The Nedd8-Activating Enzyme Inhibitor MLN4924 Induces ... · Nedd8-activating enzyme (NAE) is critical for neddylation of CRL complexes and their growth-promoting function. Recently,

Therapeutics, Targets, and Chemical Biology

The Nedd8-Activating Enzyme Inhibitor MLN4924 InducesAutophagy and Apoptosis to Suppress Liver Cancer CellGrowth

Zhongguang Luo1,3, Guangyang Yu1, Hyuk Woo Lee7, Lihui Li1, Lingyan Wang4, Dongqin Yang1,3,Yongfu Pan1, Chan Ding5, Jing Qian1, Lijun Wu3, Yiwei Chu1,2, Jing Yi6, Xiangdong Wang4,Yi Sun8, Lak Shin Jeong7, Jie Liu1,3, and Lijun Jia1,2,4

AbstractPosttranslational neddylation of cullins in the Cullin-Ring E3 ligase (CRL) complexes is needed for proteolytic

degradation of CRL substrates, whose accumulation induces cell-cycle arrest, apoptosis, and senescence. TheNedd8-activating enzyme (NAE) is critical for neddylation of CRL complexes and their growth-promotingfunction. Recently, the anticancer small molecule MLN4924 currently in phase I trials was determined to be aninhibitor of NAE that blocks cullin neddylation and inactivates CRL, triggering an accumulation of CRL substratesthat trigger cell-cycle arrest, apoptosis, and senescence in cancer cells. Here, we report thatMLN4924 also triggersautophagy in response to CRL inactivation and that this effect is important for the ability of MLN4924 to suppressthe outgrowth of liver cancer cells in vitro and in vivo. MLN4924-induced autophagy was attributed partially toinhibition ofmTOR activity, due to accumulation of themTOR inhibitory proteinDeptor, as well as to induction ofreactive oxygen species stress. Inhibiting autophagy enhanced MLN4924-induced apoptosis, suggesting thatautophagy is a survival signal triggered in response to CRL inactivation. In a xenograft model of human livercancer, MLN4924 was well-tolerated and displayed a significant antitumor effect characterized by CRL inacti-vation and induction of autophagy and apoptosis in liver cancer cells. Together, our findings support the clinicalinvestigation of MLN4924 for liver cancer treatment and provide a preclinical proof-of-concept for combinationtherapy with an autophagy inhibitor to enhance therapeutic efficacy. Cancer Res; 72(13); 3360–71. �2012 AACR.

IntroductionLiver cancer, especially hepatocellular carcinoma (HCC), is

one of the most common human malignancies and the thirdleading cause of cancer death worldwide with 600,000 deathsper year (1). Moreover, liver cancer is annually diagnosed inmore than half a million people worldwide. Although surgicalresection and liver transplantation, in combination with che-

motherapies when necessary, represent curative treatmentsfor early or localized disease, approximately 80% of patientswith liver cancer with advanced disease are not permissible tosurgical resection or transplantation andhave tomainly rely ontraditional chemotherapies (2). However, the current chemo-therapy is far from satisfaction due to relatively low anticancerefficacy, severe treatment-associated adverse effects, as well asacquired drug resistance, leading to high risk of tumor recur-rence and poor long-term survival. The dilemma makes anurgent necessity to identify novel anticancer targets anddevelop new therapeutic agents with efficient and selectiveanticancer efficacy to improve the treatment of liver cancer.

The Cullin-Ring ligases (CRL), also known as SKP1-Cullin-F-box (SCF) E3 ligases for its foundingmember (3), are the largestmultiunit ubiquitin ligase family in cells. CRL/SCF E3 ligases(CRL/SCF) are in charge of the degradation of about 20% ofubiquitinated cellular proteins to regulate diverse biologicprocesses (4, 5), whereas its dysfunction leads to carcinogen-esis. Recently, CRL/SCF is identified as a promising anticancertarget based on the following findings (6, 7): (i) Some essentialcomponents of CRL/SCF such as F-box protein SKP2 (8, 9) andRING-finger protein RBX1/ROC1 serve as oncoproteins and arespecifically overexpressed in human cancers (10); (ii) Knock-down of these oncogenic proteins via siRNA silencing signif-icantly suppresses cancer cell growth in vitro and in vivo (10,11); and (iii) Inactivation of CRL/SCF via small-molecule

Authors' Affiliations: 1Department of Immunology, Shanghai MedicalCollege, 2BiotherapyResearchCenter, 3Department ofDigestiveDiseases,Huashan Hospital, 4Biomedical Research Center, Zhongshan Hospital,Fudan University, Shanghai; 5Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences; 6Department of Biochemistryand Cell Biology, Institutes of Medical Sciences, Shanghai Jiao TongUniversity School of Medicine, Shanghai, China; 7Department of Bioin-spiredScience andCollege of Pharmacy, EwhaWomansUniversity, Seoul,Korea; and 8Division of Radiation and Cancer Biology, Department ofRadiation Oncology, University of Michigan, Ann Arbor, Michigan

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Lijun Jia, Fudan University, 130 Dong-An Road,Shanghai 200032, China. Phone: 86-21-54237751; Fax: 86-21-54237751;E-mail: [email protected]; Jie Liu. Phone: 86-21-52888236; Fax:86-21-52888236; E-mail: [email protected]; and Lak Shin Jeong.Phone: 82-2-3277-3466; Fax: 82-2-3277-2851; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-12-0388

�2012 American Association for Cancer Research.

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inhibitors, such as MLN4924, showed striking anticancer effi-cacy with good tolerance (4, 12).Neddylation, a process of adding ubiquitin-like molecule

Nedd8 to target proteins, is a new type of protein posttrans-lationalmodification (13). The reaction involves the successiveaction of Nedd8-activating enzyme E1 (NAE), Nedd8-conju-gating enzyme E2 (Ubc12), andNEDD8-E3 ligase (13). Intensivestudies showed that the activation of CRL/SCF requires ned-dylationmodification of its essential subunit Cullin (4). Recent-ly, MLN4924, a specific inhibitor of NAE, was discovered via ahigh-throughput screening (4, 14). Because of its significantanticancer efficacy in preclinical studies, MLN4924 has beenadvanced into several phase I clinical trials for several solidtumors and hematologic malignancies (15). In mechanism,MLN4924 inhibits NAE activities by binding to NAE at theactive site to forma covalent Nedd8-MLN4924 adduct (16). As aresult, cullin neddylation is blocked and CRL/SCF is inacti-vated. By doing so, MLN4924 causes accumulation of a mass ofCRL/SCF E3 substrates (4, 17, 18), which triggers DNA rere-plication stress and DNA damage response (DDR), as well asinduces abnormal cell-cycle progression, apoptosis, and/orsenescence to suppress the growth of cancer cells in vitro andin vivo (4, 12, 19–23). Recent accumulated data suggested thatautophagy may be involved in the induction of apoptosis orsenescence upon cellular stresses (24). However, it is complete-ly unknown whether MLN4924 regulates autophagy responseuponCRL/SCF inactivation. Moreover, the efficacy of this first-in-class agent on liver cancer still remains elusive. In this study,we showed a significant therapeutic efficacy of MLN4924 onliver cancer in vitro and in vivo by modulating autophagy andapoptosis pathways, which provides proof-of-concept evi-dence for the clinical investigation of this first-in-class anti-cancer agent in the treatment of liver cancers.

Materials and MethodsCell lines, culture, and reagentsHuman liver cancer cell lines Huh-7 and Hep G2 were

obtained from the American Type Culture Collection, andcultured in Dulbecco's Modified Eagle's Medium (Hyclone),containing 10% FBS (Biochrom AG) and 1% penicillin–strep-tomycin solution, at 37�C with 5% CO2. MLN4924 was synthe-sized as previously described (25). For in vitro studies,MLN4924 was dissolved in dimethyl sulfoxide (DMSO) andkept in �20�C. For in vivo studies, MLN4924 was dissolved in10% 2-hydroxypropyl-b-cyclodextrin (HPBCD), and the solu-tion of MLN4924 was freshly made every week and stored indark at room temperature before use.

siRNA knockdown of Deptor, ATG5, and Beclin 1Liver cancer cells were transfected with siRNA oligonucleo-

tides made by GenePharma using Lipofectamine 2000. Thesequences of siRNAs are as follows: for Deptor (26), siDeptor-1:50-GCCATGACAATCGGAAATCTA-30, siDeptor-2: 50-GCAAG-GAAGACATTCACGATT-30; for ATG5 (27), siATG5: 50-CAUCU-GAGCUACCCGGAUAUU-30; for Beclin 1 (28), siBeclin 1: 50-CAGUUUGGCACAAUCAAUATT-30; and for control scrambledsiRNA, siControl: 50-UUCUCCGAACGUGUCACGUTT-30.

ImmunoblottingCell lysates were prepared for immunoblotting, using anti-

bodies against Wee1, Cullin1 (Santa Cruz Biotechnology), p21,total CHK2 (t-CHK2), total H2A (t-H2A; Epitomics, Inc.),mTOR, p-4E-BP1 (T37/46), NonP-4E-BP1, p-CHK2 (T68), p-H2A (Ser139), p27, Atg5, Atg7, Beclin 1, cleaved caspase-3,cleaved PARP, p-Histone H3 (Ser10; Cell Signaling) and glyc-eraldehyde-3-phosphate dehydrogenase (GAPDH), LC3, andDeptor (Sigma).

Cell counting and clonogenic assayFor cell counting, cells were seeded into 24-well plates with

1� 104 cells per well. Cells were trypsinized, resuspended, andcounted with Cellometer Auto T4 (Nexcelom Bioscience) atindicated time points. For clonogenic assay, cells were seededinto a 60-mm dish with 500 cells per well and cultured for 10days. The colonies on the dish were fixed with 4% paraformal-dehyde and stained with crystal violet. The colonies with morethan 50 cells were counted.

Propidium iodide staining and fluorescence-activatedcell-sorting analysis

Cells treated with MLN4924 or DMSO were harvestedand fixed in 70% ethanol at �20�C overnight, and stainedwith propidium iodide (PI; 36 mg/mL; Sigma) containingRNase (10 mg/mL; Sigma) at 37�C for 15 minutes, thenanalyzed for apoptosis and cell-cycle profile by CyAn ADP(Beckman Coulter; ref. 10). Apoptosis was measured by thepercentage of cells in sub-G1 population. Data were ana-lyzed with ModFit LT software. The activation of caspase-3was determined by CaspGLOW Fluorescein Active Caspase-3 Staining Kit (BioVision) according to the manufacturer'sinstructions.

Establishment of Huh-7-EGFP-LC3 and Hep G2-EGFP-LC3 cell lines

Huh-7 and Hep G2 cells, stably expressing EGFP-LC3fusion protein, were established as described (29, 30). Briefly,cells were seeded in 6-well plates and transfected with 3 mgpEGFP-LC3 plasmid using Lipofectamine 2000 (Invitrogen).Cells with enhanced green fluorescent protein (EGFP) fluo-rescence were selected by MoFloXDP Cell Sorter (BeckmanCoulter) and cultured in complete cell culture mediumcontaining G418 at 200 mg/mL. The autophagy induced byMLN4924 was measured by appearance of punctate vesiclestructure and photographed under a fluorescence micro-scope (Leica).

Acridine orange stainingQuantification of autophagy by acridine orange staining was

conducted as described (31). Briefly, cells treated with orwithout MLN4924 at 0.1 mmol/L for 24 hours were trypsinizedand stained with 1 mmol/L acridine orange in PBS containing5% FBS at 37�C for 15 minutes. Cells were washed, resus-pended, and subjected to FACS assays. Green (510–530 nm)and red (650 nm) fluorescence emission from cells illuminatedwith blue (488 nm) excitation light wasmeasured by CyAnADP(Beckman Coulter).

Anti-Liver Cancer Activity by MLN4924

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Cell growth assay with CELL-IQ cell-culturing platformCELL-IQ was conducted as described (32). Briefly, the cell

numberwasmeasured by the real-time cellmonitoring system,using a Cell-IQ cell-culturing platform (Chip-Man Technolo-gies). To determine cell number of each stage during prolif-eration, Huh-7 cells were seeded in 24-well plates (1� 104 cellsper well) overnight and then treated with or without MLN4924at 0.1 mmol/L. Images of each microscopic field were capturedat 50-minute intervals for 100 hours. Cell-IQ system automat-ically calculated total cell number of each image. Each groupcontained 12 replicate microscopic fields.

In vivo antitumor effect of MLN4924Orthotopic xenograft model of liver cancer was established

by AntiCancer Biotech as described (33). Briefly, Hep G2-GFPhuman liver cancer tissue that originated from subcutaneoustumor of nude mice was harvested and carefully inspected toremove necrotic tissue. The harvested tumor tissue was thenequally divided into small pieces of 1 mm3 each. One 1-mmpiece of the above tumor tissue fragments was inserted intothe incision on the left lobe of liver of each mouse. Thetumor-bearing mice were randomized into 2 groups (10 ani-mals/group) and treated with 10% HPBCD or MLN4924(30 mg/kg, s.c.), twice a day respectively, on a 3-days-on/2-days-off schedule for 7 cycles within total 35 days (4). Thesize of tumors was measured by whole body fluorescenceimaging system twice a week, as described (30, 34). Briefly,whole body imaging of tumor-bearing animals was conductedwith an Olympus OV100 imaging system with 470 nm excita-tion light originating from an MT-20 light source. Emittedfluorescence was collected through appropriate filters config-ured on a filter wheel with a DP70 CCD camera, and imageswere captured with a PC and processed for contrast andbrightness with Paint Shop Pro 8 and CellR (34). At the endof the study, the serum of animals was collected for biochem-ical assays with Modular DDP (Roche), followed by sacrifice ofanimals. Then, tumor tissues of mice were collected, photo-graphed, and weighed. Parts of tumor tissues were homoge-nized in lysis buffer with tissue tearor for 1 minute, andcentrifuged at 10,000 � g at 4�C for 5 minutes to collect thesupernatant for immunoblotting.

Statistical analysisThe statistical significance of differences between groups

was assessed using the GraphPad Prism5 software. Theunpaired 2-tailed t test was used for the comparison of para-meters between groups. The level of significance was set atP < 0.05.

ResultsMLN4924 inhibited the growth of liver cancer cells

To evaluate the efficacy of MLN4924 on liver cancer cells,Huh-7 cells carrying mutated and inactivated p53 and Hep G2cells expressing wild-type and functional p53were treatedwithMLN4924 and subjected to cell growth analysis. As shownin Fig. 1,MLN4924 completely inhibited cullin neddylation (Fig.1A) and significantly suppressed the proliferation of Huh-7 and

Hep G2 cells, regardless of p53 status (Fig. 1B). MLN4924 alsonotably suppressed cell clonogenic survival in these cells (Fig.1C). Moreover, we applied a real-time cell monitoring systemusing a Cell-IQ cell culture platform to kinetically measure cellgrowth over time. As shown in Fig. 1D (left), MLN4924-treatedHuh7 cells only underwent slow replication for limited gen-erations, whereas control cells proliferated rapidly and reached100% confluence in cell culture dish at 100 hours after treat-ment. Consistently, the cell growth curve generated by Cell-IQmonitoring system showed significantly inhibitory effect ofMLN4924 on treated cells (Fig. 1D, right). Similar results usingCell-IQmonitoring system were obtained in Hep G2 cells (datanot shown). These findings showed a striking inhibitory effectof MLN4924 on the growth of liver cancer cells.

MLN4924 induced G2 cell-cycle arrest and apoptosis inliver cancer cells

Previous studies reported that MLN4924 inactivatesCRL/SCF, induces DDR (4, 12, 19–23), triggers cell-cycledisturbance, apoptosis, and/or senescence to suppress can-cer cell growth. Similarly, we found that MLN4924 inducedDDR in Huh-7 and Hep G2 cells, as shown by the appearanceof phosphorylated H2A and CHK2, 2 classical markers ofDDR (Fig. 2A). During the treatment, p21 and p27, 2 well-known CRL/SCF substrates (6, 21), accumulated notably(Fig. 2A), further indicating efficient inactivation of CRL/SCF by MLN4924.

Further analysis of cell-cycle profile and apoptosisrevealed that MLN4924 triggered G2–M cell-cycle arrest,followed by apoptosis in liver cancer cells (Fig. 2B andSupplementary Fig. S1). MLN4924-induced G2–M cell-cyclearrest reached the peak at 24 hours after treatment and thendecreased over time (Fig. 2B), whereas apoptosis appeared at48 hours after treatment and continued to increase overtime (Fig. 2B for sub-G1 peak and Fig. 2C for caspase-3 andPARP activation). These data suggest that arrested cells dieof apoptosis at late stage.

To determine at which phase MLN4924-treated cells werearrested, we detected the expression status of Wee1, a well-defined CRL/SCF substrate and an inhibitor of G2–M phasetransition (35), as well as p-Histone H3 (p-H3, ser10), a hall-mark of M phase cells (36). As shown in Fig. 2C, Wee1significantly accumulated, whereas p-Histone H3 sharplydecreased upon MLN4924 treatment (Fig. 2C), indicating thatMLN4924-treated cells were arrested at the G2 and failed toenter M-phase.

MLN4924 induced autophagy in liver cancer cellsAlthough MLN4924 frequently affects cell-cycle progression

and apoptosis, it is totally unknown whether it regulatesautophagy pathway. To address this, we first determined theeffect of MLN4924 on the formation of autophagosome mem-brane via detection of the conversion of LC3 I (microtubule-associated protein 1 light chain 3) to lipidated LC3 II, as well asvia classical punctuative distribution of membrane-associatedLC3 II, 2 classical hallmarks of autophagy (37, 38). As shownin Fig. 3A and B, MLN4924 indeed induced the conversion ofLC3 I to LC3 II, showed by the increasing accumulation of LC3

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II over time in Huh-7 and Hep G2 cells (Fig. 3A) and punc-tuative distribution of membrane-associated lipidated LC3 IIin Huh-7-EGFP-LC3 and Hep G2-EGFP-LC3 cells (Fig. 3B).Furthermore, we conducted cell staining with acridine orangewhich detects the formation of acidic vesicular organelle(AVO), a characteristic of autophagy (37, 38). The resultsshowed that MLN4924 treatment induced AVO accumulationsignificantly (Fig. 3C), suggesting autophagy induction. Finally,

we detected the appearance of double-membraned autopha-gosome, which contains engulfed bulk cytoplasm and cyto-plasmic organelles, as golden hallmark of autophagy (37, 38) bytransmission electronic microscopy in treated cells. As shownin Fig. 3D, the double-membraned autophagosome could beeasily observed in MLN4924-treated but not in control cells.Taken together, these results convincingly showed thatMLN4924 induced autophagy in cancer cells.

Figure 1. MLN4924 inhibited the growth of liver cancer cells. A, MLN4924 inhibited cullin1 neddylation. Huh-7 and Hep G2 cells were seeded into 100-mmdishes with 6 � 105 cells per dish, cultured overnight, treated with or without MLN4924 at 0.1 mmol/L for indicated time, then harvested and subjected toimmunoblotting (IB) using antibodies against cullin1 with GADPH as a loading control. B, MLN4924 inhibited liver cancer cell proliferation. Cells were seededinto 24-well plates with 1 � 104 cells per well, cultured overnight, and then treated with or without MLN4924 at 0.1 mmol/L. Cells were trypsinizedand cell numbers of each well were counted with Cellometer Auto T4 (Nexcelom Bioscience) at indicated time points (�, P < 0.001, n ¼ 3). C, MLN4924suppressed colony formation in liver cancer cells. Huh-7 and Hep G2 cells were seeded into 60-mmdishes with 500 cells per dish and cultured overnight andthen treated with or without MLN4924 at 0.1 mmol/L for 10 days, followed by crystal violet staining and colony counting (�, P < 0.001, n ¼ 3). D,MLN4924 inhibited liver cancer cell proliferation measured by CELL-IQ assay. Huh-7 liver cancer cells were seeded into 24-well plates with 1� 104 cells perwell and cultured overnight, and then treated with or without MLN4924 at 0.1 mmol/L. Cell proliferation at the fixed microscopic fields was continuouslyphotographed over time. Cell number of each microscopic field at different time points was imaged and counted. Data obtained from 12 differentmicroscopic fields in each group were showed with mean � SE and error bar.






MLN4924-induced autophagy is partially attributed tomTOR inactivation by Deptor accumulation and toreactive oxygen species stress

Considering that MLN4924 exerts its effects by inactivatingCRL/SCF and leading to accumulation of its substrates, we firstsearched for CRL/SCF substrates whose accumulation mayaffect autophagy pathways. Most recently, we characterized anmTOR-inhibitory protein Deptor (26) as a novel substrate ofCRL/SCF (39–41). Because inhibition ofmTOR activity inducesautophagy response, we hypothesized that CRL/SCF inactiva-tion by MLN4924 would block Deptor degradation, causing its

accumulation, and thus inhibiting mTOR activity to triggerautophagy. Indeed, we found that MLN4924 blocked the turn-over of Deptor and led to its accumulation in both Huh-7 andHep G2 cells (Fig. 4A). The accumulation of Deptor byMLN4924 resulted in a remarkable decrease in phosphorylated4E-BP1 [p-4E-BP1 (T37/46)], which was coupled with remark-able increase in nonphosphorylated 4E-BP1 (NonP-4E-BP1).Because 4E-BP1 is phosphorylated by mTOR and p-4E-BP1serves as a marker of mTOR activation (39), significantdecrease in p-4E-BP1 after MLN4924 treatment indicatedmTOR inactivation (Fig. 4A). Consistently, rapamycin, a

Figure 2. MLN4924 inducedG2 cell-cycle arrest andapoptosis inHuh-7cells. A, MLN4924 induced DDRand accumulation of p21 and p27.Huh-7 and Hep G2 cells seededinto 100-mm dishes with 6 � 105cells per dish were culturedovernight, treated with or withoutMLN4924 at 0.1 mmol/L forindicated time, then harvested andsubjected to IB analysis usingantibodies against p21, p27, p-H2A, p-CHK2, t-H2A, t-CHK2 withGADPH as a loading control. B,MLN4924 induced cell-cycle arrestand apoptosis. Huh-7 cells seededinto 60-mm dishes with 1 � 105cells per dish were culturedovernight, treated with or withoutMLN4924 at 0.1 mmol/L, andsubjected to PI staining and FACSanalysis at indicated time points.The percentage of cells at the G2–M phase and sub-G1 phase wasindicated in figures. C,immunoblotting analysis ofproteins after MLN4924 treatment.Huh-7 and Hep G2 cells seededinto 100-mm dishes with 6 � 105cells per dish were culturedovernight, treated with or withoutMLN4924 at 0.1 mmol/L forindicated time, and subjected to IBanalysis using antibodies againstWee1, p-Histone H3, cleavedCaspase-3 (cleaved Casp-3), andcleaved PARP with GADPH as aloading control.

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well-known small-molecule inhibitor of mTOR, also decreasedp-4E-BP1 and triggered autophagy in these cells (data notshown).To determine whether Deptor accumulation contributed to

autophagy response upon CRL/SCF inactivation directly, theexpression of Deptor in MLN4924-treated cells was down-regulated via siRNA silencing (Fig. 4B). As a result, Deptorknockdown significantly restored the phosphorylation of 4E-BP1 after MLN4924 treatment and attenuated the conversionof LC3 I to LC3 II in liver cancer cells, especially in Hep G2 cells(Fig. 4B). However, we found that Deptor siRNA silencing couldnot fully block the conversion of LC3 I to LC3 II upon CRL/SCFinactivation (Fig. 4B), suggesting that Deptor accumulation isnecessary but not sufficient to MLN4924-triggered autophagy.Recently, it was reported that MLN4924 enhances the gen-

eration of reactive oxygen species (ROS) and induces ROSstress in cancer cells (20, 42). Considering that ROS stresscould serve as a potential trigger of autophagy (42), we deter-mined whether MLN4924-induced autophagy in liver cancer

cells is also attributed to ROS stress by addingN-acetyl cysteine(NAC), a classical ROS scavenger, to cell culture medium (43).As the result, NAC significantly blocked the conversion of LC3 Ito LC3 II induced by MLN4924 in both Huh-7 and Hep G2 cells(Supplementary Fig. S2), suggesting that ROS stress may beinvolved in autophagy response upon CRL/SCF inactivation.

Autophagy occurred prior to apoptosis and played anantiapoptotic role during MLN4924 treatment

The elucidation of the cross-talk between autophagy andapoptosis, 2 programmed cellular responses against unfavor-able stresses, has become an important object for effectivecancer treatment (24). Here, we first determined which hap-pening first occurred upon MLN4924 treatment, autophagy orapoptosis. To address this, the kinetics of autophagy andapoptosis responses upon treatment was evaluated by calcu-lating the percentage of cells displaying punctuative distribu-tion of EGFP-LC3 for autophagy and sub-G1 phase distributionfor apoptosis, respectively. As shown in Fig. 5A and B,

Figure 3. MLN4924 induced autophagy in liver cancer cells. A, autophagy responsemeasured by LC3 I to LC3 II conversion. Cells treatedwithMLN4924 at 0.1mmol/L for 0, 24, 48, and 72 hours were harvested and subjected to IB analysis. B, autophagy response measured by appearance of punctate vesiclestructure. Stably expressing EGFP-LC3 Huh-7 and Hep G2 cells were treated with the MLN4924 at 0.1 mmol/L for 72 hours and photographed underfluorescent microscope. Cells with punctate vesicle structures of EGFP-LC3 were counted as autophagy-positive cells. C, autophagy measured by acridineorange staining. Cells were seeded in 60-mm dishes with 1 � 105 cells per dish, treated with or without MLN4924 at 0.1 mmol/L for 24 hours, and thensubjected to acridine orange staining and FACS assays, as described in Materials and Methods. D, autophagy measured by transmission electronmicroscopy. Cells were treated with or without MLN4924 at 0.1 mmol/L for 72 hours, and then fixed for analysis with transmission electron microscopy.Arrowheads denote typical appearance of double-membraned autophagosomes, which contained engulfed bulk cytoplasm and cytoplasmicorganelles. Scale bar, 500 nm.






autophagy started to occur as early as 24 hours after MLN4924treatment, whereas apoptosis started to appear at 48 hoursupon treatment in bothHuh-7 andHepG2 cells. Once initiated,both autophagy and apoptosis continued to increase over timeand reached the peak at 96 hours after treatment (Fig. 5A andB). The findings suggested that autophagy and apoptosis occursequentially in liver cancer cells upon MLN4924 exposure.

To further determine the effect of autophagy response onapoptosis induction, we first tried to block the autophagypathway via siRNA silencing of autophagy genes ATG5 andBeclin 1 and evaluated its effect on apoptosis. As shown in Fig.5C, partial blockage of autophagy by siRNA knockdown ofATG5 and Beclin 1 enhanced apoptosis response uponMLN4924 treatment, as measured by caspase-3 activation inHep G2 cells. To further confirm the result, we first used a pairof mouse embryonic fibroblast (MEF) cells, MEF-Atg5-WT(autophagy-competent) versus MEF-Atg5-KO (autophagy-deficient) cells. As shown in Fig. 5D, MLN4924 induced autop-hagy response only in MEF-Atg5-WT but not in MEF-Atg5-KOcells (Fig. 5D). Moreover, apoptosis could be obviously inducedby MLN4924 even at a lower concentration (1 mmol/L) inautophagy-deficient Atg5-KO but not autophagy-competentAtg5-WT cells. Moreover, the intensity of apoptosis activationin autophagy-deficient cells was much stronger than that inautophagy-competent cells upon MLN4924 treatment at anydose (1–10 mmol/L; Fig. 5D). Similar result was obtained fromautophagy-deficient Atg7-KO MEF cells (Supplementary Fig.

S3). These findings suggest that autophagy response inducedby MLN4924 plays an antiapoptotic role.

MLN4924 suppressed tumor growth in vivo by inducingautophagy and apoptosis

Having established that MLN4924 suppressed the growthof liver cancer cells by modulating autophagy and apoptosisin vitro, we next evaluated the in vivo antitumor activity ofMLN4924 and elucidated potential mechanisms. MLN4924was administered to Hep G2-GFP orthotopic xenografts, andthe kinetic growth of tumors was monitored via a fluores-cence-based imaging system (34). As shown in Fig. 6A and B,MLN4924-treated tumors progressed slowly, whereas con-trol tumors grew rapidly over time, as shown by represen-tative kinetic images of tumors (Fig. 6A) and tumor growthcurve (Fig. 6B, n ¼ 10). At the end point of MLN4924treatment (35th day), tumors of both treated and controlgroups were collected, imaged (Fig. 6C), and weighed (Fig.6D). As shown in Fig. 6C, the size of control tumors wasmuch larger than that of MLN4924-treated tumors. Consis-tently, the weight of control tumors was significantly higherthan that of treated tumors (Fig. 6D). During the wholetreatment, no obvious treatment-related toxicity againstbody weight, liver function, and kidney function of animalswas observed (Fig. 6E and Supplementary Fig. S4). Thesefindings showed that MLN4924 has impressive anti-livercancer activity in vivo and is well tolerated in mice.

Figure 4. MLN4924-inducedautophagy was partially attributedto Deptor accumulation. A, Deptoraccumulation and mTOR pathwayinactivation by MLN4924. Cellswere treated with MLN4924 at 0.1mmol/L for indicated time, followedby IB analysis to probe indicatedproteins with GADPH as a loadingcontrol. B, attenuation ofMLN4924-induced autophagy byDeptorsiRNA silencing. Cells weretransfected with siRNA targetingDeptor (siDeptor) or scrambledcontrol siRNA (siCont) for 48 hoursand subjected to MLN4924treatment at 0.1 mmol/L for 72hours, followed by IB analysisusing indicated antibodies withGADPH as a loading control.

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To explore the in vivo anticancer mechanism of MLN4924,we extracted proteins from treated and control tumors anddetermined the inactivation status of CRL/SCF and theinduction of autophagy and apoptosis. As shown in Fig.6F, MLN4924 significantly inhibited cullin neddylation andled to accumulation of CRL/SCF substrates p21 and p27,indicating the efficient inactivation of CRL/SCF in vivo aftertreatment. Consistently, MLN4924 induced autophagy asshown by the accumulation of Deptor and conversion ofLC3 I to LC3 II. Meanwhile, it triggered apoptosis as shownby enhanced activation of PARP and caspase-3 in treatedtumors. Taken together, these findings showed that

MLN4924 exploited the similar anticancer mechanisms invitro and in vivo.

DiscussionLiver cancer, one of the most common humanmalignancies

with high recurrence rate and poor long-term survival (2), callsfor novel therapeutic agents with high efficacy and low toxicity.Recently, CRL/SCF has been recognized as a promising anti-cancer target (6, 7). The effort to discover small-moleculeinhibitors against CRL/SCF led to the discovery of MLN4924(4). MLN4924 functions as a specific inhibitor of NAE and

Figure 5. Autophagy occurredbefore apoptosis andprevented cellsfrom apoptosis induced byMLN4924. A and B, autophagyoccurred before apoptosis. Huh7-EGFP-LC3 or Hep G2-EGFP-LC3cells seeded in 24-well plates with1 � 104 cells per well were culturedovernight and treated with or withoutMLN4924 at 0.1 mmol/L for indicatedtime. Autophagy was evaluated byappearance of punctate vesiclestructure with fluorescencemicroscope (A), whereas apoptosiswas evaluated by cell populations insub-G1 phase with PI staining andFACS (B). C, blocking of autophagypathwaybyATG5andBeclin 1 siRNAsilencing amplified MLN4924-induced apoptosis. Cells weretransferred with siRNAs againstATG5 and Beclin 1 or control for48 hours, and then treated withMLN4924 at 0.1 mmol/L for 96 hours.Finally, cells were subjected tocaspase-3 activation assays, asdescribed in Materials and Methods.D, MLN4924 induced moreapoptosis in MEF-Atg5-KOcompared with MEF-Atg5-WT.Paired MEFs (Atg5-WT vs. Atg5-KO)were treated with MLN4924 atindicated concentrations for48 hours, followed by IB analysiswithindicated antibodies.






blocks cullin neddylation, which is required for CRL/SCFactivity (4). Because of its promising anticancer efficacy inpreclinical studies, MLN4924 has been advanced to phase Iclinical trials as a novel class of anticancer agents (15). In thispreclinical study, we showed the significant anticancer effect ofMLN4924 on liver cancer both in vitro and in vivo by inducingautophagy and apoptosis upon CRL/SCF inactivation.

Previous studies, including ours, showed that MLN4924inhibits cullin neddylation, inactivates CRL/SCF, and stabilizesCRL/SCF substrates, such as DNA replication licensing pro-teins Cdt1 andOrc1, induces DDR, leading to S-phase cell-cycle

arrest and cell death via apoptosis and/or senescence in solidtumor cells (4, 12, 19–23). In addition, it was recently reportedthat MLN4924 could induce the dramatic accumulation ofCRL/SCF substrate IkB-a and trigger G1 phase arrest in NF-kB–dependent activated B-cell (ABC) lymphoma cells inwhichlittle DNA rereplication was observed in treated cells (12). Inpresent study, however, we found that in both Huh-7 and HepG2 liver cancer cells, MLN4924 triggered DDR and cell-cyclearrest at G2 phase, but not S-phase or G1 phase, which wasconsistent with our recent finding that inactivation of CRL/SCF via siRNA silencing of RBX1/ROC1, a CRL/SCF essential

Figure 6. MLN4924 suppressed the growth of orthotopic xenograft model of liver cancer by modulating autophagy and apoptosis. A–B, MLN4924 inhibitedtumor growth measured by fluorescence imaging system. Nude mice bearing liver cancer xenografts with Hep G2-GFP cells were administrated withMLN4924 at 30 mg/kg s.c. twice daily on a 3-days-on/2-days-off schedule for 7 cycles. Tumor size was monitored twice a week with fluorescence imagingsystem (A). The data were converted to tumor growth curves by ModFit LT software (B). C and D, MLN4924 significantly reduced tumor volume. Mice weresacrificed at 35th day after treatment (the end of study, n ¼ 10). Tumor tissues of mice were collected, photographed (C), and weighed (D; P < 0.001).E, no obvious toxicity against body weight and liver function was observed during MLN4924 treatment. Body weight of animals was measured twice a weekduring the treatment (E, top). At the end of the study, mice were sacrificed, and the serum was collected for biochemical assays. Liver function oftreatedmice was determined bymeasuring the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as described inMaterialand Methods. NS, no significance. F, MLN4924 induced autophagy and apoptosis in vivo. Tumor tissues were collected and homogenized in lysisbuffer with tissue tearor for 1 minute, then centrifuged at 10,000� g at 4�C for 5 minutes. After centrifugation, the supernatant was used for IB analysis withindicated antibodies. GAPDH was used as a loading control. P < 0.001, n ¼ 10.

Luo et al.





component, triggered G2 phase arrest as a result of activatingDNA damage checkpoint in multiple cancer cells (44).Interestingly, inactivation of CRL/SCF via either MLN4924treatment or RBX1/ROC1 siRNA silencing led to significantaccumulation of CRL/SCF substrate Wee1, which serves as aninhibitor of cell entry from G2 into M phase (35). Takentogether, these findings suggested that different cell-cyclearrest induced by MLN4924 in different cell lines may resultfrom the accumulation of different CRL/SCF substrates in cellline–dependent manner.Another novel finding of this study is thatMLN4924 induced

autophagy as a cellular response while it triggered cell-cyclearrest and apoptosis in liver cancer cells both in vitro and invivo. Actually, we found that MLN4924 also triggered autop-hagy response in other multiple human cancer cell lines,including H1299 lung cancer cells, U87 glioblastoma cells, andHela cervical cancer cells (data not shown). Moreover, wefound that inactivation of CRL/SCF via siRNA silencing of itsessential component RBX1/ROC1 triggered autophagy in abroad spectrum of cancer cells as well (unpublished data).These findings indicate that autophagy is a general phenom-enon to CRL/SCF inactivation, induced either by small-mol-ecule inhibitors (such as MLN4924) or by siRNA silencing of itsessential components (such as RBX1/ROC1).In terms of mechanism, MLN4924-induced autophagy could

be partially attributed to the accumulation ofmTOR-inhibitoryprotein Deptor (26). Three recent studies, including ours,identified Deptor as a novel substrate of SCFbTrCP E3 ubiquitinligase and the stabilization of Deptor by SCFbTrCP inactiva-tion could inhibit mTOR activity (39–41). Thus, it is anti-cipated that pharmacologic inhibition of CRL/SCF activity byMLN4924 triggered autophagy owing to Deptor accumulationand subsequent mTOR inactivation. Meanwhile, ROS stressseemed to be also involved in MLN4924-triggered autophagy,as ROS scavenger NAC could significantly block autophagyresponse upon MLN4924 treatment. Most recently, severalhundreds of new potential CRL/SCF substrates have beenidentified in MLN4924-treated cells via high-throughputproteomic approaches (17, 18, 45, 46). Some of these potentialCRL/SCF substrates, such as autophagy/Beclin-1 regulator1 (Ambra1), may also be accumulated upon CRL/SCF inacti-vation and contribute to autophagy response directly (18, 47).Thus, MLN4924 probably induces autophagy response bymodulating several signaling pathways, which are regulatedby different CRL/SCF substrates. Further studies by evaluatingthe effect of high-throughput siRNA silencing of potential SCFsubstrates on autophagy induction upon MLN4924 treatmentshould be helpful to address this issue.As an important cellular response, autophagy plays a key

role in the regulation of cell survival during diverse stresses.Some studies show that autophagy serves as a prosurvivalmechanism against unfavorable conditions, whereas othersshow that autophagy can cause cell death (known as type IIprogrammed cell death; 48, 49). The reason for this obviousdiscrepancy about the role of autophagy in regulating cellsurvival is rather complex, and may be attributed to manyfactors, such as the type of cellular stresses, the time and extentof autophagy induction, as well as cell lines used (48, 49). In this

study, MLN4924-induced autophagy was a survival signal, andblockage of autophagy pathway enhanced cell apoptosis. Con-sistently, we found that inactivation of CRL/SCF by RBX1/ROC1 siRNA silencing also triggered autophagy, which played aprosurvival and antiapoptotic role in liver cancer cells (unpub-lished data). The findings showed that the pharmaceutical orgenetic inhibition of CRL/SCF activity triggers protectiveautophagy responses, and blockage of autophagy may serveas a promising strategy to enhance MLN4924-induced sup-pression of cancer cell growth by amplifying apoptosis.

Furthermore, we showed in vivo anticancer effects ofMLN4924 using a physiologically relevant orthotopic xenograftmodel of liver cancer. As it worked in vitro, MLN4924 signif-icantly inhibited cullin neddylation, inactivated CRL/SCFactivity, leading to the accumulation of CRL/SCF substrates(such as p21, p27, and Deptor) in vivo. Importantly, mechanismanalysis revealed that MLN4924 induced both autophagy andapoptosis in tumors, suggesting that MLN4924 worked viasimilar mechanisms in vitro and in vivo. Encouragingly,MLN4924 treatment was well-tolerated in mice during entireexperimental periods. The high safety of MLN4924 should beattributed to its improved selectivity as MLN4924 only blocksthe degradation of a specific subset of substrates regulated byCRL/SCF (4, 15). In contrast, the first and only U.S. Food andDrug Administration (FDA)-approved proteasome inhibitorbortezomib blocks the degradation of all substrates regulatedby ubiquitin-proteasome system, resulting in severe generaltoxicity (50).

Our findings from this study can be summarized as follows:MLN4924 induces cell-cycle arrest and apoptosis in p53-inde-pendent manner to inhibit liver cancer cell growth. During theprocess, it also triggers prosurvival autophagy responsemainlyby Deptor accumulation. The blockage of autophagy pathwaysensitizes liver cancer to MLN4924-induced apoptosis (Fig. 7).Moreover, MLN4924 displayed striking in vivo antitumor

MLN4924

CRL/SCF

DDRDeptor, ROS, etc

Autophagy Apoptosis

Cell survival

siATG5/siBeclin 1ATG5/7 deletion

Figure 7. Working model. MLN4924 induced DDR and apoptosis tosuppress liver cancer cell growth in vitro and in vivo, whereas it triggeredautophagy as a survival signal due to Deptor accumulation and ROSstress. Blockage of autophagy via siRNA silencing or genetic deletion ofautophagy essential genes sensitized cancer cells to MLN4924-inducedapoptosis.






effects via a similar anticancer mechanism with high safety.These findings indicate a great value for future clinical inves-tigation of MLN4924 for the treatment of liver cancer, andprovide a piece of proof-of-concept evidence for potentialcombination therapy with MLN4924 and autophagy inhibitorfor enhanced cancer cell killing.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: C. Ding, X.Wang, Y. Sun, J. Liu, L. JiaDevelopment of methodology: Z. Luo, H.W. Lee, L.S. Jeong, J. Liu, L. JiaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Z. Luo, L. Li, L. Wang, J. Qian, L. Wu, Y. Chu, J. Yi, L.S.Jeong, J. Liu, L. JiaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Z. Luo, L. Wang, D. Yang, J. Yi, Y. Sun, J. Liu, L. JiaWriting, review, and/or revision of themanuscript: Z. Luo, Y. Pan, Y. Sun, J.Liu, L. Jia

Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): G. Yu, L. Wu, Y. Chu, J. Liu, L. JiaStudy supervision: J. Liu, L. Jia

AcknowledgmentsThe authors thank Noboru Mizushima and Yeguang Chen for Atg5�/� and

Atg5þ/þ MEFs and Masaaki Komatsu for Atg7�/� and Atg7þ/þ MEFs.

Grant SupportThis work is supported by National Natural Science Foundation Grant of

China (81172092), National Basic Research Program of China (973 program,2012CB910302) and National Natural Science Foundation Grant of China(31071204 to L. Jia), and National Natural Science Foundation Grant of China(91129702, 81125001) to J. Liu.

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 indicate thisfact.

Received February 10, 2012; revised April 3, 2012; accepted April 23, 2012;published OnlineFirst May 4, 2012.

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2012;72:3360-3371. Published OnlineFirst May 4, 2012.Cancer Res Zhongguang Luo, Guangyang Yu, Hyuk Woo Lee, et al. Autophagy and Apoptosis to Suppress Liver Cancer Cell GrowthThe Nedd8-Activating Enzyme Inhibitor MLN4924 Induces

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