Human Elongation Factor 4 Regulates Cancer Bioenergetics ...Molecular Cell Biology Human Elongation Factor 4 Regulates Cancer Bioenergetics by Acting as a Mitochondrial Translation

Molecular Cell Biology

Human Elongation Factor 4 Regulates CancerBioenergetics by Acting as a MitochondrialTranslation SwitchPing Zhu1,2, Yongzhang Liu3, Fenglin Zhang4, Xiufeng Bai1,2, Zilei Chen1,3,Fugen Shangguan1,3, Bo Zhang4, Lingyun Zhang1, Qianqian Chen2,4, Deyao Xie5,Linhua Lan3, Xiangdong Xue6, Xing-Jie Liang6, Bin Lu3, Taotao Wei2,4, and Yan Qin1,2

Abstract

Mitochondria regulate cellular bioenergetics and redox statesand influence multiple signaling pathways required for tumori-genesis. In this study, we determined that the mitochondrialtranslation elongation factor 4 (EF4) is a critical component oftumor progression. EF4 was ubiquitous in human tissues withlocalization to the mitochondria (mtEF4) and performed qualitycontrol on respiratory chain biogenesis. Knockout of mtEF4induced respiratory chain complex defects and apoptosis, whileits overexpression stimulated cancer development. In multiple

cancers, expression of mtEF4 was increased in patient tumortissues. These findings reveal thatmtEF4 expressionmay promotetumorigenesis via an imbalance in the regulation of mitochon-drial activities and subsequent variation of cellular redox. Thus,dysregulated mitochondrial translation may play a vital rolein the etiology and development of diverse human cancers.

Significance: Dysregulated mitochondrial translation drivestumor development and progression. Cancer Res; 78(11); 2813–24.�2018 AACR.

IntroductionMitochondria are ancient bacterial symbionts that contain their

own DNA (mitochondrial DNA, mtDNA), RNA, and proteinsynthesis systems. Mammalian mtDNA encodes 13 polypeptidesof the core set of oxidative phosphorylation (OXPHOS) com-plexes involved in generating, maintaining, and utilizing the

mitochondrial inner membrane proton gradient to generateATP (1). These polypeptides are synthesized in themitochondrialmatrix by the 55S ribosome system, which is maintainedindependently by the organelle (2, 3). Mitochondrial translation(mt-translation) plays a central role in the control of mitochon-drial respiration machinery (4–6); however, the precisemechanisms involved in the regulation of mt-translation haveremained elusive.

Approximately 90 years ago, cancer development was postu-lated to originate from mitochondrial defects and subsequentaerobic glycolysis, termed the Warburg effect (7, 8). However,more recent studies suggest that functional mitochondria areessential for the intra- and intercellular communications of cancercells, and for the reprogramming of adjacent stromal cells, tomanipulate themicroenvironment for optimal cancer growth (9).More importantly, mitochondrial signaling has been identified asplaying a key role in the formation of drug-resistant tumors. Theinhibition of mitochondrial signaling is a new antitumor strategywith high potential (10). This reflects the paradox in cancerbiology, that many opposite biochemical properties can enhancetumor development similarly in diverse cellular contexts. There isincreasing evidence that various functional perturbations ofmito-chondria appear to generate opposite effects on tumor growth, forexample, mutations in the components of the tricarboxylic acid(TCA) cycle including isocitrate dehydrogenase (IDH), fumaratehydratase (FH), and succinate dehydrogenase (SDH) enhancetumorigenesis (11–15), while the loss of multiple electron trans-port chain (ETC) complexes diminishes cancer (16–18). There-fore, both enhanced and compromised mitochondrial functionsmay contribute to tumorigenesis via distinct mechanisms.

Elongation factor 4 (EF4, LepA) is one of the most commonlyconserved proteins in nearly all known genomes (19). All EF4homologs have approximately 600 amino acid (aa) residues,

1Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromole-cules, Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District,Beijing, China. 2University of Chinese Academy of Sciences, Beijing, China.3Attardi Institute of Mitochondrial Biomedicine, School of Laboratory Medicineand Life Sciences, Wenzhou Medical University, Wenzhou, China. 4NationalLaboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy ofSciences, Chaoyang District, Beijing, China. 5Departments of CardiothoracicSurgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou,China. 6Laboratory of Controllable Nanopharmaceuticals, CAS Key Laboratoryfor Biomedical Effects of Nanomaterials and Nanosafety, National Center forNanoscience and Technology of China, Zhongguancun, Beijing, China.

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

P. Zhu, Y. Liu, and F. Zhang contributed equally to this article.

Corresponding Authors: Yan Qin, Key Laboratory of RNA Biology, CAS Centerfor Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academyof Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China. Phone: 86-10-64888434; Fax: 86-10-6487-1293; E-mail: [email protected]; Taotao Wei,National Laboratory of Biomacromolecules, CAS Center for Excellence inBiomacromolecules, Institute of Biophysics, Chinese Academy of Sciences,Beijing 100101, China. Phone: 86-10-64888515; E-mail: [email protected];and Bin Lu, Attardi Institute of Mitochondrial Biomedicine, School of LaboratoryMedicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035,China. Phone: 86-577-86699291; E-mail: [email protected].

doi: 10.1158/0008-5472.CAN-17-2059

�2018 American Association for Cancer Research.

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which are composed of 5 structural domains. All eukaryotichomologs have a putative mitochondrial targeting signal(MTS) in their N-termini (20, 21). In bacteria, EF4 functions asa translation factor that catalyzes the elongating ribosomeone-codon back-movement along an mRNA, and thus maintainstranslation fidelity under stress conditions (19, 22–24). However,the physiologic role of EF4 in human cells has remained elusive.More importantly, the reasoning behind the dependence ofmt-translation on EF4 is not known. This study investigated thepotential regulatory role of human EF4 in mt-translation andrevealed its direct contribution to tumor development, sheddinglight on its biological functions.

Materials and MethodsPatients and tissue collection

The tissue samples were collected in the First AffiliatedHospitaland the Second Affiliated Hospital of Wenzhou Medical Univer-sity (Wenzhou, China) between 2005 and 2013. This study wasapproved by the Board and Ethical Committee of WenzhouMedical University. All patients participated in this study provid-ed written informed consents in accordance with the Declarationof Helsinki. Each pair of normal and cancerous tissue wasobtained from the same patient without radiotherapy or chemo-therapy prior to the operation.

Cell culturesThe HFFs, HeLa, MDA-MB-231, HepG2, H1299, H460, A549,

SK-MES-1, 16HBE and PASMC cell lines were obtained fromATCC (2010) and grown in high-glucose DMEM, and the K562and PC3 cell lines (ATCC, 2010) in RPMI1640medium, HFL-1 inF12K medium, supplemented with 10% FBS. All cells wereauthenticated via STR profiling. Mycoplasma was often checkedusing the TransDetect Luciferase Mycoplasma Detection Kit pur-chased from Beijing TransGen Biotech.

RNA extraction and RT-PCRTotal RNA was extracted using RNeasy Mini Kit (Qiagen). The

first strand of cDNA was synthesized using M-MLV reverse tran-scriptase (Promega). GAPDH or b-actin was chosen as an internalcontrol for normal tissue or cancer cells, respectively. Primers usedare listed in the Supplementary Table. RT-PCR reactions wereperformed in an ABI StepOne Plus Real-Time PCR System(Life Technologies).

Western blottingTissue samples were homogenized using tissue lysis buffer

(1 mmol/L NaF, 1 mmol/L Na3VO4, and inhibitor cocktail).Protein concentrations were quantified using the BCA ProteinAssay Kit (Thermo Scientific). Each sample equivalent of 20 mgtotal protein was separated by 8% SDS-PAGE gels followed byelectrophoretic transfer onto polyvinylidene difluoride mem-brane. Then the membranes were blocked and probed withprimary antibodies and secondary antibodies. Detailed informa-tion of the antibodies used is listed in the Supplementary Table.

MtEF4 protein stabilityHeLa cells were cultured in 6-cm dishes and 50 mg/mL cyclo-

heximidewas added to inhibit cytoplasmic translation. Cells wereharvested at 0, 12, 24, and 36hours and cell lysateswere separatedby SDS-PAGE and detected by Western blotting.

Knockdown and overexpression of mtEF4Knockdown (KD) of mtEF4 to generate HeLa-si or H1299-si

was performed by transfecting HeLa or H1299 cells with100 nmol/L of siRNA using Lipofectamine 2000 (Invitrogen).Knockdown of mtEF4 to generate HeLa-sh or H1299-sh wasimplemented through lentiviral transduction. The targetsequenceswere listed in Supplementary Table. For overexpressionof mtEF4, HeLa cells were transiently transfected with plasmidsthat carried the full-length mtEF4 coding sequence, followed byGFP using Lipofectamine 2000. Stable-transfected mtEF4 KD andexpression cells were selected by flow cytometry for cell sorting.

Trypsin protection analysisAssays were performed as described previously (25) with

modification. Isolated mitochondria were either kept on ice ascontrol or treated with trypsin (100mg/mL) alone or with trypsin(30 mg/mL) plus Triton X-100 (1%). Treated mitochondria ormitoplasts were collected by centrifugation at 18,000 � g for3 minutes and the pellets were resuspended in SDS-PAGE samplebuffer for Western blotting.

Transmission electron microscopyCells were fixed in 2.5% glutaraldehyde overnight at 4�C and

postfixed in 1.0 % osmium tetroxide for 1 hour, dehydratedthrough a graded series of acetone, and embedded in resin. Theultrathin section was stained with uranyl acetate and examinedusing a Hitachi 7500 transmission electron microscope.

Oxygen consumption and extracellular acidification ratesA total of 4�104 cellswere grown in eachwell of SeahorseXF24

cell culture microplate for 16 hours in complete medium(90–100% confluent at measurement). The oxygen consumptionrate (OCR) and extracellular acidification rate (ECAR) were,respectively, assayed with the XF Cell Mito Stress Test Kit andthe XF Glycolysis Stress Test Kit, coupled with the XF24 extracel-lular flux assay kit, and was recorded by XF24-3 Extracellular FluxAnalyzer (Seahorse Bioscience) according to the manufacturer'soperation manual and instructions. All OCR and ECAR measure-ments were normalized with well-by-well protein contents.

TCA metabolites assayCells were grown to 80% confluency on 10-cm dishes before

collection.Metabolites were extracted from cells by adding 0.5mL80:20 methanol: water solution to 1 � 106 cells, followed byvortexing for 10 seconds, incubation at 4�C for 10 minutes, andcentrifugation at 13,000� g for 10 minutes. The supernatant wasthen transferred to LC-MS vials for analysis. Agilent 6400 TripleQuad LC-MSmass spectrometer was used (Agilent Technologies).TCA cycle intermediates were monitored by tandem MS usingmultiple reaction monitoring mode.

Blue native gel and in-gel activityThe isolated mitochondria were solubilized, centrifuged and

loaded onto a 4%–13% gradient blue native electrophoresis gel.After blue native electrophoresis, n-Dodecyl b-D-maltoside(DDM-treated samples were subjected to IGA analysis. ComplexI IGA (CI-IGA) was visualized by incubating the gel with0.5 mmol/L nitrotetrazolium blue (NBT) and 5 mmol/L NADHin 50 mmol/L Tris-HCl (pH 7.4) at room temperature for 60minutes. Complex II IGA (CII-IGA) was visualized by incubatingthe gel with 4.5 mmol/L EDTA, 10 mmol/L KCN, 0.2 mmol/L

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phenazine methasulfate (PMS), 84 mmol/L succinic acid and 50mmol/L NBT. Complex IV IGA (CIV-IGA) was visualized byincubating the gel with 0.1% (w/v) 3,30-diaminobenzidine(DAB), 0.1% (w/v) cytochrome c, and 24 U/mL catalase in 50mmol/L Tris-HCl (pH 7.4) at 37�C for 3–6 hours. Complex V IGA(CV-IGA) was visualized according to the following procedure: acouple of times in water; incubate in 50mmol/L glycine (pH 8.6)for 1 hour; prepare reactionmix in the following order: 35mmol/L Tris, 270 mmol/L glycine, 14 mmol/L MgSO4, 5 mmol/L ATP,adjust pH to 7.8, add 0.2% Pb(NO3)2, and incubate the gel inreaction mix at 37�C for 3–6 hours.

Estimation of mitochondrial ROS productionCells were plated in 35-mm glass bottom dish (In Vitro Sci-

entific), pretreated with or without mitochondria-targeted anti-oxidant MitoTEMPO (Sigma) at 20 mmol/L for 60 minutes. ROSproduction was measured using MitoSOX Red (Thermo FisherScientific) at 5mmol/L for 10minutes. Imageswere acquired usinga confocal microscopy (Nikon).

Detection of mitochondrial membrane potentialHeLa and H1299 suspension were incubated in medium con-

taining 5 mg/mL JC-1 at 37�C for 20 minutes, washed twice withPBS, and resuspended. TheMMPwasmeasured on a FACSCaliburFlow Cytometer (BD Biosciences) and the data were presented asthe red/green fluorescence ratio.

ATP assayThe amount of ATP was measured by the luciferin–luciferase

method using an ATP Detection Kit (Roche Applied Science). TheATPlite assay procedure was carried out according to the manu-facturer's instructions. The luminescence emitted from the ATP-dependent luciferase. Reaction was measured using a Lumin-ometer (Perkin Elmer).

Detection of reactive oxygen speciesCells cultured in 6-well culture plates were washed with PBS

and incubated with 10 mmol/L DCFH-DA (Invitrogen) in DMEMat 37�C for 30 minutes in dark. After washing three times, thecells were trypsinized and analyzed by flow cytometry (BDBiosciences) using Cell Quest software.

Tumor growth assaysMice weremaintained in a specific pathogen-free environment,

and all experiments withmicewere done following the guidelinesfor experimental animals and were approved by the InstitutionalAnimal Care and Use Committee of Institute of Biophysics,Chinese Academy of Sciences (IACUC-IBP). For xenograft assays,H1299-sh1, A549-sh2, H1299-OE, HeLa-sh, or -OE cells wereinjected subcutaneously into the flank of 6- to 8-week-old Balb/cnude mice. Tumor volume was calculated using the formula:volume ¼ length � width � width/0.5. Five to six weeks afterimplantation, mice were anesthetized for imaging and sacrificed.All procedures involving animals and their care were approved bythe animal ethics committee of the Institute of Biophysics at theChinese Academy of Sciences.

In situ ROS detectionThe tumor-bearing nude mice were intraperitoneal anesthe-

tized and ROS Brite 700 (AAT Bioquest) was injected into tumortissue according to the product instructions. The in vivo fluores-

cence imaging was performed by using the Maestro ImagingSystem (CRi).

Cell proliferation and apoptosisA total of 4 � 103 cells were seeded on 96-well plates and

cultured overnight. To induce hypoxia, cells were rendered in 1%O2 and 5% CO2-balanced N2 at 37�C. The level of oxygen wasverified using a gas monitor (SKC). Cell proliferation was ana-lyzed using MTT (570 nm). After the cells were treated withetoposide for 24 hours, apoptosis was determined by Hoechststaining and by analysis of cleaved caspase-3, cleaved PARP, andthe release of cytochrome c.

Binding assayThe binding of EF4 and mtEF4 to the 70S ribosome and

their dissociation constants were calculated as described previ-ously (19).

Functional identification of mtEF4 in the translation systemAssayswere performed as described previously (19), except that

either EF4 (E. coli) or mtEF4 (human) proteins were loaded intothe reactions.

Statistical analysisAll statistical analyses were performed using the GraphPad

Prism Software (GraphPad Software). For comparisons, Studentt test (normal distribution), log-rank test, and Wilcoxon signed-rank test were performed as indicated. Statistical significancerefers to �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

ResultsHuman EF4 is localized to mitochondria and interacts with mtribosomes

The human ef4 gene, originally named guf1 (26), has a con-served domain architecture. Human EF4 shares 48% amino acid(aa) identity with its E. coli counterpart (Supplementary Fig. S1A),possessing 665 aa residues and a 61 aa MTS (SupplementaryFig. S1B).Mitochondrial localization of humanEF4was predictedin silica (Supplementary Fig. S1B), and then experimentally val-idated by fluorescence imaging and subcellular fractionation,followed by trypsin digestion. In Fig. 1A, EF4 was colocalizedwith Mito-DsRed, a discosoma red fluorescent protein fused to amitochondrial targeting sequence. In a trypsin protection assay(25), EF4 was colocalized with NDUFA9 and HSP60, indicatingits location in the mitochondrial lumen (SupplementaryFig. S1C). These data suggest that EF4 is a bonafidemitochondrialprotein, therefore it was named mtEF4.

Super-resolution fluorescence microscopy showed that mtEF4is an mt-translation factor associated with mitochondrial ribo-somes (mt-ribosomes), based on the observation that it wasmerged with mitochondrial ribosomal protein S18b (Fig. 1B).The molecular mechanisms of action of mtEF4 on the ribosomewere studied in a bacterial translation system, a model similar tomt-translation (27). The dissociation constant (Kd) betweenmtEF4 and 70S ribosome was equal to that of bacterial EF4(2.03 mmol/L vs. 1.97 mmol/L, Fig. 1C), and it strongly catalyzedthe multiple turnover of GTP hydrolysis (Fig. 1D). In a back-translocase assay, mtEF4, like bacterial EF4, did not affect tRNAbinding but decreased the puromycin reaction of the posttran-slocational (POST) state to 0.17 (Fig. 1E). These results

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Figure 1.

EF4 interacts with mitochondrialribosome and its expression levelsinfluence the respiration–glycolysisratio. A, Subcellular localization ofEF4 to the mitochondria. Cells werecotransfected with an EGFP-taggedEF4 expression vectorpcDNA3.1-mtEF4 (green) and amitochondrial-targetingpDsRed2-Mito plasmid (red). B,Colocalization of mtEF4 withmitochondrial ribosomes. Cells weretransfected with an EGFP-taggedmtEF4 expression plasmid (mtEF4) ormock vehicle (Vector). MRPS18b,mitochondrial ribosomal protein S18b.C, Dissociation constants (Kd) of E.coli EF4 (black) or mtEF4 (red) to 70Sribosomes. D, Uncoupledribosome-dependent GTPase activityof EF4 (black) and mtEF4 (red). TheGTPase activity is given as GTPhydrolyzed in 5 minutes per ribosomeand is plotted as a function of themolar ratio of factor to ribosome. E,Binding of tRNA and reaction ofpuromycin in pre- (PRE, tRNAs inA- and P- sites) and post- (POST,tRNAs in P- and E- sites)translocational complexes incubatedwith mtEF4 and GTP. n, the number ofreactions per ribosome.[32P]tRNAMet

f, blue. Ac[3H]Phe-tRNA,

cyan. F, KD of mtEF4 inhibitedmitochondrial respiration butpromoted glycolysis. OCR of thecancer cells transfected withmtEF4-targeting siRNA (si) or shRNA(sh). G, ECAR of the cancer cellstransfected with mtEF4-targetingsiRNA (si) or shRNA (sh). H, Basalrespiration of the cancer cellstransfected with mtEF4-targetingsiRNA (si) or shRNA (sh; mean � SD;n ¼ 3; � , P < 0.05, �� , P < 0.01,�� , P < 0.001; Student t test). I, GM/GCof the cancer cells transfectedwithmtEF4-targeting siRNA (si) or shRNA(sh). GM, glucose metabolism; GC,glycolytic capacity (mean� SD; n¼ 7;� , P < 0.05, ��, P < 0.01, �� , P < 0.001;Student t test). J and K,Overexpression (OE) of mtEF4increased mitochondrial respirationand inhibited glycolysis. The OCR (J)and the ECAR (K) of HeLa cellstransfected with pcDNA3.1-mtEF4(mean � SD; n ¼ 7; � , P < 0.05,�� , P < 0.01, �� , P < 0.001;Student t test).

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demonstrate that mtEF4 is likely to function in a similar mannerto EF4 (19, 24, 28) in themitochondrialmatrix, andhence controlmt-translation in the biogenesis of respiratory complexes.

Changes of mtEF4 expression levels resulted in an alteredcomposition of cellular biogenetics

To explore the physiologic role of mtEF4, KD ofmtEF4 inHeLa(cervix) and H1299 (lung) cancer cells with either siRNA orshRNA targeting mtEF4 mRNA (Supplementary Fig. S1D–S1F),and overexpression (OE) ofmtEF4 with anmtEF4 overexpressionplasmid (Supplementary Fig. S1G and S1H) was carried out.Scrambled RNA or empty vector were used as negative controls(NC). Respiration and glycolysis was measured in these cells.mtEF4 KD caused a decrease in basal OCRs (Fig. 1F) and acompensatory increase in basal ECAR. Although the magnitudeof increased ECAR was statistically significant, it was modest (Fig.1G).When treatedwith the uncoupler p-trifluoromethoxyphenyl-hydrazoydrazon (FCCP), the cells exhibited adistinct reduction inmaximal respiration (Fig. 1H) and an average increase of 0.9-foldin total glucose metabolism (Fig. 1I) without distinct variation ofTCA metabolites (Supplementary Fig. S1I). These data reflectOXPHOS impairment and a compensatory enhancement of cellglycolysis uponmtEF4KD. In comparison, upregulationofmtEF4significantly increased the OCR (Fig. 1J) and decreased the ECAR(Fig. 1K).

Together, these results demonstrate that both HeLa andH1299cancer cells possess developed mitochondrial systems; KD ofmtEF4 caused inhibition of respiration and glycolysis activation,where mtEF4 upregulation improved respiration and suppressedglycolysis. These data are consistent with the proofreadingfunction of EF4 in translation, as was established earlier inbacteria (19), indicating that mtEF4 plays a similar role withinthe mitochondria of human cells. To better understand therelationship between mtEF4 and respiration in cancer cells,the impact of mtEF4 dysregulation on respiratory complexes inHeLa-KD and -OE cells was assayed.

mtEF4 expression levels determined the amount of respiratorychain complexes

Confocal imaging indicated that the mitochondria of KD cellswere highly fragmented (Fig. 2A). In contrast to the long mito-chondrialfilaments seen inNC samples,mitochondria inKD cellswere shorter andmore globular, exhibiting clear structural abnor-malities. Using electronmicroscopy, mitochondria in NC and KDcells were shown to be completely different in terms of size,abundance, and cristae morphology. Specifically, KD mitochon-dria were smaller and more globular, but exhibited a higherabundance relative to NC samples. Most strikingly, these mito-chondria were almost completely depleted of cristae, which is animportant sign of respiratory chain biogenesis dysfunction(Fig. 2B and C). Therefore, respiratory chain complexes (RCC)were quantitatively and qualitatively assessed in HeLa cells. Thetranscripts of nuclear-encoded (Supplementary Fig. S2A and S2B)and mitochondrial-encoded (Supplementary Fig. S2C) RCC sub-unitswere largely unchanged inKDcells.However, when an equalamount of mitochondrial protein was loaded on a blue native gel(BNG), all complexes vanished inKDmitochondria (Fig. 2D). Forconfirmation, the amount of each complex was quantified byWestern blotting (BNG-WB). Results clearly showed that theamount of each complex decreased dramatically (Fig. 2E). Next,the in-gel enzymatic activity (IGA) of each complex was tested.

Consistent with the above results, little IGA was detected incomplexes I, II and IV, and complex V showed significantly lessactivity (Fig. 2F). Quantification of the BNG-WB and the IGAshowed that the RCCs in KD cells were 80% reduced, on average(Fig. 2G). Because KD of mtEF4 reduced the amount of RCC, thepotential for overexpression ofmtEF4 to improve RCC generationand OXPHOS in cancer cells was tested. BNG-WB and IGAanalysis of cells overexpressing mtEF4 provided supportive data.In HeLa-OE, each RCC was upregulated quantitatively andqualitatively (Fig. 2H and I). The cellular bioenergetic status wasthen determined by measuring the ATP content in HeLa-KD and-OE cells. KD cells exhibited ATP reduction, while ATP biosyn-thesis was promoted in HeLa-OE cells (Fig. 2J; SupplementaryFig. S2D). Similar results were achieved in the measurement ofmitochondrial membrane potential (MMP; SupplementaryFig. S2E). Reactive oxygen species (ROS) detection assay bymitoSOX exhibited more ROS production in KD cells than innegative controls. Upon treatment with the mitochondrial spe-cific superoxide scavenger mitoTEMPO, mtROS signals decreasedsignificantly, confirming the mitochondrial origin of the ROSfollowing KD of mtEF4 (Fig. 2K).

However, when the MTS of mtEF4 was deleted (mtEF4�MTS,Supplementary Fig. S2F), its localization in the cytoplasm (Fig.2L) and overexpression in HeLa cells (Supplementary Fig. S2G)abolished the phenotypes observed in HeLa-OE. Namely, thegrowth rate and cellular parameters of MMP, ATP, and ROS wereat the same levels as in HeLa cells transfected with the emptyvector, butmuch lower than inHeLa-OE (Fig. 2M; SupplementaryFig. S2H).

Collectively, these observations suggest that KDorOEofmtEF4induced the cancer cells to undergo disparate, specific bioener-getic pathway(s). This also suggests that mitochondrial targetingof mtEF4 is essential for the protein to execute its biologicaleffects. The next step was to reveal the molecular connectionsbetween the aberrant-mtEF4–induced bioenergetic variations andcancer cell fate, by using in vivo and in vitro approaches.

mtEF4 knockdown induced apoptosis, while overexpressionpromoted tumor progression

To ascertain the effect of mtEF4 over- or under-expression incancer cells, mtEF4 expression was analyzed in multiple cancercell lines and comparedwith normal cells, that is, human foreskinfibroblasts (HFF). In contrast to the very low level of mtEF4 inHFFs, HeLa cells expressed a medium level of mtEF4, whileH1299 cells exhibited high expression (Fig. 3A). When the anal-ysis was extended to a sizable panel of lung cancer cell lines,including NCI-H460, A549, and SK-MES-1, a higher level ofmtEF4 was observed in every tested cell when compared withHeLa cells (Fig. 3A). Therefore, the molecular effects of mtEF4expressionon cancer in both lung and cervical cancer cell lineswastested.

mtEF4 was knocked down in H1299 and A549 cells using twoindependent shRNAs (shRNA1 and shRNA2), and the resultingcells were injected into nude mice. When compared with emptyvector (negative control, NC), both mtEF4 shRNAs reduced thetumorigenic potential in nude mice (Fig. 3B–D, left and middlecolumns). In contrast, augmentation of mtEF4 expression inH1299 cells increased tumorigenicity (Fig. 3B–D, right columns).Similar results were observed in the tumor formation assays withHeLa cells (Supplementary Fig. S3A and S3B). To confirm theseresults, the growth rate of the cells under hypoxic conditions






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was measured in vitro. Consistently, decreased cell growth wasobserved for HeLa-sh, but not for NC (Supplementary Fig. S3C).In contrast, the OE cells exhibited increased proliferation underthe same conditions (Supplementary Fig. S3D). When cells weretreated with the apoptosis-inducing drug etoposide, apoptoticnuclei were only observed in the KD HeLa-sh and -si cells(Supplementary Fig. S3E). Caspase-3 cleavage was also detectedin KD but not OE cells, which verified the activation of apoptosis(Supplementary Fig. S3F). In KD cells, PARP cleavage and cyto-chrome c release from the mitochondria to the cytoplasm werealso detected (Supplementary Fig. S3G), providing further evi-dence of apoptosis. Together, these results indicate that mtEF4upregulation promoted cancer cell growth under hypoxic condi-tions, while its reduction caused growth retardation and a highsensitivity to apoptotic stimulus.

Next, theprotein levels ofmtEF4 andRCC subunits in xenografttumors were measured, and the expected under- and overexpres-sion of mtEF4 was observed in respective cells (Fig. 3E and F;Supplementary Fig. S3H and S3I). The quantitative changes ofRCC subunits exhibited very similar patterns in response toaltered mtEF4 expression. This suggests that the potential regu-latory role of mtEF4 in the RCC of cancer cells strongly correlateswith expression levels. This was further explored in vivo.

mtEF4 levels influenced redox status in vivoA real-time imaging system was optimized to detect cellular

ROS levels in vivo (Fig. 4A). H1299 cells with mtEF4 inhibition oroverexpression (H1299-sh and -OE) were transplanted into nudemice, and in situ signals were detected with a specific dye formitochondria-generated ROS via fluorescence microscopy.Higher ROS signals were detected in H1299-sh xenograft tumortissues than in NC tissues (Fig. 4B and C), and the tumor mass ofthe NC tissues was greater than in the H1299-sh tissues (Fig. 4D).In contrast, lower ROS signals were recorded in the H1299-OEtumor tissues compared with the NC tissues on the contralateralside (Fig. 4E and F), although the H1299-OE–derived tumorswere much larger (Fig. 4G). These data demonstrate that dimin-ishing mtEF4 decreases tumorigenesis via higher ROS-inducedtoxicity, as observed in vitro (Fig. 2K). However, mtEF4 over-expression provoked tumor development via reduced ROS signalsin the xenograft tissues, an opposite result from what was seenin vitro.

In vitromtEF4 is expressed ubiquitously in human tissues, andupregulation in various cancers reflects its oncogenic role

mtEF4 was detected ubiquitously in all normal tissues in ahuman multiple tissue cDNA library (Fig. 5A), and in human

normal and cancerous tissues (Fig. 5B).mtEF4mRNA and proteinwere also detected in various normal and cancer cell lines (Fig. 5Cand D). In HeLa cells, mtEF4 was stable for a long period of timeinside the cell (Supplementary Fig. S4A). To examine the precisenature of the cellular location and expression in cancer tissues,Western blot and IHC studieswere carried out. Normal and cancertissue samples from patients diagnosed with lung, esophageal,bladder, and cervical cancers were analyzed. In cancer tissues,mtEF4 was confirmed in the mitochondria (SupplementaryFig. S4B). Compared with normal tissues adjacent to the tumorof the same patient, the cancer samples exhibited highly increasedlevels of mtEF4 expression (Fig. 5E; Supplementary Fig. S4C).For semiquantification of mtEF4 protein and mRNA levels in thefour types of cancer, Western blotting and qPCR assays wereconducted with tissue extracts from patient samples. The resultsdemonstrated that in all four cancers, the levels of mtEF4 protein(Fig. 5F) andmRNA (Fig. 5G) were significantly higher relative tonormal tissues. In the human Cancer Genome Atlas (TCGA),mtEF4 is upregulated in various cancer tissues; specifically, it ismarkedly increased in lung cancers. These results reflect thatthere is a positive correlation between mtEF4 levels and well-established cancers.

DiscussionMitochondrial proteostasis is a microcosm of cytoplasmic

proteostasis, which refers to a sophisticated balance betweenprotein synthesis, folding, assembly, and degradation (29). Thereis growing evidence that ribosomes serve as a hub for cotransla-tional folding, chaperone recruitment, degradation, and the stressresponse (30). Thus, in addition to the conventional qualitycontrol systems, including chaperones and proteasomes, theribosome and its associated factors have emerged as key playersin proteostasis (31–33). High translation speed, mainly referringto the fast translation elongation cycles, increases translationalproductivity but negatively affects both translation fidelity andcotranslational folding. To control the translation speed, EF4evolved to transiently brake the ribosome on the mRNA and tofacilitate cotranslational folding of the nascent peptides (34). Thisstudy demonstrated that humanmt-translation retains EF4 for thesame reason as its bacterial counterpart, to maintain translationwith high fidelity and to allow sufficient cotranslational folding.Hence, the availability of the appropriate amount of mtEF4 iscrucial to maintain mt-translation in the biogenesis of functionalRCCs.

Mitochondrial RCC subunits have dual genetic origins, and thecotranslational assembly of functional RCCs requires the

Figure 2.mtEF4 expression levels determine the amount of respiratory chain complexes and cellular energy level. A, mtEF4 KD altered the mitochondrial morphology.Cells were transfected with pDsRed2-Mito and mtEF4-targeting shRNA expression plasmids (sh-mtEF4) or mock vehicle (NC). B, Electron microscopepictures of HeLa NC (scramble RNA) and siRNA-transfected (si) cells. Arrows, mitochondria. C, Mitochondrial length and width, mitochondrial number pergiven area, and abundance of cristae. AU, arbitrary unit. Mean � SD; n ¼ 7; � , P < 0.05, �� , P < 0.01; Student t test. D–G, Quantitative and qualitative analysisof eachRCC fromHeLa-NC and -si cells.D,BNGof isolatedmitochondria treatedwithDDM.E,BNG-WBdetectedby specific antibodies against complex I, II, III, IV, andV. TOM20, internal control. F,BNGand IGA of complex I, II, IV, andV.G,Quantification of BNG-WBand IGA. Mean� SD; n¼ 3; � , P <0.05, �� , P <0.001; Student t test.H and I, Quantitative and qualitative analyses of each RCC from HeLa-NC and -OE cells. Mean � SD; n ¼ 3; � , P < 0.05, �� , P < 0.01, �� , P < 0.001; Student t test. J,Relative ATP content in HeLa-si, -sh, and -OE cells. Mean � SD; n ¼ 10; �� , P < 0.01, �� , P < 0.001; Student t test. K, ROS content indicated by mitoSOX stainingin HeLa -sh cells treatedwithmitoTEMPO. NC, negative controls. L andM,MTS-depletedmtEF4 is not localized to themitochondria and its overexpression abolishesthe phenotypes ofHeLa-OE.L, LackofmtEF4�MTS-EGFP localization tomitochondria.mtEF4withoutMTS (mtEF4�MTS)were taggedwith anEGFP at theC-terminus,while the empty vector was used as control (Vector). The morphology of the mitochondria (MitoDsRed) remained unchanged. M, Relative MMP, ATP, andROS levels assayed on the HeLa cells transfected with empty vector (Vector) or mtEF4�MTS overexpression vector (mtEF4�MTS; mean� SD; n¼ 3; Student t test).






coordinated expression of respiratory subunits encoded by bothnDNA and mtDNA. This process strictly depends on cooperationbetween the cytoplasmic and mitochondrial translation systems(35). However, the translation rate of the two systems is very

different. The mitochondrial system mimics its bacterial counter-part (36), which possesses an optimal elongation speed ofapproximately 20 amino acids polymerized per second per ribo-some (37). In contrast, cytoplasmic translation in eukaryotes is

Figure 3.

mtEF4 KD inhibits tumor growth,while its overexpression promotes theprocess. A, Western blot analysis ofmtEF4 protein in multiple lung cancercell lines, with quantified resultsshown on the right. Mean � SD; n ¼ 3;� , P < 0.05, �� , P < 0.01; Student t test.B, Resected tumors from nude miceinjected with H1299-sh1, A549-sh2,and H1299-OE cells. Scale bar in cm. C,Tumor development curves based ontumor mass at different stages. NC,negative control of H1299 or A549cells treated with the empty vector; shand OE, cancer cells treated withmtEF4 shRNA or transfected with themtEF4 expression unit. Mean � SD;n ¼ 12; � , P < 0.05, �� , P < 0.01,�� , P<0.001; Student t test.D,Weightof xenograft tumor tissues at the endstage of the assay. Mean� SD; n¼ 12;� , P < 0.05, �� , P < 0.01, �� , P < 0.001;Student t test. E and F, Western blotanalysis of mtEF4 and RCC subunits inH1299 xenograft tumors, withquantified results shown on the right.Mean � SD; n ¼ 3; �, P < 0.05,�� , P < 0.01, �� , P < 0.001;Student t test.

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slower (38). Considering that EF4 is the only translation decel-erator with high evolutionary conservation, the reason for itspreservation in mt-translation may be the requirement for alower speed mitochondrial ribosome to match its counterpartin the cytoplasm. At basal expression levels, EF4 maintainsmt-translation speed–quality balance for the biogenesis of func-tional RCCs.

Previous studies have shown that in E. coli, EF4 plays a mul-tifaceted role during protein biosynthesis and proteinmaturationprocesses, including peptide bond isomerization and proteinfolding (19, 32, 33). Appropriate levels of EF4 are paramount

for maintaining a fine balance between translational efficiencyand fidelity. Although low-level expression of E. coli EF4 results ina synthesis of abundant proteins, these proteins are prematureand cannot undergo cotranslational folding or furthermaturationto obtain a functional conformation, and thus will be degradedrapidly. Upregulation of EF4 improves translation to producemore high-quality proteins (19). In this study, human EF4expression at various levels yielded similar results as those shownfor E. coli. One difference this investigation revealed was that noabundant synthesis of premature RCC subunits was observed inHeLa-KD cells. This might be due to an immediate degradation of

Figure 4.

Effects of altered mtEF4 levels oncellular redox status and tumorgrowth in H1299 xenograft tissues.A, Strategy for detecting the cellularredox state in situ by real-timeimaging with xenograft tumor tissuesstained by ROS detection dyes.B, Real-time imaging of ROS inH1299-NC and –sh tumor tissues.Representative images in the bright(leftmost) and dark field (from left toright, lying on the right side, on thestomach, and on the left side) of thetreatedmouse.C,Quantification of theaverage fluorescent signal in theregion of interest (ROI). Mean � SD;n ¼ 7; �� , P < 0.01; Student t test.D, Resected tumors from nudemice ofB, with the weight of xenograft tumortissues at the end stage shown on theright in each case. Scale bar in cm.Mean� SD; n¼ 7; �� , P < 0.01; Studentt test. E, Real-time imaging of ROS inH1299-NC and –OE tumor tissues. F,Quantification of the averagefluorescent signal in the ROI.Mean� SD; n¼ 7; � , P <0.05; Student ttest. G, Resected tumors from nudemice of E,with theweight of xenografttumor tissues at the end stage shownon the right in each case. Scale barin cm. Mean� SD; n¼ 7; �� , P < 0.001;Student t test.






Figure 5.

Human EF4 is significantly upregulated in lung, esophageal, bladder, and cervical cancers. A, mtEF4 mRNA detected in all tested human tissues.B, mtEF4 mRNA levels in representative normal and cancerous tissues. C, mtEF4 mRNA levels in the tested cancer cell lines. D, mtEF4 protein levels in thenormal and cancer cell lines. The normal cell lines human fetal lungfibroblast-1 (HFL-1), 16 humanbronchial epithelial cells (HBE), andpulmonary artery smoothmusclecells (PASMC) were analyzed in comparison with lung cancer cell H1299. E, IHC of adjacent normal tissues versus cancerous tissues from cancer patients. F and G,Relative quantification of mtEF4 protein (F) and mRNA (G) level usingWestern blot analysis and qRT-PCR assays, respectively. Mean� SD; � , P < 0.05, �� , P < 0.01,�� , P < 0.001.


Zhu et al.




these subunits when they were not assembled into the RCCcomplex, as was observed in various earlier mitochondrial studies(refs. 2, 39, 40).

Low and high levels of mtEF4 cause different types of speed–quality imbalance, and hence induce the activation of differentbioenergetic pathways in cancer cells. At low levels of mtEF4, mt-translation is increased at the cost of low translational fidelity andshortened cotranslational folding. The resulting low-quality RCCseither undergo degradation or synthesize less ATP andmore ROS,thus rendering the cell prone to apoptosis. When mtEF4 expres-sion is high, RCC production is increased, leading to elevatedcellular contents of both ATP and ROS, the two major energy andsignaling molecules in cells (41).

Upregulation ofmitochondrial DNA replication, transcription,and translationmachineries has been reported to promote tumor-igenesis by providing increased ATP supply to tumor tissues incertain cases (reviewed in refs. 16, 42). These findings suggest thata healthy mitochondrial system may also benefit tumor growth.In addition, upregulation of mt-translational factors may com-pensate for respiratory dysfunction induced by mt-translationdefects (43). Therefore, functional OXPHOS may be the aim ofmtEF4 overexpression in the cancer cells examined in this study. Ithas been well characterized that mitochondrial metabolismreprogramming (44), specifically the inhibition ofmitochondrialbioenergetic capacity by biguanides (45), and the recovery ofmitochondria-dependent apoptosis (46–48) are the most func-tional recipes for cancer treatment. Therefore, the specific down-regulation of mtEF4 expression in the tumor tissues of the cancerpatients could be a promising new therapy for cancer treatment.

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

Authors' ContributionsConception and design: Y. Liu, L. Zhang, B. Lu, T. Wei, Y. QinDevelopment of methodology: Y. Liu, F. Zhang, F. Shangguan, B. Zhang,X.-J. Liang, B. Lu, T. WeiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. Zhu, Y. Liu, F. Zhang, Z. Chen, F. Shangguan,L. Zhang, Q. Chen, D. Xie, L. Lan, X. Xue, X.-J. Liang, B. LuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Liu, F. Zhang, X.F. Bai, F. Shangguan, Q. Chen,D. Xie, L. Lan, X.-J. Liang, B. Lu, T. Wei, Y. QinWriting, review, and/or revision of the manuscript: Y. Liu, X.F. Bai, L. Zhang,B. Lu, T. Wei, Y. QinAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Liu, F. Shangguan, L. Zhang, X.-J. Liang, B. LuStudy supervision: Y. Liu, X.-J. Liang, B. Lu, Y. Qin

AcknowledgmentsThis work was supported by Strategic Priority Research Programs (Category

A) of the Chinese Academy of Sciences XDA12010313, Key Research ProgramofFrontier Sciences, CAS (grant no. QYZDB-SSW-SMC028, National Key R&DProgram of China 2017YFA0205501 and National Natural Science Foundationof China 81602783, 31671175, 31070710, and 31570772, andNatural ScienceFoundation of Zhejiang Province LY17C070005. We thank Profs. F. Yang(IBP, CAS), K.H. Nierhaus (MPI), and J. Xu (TMU) for discussions, X.F. Guo,X.D. Zhao, J. Hao, Y.Dong, S.G. Li, Y. Teng, Y. Feng, Y.X. Jia, L. Sun, J.F.Hao,G.Z.Shi, X. Shi, L. Zhou, S. Yang, M.F. Wang from IBP, CAS, Y.B. Tian (IGDB), X.Y.Sheng (CNU), Y. Tang, andD.Q. Zhang fromNJMU,W. Liu (TUST) andC. Zhao(TMU) for technical assistance. Y. Qin and P. Zhu hold a patent (China patentno. 201210566942.7, Filed: Dec.24, 2012) on the technology described in thispublication.

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 July 14, 2017; revised January 1, 2018; accepted March 20, 2018;published first March 23, 2018.

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2018;78:2813-2824. Published OnlineFirst March 23, 2018.Cancer Res Ping Zhu, Yongzhang Liu, Fenglin Zhang, et al. Acting as a Mitochondrial Translation SwitchHuman Elongation Factor 4 Regulates Cancer Bioenergetics by

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