CHCHD2 Is Co-Amplified with EGFR in NSCLC and Regulates Mitochondrial Function and Cell Migration

Author Manuscript Published OnlineFirst on March 17, 2015; DOI: 10.1158/1541-7786.MCR-14-0165-T Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

CHCHD2 is Co-amplified with EGFR in NSCLC and Regulates Mitochondrial Function and Cell Migration

Yuhong Wei,1 Ravi N. Vellanki, 2,3 Étienne Coyaud,3 Vladimir Ignatchenko,3 Lei Li,1,3 Jonathan R. Krieger,1 Paul Taylor,1 Jiefei Tong,1 Nhu-An Pham,2,3 Geoffrey Liu,3,4 Brian Raught,3,6 Bradly G. Wouters,2,3,6,7 Thomas Kislinger,3,6 Ming Sound Tsao,2,3,8 and Michael F. Moran1,3,5 *

1Program in Molecular Structure and Function, Hospital For Sick Children, Toronto, Canada; 2Campbell Family Institute for Cancer Research, 3Princess Margaret Cancer Centre, 4Department of Medical Oncology, University Health Network; Departments of 5Molecular Genetics, 6Medical Biophysics, 7Radiation Oncology, and 8Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada

Running Title: Functional roles of CHCHD2 in NSCLC

Keywords: CHCHD2, non-small cell lung carcinoma, label-free quantitative proteomics, interactome, mitochondrial respiration, migration/proliferation

Abbreviations: CHCHD2, coiled-coil helix coiled coil helix domain containing 2; NSCLC, non-small cell lung carcinoma; LC-MS/MS, liquid chromatography-tandem mass spectrometry; SRM, selected reaction monitoring; AP-MS, affinity purification coupled to mass spectrometry; BioID, biotin identification; GO, Gene Ontology; OCR, oxygen consumption rate; ECAR, extracellular acidification rate; FCCP: carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone

*Corresponding author: M.F. Moran Hospital For Sick Children 686 Bay Street, Toronto, ON, M5G 0A4 Tel. 001 647 235-6435; E-mail: [email protected]

Grant Support: This work was supported through funding provided by the Canada Research Chairs Program (TK and MFM), the Ontario Research Fund (MST), and the Canadian Institutes of Health Research (MFM).

Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 17, 2015; DOI: 10.1158/1541-7786.MCR-14-0165-T Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

There are no potential conflicts of interest.

Word count: 5443 (5189 not including title pages) Total Tables/Figures: 7 (6 figures + 1 table) Supplementary Files: 4 supplementary figures + 5 supplementary tables

ABSTRACT

Coiled-coil helix coiled-coil helix domain-containing 2, a mitochondrial protein, encoded

by CHCHD2 is located at chromosome 7p11.2 and proximal to the EGFR gene. Here,

bioinformatic analyses revealed that CHCHD2 is consistently co-amplified with EGFR in

non-small cell lung carcinoma (NSCLC). In addition, CHCHD2 and EGFR protein

expression levels were positively correlated and up-regulated relative to normal lung in

NSCLC tumor-derived xenografts. Knockdown of CHCHD2 expression in NSCLC cells

attenuated cell proliferation, migration, and mitochondrial respiration. CHCHD2 protein-

protein interactions were assessed by the complementary approaches of affinity

purification mass spectrometry and in vivo proximity ligation. The CHCHD2 interactome

includes the apparent hub proteins C1QBP (a mitochondrial protein) and YBX1 (an

oncogenic transcription factor), and an overlapping set of hub-associated proteins

implicated in cell regulation.

Implications: CHCHD2 influences mitochondrial and nuclear functions and contributes

to the cancer phenotype associated with 7p11.2 amplification in NSCLC.

INTRODUCTION

Lung cancer is the most common cause of cancer mortality in the world (1). Eighty five

percent of lung cancers are non-small cell lung carcinoma (NSCLC), a heterogeneous

group composed of two main subtypes, squamous cell carcinoma (SCC) and

adenocarcinoma (ADC) (2). NSCLC are characterized by somatic genetic alterations that

recur according to histology, and which include oncogenic driver mutations that in some

instances are associated with genomic amplification or deletion (3-6). For example, in

NSCLC recurrent amplification is observed in the 7p11 region encoding the drug target

EGFR (3, 6, 7). Indeed, EGFR gene copy number is a significant molecular predictor of a

differential survival benefit from treatment of NSCLC with the EGFR inhibitor erlotinib

(8). Despite advances in the identification and pharmacological modulation of cancer

drivers such as the EGFR, and improved responses to combination therapies, NSCLC

outcomes remain poor. Contributing to this, tumor heterogeneity and genetic complexity

are significant factors that confound NSCLC tumor classification and treatment (2-4, 9).

Regions of DNA amplification can be up to 1 Mb in size, and therefore often

encompass neighboring genes in addition to the suspected driver gene (10-12). It has

been suggested that genes co-amplified within a given amplicon could be functionally

relevant (12) and that co-amplified genes may cooperate to promote tumor progression

(2, 10). Indeed, in a series of NSCLC-derived cell lines several genes mapping proximal

to EGFR including CHCHD2 have been reported as co-amplified and with elevated

mRNA expression (13). Similarly, a recent integrated omic analysis of NSCLC indicated

consistent upregulation of proteins genetically linked to EGFR within the recurrent

7p11.2 amplicon, including the chaperonin subunit CCT6A, and CHCHD2 (14).

However, the genes frequently co-amplified with EGFR including CHCHD2 and their

protein products have not been systematically investigated for roles in NSCLC.

According to The Human Protein Atlas, the CHCHD2 gene product is a widely

expressed 16.7-kDa mitochondrion-localized protein that is upregulated in lung cancer

(15). CHCHD2 consists of an N-terminal mitochondrion localization sequence and a

strongly conserved C-terminal CHCH domain, which is characterized by twin CX9C

motifs in a CX9CXnCX9C configuration. Twin CX9C proteins such as CHCHD2 have

been proposed to function as scaffolding proteins in the mitochondrion, and have been

identified as part of respiratory chain complexes, cytochrome c oxidase (COX) assembly

factors, or involved in the maintenance of mitochondrion structure and function (16-18).

Depletion of CHCH proteins decreases cellular oxygen consumption and ATP production

(16, 18, 19). Recently, CHCHD2 was predicted through computational expression

screening, and then experimentally validated, as a regulator of oxidative phosphorylation

(oxphos) (20). It was further demonstrated that CHCHD2 could bind to the promoter of

COX subunit 4 isoform 2 (COX4I2) and interact with two other transcription factors

(RBPJ, CXXCC5) in regulating COX4I2 gene expression under the influence of oxygen

concentration (21). The CHCHD2 cDNA was found to promote cell migration and alter

cell adhesion when ectopically overexpressed in NIH3T3 fibroblasts (22). Dysregulated

metabolism and altered migration/invasion are pivotal factors for tumor initiation and

progression (23). Therefore, the EGFR-linked CHCHD2 gene product is implicated as a

factor contributing to the cancer phenotype in NSCLC.

In this study the role of CHCHD2 in NSCLC was addressed by analysis of

primary NSCLC-derived xenografts, and tumor-derived cell lines. An integrative

approach including measurement and modulation of CHCHD2 protein expression,

affinity purification-mass spectrometry (AP-MS), in vivo proximity ligation, and

computational methods were used to gain insight into the interactions and function of

CHCHD2. The results suggest that CHCHD2 functions in the regulation of NSCLC cell

growth, migration and mitochondrial function via complex protein-protein interaction

networks.

MATERIALS and METHODS

Cells, Constructs and Reagents

The established NSCLC cell line HCC827 and the xenograft derived lung primary cell

line LPC43 (Table S1) were maintained in RPMI 1640 supplemented with 10% fetal

bovine serum, 50 U/ml penicillin and 50μg/ml streptomycin (Invitrogen). HEK293 cells

were grown in DMEM with 10%FBS and penicillin/streptomycin. All cells were cultured

o at 37 C in a 5% CO2 -humidified incubator.

The plasmid containing human CHCHD2 full-length cDNA was obtained from

the SPARC BioCentre (Hospital for Sick Children, Toronto). To generate a C-terminal

Flag-tagged construct, CHCHD2 cDNA was amplified by PCR using specific primers

with a linker encoding the Flag tag (Table S1). The PCR product was subcloned into a

pcDNA3.1-Myc/His expression vector (Invitrogen). All expression vectors were

sequenced to confirm their authenticity.

Antibodies used were rabbit polyclonal anti-CHCHD2 (HPA027407, Sigma-

Aldrich), mouse monoclonal anti-FLAG M2 (Sigma-Aldrich), rabbit polyclonal anti-

EGFR (SC-03, Santa Cruz), rabbit monoclonal anti-C1QBP (6502S, Cell Signaling) and

rabbit monoclonal anti-YB1 (9744, Cell Signaling). All other reagents were purchased

from Sigma-Aldrich unless otherwise specified.

Generation of CHCHD2 Knockdown and Rescue Stable Cell Lines

Stable knockdown at the protein level was achieved by using shRNAi: pGIPZ-GFP-

human CHCHD2-shRNAmir (Clone ID: V2LHS_97752) and pGIPZ-GFP-scrambled

shRNAmir lentiviral vectors were sourced from Open Biosystems. Lentiviral particles,

prepared by SPARC BioCentre, were used to infect target cells (HCC827, LPC43)

following standard protocols. The stably transduced CHCHD2 knockdown (KD) and

non-silencing control (NSC) cell lines were selected by growth in medium containing

2μg/mL puromycin. The rescue/over-expression cell lines (RES/OE) were established by

ectopic expression of an shRNA-resistant human CHCHD2 cDNA in the KD lines:

CHCHD2 full-length cDNA, resistant to pGIPZ-CHCHD2 shRNAmir by introduction of

a silent mutation, was subcloned into pLX303 (from Dr. Sergio Grinstein, Hospital for

Sick Children), and used to prepare virus particles. Transduced, blasticidin-resistant

CHCHD2-KD cells (derived from HCC827 and LPC43) were selected and verified for

CHCHD2 expression.

Affinity Purification and Western Blotting

For the isolation of immunoprecipitates (IPs) containing CHCHD2 protein

complexes, HEK293 cells were transiently transfected with Flag-tagged CHCHD2. Cells

were lysed in NP-40 buffer (50

mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 1% (v/v) Nonidet P-40, 100 mM NaF, 1

mM sodium orthovanadate, and protease inhibitors) (24), clarified and subject to

immunoaffinity purification by using immobilized anti-FLAG M2 agarose beads (Sigma-

Aldrich). For co-immunoprecipitation (co-IP), immune complexes were eluted from

beads by using 2X Laemmli sample buffer, followed by western blotting. Western

blotting of total cell protein extracts (15 µg protein/sample) or IP eluates involved SDS-

PAGE followed by electrophoretic transfer to nitrocellulose membranes (Amersham

Bioscience), blocking, and probing with primary and secondary antibodies as previously

described (24). For IP followed by LC-MS/MS, isolated immune complexes were

dissociated by using 0.15% trifluoroacetic acid (TFA), neutralized, and then immediately

processed for downstream proteomic analysis (38).

Immunofluorescence staining and confocal microscopy

LPC43 and Hela cells were grown on coverslips for 24 h. For transient CHCHD2-flag

expression, Hela cells were transfected by using Lipofectamine reagent (Life

Technologies), and then incubated for 24 h. For CHCHD2 imaging, cells were grown on

coverslips submerged in a 24-well plate until sub-confluent. The mitochondria of live

cells were stained with MitoTracker Red CMXRos (red color) for 30 min (37 °C, 5%

CO2). After paraformaldehyde fixation, cells were permeabilized with 0.2% Triton X-

100, incubated with rabbit antibody to CHCHD2 (Sigma-Aldrich. HPA027407) and then

detected with Alexa Fluor® 647-conjugated secondary antibodies (far-red). Confocal

microscopy was performed by using a Zeiss LSM 510 META laser scanning microscope

equipped with a 64X objective. Green fluorescence was an indication of lentivirus

infection. Counterstaining of DNA with 4’,6-diamidino-2-phenylindole (DAPI) was used

to localize cell nuclei (blue). Additional details on experimental protocols were published

previously (24, 25).

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Analysis

MS sample preparation was performed essentially as described previously (26). Tryptic

peptides were concentrated and purified on homemade C18 columns or C18 StageTips

(Thermo Fisher Scientific) before LC-MS/MS. For MS analysis, peptides were separated

by reverse-phase chromatography using a nanoflow UPLC system (Thermo Fisher

Scientific) with a 240-min linear gradient. The UPLC system was coupled to an Orbitrap

Elite MS instrument (Thermo Fisher Scientific), and peptides were fragmented by

collision-induced dissociation (CID).

MS Data Processing and Analysis

Raw MS files acquired from the Orbitrap-Elite were processed by MaxQuant software

(version 1.3.0.5) according to the standard workflow (27). MS/MS spectra were searched

against the UniProt human proteome (release 2013_03_06) containing 87,656 entries

(including common contaminants) by using the Andromeda search engine (28). For

statistical evaluation of the data, a false discovery rate of 0.01 was set for peptide and

protein identification.

MS data related to NSCLC primary tumors was accessed from ProteomeXchange

(http://www.proteomexchange.org) with the data set identifier PXD000853, and analyzed

by using MaxQuant as described in Li et al. (14). Protein LFQ (label free quantification)

intensity obtained from MaxQuant was chosen as the quantitative value representing

protein abundance, and used for calculation of protein differential expression. For

quantitative proteomics, protein LFQ intensities were exported and statistical analysis

was performed by using the R software package. For quantitative mass spectrometric

analysis of anti-Flag IPs, protein LFQ intensities across different samples were first

normalized according to the intensities of the bait protein CHCHD2 in each sample.

Normalized LFQ intensities were then used for determination of specific protein-protein

interactions by using Perseus software 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 BioID method (29) was

employed. By this approach, proteins in the vicinity of the FlagBirAR118G –CHCHD2

fusion protein were covalently modified by biotin in vivo, followed by MS identification

of biotinylated proteins isolated by streptavidin-based affinity capture in vitro. In brief,

full-length CHCHD2 coding sequence was subcloned into the pcDNA5/FRT/TO-

FlagBirA* expression vector. HEK293T-REx cells stably expressing FlagBirA*-

CHCHD2 or CHCHD2-BirA*Flag were generated by using the Flp-In system

(Invitrogen). Cells were incubated for 24 h in complete medium supplemented with 1

μg/mL tetracycline (Sigma-Aldrich) and 50 μM biotin (Bioshop), and then lysed in RIPA

buffer(50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton x-100, 1% Sodium

deoxycholate, 0.1% SDS, 1 mM EDTA, Protease Inhibitors). Clarified cell lysates were

subject to streptavidin affinity purification followed by trypsin digestion and MS

analysis, essentially as described previously (30). Detailed information is included in

Supplementary Methods.

CHCHD2 Quantification by Selected Reaction Monitoring (SRM)-MS

SRM was performed as described (26) with slight modifications. SRM peak intensities

were calculated by using Skyline software (31), and signal intensity of each peptide was

the average of two technical replicates. Total intensities from three CHCHD2 peptides

(Table S2) were summed and multiplied by a correction factor, which was determined by

normalization of the sum of MYH9 and ACTG total peak intensities across the different

samples.

Cell Proliferation and Migration Assays

Real-time cell proliferation and migration activities were monitored by using the

xCELLigence system (AECA Biosciences) according to manufacturer’s instructions. For

growth curves, cells were seeded in triplicate into 96-well E-plates, and the cell index,

reflecting electrical impedance was measured every 15 min throughout the experiment.

For migration, cells were serum-starved for 16 h, trypsinized, resuspended in serum-free

media and then seeded in triplicate on the top chamber of CIM-16 plates. Serum-

containing medium was added to the lower chamber as a migratory stimulant. Cells were

allowed to settle for 30 min before measurements were taken. The cell index was

collected at 5 min intervals. Proliferation and migration rates were determined from the

slope of cell index curve.

Oxygen Consumption and Extracellular Acidification Measurement

To measure mitochondria function in intact cells, a Seahorse Bioscience XF96

Extracellular Flux Analyzer was used. Briefly, cells were seeded in XF96-well

microplates (25,000/well) with RPMI-1640 complete medium, and incubated for 16 h.

The pH of medium was adjusted to 7.4, and final concentrations of glucose and glutamine

were 11 mM and 2 mM, respectively. Cells were subsequently washed and restored with

bicarbonate-free medium and incubated in a CO2-free incubator for 2 h. Oxygen

consumption rate and extracellular acidification rate were measured under basal

conditions, in the presence of the ATP-synthase inhibitor Oligomycin (1 μM) and the

mitochondrion uncoupling agent, FCCP (500 nM). Data were normalized according to

the number of seeded cells.

Statistical Analysis and Bioinformatics

The significance of difference between groups was determined by the Student’s t test

using R software or by one-way ANOVA followed by Bonferroni post hoc test using

GraphPad Prism 3.0 (GraphPad Software Inc.), unless stated otherwise.

Gene ontology enrichment analysis was performed by using DAVID

bioinformatics resources (32). A p value less than 0.05 was considered statistically

significant. Protein interaction network analysis was based on literature investigations

and the Biological General Repository for Interaction Datasets (BioGRID) (33).

Cytoscape version 3.0 (34) was used for visualization of protein interaction networks.

RESULTS

Correlated expression of EGFR and CHCHD2 in NSCLC

As EGFR overexpression in NSCLC is often a consequence of gene amplification (35),

we examined whether the EGFR-proximal gene CHCHD2 is co-amplified. Data on

somatic copy number variation (SCNV) in lung adenocarcinoma (LADC) and lung

squamous cell carcinoma (LSCC) were retrieved from The Cancer Genome Atlas

(TCGA) data portal (www.cancergenome.nih.gov) and through cBioProtal (36). This

indicated that CHCHD2 is indeed frequently co-amplified along with EGFR in both

subtypes, and with a strong tendency towards co-occurrence (Odds Ratio >>10) (Fig.

1A). As shown in Fig. 1A, it is apparent that EGFR as expected, but not CHCHD2, is in

some instances mutated in addition to being amplified. We recently reported that EGFR

and CHCHD2 protein levels are upregulated in primary NSCLC (14). Fig. 1B shows that

the levels of EGFR and CHCHD2 are consistently elevated relative to normal lung in a

set of eleven NSCLC patient-derived xenograft (PDX) tumors (listed in Table S1), and

show a strong correlation in their expression levels, with a coefficient of determination

(Pearson R2) >0.6 (Fig. 1C). Ectopic over-expression of EGFR in HEK293 cells, an

established model system for EGFR regulation (e.g. 37), did not up-regulate endogenous

CHCHD2 protein expression concomitantly (Fig. 1D). This suggests that the correlated

expression of the two gene products that occurs at the protein level, at least in NSCLC

contexts, extends beyond their genetic linkage to the protein level, but, as shown by the

HEK293 experiment (Fig. 1D) does not appear to reflect increased synthesis or stability

of CHCHD2 as a product of elevated EGFR expression. Western blot analysis of four

NSCLC cell lines, including LPC43 and LPC72, which were generated from NSCLC

PDX sample A6 (see Table S1), and the established cell lines RVH6849 and HCC827,

showed that EGFR and CHCHD2 were co-expressed in vitro (Fig. S1).

Modulation of CHCHD2 expression alters NSCLC cell proliferation and migration

The co-amplification and elevated protein expression of EGFR and CHCHD2 in NSCLC

led us to consider whether CHCHD2 expression contributes to key aspects of the cancer

phenotype including cell proliferation and migration. The NSCLC cell lines HCC827 and

PDX A6-derived LPC43 were used to generate cell lines stably expressing (i) non-

silencing control shRNA (NSC), (ii) shRNA directed against human CHCHD2 (KD,

knockdown), and (iii) CHCHD2 knockdown cells “rescued” with ectopic over-expression

of an shRNA-resistant human CHCHD2 cDNA (RES/OE). CHCHD2 protein in LPC43-

derived cells, was mainly localized in mitochondria, as determined by co-localization

with MitoTracker, whereas CHCHD2 staining was minimal on KD cells (Fig. 2).

Consistent with the cell imaging results, quantification of CHCHD2 by SRM-MS

analysis of 3 different CHCHD2 peptide ions confirmed that protein expression was

greatly reduced in KD and increased in RES/OE lines relative to NSC cells (Fig. 3A). In

HCC827 KD cells, the level of CHCHD2 was 7% of the level measured in NSC, and in

RES/OE the amount of CHCHD2 was approximately 2.4-fold greater than NSC. In

LPC43 the amount of CHCHD2 in KD was 12% of that seen in NSC, and RES/OE

expressed 6.7-fold more than RES (Fig. 3A).

The impact of CHCHD2 depletion and overexpression on NSCLC cell

proliferation and migration were considered. Cell proliferation kinetics were measured in

real-time by monitoring the electrical impedance of the substrate surface, which is

affected by the area of cell contact, and hence cell number (Fig. 3B). In order to measure

cell migration, a trans-well migration assay based on cell surface impedance was used

(Fig. 3C). In HCC827, there was not a significant change in cell proliferation when

CHCHD2 was knocked down, but there was a significant increase in the over-expressing

RES/OE cells compared with KD. In LPC43, CHCHD2 knockdown significantly reduced

the rate of proliferation, but proliferation remained impaired in the RES/OE cells. In

HCC827 and LPC43 migration was significantly decreased in CHCHD2 KD cells. This

effect was not recued by in HCC827 RES/OE, but was in LPC43. Therefore, in both cell

types HCC827 and LPC43, measures of proliferation and migration associated with

CHCHD2 expression showed similar trends towards decreases in KD compared with

NSC and/or RES/OE.

Mitochondrial respiration as a function of CHCHD2 in NSCLC

Metabolic capacity in NSC, KD, and RES/OE cells was assessed by using an

extracellular flux analyzer. The parameters measured were basal oxygen consumption

rate (OCR), an index of oxphos, along with FCCP-stimulated maximal oxygen

consumption, and mitochondrial reserve capacity. In both cell types (HCC827 and

LPC43) basal and maximal OCR and mitochondrial reserve capacity were significantly

decreased in KD cells compared with NSC controls (Fig 4A and 4B). In both of the

RES/OE cell types these parameters showed a general trend intermediate between control

NSC and KD values, but were not significantly different from either. This suggests that in

terms of mitochondrial function, over-expression of CHCHD2 in RES/OE cells did not

achieve a true “rescue” effect. Analysis of additional RES clones with CHCHD2

expression closer to endogenous levels might address this possibility. Basal extracellular

acidification rate (ECAR), a marker for glycolysis was not significantly changed as a

function of CHCHD2 in the two cell lines (Fig. S2).

The effect of CHCHD2 expression on NSCLC proteomes

To elucidate the molecular changes that contribute to the phenotypes observed, MS-based

quantitative proteome analyses were performed to identify proteins differentially

expressed as a function of CHCHD2 expression in the LPC43- and HCC827-based, NSC,

KD, and RES/OE cell lines. Three independent analyses of each cell line was completed

and in aggregate a total of 4308 non-redundant proteins were identified and quantified.

An overlap of 70-75% (at the protein level) was observed between the independent

replicate experiments (Fig. S3). After pooling the respective NSC, KD, and RES/OE

datasets derived from LPC43 and HCC827, a majority of proteins (3249, 75%) was

shared by the NSC, KD and RES/OE groups (Fig. 5A). Statistical analysis (paired t-test)

identified 205 proteins as significantly differentially expressed (p <0.05) between KD and

NSC cells (Table S3). Gene ontology (GO) analysis revealed that the differentially

expressed proteins are implicated in diverse biological processes (Fig. 5B), and

distributed across various cellular compartments, primarily mitochondrion, nuclear

lumen, ribonucleoprotein complex, and cytoskeleton (Fig. S3).

In order to more stringently identify candidates whose expression levels changed

upon CHCHD2 depletion, the list of differentially expressed proteins was filtered to

include only proteins showing ≥2-fold expression difference between NSC and KD, but

not between NSC and RES/OE. This produced a smaller set of 13 proteins, excluding

CHCHD2, regarded as CHCHD2 regulated proteins (Table 1). Ten proteins showed

parallel expression changes with CHCHD2 expression: down-regulation in CHCHD2 KD

cells, and recovered expression in CHCHD2 RES/OE cells (Table 1). This set includes

two protein kinases: The tyrosine-specific anaplastic lymphoma kinase (ALK), and the

MAP2K4-encoded dual specificity enzyme known as mitogen-activated protein kinase

kinase 4 (MKK4). Substrates activated by MKK4 include the MAP kinases JNK1 and -2

and the stress-activated MAP kinase p38 (38). By contrast, the expression levels 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 Interactome

As a putative scaffolding protein and transcription factor, CHCHD2 is expected to

function through protein-protein interactions. To test this, CHCHD2-associated proteins

were characterized by affinity purification mass spectrometry (AM-MS). C-terminal

Flag-tagged CHCHD2 constructs were ectopically expressed in transiently transfected

cells, which were verified by immunoblot and immunostaining analysis (Fig. S4). Anti-

Flag immunoprecipitates (IPs) were prepared from cells expressing epitope-tagged

CHCHD2 and control cells not expressing ectopic CHCHD2. The criterion for a specific

CHCHD2-interacting protein was differential recovery relative to the negative control

(fold enrichment ≥ 32, and p ≤ 0.01; Fig. 6A, and Fig. S4). By this criterion 58 proteins

were identified as binding directly or indirectly to CHCHD2 (Table S4).

To further validate the CHCHD2 interacting proteins, a recently developed

proximity-dependent in vivo biotinylation method termed BioID (29) was applied in

combination with MS to identify proteins that interact or come into close proximity with

CHCHD2 within cells. Following the BioID methodology, CHCHD2 was expressed in

transfected cells as a fusion protein including the promiscuous biotin protein ligase

domain BirA* fused to the carboxyl end of CHCHD2. A total of 65 proteins that became

biotinylated in vivo due to their proximity with CHCHD2-BirA* passed stringent criteria

applied by using SAINT software (39) (Fig. 6A and Table S5). Seven proteins were

identified by both the AP-MS and proximity ligation methods, suggesting they are

central, or at least high-confidence CHCHD2-associated proteins (Fig. 6B). Among the

remaining CHCHD2-interacting proteins that were identified by only one of the methods

(i.e. AP-MS or BioID), interrogation of BioGRID (33) verified eight as having

interactions with one or more of the seven high-confidence CHCHD2-associated

proteins. These proteins were organized and visualized by using Cytoscape tools (Fig.

6B) (34). This revealed two highly connected protein sub-clusters that were centered on

proteins C1QBP and YBX1, suggesting that they may function as hubs within a

CHCHD2 network. The protein CAND1 was common to both the C1QBP and YBX1

complexes. It is reportedly a transcription factor, and also an F-box protein exchange

factor, influencing the association of diverse F-box proteins in SCF ubiquitin 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

were identified by both AP-MS and proximity ligation including the uncharacterized

protein coiled-coil domain containing 71-like (CCDC71L); cytidine monophosphate N-

acetylneuraminic acid synthetase (CMAS), the inner nuclear membrane protein LBR; the

putative RNA helicase DHXS7; and the nuclear chaperone THOC4 (encoded by

ALYREF). Two previously reported CHCHD2-interacting proteins RPL24 and

polyubiquitin-C (UBC) (41) also were detected by AP-MS, but did not pass the selection

criteria. Further support for the interaction of CHCHD2 with YBX1 and C1QBP was

obtained by demonstration of associations between ectopic CHCHD2 and endogenous

YBX1 and C1QBP by western blot analysis of CHCHD2-Flag IPs recovered from

transfected HEK293 cells (Fig. 6C).

DISCUSSION

Recurrent cancer-associated amplifications and deletions are logically thought to be

driven by changes in expression of encoded oncogenes and tumor suppressors,

respectively. Integrated analysis of NSCLC proteomes and somatic gene copy number

variation suggests that genomes are organized such that co-amplified genes are

functionally linked to the cancer phenotype (14). Co-amplification of EGFR and

CHCHD2 has been reported in NSCLC (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 EGFR and CHCHD2 are indeed co-amplified in

both the ADC and SCC subtypes of NSCLC. Consistent with this trend, we found that

CHCHD2 and EGFR protein expression levels are elevated relative to normal lung and

are positively correlated with each other in primary NSCLC xenografts. However, in

HEK293 cells CHCHD2 protein expression was not increased as a product of elevated

ectopic EGFR expression. This negates a model, at least in the HEK293 system, wherein

CHCHD2 protein expression is itself elevated as a product of EGFR protein expression.

While we do not yet understand the mechanisms regulating EGFR or CHCHD2 protein

levels, the observations that both proteins appear consistently upregulated in NSCLC

(Fig. 1B, and ref. 14) provided a rationale for experiments to address whether CHCHD2

contributes to the transformed cell phenotype.

Cancer metabolism is known to affect cell migration (44). CHCHD2 was

implicated as a regulator of oxphos (20), and ectopic CHCHD2 promoted NIH3T3 cell

migration (22). Consistent with these observations, we found that cell migration and

proliferation rates in the NSCLC cell lines HCC827 and LPC43 were stimulated by

CHCHD2 expression. Furthermore, knockdown of CHCHD2 significantly attenuated

mitochondrial respiratory activity, typified by reduced basal and maximum OCR, and

diminished mitochondrial reserve capacity. In breast cancer cells loss of reserve capacity

is linked to apoptosis (45), and maintenance of reserve capacity positively correlates with

rapid proliferation (46). Therefore, we speculate that the impaired NSCLC cell

proliferation associated with CHCHD2 knockdown was at least in part due to the

measured reduction in mitochondrial reserve capacity. Consistent with this, our

comprehensive analysis of NSCLC cell proteomes revealed proteins that changed in

abundance as a function of CHCHD2 knockdown and rescue, and including significant

enrichment for biological processes involved in cell proliferation and migration (i.e. GO

categories cell proliferation, cell cycle, and cell projection morphogenesis). For example,

Agrin protein expression increased 2-fold upon CHCHD2 knockdown and this effect was

reversed by CHCHD2 rescue. Over-expression of Agrin increased substrate adhesion in

COS7 cells (47). We therefore speculate that elevated Agrin may have stimulated cell

adhesion, and consequently hindered cell migration, in response to CHCHD2

knockdown. These results are consistent with observations of increased cell motility

associated with EGFR amplification (48), and suggest that co-amplification of CHCHD2

may contribute to this cancer-associated phenomenon.

The receptor tyrosine kinase ALK (anaplastic leukemia kinase) is activated as a

consequence of somatic chromosomal rearrangements in a subset (3%-7%) of ADC-

subtype NSCLC (49). Knockdown of CHCHD2 was accompanied by reduced expression

of ALK, however, to the best of our knowledge wild type ALK, which is known to

function in brain development and neural function, has not been implicated as an

oncogenic driver. Interestingly, recent observations of concomitant EGFR mutation and

ALK gene rearrangement has suggested that coordinated activation of the signaling

networks of EGFR and ALK may be important in lung ADC (50). While not tested in this

study, our results suggest that co-amplification of CHCHD2 and EGFR may be linked to

ALK.

In addition to Agrin, the expression of other proteins was affected by CHCHD2

protein levels (Table 1). WDR20 displayed increased expression upon CHCHD2

depletion and dropped back towards control levels after CHCHD2 was re-expressed.

WDR20 interacts with and stimulates the activity of ubiquitin-specific protease USP12

and also interacts with USP46 (51). USP12 regulates Notch signaling (52) and USP46 is

a tumor suppressor (53). However, this study did not address mechanisms through which

CHCHD2 might affect protein production, modifications, or stability. The proteins that

changed in abundance in cells depleted of CHCHD2 relate to several different cellular

functions and subcellular localizations other than mitochondrion.

The CHCHD2 interactome was defined by the complementary approaches of

AM-MS and proximity ligation. This analysis revealed two highly connected sub-

networks centered on putative hub proteins C1QBP and YBX1. The mitochondrial

protein C1QBP is upregulated in a variety of neoplasms including lung cancer (43, 54),

and has pro-migration activity in cancer cell lines (43, 54). Moreover, C1QBP has been

shown to promote cancer cell proliferation and resistance to cell death (54). YBX1 is an

oncogenic transcription factor with roles in cell proliferation, invasion, and metastasis as

well as energy metabolism (55). It was suggested that nuclear translocation of YBX1

could induce up-regulation of EGFR expression and a more aggressive NSCLC

phenotype (56). A recent report further revealed YBX1 as a convergent hub for

lysophosphatidic acid and EGF signaling, and activation 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” within the CHCHD2

interactome, we propose that CHCHD2 functions at least in part through interactions with

C1QBP and YBX1. Our non-quantitative indirect immunofluorescence microscopic

analysis most obviously depicted a mitochondrial localization of CHCHD2, which may

reflect its major subcellular localization under the conditions of our analysis, but may

also mean that non-mitochondrial CHCHD2 was below our level of detection. The

changes in protein levels in response to CHCHD2 knockdown (Table 1), could be

downstream effects stemming from the loss of CHCHD2 function in mitochondria,

and/or a consequence of changes in gene expression related to the noted transcription

factor activity of CHCHD2 (21), and possibly involving its interactions with YBX1. In

conclusion, our analyses indicate that CHCHD2 gene copy number and protein levels are

linked with EGFR in NSCLC. CHCHD2 participates in mitochondrial and extra-

mitochondrial protein-protein interactions and is an effector of cell proliferation,

migration, and respiration. Therefore, along with EGFR, it should be considered as a

driver of 7p11.2 amplification and the cancer phenotype.

ACKNOWLEDGEMENTS

We thank Drs. Sergio Grinstein and Robert Rottapel for DNA constructs. We gratefully

acknowledge The Cancer Genome Atlas Research Network and their research groups and

specimen donors for allowing access to datasets.

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Table 1. Proteins differentially expressed between CHCHD2 knockdown (KD) and non-silenced control cells (NSC)

Protein p Gene Name Identifier Protein Description Log2[KD:NSC]

CHCHD2 Q9Y6H1 Coiled-coil-helix-coiled-coil-helix domain- 0.000 -4.652 containing protein 2 0.043 -2.422 HIP1R O75146 Huntingtin-interacting protein 1-related 0.006 -1.436 UFL1 O94874 E3 UFM1-protein ligase 1 0.036 -1.326 CTPS2 Q9NRF8 CTP synthase 2 0.012 -1.274 GSTT1 P30711 Glutathione S-transferase theta-1 0.012 -1.216 MFAP1 P55081 Microfibrillar-associated protein 1

ACOT1/2 P49753 Acyl-coenzyme A thioesterase (1-cytoplasm, 2- 0.030 -1.124 mitochondrial)

MAP2K4 P45985 Dual specificity mitogen-activated protein 0.023 -1.123 kinase kinase 4 0.048 -1.094 QTRT1 Q9BXR0 Queuine tRNA-ribosyltransferase 0.015 -1.061 AATF Q9NY61 Protein AATF 0.014 -1.024 ALK Q9UM73 ALK receptor tyrosine kinase 0.001 1.016 AGRN O00468 Agrin 0.047 1.529 WDR20 E7EUY8 WD repeat-containing protein 20

CLPP Q16740 Putative ATP-dependent Clp protease 0.027 1.610 proteolytic subunit, mitochondrial

FIGURE LEGENDS

Figure 1. CHCHD2 and EGFR protein expression in non-small cell lung carcinoma

(NSCLC)

(A) Amplification of EGFR and CHCHD2 genes in NSCLC subtypes. Shown are

OncoPrint outputs (cBioPortal for Cancer Genomics; www.cbioportal.org) where each

bar represents a tumor that was found to contain a DNA alteration (amplification,

deletion, mutation, as indicated) in EGFR and CHCHD2 in the indicated NSCLC

subtypes; note that aligned bars represent the same tumor. Data for lung

adenocarcinoma (LADC) and lung squamous cell carcinoma (LSCC) are from The

Cancer Genome Atlas resource (TCGA), obtained through cBioPortal. For LADC 40 of

230 cases (17%) contained a DNA alteration in the EGFR gene, while the incidence for

CHCHD2 was 7%. In 13/15 cases the genes were co-amplified; Odds Ratio =101.8,

indicating a strong tendency towards co-occurrence of 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 =infinite, indicating a strong tendency towards co-occurrence of

EGFR and CHCHD2 amplification. EGFR as expected, but not CHCHD2, is in some

instances mutated in addition to being amplified, as indicated 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 were

highly correlated as indicated by correlation of determination analysis (R2), as indicated.

After removing sample S3 which was the only sample with a cytogenetically defined

amplification of EGFR/7p11.2, the two proteins remained moderately correlated in their

expression level (R2 =0.35). (D) Western blot analysis of CHCHD2 and EGFR

expression in HEK293 cells (control), or this cell type stably expressing ectopic EGFR

(EGFR). Transferrin receptor (TfR) was detected as an input control.

Figure 2. Expression and localization of CHCHD2 in NSCLC cells

Fluorescence confocal microscopy was used to determine the localization of endogenous

and ectopic CHCHD2 in LPC43 cells. CHCHD2 knockdown (KD), rescue/overexpressed

(RES/OE) and control (NSC) cells were grown on coverslips until sub-confluent, and

then live cells were stained for mitochondria by using MitoTracker (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 lentiviral vector, was used as a marker of transduced cells (upper left

panel). DAPI staining was used to stain nuclei in some samples. Pink indicates co-

localization of mitochondria and CHCHD2 in NSC and RES/OE cells (right-middle, and

right-lower panels), which was largely absent in KD cells (lower-left panel). Scale bars

are 15 μm.

Figure 3. SRM-MS quantification of CHCHD2.

(A) CHCHD2 protein was measured in HCC827 and LPC43 NSC, KD, and RES/OE

cells by using SRM-MS. The effect of CHCHD2 protein expression on cell proliferation

(B) and cell migration was measured (C). Rates, expressed in arbitrary units (a.u.) per

hour, were calculated from the slopes of cell index curves. Data are mean SD for 3

independent peptides (panel A), or ±SEM from n ≥3 independent experiments (panels B

and C). * p <0.05

Figure 4. Dependence of mitochondrial respiration on CHCHD2 protein expression.

Control (NSC), knockdown (KD) and rescue/over-expression (RESOE) cells were seeded

in specialized microplates and cultured for 16 h. Cells were then switched to bicarbonate-

free medium and mitochondrial function was assessed using sequential injection of

oligomycin and FCCP. Shown are representative OCR curves and summarized

quantification of basal OCR, maximal OCR, and reserve capacity of (A) HCC827 and

(B) LPC43. Results are mean ± SEM from 3 independent experiments.

** p <0.01; * p <0.05.

Figure 5. Proteome changes associated with CHCHD2 expression in NSCLC cells.

(A) Venn diagram depicting the number of non-redundant proteins identified in

CHCHD2 knockdown (KD), rescue/over-expression (RES/OE) and control (NSC)

groups. Label-free quantitative proteome analysis was performed on six NSCLC cell

lines (NSC, KD, and RES/OE derivatives of HCC827 and LPC43). (B) Shown are

enriched GO biological process terms from proteins differentially expressed between

CHCHD2 knockdown and control cells.

Figure 6. Analysis of CHCHD2 interactome by MS approach. (A) Schematic

depiction of the experimental plan for the identification of CHCHD2 associated proteins.

In one arm, CHCHD2 associated proteins were identified by affinity purification (AP)-

MS analysis of proteins recovered by anti-Flag immunoprecipitation of ectopically

expressed CHCHD2-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 fusion

protein 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

interacting proteins. Lysates and anti-Flag IPs from transfected HEK293 cells expressing

ectopic CHCHD2-Flag and control cells not expressing CHCHD2-Flag were analyzed by

SDS-PAGE and immuno-blotting. Expression of YBX1 and C1QBP was confirmed by

analysis of lysates (lanes 1 and 2). Both proteins were recovered by co-IP with

CHCHD2-Flag (lane 4), but not detected in anti-Flag IPs from control cells (lane 3).

CHCHD2 is Co-amplified with EGFR in NSCLC and Regulates Mitochondrial Function and Cell Migration

Yuhong Wei, Ravi N. Vellanki, Etienne Coyaud, et al.

Mol Cancer Res Published OnlineFirst March 17, 2015.

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