Tumor Biology and Immunology

ME1 Regulates NADPH Homeostasis to PromoteGastric Cancer Growth and MetastasisYun-Xin Lu1,2, Huai-Qiang Ju1, Ze-Xian Liu1, Dong-Liang Chen1,2, Yun Wang1,2,Qi Zhao1, Qi-Nian Wu1,2, Zhao-lei Zeng1, Hai-Bo Qiu1,3, Pei-Shan Hu1,Zhi-Qiang Wang1,2, Dong-Sheng Zhang1,2, Feng Wang1,2, and Rui-Hua Xu1,2

Abstract

Genomic alterations of tumor suppressorsoften encompass collateral protein-codinggenes that create therapeutic vulnerabilityto further inhibition of their paralogs. Here,we report that malic enzyme 2 (ME2) is fre-quently hemizygously codeleted withSMAD4 in gastric cancer. Its isoenzymeME1was upregulated to replenish the intracellu-lar reducing equivalent NADPH and tomaintain redox homeostasis. Knockdownof ME1 significantly depleted NADPH,induced high levels of reactive oxygen spe-cies (ROS), and ultimately cell apoptosisunder oxidative stress conditions, such asglucose starvation and anoikis, in ME2-underexpressed cells. Moreover, ME1 pro-moted tumor growth, lung metastasis, andperitoneal dissemination of gastric cancerin vivo. Intratumoral injection ofME1 siRNAsignificantly suppressed tumor growth incell lines and patient-derived xenograft–based models. Mechanistically, ME1 was transcriptionally upregulated by ROS inan ETV4-dependent manner. Overexpression of ME1 was associated with shorter overall and disease-free survival in gastric cancer.Altogether, our results shed light on crucial roles of ME1-mediated production of NADPH in gastric cancer growth and metastasis.

Significance: These findings reveal the role of malic enzyme in growth and metastasis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/8/1972/F1.large.jpg. Cancer Res; 78(8); 1972–85.�2018 AACR.

IntroductionMultifaceted sequencing has revealed an unprecedentedly

detailed blueprint for gene amplification or deletion in human

genomes as well as in other mammals (1). However, vasttargeted therapies often focused on the amplified, overex-pressed, or mutant-driving oncoproteins (2, 3), whereas thedeleted, underexpressed, or mutant-inactivated tumor suppres-sors received less attention (4). Recently, strategies such assynthetic lethality have been proposed to exploit genomic lossof suppressor genes, as these events often occur at large regionsthat may encompass critical fundamental housekeeping genesthat are essential for cell growth and survival (4–8). Cancer cellsmay sometimes tolerate these stresses by rewiring the informa-tion flow into functionally redundant paralogs that maintainthese essential cellular reactions (6, 8, 9). When several otherhomologous genes serving overlapping functions were shutdown by inhibitors, the cells would experience lethal strike(4, 6, 10). Genetic and pharmacologic studies have evidencedthe therapeutic exploit of collateral deletion in the tumor-suppressive loci (4, 10–12). One notable example is thathemizygous deletion of TP53 in colorectal cancer necessarilyled to high vulnerability to inhibition of the neighboring genePOLR2A (5).

Glutamine

Aspartate

Malate

Pyruvate

ME1 ME1

NADPH

Stomach

ME1 maintains redox homeostasis to support cell survival under nutrient deprivation and anchorage-independent growthwhen its paralogue ME2 is codeleted with SMAD4.

Promotes cell survivaland growth in nutrient-

deprived regions

Promotes anchorage-independent growth

for metastasis

Compensation

SMAD4 ME2

Deletion

Nucleus

Cytoplasm

NADP+

ETV4

ETV4 RE ME1ETV4

ETV4 RE ME1

ROS

© 2018 American Association for Cancer Research

1Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology inSouth China, Collaborative Innovation Center for Cancer Medicine, Guangzhou,China. 2Department of Medical Oncology, Sun Yat-sen University Cancer Center,Guangzhou, China. 3Department of Gastric and Pancreatic Surgery, Sun Yat-senUniversity Cancer Center, Guangzhou, China.

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

Corrected online July 11, 2019.

Y.-X. Lu, H.-Q. Ju, and Z.-X. Liu contributed equally to this article.

Corresponding Authors: Rui-Hua Xu, Sun Yat-sen University Cancer Center, 651Dongfeng Load East, Guangzhou, Guangdong 510060, China. Phone: 8620-8734-3295; Fax: 8620-8734-3333; E-mail: xurh@sysucc.org.cn; and FengWang,wangfeng@sysucc.org.cn

doi: 10.1158/0008-5472.CAN-17-3155

�2018 American Association for Cancer Research.

CancerResearch

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Malic enzymes are responsible for oxidative decarboxylationof malate to pyruvate, which is the primary substrate supportingthe tricarboxylic acid (TCA) cycle and the major source ofintracellular reducing equivalents (13, 14). In human cells,malicenzymes are encoded by three homologous genes (15, 16),including ME1, which is located in the cytoplasm and enzymicactivities require NADP; malic enzyme 2 (ME2), which is locatedin the mitochondrion and enzymic activities require NAD; andME3, which is located in the mitochondrion and enzymic activ-ities require NADP. These enzymes are widely distributed innature and have highly conserved sequences and similar struc-tural topologies across different species, suggesting that theyhaveimportant biological functions (17).

For chemical work, there is an equally important role for ATPand NADPH, which powers the redox defense and reductivebiosynthesis (18). Tumor cells reprogram theirmetabolic patternsto satisfy the needs of rapid cell proliferation at the expense ofoverproduced reactive oxygen species (ROS), which requiresplenty of NADPH supplementation (19). By recycling the TCAintermediate malate into the common carbon source pyruvate,malic enzymes may have a regulatory role in satisfying cellulardemand for reducing equivalents, energy, and biosynthetic pre-cursors (16). Malic enzymes are essential for NADPH productionfrom both the oxidative pentose phosphate pathway (16) andglutamine metabolism (20) and thus have been evaluated astherapeutic targets. It has been reported that ME1 producesNADPH at levels as high as those produced by G6PD in thepentose phosphate pathway shunt (18). Repression of ME1 orME2 results in altered metabolism (13, 14), reduced cell growth,migration, and elevated ROS level in nasopharyngeal carcinoma(13) and non–small cell lung cancer (21).

To mine the therapeutic targets with collateral lethality ingastric cancer, we analyzed The Cancer Genome Atlas (TCGA)and Cancer Cell Line Encyclopedia (CCLE) databases and foundthat the housekeeping gene ME2 was frequently codeleted orcounderexpressed with the tumor suppressor gene SMAD4 ingastric cancer. During energy stress such as glucose deprivation,anchorage-independent growth, and solid tumor formationin vivo, ME1 played essential roles in supplying NADPH forelimination of intracellular ROS when its paralog ME2 wassuppressed due to coalteration with SMAD4.

Materials and MethodsCell culture

GES1, AGS, SGC7901 (originally purchased from ATCC onJuly 2014) and SNU216, BGC823, HGC27, MGC803, NUGC4,MKN45, MKN74 (originally purchased from the Institute ofBasic Medical Sciences of the Chinese Academy of MedicalSciences on June 2014) were cultured in RPMI1640 or DMEMmedium (Invitrogen) supplemented with 10% FBS (HyClone)at 37�C with 5% CO2 according to the suppliers' instructions.Glucose-free RPMI1640 medium (GIBCO/Thermo Fisher Sci-entific, cat. no. 11879020) supplemented with 10% dialyzedFBS (GIBCO/Thermo Fisher Scientific, cat. no. 26400-044) wasused for glucose deprivation assays. All cells were tested neg-ative for mycoplasma and authenticated by short tandemrepeat DNA fingerprinting at the Medicine Lab of the ForensicMedicine Department of Sun Yat-sen University (Guangzhou,China). All cell lines have not been passaged for more than 6months in our study after resuscitation.

Bioinformatic analysisGene copy number and corresponding gene expression

was analyzed using data obtained from CCLE (http://www.broadinstitute.org/ccle) and TCGA (http://www.cbioportal.org/portal/) according to previously described methods (11).We searched potential target gene in the proximity of SMAD4gene and analyzed its codeletionwith SMAD4 in gastric cancer asin a recent report (6).

Protein extraction, immunoblotting, and antibodiesProteins were extracted with the RIPA lysis buffer (Cell Signal-

ing Technology, cat. no. 9806). Briefly, scrapped cells were col-lected after centrifugation at 2,000 rpm for 3 minutes. Then, thepelleted cells were lysed in RIPA buffer containing proteinase andphosphatase inhibitors for 15 minutes on ice. The supernatantwas transferred to anew tube, and the protein concentrationsweremeasured using BCA Protein Assay Kit (Thermo Fisher Scientific,cat. no. 23225). SDS-PAGE and immunoblotting was performedas described previously (22). The following antibodies wereused: ME1 (Abcam, cat. no. ab97445); ME2 (Abcam, cat. no.ab139686); ME3 (Abcam, cat. no. ab172972); vinculin (Abcam,cat. no. ab129002); G6PD (Abcam, cat. no. ab993); PHGDH(Abcam, cat. no. ab211365); Flag (Abcam, cat. no. ab49763);E-cadherin (Cell Signaling Technology, cat. no. 3195);N-cadherin(Cell Signaling Technology, cat. no. 13116); cleaved PARP (CellSignaling Technology, cat. no. 5625); cleaved caspase-3 (CellSignaling Technology, cat. no. 9664); ETV4 (Aviva SystemsBiology, cat. no. ARP32263_P050); ETV4 (Lifespan Biosciences,cat. no. LS-B1527); and b-actin (Cell Signaling Technology,cat. no. 4970).

RNA extraction and qPCR analysisTotal RNA was isolated from cells or tissues by TRIzol Reagent

(Invitrogen, cat. no. 15596018). One microgram of RNA for eachsample was reversed to cDNA by a Prime Script RT Master Mix Kit(Takara, cat. no. RR036Q), and 1 mL cDNAwas used as a templateto performqPCRwithGoTaq qPCRMasterMix (Promega, cat. no.A6002) according to the manufacturer's instructions. Primersused in our study were listed in Supplementary Table S1.

Tissue specimens and clinicopathologic characteristicsThe total 207paraffin-embedded, archived gastric samplesused

in this study were histopathologic and clinically diagnosed at theSun Yat-sen University Cancer Center between 2007 and 2009.Written informed consent was obtained from all patients, and nopatient received any chemo- or radiotherapy prior to surgery. Theuse of clinical specimens for research purposes was conducted inaccordance with the Declaration of Helsinki and approved by theethical committee of Sun Yat-sen University Cancer Center. Theclinicopathologic characteristics of the samples are summarized inSupplementary Table S2. All patients were followed up regularlyafter the operation at 3-month intervals. The median follow-uptime was 49 months (range, 3–102 months). Fifty freshly collect-ed gastric cancer tissues andmatched adjacent nontumoral gastrictissues from the same patient were frozen and stored in liquidnitrogen until required for RNA or protein extraction.

IHC and TUNEL analysisIHC assays were conducted as reported previously (5). Briefly,

the sections were deparaffinized and rehydrated before they were

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heated at a subboiling temperature in sodium citrate buffer(pH 6.0) for 10 minutes with a microwave oven for antigenretrieval. Samples were then incubated with 3% hydrogen per-oxide for 10minutes to block endogenous peroxidase activity andthen with antibody against ME1 (Abcam, ab97445, 1:500), ME2(Abcam, cat. no. ab139686), Ki-67 (Cell Signaling Technology,9129, 1:500) or cleaved caspase-3 (Cell Signaling Technology,9664, 1:1,000) at 37�C for 1 hour. The IHC Kit (Dako, cat. no.K5007) including second antibody andDAB substratewas used todetect protein expression according to the manufacturer's proto-col. Counterstaining color was carried out using hematoxylin.Assessments of the staining were scored by two experiencedpathologists blinded to the patients' identity and clinical status.In discrepant cases, a pathologist reviewed the cases and reachedthe consensus. Expression level was determined according to ourprevious report (23). The terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) assays were performedwith the In Situ Cell Death Detection Kit (Roche, cat. no.11684795910) according to the manufacturer's instructions.

ROS, cell apoptosis detection, and measurement of NADPHROS levels were determined as described previously (24).

Briefly, cells cultured in glucose-deprived medium or in matrixdetachment conditions were incubated with 10 mmol/L 20,70-dichlorodihydrofluorescein diacetate (H2-DCFDA, Thermo Fish-er Scientific, cat. no. D399) at 37�C for 30minutes. Afterward, thecells were collected, washed twice in ice-cold PBS, and resus-pended in PBS. Fluorescence was immediately measured using aFACScan FlowCytometer (Beckman-Coulter). For apoptosis anal-ysis, cells were collected and stained with Annexin V-FITC and PI(4A Biotech Co. cat. no. FXP018) before measurement with flowcytometer. The intracellular levels of NADPH, total NADP, andGSH were measured with the NADP/NADPH-Glo Kit (Promega,cat. no. G9081) or GSH/GSSG-Glo Kit (Promega, cat. no. V6612)according to the manufacturer's instructions.

Vectors, siRNAs, cell transfection, and lentivirus productionME1 vectors including wild-type (cat. no. EX-T8139-Lv121)

and silent mutation (cat. no. CS-T8139-Lv121-1) were purchasedfrom GeneCopoeia, Inc. The siRNAs targeting ME1 (targetingsequences: GGGCATATTGCTTCAGTTC, GAGAGACAGCAATT-GAACA) and ME2 (targeting sequence: CCCAGTATGGACA-CATCTTTA) were synthesized by RiboBio. The siRNAs targetingG6PD and PHGDH were purchased from RiboBio. All the trans-fection experiments were conducted with Lipofectamine 3000(Thermo Fisher Scientific, cat. no. L3000015) as recommended.

SGC7901, MGC803, and HGC27 cells were transfected withlentiviruses containing ME1 shRNA (GenePharma, cat. no. 201613971), and stable cell lines were obtained after treatment with3 mg/mL puromycin for 3 to 5 days. Knockdown lentivirustargeting ME2 was constructed as reported previously (16) andtransfected into HGC27 cells.

Anoikis and soft agar colony formation assayAnoikis was induced by plating cells (2 � 106) on ultralow

attachment 6-well plate (Sigma, cat. no. CLS3471-24EA). For softagar colony formation assay, cell suspension was mixed with0.7% soft agar in 2�DMEM containing 20% FBS in equal volumeand layered in triplicate onto 1.4% solidified agar in 2�DMEMcontaining 20% FBS. After 10- to 14-day culture, colonies werecounted under microscopy and photographed.

Reporter assayThe dual reporter construct expressing Gaussia luciferase under

the human ME1 promoter, and secreted alkaline phosphatase(SEAP) under the CMV promoter (used for transfection normal-ization) was from GeneCopoeia, Inc (cat. no. HPRM23418-PG04). The indicated cells were plated 18 hours before transfec-tion in 24-well plates and transiently transfected with 500 ng ofthe reporter plasmid using Lipofectamine 3000. Plasmids con-taining ETV4 open reading frame or siRNAs targeting ETV4 weretransfected 24 hours later using Lipofectamine 3000. The lucif-erase activity was determined according to the manufacturer'sinstructions (GeneCopoeia, Inc., cat. no. LF032) and normalizedto that of the SEAP activity.

Chromatin immunoprecipitation assayThe chromatin immunoprecipitation (ChIP) assay was per-

formedwith an EZ-ChipKit (Millipore, cat. no. 17-371) followingthemanufacturer's instruction as described previously (16). 293Tcells were grown to 80% confluence, and crosslinking was per-formed with 1% formaldehyde for 10 minutes. The cell lysateswere sonicated to shear DNA to sizes of 300 to 1,000 bp. Equalaliquots of chromatin supernatants were incubated with anti-ETV4 or anti-IgG antibody (Millipore) overnight at 4�C withrotation. After reverse cross-link of protein/DNA complexes tofree DNA, RT-PCR was carried out using the specific primer(forward: 50-ACACCTGTCAGTTTCTACAGA-30, reverse: 50-CAT-TATTCAGAGAGAGCAGTGG-30) detecting the ETV4-binding siteon ME1 promoter region.

In vivo tumorigenesis and metastasis assaysAll female BALB/c nudemice (4–5weeks old) used in our study

were purchased from the Beijing Vital River Laboratory AnimalTechnology Co., Ltd. and housed in specific pathogen-free units.Subcutaneous mice model was performed as reported previously(22, 23).

For lungmetastasis model, 5� 106 cells resuspended in 100 mLof sterile PBS were injected into the tail veins of nude mice. Lungcolonization was monitored at the indicated time point afterintraperitoneal injectionof D-luciferin (Goldbio, cat. no. LUCK-1)with a Xenogen IVIS 100 bioluminescent imaging system. Sixtydays later, mice were sacrificed with cervical dislocation, and thelungs were dissected out and paraffin embedded to histopatho-logically examine the metastatic locus.

Peritoneal dissemination ability of gastric cancer cells wasevaluated through intraperitoneal injection. In brief, SGC7901(NC, sh#1, sh#2) cells (5 � 106) in 0.5 mL of PBS were injectedinto the peritoneal cavity of BALB/c nude mice. Mice were care-fully monitored until they were killed at 60 days after injection.Colon metastasis was examined and recorded.

Patient-derived xenograft models and in vivo siRNA treatmentThe patient-derived xenograft (PDX)–bearing male nude mice

model was raised and passaged as described previously (23, 25).In brief, patient-derived tumor materials were collected in culturemedium and transferred to the animal houses on wet ice within 1hour after resection. Upon arrival, necrotic and supporting tissueswere carefully removed using sterilized surgical blades. The tumorgross was cut into different fragments for several purposes, flashfrozen, paraffin embedding for histopathologic analysis. Onethird was flash frozen for protein extraction or stored at �80�C

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for genomic profiling genomic typing and one third was fixed in10% neutral-buffered formalin for histopathologic examination.The rest was implanted subcutaneously into the flank region offemale nude mice, and the incision was closed with surgicalsuture. Successfully engrafted tumor models were then passagedand banked in liquid nitrogen after three passages in mice.

For siRNA treatment analysis, PDX-bearingmice were preparedafter subcutaneous incubation of gastric cancer tumor mass.Cholesterol-modified ME1 siRNA or control siRNA (RiboBio,5 nmol/kg) dissolved in diluted water were intratumorallyinjected every 3 days for 18 days. All animal experiments werecarried out in accordancewith theNIHGuide for theCare andUseof Laboratory Animals with the approval from the InstitutionalAnimal Care and Use Committee of Sun Yat-Sen University.

Statistical analysisAll in vitro experiments were repeated three times or more, and

data are presented as mean � SD unless otherwise indicated. TheStudent t test assumed two-tailed distributions to calculate sta-tistical significance between groups. Survival curves were gener-atedusing theKaplan–Meiermethod and comparedusing the log-rank tests. The independent prognostic factors were identified bythe Cox proportional hazards regression model. ROC curve wasgenerated with Medcalc software. Differences were analyzed byGraphPad Prism 5 and P values less than 0.05 were considered toreach statistical significance.

ResultsDownregulation of ME2 in gastric cancer due to genomicalterations

Genomic alteration of SMAD4 occurs frequently in humancancers (Supplementary Fig. S1A). We screened nearby protein-coding genes located with SMAD4 and found that ME2 is posi-tioned approximately 100 kb upstream to SMAD4 locus (Fig. 1A).Consistently, frequent genomic deletion ofME2 was observed inhuman cancers (Supplementary Fig. S1B). Although homozygousdeletion of SMAD4 and ME2 was found only in 4.76% of TCGAstomach tissues, 162 of 414 (39.23%) cases bear hemizygousdeletion of SMAD4 and ME2 (Fig. 1A and B). Moreover, copynumber and transcriptomic analyses in TCGA gastric cancerdatabase showed positive correlations between SMAD4 andME2at both the DNA and mRNA level (Fig. 1C and D). These positivecorrelations were also validated in pan-cancer cell lines from theCCLE database (Fig. 1E and F). To address whether genomic losscould lead to alteration of gene expression, we compared copynumbers of SMAD4 and ME2 with corresponding mRNA level.Analysis of CCLE databases revealed that expression of SMAD4andME2was tightly correlated with their gene copy number (Fig.1GandH). In apanel of gastric cancer cell lines, expressionofME2mirrored that of SMAD4atmRNAandprotein level (Fig. 1I and J).Importantly, ME2 and SMAD4 were downregulated in the major-ity of gastric cancer cells, except for that inHGC27 cells, comparedwith that of GES1 cells (Fig. 1I and J). Our data indicated thatME2was suppressed in gastric cancer cells, probably due to hemizygouscodeletion with SMAD4.

ME1 is essential for survival of gastric cancer cells under glucosedeprivation

Malic enzymes replenish the TCA cycle and produce the majorantioxidant NADPH via oxidation of malate to pyruvate (16, 19)

and are redundant metabolic pathway due to three isoenzymes(Fig. 2A). Because ME2 was suppressed in gastric cancer, wetherefore focused on its paralogs including ME1 and ME3. West-ern blot assays showed remarkable increase in protein levels ofME1 in themajority of gastric cancer cell lines compared with thatin GES1 cells, whereas ME3 was only detected in MKN74 andBGC823 cells (Fig. 2B). To explore collateral lethality of ME2genomic alteration in gastric cancer, lentiviruses containing shorthairpin RNAs targeting ME1 were introduced into ME2-down-regulated SGC7901 andMGC803 cells (Supplementary Fig. S2A).Surprisingly, intracellular NADPH as well as GSH levels were notaffected after ME1 knockdown (Fig. 2C; Supplementary Fig. S2B).We hypothesized that the resting ME2 was sufficient for NADPHproduction in this condition. However, cancer cells often expe-rience nutrition stress due to insufficient vascularization (26, 27).Tomimic in vivo situation, we cultured cells in glucose deprivationmedium and found that NADPH level in knockdown cells wassignificantly decreased compared with that in control cells (Fig.2D). Glucose deprivation induced significantly elevated apopto-tic percentage of SGC7901 andMGC803 cells after knockdown ofME1 (Fig. 2E), which could be restored by reintroduction of ME1vector with silent mutation (Supplementary Fig. S2C and S2D) orpretreatment with NAC (Fig. 2F; Supplementary Fig. S2E). How-ever, HGC27 cells with elevated ME2 expression (Fig. 1J) wereresistant to glucose deprivation, which could be completelyabrogated by simultaneous silence of ME1 and ME2 (Fig. 2G;Supplementary Fig. S2F). Intracellular ROS level was remarkablyelevated after knockdown ofME1 in SGC7901 andMGC803 cells(Fig. 2H), whereas increase of ROS (Fig. 2I) and decrease ofNADPH level (Fig. 2J) in HGC27 cells was only observed aftercoinhibition of ME1 and ME2. These data showed that duringglucose deprivation, ME1 enables survival of gastric cancer cellswith underexpressed ME2.

ME1 mediates anoikis resistance of gastric cancerLike glucose starvation, matrix detachment elicits energy stress

(9) evidenced by elevated H2O2 levels (Fig. 3A), which werecorrelated to the extent of NADPH depletion (Fig. 3B). Wetherefore tested whether ME1 was required for redox regulationduring anchorage-independent growth, a hallmark of cancermetastasis (28). Matrix detachment significantly increased intra-cellularH2O2 anddecreased theNADPH level inME1knockdownSGC7901 and MGC803 cells in comparison with control cells(Fig. 3C and D). Knockdown of ME1 in SGC7901 and MGC803cells significantly suppressed colonies in soft agar (Fig. 3E).Apoptotic assays further confirmed ME1 mediated anoikis resis-tance (Fig. 3F). Enforced expression with silent mutated ME1 orpretreatment with NAC significantly attenuated anoikis of ME1knockdown cells (Fig. 3F). During suspension culture, HGC27cells with ME1 knockdown showed resistance to anoikis, andsignificantly elevated apoptosis was observed after concomitantsilence ofME2 (Fig. 3G–I).However, neithermigration ability norepithelial–mesenchymal transition markers of gastric cancer cellsshowed differences following ME1 knockdown (SupplementaryFig. S3A and S3B). As serine metabolism and pentose phosphateshunt also provided substantial NADPH in cancer cells, we nextexamined the possible influence of these pathways on gastriccancer cell survival under glucose-deprived or anchorage-inde-pendent conditions. Knockdown of PHGDH or G6PD resulted inelevated apoptosis percentage in SGC7901 and MGC803 cellsunder glucose-deprived or anchorage-independent conditions

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Figure 1.

ME2 is suppressed in gastric cancer due to codeletion with SMAD4. A, Ideogram of chromosome 18 showing close proximity (<200 kb) of ME2 to SMAD4.Copy number alteration (CNA) of SMAD4 in gastric cancer.B, Frequencies of ME2 and SMAD4 copy number alteration in TCGAgastric cancer samples.C,Correlationof SMAD4 and ME2 copy number alteration in TCGA gastric cancer samples. D, Correlation of SMAD4 and ME2 mRNA level in TCGA gastric cancersamples.E,Correlation of SMAD4 andME2 copy number alteration in CCLE cancer cell lines. F,Correlation of SMAD4 andME2mRNA level in CCLE cancer cell lines.G,Correlation of SMAD4 CNA and mRNA level in CCLE cancer cell lines as well as in gastric cancer cell lines. H, Correlation of ME2 copy number alterationandmRNA level in CCLE cancer cell lines as well as in gastric cancer cell lines. I, qPCR assays of ME2 and SMAD4 in a panel of gastric cancer cells and GES1 epithelialcells. J, Immunoblots ofME2 andSMAD4 in apanel of gastric cancer cells andGES1 epithelial cells. Pearson correlation coefficient (r) andP values are displayed inB–H.

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Figure 2.

ME1 is essential for cell survival during glucose deprivation. A, Overview of malic enzyme reaction. B, Immunoblots of ME1 and ME3 in a panel of gastric cancer cellsand GES1 epithelial cells. Vinculin was used as a loading control. C, Measurement of NADPH/NADPþ in the indicated cells (control, sh#NC; ME1 knockdown, sh#1or sh#2; knockdown-resistant ME1 vector, sh#1þR) cultured in normalmedium.D,Measurement of NADPH/NADPþ in the indicated cells cultured in glucose deprivationmedium.E,Brightfield imagesof SGC7901 andMGC803cells cultured inglucose deprivationmediumafter knockdownofME1.F,Cell apoptosis of indicated cells culturedin glucose deprivation medium. Representative images and quantification data are shown. G, Brightfield images and apoptotic percentage of HGC27 cells afterknockdown of ME1 and ME2 cultured in glucose deprivation medium. H, Measurement of ROS level in SGC7901 and MGC803 cells cultured in glucose deprivationmedium after knockdown of ME1. Measurement of ROS (I) and NADPH/NADPþ (J) in HGC27 cells after knockdown of ME1 and ME2 cultured in glucose deprivationmedium. All error bars represent the SD of at least three replicates from two independent experiments. P values were determined by two-tailed t test.

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Figure 3.

ME1 mediates anoikis resistance in gastric cancer. Intracellular ROS (A) or NADPH/NADPþ (B) level of indicated cells cultured in attached or detachedconditions. C, Intracellular ROS level in SGC7901 and MGC803 cells cultured in detached conditions was measured after knockdown of ME1. D, NADPH/NADPþ

level in the indicated cells cultured in detached conditions was measured. E, Soft agar colony formation assays in SGC7901 and MGC803 cells afterknockdown of ME1. F, Representative histograms depicting apoptosis and apoptotic rate of indicated cells after 48 hours of suspension, as determined by flowcytometry. G, Apoptotic rate of HGC27 cells after 72 hours of suspension. H, Immunoblots of cleaved PARP (c-PARP) in HGC27 cells after 72 hours ofsuspension. I, Intracellular ROS level in HGC27 cells cultured in detached conditions. All error bars represent the SD of at least three replicates from twoindependent experiments. P values were determined by two-tailed t test.

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(Supplementary Fig. S3C and S3D). However, changes in apo-ptosis were not as obvious as that in ME1 knockdown cells(Supplementary Fig. S3D). These data clearly supported thenotion that ME1 could protect gastric cancer cells from anchor-age-independent growth.

ME1 is required for tumor growth, lung metastasis, andperitoneal dissemination of gastric cancer in vivo

Knockdown of ME1 in SGC7901 cells significantly suppressedtumor growth in vivo as evidenced by slowed growth curve andreduced xenograft weight (Fig. 4A–C). Moreover, immunostain-ing assays of Ki-67, TUNEL, and cleaved caspase-3 indicated thattumors formed by negative control cells showed characteristic ofrapid proliferation and less apoptosis than that formed by ME1knockdown cells (Fig. 4D). However, knockdown ofME1 or ME2alone in HGC27 cells showed no effects on tumor growth in themice, whereas simultaneous ablation of ME1 and ME2 signifi-cantly suppressed tumor growth in the subcutaneous mice model(Supplementary Fig. S4A and S4D),whichwas consistentwith thein vitro results. To analyze anoikis in vivo, gastric cancer cells wereinjected into the tail vein, and fluorescence imaging was used tomonitor lung metastasis. The majority of gastric cancer cellsdiminished 48 hours after injection (Fig. 4E). However,SGC7901/sh#NC cells formed remarkably large and excessivelung metastatic diseases than SGC7901/sh#1 or SGC7901/sh#2cells (Fig. 4E). Hematoxylin and eosin staining of dissected lungsshowed significantly more metastasis nodules in the controlgroup compared with that in the knockdown groups (Fig. 4F).IHC analysis of the lung metastases confirmed effective knock-down of ME1 with no effects on ME2 (Supplementary Fig. S5A).Moreover, knockdownofME1 significantly suppressedmetastasisin the intestinal wall after peritoneal injection of gastric cancercells (Fig. 4G andH). Altogether, our results indicated that ME1 iscritical for gastric cancer growth and metastasis.

Intratumoral knockdown of ME1 suppresses growth of gastriccancer in vivo

To further explore whether ME1 could be used as a therapeutictarget in gastric cancer, we assessed the antitumor activity of ME1targeting siRNA in mice bearing SGC7901 cells xenograftedtumors and three PDXs (PDX#1–3). These PDX models werecharacterized with decreased ME2 and increased ME1 expressionin the tumor tissues compared with corresponding normal tissues(Supplementary Fig. S5B). When the tumor volume reachedapproximately 50 mm3, siRNAs targeting ME1 were injectedintratumorally once every other day. The growth of tumors treatedwith siRNAs was significantly suppressed in SGC7901 cell–basedxenografts and in three PDXs compared with control group (Fig.5A–F). The tumors developed from the ME1 siRNAs treatmentgroup displayed lower Ki-67 and ME1 staining, elevated TUNELsignal, and cleaved caspase-3 expression than that in controlgroup (Fig. 5G). As PDX models closely resemble the biologicalcharacteristics and genomic landscape of human cancers at thepopulation level (25), our study provides clear preclinical cluesfor developing ME1 inhibitors in gastric cancer.

ETV4 upregulates ME1 expression in gastric cancerTumor cells often experience oxidative stress, which could

facilitate tumor growth by causing genomic instability and repro-gramming cancer cell metabolism (29). Glucose restriction andanchorage-independent growth induced elevated H2O2 levels

(Fig. 6A). We therefore asked whether glutaminolysis pathwaymediated by ME1 was activated by ROS overproduction. Asshown in Fig. 6B and C, expression of ME1 was upregulatedunder glucose deprivation medium or in matrix-detached con-ditions at both the mRNA and protein level, and this effect wasreversed by the antioxidant NAC, indicating a transcriptionalregulation manner of ME1 by ROS. To this end, we searchedtranscription factors with potential binding capacity with ME1in JASPAR database and found ETV4 response elements in theME1 promoter (Fig. 6D). Interestingly, ETV4 mRNA and pro-tein levels mirrored that of ME1 during ROS stress (Fig. 6C;Supplementary Fig. S6A). Overexpression of ETV4 in SGC7901and MGC803 cells increased ME1 protein levels (Fig. 6E),whereas knockdown of ETV4 in SGC7901 and MGC803 cellsby siRNAs reduced both mRNA and protein levels of ME1 (Fig.6F; Supplementary Fig. S6B). Upregulation of ME1 induced byROS was blocked by ETV4 depletion in SGC7901 and MGC803cells (Fig. 6G). Importantly, as in the case with ME1, knock-down of ETV4 sensitized SGC7901 and MGC803 cells toglucose deprivation and detached conditions (SupplementaryFig. S6C). These results suggest that ME1 was transcriptionallyupregulated by ROS/ETV4 during energy stress conditions ingastric cancer cells.

To determine whether ROS/ETV4 axis upregulatedME1 expres-sion transcriptionally, we cloned 1.2 kb of genomic DNAupstream of the transcription start site of the ME1 gene into aluciferase reporter plasmid. Redox stress significantly increasedthe luciferase activity ofME1 promoter in SGC7901 andMGC803cells,which couldbe abrogatedbyNACpretreatment (Fig. 6H).Asshown in Fig. 6I, enforced ETV4 expression induced elevatedreporter activity in SGC7901 and MGC803 cells. Knockdown ofETV4 in SGC7901 and MGC803 cells reduced transcriptionalactivity of ME1 reporter (Fig. 6J). ChIP-PCR analysis furthershowed direct binding of ETV4 with ME1 promotor (Fig. 6K).

Combination of ETV4 and ME1 predicts poor prognosis ingastric cancer

To investigate the biological role of ME1 in human gastriccancer progression, IHC was performed to examine the proteinexpression level ofME1 in 207 cases of paraffin-embedded gastrictissues (Supplementary Fig. S7A). ME1 expression was signifi-cantly increased in distant organ metastasis (M) and lymph nodemetastasis (Ln) tissues compared with adjacent normal tissues(ANT) and paired primary tumor tissues (T; Fig. 7A and B),supporting potential link between ME1 expression and gastriccancer metastasis. Consistently, ME1 expression was markedlyoverexpressed in the gastric cancer tissues compared with pairednormal gastric tissues at protein (Fig. 7A and B) and mRNA level(Fig. 7C).

To determine the clinical relevance of ME1 in gastric cancer,archived patients were divided into high expression and lowexpression group according to immunoscoring of ME1. Statisticalanalyses revealed that expression of ME1 was significantly corre-lated with differentiation state (P ¼ 0.046), but not with otherclinical parameters, including age, gender, tumor size, lymphnode metastasis, venous invasion, perineural invasion, andtumor–node–metastasis (TNM) stage (Supplementary TableS2). In addition, Kaplan–Meier survival analysis and log-ranktest showed that ME1 overexpression was correlated with shorteroverall survival and disease-free survival (P < 0.001; Fig. 7D).Univariate and multivariate analyses indicated that only

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Figure 4.

KnockdownofME1 inhibits growth andmetastasis of gastric cancer in vivo.A, Tumor volumeprogression of xenografted subcutaneous SGC7901 (sh#NC, sh#1, sh#2)cells (n ¼ 6). Tumor growth curves were measured after injection, and tumor diameters were measured every 3 days. The values were given as mean � SD.B, Photograph of dissected xenografts. C,Weight of dissected xenografts was recorded.D, Representative immunostaining of Ki-67, TUNEL, and cleaved caspase-3(c-Cas3) in xenografted tumors. Scale bar, 50 mm. E, Representative luciferase imaging of lung metastatic cells in nude mice after knockdown of ME1. F,Representative results of hematoxylin and eosin staining (left) of metastatic lung nodules frommice injected with ME1 knockdown and control SGC7901 cells via thetail vein. Metastatic nodules under naked eyes or microscope were counted and recorded (right).G, SGC7901 cells were injected intraperitoneally andmetastases inthe colonicwallwas recorded60days later.H,Dissected colonswerephotographedandmetastatic nodules are indicated (arrows). Hematoxylin andeosin stainingofcolon was performed and metastatic numbers were recorded. All error bars represent the SD of results from 6 mice. P values were determined by two-tailed t test.

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Figure 5.

Intratumoral silencing of ME1 suppressed gastric cancer growth in vivo. A, Effect of intratumoral ME1 knockdown on tumor growth in mice after injection ofSGC7901 cells. B, Dissected xenografts after intratumoral silence of ME1 were photographed. C, Tumor weights of xenografts were recorded. D–F, Effects ofintratumoral ME1 knockdown on three PDXmodels. G, Hematoxylin and eosin (H&E) and immunostaining of Ki-67, ME1, TUNEL, and c-Cas3 in cell line or PDX-basedxenografts. Scale bar, 50 mm. All error bars represent the SD of results from 5 mice. P values were determined by two-tailed t test.

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Figure 6.

ETV4 upregulates ME1 expression in gastric cancer. A, Intracellular ROS level in SGC7901 and MGC803 cells cultured in glucose deprivation medium or indetached conditions. B, The ME1 mRNA level in SGC7901 and MGC803 cells cultured in glucose deprivation medium or in detached conditions was measured.C, Immunoblots of ETV4 and ME1 in SGC7901 and MGC803 cells cultured in glucose deprivation medium or in detached conditions. D, ETV4 DNA-binding sites arepresented in the human ME1 promoter region. E, Immunoblots of ETV4 and ME1 in SGC7901 and MGC803 cells after enforced expression of ETV4. F, Immunoblots ofETV4 and ME1 in SGC7901 and MGC803 cells after siRNA-mediated knockdown of ETV4. G, Immunoblots of ETV4 and ME1 in SGC7901 and MGC803 cells cultured inglucose deprivation medium after siRNA-mediated knockdown of ETV4. H, Dual-luciferase reporter assays in SGC7901 and MGC803 cells cultured in glucosedeprivationmedium or in detached conditions. I,Dual-luciferase reporter assays in SGC7901 and MGC803 cells after enforced expression of ETV4. J,Dual-luciferasereporter assays in SGC7901 and MGC803 cells after knockdown of ETV4. K, ChIP-PCR in 293T cells demonstrating ME1 promoter occupancy by ETV4. Data arerepresentative of two independent experiments. All error bars represent the SD of at least three replicates from two independent experiments. P values weredetermined by two-tailed t test.

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Figure 7.

ETV4/ME1 axis is overactivated in gastric cancer. A, Representative staining showed upregulated expression of ME1 protein in gastric tumor tissues compared withcorresponding nontumorous tissues. Scale bar, 100 mm. B, Immunoscoring of ME1 in adjacent normal tissues (ANT), gastric cancer tissues (T), distant organmetastasis (M), and lymph node metastasis (Ln). C, The ME1 mRNA expression in 50 paired gastric tumor samples and corresponding nontumorous tissues.D, Kaplan–Meier analysis of overall survival or disease-free survival curves for gastric cancer patients with low versus high expression of ME1. E, Immunoblots ofETV4 and ME1 in eight freshly collected gastric cancer samples. F, The relative protein expression levels in eight freshly collected gastric cancer samples werequantified by comparing the gray level of each band using ImageJ Software. G, Correlations between mRNA level of ETV4 and ME1 in 50 freshly collectedgastric cancer samples. H, Correlations of ETV4 and ME1 protein expression in gastric cancer tissues based on immunoscoring. I, Gastric cancer patients weredivided into three groups according to immunoscoring of ETV4 and ME1, and overall survival curve was generated with Kaplan–Meier methods. J, Receiveroperating characteristic (ROC) curve analysis of ME1 [area under a curve (AUC)¼ 0.587 (95% CI, 0.517–0.655)] or ETV4 [AUC¼0.672 (95% CI, 0.603–0.735)] singlescoring or combinational scoring [AUC ¼ 0.785 (95% CI, 0.722–0.839)]. P values were determined by two-tailed t test. K, Proposed working model of thecurrent study.

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upregulated ME1 expression and TNM stage were independentprognostic factors for outcome in gastric cancer (P < 0.001;Supplementary Table S3).

We further analyzed the expression of ETV4 and ME1 in eightfreshly collected gastric cancer samples. Western blot analysisindicated that both ETV4 andME1 were significantly upregulatedin the eight tumor samples examined, compared with the pairedadjacent noncancerous tissues from the samepatients (Fig. 7E). Inaddition, ETV4 expression was positively correlated with ME1expression at protein level (P¼ 0.0043, r¼ 0.6727) as analyzed inthe eight samples (Figs. 7E and F; Supplementary Fig. S7B). In 50freshly collected clinical gastric cancer samples, ETV4 mRNAexpression was statistically correlated with the mRNA levels ofME1 (P < 0.001, r¼ 0.6298; Fig. 7G). Figure 7H showed that therewas a significant positive correlation between ETV4 expressionand ME1 (P ¼ 0.035) in the 207 gastric cancer samples. Samplesthat had lower level of ETV4 expression also had a lower ME1expression, whereas samples that had higher level of ETV4 expres-sion had a higher ME1 expression (Supplementary Fig. S7C). Inour patient cohort, overexpression of ETV4 was significantlyassociated with outcome of gastric cancer (Supplementary Fig.S7D). On the basis of ETV4 and ME1 expression, gastric cancerpatients were categorized into three groups with different risks ofdisease progression or death. Patients with high expression ofboth ETV4 and ME1 showed the worst outcome (Fig. 7I; Supple-mentary Fig. S7E). Moreover, combination of ETV4 and ME1immunostaining showed higher predictive value than eitherparameter alone of gastric cancer patients survival in ROC curveanalysis (Fig. 7J).

DiscussionHomozygous deletion of SMAD4 was frequently identified in

nearly one third of pancreatic cancer cases (30), and loss ofneighboring housekeeping genes often leads to collateral lethality(4, 6). However, neither homozygous deletion (31) nor inacti-vation mutation (32) of SMAD4 was reported to be tightlyassociated with gastric cancer, although knockout mice haveclearly demonstrated its tumor-suppressive functions in the gas-trointestinal tract (33). Our analysis identified frequent hemizy-gous deletion of SMAD4 in gastric cancer and concurrent under-expression ofME2, which leads to high dependency of cancer cellsto ME1 in energy stress conditions.

Cancer cells often require much more NADPH supplemen-tation for redox hemostasis, lipid oxidation, and biomolecularsynthesis than their normal counterparts (19, 34, 35). It ispredicted that intracellular NADPH comes from the oxidativepentose phosphate pathway (�30%), the glutaminolysis fluxthrough malic enzymes (�30%), and the methylenetetrahy-drofolate dehydrogenase–mediated folic metabolism (�40%)in proliferating cells (18). We previously reported that disrup-tion of G6PD-gated pentose phosphate pathway resulted inmarked reduction in NADPH and enhanced sensitivity to ROSstresses (23). In this study, we focused on malic enzymes familyand found that ME2 was downregulated in gastric cancer due toconcomitant genomic deletion with SMAD4 (6, 32). Cellscould tolerate hemizygous deletion of ME2 as the redundantparalog ME1 provided metabolic anaplerosis for reducingequivalents and TCA substrates. However, metabolic stressdeveloped when tumor growth exceeded the ability of availablevasculature to supply tumor cells with oxygen and nutrients,

which was a common impediment to tumor growth (9) andtotally distinguished them from in vitro culture system. Whenits paralog ME2 was suppressed, ME1 showed a key function inproviding NADPH for glutathione regeneration and ROS elim-ination, which was critical for gastric cancer cell survival underenergy stress conditions, such as glucose limitations, anchor-age-independent growth, and solid tumor formation in vivo.Moreover, ME1 was transcriptionally upregulated by ROS in anETV4-dependent manner. Our results provided comprehensiveinsights into the redundant roles of ME1 in gastric cancertumorigenesis and metastasis (Fig. 7K).

Expression of ME1 was known to be regulated by well-knownoncogenes or tumor suppressors such as KRAS (20, 35) or TP53(16). Overexpression of ME1 was reported to predict poorprognosis of hepatocellular carcinoma (36) and to conferradiation resistance in lung cancer (21). Suppression of ME1led to glucose addiction of nasopharyngeal cancer (13) andcolorectal cancer cells (14), which was further confirmed in ourstudy. However, Zheng FJ and colleagues found that enzymicactivity and protein level of ME1 was induced by excessivecarbohydrate supplementation, including glucose and pyruvate(13), whereas upregulation of ME1 was observed after glucosedeprivation in our study, indicating a tissue-specific regulationpattern. Moreover, aberrant expression of ME1 was associatedwith poor prognosis in our patient cohort. Taken together,these findings suggested that ME1 has an important functionin the growth and survival of cancer cells and that it could beused as a drug target in cancer therapy. We next exploredtherapeutic potential of ME1 inhibition in cell line–based aswell as PDX models via in vivo siRNA treatment. Silence of ME1significantly suppressed tumor growth and induced elevatedcell apoptosis. Importantly, a recent report has revealed a panelof small molecules that could inhibit activities of malicenzymes (17). However, further studies are needed to confirmthe clinical benefit of these inhibitors.

In this study, we provide the genetic and pharmacologic evi-dences that ME1 inhibition is lethal in cells with collateral loss ofME2 due to hemizygous deletion of SMAD4, whereasME2-intactcells could rely onME2 to undergo glutaminolysis and to provideNADPH for cell survival under redox stress conditions. Inhibitionof ME1 would be a promising therapeutic alternative in gastriccancer treatment.

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

Authors' ContributionsConception and design: H.-Q. Ju, R.-H. XuDevelopment of methodology: Y.-X. Lu, H.-Q. Ju, Y. Wang, P.-S. Hu,D.-S. Zhang, F. Wang, R.-H. XuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y.-X. Lu, H.-Q. Ju, D.-L. Chen, Y. Wang, Q.-N. Wu,H.-B. Qiu, Z.-Q. Wang, D.-S. ZhangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y.-X. Lu, H.-Q. Ju, Z.-X. Liu, Q. Zhao, Q.-N. Wu,R.-H. XuWriting, review, and/or revision of themanuscript: Y.-X. Lu, Z.-X. Liu, R.-H. XuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y.-X. Lu, Y. Wang, Q. Zhao, Z.-l. Zeng,Z.-Q. Wang, D.-S. Zhang, F. Wang, R.-H. XuStudy supervision: F. Wang, R.-H. XuOther (assistance in generation of PDX models): Y. Wang

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AcknowledgmentsThis research was supported by National High Technology Research and

Development Program of China (863 Program), China (No. 2015AA020103 toR.-H. Xu), National Natural Science Foundation of China (nos. 81602137 toH.-Q. Ju; 81572392 to Z.-L. Zeng; 31501069 to Z.-X. Liu), Natural ScienceFoundation of Guangdong Province (nos. 2017A030313485 to H.-Q. Ju;2014A030312015 to R.-H. Xu), and Science and Technology Program ofGuangdong (no. 2015B020232008 to R.-H. Xu).

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 October 14, 2017; revised December 6, 2017; accepted January 18,2018; published online April 15, 2018.

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Correction

Correction: ME1 Regulates NADPHHomeostasis to Promote Gastric CancerGrowth and MetastasisYun-Xin Lu, Huai-Qiang Ju, Ze-Xian Liu, Dong-Liang Chen,Yun Wang, Qi Zhao, Qi-Nian Wu, Zhao-lei Zeng, Hai-Bo Qiu,Pei-Shan Hu, Zhi-Qiang Wang, Dong-Sheng Zhang,Feng Wang, and Rui-Hua Xu

In the original version of this article (1), the immunostaining of Ki-67 (si#ME1 groupof PDX#2 and PDX#3) in Fig. 5G was inadvertently duplicated during data arrange-ment. The image has been replaced with the intended image for Fig. 5G. The errordoes not affect the conclusion and has been corrected in the latest online HTML andPDF versions of the article. The authors regret this error.

Reference1. Lu YX, Ju HQ, Liu ZX, Chen DL, Wang Y, Zhao Q, et al. ME1 regulates NADPH homeostasis to

promote gastric cancer growth and metastasis. Cancer Res 2018;78:1972–85.

Published online July 15, 2019.Cancer Res 2019;79:3789doi: 10.1158/0008-5472.CAN-19-1611�2019 American Association for Cancer Research.

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