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Genomics

CHCHD2 Is Coamplified with EGFR in NSCLC andRegulates Mitochondrial Function and CellMigrationYuhong Wei1, Ravi N. Vellanki2,3, �Etienne Coyaud3, Vladimir Ignatchenko3, Lei Li1,3,Jonathan R. Krieger1, Paul Taylor1, Jiefei Tong1, Nhu-An Pham2,3, Geoffrey Liu3,4,Brian Raught3,5, Bradly G.Wouters2,3,5,6, Thomas Kislinger3,5, Ming Sound Tsao2,3,7, andMichael F. Moran1,3,8

Abstract

Coiled-coil-helix-coiled-coil-helix domain-containing 2, amitochondrial protein, encoded by CHCHD2 is located atchromosome 7p11.2 and proximal to the EGFR gene. Here,bioinformatic analyses revealed that CHCHD2 is consistentlycoamplified with EGFR in non–small cell lung carcinoma(NSCLC). In addition, CHCHD2 and EGFR protein expressionlevels were positively correlated and upregulated relative tonormal lung in NSCLC tumor-derived xenografts. Knockdownof CHCHD2 expression in NSCLC cells attenuated cell prolif-eration, migration, and mitochondrial respiration. CHCHD2protein–protein interactions were assessed by the complemen-

tary approaches of affinity purification mass spectrometry andin vivo proximity ligation. The CHCHD2 interactome includesthe apparent hub proteins C1QBP (a mitochondrial protein)and YBX1 (an oncogenic transcription factor), and an over-lapping set of hub-associated proteins implicated in cellregulation.

Implications: CHCHD2 influences mitochondrial and nu-clear functions and contributes to the cancer phenotype asso-ciated with 7p11.2 amplification in NSCLC. Mol Cancer Res;13(7); 1119–29. �2015 AACR.

IntroductionLung cancer is the most common cause of cancer-related

mortality in the world (1). Eighty-five percent of lung cancersare non–small cell lung carcinoma (NSCLC), a heterogeneousgroup composed of twomain subtypes, squamous cell carcinoma(SCC) and adenocarcinoma (ADC; ref. 2). NSCLC are character-ized by somatic genetic alterations that recur according to histol-ogy, and which include oncogenic driver mutations that insome instances are associated with genomic amplification ordeletion (3–6). For example, in NSCLC, recurrent amplificationis observed in the 7p11 region encoding the drug target EGFR(3, 6, 7). Indeed, EGFR gene copy number is a significant molec-ular predictor of a differential survival benefit from treatment of

NSCLC with the EGFR inhibitor erlotinib (8). Despite advancesin the identification and pharmacologic modulation of cancerdrivers such as the EGFR, and improved responses to combi-nation therapies, NSCLC outcomes remain poor. Contributingto this, tumor heterogeneity and genetic complexity are signif-icant factors that confound NSCLC tumor classification andtreatment (2–4, 9).

Regions of DNA amplification can be up to 1 Mb in size, andtherefore often encompass neighboring genes in addition to thesuspected driver gene (10–12). It has been suggested that genescoamplified within a given amplicon could be functionally rel-evant (12) and that coamplified genes may cooperate to promotetumor progression (2, 10). Indeed, in a series of NSCLC-derivedcell lines, several genes mapping proximal to EGFR, includingCHCHD2, have been reported as coamplified and with elevatedmRNA expression (13). Similarly, a recent integrated omic anal-ysis of NSCLC indicated consistent upregulation of proteinsgenetically linked to EGFR within the recurrent 7p11.2 amplicon,including the chaperonin subunit CCT6A, and CHCHD2 (14).However, the genes frequently coamplified with EGFR, includingCHCHD2, and their protein products have not been systemati-cally investigated for roles in NSCLC.

According to The Human Protein Atlas, the CHCHD2 geneproduct is a widely expressed 16.7-kDa mitochondrion-localizedprotein that is upregulated in lung cancer (15). CHCHD2 consistsof an N-terminal mitochondrion localization sequence anda strongly conserved C-terminal CHCH domain, which is char-acterized by twin CX9C motifs in a CX9CXnCX9C configuration.Twin CX9C proteins, such as CHCHD2, have been proposed tofunction as scaffolding proteins in the mitochondrion, andhave been identified as part of respiratory chain complexes,

1Program in Molecular Structure and Function, Hospital For Sick Chil-dren,Toronto, Ontario, Canada. 2Campbell Family Institute for CancerResearch, Toronto, Ontario, Canada. 3Princess Margaret Cancer Cen-tre, Toronto, Ontario, Canada. 4Department of Medical Oncology,University Health Network,Toronto, Ontario, Canada. 5Department ofMedical Biophysics, University of Toronto, Toronto, Ontario, Canada.6Department of Radiation Oncology, University of Toronto, Toronto,Ontario, Canada. 7Department of Laboratory Medicine and Pathobi-ology,UniversityofToronto,Toronto,Ontario,Canada. 8DepartmentofMolecular Genetics, University of Toronto, Toronto, Ontario, Canada.

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

Corresponding Author: Michael F. Moran, Hospital For Sick Children, 686 BayStreet, Toronto, ON, M5G 0A4, Canada. Phone: 647-235-6435; Fax: 416-813-5993; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-14-0165-T

�2015 American Association for Cancer Research.

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cytochrome c oxidase (COX) assembly factors, or involved in themaintenance of mitochondrion structure and function (16–18).Depletion of CHCH proteins decreases cellular oxygen consump-tion and ATP production (16, 18, 19). Recently, CHCHD2 waspredicted through computational expression screening, and thenexperimentally validated, as a regulator of oxidative phosphory-lation (oxphos; ref. 20). It was further demonstrated thatCHCHD2 could bind to the promoter of COX subunit 4 isoform2 (COX4I2) and interact with two other transcription factors(RBPJ, CXXCC5) in regulating COX4I2 gene expression underthe influence of oxygen concentration (21). The CHCHD2 cDNAwas found to promote cellmigration and alter cell adhesionwhenectopically overexpressed in NIH3T3 fibroblasts (22). Dysregu-lated metabolism and altered migration/invasion are pivotalfactors for tumor initiation and progression (23). Therefore, theEGFR-linked CHCHD2 gene product is implicated as a factorcontributing to the cancer phenotype in NSCLC.

In this study, the role of CHCHD2 in NSCLC was addressed byanalysis of primary NSCLC-derived xenografts, and tumor-derived cell lines. An integrative approach including measure-ment and modulation of CHCHD2 protein expression, affinitypurification-mass spectrometry (AP-MS), in vivo proximity liga-tion, and computational methods was used to gain insight intothe interactions and functionofCHCHD2. The results suggest thatCHCHD2 functions in the regulation of NSCLC cell growth,migration, and mitochondrial function via complex protein–protein interaction networks.

Materials and MethodsCells, constructs, and reagents

The established NSCLC cell line HCC827 and the xenograftderived lung primary cell line LPC43 (Supplementary Table S1)were maintained in RPMI-1640 supplemented with 10% fetalbovine serum, 50 U/mL penicillin, and 50 mg/mL streptomycin(Invitrogen). HEK293 cells were grown in DMEM with 10% FBSand penicillin/streptomycin. All cells were cultured at 37�C in a5% CO2-humidified incubator.

The plasmid containing human CHCHD2 full-length cDNAwas obtained from the SPARC BioCentre (Hospital for SickChildren, Toronto, ON, Canada). To generate a C-terminalFlag-tagged construct, CHCHD2 cDNA was amplified by PCRusing specific primers with a linker encoding the Flag tag (Sup-plementary Table S1). The PCR product was subcloned into apcDNA3.1-Myc/His expression vector (Invitrogen). All expressionvectors were sequenced to confirm their authenticity.

Antibodies used were rabbit polyclonal anti-CHCHD2(HPA027407; Sigma-Aldrich), mouse monoclonal anti-FLAGM2 (Sigma-Aldrich), rabbit polyclonal anti-EGFR (SC-03; SantaCruz Biotechnology), rabbit monoclonal anti-C1QBP (6502S;Cell Signaling Technology) and rabbit monoclonal anti-YB1(9744; Cell Signaling Technology). All other reagents were pur-chased from Sigma-Aldrich unless otherwise specified.

Generationof CHCHD2knockdown and rescue stable cell linesStable knockdown at the protein level was achieved by using

shRNAi: pGIPZ-GFP-human CHCHD2-shRNAmir (Clone ID:V2LHS_97752) and pGIPZ-GFP-scrambled shRNAmir lentiviralvectors were sourced from Open Biosystems. Lentiviral particles,prepared by SPARC BioCentre, were used to infect target cells(HCC827, LPC43) following standard protocols. The stably trans-

duced CHCHD2 knockdown (KD) and nonsilencing control(NSC) cell lines were selected by growth in medium containing2 mg/mL puromycin. The rescue/overexpression cell lines (RES/OE) were established by ectopic expression of an shRNA-resistanthuman CHCHD2 cDNA in the knockdown lines: CHCHD2 full-length cDNA, resistant to pGIPZ-CHCHD2 shRNAmir by intro-duction of a silent mutation, was subcloned into pLX303(from Dr. Sergio Grinstein, Hospital for Sick Children), and usedto prepare virus particles. Transduced, blasticidin-resistantCHCHD2-KD cells (derived from HCC827 and LPC43) wereselected and verified for CHCHD2 expression.

Affinity purification and Western blottingFor the isolation of immunoprecipitates (IPs) containing

CHCHD2 protein complexes, HEK293 cells were transientlytransfected with Flag-tagged CHCHD2. Cells were lysed in NP-40 buffer [50 mmol/L Tris–HCl, pH 7.5, 150 mmol/L sodiumchloride, 1% (v/v) Nonidet P-40, 100 mmol/L NaF, 1 mmol/Lsodium orthovanadate, and protease inhibitors; ref. 24], clar-ified, and subject to immunoaffinity purification by usingimmobilized anti-FLAG M2 agarose beads (Sigma-Aldrich).For coimmunoprecipitation (co-IP), immune complexes wereeluted from beads by using 2X Laemmli sample buffer, fol-lowed by Western blotting. Western blotting of total cell pro-tein extracts (15 mg protein/sample) or IP eluates involvedSDS–PAGE followed by electrophoretic transfer to nitrocellu-lose membranes (Amersham Bioscience), blocking, and prob-ing with primary and secondary antibodies as previouslydescribed (24). For IP followed by liquid chromatography-tandem mass spectrometry (LC/MS-MS), isolated immunecomplexes were dissociated by using 0.15% trifluoroacetic acid(TFA), neutralized, and then immediately processed for down-stream proteomic analysis (25).

Immunofluorescence staining and confocal microscopyLPC43 and Hela cells were grown on coverslips for 24 hours.

For transient CHCHD2-flag expression, Hela cells were trans-fected by using Lipofectamine reagent (Life Technologies), andthen incubated for 24 hours. For CHCHD2 imaging, cells weregrown on coverslips submerged in a 24-well plate until subcon-fluent. The mitochondria of live cells were stained with Mito-Tracker Red CMXRos (red color) for 30minutes (37�C, 5%CO2).After paraformaldehyde fixation, cells were permeabilized with0.2% Triton X-100, incubated with rabbit antibody to CHCHD2(Sigma-Aldrich; HPA027407) and then detected with Alexa Fluor647–conjugated secondary antibodies (far-red). Confocalmicros-copy was performed by using a Zeiss LSM 510 META laserscanning microscope equipped with a �64 objective. Greenfluorescence was an indication of lentivirus infection. Counter-staining of DNAwith 40,6-diamidino-2-phenylindole (DAPI) wasused to localize cell nuclei (blue). Additional details on experi-mental protocols were published previously (24, 26).

Liquid chromatography-tandem mass spectrometry analysisMS sample preparation was performed essentially as described

previously (27). Tryptic peptides were concentrated and purifiedon homemade C18 columns or C18 StageTips (Thermo FisherScientific) before LC/MS-MS. For MS analysis, peptides wereseparated by reverse-phase chromatography using a nanoflowUPLC system (Thermo Fisher Scientific) with a 240-minute lineargradient. The UPLC system was coupled to an Orbitrap Elite MS

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instrument (Thermo Fisher Scientific), and peptides were frag-mented by collision-induced dissociation (CID).

MS data processing and analysisRawMS files acquired from theOrbitrap-Elite were processed by

MaxQuant software (version 1.3.0.5) according to the standardworkflow (28). MS/MS spectra were searched against the UniProthuman proteome (release 2013_03_06) containing 87,656 entries(including common contaminants) by using the Andromedasearch engine (29). For statistical evaluation of the data, a falsediscovery rate of 0.01was set for peptide and protein identification.

MS data related to NSCLC primary tumors were accessed fromProteomeXchange (http://www.proteomexchange.org) with thedataset identifier PXD000853, and analyzed by using MaxQuantas described in Li and colleagues (14). Protein LFQ (label-freequantification) intensity obtained fromMaxQuant was chosen asthe quantitative value representing protein abundance, and usedfor calculation of protein differential expression. For quantitativeproteomics, protein LFQ intensities were exported and statisticalanalysis was performed by using the R software package. Forquantitative mass spectrometric analysis of anti-Flag IPs, proteinLFQ intensities across different samples were first normalizedaccording to the intensities of the bait protein CHCHD2 in eachsample. Normalized LFQ intensities were then used for determi-nation of specific protein–protein interactions by using Perseussoftware tools available in the MaxQuant environment.

Identification of CHCHD2 interactions by proximity-dependent biotinylation (BioID) and MS

To identify proximate and interacting proteins in living cells,the BioIDmethod (30)was used. By this approach, proteins in thevicinity of the FlagBirAR118G–CHCHD2 fusion protein were cova-lently modified by biotin in vivo, followed byMS identification ofbiotinylated proteins isolated by streptavidin-based affinity cap-ture in vitro. In brief, full-length CHCHD2 coding sequence wassubcloned into the pcDNA5/FRT/TO-FlagBirA� expression vector.HEK293T-REx cells stably expressing FlagBirA�-CHCHD2 orCHCHD2-BirA�Flag were generated by using the Flp-In system(Invitrogen). Cells were incubated for 24 hours in completemedium supplemented with 1 mg/mL tetracycline (Sigma-Aldrich) and 50 mmol/L biotin (Bioshop), and then lysed in RIPAbuffer(50mmol/L Tris–HClpH7.4, 150mmol/LNaCl, 1%TritonX-100, 1% sodium deoxycholate, 0.1% SDS, 1 mmol/L EDTA,protease inhibitors). Clarified cell lysates were subject to strepta-vidin affinity purification followed by trypsin digestion and MSanalysis, essentially as described previously (31). Detailed infor-mation is included in Supplementary Methods.

CHCHD2 quantification by selected reaction monitoring-MSSelected reaction monitoring (SRM) was performed as

described (27) with slight modifications. SRM peak intensitieswere calculated by using Skyline software (32), and signal inten-sity of each peptide was the average of two technical replicates.Total intensities from three CHCHD2 peptides (SupplementaryTable S2) were summed and multiplied by a correction factor,whichwas determined by normalization of the sumofMYH9 andACTG total peak intensities across the different samples.

Cell proliferation and migration assaysReal-time cell proliferation and migration activities were mon-

itored by using the xCELLigence system (AECA Biosciences)

according to the manufacturer's instructions. For growth curves,cells were seeded in triplicate into 96-well E-plates, and the cellindex, reflecting electrical impedance was measured every 15minutes throughout the experiment. For migration, cells wereserum-starved for 16 hours, trypsinized, resuspended in serum-free media and then seeded in triplicate on the top chamber ofCIM-16plates. Serum-containingmediumwas added to the lowerchamber as a migratory stimulant. Cells were allowed to settle for30 minutes before measurements were taken. The cell index wascollected at 5-minute intervals. Proliferation and migration rateswere determined from the slope of cell index curve.

Oxygen consumption and extracellular acidificationmeasurement

To measure mitochondria function in intact cells, a SeahorseBioscience XF96 Extracellular Flux Analyzer was used. Briefly, cellswere seeded in XF96-well microplates (25,000/well) with RPMI-1640 complete medium, and incubated for 16 hours. The pH ofmedium was adjusted to 7.4, and final concentrations of glucoseand glutamine were 11mmol/L and 2mmol/L, respectively. Cellswere subsequently washed and restored with bicarbonate-freemedium and incubated in a CO2-free incubator for 2 hours.Oxygen consumption rate and extracellular acidification rate weremeasured under basal conditions, in the presence of the ATP-synthase inhibitor oligomycin (1 mmol/L) and the mitochondri-on uncoupling agent, FCCP (500 nmol/L). Data were normalizedaccording to the number of seeded cells.

Statistical analysis and bioinformaticsThe significance of difference between groups was determined

by the Student t test using R software or by one-way ANOVAfollowed by Bonferroni post hoc test using GraphPad Prism 3.0(GraphPad Software Inc.), unless stated otherwise.

Gene ontology (GO) enrichment analysis was performed byusing DAVID bioinformatics resources (33). A P value less than0.05 was considered statistically significant. Protein interactionnetwork analysis was based on literature investigations and theBiological General Repository for Interaction Datasets (BioGRID;ref. 34). Cytoscape version 3.0 (35) was used for visualization ofprotein interaction networks.

ResultsCorrelated expression of EGFR and CHCHD2 in NSCLC

As EGFR overexpression in NSCLC is often a consequence ofgene amplification (36), we examined whether the EGFR-proxi-mal gene CHCHD2 is coamplified. Data on somatic copy numbervariation (SCNV) in lung adenocarcinoma (LADC) and lung SCC(LSCC) were retrieved from The Cancer Genome Atlas (TCGA)data portal (www.cancergenome.nih.gov) and through cBioPro-tal (37). This indicated that CHCHD2 is indeed frequently coam-plified along with EGFR in both subtypes, and with a strongtendency toward cooccurrence (odds ratio >>10; Fig. 1A). Asshown in Fig. 1A, it is apparent that EGFR as expected, but notCHCHD2, is in some instances mutated in addition to beingamplified. We recently reported that EGFR and CHCHD2 proteinlevels are upregulated in primary NSCLC (14). Figure 1B showsthat the levels of EGFR and CHCHD2 are consistently elevatedrelative to normal lung in a set of 11 NSCLC patient-derivedxenograft (PDX) tumors (listed in Supplementary Table S1), andshow a strong correlation in their expression levels, with a

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coefficient of determination (Pearson R2) > 0.6 (Fig. 1C). Ectopicoverexpression of EGFR in HEK293 cells, an established modelsystem for EGFR regulation (e.g., 38), did not upregulate endog-enous CHCHD2 protein expression concomitantly (Fig. 1D). Thissuggests that the correlated expression of the two gene productsthat occurs at the protein level, at least in NSCLC contexts, extendsbeyond their genetic linkage to the protein level, but, as shown bythe HEK293 experiment (Fig. 1D), does not appear to reflectincreased synthesis or stability of CHCHD2 as a product ofelevated EGFR expression. Western blot analysis of four NSCLCcell lines, including LPC43 andLPC72,whichwere generated fromNSCLC PDX sample A6 (see Supplementary Table S1), and theestablished cell lines RVH6849 and HCC827, showed that EGFRand CHCHD2 were coexpressed in vitro (Supplementary Fig. S1).

Modulation of CHCHD2 expression alters NSCLC cellproliferation and migration

The coamplification and elevated protein expression of EGFRand CHCHD2 in NSCLC led us to consider whether CHCHD2expression contributes to key aspects of the cancer phenotype,including cell proliferation and migration. The NSCLC cell linesHCC827 and PDX A6-derived LPC43 were used to generate celllines stably expressing (i)NSC shRNA, (ii) shRNAdirected againsthuman CHCHD2 (knockdown), and (iii) CHCHD2 knockdowncells "rescued" with ectopic overexpression of an shRNA-resistanthuman CHCHD2 cDNA (RES/OE). CHCHD2 protein in LPC43-derived cells, was mainly localized in mitochondria, as deter-mined by colocalization with MitoTracker, whereas CHCHD2staining was minimal on knockdown cells (Fig. 2). Consistentwith the cell imaging results, quantification of CHCHD2 by SRM-MS analysis of three different CHCHD2 peptide ions confirmedthat protein expression was greatly reduced in knockdown andincreased in RES/OE lines relative to NSC cells (Fig. 3A). InHCC827 knockdown cells, the level of CHCHD2 was 7% of thelevel measured in NSC, and in RES/OE the amount of CHCHD2was approximately 2.4-fold greater than NSC. In LPC43 theamount of CHCHD2 in knockdown was 12% of that seen inNSC, and RES/OE expressed 6.7-fold more than RES (Fig. 3A).

The impact of CHCHD2 depletion and overexpression onNSCLC cell proliferation and migration were considered. Cellproliferation kinetics were measured in real-time by monitoringthe electrical impedance of the substrate surface, which is affectedby the area of cell contact, and hence cell number (Fig. 3B). In orderto measure cell migration, a Transwell migration assay based oncell-surface impedance was used (Fig. 3C). In HCC827, there wasnot a significant change in cell proliferation when CHCHD2 wasknocked down, but there was a significant increase in the over-expressing RES/OE cells compared with knockdown. In LPC43,CHCHD2 knockdown significantly reduced the rate of prolifera-tion, but proliferation remained impaired in the RES/OE cells. InHCC827 and LPC43, migration was significantly decreased inCHCHD2 knockdown cells. This effect was not recued by inHCC827 RES/OE, but was in LPC43. Therefore, in both cell typesHCC827 and LPC43, measures of proliferation and migrationassociatedwithCHCHD2expression showedsimilar trends towarddecreases in knockdown compared with NSC and/or RES/OE.

Mitochondrial respiration as a function of CHCHD2 in NSCLCMetabolic capacity in NSC, knockdown, and RES/OE cells was

assessed by using an extracellular flux analyzer. The parametersmeasured were basal oxygen consumption rate (OCR), an index

Figure 1.CHCHD2 and EGFR protein expression in NSCLC. A, amplificationof EGFR and CHCHD2 genes in NSCLC subtypes. Shown are OncoPrintoutputs (cBioPortal for Cancer Genomics; www.cbioportal.org) whereeach bar represents a tumor that was found to contain a DNAalteration (amplification, deletion, mutation, as indicated) in EGFR andCHCHD2 in the indicated NSCLC subtypes; note that aligned barsrepresent the same tumor. Data for LADC and LSCC are from TCGAresource, obtained through cBioPortal. For LADC, 40 of 230 cases (17%)contained a DNA alteration in the EGFR gene, while the incidence forCHCHD2 was 7%. In 13 of 15 cases, the genes were coamplified; oddsratio ¼ 101.8, indicating a strong tendency toward cooccurrenceof EGFR and CHCHD2 amplification. For LSCC 16 of 178 cases (9%)contained EGFR amplification or mutation, and an equal number of cases(13) contained amplification of both EGFR and CHCHD2. Odds ratio ¼infinite, indicating a strong tendency toward cooccurrence ofEGFR and CHCHD2 amplification. EGFR as expected, but not CHCHD2, isin some instances mutated in addition to being amplified, asindicated by the green features. B, LC/MS-MS analysis of 11 human tumor-derived NSCLC xenografts (as indicated; A, adenocarcinoma; S,squamous cell carcinoma). C, EGFR and CHCHD2 expression werehighly correlated as indicated by correlation of determination analysis(R2), as indicated. After removing sample S3 that was the only samplewith a cytogenetically defined amplification of EGFR/7p11.2, the twoproteins remained moderately correlated in their expression level(R2 ¼ 0.35). D, Western blot analysis of CHCHD2 and EGFR expressionin HEK293 cells (control), or this cell type stably expressing ectopicEGFR (EGFR). Transferrin receptor (TfR) was detected as an inputcontrol.

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of oxphos, along with FCCP-stimulated maximal oxygen con-sumption, and mitochondrial reserve capacity. In both cell types(HCC827 and LPC43) basal and maximal OCR and mitochon-drial reserve capacity were significantly decreased in knockdowncells compared with NSC controls (Fig. 4A and B). In both of theRES/OE cell types, these parameters showed a general trendintermediate between control NSC and knockdown values, butwere not significantly different from either. This suggests that interms of mitochondrial function, overexpression of CHCHD2 inRES/OE cells did not achieve a true "rescue" effect. Analysis ofadditional RES clones with CHCHD2 expression closer to endog-enous levels might address this possibility. Basal extracellularacidification rate (ECAR), a marker for glycolysis was not signif-icantly changed as a function of CHCHD2 in the two cell lines(Supplementary Fig. S2).

The effect of CHCHD2 expression on NSCLC proteomesTo elucidate the molecular changes that contribute to the

phenotypes observed, MS-based quantitative proteome analyseswere performed to identify proteins differentially expressed as afunction of CHCHD2 expression in the LPC43- and HCC827-based, NSC, knockdown, and RES/OE cell lines. Three indepen-dent analyses of each cell line were completed and in aggregate atotal of 4,308 nonredundant proteins were identified and quan-tified. An overlap of 70% to 75% (at the protein level) wasobserved between the independent replicate experiments (Sup-plementary Fig. S3). After pooling the respective NSC, knock-down, and RES/OE datasets derived from LPC43 and HCC827, amajority of proteins (3,249; 75%) was shared by the NSC,knockdown, and RES/OE groups (Fig. 5A). Statistical analysis(paired t test) identified 205 proteins as significantly differentiallyexpressed (P < 0.05) between knockdown and NSC cells (Sup-plementary Table S3). GO analysis revealed that the differentiallyexpressed proteins are implicated in diverse biologic processes(Fig. 5B), and distributed across various cellular compartments,primarily mitochondrion, nuclear lumen, ribonucleoproteincomplex, and cytoskeleton (Supplementary Fig. S3).

In order to more stringently identify candidates whose expres-sion levels changed upon CHCHD2 depletion, the list of differ-entially expressed proteins was filtered to include only proteinsshowing �2-fold expression difference between NSC and knock-down, but not betweenNSCandRES/OE. This produced a smallerset of 13 proteins, excluding CHCHD2, regarded as CHCHD2regulated proteins (Table 1). Ten proteins showed parallel expres-sion changes with CHCHD2 expression: downregulation inCHCHD2 knockdown cells, and recovered expression inCHCHD2 RES/OE cells (Table 1). This set includes two proteinkinases: the tyrosine-specific anaplastic lymphoma kinase (ALK),and the MAP2K4-encoded dual specificity enzyme known asmitogen-activated protein kinase kinase 4 (MKK4). Substratesactivated by MKK4 include the MAP kinases JNK1 and -2 and thestress-activated MAP kinase p38 (25). In contrast, the expressionlevels of three proteins, including ATP-dependent Clp protease(CLPP), WD repeat-containing protein 20 (WDR20) and Agrin(AGRN), were increased upon CHCHD2 knockdown (Table 1).

Proteomic analysis of the CHCHD2 protein interactomeAs a putative scaffolding protein and transcription factor,

CHCHD2 is expected to function through protein–protein inter-actions. To test this, CHCHD2-associated proteins were charac-terized by affinity purification mass spectrometry (AM-MS). C-terminal Flag-tagged CHCHD2 constructs were ectopicallyexpressed in transiently transfected cells, which were verified byimmunoblot and immunostaining analysis (Supplementary Fig.S4). Anti-Flag IPs were prepared from cells expressing epitope-tagged CHCHD2 and control cells not expressing ectopicCHCHD2. The criterion for a specific CHCHD2-interacting pro-tein was differential recovery relative to the negative control (foldenrichment � 32, and P � 0.01; Fig. 6A; Supplementary Fig. S4).By this criterion, 58 proteins were identified as binding directly orindirectly to CHCHD2 (Supplementary Table S4).

To further validate the CHCHD2-interacting proteins, a recentlydeveloped proximity-dependent in vivo biotinylation methodtermedBioID (30)was applied in combinationwithMS to identifyproteins that interact or come into close proximity with CHCHD2within cells. Following the BioID methodology, CHCHD2 wasexpressed in transfected cells as a fusion protein including the

Figure 2.Expression and localization of CHCHD2 in NSCLC cells. Fluorescence confocalmicroscopy was used to determine the localization of endogenous andectopic CHCHD2 in LPC43 cells. CHCHD2 knockdown, rescue/overexpressed(RES/OE) and control (NSC) cells were grown on coverslips until sub-confluent, and then live cells were stained for mitochondria by usingMitoTracker (red). After fixation, permeabilized cells were probed with anti-CHCHD2 and an Alexa Fluor 647 secondary antibody (far-red; imaged cyan).Green fluorescent protein (GFP), encoded by the shRNA pGIPZ lentiviralvector, was used as a marker of transduced cells (top left). DAPI staining wasused to stain nuclei in some samples. Pink indicates colocalization ofmitochondria and CHCHD2 in NSC and RES/OE cells (right-middle, and right-bottom), which was largely absent in knockdown cells (bottom-left). Scalebars are 15 mm.






promiscuous biotin protein ligase domain BirA� fused to thecarboxyl end of CHCHD2. A total of 65 proteins that becamebiotinylated in vivo due to their proximity with CHCHD2-BirA�

passed stringent criteria applied by using SAINT software (Fig. 6Aand Supplementary Table S5; ref. 39). Seven proteins were iden-tified by both the AP-MS and proximity ligationmethods, suggest-ing they are central, or at least high-confidence CHCHD2-associ-ated proteins (Fig. 6B). Among the remaining CHCHD2-interact-ing proteins that were identified by only one of the methods (i.e.,AP-MS or BioID), interrogation of BioGRID (34) verified eight ashaving interactions with one ormore of the seven high-confidenceCHCHD2-associated proteins. These proteins were organized andvisualized by using Cytoscape tools (Fig. 6B; ref. 35). This revealedtwo highly connected protein subclusters that were centered onproteins C1QBP and YBX1, suggesting that they may function ashubs within a CHCHD2 network. The protein CAND1 was com-mon to both the C1QBP and YBX1 complexes. It is reportedly atranscription factor, and also an F-box protein exchange factor,influencing the association of diverse F-box proteins in SCF ubi-quitin ligase complexes (40). In addition to C1QBP and YBX1,

which were previously reported as CHCHD2-interacting proteins(22, 41–43), five other CHCHD2-interacting candidates wereidentified by both AP-MS and proximity ligation including theuncharacterized protein coiled-coil domain containing 71-like(CCDC71L); cytidine monophosphate N-acetylneuraminic acidsynthetase (CMAS), the inner nuclear membrane protein LBR; theputative RNA helicase DHXS7; and the nuclear chaperone THOC4(encoded by ALYREF). Two previously reported CHCHD2-inter-acting proteins RPL24 and polyubiquitin-C (UBC; ref. 41) alsowere detected by AP-MS, but did not pass the selection criteria.Further support for the interaction of CHCHD2 with YBX1 andC1QBP was obtained by demonstration of associations betweenectopic CHCHD2 and endogenous YBX1 and C1QBP by Westernblot analysis of CHCHD2-Flag IPs recovered from transfectedHEK293 cells (Fig. 6C).

DiscussionRecurrent cancer-associated amplifications and deletions are

logically thought tobedrivenby changes in expressionof encoded

Figure 3.SRM-MS quantification of CHCHD2.A, CHCHD2 protein was measured inHCC827 and LPC43 NSC, knockdown,and RES/OE cells by using SRM-MS.The effect of CHCHD2 proteinexpression on cell proliferation (B) andcell migration was measured (C).Rates, expressed in arbitrary units (a.u.) per hour, were calculated from theslopes of cell index curves. Data aremean �SD for three independentpeptides (A), or �SEM from n � 3independent experiments (B and C).� , P < 0.05.

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oncogenes and tumor suppressors, respectively. Integrated anal-ysis of NSCLC proteomes and somatic gene copy number vari-ation suggests that genomes are organized such that coamplifiedgenes are functionally linked to the cancer phenotype (14).Coamplification of EGFR and CHCHD2 has been reported inNSCLC (13) and other cancer types, including glioma (12).However, not all amplified genes in tumors are overexpressed(12). Our analysis of TCGA datasets clearly indicates that EGFRand CHCHD2 are indeed coamplified in both the ADC and SCCsubtypes of NSCLC. Consistent with this trend, we found thatCHCHD2andEGFRprotein expression levels are elevated relativeto normal lung and are positively correlated with each other in

primary NSCLC xenografts. However, in HEK293 cells, CHCHD2protein expression was not increased as a product of elevatedectopic EGFR expression. This negates a model, at least in theHEK293 system, wherein CHCHD2 protein expression is itselfelevated as a product of EGFR protein expression. Althoughwe donot yet understand themechanisms regulating EGFRor CHCHD2protein levels, the observations that both proteins appear con-sistently upregulated in NSCLC (Fig. 1B; ref. 14) provided arationale for experiments to address whether CHCHD2 contri-butes to the transformed cell phenotype.

Cancer metabolism is known to affect cell migration (44).CHCHD2 was implicated as a regulator of oxphos (20), and

Figure 4.Dependence of mitochondrialrespiration on CHCHD2 proteinexpression. Control (NSC),knockdown, and rescue/overexpression (RESOE) cells wereseeded in specialized microplates andcultured for 16 hours. Cells were thenswitched to bicarbonate-free mediumand mitochondrial function wasassessed using sequential injection ofoligomycin and FCCP. Shown arerepresentative OCR curves andsummarized quantification of basalOCR, maximal OCR, and reservecapacity of (A) HCC827 and (B)LPC43. Results are mean � SEM fromthree independent experiments.�� , P < 0.01; � , P < 0.05.






ectopic CHCHD2 promoted NIH3T3 cell migration (22). Con-sistent with these observations, we found that cell migration andproliferation rates in the NSCLC cell lines HCC827 and LPC43were stimulated by CHCHD2 expression. Furthermore, knock-down of CHCHD2 significantly attenuated mitochondrial respi-ratory activity, typified by reduced basal andmaximumOCR, anddiminished mitochondrial reserve capacity. In breast cancer cells,loss of reserve capacity is linked to apoptosis (45), and mainte-nance of reserve capacity positively correlates with rapid prolif-eration (46). Therefore, we speculate that the impaired NSCLCcell proliferation associated with CHCHD2 knockdown was, atleast in part, due to the measured reduction in mitochondrialreserve capacity. Consistent with this, our comprehensive analysisof NSCLC cell proteomes revealed proteins that changed inabundance as a function of CHCHD2 knockdown and rescue,and including significant enrichment for biologic processesinvolved in cell proliferation and migration (i.e., GO categories

cell proliferation, cell cycle, and cell projection morphogene-sis). For example, Agrin protein expression increased 2-foldupon CHCHD2 knockdown and this effect was reversed byCHCHD2 rescue. Overexpression of Agrin increased substrateadhesion in COS7 cells (47). We therefore speculate thatelevated Agrin may have stimulated cell adhesion, and conse-quently hindered cell migration, in response to CHCHD2knockdown. These results are consistent with observations ofincreased cell motility associated with EGFR amplification(48), and suggest that coamplification of CHCHD2 may con-tribute to this cancer-associated phenomenon.

The receptor tyrosine kinase ALK is activated as a conse-quence of somatic chromosomal rearrangements in a subset(3%–7%) of ADC-subtype NSCLC (49). Knockdown ofCHCHD2 was accompanied by reduced expression of ALK;however, to the best of our knowledge, wild-type ALK, whichis known to function in brain development and neural

Figure 5.Proteome changes associatedwith CHCHD2 expressionin NSCLC cells. A, Venn diagram depicting the numberof nonredundant proteins identified in CHCHD2knockdown, rescue/overexpression (RES/OE), andcontrol (NSC) groups. Label-free quantitativeproteome analysis was performed on six NSCLC celllines (NSC, knockdown, and RES/OE derivatives ofHCC827 and LPC43). B, enriched GO biologic processterms from proteins differentially expressed betweenCHCHD2 knockdown and control cells.

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function, has not been implicated as an oncogenic driver.Interestingly, recent observations of concomitant EGFR muta-tion and ALK gene rearrangement has suggested that coordi-nated activation of the signaling networks of EGFR and ALKmay be important in lung ADC (50). Although not tested in this

study, our results suggest that coamplification of CHCHD2 andEGFR may be linked to ALK.

In addition to Agrin, the expression of other proteins wasaffected by CHCHD2 protein levels (Table 1). WDR20 displayedincreased expressionuponCHCHD2depletion anddroppedback

Table 1. Proteins differentially expressed between CHCHD2 knockdown and NSC

Gene name Protein identifier Protein description P Log2[KD:NSC]

CHCHD2 Q9Y6H1 Coiled-coil-helix-coiled-coil-helix domain-containing protein 2 0.000 �4.652HIP1R O75146 Huntingtin-interacting protein 1-related 0.043 �2.422UFL1 O94874 E3 UFM1-protein ligase 1 0.006 �1.436CTPS2 Q9NRF8 CTP synthase 2 0.036 �1.326GSTT1 P30711 Glutathione S-transferase theta-1 0.012 �1.274MFAP1 P55081 Microfibrillar-associated protein 1 0.012 �1.216ACOT1/2 P49753 Acyl-coenzyme A thioesterase (1-cytoplasm, 2-mitochondrial) 0.030 �1.124MAP2K4 P45985 Dual specificity mitogen-activated protein kinase kinase 4 0.023 �1.123QTRT1 Q9BXR0 Queuine tRNA-ribosyltransferase 0.048 �1.094AATF Q9NY61 Protein AATF 0.015 �1.061ALK Q9UM73 ALK receptor tyrosine kinase 0.014 �1.024AGRN O00468 Agrin 0.001 1.016WDR20 E7EUY8 WD repeat-containing protein 20 0.047 1.529CLPP Q16740 Putative ATP-dependent Clp protease proteolytic subunit, mitochondrial 0.027 1.610

Figure 6.Analysis of CHCHD2 interactome by MS approach. A, schematic depiction of the experimental plan for the identification of CHCHD2 associated proteins. In onearm, CHCHD2-associatedproteinswere identifiedbyAP-MS analysis of proteins recoveredby anti-Flag immunoprecipitationof ectopically expressedCHCHD2-Flag.In the other arm, the BioID method was used to characterize by MS proteins that became covalently modified by ubiquitin in cells expressing a fusionprotein of CHCHD2 linked to the promiscuous ubiquitin ligase BirA*. Seven proteins in addition to the CHCHD2 bait proteins were identified by both approaches.B, network analysis of the CHCHD2 interactome. Proteins and their interactions are shown as nodes and edges. Interacting proteins were identified by AP-MS(light purple), BioID (orange), or both methods (pink), as indicated. Solid black lines indicate interactions also present in the BioGRID database search(http://thebiogrid.org). The dashed boxes encompass two potential CHCHD2-associated complexes. C, Western blot validation of CHCHD2 interactingproteins. Lysates and anti-Flag IPs from transfected HEK293 cells expressing ectopic CHCHD2-Flag and control cells not expressing CHCHD2-Flag wereanalyzed by SDS-PAGE and immuno-blotting. Expression of YBX1 and C1QBP was confirmed by analysis of lysates (lanes 1 and 2). Both proteins were recoveredby co-IP with CHCHD2-Flag (lane 4), but not detected in anti-Flag IPs from control cells (lane 3).






toward control levels after CHCHD2 was reexpressed. WDR20interacts with and stimulates the activity of ubiquitin-specificprotease USP12 and also interacts with USP46 (51). USP12regulates Notch signaling (52) and USP46 is a tumor suppressor(53). However, this study did not address mechanisms throughwhich CHCHD2 might affect protein production, modifications,or stability. The proteins that changed in abundance in cellsdepleted of CHCHD2 relate to several different cellular functionsand subcellular localizations other than mitochondrion.

The CHCHD2 interactome was defined by the complementaryapproaches of AM-MS and proximity ligation. This analysisrevealed two highly connected subnetworks centered on putativehub proteins C1QBP and YBX1. The mitochondrial proteinC1QBP is upregulated in a variety of neoplasms, including lungcancer (43, 54), and has promigration activity in cancer cell lines(43, 54). Moreover, C1QBP has been shown to promote cancercell proliferation and resistance to cell death (54). YBX1 is anoncogenic transcription factor with roles in cell proliferation,invasion, and metastasis as well as energy metabolism (55). Itwas suggested that nuclear translocation of YBX1 could induceupregulation of EGFR expression and a more aggressive NSCLCphenotype (56). A recent report further revealed YBX1 as aconvergent hub for lysophosphatidic acid and EGF signaling, andactivation of YBX1 contributed to ovarian cancer cell invasion(57). Considering the multifaceted roles of C1QBP and YBX1,together with our observation that they present as "hubs" withinthe CHCHD2 interactome, we propose that CHCHD2 functions,at least in part, through interactions with C1QBP and YBX1. Ournonquantitative indirect immunofluorescence microscopic anal-ysis most obviously depicted a mitochondrial localization ofCHCHD2, which may reflect its major subcellular localizationunder the conditions of our analysis, butmay alsomean that non-mitochondrial CHCHD2 was below our level of detection. Thechanges in protein levels in response to CHCHD2 knockdown(Table 1), could be downstream effects stemming from the loss ofCHCHD2 function in mitochondria, and/or a consequence ofchanges in gene expression related to the noted transcriptionfactor activity of CHCHD2 (21), and possibly involving its inter-actions with YBX1. In conclusion, our analyses indicate that

CHCHD2 gene copy number and protein levels are linked withEGFR in NSCLC. CHCHD2 participates in mitochondrial andextra-mitochondrial protein–protein interactions and is an effec-tor of cell proliferation, migration, and respiration. Therefore,along with EGFR, it should be considered as a driver of 7p11.2amplification and the cancer phenotype.

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

Authors' ContributionsConception anddesign:Y.Wei, B. Raught, B.G.Wouters, T. Kislinger,M.S. Tsao,M.F. Moran

Development of methodology: Y. Wei, R.N. Vellanki, �E. Coyaud, J. TongAcquisition of data (provided animals, acquired and managed patients,

provided facilities, etc.): Y. Wei, �E. Coyaud, J.R. Krieger, P. Taylor, N.-A. Pham,G. Liu, B. Raught, M.S. TsaoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Wei, R.N. Vellanki, V. Ignatchenko, L. Li,J.R. Krieger, P. Taylor, M.S. Tsao, M.F. MoranWriting, review, and/or revision of the manuscript: Y. Wei, R.N. Vellanki,N.-A. Pham, G. Liu, B.G. Wouters, M.S. Tsao, M.F. MoranAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Wei, J. Tong, N.-A. Pham, G. Liu, M.S. TsaoStudy supervision: M.S. Tsao, M.F. Moran

AcknowledgmentsThe authors thank Drs. Sergio Grinstein and Robert Rottapel for DNA

constructs. We gratefully acknowledge TCGA Research Network and theirresearch groups and specimen donors for allowing access to datasets.

Grant SupportThis work was supported through funding provided by the Canada

Research Chairs Program (to T. Kislinger and M.F. Moran), the OntarioResearch Fund (to M.S. Tsao), and the Canadian Institutes of Health Research(M.F. Moran).

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 May 27, 2014; revised March 6, 2015; accepted March 7, 2015;published OnlineFirst March 17, 2015.

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2015;13:1119-1129. Published OnlineFirst March 17, 2015.Mol Cancer Res Yuhong Wei, Ravi N. Vellanki, Étienne Coyaud, et al. Mitochondrial Function and Cell MigrationCHCHD2 Is Coamplified with EGFR in NSCLC and Regulates

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