (data not shown). A similar induction of deletion of NEDP1 homologs in S. Pombe and MDMX, an MDM2 structural homolog that lacks E3 Drosophila did not result in accumulation of neddylated ligase activity, was not observed with increasing Cul1 or Cul3 (Zhou and Watts, 2005; Chan et al., 2008). amounts of T7-NEDD8 (Figure 1b). However, However, a significant accumulation of as yet to-be- MDM2(C464A) RING finger mutant that lacks E3 identified neddylated substrates was noted in both ligase activity was modified, albeit to a lesser extent, by model systems upon deletion of NEDP1 homologs. NEDD8 and its expression level was increased by Thus, we asked whether NEDP1 had a role in MDM2 ectopic expression of NEDD8 (Supplementary deneddylation. Ectopic expression of myc-NEDP1, but Figure 1). Therefore, we asked whether the known not HA-CSN5, markedly attenuated the levels of NEDD8 E2-conjugating enzyme Ubc12 modulates neddylated MDM2 in a dose-dependent manner under MDM2 levels. HEK293A cells transfected with increas- conditions that favored robust MDM2 neddylation ing amounts of His–Ubc12 showed a dose-dependent (Figure 2a). Furthermore, cysteine protease activity of increase in MDM2 (Figure 1c, left panels). However, NEDP1 is required for MDM2 deneddylation as the cells expressing increasing amounts of the dominant- catalytically defective myc-NEDP1(C163A) mutant negative Ubc12(C111S), which forms a stable interac- failed to deneddylate MDM2 (Figure 2b). Importantly, tion with NEDD8 (Figure 1c, bottom right panel) and expression of NEDP1 decreased MDM2 levels while an therefore has limited ability to transfer NEDD8 to ubiquitin-specific isopeptidase HAUSP increased target substrates, showed a striking dose-dependent MDM2 levels (Figure 2c). Moreover, ectopic expression decrease in MDM2 (Figure 1c, right panels). We next of myc-NEDP1 appeared to accelerate the turnover of asked whether the observed changes in MDM2 expres- endogenous MDM2 in contrast to the myc-NEDP1(- sion levels were because of alterations in MDM2 protein C163A) mutant (Figure 2d). These results show that stability. Endogenous MDM2 half-life was approxi- deneddylation destabilizes MDM2. mately 30–40 min in cells expressing T7-NEDD8DGG mutant or an empty plasmid (Figure 1d). In contrast, cells ectopically expressing T7-NEDD8 showed a near Chemotherapy increases NEDP1 levels threefold increase in MDM2 half-life to 1.6 h MDM2 destabilization in response to DNA-damaging (Figure 1d). Similar results were observed in U2OS agents has been shown to have a critical role in p53 osteosarcoma cell line (Supplementary Figure 2). activation (Toledo and Wahl, 2006). Recently, the Notably, only a minor fraction of MDM2 was observed activity of COP9 signalosome deneddylating complex to be neddylated at a given time (see Figure 1a). was shown to increase in response to apoptotic stimuli However, conjugation and de-conjugation of ubiquitin whereas ubiquitin E2 enzyme Ubc5b/c protein levels and ubiquitin-like proteins such as NEDD8 and SUMO were shown to decrease in response to doxorubicin is a dynamic process and hence, a greater proportion of treatment (Saville et al., 2004; Hetfeld et al., 2008). MDM2 is likely subjected to NEDD8 modification. In Considering the present observation that the NEDD8 support of this notion, recent studies have shown that pathway modulates MDM2 stability, we asked whether mutants lacking the NEDP1 homologs in S. Pombe and DNA-damaging chemotherapeutic agents regulate the Drosophila significantly increased the profile of neddy- components of the NEDD8 pathway, in particular lated proteins that otherwise would not have been NEDP1 and Ubc12. Although we did not observe detected (Zhou and Watts, 2005; Chan et al., 2008). discernible changes in Ubc12 levels in a variety of cell These results suggest that NEDD8 conjugation pro- lines following treatment with several chemotherapy motes MDM2 protein stabilization. drugs (data not shown), NEDP1 proteins levels were consistently induced in several cancer cell lines in response to multiple chemotherapeutic agents, including NEDP1-mediated deneddylation promotes MDM2 topoisomerase inhibitors doxorubicin and camptothecin destabilization and the radiomimetic neocarzinostatin (Figure 3a, A corollary prediction is that deneddylation would lead Supplementary Figure 3 and data not shown). Notably, to MDM2 destabilization. The two most commonly the increase in NEDP1 levels corresponded with studied NEDD8-specific proteases are the COP9 signa- elevated p53 and p53-target gene products PUMA and losome complex, in which CSN5/JAB1 possesses the p21, which were consistent with diminishing MDM2 protease activity and the more recently identified expression (Figure 3a). In addition, the rise in NEDP1 NEDP1 (Gan-Erdene et al., 2003; Mendoza et al., upon chemotherapy treatment paralleled the appearance 2003; Wu et al., 2003). The importance of COP9 of cleaved PARP, a marker of apoptosis (Figure 3a). In signalosome-dependent regulation of Cullin family support of a role of NEDP1 in activating p53, ectopic members is well established. CSN5 was recently shown expression of myc–NEDP1 decreased MDM2 levels and to stabilize MDM2 by inhibiting its autoubiquitylation correspondingly increased p53 and p53-target PUMA function (Zhang et al., 2008), which suggests that CSN5 levels in MCF7 cells treated with doxorubicin is an unlikely candidate to promote MDM2 deneddyla- (Figure 3b). Furthermore, doxorubicin treatment of tion. NEDP1 can also deneddylate Cullins in vitro, but p53À/À SAOS2 osteosarcoma cells increased NEDP1 NEDP1 shows three orders of magnitude less activity in levels (Supplementary Figure 3c, d, and e) and ectopic

Oncogene NEDP1 promotes MDM2 destabilization IR Watson et al 300

Figure 2 NEDP1-mediated deneddylation decreases MDM2 stability. (a) HEK293 cells were transfected with plasmids encoding human MDM2, T7-NEDD8 and increasing amounts of wild-type myc–NEDP1 or HA-CSN5 (kindly provided by Dr Xin Chen). Cells were lysed under denaturing conditions, equal amounts of whole cell extracts were resolved by SDS–PAGE and immunoblotted with the indicated antibodies. (b) HEK293 cells transfected with plasmids encoding human MDM2, T7-NEDD8 and increasing amounts of wild-type myc–NEDP1 or myc–NEDP1(C163A), and western blot analysis was performed as in (a). pcDNA3–myc–NEDP1 and the pcDNA3–myc–NEDP1(C163A) plasmids were generated from templates kindly provided by Dr Ronald T. Hay by PCR with primers (50-GAC GAC AAG CTT GCC ACC ATG GAG CAG AAG CTG ATC TCC GAG GAG GAC CTG GAA TTC GAC CCC GTA GTC TTG AGT TAC-30 and 50-GAC GAC CTC GAG CTA CTT TTT AGC AAG TGT GGC-30), digested with HindIII and Xho1 and ligated in-frame into a pcDNA3.1 þ vector. (c) H1299 cells were transfected with plasmids encoding human MDM2, NEDP1 or the deubiquitinating enzyme myc–HAUSP (kindly provided by Dr Roger Everett). Cells were lysed, resolved by SDS–PAGE and immunoblotted with the indicated antibodies. (d) U2OS cells were transfected with empty plasmids (MOCK) or plasmids encoding myc–NEDP1 or myc–NEDP1(C163A). Cells were treated with 40 mg/ml cycloheximide (CHX) (Simga) for the indicated time periods, lysed and equal amounts of lysates resolved by SDS–PAGE and immunoblotted with the indicated antibodies. IB: immunoblot.

expression of p53 in p53À/À H1299 lung adenocarci- NEDP1 modulates p53-apoptotic response noma cells had a negligible effect on NEDP1 expression to chemotherapy in contrast to a known p53 target p21 (Supplementary The efﬁcacy of chemotherapy is dependent on a Figure 3a), suggesting that NEDP1 is not a target of successful execution of p53-mediated apoptosis to over- p53. In addition, the induction of NEDP1 was similar in ride the survival signals acquired by cancer cells. For AT cells (GM16666) and AT isogenic clonal cells example, tumors harboring p53 mutations are associated reconstituted with wild-type ATM (GM16667) (Supple- with chemoresistance and predict a considerably worse mentary Figure 3f, g and h). These results suggest that patient prognosis in comparison with cancers with wild- the induction of NEDP1 does not require p53 or ATM type p53 (Lowe, 1995). A prediction then is that forced and support the notion that the activation of p53 upon downregulation of NEDP1, which we show here to be chemotherapy treatment is in part due to the induction induced in response to DNA-damaging chemotherapies, of NEDP1-mediated destabilization of MDM2. would promote chemoresistance because of enhanced

Oncogene NEDP1 promotes MDM2 destabilization IR Watson et al 301 p53 target gene, and as a result MDM2 regulates p53 activity via an autoregulatory feedback. Thus, while the initial MDM2 stabilization upon NEDP1 knockdown leads to increased downregulation of p53, the resulting ultimately attenuates MDM2 levels at later time points (Figure 4a and c). These results suggest that NEDP1 has a critical role in activating p53 apoptotic response to DNA damage by modulating the levels of MDM2. An essential response to DNA damage is the uncoupling of MDM2-mediated negative regulation of p53, enabling p53-dependent DNA repair, cell cycle arrest or apoptosis. For example, in response to radiomimetic drug neocarzinostatin, MDM2 becomes phosphorylated by ATM, which abrogates MDM2 binding to the deubiquitylating enzyme HAUSP, resulting in increased ubiquitylation and degradation of MDM2 (Khosravi et al., 1999; Stommel and Wahl, 2004; Meulmeester et al., 2005). In response to UV, SUMO-specific protease SUSP4 desumoylates MDM2, which enhances MDM2 autoubiquitylation and destabilization (Lee et al., 2006). Intriguingly, these mechanisms regulating MDM2 destabilization occur early in the DNA damage response ranging from 0.5–2 h (Stommel and Wahl, 2004; Meulmeester et al., 2005; Lee et al., 2006). We show here that MDM2 destabilization is mediated through increased NEDP1-mediated deneddylation of MDM2 in response to chemotherapy-induced DNA damage (see Figure 4d). The sustained induction of NEDP1 ranges from 8–72 h post-treatment. There- fore, we appear to have identified a novel mechanism of p53 activation that occurs relatively late in response to DNA damage. p53 is also a target of NEDD8 and p53 neddylation has been shown to inhibit p53 transactiva- tion function. Similar to temporal induction of NEDP1, Xirodimas et al. showed that a decrease in p53 neddylation occurs through an unknown mechanism at later time points (post 8 h) following UV treatment (Xirodimas et al., 2004). However, whether NEDP1 Figure 3 Chemotherapy increases NEDP1-mediated p53 activa- directly deneddylates p53 in response to DNA damage is tion. (a) MCF7 breast adenocarcinoma cells were treated with 0.1 mM not known. Recent studies have also identified putative doxorubicin (DOX) (Sigma) (top panel) and U2OS osteosarcoma NEDD8 substrates involved in DNA repair and cells were treated with 300 ng/ml neocarzinostatin (NCS) (Sigma) replication, including mismatch repair enzymes and (bottom panel). Cells were harvested at the indicated time points, replicative helicases (Jones et al., 2008; Xirodimas lysed and equal amounts of whole cell extract were resolved by SDS– PAGE and immunoblotted with the indicated antibodies. (b)MCF7 et al., 2008). Thus, it is possible that NEDP1 has cells were transfected with empty plasmid (MOCK) or plasmid- important functions in the regulation of as yet-to-be- encoding myc–NEDP1 and treated 48 h post-transfection with defined components of the DNA damage response. 0.1 mM DOX (Sigma). Cells were harvested at the indicated time Identification of biological stimuli that regulate points, lysed and equal amounts of whole cell extracts were resolved by SDS–PAGE and immunoblotted with the indicated antibodies. components of the NEDD8 pathway is one of the The left and right panels are derived from identical exposure of the outstanding questions in the field (Rabut and Peter, same immunoblot. 2008). Here, we show the expression of an NEDD8 isopetidase NEDP1 is induced by DNA-damaging MDM2-mediated suppression of p53. In support, siRNA- chemotherapeutic agents, and that induction in NEDP1 mediated knockdown of endogenous NEDP1 promoted expression occurs in a variety of tumor cell lines MDM2 stabilization, decreased p53 levels and activa- upon treatment with several chemotherapeutic drugs. tion and resulted in enhanced chemoresistance of MCF7 Our findings suggest that the MDM2–p53 axis is a cells in response to doxorubicin treatment as measured crucial target of NEDP1 and that NEDP1-mediated by reduced levels of cleaved PARP and TUNEL activation of p53 through MDM2 destabilization is a positivity (Figure 4 and Supplementary Figure 4a). common event promoting chemotherapy-induced cell Similar results were observed in HeLa cervical and 786- death. Notably, MDM2 is overexpressed in numerous O renal carcinoma cells possessing wild-type p53 cancers by a variety of gene amplification-independent (Supplementary Figure 4b). Notably, MDM2 itself is a mechanisms (Levav-Cohen et al., 2005). Interestingly,

Oncogene NEDP1 promotes MDM2 destabilization IR Watson et al 302

NEDP1 is located on chromosome 15q23 and genetic et al., 2002; Koschny et al., 2006; Huang et al., 2008; aberrations have been reported in this region in non- Suzuki et al., 2008). However, it remains an outstanding small cell lung cancers, gliomas, oligodendrigliomas question as to whether NEDP1 loss has an oncogenic and diffuse-type tenosynovial giant cell tumors (Paunu role in mediating MDM2 overexpression in cancer.

Oncogene NEDP1 promotes MDM2 destabilization IR Watson et al 303 Figure 4 siRNA-mediated downregulation of NEDP1 enhances chemoresistance. (a) MCF7 cells were transfected with siNEDP1-1 (50-GGAUGUACGUGAUAUGUAAdUdU-30 and 50-UUACAUAUCACGUACAUCCdUdU-30) or siCONTROL oligos (siGEN- OME RISC-Free Control siRNA) (Dharmacon, Lafayette, CO, USA) as previously described (Lau et al., 2007). Cells were treated 48 h post-transfection with 0.1 mM doxorubicin (DOX), harvested at the indicated time points, lysed, equal amounts of whole cell extract resolved by SDS–PAGE and immunoblotted with the indicated antibodies. (b) MCF7 cells were transfected with a second siNEDP1-2 oligo (50CAGAGAAACUGGAGGCUUUdUdU-30 and 50-AAAGCCUCCAGUUUCUCUGdUdU-30). Forty-eight hours post- transfection, cells were treated with 2 mM DOX for 48 h, trypsinized and 5 Â 104 cells were mounted on glass slides by cytospin centrifugation. TUNEL assays were performed according to manufacturer’s protocol (Promega, Madison, WI, USA) and images were taken by an inverted epifluorescence microscope (Zeiss Axiovert 200). TUNEL- and DAPI-positive cells were counted using Volocity software (Improvision, Coventry, UK) and confirmed by manual counting (left panel). Similar results were observed with additional doses of DOX. Equal amounts of whole cell extracts generated from the remaining cells were resolved by SDS–PAGE and immunoblotted with the indicated antibodies (right panel). (c) MCF7 cells were transfected with siNEDP1-2 or siCONTROL oligos and treated 48 h post-transfection with 0.1 mM DOX. Cells were harvested at the indicated time points and western blot analysis performed (left panel). Cells harvested at 72 h time point were lysed and visualized by immunoblotting with the indicated antibodies (right panel). (d) A model of NEDP1-mediated activation of p53 apoptotic response. Steady-state MDM2 levels are regulated in part by the NEDD8 pathway mediated by the E2-conjugating enzyme Ubc12 and/or other unknown factor(s) and the NEDD8-specific isopeptidase NEDP1. In response to DNA-damage, NEDP1 levels increase to mediate MDM2 deneddylation, resulting in MDM2 destabilization and increased p53 activation to ultimately trigger apoptosis.

Conﬂict of interest and Loretta Lau for helpful discussions. We thank Drs Stephen Meyn and Paul Bradshaw for providing AT reagents. The authors declare no conﬂict of interest. This work was supported by funds from the Canadian Cancer Society Research Institute (CCSRI 018460 to MO and 018054 to MSI). IRW is a recipient of the Hospital for Sick Children Acknowledgements Foundation Student Scholarship and the Canadian Institutes of Health Research (CIHR) Canada Graduate Scholarship. We thank Lynn Cheng, Joanne Lau, Roxana Sufan and Ryan OR is a recipient of the CIHR Fellowship. MO and MSI are Russell for their technical assistance. We thank Drs Uri Tabori Canada Research Chairs.

References

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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