Targeting Adenine Nucleotide Translocase-2 (ANT2) to ... · ANT2 was inhibited by transfecting cells with an ANT2-speciﬁc shRNA. ANT2 expression was elevated in the H1975 and HCC827/GR

Cancer Biology and Signal Transduction

Targeting Adenine Nucleotide Translocase-2(ANT2) to Overcome Resistance to EpidermalGrowth Factor Receptor Tyrosine Kinase Inhibitorin Non–Small Cell Lung CancerJi-Young Jang1,2, Yong-goo Kim1,2, Soo Jeong Nam1,2,3, Bhumsuk Keam4,Tae Min Kim4, Yoon Kyung Jeon1,2,3, and Chul Woo Kim1,2,3

Abstract

EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy hasachieved favorable clinical outcomes in non–small cell lungcancer (NSCLC) patients with EGFR mutations. However,patients eventually develop resistance to EGFR-TKIs by severalmechanisms. Adenine nucleotide translocase-2 (ANT2) is anoncogenic mitochondrial membrane–associated protein. Weinvestigated the therapeutic potential of ANT2 inhibition toEGFR-TKI resistance in NSCLC using gefitinib-sensitive (PC9and HCC827) and gefitinib-resistant (H1975 and HCC827/GR) NSCLC cell lines. ANT2 was inhibited by transfecting cellswith an ANT2-specific shRNA. ANT2 expression was elevated inthe H1975 and HCC827/GR cells compared with the PC9 andHCC827 cells. ANT2 upregulation in gefitinib-resistant cellswas associated with increased SP1 binding to the ANT2 pro-moter. ANT2-specific shRNA decreased NSCLC cell viability.

Moreover, ANT2-specific shRNA sensitized the H1975 andHCC827/GR cells to gefitinib, accompanied by HSP90 andEGFR downregulation. ANT2-specific shRNA also inactivatedthe PI3K/Akt signaling pathway in the H1975 and HCC827/GRcells, which was mediated by the suppression of miR-221/222levels and by the subsequent restoration of PTEN. In EGFR-TKI–treated NSCLC patients, ANT2 expression was higher inpatients exhibiting poor responses compared with patientsshowing excellent responses. Furthermore, ANT2 expressionincreased in tumor tissues biopsied after acquiring gefitinibresistance compared with tissues before gefitinib treatment.These findings suggest that ANT2 overexpression contributesto EGFR-TKI resistance in NSCLC and that ANT2 targeting maybe considered a novel strategy for overcoming this resistance.Mol Cancer Ther; 15(6); 1387–96. �2016 AACR.

IntroductionLung cancer is the most common cause of cancer-related

deaths worldwide, and non–small cell lung cancer (NSCLC)accounts for 85% of lung cancers (1). Adenocarcinoma is themost common NSCLC histology, and its incidence is contin-ually increasing (2). Because more than half of the NSCLCpatients present at advanced stages of the disease, systemictreatments such as chemotherapy are important for managingNSCLC (3). In recent decades, the management of patients with

NSCLCs, particularly adenocarcinomas, has shifted towardpersonalized targeted therapy based on the genetic status ofthe tumor cells (4). The most common druggable geneticalterations in NSCLC are EGFR mutations, which are observedin approximately 60% of East-Asian patients with adenocarci-nomas and in 30% of Western adenocarcinoma patients (5).Mutated EGFR activates the PI3K/Akt, JAK/STAT, and MAPK/ERK pathways and contributes to the carcinogenesis of NSCLC(6–8). Targeted therapies using EGFR tyrosine kinase inhibitors(TKI) have prolonged the survival of pulmonary adenocarci-nomas patients with EGFR mutations (9). However, the clinicaldrawback of EGFR-targeted therapy is that EGFR-TKI resistanceeventually develops in all patients (10). Approximately halfof the acquired resistance to EGFR-TKIs is caused by a second-ary gatekeeper mutation in the EGFR tyrosine kinase domain(i.e., the T790M mutation in EGFR exon 20; ref. 11). Othermechanisms that contribute to acquired EGFR-TKI resistanceinclude MET amplification, small-cell carcinoma transforma-tion, epithelial–mesenchymal transition, and HER2 ampli-fication (12–15). However, the mechanisms of EGFR-TKI resis-tance remain unclear for 20% to 40% of patients (10), warrant-ing the identification of EGFR-TKI resistance mechanisms toovercome acquired EGFR-TKI resistance.

Adenine nucleotide translocase (ANT) is a protein abundantin the inner mitochondrial membrane involving in mitochon-drial oxidative phosphorylation and glycolytic metabolism

1Tumor Immunity Medical Research Center, Cancer Research Center,Seoul National University College of Medicine, Seoul, South Korea.2Tumor Microenvironment Global Core Research Center, SeoulNational University, Seoul, South Korea. 3Department of Pathology,Seoul National University Hospital, Seoul National University Collegeof Medicine, Seoul, South Korea. 4Department of Internal Medicine,Seoul National University Hospital, Seoul National University Collegeof Medicine, Seoul, South Korea.

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

CorrespondingAuthor:ChulWooKim, Department of Pathology, Seoul Nation-al University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 110-799,South Korea. Phone: 822-3668-7651; Fax: 822-743-8301; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0089

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

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on June 22, 2020. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst February 16, 2016; DOI: 10.1158/1535-7163.MCT-15-0089

http://mct.aacrjournals.org/

(16–18). ANT also interacts with BCL2 family proteins andregulates mitochondrial membrane permeability and mito-chondria-mediated apoptosis (16–20). Four isoforms of ANT,encoded by ANT1, ANT2, ANT3, and ANT4 genes, have beenidentified in humans. The expressions of ANT isoforms differsdepending on the tissue and cell type, developmental stage, andcellular proliferation status. Specifically, ANT3 is ubiquitouslyexpressed in all tissues at low levels or at a degree correlatingwith the level of oxidative metabolism. ANT1 is abundantlyexpressed in terminally differentiated cells. In contrast, ANT2 ishighly expressed in undifferentiated cells and tissues withproliferating and regenerating abilities including lymphocytesand the kidneys and liver (16, 17, 21). ANT2 expression is alsohigher than ANT1 expression in several types of human cancercells (17, 18, 22, 23). Notably, ANT1 and ANT3 play proa-poptotic roles; however, ANT2 displays an antiapoptotic func-tion in cancer cells by inhibiting mitochondrial membranepermeabilization (17, 19, 23, 24). Furthermore, ANT2 wasdemonstrated to promote glycolytic metabolism in cancer cellsunder aerobic and anaerobic conditions (25). These observa-tions have suggested that ANT2 functions as an oncogenicmolecule. Consistently, silencing ANT2 led to an increasein mitochondrial membrane potential and to the chemo-sensitization of cancer cells (22). In our previous studies,ANT2 knockdown by short hairpin RNA (shRNA) showedanticancer effects via diverse mechanisms in breast and livercancer cells (23, 26–30). Thus, we hypothesized that ANT2might be involved in the resistance to EGFR-TKI in NSCLCcells and investigated the therapeutic potential of targetingANT2 to overcome acquired EGFR-TKI resistance.

Materials and MethodsCell lines and reagents

EGFR-TKI–sensitive human lung adenocarcinoma cell lines,PC9 and HCC827 (both having EGFR exon 19 deletion muta-tions) and EGFR-TKI–resistant human lung adenocarcinomacell lines, H1975 (having both EGFR L858R and T790M muta-tion) and HCC827/GR cells, which were induced to haveacquired MET amplification (31), were used. PC9 was kindlyprovided by Prof. Mayumi Ono (Kyushu University, Fukuoka,Japan), and HCC827 and HCC827/GR cells were kindly pro-vided by Prof. Jae Cheol Lee of the Asan Medical Center (Seoul,South Korea). H1975, and other lung adenocarcinoma celllines, A549, H1666, and H522 (all EGFR wild-type), werepurchased from ATCC. These cell lines were obtained from2012 to 2014, and authenticated by morphology and growthcharacteristics, and frozen. All cells were also routinely screenedfor the absence of mycoplasma. PC9, H1975, A549, H1666,and H522, and HCC827 cells were cultured in DMEM supple-mented with 10% FBS, 100 U/mL penicillin, and 100 mg/mLstreptomycin at 37�C in a humidified incubator. HCC827/GRcells were cultured under identical conditions, with the addi-tion of 1 mmol/L gefitinib (BioVision). Lonidamine was pur-chased from R&D Systems.

Construction of expression vectorThe ANT2-specific shRNA expression vector was constructed

as reported previously (23). Briefly, an ANT2-specific siRNA(50-GCAGAUCACUGCAGAUAAGTT-30) complementary toexon 2 of ANT2 (Genbank accession number: NM001152) was

synthesized (Bioneer) to achieve the specific downregulationof ANT2. DNA vectors expressing the shRNA forms of thesiRNAs were then generated using pSilencer 3.1-H1 puro plas-mids with a TTCAAGAGA linker sequence to form loopedstructures (Ambion). A scramble (sc) shRNA (Ambion) withno homology to human genes was used as a control shRNA.The HSP90 expression vector (pCMV6-entry) with TruORFGold expression-validated cDNA clone (OriGene Techno-logies) was purchased. Dominant-negative PI3K (dnPI3K) wasprovided by Prof. Yell Cheol Kang of Yonsei University (Seoul,South Korea).

Transfection of plasmid, siRNA, and miRNA inhibitor ormimics

Cells were plated in either 6-well plates (2 � 105 cells/well) or100-mm dishes (2 � 106 cells) and allowed to adhere for 24hours. Then, the cells were transfected with either ANT2-specificshRNA (ANT2 shRNA) or sc shRNA vectors using Lipofectamine2000 (Invitrogen) in Opti-MEM media (QIAGEN). The trans-fected cells were cultured for 6 hours, and the medium wasreplaced with fresh medium supplemented with 10% FBS. Thecells were harvested 24 to 48 hours after transfection. In addition,ANT2-specific siRNA (50-GCAGAUCACUGCAGAUAAGdTdT-30),sc siRNA (50-CCUACGCCACCAAUUUCGUdTdT-30), or HSP90-specific siRNA (50-ACAAGAAGAAGAAGAAGAAdTdT-30; Bio-neer) was transfected into the cells using the same method.mir-221 inhibitor (50-AGCUACAUUGUCUGCUGGGUUUC-30)and miR-222 inhibitors (50-AGCUACAUCUGGCUACUGGGU-30), which were chemically modified and optimized nucleic acidsdesigned to specifically target the miRNA molecules, were pur-chased from GenePharma and transfected as described above.

Protein–DNA binding assayNuclear extracts were prepared from cells and subjected to

protein–DNA binding assays using an EpiQuik General Pro-tein–DNA Binding Assay Kit (Colorimetric; Epigentek) to detectthe binding of Sp1 to the ANT2 promoter DNA sequence. ADNA probe corresponding to the Sp1-binding sequence inthe ANT2 promoter was constructed as follows: 50-CAGAC-CCCGCCCCGGCCCCGCCTCCGAC-30.

ChIP-qPCR assayChIP-qPCR assay was performed using ChIP Abþ Sp1 - ChIP

validated antibody and primer set (Merck Millipore). In brief,chromatin from cells was cross-linked with 1% formaldehyde,sheared to an average size of approximately 500 bp, and thenimmunoprecipitated with anti-Sp1 antibody. Putative bindingsites of Sp1 within the human ANT2 promoter region wereidentified by TFSEARCH. Two potential Sp1-binding sites wereamplified and monitored by real-time quantitative PCR method.

Cell viability and apoptosis assayCell viability was measured using Cell Counting Kit-8

(CCK8) assays (Dojindo Molecular Technologies) accordingto the manufacturer's protocol. All of the experiments wererepeated at least three times in triplicate and the results arepresented as the mean � SD. Apoptosis assay was performed byAnnexin V and PI staining (BD Pharmigen) followed by flowcytometry analysis (Epics XL; Beckman Coulter).

Jang et al.

Mol Cancer Ther; 15(6) June 2016 Molecular Cancer Therapeutics1388




Western blottingTotal cellular protein was extracted using lysis buffer (5

nmol/L ethylenediamine tetra-acetic acid, 300 nmol/L NaCl,0.1% NP-40, 0.5 nmol/L NaF, 0.5 nmol/L Na3VO4, and 0.5nmol/L phenylmethylsulfonyl fluoride, as well as 10 mg/mLeach of aprotinin, pepstatin, and leupeptin; Sigma-Aldrich). Intotal, 50 mg of protein was subjected to 10% SDS-PAGE, and theresolved proteins were transferred to polyvinylidene difluoridemembranes (Millipore), which were then incubated with anti-bodies directed against ANT2, phospho-Akt (P-Akt) or Akt (CellSignaling Technology) or with antibodies directed againstHSP90, EGFR, MET, PTEN, cleaved PARP, or b-actin (SantaCruz Biotechnology). The immunoblots were visualized usingan enhanced chemiluminescence detection system (AmershamPharmacia Biotech).

Quantitative reverse transcription PCRTotal RNA was extracted from the cells using TRIzol reagent

(Life Technologies) and reverse transcribed using a PrimeScriptFirst Strand cDNA Synthesis Kit (Takara Bio). GAPDH was usedas the internal control for mRNA expression. To evaluate themiR-221/222 levels, the extracted total RNA was subjected tocDNA synthesis and quantitative reverse transcription PCR(qRT-PCR) analysis using Mir-X miRNA First Strand Synthesisand SYBR qRT-PCR Kits (Clontech Laboratories). U6-specific

snRNA (catalog no. 4373381, Applied Biosystems) was usedas an internal control for miRNA expression. The sequences ofthe primers used for qRT-PCR to analyze the miRNAs andmRNAs are as follows: for ANT2: forward, 50-ACGTGTCTGTG-CAGGGTATT-30, and reverse, 50-GTGTCAAATGGATAGGAAG-30; for PTEN: forward, 50-CGAACTGGTGTAATGATATGT-30 andreverse 30-CATGAACTTGTCTTCCCGT-30; for GAPDH, forward50- CCCTTCATTGACCTCAACTA-30, and reverse, 50- ACGATAC-CAAAGTTGTCATG-30; for miR-221: forward, 50-CGGCGGTA-GCTACATTGTCTGCTG-30, and, reverse, 50-CCAGTGCAGGGT-CCGAGGTAT-30; for miR-222: forward, 50-CGGCGGTAGCTA-CATCTGGCTA-30, and reverse, 50- CCAGTGCAGGGTCCGAGG-TAT-30. The PCR reactions were performed using a Step OnePlus thermal cycler (Applied Biosystems) in triplicate. Therelative changes in gene expression were calculated using thefollowing formula, and the data are represented as fold upre-gulation/downregulation: the fold change is indicated as2�DDCt, where DDCt ¼ (Ct of gene of interest, treated � Ct ofinternal control, treated) � (Ct of gene of interest, control � Ct

of internal control gene, control; Ct, the threshold cyclenumber).

Intracellular ATP assayIntracellular ATP assays were performed using CellTiter-Glo

Luminescent Cell Viability Assay Kits (Promega), which emits

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Figure 1.ANT2 expression is elevated in in EGFR-TKI–resistant NSCLC cells through increased transcription by Sp1. A, the levels of ANT2 expression were evaluatedin various NSCLC cell lines that are known to be sensitive (PC9 and H1666) and resistant (A549, H1975, and H522) to EGFR-TKI, using qRT-PCR andWestern blotting. The bar graph shows the relative expression level (2�DDCt) of ANT2 in each cell line compared with that of PC9 cells. B and C, the levelsof ANT2 expression were evaluated and compared in gefitinib-sensitive PC9 cells (EGFR E19 del), gefitinib-resistant H1975 cells (EGFR L858R andT790M), gefitinib-sensitive HCC827 cells (EGFR E19 del), and gefitinib-resistant H827/GR cells (EGFR E19 del and MET amplification) by qRT-PCR (B)and Western blotting (C). The relative expression levels (2�DDCt) of ANT2 in H1975, HCC827, and HCC827/GR cells were compared with those of PC9 cellsby qRT-PCR. D, nuclear extracts from PC9, H1975, HCC827, and HCC827/GR cells were subjected to protein–DNA binding assays and ChIP-qPCR assayto detect the binding of Sp1 to the ANT2 promoter. The values presented in all of the histograms are the mean values � SD. � , P < 0.05; ��, P < 0.005,statistically significant differences as determined by unpaired Student t tests.

Targeting ANT2 in EGFR-TKI–Resistant NSCLC

www.aacrjournals.org Mol Cancer Ther; 15(6) June 2016 1389




light upon interactions with ATP and luciferin. In brief,lyophilized enzyme/substrate mixtures (250 mL) were trans-ferred to opaque 96-well microplates containing cell lysates.The plates were incubated at room temperature for 10 min-utes to stabilize the luminescence signals, which were thenquantified using an Orion Luminometer (Berthold DetectionSystems).

Immunohistochemical analysis of human lung cancertissues

From the registry of lung cancer patients who underwentsurgical resection for lung cancer and were treated with gefitinibfor recurrent or metastatic disease at Seoul National UniversityHospital (SNUH; Seoul, South Korea), 84 patients who har-bored EGFR mutation were collected. One medical oncologist(B. Keam), who was unaware of the ANT2 statuses of thetumors, reviewed the radiologic images by RECIST 1.0 criteriato classify the responses as complete response (CR), partialresponse (PR), stable disease (SD), or progressive disease(PD). Progression-free survival (PFS) was defined as the timefrom the beginning of EGFR-TKI administration to confirmeddisease progression or death. The 84 patients were divided intothree groups: (i) poor responders, (ii) conventional responders,and (iii) excellent responders. The poor responders weredefined as PD or SD without durable (�6 months) PFS (32).The excellent responders were defined as CR or PR with pro-longed PFS (�12 months). The conventional responders weredefined as neither poor nor excellent responders. Finally, 10cases with poor responses to gefitinib and 9 cases with excellentresponses to gefitinib were selected. All 19 patients had EGFR-TKI–sensitive mutations, including EGFR exon 19 deletions (n¼ 9), and L858R (n ¼ 9) or T854I mutations (n ¼ 1) in EGFR

exon 21. Formalin-fixed paraffin-embedded tumor tissueblocks from these patients were subjected to immunohis-tochemistry (IHC) using an anti-ANT2 antibody (CreativeBiomart). IHC was performed using the CSA II Biotin-freeTyramide Signal Amplification System (DAKO Cytomation)and the results were interpreted by a pathologist (Y.K. Jeon)who was blinded to the treatment response. The ANT2 IHC wasgraded as follows: 0, negative or staining in less than 10% oftumor cells; 1, weak; 2, moderate; and 3, strong staining in �10% of tumor cells. This study was approved by the Institu-tional Review Board of SNUH (H-1401-031-548).

Statistical analysisThe data were analyzed using Student t tests (SPSS version 21;

IBM Corp.). P < 0.05 was considered statistically significant.

ResultsANT2 expression was upregulated in EGFR-TKI–resistantNSCLC cells

First, we screened the ANT2 expression in several NSCLC celllineswith variable sensitivity to EGFR-TKI. As shown in Fig. 1A–C,ANT2 expression was lower in EGFR-TKI–sensitive cell lines(including PC9 and HCC827) and higher in EGFR-TKI–resistantcell lines (including A549, H522 H1975, and HCC827/GR) atboth mRNA and protein levels.

Because Sp1 is the most important transcription factorfor human ANT2 (33), we assessed the binding of Sp1 to theANT2 promoter using protein–DNA binding assays andChIP-qPCR assay (Fig. 1D). Sp1 binding to the ANT2 promoterwas significantly elevated in H1975 and HCC827/GRcells compared with that in PC9 and HCC827 cells. Together,

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Figure 2.The inhibition of ANT2 suppresses cell viability and restores gefitinib sensitivity in EGFR-TKI–resistant NSCLC cells. A, PC9, H1975, HCC827, and HCC827/GRcells were transfected with ANT2 shRNA or sc shRNA. At 24 hours after transfection, the cells were treated with gefitinib (50 nmol/L) or DMSO as acontrol and subsequently incubated for an additional 24 hours. Then, cell viability was measured using CCK8 assays and presented as a relative valuecompared with that of sc shRNA-treated cells. The values presented in all of the histograms are the mean values � SD. � , P < 0.05; �� , P < 0.005,statistically significant differences as determined by unpaired Student t tests; n.s., not significant. B, at 24 hours after transfection with ANT2 shRNAor sc shRNA, the cells were treated with gefitinib (50 nmol/L) or DMSO as a control and subsequently incubated for an additional 24 hours. Westernblotting was then performed to determine the level of cleaved PARP.

Jang et al.





these data suggest that ANT2 expression is upregulated inEGFR-TKI–resistant NSCLC cells at the transcriptional level,which may be mediated by increased binding of Sp1 to theANT2 promoter.

Inhibition of ANT2 suppresses cell viability and restoresgefitinib sensitivity in EGFR-TKI–resistant NSCLC cells

Next, it was determined whether ANT2 shRNA would sup-press the viability of EGFR-TKI–resistant NSCLC cells withelevated ANT2 expression. The IC50 of gefitinib was 50 and10 nmol/L in PC9 and HCC827 cells, respectively, but as muchas 1,000-fold higher (10 mmol/L) in H1975 and HCC827/GRcells (Supplementary Fig. S1). Suppression of ANT2 by ANT2shRNA was assured by Western blot analysis (SupplementaryFig. S2). As shown in Fig. 2A, ANT2 shRNA transfection ofPC9 and HCC827 cells decreased cell viability by 20%, whereasANT2 shRNA transfection of H1975 and HCC827/GR cellsdecreased cell viability by 40% and 50%, respectively. Thecombined treatment of ANT2 shRNA and gefitinib exhibitedan additive effect on decrease of cell viability in PC9 and

HCC827 cells as well as in H1975 and HCC827/GR cells(Fig. 2A). Furthermore, apoptosis of H1975 and HCC827/GRcells was much increased by combined gefitinib and ANT2shRNA treatment as shown by PARP cleavage (Fig. 2B) andAnnexin V/PI staining (Supplementary Fig. S3). As a pharma-cologic ANT2 inhibitor, we used lonidamine, which is a mito-chondrial hexokinase inhibitor provoking a disruption of themitochondrial transmembrane potential similar to the ANT2shRNA-induced apoptosis. Lonidamine also compromised cellsurvival in lung cancer cells along with gefitinib (Supplemen-tary Fig. S4). Taken together, these data suggested that ANT2knockdown by ANT2 shRNA might effectively suppress theviability of gefitinib-resistant NSCLC cells and overcome gefi-tinib resistance mediated by T790M mutations and by METamplification.

EGFR-TKI sensitization by ANT2 inhibition is mediated by thesuppression of HSP90 and EGFR expression

To address the mechanism by which ANT2 shRNA sensitizesH1975 and HCC827/GR cells to gefitinib, we focused on

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Figure 3.ANT2 inhibition sensitizes NSCLC cells to gefitinib by suppressing the expression of HSP90 and EGFR. A, cells were transfected with ANT2 shRNA or sc shRNAfor 24 hours and then lysed to quantify total intracellular ATP levels. The results are tabulated as relative ATP production by normalizing luminescenceunits to total protein levels. B, at 24 hours after transfection with ANT2 shRNA or sc shRNA, Western blotting was performed to determine the levelsof HSP90, EGFR, P-Akt, Akt, and MET. C, H1975 and HCC827/GR cells were transfected with ANT2 siRNA, HSP90 siRNA, or sc siRNA for 24 hours, and then treatedwith gefitinib. After an additional 24-hour incubation, cell viability was measured using CCK8 assays and presented as a relative value compared with thatof sc siRNA-treated cells. D, H1975 and HCC827/GR cells were transfected with ANT2 shRNA or sc shRNA with/without HSP90 expression vector. At 24 hoursafter transfection, the cells were treated with gefitinib (50 nmol/L) or DMSO as a control and subsequently incubated for an additional 24 hours. Then, cellviability was measured using CCK8 assays. The values presented in all of the histograms are the mean values � SD. � , P < 0.05; ��, P < 0.005, statisticallysignificant differences as determined by unpaired Student t tests; n.s., not significant.






HSP90 expression. As shown in Fig. 3A, ANT2 shRNA decreasedthe intracellular ATP level in NSCLC cells in a degree propor-tionate to the ANT2 expression level (i.e., greater in H1975 andHCC827/GR cells compared with PC9 and HCC827 cells).ANT2 shRNA also suppressed HSP90 expression in theseNSCLC cells, which was accompanied by the downregulationof EGFR expression (Fig. 3B). However, the levels of Akt andMET, which are other HSP90 client proteins, were not affectedby ANT2 shRNA (Fig. 3B).

To determine whether ANT2 shRNA-mediated HSP90 down-regulation could be involved in the sensitivity of H1975 andHCC827/GR cells to gefitinib, we knocked downHSP90 using anHSP90 siRNA and evaluated cell viability after combined treat-ment with gefitinib. The transfection of ANT2 siRNA and HSP90siRNA reduced the expression of ANT2 and HSP90 by approxi-mately 30% inH1975 andHCC827/GR cells (Supplementary Fig.

S5). ANT2 siRNA and HSP90 siRNA transfection led to approx-imately 30% and 10% decreases in cell viability in H1975 andHCC827/GR cells, respectively (Fig. 3C). In addition, combinedtreatment of gefitinib and ANT2 siRNA or HSP90 siRNA trans-fection further suppressed cell viabilities in these cells (Fig. 3C).Moreover, rescue of HSP90 expression in ANT2 shRNA–trans-fected cells restored the cell viability after gefitinib treatment (Fig.3D). These data suggested that the ANT2 shRNA-mediated down-regulation of HSP90 might be involved in the restoration ofgefitinib sensitivity in EGFR-TKI–resistant NSCLC cells. Mean-while, the degree of suppression of cell viability by combinedHSP90 siRNA and gefitinib was much lower compared with thatobserved by combined ANT2 siRNA and gefitinib treatment (Fig.3C). Thus, we hypothesized that mechanisms other than HSP90suppression might contribute to the restoration of gefitinib sen-sitivity by ANT2 shRNA in EGFR-TKI–resistant NSCLC cells.

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Figure 4.ANT2 inhibition sensitizes NSCLC cells to gefitinib by downregulating the levels of miR-221/222, consequently restoring PTEN expression and inactivatingthe PI3K/Akt pathway. A and B, the expression levels of miR-221 and miR-222 and PTEN were assessed by qRT-PCR in EGFR-TKI–sensitive (PC9 andHCC827) and EGFR-TKI–resistant (H1975 and HCC827/GR) cells. The relative expression levels (2�DDCt) of these molecules were compared with those ofPC9 cells. Western blotting was performed to determine the levels of PTEN. C and D, H1975 and HCC827/GR cells were transfected with miR-221 ormiR-222 inhibitors or negative inhibitors, and the changes in PTEN levels were measured by qRT-PCR and Western blotting. The relative expressionlevels (2�DDCt) of PTEN were compared with those of H1975 cells transfected with negative inhibitors. E–G, At 24 hours after transfection with ANT2 shRNAor sc shRNA, the expression levels of miR-221 and miR-222 (E) and PTEN (F) were measured by qRT-PCR. The relative expression levels (2�DDCt) ofthese molecules were compared with those of cells transfected with sc shRNA. PTEN expression was also assessed by Western blotting (G). H, H1975and HCC827/GR cells were transfected with ANT2 shRNA, dominant-negative PI3K (dnPI3K) vector, or sc shRNA for 24 hours, and then treated withgefitinib. After an additional 24-hour incubation, cell viability was measured via CCK8 assays and presented as a relative value compared with thatof sc shRNA-transfected cells. The values presented in all of the histograms are the mean values � SD. � , P < 0.05; �� , P < 0.005, statistically significantdifferences as determined by unpaired Student t tests; n.s., not significant.

Jang et al.





EGFR-TKI sensitization by ANT2 inhibition is mediated bythe downregulation of miR-221/222, and the restorationof PTEN and the subsequent inactivation of the PI3K/Aktpathway

We focused on the PI3K/Akt pathway as another mechanismunderlying the restoration of gefitinib sensitivity mediated byANT2 shRNA because P-Akt was much decreased by ANT2shRNA in NSCLC cells (Fig. 3B). Because the Akt level was notaltered by ANT2 shRNA (Fig. 3B), we hypothesized that ANT2shRNA might affect the regulatory molecules in the PI3K/Aktpathway, which are representative by PTEN. An examination ofthe miRNA profile after ANT2 shRNA transfection in A549 cellsrevealed that miR-221 and 222 were most markedly down-regulated (Supplementary Fig. S6), and a database searchindicated that miR-221 and 222 were able to target PTEN.Consistently, miR-221 and 222 levels were much higher andPTEN expression was significantly lower and in H1975 andHCC827/GR cells compared with those in PC9 and HCC827cells (Fig. 4A and B). Furthermore, transfection with miR-221or 222 inhibitors in H1975 and HCC827/GR cells resulted inthe upregulation of PTEN (Fig. 4C and D).

Moreover, ANT2 shRNA transfection significantly downregu-lated miR-221 and 222 levels in both H1975 and HCC827/GRcells (Fig. 4E), which was accompanied by PTEN upregulation atboth the mRNA and protein levels (Fig. 4F and G). Furthermore,the transfection of H1975 and HCC827/GR cells with a domi-nant-negative PI3K (dnPI3K) construct suppressed cell viability,suggesting the PI3K/Akt pathway might be important for thesurvival of these cells (Fig. 4H). Together, these data suggested

that ANT2 inhibition downregulated miR-221 and 222 expres-sions, subsequently restoring PTEN expression and inactivatingthe PI3K/Akt signaling pathway, which might contribute to thesensitization of H1975 and HCC827/GR cells to gefitinib, alongwith other mechanisms.

ANT2 overexpression in NSCLC tissues from patients showingresistance to EGFR-TKI therapy

Finally, we evaluated ANT2 expression in human NSCLCtissues by IHC to determine whether ANT2 overexpression isrelated to the responsiveness of NSCLC patients to gefitinib. Asshown in Fig. 5A, ANT2 overexpression was more frequentlyobserved in patients who showed poor responses to gefitinibtherapy than in patients who showed excellent responses. Fur-thermore, ANT2 expression was frequently (4 of 7, 57%)increased in tumor tissues biopsied from patients with acquiredresistance to gefitinib compared with paired tumor biopsiesobtained before gefitinib treatment. However, ANT2 expressionwas maintained in two samples and decreased in 1 of 7 patients(Fig. 5B).

DiscussionThis study demonstrated that ANT2 expression was upregu-

lated in EGFR-TKI–resistant NSCLC cells and that ANT2 knock-down by ANT2 shRNA could suppress cell viability in ANT2-overexpressing NSCLC cells and restore the EGFR-TKI sensitivityof NSCLC cells with acquired resistance to EGFR-TKIs. Moreover,ANT2 shRNA was revealed to overcome EGFR-TKI resistance by

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Figure 5.Expression levels of ANT2 in tumor tissues from NSCLC patients treated with gefitinib. A, ANT2 expression was determined using IHC in tumor tissuesfrom NSCLC patients who showed good primary responses (n ¼ 9) or poor responses to gefitinib (n ¼ 10). The level of ANT2 IHC was presented asthe IHC grade. The statistical significance in difference in the ANT2 IHC grade between good responder and poor responder was determined usingunpaired Student t test, which was nonsignificant (ns). B, ANT2 expression was compared in paired tumor tissues obtained before gefitinib treatment("before gefitinib") and after acquiring resistance to gefitinib ("after gefitinib") using IHC. Changes in IHC grade are depicted in each patient (n ¼ 7),and representative IHC images from patient 5 are shown below. The statistical significance in the ANT2 IHC grade before gefitinib treatment and afteracquiring resistance to gefitinib was determined using paired Student t test, which was nonsignificant (ns).






various mechanisms, as illustrated in Fig. 6, including the deple-tion of intracellular ATP, the degradation of HSP90, the down-regulation of EGFR, and the suppression of the PI3K/Akt signalingpathway through PTEN restoration.

ANT2 mRNA expression was consistently upregulated inseveral cancer cells, but not in non-neoplastic cells (17, 22,23, 29). Moreover, ANT2 mRNA was overexpressed in primarycancer tissues, including lung tumors, but not in normal tissues(22). We previously screened the ANT2 mRNA levels in variouscancer cell lines, and found that lung cancer cells also over-expressed ANT2 (23). In this study, we demonstrated that ANT2mRNA and protein expression is upregulated in EGFR-TKI–resistant NSCLC cells compared with EGFR-TKI–sensitive cells.Of note, ANT2 overexpression was observed in NSCLC cellsthat achieved resistance to EGFR-TKIs by variable mechanisms,including the T790M mutation and MET amplification. Fur-thermore, ANT2 expression was also higher in NSCLC tissuesfrom patients who showed primary or acquired resistance toEGFR-TKIs. In this study, ANT2 upregulation in EGFR-TKI–resistant NSCLC cells was mediated by Sp1 at a transcriptionallevel and the activated EGFR signaling in human glioma cellswas previously shown to enhance the activity of the Sp1transcription factor (34). Thus, we speculated that persistentEGFR activation in EGFR-TKI–resistant NSCLC cells mightcontribute to the activation of Sp1 and its binding to thepromoter region of and subsequent transcription of ANT2.

ANT2 is important for glycolytic metabolism, which is acharacteristic feature of energy metabolism in many tumors(25). In addition, ANT2 plays an antiapoptotic role by main-taining the mitochondrial membrane potential (16, 17). Theabove-mentioned unique expression pattern and the biologicfunction of ANT2 suggested a protumoral role of ANT2; there-fore, ANT2 was considered as a promising therapeutic target forcancer treatment. Previously, ANT2 silencing by ANT2 siRNAresulted in chemosensitization to lonidamine in HeLa cells (22).We also previously reported that ANT2 knockdown by ANT2shRNA suppressed cell growth, proliferation, and invasion andinduced apoptotic cell death in breast and liver cancer cells. Ofnote, this antitumor effect of ANT2 shRNA was mediated byvariable mechanisms including the depletion of ATP, the repres-sion of HSP90, the regulation of miRNA targeting signalingsuppressive molecules, and the regulation of epigenetic path-ways (23, 26–28, 30). ANT2 shRNA also enhanced chemosen-sitivity in stem cell-like breast cancer cells (29). In this study,ANT2 shRNA compromised the viability of NSCLC cells, and thedegree of cytotoxicity was proportionate to the level of ANT2expression, which suggested that a specific cytotoxic effect waselicited by ANT2 shRNA. Moreover, using gefitinib-resistantNSCLC cell lines having two most mechanism underlyingacquired resistance to EGFR-TKI, that is, T790M (H1975) orMET amplification (HCC827/GR), we demonstrated that ANT2-specific shRNA overcome EGFR-TKI resistance in NSCLC.

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Jang et al.




Recently, many strategies to overcome EGFR-TKI resistancehave been developed in vitro and in vivo, and several agents areundergoing clinical trials (10). Among these agents, HSP90 inhi-bitors have shown favorable results for sensitization to EGFR-TKIsin NSCLC; the effects of these drugs are mediated by downregula-tion of HSP90 client proteins including EGFR, MET, Akt, andErbB3 (35, 36). Previously, we demonstrated that ANT2 shRNAdownregulated HER2 expression in breast cancer cells, therebyexerting an antitumor effect. This effect wasmediated by the ANT2shRNA–induced depletion of intracellular ATP and the subse-quent suppression of HSP90 function (27). In this study, thetransfection of H1975 and HCC827/GR cells with an ANT2-specific shRNA led to ATP depletion and HSP90 downregulation,which was accompanied by EGFR suppression. Although HSP90inhibitors such as 17-allylamino-17-demethoxygeldanamycin(17-AAG) are known to be functional inhibitors of HSP90,reduction in HSP90 levels by 17-AAG have been reported previ-ously (37). However, the mechanism by which ANT2 shRNA ledto the suppression of HSP90 levels must be elucidated by furtherstudy. Unexpectedly, the levels of other HSP90 client proteins(Akt and MET) were not affected by ANT2 shRNA, even in MET-amplified HCC827/GR cells. However, the P-Akt level wasmarkedly downregulated. These observations suggest that ANT2shRNA–mediated sensitization to gefitinib cannot be fullyexplained by HSP90 suppression.

Meanwhile, it was observed that ANT2 shRNAmarkedly down-regulated P-Akt expression in both gefitinib-resistant and gefiti-nib-sensitive cell lines. In addition, P-Akt expression was elevatedin gefitinib-resistant H1975 and HCC827/GR cells comparedwith gefitinib-sensitive PC9 and HCC827 cells, whereas PTENlevels were lower in gefitinib-resistant cells but higher in gefitinib-sensitive cells. Furthermore, ANT2 shRNA led to the restoration ofPTEN expression in H1975 and HCC827/GR cells. Thus, toidentify the regulatory mechanism of PTEN by ANT2 shRNA, weperformed screening for miRNAs whose expression was changedby ANT2 shRNA. By doing this, we demonstrated that ANT2shRNA suppressed miR-221 and miR-222 with the concomitantupregulation of PTEN and the inhibition of PI3K/Akt signaling inthe EGFR-TKI–resistant H1975 andHCC827/GR cell lines, whichmight underlie a mechanism associated with ANT2 shRNA-

induced cell death. Consistently, PI3K/Akt pathway inhibitionhas been suggested as one approach to overcome EGFR-TKIresistance in NSCLC (38, 39), and this inhibition might providea rationale for ANT2 shRNA as a strategy to restore the respon-siveness of EGFR-TKI-resistant cells to EGFR-TKI.

In conclusion, this study demonstrated that ANT2 overexpres-sion might contribute to resistance to EGFR-TKIs in NSCLC andthat ANT2 knockdown using ANT2 shRNA can suppress cellviability and overcome EGFR-TKI resistance in NSCLC cells bysuppressing HSP90 and downregulating the PI3K/Akt pathway.Thus, targeting ANT2 may be considered a novel therapeuticstrategy to overcome primary or acquired resistance to EGFR-TKIsin NSCLC.

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

Authors' ContributionsConception and design: J.-Y. Jang, C.W. KimDevelopment of methodology: Y.K. JeonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B. Keam, T.M. KimAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.-Y. Jang, S.J. Nam, T.M. Kim, C.W. KimWriting, review, and/or revision of the manuscript: J.-Y. Jang, B. Keam,Y.K. Jeon, C.W. KimAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.-Y. Jang, Y.-G. Kim, B. Keam, T.M. KimStudy supervision: C.W. Kim

Grant SupportThis study was supported by the Basic Science Research Program (grant no.

NRF-2013R1A1A2057622) and the Global Core Research Center (GCRC; grantno. 2012-0001190) through the National Research Foundation (NRF) fundedby the Ministry of Education, Science and Technology (MEST), Republic ofKorea. All the authors were supported by these grants.

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 January 30, 2015; revised January 17, 2016; accepted February 2,2016; published OnlineFirst February 16, 2016.

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2016;15:1387-1396. Published OnlineFirst February 16, 2016.Mol Cancer Ther Ji-Young Jang, Yong-goo Kim, Soo Jeong Nam, et al.

Small Cell Lung Cancer−Inhibitor in Non Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Targeting Adenine Nucleotide Translocase-2 (ANT2) to Overcome

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Targeting Adenine Nucleotide Translocase-2 (ANT2) to ... · ANT2 was inhibited by transfecting cells with an ANT2-speciﬁc shRNA. ANT2 expression was elevated in the H1975 and HCC827/GR