CBS9106-Induced CRM1 Degradation Is Mediated by Cullin Ring … · Cancer Biology and Signal Transduction CBS9106-Induced CRM1 Degradation Is Mediated by Cullin Ring Ligase Activity

Cancer Biology and Signal Transduction

CBS9106-Induced CRM1 Degradation Is Mediated by CullinRing Ligase Activity and the Neddylation Pathway

Naoya Saito1, Keiichi Sakakibara1, Takuji Sato1, JonathanM. Friedman1, DonaldW. Kufe2, Daniel D. VonHoff3,and Takumi Kawabe1

AbstractChromosome region maintenance 1 (CRM1) mediates the nuclear export of proteins and mRNAs, and is

overexpressed in various cancers. Recent studies have also reported thatCRM1protein expression is a negative

prognostic factor in patients with cancer. Therefore, CRM1 is considered a potential target for anticancer

therapy. Our previous study demonstrated that CBS9106, a synthetic small-molecular inhibitor of CRM1,

decreases CRM1 protein through proteasomal degradation without affecting CRM1 mRNA levels. However,

the mechanism by which CRM1 is degraded is not well understood. Here, we demonstrate a novel signaling

pathway that plays an important role in CBS9106-induced CRM1 degradation. We found that MLN4924, a

selective inhibitor of NEDD8-activating enzyme (NAE), effectively inhibits cullin neddylation and attenuates

CBS9106-induced CRM1 degradation in a time- and dose-dependent manner. MLN4924 also attenuated

CBS9106-induced nuclear accumulation of Ran-binding protein 1 (RanBP1), cell growth inhibition, and

apoptosis. Furthermore, RNAi-mediated knockdown of neddylation pathway proteins (NEDD8 and UBA3)

or cullin ring ligase (CRL) component protein (Rbx1) attenuated CRM1 protein degradation andG1 phase cell-

cycle arrest by CBS9106. Knockdown of CSN5 or CAND1 also partially inhibited CBS9106-induced CRM1

degradation. These findings demonstrate that CBS9106-induced CRM1 degradation is conferred by CRL

activity involving the neddylation pathway, and that this response to CBS9106 leads to cell growth inhibition

and apoptosis. Mol Cancer Ther; 13(12); 3013–23. �2014 AACR.

IntroductionChromosome region maintenance 1, CRM1, was orig-

inally identified in yeast as a gene required for maintain-ing chromosome structure (1) . CRM1 protein is nowknown to be a nuclear export receptor belonging to thekaryopherin b family of transport receptors (2). CRM1binds to different cargo proteins bearing leucine-richnuclear export sequences leading to nuclear export ofcargo proteins andmRNAs (3, 4). Exported cargo proteinsinclude tumor suppressors, such asp53, BRCA1, survivin,nucleophosmin, and adenomatous polyposis coli thatshuttle between the nucleus and cytoplasm (5) . CRM1is overexpressed in various malignancies, such as breast,cervical, ovarian, and pancreatic cancers, glioma, andosteosarcoma (6). Knockdown of CRM1 protein via

siRNA causes cell growth inhibition and apoptosis incancer cells, whereas normal tissues are unaffected byCRM1 downregulation (7). Thus, CRM1-mediated nucle-ar export is considered a potential target for treatingcancers.

The natural product leptomycin B (LMB)was identifiedas the first specific inhibitor of CRM1 (8). LMB bindscovalently to a specific cysteine residue on CRM1(Cys528), prevents complex formation between CRM1and cargo proteins, and effectively blocks nuclear exportof cargo proteins (9). LMB exhibits strong antitumoractivity in vitro against various cancer cells; however, aphase I trial of LMB had to be discontinued because ofsignificant toxicity and apparent lack of efficacy (10, 11).

We have identified CBS9106 as a novel CRM1 inhib-itor that, unlike LMB, contributes to CRM1 proteindegradation without affecting CRM1 mRNA levels.CBS9106-induced CRM1 protein degradation was coun-teracted by bortezomib, indicating that a proteasome-dependent degradation pathway was involved in theprocess (12). Subsequently, CRM1-selective inhibitors ofnuclear export, such as KPT-185 and KPT-330, werereported by some groups in collaboration with Karyo-pharm therapeutics (13–15). Like CBS9106, both KPT-185 and KPT-330 induce proteasome-dependent CRM1protein degradation, but both also promoted CRM1mRNA expression (14).

1CanBas Co., Ltd., Numazu, Japan. 2Dana-Farber Cancer Institute, Har-vard School, Boston, Massachusetts. 3Translational Genomics ResearchInstitute (TGen), Phoenix, Arizona.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Takumi Kawabe, CanBas Co., Ltd., 2-2-1 Ote-machi, Numazu, Shizuoka 410-0801, Japan. Phone: 81-55-954-3666;Fax: 81-55-954-3668; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-14-0064

�2014 American Association for Cancer Research.

MolecularCancer

Therapeutics

www.aacrjournals.org 3013

on November 18, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst September 24, 2014; DOI: 10.1158/1535-7163.MCT-14-0064

http://mct.aacrjournals.org/

The ubiquitin–proteasome system regulates diversebiologic processes by controlling the degradation ratesof various cellular proteins (16, 17). Proteasome inhibitionis recognized as a novel therapeutic approach for humancancer and several proteasome inhibitors have beendeveloped for this purpose, including bortezomib, carfil-zomib, andMG132 (18, 19). In particular, bortezomib wasthe first to enter clinical use (20). Carfizomib, a second-generation proteasome inhibitor that is structurally andmechanistically different from bortezomib, has also beenapproved for treatment of patients with relapsed/refrac-tory multiple myeloma (21).

Neddylation is a process of protein modification by theubiquitin-like NEDD8 protein and is one of the pathwaysthat lead to proteasome-dependent protein degradation.The cullin family of E3 ubiquitin ligase scaffolds has beencharacterized as the major class of substrates for neddyla-tion. Neddylation is required for conformational changesin cullin that are necessary for regulating the activity ofcullin ring ligases (CRL). The components of the neddyla-tion pathway include NEDD8-activating enzyme E1(NAE), NEDD8-conjugating enzyme E2 (Ubc12), andNEDD8 E3 ligase. MLN4924 was recently discovered asa first-in-class, potent, and selective inhibitor of NAE andis currently in phase I clinical trials for both solid tumorand hematologic malignancies (22, 23).

CRLs are the largest family of E3 ubiquitin ligasestargeting approximately 20% of ubiquitinated proteinsubstrates for 26S proteasomal degradation (22). CRLsconsist of multisubunits containing cullin (Cul 1, 2, 3, 4A,4B, 5, and 7) and the RING-finger protein Rbx (24). CRLactivity is regulated in vitro by the highly conservedCOP9signalosome (CSN) protein complex and by cullin-asso-ciated and neddylation-dissociated protein 1 (CAND1;refs. 25–27). Neddylation is reversed by the eight-subunitCSN complex (CSN1–CSN8) via specific isopeptidase-dependent cleavage of NEDD8 from the cullin subunitof CRLs (25). CAND1 binds to the unneddylated form ofall cullins and inhibits E3 ligase activity of CRL (28, 29).However, depletion of CSN or CAND1 from cells leads todecreased CRL function (25). These apparent paradoxeswere reconciled by some several reports. Certain groupshave proposed that depletion of CSNprevents cleavage ofNEDD8 from cullins and that the resulting NEDD8-acti-vated cullins inducedegradation of several F-boxproteins(30–32). Other groups have proposed that depletion ofCAND1 prevents displacement of Skp1 from cullins, andthereby prevents newF-box proteins frombeing recruitedto preexisting cullins (33–35).

In this study, we have identified that CBS9106 inducesCRL activity involving the neddylation pathway, andthereby degradation of CRM1 protein.

Materials and MethodsCell lines and reagents

Human colon carcinoma cell lines, HCT116 (p53 wt,CCL-247), HT29 (p53 mt, HTB-38), HCT15 (p53 mt, CCL-

225), RKO (p53 wt, CRL-2577), and RKO/E6 (functionalp53-deficient, CRL-2578)were purchased from theATCC.Cell lines were authenticated by short tandem repeatanalysis. Cell lines were initially grown and low passagestocks were cryopreserved in liquid nitrogen. Cells werereplaced from frozen stocks after 3 months continuousculture. HCT116 and HT29 cells were cultured in McCoy5amedium (Gibco Life Technologies) containing 10% FBS(Gibco Life Technologies) and 1% penicillin/streptomy-cin (PS; Gibco Life Technologies). HCT15 cells were cul-tured in RPMI-1640 medium (Sigma-Aldrich) containing10% FBS, 1% PS, 10 mmol/L Hepes, 1 mmol/L sodiumpyruvate (Sigma-Aldrich), and 4.5 g/L D-glucose. RKOand RKO/E6 cells were cultured in Minimum EssentialMedium Eagle (Sigma-Aldrich) containing 10% FBS, 1%PS, 1 mmol/L sodium pyruvate, and 2 mmol/L L-gluta-mine (Sigma-Aldrich). The cells were grown at 37�C inhumidified atmosphere consisting of 5%CO2 and 95% air.CBS9106 was manufactured by Mercachem. MLN4924was purchased from Active Biochem. LMB was pur-chased from Santa Cruz Biotechnology. Bortezomib waspurchased from Selleck Chemicals. MG132 and 3-Methy-ladenine (3-MA) were purchased from Calbiochem. Bafi-lomycin A1 was purchased from Sigma-Aldrich.

Cell extracts and Western blot analysisTo prepare extracts from cell culture, cells werewashed

with PBS and resuspended in lysis buffer [20 mmol/LTris-HCl (pH 8), 150 mmol/L NaCl, 1 mmol/L EDTA, 1mmol/L EGTA, 0.5% (v/v) Nonidet P40, complete pro-tease inhibitor cocktail (Roche), PhosSTOP phosphataseinhibitor cocktail (Roche)]. Cells were vortexed with peri-odic cooling on ice for 1 hour and then centrifuged at13,000 rpm for 10 minutes (4�C). Cell extracts were resus-pended in a loading buffer and stored at �80�C untilanalysis by SDSPAGE and Western blotting. Proteinswere separated by electrophoresis on the Mini-proteanTGX gel (Bio-Rad) and transferred to Immobilon-P poly-vinylidene difluoride membranes (Millipore). Antibodiesagainst CRM1 (H300), CyclinB1 (H433), Ub (P4D1), UBA3(E5), CAND1 (TIP120A, A13), Skp2 (H435), p27 (F8), orb-actin (C4) were purchased from Santa Cruz Biotechnol-ogy.Antibodies againstNEDD8 (2754), p53 (9282), LC3-I/II (12741), SUMO-1 (4930), SUMO-2/3 (4971), Rbx1 (4397),CSN5 (COPS5, 6895), and rabbit IgG, aswell asmouse IgGconjugated with horseradish peroxidase, were purchasedfrom Cell Signaling Technology.

Cell viability and cell growth assaysHCT116 cells (2 � 103) were seeded on 96-well plates

and incubated for 24 hours. After incubation withCBS9106, LMB, orMLN4924, cell viability and cell growthwere analyzed with a Cell Counting Kit-8 (Dojindo).Reagent solution was added to each well, the 96-wellplates were incubated for 3 hours at 37�C, and the absor-bance at 450 nm (OD450) was measured using a Sunrisebasic microplate reader (Tecan Austria GmbH), in accor-dance with the manufacturer’s instructions.

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Mol Cancer Ther; 13(12) December 2014 Molecular Cancer Therapeutics3014




Cell-cycle analysisHCT116 cells were washed in PBS and harvested with

trypsinization. Cells were stained in propidium iodide/RNase buffer and analyzed by FACSCalibur flow cyt-ometer using CellQuest software (Becton Dickinson).Annexin V–FITC (Trevigen) was used to stain apoptoticcells.

ImmunofluorescenceApproximately 5 � 103 cells were seeded onto CELL-

view glass bottom dishes (Greiner Bio-One) and grownovernight at 37�C. Cells were treated with CBS9106 (100nmol/L), LMB (0.5 nmol/L), and/or MLN4924 (50nmol/L) at 37�C for 24 hours. Cells were fixed with4% paraformaldehyde phosphate buffer solution(Wako) for 30 minutes at room temperature, permeabi-lized with 0.1% Triton X-100 in PBS for 15 minutes andthen blocked with 4% Block Ace (DS Pharma Biomed-ical) for 1 hour at room temperature followed by specificantibody treatment. Goat-anti–RanBP1 antibodies (C19;Santa Cruz Biotechnology) or Goat-anti–CRM1 antibo-dies (C20; Santa Cruz Biotechnology) were used at a 1:50dilution. Alexa488-conjugated donkey anti-goat antibo-dies (Molecular Probes Life Technologies) were used ata 1:500 dilution. Cellular DNA was stained with 1 mg/mL Hoechst33342 (Dojindo). The stained cells werevisualized on a FluoView FV10i confocal microscope(Olympus) equipped with a �60 oil immersion lens.Images were processed using FV10-ASW (Version 2.1)software (Olympus).

siRNA nucleofectionHCT116 cells (2 � 106) were resuspended in 100 mL of

the Cell Line Solution Kit V (Lonza). One mmol/L siGEN-OME SMARTpool siRNA (Dharmacon) was added andthe cells were immediately transfected using the Nucleo-fector device (Lonza; programD-032). Of note, 2mL of thepreequilibrated culture medium was then added and thecells were cultured in the 6-well plate for 48 or 72 hours.Target gene–specific siRNAs were obtained from Dhar-macon as follows: NEDD8 (M-020081), UBA3 (M-005249),RBX1 (M-004087), CSN5 (COPS5,M-005814), CAND1 (M-015562), and siGENOME Nontargeting siRNA Pool (D-001206).

Statistical analysisData were analyzed using the t test (Student t test or

Welch t test). Data are expressed as the mean � SD. AP value of <0.05 was considered significant.

ResultsMLN4924 prevents CBS9106-induced CRM1reductionCBS9106 is a CRM1 inhibitor identified through cell-

cycle–based phenotypic screening (Fig. 1A). CBS9106treatment leads to reduction of CRM1 protein througha proteasome-mediated degradation pathway. However,the detailed mechanism responsible for CRM1 degrada-

tion is unclear. In this study, we focused on the neddyla-tion pathway involving CRL activity. Experiments wereperformed using MLN4924, a specific NAE inhibitor thatblocks this first step of the neddylation pathway. HCT116cells were treated with CBS9106, and the levels ofCRM1 protein were analyzed by Western blotting usingan anti-CRM1 antibody. As previously reported (12) andas shown in Fig. 1B, 100 nmol/L CBS9106 decreasedCRM1 protein expression in a time-dependent manner.Decreased levels of CRM1 protein were clearly observedat 4 to 8 hours of treatment (lanes 3–5). In contrast, asimilar decrease was not observed upon concomitanttreatment with MLN4924 (lanes 8–10). MLN4924 targetsthe neddylation pathway, which is upstream of the 26Sproteasome in the ubiquitin–proteasome pathway (36).As shown in lanes 6 to 10 in Fig. 1B, neddylation of cullinwas inhibited byMLN4924, even at the shortest exposuretime (2 hours, lane 7). The expression level of p53, one ofthe cargoes of CRM1, was increased upon treatment withCBS9106 alone (lanes 3–5) orwithCBS9106plusMLN4924(lanes 8–10).

Figure 1C shows the dose-dependent effects ofMLN4924 in preventing the reduction of CRM1 byCBS9106 inHCT116 cells. The expression ofCRM1proteinwas significantly reduced by treatment for 24 hours with100 nmol/L CBS9106 (lane 6). In contrast, the CBS9106-induced decrease inCRM1was attenuatedwith 5 nmol/LMLN4924 (lane 8), the concentration at which decreasedneddylation of cullins began to appear. The levels of p53were increased by treatment with 50 nmol/L MLN4924(lane 5). However, stabilization of p53 by CBS9106 wasmodestly decreased by 5 and 10 nmol/LMLN4924 (lanes8 and 9).

Similar results were obtained with MM.1S multiplemyeloma cells, a cell line more sensitive to CBS9106treatment than HCT116 (Supplementary Fig. S1). Treat-ment of MM.1S cells with MLN4924 also inhibited theaccumulation of cleaved PARP, an apoptosis marker thatincreases in the presence of CBS9106.

MLN4924 is known to selectively inhibit NAE relativeto related enzymes, such as SUMO-activating enzymeand ubiquitin-activating enzyme (22). Therefore, thesefindings indicate that CBS9106-induced CRM1 degra-dation is conferred by the NAE-mediated neddylationpathway.

CBS9106-induced CRM1 degradation is dependenton the neddylation pathway, but not on autophagy/lysosome or p53

To investigate the degradation pathway of CRM1 byCBS9106, HCT116 cells were treated with various inhibi-tors, including MLN4924, ubiquitin–proteasome inhibi-tors (MG132 and bortezomib) and autophagy/lysosomeinhibitors (3-MA and bafilomycin A1). As shownin Fig. 2A, MLN4924, MG132, and bortezomib inhibitedCBS9106-induced CRM1 degradation. In addition, theresults show that MLN4924 treatment was not associatedwith increases in polyubiquitinated signals as compared

CBS9106-Induced CRM1 Degradation Requires CRL Activity

www.aacrjournals.org Mol Cancer Ther; 13(12) December 2014 3015




with that obtainedwith the proteasome inhibitors,MG132or bortezomib (Fig. 2A, lanes 3–8).

In contrast, 3-MA and bafilomycin A1 did not inhibitCRM1 degradation by CBS9106 (Fig. 2A and Supplemen-tary Fig. S6). Disruption of autophagy can be inducedeither before (3-MA) or after (bafilomycinA1) the LC3-I toLC3-II conversion. Furthermore, p62 protein is a usefulmarker for the induction of autophagy (37). As shown inSupplementary Fig. S6, 3-MA treatment prevented p62reduction. These results indicate that CBS9106-inducedCRM1 degradation depends on a neddylation and not onautophagy.

Inhibition of CBS9106-induced CRM1 degradation byMLN4924 was also confirmed in the HT29 and HCT15colon cancer cell lines (Fig. 2B). As shown in Fig. 2B,CBS9106-induced stabilization of p53 was reduced byMLN4924. Therefore, CRM1 degradation appears to beimportant for the CRM1 inhibitory activity of CBS9106.To confirm this hypothesis, we analyzed the effect ofMLN4924 on p53 stabilization caused by CRM1 inhibi-tors. Treatment of RKO cells with CBS9106 led to sta-bilization of p53 protein that could be inhibited byMLN4924 (Fig. 2C). Another CRM1 inhibitor, LMB also

led to the stabilization of p53 protein, but this stabili-zation increased further upon addition of MLN4924(Fig. 2C). As expected, stabilization of p53 protein wasnot found in RKO/E6 cells (Fig. 2C). These results usingtwo different CRM1 inhibitors show that the inhibitoryeffect of MLN4924 is specific to CBS9106 and furthersupport the importance of CRM1 degradation to theactivity of CBS9106.

Inhibition of neddylation partially protects HCT116cells from CBS9106-induced cell growth inhibitionand apoptosis

The effect of CBS9106 and MLN4924 on the cellviability and growth of HCT116 cells was evaluated bythe Cell Counting Kit-8. In this experiment, the dose ofMLN4924 was decreased from 50 to 10 nmol/L to avoidthe induction of apoptosis observed at the higher con-centration. A single treatment with 10 nmol/L ofMLN4924 inhibited neddylation of cullins withoutinducing apoptosis, whereas 50 nmol/L of MLN4924alone induced apoptosis (subG1 17.0%) starting at 72hours (Supplementary Fig. S2). MLN4924 induces DNAdamage and apoptosis through DNA re-replication

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Figure 1. CBS9106-induced CRM1 degradation is prevented by MLN4924 in time- and dose-dependent manner in HCT116 cells. A, chemical structure ofCBS9106. B, HCT116 cells are treated with CBS9106 (100 nmol/L) and/or MLN4924 (50 nmol/L) for indicated period (0–8 hours). C, HCT116 cells are treatedwith increasing concentrations of MLN4924 (0, 1, 5, 10, or 50 nmol/L) and/or CBS9106 (100 nmol/L) for 24 hours. B and C, protein levels of CRM1,neddylated cullin (CulNEDD8), p53, and b-actin are determined by Western blot analysis. The measured band density was normalized relative to that of thecontrol sample, with the control value set to 1. b-Actin is used as a loading control.

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(38, 39). In this context, treatment with 50 nmol/LMLN4924 for 72 hours increased DNA content greaterthan 4-fold (Supplementary Fig. S2).The viability of HCT116 cells at 72 hours was signifi-

cantly reduced by 100 nmol/L CBS9106, but recoveredfrom 24.9% to 61.3% upon adding 10 nmol/L MLN4924(Fig. 3A). In contrast, LMB, which does not reduce CRM1protein, leads to a reduction of cell viability that was notreversed by the addition of MLN4924 (Fig. 3B). Theinhibition of cell growth by 100 nmol/L CBS9106 waspartially rescued by adding 10 nmol/L MLN4924, withthe degree of rescue increasing steadily over time(Fig. 3C). However, the inhibition of cell growth by 0.5

nmol/L LMB was not rescued by adding 10 nmol/LMLN4924.

Cell-cycle distribution was evaluated by flow cytome-try with the subG1 population reflecting the induction ofapoptosis. The subG1 fraction increased to 29.3% after 72hours with 200 nmol/L CBS9106 treatment, but wasreduced to 17.4% by adding 10 nmol/L MLN4924 (Fig.3D). In contrast, cotreatment of 0.3 nmol/Lwith 10 nmol/L MLN4924 tended to enhance apoptosis. Similar patternwas obtained using FITC-labeled Annexin V (Fig. 3E).Results similar to those for HCT116 were obtained forMM.1S (Supplementary Fig. S3), HT29, and HCT15 (Sup-plementary Fig. S4) cells. These findings indicate that

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Figure 2. CBS9106-induced downregulation of CRM1 depends on a neddylation pathway, but is independent of autophagy or p53 status. A, effect ofvarious inhibitors on CBS9106-induced CRM1 protein degradation. Each inhibitor is added to HCT116 cells in the presence and absence of CBS9106(100 nmol/L). HCT116 cells are treated with MLN4924 (10 nmol/L), MG132 (5 mmol/L), bortezomib (20 nmol/L), 3-MA (10 mmol/L), or bafilomycinA1 (50 nmol/L) for 24 hours. B, three colon cancer cell lines, HCT116, HT-29, and HCT15, are treated with MLN4924 (10 or 50 nmol/L) and/orCBS9106 (100 nmol/L) for 24 hours. C, RKO and RKO/E6 are treated with CBS9106 (200 nmol/L), LMB (3 nmol/L), or MLN4924 (50 nmol/L) either aloneor in combination for 24 hours. A to C, protein levels of CRM1, CulNEDD8, p53, ubiquitin (poly-Ub), LC3-I/II, and b-actin are determined by Western blotanalysis.






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Figure 3. The cell growth inhibition and apoptosis by CBS9106 is partially suppressed by MLN4924 in HCT116 cells. A, HCT116 cells are treated withCBS9106 (50 or 100 nmol/L) or MLN4924 (1 or 10 nmol/L) either alone or in combination for 72 hours (n ¼ 6). Cell viability is determined by the CellCounting Kit-8. B, HCT116 cells are treated with LMB (0.3 or 0.5 nmol/L) or MLN4924 (1 or 10 nmol/L) either alone or in combination for 72 hours (n ¼ 6).Cell viability is determined by the Cell Counting Kit-8. C, HCT116 cells are treated with CBS9106 (100 nmol/L), LMB (0.5 nmol/L), and/or MLN4924 (1 or10 nmol/L) for indicated period (24–72 hours, n ¼ 6). Cell growth is assessed at indicated times using the Cell Counting Kit-8. D, the apoptoticpopulation (indicated by subG1) is measured by flow cytometry after staining with propidium iodide. HCT116 cells are treated with CBS9106 (100 or200 nmol/L), LMB (0.3 nmol/L), and/or MLN4924 (1 or 10 nmol/L) for 72 hours (n ¼ 3). E, Annexin V–positive apoptosis cells are measured by flowcytometry (n ¼ 3); Data, the mean � SD; �, P < 0.05.

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MLN4924 attenuates the effects of CBS9106 on cell growthinhibition and cell death.

Nuclear localization of RanBP1 by CBS9106 isprevented by MLN4924Ran-binding protein 1 (RanBP1) shuttles between the

nucleus and cytoplasm, but is predominantly localized tothe cytoplasm under normal culture conditions. Nuclearaccumulation of RanBP1 protein is used as a surrogatemarker for CRM1 inhibition due to its ubiquitous expres-sion and rapid CRM1-dependent shuttling (10). We andother groups have previously reported the nuclear accu-mulation of RanBP1 induced by CRM1 inhibitors asobserved by immunofluorescence microscopy (12, 40).To confirm the inhibitory effect of MLN4924 againstCBS9106, the subcellular localization of RanBP1 was ana-lyzed when HCT116 cells were treated with MLN4924and/or CBS9106 for 24 hours (Fig. 4). RanBP1 localized tothe cytoplasm in control (DMSO treated) and MLN4924-treated cells; however, CBS9106 or LMB treatmentinduced nuclear accumulation of RanBP1 (Fig. 4A).More-over, cotreatment of CBS9106 with MLN4924 preventedthe nuclear localization of RanBP1 (Fig. 4A). CRM1 pro-tein is mainly localized in the nucleus in the control(DMSO treated) and MLN4924-treated cells. CBS9106treatment reduced the overall level of CRM1 protein,and this response was prevented by adding MLN4924

(Fig. 4B). The localization of RanBP1 and CRM1 by LMBwas not affected by MLN4924. These results show thatMLN4924 attenuates CBS9106-induced nuclear accumu-lation of RanBP1, as well as CRM1 degradation. Similarresults were obtained for MM.1S cells (SupplementaryFig. S5).

Knockdown of NEDD8, UBA3, or Rbx1 preventsCRM1 degradation by CBS9106

To determine more directly whether CRM1 proteindegradation is mediated by a neddylation pathway,siRNA knockdown experiments were performed inHCT116 cells to reduce the expression levels of NEDD8or UBA3, two major effectors in the neddylation path-way or of Rbx1, an essential component of CRLs. Asshown in Fig. 5A, knockdown of the endogenousNEDD8 remarkably inhibited cullin neddylation andCBS9106-induced CRM1 degradation. The knockdownof NEDD8 also stabilized p53 and cyclin B1 (Fig. 5A). Inaddition, the expression levels of the SUMOylationproteins SUMO1 and SUMO2/3 were not affected(Fig. 5A). Flow-cytometry analysis of control siRNA–transfected cells showed that the G1 phase fractionincreases significantly from 51.9% (Fig. 5B, lane 1) to71.9% (Fig. 5B, lane 2) upon treatment with 100 nmol/LCBS9106. However, NEDD8 siRNA–transfected cellsexhibited a more modest increase in G1 phase fraction

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Figure 4. Nuclear export inhibitionis canceled by MLN4924treatment. HCT116 cells aretreated with CBS9106(100 nmol/L), LMB (0.5 nmol/L),and/orMLN4924 (50 nmol/L) for 24hours.A, cells arefixedandstainedfor RanBP1 (cytoplasmic protein,green, left) and nucleic acid(Hoechst33342 stain, blue,middle). Merged images areshown at right. B, cells are fixedand stained for CRM1 (nuclearprotein, green, left) and nucleicacid (middle). Merged images areat right. White bar, 20 mm.






[from 47.9% (Fig. 5B, lane 7) to 54.5% (Fig. 5B, lane 8)]upon 100 nmol/L CBS9106 treatment. These resultsindicate that CBS9106-induced G1 phase cell-cyclearrest is suppressed, albeit partially, by reducing thelevel of NEDD8 protein.

Reducing the level of UBA3, an E1 component of theneddylation pathway, strongly inhibits the induction ofCRM1 protein degradation by CBS9106 treatment (Fig.5C). Similarly, depletion of Rbx1 inhibits the CBS9106-induced degradation of CRM1 protein (Fig. 5D). These

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Figure 5. Silencing of NEDD8 inhibits CRM1 degradation and G1 phase cell-cycle arrest by CBS9106. A, HCT116 cells are nucleofected with control siRNA(siCont; 250 nmol/L) or NEDD8 siRNA (siNEDD8; 10, 50, or 250 nmol/L) for 72 hours. Thereafter, cells are treated with or without CBS9106 (100 nmol/L) for 24hours. Protein levels of NEDD8, CulNEDD8, CRM1, cyclinB1, p53, SUMO1, SUMO2/3, and b-actin are determined by Western blot analysis. B, cell samplesare obtained fromA.Cell-cycle distributions aremeasuredbyflowcytometry after stainingwithpropidium iodide (n¼3).C,HCT116 cells are nucleofectedwithcontrol siRNA (siCont; 1 mmol/L) or UBA3 siRNA (siUBA3; 1 mmol/L) for 72 hours. The nucleofected cells are treated with increasing concentrations ofCBS9106 (CBS; 12.5, 25, 50, or 100 nmol/L) for 24 hours. D, HCT116 cells expressing control siRNA (siCont; 1 mmol/L) or Rbx1 siRNA (siRbx1; 1 mmol/L) areassessed similarly to those in C. C and D, protein levels of UBA3, Rbx1, CRM1, or b-actin are determined by Western blot analysis.

Saito et al.





findings suggest that CRL activity involving the neddyla-tion pathway contributes to CBS9106-induced CRM1 pro-tein degradation.

Knockdown of CSN5 and CAND1 partially inhibitsCRM1 degradation by CBS9106CRL activity is also regulated by cycles of CSN-medi-

ated CRL deneddylation and CAND1 binding (25–27).Depletion of CSN or CAND1 prevents the formation ofCRL complexes and inhibits CRL activity (25). CSN5encodes the catalytic component of the CSN and knock-down of this molecule induces functional dysregulationof the CSN (30–32). To investigate whether CRM1 pro-tein degradation is mediated by CRLs, we knockeddown CSN5 or CAND1 in HCT116 cells using siRNA.As shown in Fig. 6, knockdown of the endogenousCSN5 or CAND1 partially inhibited CRM1 degradationby CBS9106. Deletion of CSN5 destabilizes Skp2 andleads to increased cullin neddylation. In turn, the desta-bilization of Skp2 reduces CRL activity. These findingssuggest that CBS9106-induced CRM1 degradation is

mediated by CRL activity involving the neddylationpathway.

DiscussionIn the present study, we investigated how CBS9106

promotes the degradation of CRM1 protein. The seminalfindings of this work are: (i) CBS9106-induced CRM1degradation is inhibited by MLN4924; (ii) CBS9106-induced cell growth inhibition, apoptosis, and nuclearlocalization of RanBP1 are also inhibited by MLN4924;and (iii) knockdown of NEDD8, UBA3, or CRL-relatedproteins prevent CRM1 degradation by CBS9106. Thesefindings indicate that CBS9106-mediated degradation ofCRM1 is dependent on CRL activity involving the ned-dylation pathway.

MLN4924 is known to induce autophagy in multiplehuman cancer cell lines (37, 41, 42). MLN4924-inducedautophagy derives partially from the inhibition of mTORactivity, caused by accumulation of CRL substrates suchas DEPTOR and HIF1a. Therefore, autophagy inducedby MLN4924 may have led to its inhibitory effect againstCBS9106 activity. However, low doses of MLN4924were able to inhibit CBS9106 activity without inducingautophagy, and inhibitors of autophagy did not affect thereversal ofCBS9106 activity byMLN4924 (SupplementaryFig. S6).MLN4924-induced autophagywasdemonstratedby accumulation of HIF1a and degradation of p62(37). High doses of MLN4924-induced accumulation ofHIF1a, but low doses of MLN4924 did not. Furthermore,the degradation of p62 was induced by high doses ofMLN4924, but it was blocked by autophagy inhibitor 3-MA. Incidentally, p62 contains a nuclear export signal andshuttles between nuclear and cytoplasm. CRM1 inhibitorCBS9106 treatment induced stabilization of p62 inHCT116 cells. In addition, a variety of autophagy inhibi-tors, including 3-MA and bafilomycin A1 and mTORinhibitor/autophagy activator CCI779, did not affectCBS9106-induced CRM1 degradation and RanBP1 nucle-ar accumulation (Fig. 2A, data not shown). Therefore, weconclude that the inhibitory activity of MLN4924 againstCBS9106-induced degradation of CRM1 is unrelated toautophagy.

Knockdown of Rbx1 protein by siRNA reversedCBS9106-induced CRM1 degradation (Fig. 5D). Thisresult indicates that CRM1 degradation caused byCBS9106 treatment depends on CRL activity. Cullin pro-teins complexes composed of seven cullin members donot have catalytic activity on their own, but instead serveas scaffolds that facilitate the assembly of multimeric E3ligase complexes for transferring ubiquitin from E2-con-jugating enzyme to its substrate (43). To identify CRLcomponents required for CRM1 degradation, siRNA-mediated knockdown of each cullin component was per-formed. Notably, individual siRNA knockdown experi-ments for each of six cullin members (Cul1, 2, 3, 4A,4B, and 5) had no effect on CRM1 degradation byCBS9106. Milhollen and colleagues (39) reported that

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Figure 6. CRM1 protein degradation by CBS9106 is mediated by CRLs.HCT116 cells are nucleofected with control siRNA (siCont; 1 mmol/L),CSN5 siRNA (siCSN5; 1 mmol/L), or CAND1 siRNA (siCAND1; 1 mmol/L)for 48 hours. Thereafter, cells are treated with or without CBS9106(100 nmol/L) for 24 hours. Protein levels of CRM1, CSN5, CAND1,CulNEDD8, p27, Skp2, and b-actin are determined by Western blotanalysis. The measured band density was normalized relative to that ofthe control sample, with the control value set to 1.






triple depletion of Cul1 and Cul4A/B is required torecapitulate the DNA rereplication phenotype observedwith MLN4924 treatment. Therefore, the silencing of asingle gene might not be sufficient to detect an inhibitoryeffect on induction of CRM1 degradation by CBS9106.Further analysis is, therefore, required to identify thecullins that are necessary for CBS9106-induced CRM1degradation.

NEDD8 can conjugate directly not only to cullins butalso to other target proteins such as p53, pVHL, EGFR,and ribosomal proteins (44–46). Protein neddylationoccurs at lysine residues within target proteins and reg-ulates its substrate activity. The CRM1 protein containsmultiple lysine residues, and some of which are possibleneddylation sites. Computer analysis suggests thatthe CRM1 Lys139–containing sequence (LVQILKQE), issimilar to a reported cullin neddylation consensussequence ([IL][VIT][RQ][IS][MLV]K[MAS][RHE]; refs. 47,48). However, exogenously expressedCRM1 proteinwitha Lys to Ala point mutation at residue 139 (CRM1 K139A)was degraded as extensively as the wild-type (data notshown). Further studies will be needed to confirmwheth-er the CRM1 protein is a substrate of neddylation.

The activity of CBS9106 mainly depends on an induc-tion of the CRM1 degradation, but not completely. Asshown in Figs. 3A and4A,MLN4924 couldnot completelyrestore cell viability and nuclear shuttling activity eventhough MLN4924 prevents CRM1 degradation. In addi-tion, MLN4924 could not completely suppress CBS9106-induced apoptosis in Fig. 3D andE. The implication is thatCBS9106 exhibits residual anticancer activity withoutobservable CRM1 degradation. Furthermore, CBS9106and LMB bind to the same cysteine residue on the CRM1protein, CBS9106 reversibly and LMB irreversibly. Sunand colleagues (49) showed that hydrolysis of LMB byCRM1 causes irreversible covalent conjugation. LMB,which is hydrolyzed, has no CRM1 degradation activity,but KPT-185, which is not hydrolyzed, has it. LikeCBS9106, KPT-185 binds to CRM1 by reversible covalent

conjugation and attenuates CRM1 protein (14). Thus, thereversible reactivity against CRM1 may be related to theCRM1 degradation.

In conclusion, this study provides the first evidence thatCBS9106-induced CRM1 degradation depends on CRLactivity involving the neddylation pathway (Supplemen-tary Fig. S7). As expected, MLN4924 attenuated tumorgrowth inhibition by CBS9106 in vivo in a preliminaryxenograft experiment (Supplementary Fig. S8). This studyindicates that inhibitors against the CRL-dependent pro-teasome pathway may reduce the efficacy of CBS9106.Importantly, CRM1 protein degradation plays importantroles in CBS9106-induced responses such as cell growthinhibition and cell death. Therefore, the expression levelofCRM1protein appears to be a suitable surrogatemarkerfor CBS9106 activity.

Disclosure of Potential Conflicts of InterestD.W. Kufe and D.D. VonHoff are consultant/advisory board members

for CanBas. T. Kawabe is the president and CEO at CanBas Co., Ltd. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception anddesign:N. Saito, K. Sakakibara, D.D. VonHoff, T. KawabeDevelopment ofmethodology:N. Saito, K. Sakakibara, T. Sato, T. KawabeAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N. Saito, T. SatoAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis):N. Saito, K. Sakakibara, T. Sato, J.M. Fried-man, T. KawabeWriting, review, and/or revision of the manuscript: N. Saito, K. Sakaki-bara, T. Sato, J.M. Friedman, D.W. Kufe, D.D. VonHoff, T. KawabeStudy supervision: N. Saito, D.W. Kufe, D.D. VonHoff, T. Kawabe

AcknowledgmentsThe authors thank all the colleagues who have contributed to these

projects.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 21, 2014; revisedAugust 22, 2014; accepted September9, 2014; published OnlineFirst September 24, 2014.

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2014;13:3013-3023. Published OnlineFirst September 24, 2014.Mol Cancer Ther Naoya Saito, Keiichi Sakakibara, Takuji Sato, et al. Ligase Activity and the Neddylation PathwayCBS9106-Induced CRM1 Degradation Is Mediated by Cullin Ring

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CBS9106-Induced CRM1 Degradation Is Mediated by Cullin Ring … · Cancer Biology and Signal Transduction CBS9106-Induced CRM1 Degradation Is Mediated by Cullin Ring Ligase Activity