endostatin has atpase activity, which mediates its ... · has novel atpase activity, which mediates...

Cancer Biology and Signal Transduction

Endostatin Has ATPase Activity, WhichMediates Its Antiangiogenic and AntitumorActivitiesShan Wang1,2,3, Xin-an Lu1,2,3, Peng Liu1, Yan Fu1,2,3, Lin Jia1,2,3, Shunli Zhan1, andYongzhang Luo1,2,3

Abstract

Endostatin is an endogenous angiogenesis inhibitor withbroad-spectrum antitumor activities. Although the molecularmechanisms of endostatin have been extensively explored, theintrinsic biochemical characteristics of endostatin are notcompletely understood. Here, we revealed for the first time thatendostatin embedded novel ATPase activity. Moreover, mutagen-esis study showed that the ATPase activity of endostatin mutantspositively correlated with effects on endothelial cell activities andtumor growth. E-M, an endostatin mutant with higher ATPaseactivity than that of wild-type (WT) endostatin, significantlyincreased endostatin-mediated inhibitory effects on endothelialcell proliferation, migration, tube formation, and adhesion. Invivo study showed that E-M displayed enhanced antitumor effects

compared with WT. On the other hand, K96A, K96R, and E176A,endostatin mutants with lower ATPase activities than that of WT,showed reduced or comparable effects on targeting both in vitroendothelial cell activities and in vivo tumor angiogenesis andtumor growth. Furthermore, endostatin and itsmutants exhibiteddistinct abilities in regulations of gene expression (Id1, Id3), cellsignaling (Erk, p38, and Src phosphorylation), and intracellularATP levels. Collectively, our study demonstrates that endostatinhas novel ATPase activity, which mediates its antiangiogenic andantitumor activities, suggesting that construction of endostatinanalogues with high ATPase activity may provide a new directionfor the development of more potent antiangiogenic drugs. MolCancer Ther; 14(5); 1192–201. �2015 AACR.

IntroductionAntiangiogenic agents have been widely considered as the

fourth modality in cancer treatment, together with surgery, che-motherapy, and radiotherapy. Endostatin, as an endogenousantiangiogenic inhibitor (1), exhibits potent antitumor activitiesin variousmousemodels (2) and receives CFDA (China Food andDrug Administration) approval as a new drug for the treatment ofnon–small cell lung cancer (NSCLC) patients in clinic (3). Thisdrug is widely used in China and shows profound clinical efficacy(4–6). Accumulating evidence demonstrates that the N-terminalintegrity and correct folding are essential to guarantee the struc-tural stability and biological functions of endostatin (7, 8).

The detailed mechanisms underlying antitumor activities ofendostatin have been extensively studied. Endostatin specificallydiminishes the proliferation, migration, and tube formation of

endothelial cells in vitro (1, 9). It can initiate endothelial cellapoptosis through the induction of VDAC1 phosphorylation,which facilitates the mitochondrial permeability transition pore(mPTP) opening (10). Moreover, it also binds to cell surfacereceptors such as integrins (11), glypicans (12), laminin (13) andnucleolin (14), and thereby regulates a myriad of signalingcascades. For example, endostatinbinding to integrina5b1 resultsin the inhibition of the FAK–c-Raf–MEK1/2–p38–ERK1 MAPKpathway (15). Besides, the internalization of endostatin by endo-thelial cells is important, if not essential, for the physiologicfunctions of endostatin (16). Enhancing endostatin uptake bycholesterol-chelating agents or addition of a macromoleculetransduction domain (MTD) significantly increases the therapeu-tic efficacy on animal models (17, 18). Abdollahi and colleagues(19) reported that approximately 12% of the human genome ismodulated by endostatin, therefore tipping the dynamic angio-genic balance toward the inhibition part.

ATPases play distinct roles in a lot of cellular processes, includ-ing DNA replication, protein synthesis, protein folding, proteol-ysis, and membrane fusion (20). They have evolved differentstrategies to recognize ATP and elicit ATPase activities. ATPaseshave twowell-knownWalkermotifs. TheWalker Amotif, the best-known motif associated with ATP binding, has a common nucle-otide-recognition sequence: GXXXXGK(T/S) (where X is anyamino acid residue; ref. 21). The Walker B motif contains aconserved Asp or Glu residue preceded by a consecutive sequenceof four hydrophobic residues (hhhhD/E), with this acidic residuecoordinating an Mg2þ ion (or Mn2þ or Ca2þ) essential for ATPcatalysis (21–23). Mutations of key residues in the Walker motifsalmost abolish ATPase activities and disrupt normal functions

1The National Engineering Laboratory for Anti-tumor Protein Thera-peutics, School of Life Sciences, Tsinghua University, Beijing, China.2Beijing Key Laboratory for Protein Therapeutics, School of LifeSciences, Tsinghua University, Beijing, China. 3Cancer Biology Labo-ratory, School of Life Sciences, Tsinghua University, Beijing, China.

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

S. Wang, X. Lu, and P. Liu contributed equally to this article.

Corresponding Author: Yongzhang Luo, School of Life Sciences, TsinghuaUniversity, Room 310, New Biology Building, Beijing 100084, China. Phone:86-10-6277-2897; Fax: 86-10-6279-4691; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-14-0836

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

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(24–26). For example, a point mutation (K368R) at the Walker Amotif of PKCa diminishes the kinase activity and interferes withits downstream signaling (25).

Despite comprehensive investigations, the precisemechanismsof endostatin actions still remain elusive. In this study, we firstdiscovered novel ATPase activity of endostatin, which has neverbeen reported. Endostatin mutants with decreased ATPase activ-ities (K96A, K96R, and E176A) exhibited reduced or comparablelevels in the inhibition of endothelial cell activities in vitro andantitumor efficacy in vivo compared with wild-type (WT) endo-statin. However, E-M, the endostatin mutant with higher ATPaseactivity, showed the more potent antitumor and antiangiogeniceffects. Collectively, our study demonstrates that endostatin hasnovel ATPase activity, which mediates the antiangiogenic andantitumor effects of endostatin.

Materials and MethodsPlasmids, reagents, and Abs

Human endostatin was subcloned into pET30a(þ) vector(Addgene), and endostatin mutants (K96A, K96R, E176A, andE-M) were constructed using Fast Mutagenesis System (TransGenBiotech) and verified by sequencing (Life Technologies). Recom-binant proteins, providedbyProtgenCo, Ltd.,were expressed inE.coli in high densities and cells were harvested and lysed. Theinsoluble part of the lysates containing the inclusion bodies wascollected and purified. The inclusion bodies were subsequentlyrenatured and purified by ion exchange chromatography. Theconcentrations of recombinant proteins were measured using thePierce BCA Protein Assay Kit (Thermo Fisher Scientific).

Anti-endostatin antibody was from laboratory stock. We pur-chased primary antibodies specifically recognizing GAPDH, Erk,p-Erk, p38, p-p38 (Santa Cruz), pro caspase-3, cleaved caspase-3,Src, p-Src, Id1, Id3 (Cell Signaling Technology), and CD31(Abcam). Secondary antibodies were obtained from Santa CruzBiotechnology.

ATP, ATP-g-S, and ADP-b-S were obtained from Sigma-Aldrich,and GTP, CTP, UTP, AMP, and ADP were from Sangon BiotechCo., Ltd.

Cell culturePrimary HUVECs were cultured in endothelial cell medium

(ScienCell), and used at passages 2 to 7. Culture of A549-GFP cellsandHMECswasmaintained inDMEMand10%FBS (Wisent). Allcell lines were purchased from Cell Resource Center, ChinaInfrastructure of Cell Line Resources within 6 months of thebeginning of the project and validated by the supplier, exceptfor A549-GFP cells, which were developed by stable transfectionof GFP and were not genetically authenticated.

ATPase activity assayThe malachite green assay was used to determine the ATPase

activity of endostatin by measuring the release of inorganicphosphate as previously described (27). Briefly, recombinantproteins (typically 5 mg) and ATP or NTP/ADP/AMP/ATP-g-S/ADP-b-S (final 1 mmol/L) were incubated in imidazole-Cl buffer(10 mmol/L imidazole-Cl, 75 mmol/L KCl, 0.2 mmol/L EDTA, 3mmol/L MgCl2, pH 7, final volume of 100 mL) at 37�C for 60minutes. Subsequently, HCl/Mo was added and 2 minutes later0.042% malachite green and H2SO4 were added. Absorbance at650 nm was detected after incubation for 30 minutes. pH depen-

dence was evaluated using imidazole-Cl buffer at pH 4.5 to 8.5.Bivalent cation dependence was assessed using solutions ofMgCl2, CaCl2, CuCl2, MnCl2, and ZnCl2. For the determinationof enzyme kinetics, ATP concentrations ranging from6.25mmol/Lto 2 mmol/L were used and Michaelis–Menten calculations wereperformed with GraphPad Prism 5 software (GraphPad Software,Inc.).

ATP bioluminescent assay was performed with the ATP Biolu-minescent Assay Kit (Sigma-Aldrich) according to the manufac-turer's protocol.

ATP-binding assayProteins in Tris buffer (10 mmol/L Mg2þ and 100 mmol/L

NaCl, pH 7.5) were incubated with high-affinity ATP-agarose(Innova) at 4�C for 1 hour, and then washed three times withthe same Tris buffer. The pelleted resin was mixed with reducingSDS-PAGE loading buffer for immunoblotting (28).

Tryptophan emission fluorescenceTryptophan emission fluorescence spectra of endostatin and its

mutants were measured by a Hitachi F-4500 spectrophotometerequipped with a temperature controlled liquid system asdescribed previously (8). Briefly, the concentrations of proteinsin imidazole-Cl buffer were 1 mmol/L, and allmeasurements werecarried out at 37�C, pH 7.4.

Western blot analysisSamples were mixed with reducing SDS-PAGE loading buffer,

boiled at 100�C for 15 minutes, subjected to SDS-PAGE, andtransferred onto a polyvinylidene difluoride membrane (Milli-pore). The membrane was blocked in TBST (20 mmol/L Tris, 150mmol/LNaCl, and 0.1%Tween 20) plus 5% to 10%dried non-fatskimmed milk for 30 minutes at room temperature. The mem-brane was incubated with the indicated primary antibodies inTBST and 1% dried non-fat skimmed milk for at least 2 hours atroom temperature or overnight at 4�C, washed three times withTBST for 5 minutes each time at room temperature, and thenincubated with corresponding horseradish peroxidase–conjugat-ed secondary antibodies for 60 minutes at room temperature.Following five washes with TBST, immunoreactive bands weredetected. Images were scanned using Bio-5000 plus (Microtek)and quantified with Image J (NIH).

Quantitative RT-PCRTotal RNA was isolated using Direct-zol RNA MiniPrep (Zymo

Research), and cDNAwas synthesized using the First Strand cDNASynthesis Kit (Fermentas). Quantitative RT-PCR (qRT-PCR) wasconducted using the Brilliant II SYBR Green qRT-PCR Master MixKit (Stratagene).

Cell viability assayA total of 2 � 103 HUVECs were seeded in sextuplicate in a

96-well plate and 24 hours later, endothelial cells were treatedwith 20 mg/mL indicated proteins in endothelial cell basalmedium containing 1% FBS for 72 hours. Cell viability wasassessed by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazoli-um bromide (MTT) as described previously (29). Absorbance at450 nm was obtained by Varioskan Flash (Thermo FisherScientific).

Endostatin Has Novel ATPase Activity

www.aacrjournals.org Mol Cancer Ther; 14(5) May 2015 1193




Transwell migration assay and tube formation assayTranswell migration assay and tube formation assay were

performed as described previously (29). Images were photo-graphed with an Olympus IX71 optical microscope (Olympus).The number ofmigrated cells and tube lengthwas quantifiedwithImage-Pro Plus 6.0 software (Media Cybernetics).

Cell adhesion assayThe 48-well plate was coated with Matrigel for 1 hour, washed

for two times, and chilled on ice for 1 to 2 minutes. Subsequentlyequivalent cells were added to each well with 20 mg/mL indicatedproteins and incubated at 37�C for 30minutes. Plateswere shakedfor 10 to 15 seconds and washed for two to three times. Adherentcells were photographed with an Olympus IX71 optical micro-scope and counted.

Measurement of cellular ATP levelsHMECs were treated with 20 mg/mL indicated proteins for 24

hours. Cellular ATP levels were detected as previously described(30).

Endostatin internalization assaysHUVECs were treated with 5 mg/mL endostatin or endostatin

mutants at 37�C for 1 hour. Next, cells were washed with acidicbuffer (pH 3.5) and ice-cold PBS to remove cell surface-bindingendostatin. Subsequently, the cells were examined for endostatininternalization by Western blot analysis.

Animal studiesA549-GFP tumor cells (106) were mixed with Matrigel (Becton

Dickinson) and inoculated into the axilla of nude mice s.c. Oncethe tumor volume approached 0.1 cm3, mice were randomlygrouped (n ¼ 6/group) and i.v. treated with PBS, WT endostatin,K96A, K96R, E176A, or E-M (12 mg/kg) every 2 days. Tumorvolumes weremeasured every other day and calculated as volume¼ 0.625 � length � (width)2. After 34 days, mice were sacrificed

and tumors were excised and weighed. For immunohistochem-istry analysis, tumorswerefixed in 4% formaldehyde, and embed-ded in paraffin (5 mm sections). Subsequently, these tissues werestained with Abs and diaminobenzidine, and then counter-stained with hematoxylin. Images were captured by an OlympusIX71 optical microscope (Olympus) and quantification was com-pleted using Image-Pro Plus 6.0 (Media Cybernetics).

All animal studies were approved by the Institutional AnimalCare and Use Committee of Tsinghua University (Approval no.13-LYZ6).

Statistical analysisAll data from individual experiments are represented as means

� SDs or SEMs. Comparisons were determined using two-tailedStudent t tests, and P values <0.05 were considered statisticallysignificant.

ResultsEndostatin has novel ATPase activity

Walker motifs are found in a broad range of ATPases, andthereby provide predictive value for ATPases (31). Throughdetailed examination of human endostatin sequence, we foundthat endostatin comprises a Walker A motif variant (GXXGXXK;ref. 32) and a Walker B motif (Fig. 1A, I and II), suggesting thatendostatin may display ATPase activity. Besides, Walker A motif–containing proteins are also frequently found to consist of aRX(2-3)R motif that interacts with the adenine base of ATP witha conserved Arg residue (23). Two RX(2-3)R motifs were identifiedin the endostatin sequence (Fig. 1A, IIIa and IIIb). Taken together,we proposed that endostatin might have ATPase activity. We thencompared the 3D structure of endostatin (33) with that of P97(34), an AAA-ATPase. As shown in Supplementary Fig. S1, theposition of Walker motifs on endostatin 3D structure is similar tothat of the characterized ATPase.

To validate whether endostatin indeed exhibits ATPase activ-ity, ATP binding analysis was performed. Endostatin could bind

Figure 1.Endostatin has ATPase activity.A, Walker A and B motif analyses ofendostatin amino acid sequence.B, endostatin was incubated withATP-agarose beads in the absence orpresence of ATP, and the boundproteins were detected by Westernblot analysis. C, ATP hydrolysis byendostatin in a dose-dependentmanner. D, ATP hydrolysis byendostatin in a time-dependentmanner; error bars, SDs.

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Mol Cancer Ther; 14(5) May 2015 Molecular Cancer Therapeutics1194




to ATP-agarose, and addition of excessive ATP prevented endo-statin from binding (Fig. 1B), excluding the possibility that theinteraction between endostatin and ATP-agarose is unspecific.Subsequent malachite green assay revealed that endostatinhydrolyzed ATP in a dose- and time-dependent manner (Fig.1C and D). Consistently, ATP bioluminescence assay resultsshowed that endostatin indeed directly consumed ATP (Sup-plementary Fig. S2A and S2B), whereas ATP was barely cata-lyzed by either endostatin storage buffer or angiostatin (K1-3),another endogenous angiogenic factor (Supplementary Fig.S2C and S2D). From these results, we conclude that endostatinhas novel ATPase activity.

Assays to assess the substrate specificity of the endostatinATPase showed that endostatin could also hydrolyze CTP, UTP,andGTP to an equivalent extent (Fig. 2A). In addition, endostatincould hydrolyze ADP, but not AMP (Fig. 2A), showing thatendostatin can cleave both b-g and a-b phosphodiester bonds,similar to apyrase and ecto-ATPase families (35, 36). Concordantwith these results, endostatin hydrolyzed ATP-g-S at a similar raterelative to ADP. However, ADP-b-S could not be hydrolyzed,similar to AMP (Fig. 2A).

We also observed that the endostatin's ATPase activity variedwith pH. The optimal pH of endostatin's ATPase hydrolysisreactionwas 7.0 (Fig. 2B). As a result, all of the subsequent ATPaseexperiments were performed at pH 7.0. Furthermore, divalentmetal ion dependence analysis revealed that endostatin ATPasewas dependent on Mg2þ (Fig. 2C).

Because the reaction remained linear during the initial 60minutes (Fig. 1D), the hydrolysis rate at 60 minutes was used asthe initial rate of ATP hydrolysis for Michaelis–Menten analysis.The results demonstrated aVmax of 2.117� 0.071 pmol/(min.mg)and Km of 83.36 � 11.21 mmol/L (Fig. 2D). Isolated humanHsp90 exhibits high ATPase activity with a Km of 80 mmol/L,reflecting similar affinity of endostatin and Hsp90 for ATP (37).

Taken together, our results showed that endostatin has novelATPase activity, with high affinity toward ATP.

Generation of endostatin mutantsTo probe the role of the ATPase activity of endostatin, several

mutants harboring mutations within Walker motifs were con-structed. The flexible Walker A motif contains an invariant Lysresidue. Mutation of this residue frequently leads to decreasednucleotide-binding capacity (38), therefore, mutants K96A andK96R were generated (Fig. 3A). We also aimed to construct anendostatin mutant with higher ATPase activity. Because myosinhas very high ATPase activity (39), the endostatin Walker A motifsequence was thus substituted with the myosin Walker A motifsequence, hereafter referring to this mutant as E-M (Fig. 3A). TheWalker B motif (hhhhD/E) also forms contacts with nucleotide,and the aspartate/glutamate residue is proposed to activate waterfor the hydrolysis reaction (22). Mutation of glutamate interruptsnucleotide hydrolysis, thus endostatin mutant E176A was con-structed (Fig. 3A).

Next, thesemutants were expressed in E. coli and purified by ionexchange chromatography (the purity of these proteins was ana-lyzed by SDS-PAGE gel and shown in Supplementary Fig. S3).Because endostatin consists of four tryptophan residues locatedevenly in space with all of their side chains stretching inside themolecule, therefore, tryptophan emission fluorescence can be usedto sensitively detect the changes of the tertiary structure of endo-statin (40). To rule out the possibility that mutagenesis of Walkermotifs may cause pronounced structural changes, tryptophanemissionfluorescence was applied tomonitor the tertiary structureof endostatin, K96A, K96R, E176A, and E-M. Therewas noobviouschange in maximal Trp fluorescence emission wavelengths (lmax)for all themutants comparedwith that ofWT endostatin, implyingrelatively stable core structures of endostatin mutants (Supple-mentary Fig. S4). As expected, K96A, K96R, and E176A mutants

Figure 2.Biochemical characterizations ofendostatin's ATPase activity.ATPase assays were performed withendostatin to evaluate: A, substratespecificity; B, optimal pH; C, optimaldivalent metal ion; and D, kineticanalysis. Malachite green assays wereconducted as described in Materialsand Methods; error bars, SDs.






displayed reduced ATPase activities, whereas E-M showedincreased activity relative to that of WT endostatin (Fig. 3B). Wenext sought to determine whether the differences of ATPase activ-ities of endostatinmutantswere due to distinctions in ATP bindingabilities. The ATP binding abilities of K96A, K96R, and E176Adecreased in comparison with that ofWT endostatin, whereas E-Mdisplayed an enhanced ATP binding ability (Fig. 3C).

The ATPase activity of endostatin mediates its effects onendothelial cell activities

Formany ATPases such as AAAþ ATPases, Hsp90, andDnd, theATPase activity is vital for their physiologic functions (27, 41, 42).To identify whether an intact endostatin ATPase activity isrequired for its antiangiogenic effects, we assessed the inhibitoryeffects of endostatin and its mutants on endothelial cell prolif-eration,migration, tube formation, and adhesion.WT endostatin,K96A, K96R, E176A, and E-M attenuated the endothelial cellviability by 39%, 31%, 24%, 22%, and 51%, respectively (Fig.4A). Strikingly, we noticed that endostatin mutants with lowerATPase activities showed less inhibitory ability relative to WTendostatin (P < 0.05), whereas E-M with the greatest ATPaseactivity was the most effective in suppression of cell viabilityamong the proteins tested (P < 0.001). Similar effects wereobserved in the Transwell migration assay (Fig. 4D, quantifiedin Fig. 4B). Furthermore, the tube formation activity of HUVECswas significantly reduced by endostatin and E-M, whereas K96A,K96R, and E176A treatment had no effects. The tube length wasdecreased by 21% and 48% on endostatin and E-M treatment,respectively, and this difference was statistically significant (P <0.001; Fig. 4E, quantified in Fig. 4C). Similar results were obtainedin the endothelial cell adhesion assay (Fig. 4F).

The ability of endostatin and its mutants to induce endothelialcell apoptosis was also compared by monitoring both pro andactivated (cleaved) caspase-3 levels. All the four endostatinmutants enhanced cell apoptosis to different extents. The cleavageof caspase-3 was significantly increased upon WT endostatin andK96A treatment andmoderately upregulated byK96R andE176A,whereas E-M gave rise to drastic cell apoptosis (Fig. 4G).

Altogether, the ATPase activity of endostatinmediates its effectson endothelial cell activities.

TheATPase activity of endostatin is crucial for its antitumor andantiangiogenic properties

We next questioned whether the ATPase activity of endostatinhas any role in retarding tumor growth and angiogenesis, because

it is involved in endostatin-mediated antiangiogenic propertiesin vitro. To answer this question, an A549-GFP xenograft tumormodel was established and mice received injections of PBS, WTendostatin, K96A, K96R, E176A, or E-M (12 mg/kg) every otherday. Primary tumor growth was monitored until 34 days afterimplantation. As shown in Fig. 5A, tumor volumes are signifi-cantly decreasedby endostatin and its fourmutants, amongwhichE-M leads to the most pronounced tumor growth inhibition (P <0.01 relative to WT). K96A, K96R, and E176A were less effectivecompared with WT endostatin (P < 0.05). Similar results wereobtained in tumor weight measurement (Fig. 5B, tumor photosshown in Supplementary Fig. S5). Notably, no obvious differenceof mice weight among different groups was observed, implyingthat these proteins have relatively low toxicity (data not shown).

Tumor angiogenesis was assessed by immunostaining of vas-cular endothelial cell marker CD31. It turned out that tumorangiogenesis was significantly impaired by endostatin, K96R andE-M, whereas no apparent difference was observed in K96A- andE176A-treated tumors compared with control ones (Fig. 5E,quantified in Fig. 5C). Of note, E-M administration led to reducedblood vessels in comparison with endostatin (P < 0.01). Apopto-tic cells were evaluated using immunostaining of cleaved caspase-3. Administration of endostatin, K96A, K96R, E176A, and E-M allpromoted apoptosis. More importantly, the number of cleavedcaspase-3–positive cells was significantly upregulated in E-M–

treated tumors (Fig. 5D, representative images shown in Supple-mentary Fig. S6). Moreover, neither endostatin nor its mutantsmarkedly altered the overall proliferation status of tumors (Sup-plementary Fig. S7), suggesting that these proteins did not abro-gate tumor growth by influencing tumor proliferation.

Collectively, these findings demonstrate that ATPase activity ofendostatin is crucial for its antiangiogenic and proapoptoticeffects in vivo.

Endostatin and its mutants exhibit distinct abilities inregulations of gene expression, cell signaling, and intracellularATP levels

Endostatin is implicated inmodulating multiple angiogenesis-related genes (19), thereby we sought to determine whether theATPase activity is involved in endostatin-mediated gene regula-tions. The effects of endostatin and its mutants on the mRNAlevels of Id1 and Id3 were evaluated, which are important reg-ulators of angiogenesis (43). As expected, endostatin reduced themRNA levels of Id1 and Id3. Intriguingly, K96A, K96R, and E176A

Figure 3.Generation of endostatinmutants. A, construction of endostatinmutantswithmutationswithinWalker A orWalker Bmotifs. B, relative ATPase activity of endostatinmutants compared with that of WT endostatin. C, equivalent proteins were incubated with ATP-agarose beads and the bound proteins were detected byWestern blot analysis; � , P < 0.05; �� , P < 0.001; error bars, SDs.

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exhibited impaired effects compared with WT endostatin (P <0.05), whereas E-M had the most potent inhibitory effects (Fig.6A). Concordant with qRT-PCR data, Western blot analysisshowed that E-M was still the most active in silencing proteinexpression of Id1 and Id3, and othermutants were not so efficientas WT (Fig. 6B).

Endostatin directly interacts with the VEGFR2/KDR/Flk1 recep-tor on the endothelial cells, thus blocking VEGF-induced p38MAPK and Erk activation (44). To further elucidate the molecularmechanisms why endostatin and its mutants displayed distin-guishing antitumor and antiangiogenic activities, we analyzedwhether they could block VEGF-induced MAPK/Erk and p38

activation. All the five proteins attenuated MAPK/Erk and p38phosphorylation to different extents (Fig. 6C). E-M was moreeffective than K96A, K96R, and E176A in inhibition of MAPK/Erkand p38 phosphorylation. This may explain why E-M has themost prominent antiangiogenic capability. Besides, endostatinand E-M significantly suppressed Src phosphorylation (Fig. 6C).Src kinase activity is necessary for turnover of cell-matrix adhesion(45), substantiating our data in cell adhesion assay (Fig. 4F).

ATP is the primary energy currency in the cells and controlsenergy homeostasis. Intracellular ATP levels are a core determi-nate of cell fate (46). HMEC cells were treated with 20 mg/mLendostatin, K96A, K96R, E176A, and E-M for 24 hours and

Figure 4.The inhibitory effects of endostatin and its mutants on endothelial cell activities. HUVECs were treated with 20 mg/mL indicated proteins and examined forcell proliferation at 72 hours (A), Transwell migration at 6 hours (B and D), tube formation at 6 hours (C and E); and cell adhesion assay at 1 hour (F). G, HUVECsweretreated with 20 mg/mL indicated proteins for 24 hours, then cells were collected and the pro and cleaved caspase-3 levels were determined by Western blotanalysis; � and #, P < 0.05; ##, P < 0.01; �� and ###, P < 0.001; error bars, SDs.






intracellular ATP concentration was detected as previouslydescribed (30). Following endostatin, K96A, K96R, and E176Atreatment, the ATP content was unaffected, but it was markedlyreduced in E-M–treated cells (Fig. 6D). This is also an explanationfor the most promising efficacy of E-M among these proteins invitro and in vivo. Further analysis revealed that 20 mg/mL E-Mdecreased intracellular ATP content by about 50%, whereas ele-vated concentrations could not further reduce intracellular ATPlevels (Fig. 6E). Although low concentrations of endostatin has noeffect on the intracellular ATP levels (0–20 mg/mL), increment ofendostatin amount reduced the intracellular ATP levels andshowed a dose-dependent manner (Fig. 6F).

In summary, the ATPase activity of endostatin is involved inId1, Id3 regulations and MAPK/Erk, p38, Src activation. Mean-while, these results suggest that E-Mexhibits strong antitumor andantiangiogenic effects by intracellular ATP depletion.

DiscussionEndostatin is a potent antiangiogenic factor. The work pre-

sented here is for the first time to demonstrate that endostatin hasnovel ATPase activity. Through systemic study, we elucidate thatthe ATPase activity of endostatin is critical for its antitumor andantiangiogenic behaviors both in vitro and in vivo.

Endostatin has novel ATPase activityThe discovery that endostatin exhibits novel ATPase activity is

quite surprising, which leads us to consider the functional sig-nificance of endostatin's ATPase activity and whether it is evolu-tionarily conserved. By comparing the conservation of the par-ticular Walker A and Walker B motifs within endostatin proteinsequences from six species, includingHomo sapiens,Mus musculus,Rattus norvegicus,Danio rerio, andGallus gallus,Dendrobates azureus,we noticed that only human endostatin consists of the Walker Amotif, whereas the Walker B motif exists in all species. Theintriguing fact that the Walker A motif is unique to humanendostatin out of six species suggests that this property of humanendostatin may make it more potent and its effects more com-plicated. The explanation for this phenomenon is mysterious atpresent, and merits further investigation.

Endostatin regulates intracellular ATP levelsEnergy homeostasis, the delicate balance between ATP produc-

tion and consumption, is very critical for many cellular processes(47). Several serious disorders such as cancer and obesity involvethe dysregulation of ATP balance in the cellular level and energyhomeostasis at the whole body. In this work, we present data thatshowed that 20 mg/mL E-M and 500 mg/mL endostatin both led to

Figure 5.Antitumor and antiangiogenic efficacies of endostatin and its mutants. A, tumor volume of A549-GFP xenograft over time. Mice were i.v. administratedwith PBS, endostatin, K96A, K96R, E176A, or E-M (12mg/kg) every other day;n¼ 6/group. B, tumormasswasmeasured; n¼6/group. C and E, quantified results andrepresentative images of tumor sections immunolabeled for CD31; magnification, �320. D, tumor sections were immunolabeled for cleaved caspase-3. Thenumber of apoptotic cells was counted and quantified; � and #, P < 0.05; �� and ##, P < 0.01; �� and ###, P < 0.001; error bars, SEMs.

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50% reduction of intracellular ATP levels. Given the amount ofendostatin internalized by cells (Supplementary Fig. S8A) and itscatalytic activity, it is unlikely that endostatin can directly catalyzesuch large amount of ATP in the cells. However, because endo-statin is able to affect approximately 12% of the genome, includ-ing a bunch of metabolic genes, such as HIF-1a (SupplementaryFig. S8B), aldolase, citrate synthase (19), and hexokinase 2 (10),the latter three of which are direct players in glycolysis and TCAcycle, and regulate ATP production, we thus believe that both theATPase activity and the complex gene regulations of endostatincontribute to the intracellular ATP depletion upon endostatintreatment.

A novel perspective into endostatin actions: intimatecorrelation between enzymatic and biological activities ofendostatin ATPase

On the basis of previous findings, the molecular mechanismsunderlying antiangiogenic actions of endostatin aremostly attrib-uted to four aspects: interaction with its receptors, internalizationby endothelial cells, regulations of multiple signaling pathways,and modifications of gene expression pattern (7). Our studyunravels a fifth aspect of endostatinmechanisms. We reveal novelinsights of endostatin actions by elucidating its biochemicalcharacteristic as an ATPase. Mutagenesis study demonstrates thatthe ATPase activity of endostatin participates in its inhibitory

effects on endothelial cell activities (proliferation,migration, tubeformation, and adhesion). Furthermore, endostatin low-ATPase-activity mutants show obviously reduced abilities to inhibittumor growth and angiogenesis compared with WT (Fig. 5),whereas E-M is dramatically more effective than WT endostatin.Therefore, we conclude that the biological functions of endostatinare intimately correlated with its ATPase activity. This correlationhas been well established in other ATPases. For example, AAAþ

ATPases (ATPases associated with diverse cellular activities) relyon ATP binding and hydrolysis to exert their functions (41).

Internalization of endostatin is important for its biologicalfunctions (14, 17). Consistently, Lim and colleagues (18)reported that manipulation of endostatin by addition of an MTDsignificantly increased endostatin uptake and improved its anti-tumor effects. E-M, with the fastest cellular internalization rate(Supplementary Fig. S8A), exhibited the most prominent ATPaseactivity and antitumor activity. It is plausible that enhanceduptake per se contributes to increased antitumor effects of E-Mcompared with that of WT endostatin.

Minimum endostatin sequence required for its biological activitySeveral groups have reported the minimum endostatin

sequence for its biological activity. Morbidelli and colleagues(48)demonstrated that a peptide (sequence 135–184) containingthe disulfide bridge Cys135-Cys165 has shown antiangiogenic

Figure 6.Endostatin and its mutants showed distinct abilities in regulations of gene expression, cell signaling, and intracellular ATP levels. A, the effects of endostatin and itsmutants on the mRNA levels of Id1 and Id3. B, the protein expression of Id1 and Id3 upon treatment with endostatin and its mutants for 24 hours. C, HUVECswere treatedwith indicated proteins for 1 hour, and stimulatedwithVEGF165 for 10minutes. The overall andphosphorylation levels of Erk, p38, and Srcwere examinedby Western blot analysis. D, HMECs were treated with indicated proteins for 24 hours. Intracellular ATP levels were detected using ATP bioluminescentassay. E, relative intracellular ATP levels of HMECs treated with E-M at indicated concentrations. F, relative intracellular ATP levels of HMECs treatedwith endostatinat indicated concentrations; � and #, P < 0.05; �� and ##, P < 0.01; �� and ###, P < 0.001; ns, not significant; error bars, SDs.






activities. However, this peptide was treated at the tumor periph-ery and for only 7 days. In another study (49), a minimumsequence of 27 amino acid synthetic peptide corresponding tothe N-terminal zinc–binding domain of endostatin fully mim-icked its antitumor activity. However, the peptide was adminis-trated at a higher frequency (twice a day vs. once a day for full-length endostatin). Given the above controversial reports aboutminimum sequence of endostatin, it seems more likely thatdifferent regions ormechanisms regulate distinct aspects of endo-statin activities.

Our results differ from these groups in that endostatin low-ATPase-activity mutants (K96A, K96R, and E176A) retain partialcapacity to diminish tumor growth and angiogenesis, indicatingthat endostatin activities are not exclusively mediated by itsATPase activities. We propose that the endostatin's ATPase activ-ity, together with its binding to cell surface receptors, internali-zation (Supplementary Fig. S8A), intracellular signaling path-ways, and gene-expression regulations (Fig. 6 and SupplementaryFig. S8B), all the above-mentioned functions of endostatin coop-erate and orchestrate its antitumor effects, which leads to acomplex but exquisite endostatin network.

Drug design: endostatin analogues for therapeuticsA good drug should have qualities of high stability and

efficacy. Proteins for therapeutic use can be exquisitely opti-mized through several ways, such as mutagenesis (proteinanalogues) and chemical modifications (PEGylation or acyla-tion). Although the clinical trials of the P. pastoris–expressedendostatin were terminated during phase II in the UnitedStates, an N-terminal modified E. coli–expressed endostatinwas approved by the CFDA for the treatment of NSCLC patientsin 2005 in China (3). The prominent drug design strategy ofthis drug is to covalently attach a peptide of MGGSHHHHH tothe N terminus of endostatin, thus facilitating the N-terminalintegrity of endostatin, which is essential for the antiangiogenicand antitumor activities of endostatin (7). In addition, aparallel study conducted by the Folkman laboratory revealsthat the N-terminal–modified endostatin is at least twice aspotent as P. pastoris–expressed endostatin in animal tumormodels (50). This provides an excellent example of the ana-logue approach.

In this study, we found that among the five endostatinproteins we explored, E-M not only is the most effective to

attenuate endothelial cell activities and induce apoptosis, butalso exhibits the most severe inhibitory effects on tumor growthand angiogenesis. These exciting results lead us to conclude thatE-M may be a promising therapeutic agent, and suggest thatconstruction of endostatin analogues with high ATPase activityserves as an efficient approach to optimize endostatin's ther-apeutic efficacy.

In conclusion, we demonstrate for the first time that endostatinexhibits novel ATPase activity. The results presented here contrib-ute to a systemic understanding of the link between endostatinintrinsic properties and antitumor effects, and provide a brandnew direction for the design and development of more potentantiangiogenic drugs.

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

Authors' ContributionsConception and design: S. Wang, X. Lu, Y. Fu, Y. LuoDevelopment of methodology: S. Wang, X. Lu, P. LiuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Wang, X. Lu, P. Liu, L. Jia, S. ZhanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Wang, X. Lu, Y. FuWriting, review, and/or revision of the manuscript: S. Wang, X. Lu, Y. LuoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Fu, Y. LuoStudy supervision: Y. Luo

AcknowledgmentsThe authors thank all the Luo laboratorymembers for insightful discussions.

Grant SupportY. Luo was supported by the General Programs of the National Natural

Science Foundation of China (no. 81171998), the National Science andTechnologyMajor Project for "MajorNewDrugs Innovation andDevelopment"(2013ZX09509103). Y. Fu was supported by the General Programs of theNational Natural Science Foundation of China (no. 81272529).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 6, 2014; revised March 9, 2015; accepted March 11, 2015;published OnlineFirst March 18, 2015.

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2015;14:1192-1201. Published OnlineFirst March 18, 2015.Mol Cancer Ther Shan Wang, Xin-an Lu, Peng Liu, et al. and Antitumor ActivitiesEndostatin Has ATPase Activity, Which Mediates Its Antiangiogenic

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