Mdmx Is a Substrate for the Deubiquitinating Enzyme Usp2a

N Allende-Vega, A Sparks, DP Lane and MK Saville

CR-UK Cell Transformation Research Group, Division of Molecular Medicine, Department of Surgery and Molecular Oncology, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK

It has previously been shown that ubiquitin-specific protease and MdmX are both essential negative regulators of p53 2a (USP2a) is a regulator of the Mdm2/p53 pathway. (Marine et al., 2006). Mdm2 or MdmX null mice die USP2a binds to Mdm2 and can deubiquitinate Mdm2 during embryogenesis. In both cases this lethality can be without reversing Mdm2-mediated p53 ubiquitination. Over- rescued in a p53-null background (Montes de Oca Luna expression of USP2a causes accumulation of Mdm2 and et al., 1995; Parant et al., 2001; Finch et al., 2002; promotes p53 degradation. We now show that MdmX is also Migliorini et al., 2002). Targeting Mdm2 and/or MdmX a substrate for USP2a. MdmX associates with USP2a is an approach to activate wild-type p53 for cancer independently of Mdm2. Ectopic expression of wild-type therapy (Marine et al., 2007; Toledo and Wahl, 2007; USP2a but not a catalytic mutant prevents Mdm2-mediated Wade and Wahl, 2009). Suppression of Mdm2 causes degradation of MdmX. This correlates with the ability of cell cycle arrest and in some cases apoptosis in tumour- wild-type USP2a to deubiquitinate MdmX. siRNA-mediated derived cells expressing wild-type p53 (Vassilev, 2007). knockdown of USP2a in NTERA-2 testicular embryonal Inhibiting MdmX can also have anti-tumour activity carcinoma cells and MCF7 breast cancer cells causes (Danovi et al., 2004; Gilkes et al., 2006). However, destabilization of MdmX and results in a decrease in MdmX targeting both Mdm2 and MdmX in cancer cells has protein levels, showing that endogenous USP2a participates in been observed to have a greater pro-apoptotic effect the regulation of MdmX stability. The therapeutic drug, than targeting either alone (Hu et al., 2006; Patton et al., cisplatin decreases MdmX protein expression. USP2a mRNA 2006; Wade et al., 2006; Xia et al., 2008). and protein levels were also reduced after cisplatin exposure. Mdm2 can inhibit p53 activity by binding to its The magnitude and time course of USP2a downregulation transactivation domain, and through promoting the suggests that the reduction in USP2a levels could contribute proteasomal degradation of p53 by acting as an E3 to the decrease in MdmX expression following treatment with ligase (Fang et al., 2000; Honda and Yasuda, 2000; cisplatin. Knockdown of USP2a increases the sensitivity of Michael and Oren, 2003). In mice, knockout of Mdm2 NTERA-2 cells to cisplatin, raising the possibility that increases p53 protein levels and overall transactivation suppression of USP2a in combination with cisplatin may be activity in both proliferating and post-mitotic cells. an approach for cancer therapy. However, this occurs without an apparent increase in Oncogene (2010) 29, 432–441; doi:10.1038/onc.2009.330; the specific activity of p53 (Francoz et al., 2006; Toledo published online 19 October 2009 et al., 2006). This is consistent with a predominant role of Mdm2 in normal cells of promoting p53-degradation Keywords: USP2a; MdmX; Mdm2; p53; ubiquitin; rather than transcriptional repression by direct binding deubiquitinating enzyme to p53 (Itahana et al., 2007). MdmX does not possess intrinsic ubiquitin E3 ligase activity towards p53 in vivo, but it binds to p53 and inhibits its transcriptional activity (Shvarts et al.,1996;Bottgeret al.,1999).However,thereis Introduction evidence that Mdm2–MdmX heterodimers are involved in promoting the ubiquitination of p53 (Singh et al., 2007; In response to multiple stresses including DNA damage, Uldrijan et al., 2007; Linke et al., 2008). It has also been oncogene activation and hypoxia, the tumour-suppres- suggested that MdmX may regulate the stability of p53 sor p53 is stabilized and its activity is increased leading indirectly. Mdm2 and MdmX interact through their RING to cell cycle arrest or apoptosis (Lane and Benchimol, fingers and this interaction can stabilize Mdm2 and pro- 1990; Vousden and Lu, 2002). p53 is repressed by Mdm2 mote Mdm2-mediated degradation of p53 (Tanimura et al., and its homologue MdmX, which share high sequence 1999; Gu et al., 2002). However, in mice MdmX knockout similarity particularly in the p53-binding domain and increases p53 transcriptional activity but has little or no the RING finger domains (Marine et al., 2007). Mdm2 effect on p53 levels (Marine et al., 2006). In addition, no difference in the rate of Mdm2 degradation was observed Correspondence: MK Saville, Department of Surgery and Molecular in wild-type mouse embryonic fibroblasts and mouse Oncology, University of Dundee, Ninewells Hospital and Medical embryonic fibroblasts derived from MdmX knockout mice, School, Level 6, Dundee, DD1 9SY, UK. indicating that in at least some circumstances endogenous E-mail: [email protected] Received 25 March 2009; revised 14 August 2009; accepted 15 September MdmX does not influence the half-life of Mdm2 (Francoz 2009; published online 19 October 2009 et al., 2006; Toledo et al., 2006). These observations suggest Regulation of MdmX by USP2a N Allende-Vega et al 433 that the predominant role of MdmX is to bind to p53 and Mdm2-mediated degradation of MdmX, H1299 cells repress its transcriptional activity; however, it is not clear (p53 null) were co-transfected with plasmids expressing to what extent the increased level of Mdm2 because of MdmX, Mdm2 and increasing amounts of plasmid- transcriptional activation of p53 can compensate for the expressing USP2a. We examined the effects of HAUSP loss of MdmX (Marine et al., 2006). in parallel as it has previously been shown to prevent MdmX is itself a substrate for the E3 ubiquitin ligase Mdm2-mediated degradation of MdmX (Meulmeester activity of Mdm2 (Kawai et al., 2003). Mdm2 ubiqui- et al., 2005). Mdm2 promoted MdmX degradation tinates MdmX and promotes its proteasomal degrada- (Figure 1a, lane 3 compared with lane 2). Despite tion. Upon DNA damage MdmX is rapidly degraded increasing Mdm2 protein levels, expression of USP2a via a Mdm2-dependent mechanism (Kawai et al., 2003). (lanes 4–7), like expression of HAUSP (lanes 8–11), In addition, DNA damage destabilizes Mdm2 by protected MdmX from Mdm2-mediated degradation. enhancing its proteasomal degradation (Stommel and A His box catalytic mutant of USP2a (H549A) was not Wahl, 2004). The destabilization of these two repressors able to rescue MdmX from degradation indicating that of p53 contributes to p53 activation (Wang et al., 2007). this effect is dependent on the deubiquitinating activity The deubiquitinating enzymes herpes virus-associated of USP2a (Figure 1b, lane 3). Overexpression of the ubiquitin-specific protease (HAUSP)/USP7 and ubiqui- deubiquitinating enzyme USP15 did not alter MdmX tin-specific protease 2a (USP2a) have been shown to protein levels (Figure 1c, lanes 6–8) confirming that regulate p53 by deubiquitinating components of the p53 expression of a deubiquitinating enzyme per se does not pathway. In addition, p53 can be regulated more protect MdmX from degradation. indirectly by the deubiquitinating enzyme USP5 (Dayal To investigate whether MdmX is a substrate for et al., 2009). HAUSP can deubiquitinate p53, Mdm2 USP2a we carried out an in vivo ubiquitination assay. and MdmX and overexpression of HAUSP prevents p53 degradation. However, the balance of activity of endogenous HAUSP is such that its knockdown or knockout results in destabilization of Mdm2 and MdmX and stabilization of p53 (Li et al., 2002, 2004; Cummins et al., 2004; Meulmeester et al., 2005). We have previously shown that USP2a can deubiquitinate Mdm2 without reversing Mdm2-mediated p53 ubiquitination (Stevenson et al., 2007). USP2a has oncogenic properties and its overexpression can prevent apoptosis induced by chemotherapeutic agents (Graner et al., 2004; Priolo et al., 2006). USP2a knockdown results in increased p53 protein expression and upregulation of p53 target genes (Priolo et al., 2006; Stevenson et al., 2007). USP2a mRNA is expressed in a range of normal tissues, the highest level of expression being in the testis (Lin et al., 2001; Park et al., 2002; Gousseva and Baker, 2003). USP2a is overexpressed in prostate cancer (Graner et al., 2004). This is associated with a reduction in p53 target gene expression, suggesting that USP2a overexpression could contribute to tumourigenesis by repression of p53 (Priolo et al., 2006). In this study, we show that MdmX is a substrate for USP2a. MdmX and USP2a form a complex in vitro and in vivo. Ectopically expressed USP2a deubiquitinates MdmX and prevents its Mdm2-mediated degradation. siRNA-mediated knockdown of USP2a enhances MdmX degradation, indicating that endogenous USP2a Figure 1 USP2a prevents Mdm2-mediated degradation of MdmX. (a) H1299 cells were transfected where indicated with can regulate the stability of MdmX. In addition, we expression plasmids for MdmX (6 mg), Mdm2 (3 mg) and USP2a or show that USP2a mRNA and protein levels are down- HAUSP (2.5, 5, 10 and 15 mg). Protein levels were analysed by regulated after cisplatin treatment and that knockdown western blotting. In this and subsequent experiments the level of of USP2a sensitizes cells to cisplatin. b-galactosidase (b-gal) expressed by a co-transfected plasmid was determined as an internal control (b) The deubiquitinating activity of USP2a is required for protecting MdmX from degradation. H1299 cells were transfected as shown with 6 mg of MdmX and 3 mg Results of Mdm2 expression vectors and 15 mg of constructs coding for wild-type USP2a or a catalytic mutant of USP2a (H549A). Protein USP2a deubiquitinates MdmX expression was determined by western blotting. (c) MdmX expression is not regulated by USP15. H1299 cells were transfected Ectopic expression of USP2a results in elevated levels of where indicated with expression plasmids for MdmX (6 mg), Mdm2 Mdm2 and an increase in degradation of p53 (Stevenson (3 mg) and USP2a or USP15 (5, 10, 15 mg). Protein levels were et al., 2007). To determine the effect of USP2a on analysed by western blotting.

Oncogene Regulation of MdmX by USP2a N Allende-Vega et al 434 H1299 cells were co-transfected with plasmids expressing His6-ubiquitin, Mdm2, MdmX, USP2a and HAUSP (wild-type and catalytic mutants), as indicated. The cells were treated with MG132 before harvesting to prevent the proteasomal degradation of ubiquitinated proteins. Ubiquitinated proteins were puriﬁed on Ni2 þ - agarose and ubiquitinated MdmX and Mdm2 were detected by western blotting. MdmX was ubiquitinated by Mdm2 (Figure 2, lane 2 compared with lane 1). MdmX and Mdm2 were deubiquitinated by wild-type USP2a (lane 3) but not by the USP2a catalytic site mutant H549A (lane 4). Similar results were obtained with HAUSP (lane 5) and HAUSP catalytic site mutant C223S (lane 6). These data indicate that USP2a prevents MdmX degradation by the deubiquitination of MdmX.

USP2a forms a complex with MdmX The ability of MdmX and USP2a to interact in vivo was determined by carrying out coimmunoprecipitation experiments. NTERA-2 testicular embryonal carcinoma cell lysates were incubated with anti-MdmX antibody or with rabbit IgG as a control. USP2a specifically co- precipitated with MdmX (Figure 3a). These data confirm that endogenous MdmX and USP2a can form a complex in vivo. To further explore the interaction of MdmX and USP2a bacterially expressed GST or GST–MdmX were incubated with in vitro translated (IVT) USP2a. Full- length USP2a (1-605) bound specifically to GST– MdmX (Figure 3b, lane 7). This indicates that USP2a can associate with MdmX independently of Mdm2. To map MdmX-binding sites in USP2a we used a series of deletion constructs. Deletion of both the N and C-terminus of USP2a was required to abolish binding to MdmX (lane 12). These data indicate that there are at least two binding sites for MdmX in USP2a: one between amino acids 1-200 in its N-terminal extension and one between amino acids 403–503 in its catalytic core. Similarly, deletion of both the N and C-terminus of USP2a was required to prevent its binding to Mdm2 (Stevenson et al., 2007). Figure 2 USP2a deubiquitinates MdmX in vivo. H1299 cells were transfected where indicated with 2 mg of His6-ubiquitin, 6 mgof To identify the region of MdmX involved in binding MdmX and 3 mg of Mdm2 expression vectors and 10 mgof to USP2a, we incubated GST–MdmX deletion mutants constructs coding for wild-type USP2a and HAUSP or catalytic with 35S-labelled USP2a (Figure 3c). USP2a bound to mutants of USP2a (H549A) and HAUSP (C223S). The cells were the C-terminal domain of MdmX (amino acids 393– treated with 20 mM MG132 for 4 h before harvesting. His6-tagged ubiquitinated proteins were purified using Ni2 þ -agarose beads and 490). These residues include the RING finger of MdmX analysed by western blotting for MdmX (Ub–MdmX) and Mdm2 (amino acids 437–483). The C-terminal domain of (Ub–Mdm2). Unmodified protein levels in cell lysates were MdmX consistently pulled down less USP2a than the analysed by direct western blot (lower four panels). full-length protein. This could reflect the existence of an additional binding site in MdmX or it may be due to MdmX (Figure 3d, lanes 9 and 10). Following incuba- alterations in the conformation of the C-terminal tion with Mdm2, the GST beads were washed and domain when it is expressed outside the context of the 35S-labelled USP2a was added. Pre-incubation with full-length protein. Mdm2 did not block USP2a pull down by MdmX, Mdm2 and MdmX hetero-oligomerize through their suggesting that a trimeric complex can be formed C-terminal RING finger domains (Tanimura et al., between USP2a, Mdm2 and MdmX. 1999). To determine whether Mdm2 competes for binding of USP2a to MdmX, a GST pull down assay was carried out. Full-length GST–MdmX was incubated USP2a regulates the stability of MdmX in vivo with increasing amounts of bacterially expressed Mdm2. To determine whether endogenous USP2a affects the Sufficient Mdm2 was added to saturate binding to levels of endogenous MdmX, NTERA-2 testicular

Oncogene Regulation of MdmX by USP2a N Allende-Vega et al 435

Figure 3 USP2a associates with MdmX. (a) Lysates of NTERA-2 cells were incubated with anti-MdmX antibody or control rabbit IgG. Immunoprecipitates were analysed by western blotting with anti-USP2a antibody or anti-MdmX antibodies as indicated. (b) MdmX interacts with two different domains in USP2a. 35S-methionine-labelled full-length USP2a (1-605) and N- and C-terminal deletions were incubated with GST or GST–MdmX. The upper panel shows a schematic representation of wild-type USP2a and its deletions. Bound proteins were detected by autoradiography (lower panel). (c) Mapping of USP2a-binding sites in MdmX. Full-length 35S-labelled USP2a was incubated with GST, GST–full-length MdmX (1–490) or the indicated GST–MdmX deletion mutants. Bound USP2a was detected by autoradiography (upper panel). The level of input GST and GST fusion proteins was compared by SDS– PAGE and Coomassie staining (lower panel). (d) Mdm2 does not compete for binding of USP2a to MdmX. GST–MdmX or GST alone were bound to glutathione–sepharose beads and subsequently incubated with recombinant bacterially expressed Mdm2. After 1 h, the beads were washed and 35S-labelled USP2a was added. Immobilized proteins were detected by western blotting and autoradiography. embryonal carcinoma cells and MCF7 breast cancer accumulation of endogenous wild-type p53 as observed cells were transfected with control siRNA or two siRNA previously (Stevenson et al., 2007). complementary to different sequences in USP2a. The To investigate whether the decrease in MdmX protein knockdown of USP2a was accompanied by a reduction expression following USP2a knockdown was because of in MdmX protein levels in both NTERA-2 (Figure 4a) changes in its stability, NTERA-2 and MCF7 cells were and MCF7 cells (Supplementary Figure 1a). USP2a transfected with control or USP2a siRNA and after 48 h siRNA also reduced Mdm2 protein levels and caused cycloheximide was added to inhibit protein synthesis.

Oncogene Regulation of MdmX by USP2a N Allende-Vega et al 436

Figure 4 Endogenous USP2a regulates MdmX stability. (a) NTERA-2 cells were transfected with control non-targeting siRNA or two siRNA targeting different sequences in USP2a. After 48 h, protein expression was determined by western blotting. (b and c) Forty- eight hours after transfection with control siRNA or siRNA USP2a (1) NTERA-2 cells were incubated with 20 mg/ml cycloheximide (CHX) and harvested at the indicated time points. (b) Western blots for MdmX and actin. (c) Quantiﬁcation of the western blots.

At the indicated times after the addition of cycloheximide, are consistent with an involvement of decreased USP2a the level of MdmX was analysed by western blotting. levels in destabilization of MdmX following treatment In both NTERA-2 (Figures 4b and c) and MCF7 cells with cisplatin. (Supplementary Figure 1b and c) the stability of MdmX We next directly compared the effects of USP2a was reduced by knockdown of USP2a. This is consistent knockdown and cisplatin treatment alone and in with destabilization of MdmX as a consequence of combination. Cells were transfected with control siRNA reduced deubiquitination by USP2a. These results or siRNA targeting USP2a. Forty-eight hours after indicate that endogenous USP2a regulates MdmX transfection, the cells were incubated with 20 mM protein stability. cisplatin for a further 8 h. At this time point no cell death was apparent. Cisplatin alone reduced USP2a USP2a is downregulated by cisplatin protein levels by a comparable extent to transfection Cisplatin treatment stabilizes p53, increases its trans- with siRNA targeting USP2a in both NTERA-2 cells criptional activity and causes apoptosis (Siddik, 2003). (Figure 5b, lane 2 compared with lane 3) and in MCF7 It has been shown that some p53-activating stresses cells (Supplementary Figure 2, lane 3 compared with result in a decrease in the stability of MdmX and lane 2). In addition, we observed that USP2a mRNA levels consequently a decrease in MdmX protein levels (Kawai were reduced after treatment with cisplatin (Figure 5c). et al., 2003; Gilkes et al., 2006). To investigate whether The magnitude of the cisplatin-dependent decrease in changes in USP2a expression could be involved in USP2a mRNA levels was similar to that observed regulation of MdmX following stress NTERA-2 cells following transfection with USP2a siRNA. Little or no were treated with 20 mM cisplatin for between 2 and 10 h change in MdmX mRNA levels was observed consistent as indicated (Figure 5a). We observed an increase in p53 with the principle mechanism for its decreased expres- and Mdm2 protein expression whereas the levels of sion following USP2a knockdown and DNA damage HAUSP remained constant. MdmX protein levels being destabilization of MdmX protein. These data decreased signiﬁcantly after cisplatin treatment. We also suggest that USP2a downregulation resulting from a observed a decrease in USP2a protein levels. The decrease in USP2a mRNA levels could contribute to the kinetics of the decrease in USP2a protein expression reduction in MdmX levels following DNA damage.

Oncogene Regulation of MdmX by USP2a N Allende-Vega et al 437

Figure 5 USP2a is downregulated by cisplatin. (a) NTERA-2 cells were treated with 20 mM cisplatin for the indicated times and protein expression analysed by western blotting. (b and c) NTERA-2 testicular embryonal carcinoma cells transfected with control non-targeting siRNA or siRNA USP2a (1) were treated with either 20 mM cisplatin or carrier (DMSO) for 8 h before harvesting. (b) Cisplatin and USP2a knockdown result in a comparable decrease in USP2a protein levels. Protein expression was analysed by western blotting. (c) Cisplatin decreases USP2a mRNA levels and USP2a knockdown and cisplatin in combination increase p53 transcriptional activity to a greater extent than either alone. mRNA levels of the indicated genes were quantiﬁed by real-time PCR. mRNA levels were normalized to TBP and expressed as a percentage of control (non-targeting siRNA in the absence of cisplatin). Values are means þ /À s.d. of three experiments (d) USP2a knockdown enhances the sensitivity of NTERA-2 cells to cisplatin. NTERA-2 testicular embryonal carcinoma cells were transfected with control non-targeting siRNA or siRNA USP2a (1). After 48 h the cells were treated with the indicated concentration of cisplatin for 15 h. Cell viability was measured using the CytoTox-ONE assay. Values are means þ /À s.d. of three experiments.

siRNA-mediated knockdown of USP2a and cisplatin cisplatin treatment than by either alone (Figure 5c). This in combination had a greater effect on USP2a protein indicates that together they increase p53 transactivation (Figure 5b, lane 4 and Supplementary Figure 2, lane 4) activity to a greater extent. The effect of combined and mRNA levels (Figure 5c) than either alone. USP2a USP2a knockdown and incubation with cisplatin on cell knockdown resulted in lower Mdm2 and MdmX protein viability was also investigated. Forty-eight hours after expression in both unstressed and cisplatin-treated cells. transfection, the cells were treated with the indicated This is consistent with a role of USP2a in maintaining concentration of cisplatin for a further 15 h (Figure 5d). the stability of MdmX and Mdm2. mRNA levels of the Cells transfected with the USP2a siRNA were sensitive p53 target genes Mdm2, p21 and Bax were increased to a to lower concentrations of cisplatin than the cells greater extent by combined USP2a knockdown and transfected with control siRNA.

Oncogene Regulation of MdmX by USP2a N Allende-Vega et al 438 Discussion the fingers, palm and thumb of the USP2a hand, whereas the C-terminus of ubiquitin binds into a Mdm2 is deubiquitinated by USP2a and ectopic substrate cleft at the catalytic centre. Amino acids expression of USP2a causes accumulation of Mdm2 403–503 do not include the active site Cys, QDE or His- and promotes Mdm2-mediated p53 degradation boxes but this region makes up four out of five of the (Stevenson et al., 2007). Here, we show that MdmX is ‘fingers’ which are involved in numerous contacts with also a substrate for USP2a. In addition, we observed that the ubiquitin core (Renatus et al., 2006). USP2a mRNA and protein levels are decreased after Cisplatin is a p53-activating genotoxic drug widely cisplatin treatment. This could contribute to down- used in the therapy of testicular and other cancers. regulation of MdmX following treatment with cisplatin. We observed that cisplatin treatment resulted in a MdmX and Mdm2 are two key repressors of p53. As decrease in MdmX protein expression with little or no well as promoting ubiquitination of p53, Mdm2 promotes change in MdmX mRNA levels. Strikingly, USP2a was the ubiquitination and proteasomal degradation of downregulated after cisplatin treatment. USP2a and MdmX (Kawai et al.,2003).Inthisstudy,weobserved MdmX downregulation occurred with similar kinetics. that ectopically expressed USP2a deubiquitinates MdmX In addition, the magnitude of the cisplatin-induced and consequently inhibits Mdm2-mediated MdmX de- decrease in USP2a expression was comparable with gradation. Endogenous USP2a and MdmX co-precipitate that observed following siRNA-mediated knockdown showing that they can form a complex when expressed at of USP2a. This decrease in USP2a protein levels is physiological levels. In NTERA-2 and MCF7 cells sufficient to cause destabilization of MdmX. These knockdown of endogenous USP2a causes a reduction in observations suggest that cisplatin-induced down- MdmX protein levels and stability. This is consistent with regulation of USP2a could contribute to the decrease destabilization of MdmX as a result of reduced USP2a- in MdmX levels in NTERA-2 cells resulting from mediated deubiquitination. USP2a binds to Mdm2 and treatment with the drug. is also required to maintain the stability of Mdm2 We observed that incubation with cisplatin resulted in (Stevenson et al., 2007). We observed that Mdm2 did a decrease in USP2a mRNA levels. This provides a not compete with USP2a for binding to MdmX. This mechanism for the cisplatin-induced decrease in USP2a indicates that USP2a can interact with MdmX in the protein expression. Cisplatin like other DNA-damaging presence of Mdm2, suggesting that a trimeric protein drugs can inhibit transcription. Cisplatin binds cova- complex may be formed. USP2a could thus control lently to DNA and forms intrastrand cross-links the stability of the Mdm2/MdmX complex. The ability between neighbouring purine residues. This can cause of USP2a to regulate the stability of both MdmX and stalling of RNA polymerase II at sites of damage and Mdm2 provides a way to co-ordinately regulate these sequestration of factors required for transcription (Vichi two repressors of p53. This is of relevance to the physio- et al., 1997; Cullinane et al., 1999; Siddik, 2003). Under logical regulation of p53 and makes USP2a an attractive our experimental conditions cisplatin selectively reduced potential target for p53-activating therapeutic agents. USP2a mRNA expression. Cisplatin did not change A greater effect on the viability of cancer-derived cells has b-actin and TBP mRNA levels (data not shown) and been observed when both Mdm2 and MdmX are targeted. it increased the mRNA expression of p53 target genes. The importance of suppressing both Mdm2 and MdmX Similarly, cisplatin was observed to selectively down- has been highlighted by studies involving nutlin. This regulate mRNA levels of the anti-apoptotic gene Bcl-XL compound disrupts the interaction between Mdm2 and (Campbell et al., 2006). Cisplatin can have different p53 and consequently increases the transcriptional activity effects on transcription from different promoters (Evans of p53 (Vassilev et al.,2004).However,nutlinisnot and Gralla, 1992). Studies using gene expression arrays efficient at disrupting the interaction between MdmX and have also shown that only a subpopulation of mRNA is p53 (Hu et al.,2006;Wadeet al., 2006). In several studies downregulated by cisplatin (Previati et al., 2004; Kruse it has been shown that MdmX can attenuate the effects et al., 2007). The selectivity of the effect of cisplatin on of nutlin on cell viability, in particular on apoptosis (Hu USP2a mRNA levels could arise because transcription et al., 2006; Patton et al., 2006; Wade et al., 2006; Barboza from the USP2a gene is particularly sensitive to et al., 2008; Xia et al., 2008). cisplatin–DNA adducts or the mRNA of USP2a is Two regions of USP2a are involved in binding to unstable. The decrease in USP2a mRNA levels could be MdmX. There are binding sites in the N-terminal the result of the activation of DNA-damage-sensing extension (residues 1–200) and in the catalytic core of pathways which for example regulate transcription or USP2a (residues 403–503). The presence of multiple mRNA stability (Siddik, 2003). MdmX-binding sites in USP2a could simply increase the We show that USP2a suppression and cisplatin in overall strength of the interaction. In addition, it could combination induces greater activation of p53 than allow binding of MdmX to USP2a in the correct either alone and that USP2a knockdown sensitized cells orientation for deubiquitination or facilitate regulation to the effects of cisplatin on cell viability. USP2a of the catalytic activity of USP2a by MdmX. The crystal knockdown in combination with cisplatin resulted in structure of the catalytic core of USP2 bound to bigger increases in the mRNA levels of p53-responsive ubiquitin has been determined (Renatus et al., 2006). genes involved in both cell cycle arrest (p21) and The structure of USPs bound to ubiquitin has been apoptosis (Bax) than either alone. If cisplatin-dependent likened to a hand. The core of ubiquitin interacts with p53 activation were mediated by downregulation of

Oncogene Regulation of MdmX by USP2a N Allende-Vega et al 439 USP2a then it would be anticipated that a combination at 4 1C. The beads were washed three times with NP-40 buffer, of complete ablation of USP2a expression and cisplatin eluted in 2 Â SDS sample buffer and analysed by western blotting. treatment would have a non-additive effect on the activity of p53. However, siRNA-mediated knockdown Western blotting of USP2a only partially reduced its expression and The primary antibodies used were 4B2 for Mdm2; L523 for cisplatin in combination with USP2a knockdown had a USP2a (Abgent, San Diego, CA, USA), BL1258 for MdmX greater effect on USP2a mRNA and protein levels than (Bethyl Laboratories), HAUSP (USBiological, Swampscott, MA, USA) and anti-b-galactosidase monoclonal antibody either alone. Consistent with a requirement for USP2a (200–193) (Calbiochem, Nottingham, UK). An anti-USP2a in the stabilization of Mdm2 and MdmX, knockdown of polyclonal antibody was a gift from Dr S Wing. Cell extracts USP2a resulted in lower protein levels of both Mdm2 were lysed into 2 Â SDS sample buffer. Proteins were resolved and MdmX in cells treated with cisplatin. The lower by SDS–PAGE and transferred to nitrocellulose overnight at levels of these two repressors of p53 could be responsible 25 mA. Peroxidase-coupled anti-mouse and anti-rabbit sec- for the greater effect of cisplatin and USP2a knockdown ondary antibodies were used at a dilution of 1:10 000 (Jackson in combination on the transcriptional activity of p53. Laboratories, West Grove, PA, USA). Bound antibodies were Non-genotoxic targeting of the negative regulators of detected by enhanced chemiluminescence (Amersham, Little p53 is a very attractive strategy for cancer therapy in Chalfont, UK) or using Supersignal West Dura extended tumours containing wild-type p53. This study shows duration substrate (Pierce, Cramlington, UK). that MdmX is a substrate for USP2a and indicates that In vivo ubiquitination assay through inhibition of USP2a a therapeutic agent could The ubiquitination assay relies on the transfection of cells with a destabilize MdmX. plasmid coding His6-tagged ubiquitin followed by the capture and purification of ubiquitinated proteins from cell extracts using Ni2 þ -agarose beads. This assay has been described in detail Materials and methods elsewhere (Xirodimas et al., 2001). The purified ubiquitinated proteins were analysed by western blotting. Plasmids In vitro interaction assay Plasmids expressing human Mdm2, His6-ubiquitin, USP2a and 35 HAUSP were described previously (Stevenson et al., 2007). S-labelled USP2a was mixed with bacterially expressed GST 1 pcDNA3–MdmX (human) and pcDNA3–LacZ vectors were or GST–MdmX for 2 h at 4 C. This assay has been described kindly provided by Dr AG Jochemsen. in detail elsewhere (Stevenson et al., 2007). Complexes were resolved by SDS–PAGE and bound 35S-labelled protein detected by autoradiography. Cell culture and transfection H1299 cells were cultured in RPMI medium supplemented with Cell viability assay 10% FBS and penicillin/streptomycin at 37 1Cand5%CO2.For Cell viability was measured according to the manufacturer’s transfections 5 Â 105 cells were seeded per 10 cm dish and 2 h instructions using the CytoTox-ONE assay (Promega, South- before transfection, the medium was replaced by DMEM, 10% ampton, UK). Fluorescence was measured using a fluorescence FBS. Cells were transfected using the calcium–phosphate spectrophotometer (Hitachi, Maidenhead, UK) at an excitation method with the indicated amounts of plasmids expressing wavelength of 560 nm and an emission wavelength of 590 nm. human proteins and 2 mg of plasmid-expressing b-galactosidase (b-gal) as an internal control. Cells were washed with PBS and RNA preparation and real-time PCR harvested 36 h post-transfection. Cell extracts were lysed into Total RNA was extracted using RNeasy columns (Qiagen, 2 Â SDS sample buffer and analysed by western blotting. Crawley, UK). RNA (300–600 ng) was incubated with random NTERA-2 and MCF7 cell lines, which both express wild-type primers (Promega) and Superscript II reverse transcriptase p53, were cultured in DMEM medium supplemented with (Invitrogen) to generate cDNA. Real-time PCR was carried out 10% FBS and penicillin/streptomycin at 37 1C and in 10 and with an ABI Prism 7700 sequence detector using the following 5% CO2, respectively. For transfection with siRNA, NTERA-2 protocol: 50 1Cfor2min,951C for 10 min and 40 cycles of 95 1C and MCF7 cells were plated at 1.5 Â 105 and 3 Â 105 cells/35 mm for 15 s and 60 1C for 1 min. p21, Bax, p53-responsive Mdm2-P2 dish, respectively. Transfection with single siRNA synthetic transcript and MdmX primers and 6-FAM/TAMRA-labelled duplexes (30 nM) was carried out using Oligofectamine (Invitrogen, probes were used as described previously (Giglio et al., 2005; Paisley, UK) according to the manufacturer’s instructions. Non- Stevenson et al., 2007). USP2a primers and probes were as targeting control duplex no. 2 was purchased from Dharmacon, follows: 50-CTCGTCCATCCTCCAAGAAGAG-30,50-GAGG Lafayette, CO, USA. The siRNA USP2a (1) is identical to that AGAGCTGGGACATCCTT-30 and probe 6-FAM-AGCCCAT previously described (Priolo et al., 2006; Stevenson et al., 2007) GAGGCTCCCAGTCC- TAMRA. and siRNA USP2a (2) targets the following sequence: CUCG UCCAUACUCCAAGAA. Conflict of interest Immunoprecipitation NTERA-2 cells were lysed in NP-40 buffer: 0.5% NP-40, 50 mM The authors declare no conflict of interest. HEPES pH 7.5, 100 mM NaCl, 5 mM EDTA, 1 mM DTT and complete protease inhibitor mixture (Roche Applied Science, Burgess Hill, UK). Lysates matched for protein were incu- Acknowledgements bated for 1 h with 2 mg of anti-MdmX (Bethyl Laboratories, Montgomery, TX, USA) or rabbit IgG at 4 1C. Protein G This work was funded by Cancer Research UK. Professor Sepharose beads were added and the lysate rocked for 1 h David P Lane is a Gibb fellow of Cancer Research UK.

Oncogene Regulation of MdmX by USP2a N Allende-Vega et al 440 References

Barboza JA, Iwakuma T, Terzian T, El-Naggar AK, Lozano G. transcriptional changes in mouse embryonic stem cells reveals a (2008). Mdm2 and Mdm4 loss regulates distinct p53 activities. Mol dominant p53-like response. Mutat Res 617: 58–70. Cancer Res 6: 947–954. Lane DP, Benchimol S. (1990). p53: oncogene or anti-oncogene? Genes Bottger V, Bottger A, Garcia-Echeverria C, Ramos YF, van der Eb Dev 4: 1–8. AJ, Jochemsen AG et al. (1999). Comparative study of the Li M, Brooks CL, Kon N, Gu W. (2004). A dynamic role of HAUSP p53-mdm2 and p53-MDMX interfaces. Oncogene 18: 189–199. in the p53-Mdm2 pathway. Mol Cell 13: 879–886. Campbell KJ, Witty JM, Rocha S, Perkins ND. (2006). Cisplatin Li M, Chen D, Shiloh A, Luo J, Nikolaev AY, Qin J et al. (2002). mimics ARF tumor suppressor regulation of RelA (p65) nuclear Deubiquitination of p53 by HAUSP is an important pathway for factor-kappaB transactivation. Cancer Res 66: 929–935. p53 stabilization. Nature 416: 648–653. Cullinane C, Mazur SJ, Essigmann JM, Phillips DR, Bohr VA. Lin H, Yin L, Reid J, Wilkinson KD, Wing SS. (2001). Divergent (1999). Inhibition of RNA polymerase II transcription in human N-terminal sequences of a deubiquitinating enzyme modulate cell extracts by cisplatin DNA damage. Biochemistry 38: substrate specificity. J Biol Chem 276: 20357–20363. 6204–6212. Linke K, Mace PD, Smith CA, Vaux DL, Silke J, Day CL. (2008). Cummins JM, Rago C, Kohli M, Kinzler KW, Lengauer C, Vogelstein Structure of the MDM2//MDMX RING domain heterodimer B. (2004). Tumour suppression: disruption of HAUSP gene reveals dimerization is required for their ubiquitylation in trans. stabilizes p53. Nature 428: 1 p following 486. Cell Death Differ 15: 841. Danovi D, Meulmeester E, Pasini D, Migliorini D, Capra M, Frenk R Marine JC, Dyer MA, Jochemsen AG. (2007). MDMX: from bench to et al. (2004). Amplification of Mdmx (or Mdm4) directly contributes bedside. J Cell Sci 120: 371–378. to tumor formation by inhibiting p53 tumor suppressor activity. Marine JC, Francoz S, Maetens M, Wahl G, Toledo F, Lozano G. Mol Cell Biol 24: 5835–5843. (2006). Keeping p53 in check: essential and synergistic functions of Dayal S, Sparks A, Jacob J, Allende-Vega N, Lane DP, Saville MK. Mdm2 and Mdm4. Cell Death Differ 13: 927–934. (2009). Suppression of the deubiquitinating enzyme USP5 causes the Meulmeester E, Maurice MM, Boutell C, Teunisse AF, Ovaa H, accumulation of unanchored polyubiquitin and the activation of Abraham TE et al. (2005). Loss of HAUSP-mediated deubiquitina- p53. J Biol Chem 284: 5030–5041. tion contributes to DNA damage-induced destabilization of Hdmx Evans GL, Gralla JD. (1992). Cisplatin-induced imbalances in the and Hdm2. Mol Cell 18: 565–576. pattern of chimeric marker gene expression in HeLa cells. Biochem Michael D, Oren M. (2003). The p53-Mdm2 module and the ubiquitin Biophys Res Comm 184:1. system. Semin Cancer Biol 13: 49–58. Fang S, Jensen JP, Ludwig RL, Vousden KH, Weissman AM. (2000). Migliorini D, Lazzerini Denchi E, Danovi D, Jochemsen A, Capillo M, Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself Gobbi A et al. (2002). Mdm4 (Mdmx) regulates p53-induced growth and p53. J Biol Chem 275: 8945–8951. arrest and neuronal cell death during early embryonic mouse Finch RA, Donoviel DB, Potter D, Shi M, Fan A, Freed DD et al. development. Mol Cell Biol 22: 5527–5538. (2002). mdmx is a negative regulator of p53 activity in vivo. Cancer Montes de Oca Luna R, Wagner DS, Lozano G. (1995). Rescue of Res 62: 3221–3225. early embryonic lethality in mdm2-deficient mice by deletion of p53. Francoz S, Froment P, Bogaerts S, De Clercq S, Maetens M, Nature 378: 203–206. Doumont G et al. (2006). Mdm4 and Mdm2 cooperate to inhibit Parant J, Chavez-Reyes A, Little NA, Yan W, Reinke V, Jochemsen p53 activity in proliferating and quiescent cells in vivo. Proc Natl AG et al. (2001). Rescue of embryonic lethality in Mdm4-null mice Acad Sci USA 103: 3232–3237. by loss of Trp53 suggests a nonoverlapping pathway with MDM2 to Giglio S, Mancini F, Gentiletti F, Sparaco G, Felicioni L, Barassi F regulate p53. Nat Genet 29: 92–95. et al. (2005). Identification of an aberrantly spliced form of HDMX Park KC, Kim JH, Choi EJ, Min SW, Rhee S, Baek SH et al. (2002). in human tumors: a new mechanism for HDM2 stabilization. Antagonistic regulation of myogenesis by two deubiquitinating Cancer Res 65: 9687–9694. enzymes, UBP45 and UBP69. Proc Natl Acad Sci USA 99: 9733– Gilkes DM, Chen L, Chen J. (2006). MDMX regulation of p53 9738. response to ribosomal stress. EMBO J 25: 5614–5625. Patton JT, Mayo LD, Singhi AD, Gudkov AV, Stark GR, Jackson MW. Gousseva N, Baker RT. (2003). Gene structure, alternate splicing, (2006). Levels of HdmX expression dictate the sensitivity of normal tissue distribution, cellular localization, and developmental expres- and transformed cells to Nutlin-3. Cancer Res 66: 3169–3176. sion pattern of mouse deubiquitinating enzyme isoforms Usp2-45 Previati M, Lanzoni I, Corbacella E, Magosso S, Giuffre S, Francioso and Usp2-69. Gene Expr 11: 163–179. F et al. (2004). RNA expression induced by cisplatin in an organ of Graner E, Tang D, Rossi S, Baron A, Migita T, Weinstein LJ et al. Corti-derived immortalized cell line. Hear Res 196: 8–18. (2004). The isopeptidase USP2a regulates the stability of fatty acid Priolo C, Tang D, Brahamandan M, Benassi B, Sicinska E, Ogino S synthase in prostate cancer. Cancer Cell 5: 253–261. et al. (2006). The isopeptidase USP2a protects human prostate Gu J, Kawai H, Nie L, Kitao H, Wiederschain D, Jochemsen AG et al. cancer from apoptosis. Cancer Res 66: 8625–8632. (2002). Mutual dependence of MDM2 and MDMX in their Renatus M, Parrado SG, D’Arcy A, Eidhoff U, Gerhartz B, Hassiepen functional inactivation of p53. J Biol Chem 277: 19251–19254. U et al. (2006). Structural basis of ubiquitin recognition by the Honda R, Yasuda H.. (2000). Activity of MDM2, a ubiquitin ligase, deubiquitinating protease USP2. Structure 14: 1293–1302. toward p53 or itself is dependent on the RING finger domain of the Shvarts A, Steegenga WT, Riteco N, van Laar T, Dekker P, Bazuine ligase. Oncogene 19: 1473–1476. M et al. (1996). MDMX: a novel p53-binding protein with some Hu B, Gilkes DM, Farooqi B, Sebti SM, Chen J. (2006). MDMX functional properties of MDM2. EMBO J 15: 5349–5357. overexpression prevents p53 activation by the MDM2 inhibitor Siddik ZH. (2003). Cisplatin: mode of cytotoxic action and molecular Nutlin. J Biol Chem 281: 33030–33035. basis of resistance. Oncogene 22: 7265–7279. Itahana K, Mao H, Jin A, Itahana Y, Clegg HV, Lindstrom MS et al. Singh RK, Iyappan S, Scheffner M. (2007). Hetero-oligomerization (2007). Targeted inactivation of Mdm2 RING finger E3 ubiquitin with MdmX rescues the ubiquitin/Nedd8 ligase activity of RING ligase activity in the mouse reveals mechanistic insights into p53 finger mutants of Mdm2. J Biol Chem 282: 10901–10907. regulation. Cancer Cell 12: 355–366. Stevenson LF, Sparks A, Allende-Vega N, Xirodimas DP, Lane DP, Kawai H, Wiederschain D, Kitao H, Stuart J, Tsai KK, Yuan ZM. Saville MK. (2007). The deubiquitinating enzyme USP2a regulates (2003). DNA damage-induced MDMX degradation is mediated by the p53 pathway by targeting Mdm2. EMBO J 26: 976–986. MDM2. J Biol Chem 278: 45946–45953. Stommel JM, Wahl GM. (2004). Accelerated MDM2 auto-degrada- Kruse JJ, Svensson JP, Huigsloot M, Giphart-Gassler M, Schoonen tion induced by DNA-damage kinases is required for p53 activation. WG, Polman JE et al. (2007). A portrait of cisplatin-induced EMBO J 23: 1547–1556.

Oncogene Regulation of MdmX by USP2a N Allende-Vega et al 441 Tanimura S, Ohtsuka S, Mitsui K, Shirouzu K, Yoshimura A, Vousden KH, Lu X. (2002). Live or let die: the cell’s response to p53. Ohtsubo M. (1999). MDM2 interacts with MDMX through their Nat Rev Cancer 2: 594–604. RING ﬁnger domains. FEBS Lett 447: 5–9. Wade M, Wahl GM. (2009). Targeting Mdm2 and Mdmx in cancer Toledo F, Krummel KA, Lee CJ, Liu CW, Rodewald LW, Tang M therapy: better living through medicinal chemistry? Mol Cancer Res et al. (2006). A mouse p53 mutant lacking the proline-rich domain 7: 1–11. rescues Mdm4 deﬁciency and provides insight into the Mdm2- Wade M, Wong ET, Tang M, Stommel JM, Wahl GM. (2006). Hdmx Mdm4-p53 regulatory network. Cancer Cell 9: 273–285. modulates the outcome of p53 activation in human tumor cells. Toledo F, Wahl GM. (2007). MDM2 and MDM4: p53 regulators as J Biol Chem 281: 33036–33044. targets in anticancer therapy. Int J Biochem Cell Biol 39: 1476–1482. Wang YV, Wade M, Wong E, Li Y-C, Rodewald LW, Wahl GM. Uldrijan S, Pannekoek WJ, Vousden KH. (2007). An essential function (2007). Quantitative analyses reveal the importance of regulated of the extreme C terminus of MDM2 can be provided by MDMX. Hdmx degradation for P53 activation. Proc Natl Acad Sci 104: EMBO J 26: 102–112. 12365–12370. Vassilev LT. (2007). MDM2 inhibitors for cancer therapy. Trends Mol Xia M, Knezevic D, Tovar C, Huang B, Heimbrook DC, Vassilev LT. Med 13: 23–31. (2008). Elevated MDM2 boosts the apoptotic activity of p53- Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z MDM2 binding inhibitors by facilitating MDMX degradation. Cell et al. (2004). in vivo activation of the p53 pathway by small-molecule Cycle 7: 1604–1612. antagonists of MDM2. Science 303: 844–848. Xirodimas D, Saville MK, Edling C, Lane DP, Lain S. Vichi P, Coin F, Renaud JP, Vermeulen W, Hoeijmakers JH, Moras (2001). Different effects of p14ARF on the levels of D, Egly JM. (1997). Cisplatin- and UV-damaged DNA lure the ubiquitinated p53 and Mdm2 in vivo. Oncogene 20: basal transcription factor TFIID/TBP. EMBO J 16: 7444–7456. 4972–4983.

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

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