Inhibiting DX2-p14/ARF Interaction Exerts Antitumor ... · report inspired us to examine the role...

Therapeutics, Targets, and Chemical Biology

Inhibiting DX2-p14/ARF Interaction ExertsAntitumor Effects in Lung Cancer and DelaysTumor ProgressionAh-Young Oh1, Youn Sang Jung1, Jiseon Kim2, Jee-Hyun Lee2, Jung-Hyun Cho1,Ho-Young Chun1, Soyoung Park1, Hyunchul Park3, Sikeun Lim3, Nam-Chul Ha4,Jong Sook Park5, Choon-Sik Park5, Gyu-Yong Song2, and Bum-Joon Park1

Abstract

The aminoacyl tRNA synthetase complex-interacting multi-functional protein 2 (AIMP2) splice variant designated DX2 isinduced by cigarette smoke carcinogens and is often detected inhuman lung cancer specimens. However, the function ofDX2 inlung carcinogenesis is obscure. In this study, we found that DX2expression was induced by oncogenes in human lung cancertissues and cells. DX2 prevented oncogene-induced apoptosisand senescence and promoted drug resistance by directly bind-ing to and inhibiting p14/ARF. Through chemical screening, weidentified SLCB050, a novel compound that blocks the interac-

tion between DX2 and p14/ARF in vitro and in vivo. SLCB050reduced theviabilityof human lung cancer cells, especially smallcell lung cancer cells, in a p14/ARF-dependent manner. More-over, in a mouse model of K-Ras–driven lung tumorigenesis,ectopic expressionofDX2 induced small cell andnon–small celllung cancers, both of which could be suppressed by SLCB050treatment. Taken together, our findings show how DX2 pro-motes lung cancer progression and how its activity may bethwarted as a strategy to treat patients with lung cancers exhibit-ing elevatedDX2 levels. Cancer Res; 76(16); 4791–804.�2016 AACR.

IntroductionLung cancer is one of the most common human malignan-

cies and is associated with extremely low survival rates (<10%;refs. 1, 2). However, the molecular mechanisms underlying thedevelopment of lung cancers remain unclear. Oncogenic muta-tions such as K-Ras or Her2/Neu and activation of AKT signalingby altering positive and negative regulators have been suggestedto promote lung cancers (3–7). In fact, amplification of Her2/Neu or oncogenic mutation in K-Ras is found in about 30% oflung cancer (8, 9), and loss of PTEN is also suggested as one ofimportant genetic alternation (10). Given that oncogene acti-vation induces p53-induced apoptosis or senescence via p14/ARF (11), there may be additional factors that disrupt the

functional interaction between oncogenes and p14/ARF-p53for cancer progression.

Human lung cancers are divided into two groups, small celllung cancer (SCLC) andnon–small cell lung cancer (NSCLC), thatare also divided into subgroups, adenocarcinoma, squamouscell lung carcinoma, and large-cell lung cancer. Among them,SCLC occupies 20% of lung cancer, is closely linked to smokinghabit, and is very aggressive (12–14). Moreover, until now, we donot develop proper anticancer drug against SCLC.

AIMP2 is previously known as p38/JTV-1 and cofactor ofaminoacyl-tRNA synthetase complex (15, 16). Differentially frompredicted role, housekeeping and scaffolding protein in essentialenzyme complex, AIMP2 shows diverse cellular functions such asp53 activator and substrate of Parkin (16, 17). Moreover, AIMP2knock out mouse is neonatal lethal because of defect in lungepithelial cell differentiation (15).

Recently, exon 2 skipped alternative splicing variant of AIMP2,AIMP2-DX2 (DX2) has been reported to be highly expressed inhuman lung cancer (17, 18) and tobe induced bybenzo(a)pyrene(18), a major carcinogen associated with smoking (19). Consid-ering previous literatures, DX2 would be important factor of lungcancer initiation or progression, and howDX2 contributes to lungcarcinogenesis has not been clearly demonstrated until now. Thatreport inspired us to examine the role of DX2 in human lungcancer, in particular SCLC because of tight relationship withsmoking.

In this study, we checked the expression of DX2 in several kindsof human lung cancer cell lines and revealed that it was highlyexpressed in SCLC and stabilized by various oncogenic signalingincluding K-Ras or Her2/Neu-AKT activation. Moreover, DX2blocked the oncogene-induced p14/ARF activation. Thus, wehypothesized that inhibition of DX2 would be one of plausiblecandidate for lung cancer treatment, in particular, SCLC.

1Department of Molecular Biology, Pusan National University, Busan,Republic of Korea (South). 2College of Pharmacy,ChungnamNationalUniversity, Daejeon, Republic of Korea (South). 3Forensic DNA Divi-sion, National Forensic Service, Wonju, Republic of Korea (South).4Program in Food Science and Biotechnology, College of Agricultureand Life Sciences, Seoul National University, Seoul, Republic of Korea(South). 5Division ofAllergy andRespiratoryMedicine, Department ofInternal Medicine, Soonchunhyang University Bucheon Hospital,Bucheon, Gyeonggi Do, Republic of Korea (South).

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

A.-Y. Oh, Y.S. Jung, and J. Kim equally contributed to this article.

Corresponding Authors: B.-J. Park, Pusan National University, 30 Jangjeon-dong, Geumjeong-gu, Busan 609-735, Republic of Korea (South). Phone: 82-51-510-2220; Fax: 82-51-513-9258; E-mail: [email protected]; and G.-Y. Song,[email protected]

doi: 10.1158/0008-5472.CAN-15-1025

�2016 American Association for Cancer Research.

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Materials and MethodsCell culture

A549, HCT116, H1299, and HEK293 cell lines were obtainedfrom the ATCC andmaintained in RPMI-1640 orDMEM contain-ing 10% FBS and 1% antibiotics. NSCLC cell lines (NCI-H23,NCI-H322, NCI-H358, and NCI-H460) were obtained from theATCC. SCLC cell lines (NCI-H69, NCI-H128, NCI-H209, andNCI-H146) were newly purchased from Korean Cell line Bank(KCLB) for this experiment and maintained in RPMI-1640 con-taining 10% FBS. MEF cells were isolated from 14.5 day embryosusing a standard protocol and cultured in DMEM supplementedwith 15%FBS and1%antibiotics. Cell lineswere certifiedby shorttandem repeat analysis.

Transfection of mammalian expression plasmids and siRNAThe mammalian expression plasmids encoding GFP-p14/ARF

(GFP tagged at N-terminal of full-length p14/ARF) were kindlyprovided by Dr. G. Peters (Cancer Research UK, London ResearchInstitute; ref. 20). The AIMP2-related mammalian expressionvectors (AIMP2 and DX2) and Ras (K-Ras, N-Ras and H-Ras)were obtained by Dr. S. Kim (Seoul National University, Seoul,Republic of Korea) and by Dr. S.G. Chi (Korea University, Seoul,Republic of Korea), respectively (18, 21). pVHL and Siah-1 vectorswere obtained from Dr. Y.J. Jung (Pusan National University,Busan, Republic of Korea) and from Dr. Lashuel (Qatar Founda-tion), respectively. Her2/Neu WT, CA (Constitutively Active;V659E), KD (kinase dead; K753M; ref. 22), and E3 ligases [Skp2(23), CDC4 (24)] were obtained from Addgene. For in vitro geneknockdown, siRNAs against target proteins including DX2 (18),p14/ARF (25), and control (nonsilencing; ref. 26) were generatedby costume service (Cosmo Genetech). Transfection was per-formed for 24 hours using Jet-pei (Polyplus Transfection) reagentaccording to the manufacturer's protocol. In brief, cells, seeded aday before, were washed with PBS and incubated with DNA/Jet-pei mixture for 4 hours within serum-free condition.

Western blot analysisProtein was extracted from cells in RIPA buffer (150 mmol/L

NaCl, 25 mmol/L Tris-Cl, 1% NP-40, 1% sodium deoxycholate,0.1% SDS, containing protease inhibitor cocktail). After heatinactivation with sample buffer (heated at 95�C for 7 minutes),proteins were applied to SDS-PAGE and Western blot analysis,according to general protocol (27, 28). The antibodies (Ab), usedfor this study, are purchased from Santa Cruz Biotechnology [HA(sc-7392), His (sc-8036), GFP (sc-9996), GST (sc-138), Actin (sc-1616), and p19/ARF (sc-32748)] or Millipore [Anti-p14/ARF(MAB3782)] or Sigma Aldrich [FLAG-M2 (F3165) and C-Myc(M5546)]. Anti-AIMP2 was kindly provided from Dr. S. Kim(Seoul National University).

MiceAll experimental procedures using laboratory animals were

approved by the animal care committee of Pusan National Uni-versity. DX2 (C57/BL6) and K-RasLA2 (C57/BL6) mice wereobtained from Dr. S. Kim (Seoul National University) and Dr.K. Choi (YonseiUniversity, Seoul, Republic of Korea), respectively,and double Tg mice were generated by crossing breeding of DX2and K-RasLA2 mice. Before experiment, all mice were maintainedunder temperature- and light-controlled conditions (20–23�C, 12

hour/12 hour light/dark cycle) and provided autoclaved food andwater ad libitum.

Drug treatment in vivoDK (5-month-old,N¼ 6) mice were administered with carrier,

SLCB050 (5 mg/kg), Adriamycin (1 mg/kg), and combination ofSLCB050 and Adriamycin by i.p. injection. After termination ofthe experiment of each group, mice were dissected and isolatedlung tissues. For xenograft, 1� 107 H446 cells were seeded in s.c.on nude mice. After 4 weeks, tumor-bearing mice were injectedwith Adriamycin (5 mg/kg or 1 mg/kg), SLCB050 (10 mg/kg), orcombination for 6 weeks. Every week, tumor volume and bodyweight were measured.

Histologic analysisAfter dissection of mice, tissues were fixed using 10%

formalin in PBS for 24 hours and embedded in paraffin blocksaccording to a basic tissue processing procedure. For histologicanalysis, embedded tissues were cut for 5 mm by Leica micro-tome and transferred onto adhesive-coated slides (Marienfeldlaboratory glassware). After deparaffin and rehydration, sec-tions were then stained with hematoxylin–eosin for routineexamination.

For IHC staining, rehydrated tissue sections were incubatedwith antibodies to ki-67 (Abcam; ab15580), pan-keratin (Sigma;C2931), pro-surfactant C (Millipore; AB3786), NSE (DAKO;IS612), and Her2/Neu (DAKO; A0458). Antigen retrieval wasperformed using 10 mmol/L sodium citrate (pH 6.0) 2 times at95�C for 10 minutes each, and endogenous peroxidase activitywas blocked with 3% hydrogen peroxidase for 10 minutes. Then,the slides were dehydrated following a standard procedure andsealed with cover glass using mounting solution. Terminal deox-ynucleotidyl transferase–mediated dUTP nick end labeling(TUNEL) reaction was done as described in the manual for InSitu Cell Death Detection Kit, POD (Hoffmann-La Roche Ltd.).

Chemical libraryPersonal synthetic chemicals and natural compounds library

has been described previously (27, 28). Korea chemical bank alsoprovided 8,000 chemicals for this study.

ELISATo detected p14/ARF-DX2 binding inhibitor, we generated the

screening system based on ELISA. We immobilized His-DX2recombinant protein on 96-well plate with 0.5% paraformalde-hyde. After drying and washing, we incubated GST-p14/ARF pro-teinwith0.1mmol/L chemicals (final concentration).After 1hour,plates were washed using TBS-T and incubated with anti-GSTantibody (1:10,000 for 30 minutes) and anti–mouse-IgG-horse-radish peroxidase (HRP; 1:50,000 for 1 hour). After washing twice,plates were incubated with 3,30,5,50tetramethylbenzidine (TMB)solution (Calbiochem) and stop solution (1N H2SO4). There-after, using the ELISA reader, we determined the value at450 nm. More detail information about this ELISA systemcould be obtained from our previous literature (28).

Recombinant proteins, immunoprecipitation, and GST pull-down assays

The human p14/ARF fragment (full-length) was ligatedinto the EcoRI and HindIII sites of the pGEX-TEV vector, whichis a modified vector by adding a TEV protease cleavage site to

Oh et al.

Cancer Res; 76(16) August 15, 2016 Cancer Research4792




pGEX-4T1 (Invitrogen). The recombinant proteinswere expressedin the Escherichia coli (E. coli) strain BL21 (DE3) as a GST-fusionproteins. The proteins were purified by glutathione affinity chro-matography. To address the direct binding between two proteins,agarose bead–conjugated GST (negative control) or GST-targetprotein was incubated with cell lysate or recombinant protein inRIPA buffer for 1 hour at 4�C. Immunoprecipitation (IP) assaywas performed with cell lysate or recombinant protein with RIPAbuffer. The whole lysates were incubated sequentially with appro-priated antibodies for 2 hours at 4�C and then the mixtures wereaddedproteinA/Gagarosebeads–conjugated secondary antibody(Invitrogen) for 2 hours. After incubation, mixtures were washedusing RIPA buffer 2 times. Precipitated proteins were determinedby Western blot analysis.

Immunofluorescence stainingCells grown on coated cover glasses were fixed with 100%

MeOH for 1 hour at�20�C. After washing with PBS and blockingin PBS-based buffer [containing nonrelated Goat antibody(1:500)] to eliminate nonspecific reaction, cells were incubatedwith primary antibodies (1:50�1:200; overnight at 4�C) andwithproper FITCorRhodamine-conjugated secondary antibodies for 4hours � overnight at room temperature. 4, 6-diamidino-2-phe-nylindole (DAPI) was used to stain nuclei. After washing withPBS, cover glasses were mounted withmounting solution (VectorLaboratories; H-5501) and subjected to fluorescence microscopicanalysis (Zeiss).

MTT assayTo examine cell viability or proliferation, MTT (3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assaywas performed. Cells were incubated in 0.5 mg/mL of MTTsolution for 4 hours at 37�C. After removing solution, formazanproducts were dissolved in dimethyl sulfoxide (DMSO) andmeasured by reading absorbance on a spectrophotometer at540 nm.

Senescence-associated b-galactosidase stainingCells grown on coverslips were stained by using a senescence-

associated b-galactosidase staining kit (9860S; Cell SignalingTechnology, Inc.). In brief, cells,fixedwith 4%paraformaldehyde,were incubated with staining solution for overnight at 37�Cwithout CO2. Stained cells were observed under light microscopeafter mounting with VectaMount solution (Vector Laboratories).

Human lung cancer tissue samplesEight pairs of frozen human normal and lung cancer tissue

samples were provided by a biobank in Soonchunhyang Univer-sity Bucheon Hospital (schbc-biobank-2011-003). Part of tissueswas used for extraction of protein, total RNA, and DNA, andanother part was fixedwith formalin for tissue analysis. The cDNAwas isolated from normal (N) or tumor (T) regions in the humanlung tissues of total eight cases, and then DX2 transcripts werechecked using PCR with specific primers. The primer sequenceswere as follows: for DX2 (forward 50-AACGTGCACGGCAG-GAGCTAC-30, reverse 50-CCAGCTGATAGTCTTGGCGGG-30) andfor GAPDH (forward 50-ATCTTCCAGGAGCGAGATCCC-30,revere 50-AGTGAGCTTCCCGTTCAGCTC-30). Detection of onco-genic K-Ras mutation was performed by previous literature (29).

Human patient's serum analysisHuman normal, SCLC, and NSCLC patient's sera were also

obtained from a biobank in Soonchunhyang University BucheonHospital (schbc-biobank-2011-003). To test the autoantibodiesagainst DX2, we attached recombinant DX2, Lamin A, and Snailonto nitrocellulose membrane (0.5 ng/well). Each membranewas incubated with patient's serum, diluted with blocking buffer(1:1,000, for 1 hour), and sequentially HRP-conjugated anti-human antibody (1:20,000) for 30 minutes. Associated Abs toproteins were visualized by ECL and X-ray film exposure. Moredetail procedure was available in our previous literature (28).

Analysis of tumor incidence and areaTo evaluate tumor incidence, lung tissues of each mouse was

fixed and embedded in paraffin. Five sections from each mousewere examined by three independent investigators who countedtumor. In addition, tumor area was calculated by tumor occupiedarea in total lung area using Photoshop soft wear.

Statistical analysisToobtain the statistical significance,weperformed the Student t

test. In addition, to evaluate the significant enhanced effect incombination treatment with SLCB050 and conventional antican-cer drug,we compared the simple added effect and realMTT value.

ResultsThe expression of DX2 in lung cancer tissue and cell lines

To examine the role of DX2 in lung carcinogenesis, we firstmeasured DX2 expression in human lung cancer tissues andfound that, despite strong DX2 transcription even in adjacentnoncancerous tissues, DX2 protein was detected primarily incancer tissues with K-Ras-or Her2/Neu-activation (Supplementa-ry Fig. S1A and S1B). This result implied that DX2 expression attranscription level did not relate to lung cancer progression. Weobtained similar results in human lung cancer cell line analysis(Fig. 1A). In addition, K-Ras–mutated (A549 and H460) andPTEN silent (H1299) cell lines showed the elevated DX2 expres-sion. Indeed, activated Her2/Neu, AKT, and oncogenic K-Rasincreased exogenous (Fig. 1B and C; Supplementary Fig. S1C)and endogenous DX2 expressions without alteringDX2 transcrip-tion (Fig. 1D). In addition, stabilized DX2 by oncogenes wastranslocated into nucleus (Fig. 1E). These results implied thatEGFR-AKT-K-Ras signaling cascade regulates DX2 expression andlocalization at posttranscriptional level. So, we next checked theDX2 protein stability and turnover rate. Rapid turnover of DX2within 1 hour (Fig. 1F) was extended up to 3 hours by oncogenicK-Ras (Fig. 1G) or phosphatase inhibitor (Supplementary Fig.S1D). To reveal the responsible E3 ligase, we examined the effectof several E3 ligase on AIMP2 and DX2 and found that Siah-1eliminated DX2 (Fig. 1H). Elimination of Siah-1 could increaseDX2 expression (Supplementary Fig. S1E), and Siah-1 showed thebinding ability to DX2 (Supplementary Fig. S1F). Indeed, Siah-1could promote DX2-Ubiquitinylation (Supplementary Fig. S1G).Because Siah-1 is transcriptional target of p53 (30), DX2would beregulated by p53 pathway negatively and by oncogenic signalingpositively.

DX2 provided drug resistanceBecause DX2 was stabilized by oncogenes, we speculated

that it would be related with cell growth and proliferation.

Antitumor Effect of a Novel p14/ARF-DX2 Binding Inhibitor

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Figure 1.

Oncogenic stimulations induceDX2 expression at posttranslation level.A, differential expression of DX2 in human lung cancer cell lines. H322 cells had extremely lowDX2 protein expression (red arrow) despite a transcription level (red arrow) that was similar to those in the other cell lines. In contrast, A549 and H460 cells had highDX2 protein expression. AIMP2 is indicated by the black arrows. Actin and GAPDH were used as loading controls for transcription and protein expression,respectively. B, Her2/Neu increases DX2 expression. Wild-type (WT) and constitutively active (CA) Her2/Neu but not KD Her2/Neu induced DX2. In contrast,AIMP2 was not induced by Her2/Neu. HEK293 cells were transfected with the indicated vectors for 24 hours. C, active AKT increases DX2 expression.HEK293 cells were cotransfected with AIMP2 or DX2 and AKT expression vectors for 24 hours. AKT-CA (myristoylated AKT) selectively induced DX2 expression.D, oncogenes induce endogenous DX2 in H1299. Transfection with AKT-CA (myristoylated AKT), Her2/Neu-CA, and oncogenic K-Ras into H1299 for 24 hourscould increase endogenous DX2. E, oncogenes promote nuclear localization of DX2. Although transfected DX2 was located in the cytoplasm in HEK293 cells,AKT-CA and Her2/Neu-CA promoted the translocation of DX2 into the nucleus. In contrast, AKT-KD and Her2/Neu-KD suppressed DX2 expression. F, rapidturnover of DX2. Compared with AIMP2, DX2 protein showed very short half-life (less than 1 hour). Transfected cells were incubated with cycloheximide(CHX; 100 mg/mL) for indicated times. G, oncogenic K-Ras extends the half-life of DX2. DX2-transfected HEK293 cells were cotransfected with the indicated Rasproteins. After 6-hour incubation with MG132 (10 mmol/L) to block protein degradation, cells were incubated with cycloheximide for the indicated times to blockde novo protein synthesis. However, AIMP2 did not affect its half-life by Ras transfection. H, Siah-1 inhibits DX2 expression. Compared with other E3 ligases,Siah-1 selectively inhibited DX2 expression. However, AIMP2 was not affected by E3 ligases.

Oh et al.





However, in MEF, obtained from DX2 transgenic and K-RasLA2/DX2 double transgenic (DK) mice, DX2 did not showobvious effect on cell proliferation, comparing to oncogenic K-

Ras (Supplementary Fig. S2A). Instead, DX2 expressed MEFshowed the resistance to Adriamycin- or serum starvation–induced cell death and senescence (Fig. 2A and Supplementary

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DX2 provides drug resistance. A, resistance to senescence in DX2 and DK MEF. MEF cells were cultivated in serum-free condition (SF) or in low dose of Adr-treated condition for 48 hours to induce senescence. Compared with wild-type (WT) and K-Ras MEF, which were stained by SA-b-gal and showed thetypical senescent morphology, DX2 and DK cells did not show the obvious response. B, resistance of DX2 and DX2/K-RasLA2 double transgenic (DK) cells top53-activating chemical. MEF cells were incubated with 5 mmol/L of GN25, activator of p53 for 6 hours. The obvious reduction of cell viability by GN25 in K-Ras MEFwas completely blocked in DK cells. C, Si-DX2 suppresses cell viability in a p14/ARF-dependent manner. Elimination of DX2 using si-DX2 (0, 1, 5, or 10 mg/mL)reduced the viability of H1299, H23, and H69 cells, which are p14/ARF positive. In contrast, p14/ARF-null cell lines were resistant to si-DX2. NULL and MTindicate homozygote deletion and mutant, respectively. D, differential localization of DX2 in lung cancer cell lines. Nuclear DX2 was detected in H1299 cells.The localization of AIMP2 did not differ between A549 and H1299 cells. E, DX2 blocks oncogene-induced p14/ARF. Increase of p14/ARF by Her2/Neu was abolishedby DX2 transfection. HEK293 cells were transfected with indicated vectors for 24 hours.






Fig. S2B). Indeed, DK MEF cells were completely resistant toselective p53 activator (GN25; ref. 28) on K-Ras–activatedcells (Fig. 2B). To get more clues for pathologic role of DX2on cell survival, we measured cell viability after inhibition ofDX2. We found that transfection of siRNA for DX2 (si-DX2)suppressed cell survival in a p14/ARF-dependent manner(Fig. 2C; refs. 31, 32). Because oncogenic stress can triggerp53 activation through p14/ARF (11), we hypothesized thatthe oncogenic property of DX2 is related to p14/ARF. Indeed,p14/ARF seemed to determine the localization of DX2. In p14/ARF-null cell, overexpressed DX2 was not located in nucleus(Fig. 2D), even in the same K-Ras–mutated cancer cell line,HCT116, although DX2 was able to localize in the nucleus orcytoplasm (Supplementary Fig. S2C and S2D). Moreover, DX2,but not AIMP2, could block the Her2/Neu-induced p14/ARF(Fig. 2E).

DX2 contributed to SCLC occurrenceNext, we examined the in vivo oncogenic effect of DX2 using

transgenic mouse model (18). Differentially from AIMP2knockout mice, which are neonatal lethal due to defect of lungepithelial cell differentiation (15), DX2 transgenic mice areviable, suggesting that DX2 overexpression did not affect lungcell differentiation. Although DX2 transgenic mice did notproduce tumor at 4 months old (Fig. 3A and B), comparingto K-RasLA2, DK mice bore larger tumor (Fig. 3B and Supple-mentary Fig. S3A) with low apoptotic cells (Fig. 3C). In addi-tion, besides commonly detected typical adenocarcinomas inboth K-RasLA2 and DK mice, small nuclear atypical tumorswere detected between blood vessels and airways in DK mice(Fig. 3B and Supplementary Fig. S3A). To identify these tumors,we performed the IHC staining with pan-keratin (for squamouscell cancer; ref. 33), pro-surfactant protein C (for adenocarci-noma; ref. 33), and neuron-specific enolase (NSE; for SCLC;refs. 34, 35). NSE staining was visible in the small cell tumorregion in DK-transgenic mice (Supplementary Fig. S3B). Wealso observed NSE-positive SCLC cells in tumors of DX2-trans-genic mice (6-month-old), but not in K-RasLA2 mice (Fig. S3C).Small cell cancer mass was increased following age in DX2transgenic mice (Supplementary Fig. S3D and S3E). Indeed, thetumor cells in DX2 mice had a high mitotic index and finegranular chromatin (Supplementary Fig. S3F), which are well-known cellular markers of SCLC (13). We also observed theNSE expression in primary tumor cells isolated from DX2 andDK tumors (Fig. 3D). On the basis of histologic features (Sup-plementary Fig. S3G), we analyzed tumor types in mousemodels and found that DX2 and DK mice could promoteSCLC occurrence (Fig. 3E). In fact, DX2 expression was elevatedin SCLC cell lines at translation level (Fig. 3F and G). BecauseSCLC tissues are not available, we could not directly detect theDX2 expression in human cases. Instead, because DX2 is abnor-mal gene product and many DX2-positive cancer cells wouldbe died by necrosis, we measured the DX2 autoantibody inhuman patient's sera. Although it is indirect method, we alreadytested this method for detection of cancer-specific protein.Indeed, DX2-specific autoantibody was frequently detected inSCLC sera (9/10 cases) and moderately in NSCLC (10/20 cases),whereas it was rarely produced in normal sera (1/10; Fig. 3H;Supplementary Fig. S3H; Supplementary Table S1). These re-sults suggest that DX2 is closely related with lung cancer occurr-ence and progression, in particular SCLC.

DX2 suppresses p14/ARFTo know the biological role of DX2, we tested the oncogene-

induced p14/ARF. Indeed, Her2/Neu-AKT-K-Ras signaling cas-cade can induce p14/ARF and promote oncogene-induced apo-ptosis and senescence, whereas DX2 provided the resistance tosenescence (Fig. 2A) and GN25-induced cell death in K-Ras–mutated MEF (Fig. 2B). Moreover, stabilized DX2 by oncogeneswas located in nucleus as p14/ARF-dependent manner (Figs. 1Gand 2D). First, we evaluated the expression of p19/ARF, mousep14/ARF (36, 37), in DX2-expressed MEF and found that it wasobviously reduced in DX2-expressed MEF (Fig. 4A). Similarly,elimination of DX2 could increase endogenous p14/ARF inseveral kinds of human cancer cell lines including H1299(Fig. 4B and Supplementary Fig. S4A) as well as exogenousp14/ARF inA549 (Fig. 4C andSupplementary Fig. S4B).However,si-DX2 did not increase p14/ARF expression in DX2-negative cellline (H322; Fig. 4C) and nontransformed cell lines (HEK293 andL132; Supplementary Fig. S4A). Considering the fact that si-DX2could induce exogenous p14/ARF, it would be achieved at post-translational level. In fact, si-DX2 could obviously increasep14/ARF stability (Fig. 4D) and promote nucleoplasmic location(Fig. 4E). In addition, DX2 promoted p14/ARF ubiquitination(Fig. 4F), and MG132, proteasome inhibitor, blocked the DX2-mediated p14/ARF reduction (Fig. 4G).

Direct interaction between DX2 and p14/ARFOur next question is how DX2 suppresses p14/ARF. To address

this, we first checked the interaction of them. CotransfectedDX2 and p14/ARF were recovered in nucleolus (Fig. 5A andSupplementary Fig. S4C). We could observe the physiologic inter-action of DX2 and p14/ARF through bidirectional IP analysis (Fig.5B and Supplementary Fig. S4D). Direct interaction betweenDX2 and p14/ARF was observed by in vitro GST pull-down assayusing His-DX2– or AIMP2-recombinant protein (Fig. 5C). Inter-esting feature is that AIMP2was coprecipitatedwithGST-p14whenDX2was coexisted, indicating that DX2 could bind to AIMP2. p53-GST was used for positive control (18). To avoid the nonspecificinteraction,we also checked thebinding ofDX2andGST-Smad4 orvon Hippel-Lindau tumor suppressor (VHL). However, theseproteins did not pull down DX2 (Supplementary Fig. S4E andS4F). Instead, AIMP2was precipitated with GST proteins, implyingthat AIMP2 would be sticky or multifunctional protein. Actually,AIMP2 is a cofactor of protein complex (16). These results indicatethatDX2-p14/ARF interaction is specific event.To confirmthedirectinteraction,we re-executedGSTpull-downassayusing recombinantDX2 and obtained the same result (Fig. 5D). Because p14/ARF isvery sticky protein (38), we did not exclude the nonspecific asso-ciation of DX2 and p14/ARF. So, we re-performed IP analysis withHis-AIMP2 or DX2. In this assay, we observed the selective asso-ciation of DX2 and N-terminal p14/ARF (Fig. 5E and F).

Isolation of specific binding inhibitor of DX2 and p14/ARF bychemical screening

Because DX2 blocks the oncogene-induced p14/ARF induction(Fig. 2E),we speculated that specific inhibitor againstDX2andp14/ARF bindingwould be useful for anticancer drug. So, we performedthe chemical screening (SupplementaryFig. S5A) throughmodifiedELISA system (Supplementary Fig. S5B). After testing of ELISAsystem (Fig. 6A), we screened chemical library (SupplementaryFig. S5C). Because we have screened the same library using otherprotein binding inhibitor (Pak1-PUMA binding), we excluded the

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DX2 promotes SCLC production.A, tumor incidence in 4-month-oldmice. Tumor areawas calculated from five sections per sample.B, hematoxylin–eosin staining oflung tissues from the indicated transgenic mice (4-month-old). DX2/K-RasLA2 double transgenic (DK) mice had two kinds of tumors. C, DX2 induced resistanceto apoptosis in contrast to numerous TUNEL-positive cells in tumor tissues of K-RasLA2 mice; tumor cells from DK mice were barely stained by TUNEL. D,immunofluorescence staining with NSE. To confirm that the tumors in DX2 and DK mice were SCLC tumors, tumor cells were isolated from lungs and cultured for2 weeks to eliminate primary cells such as lymphocytes. Despite serial cultivation, small cells grew well. After fixation with MeOH, cells were stained with NSE.Normal lung epithelial cells were not stained by NSE and had large nuclei. In contrast, small cells from DX2 or DK mouse tumors were strongly stained byNSE. Small cells were clearly distinguishable from normal epithelial cell (white arrows). E, Ban diagram of lung tumor spectrum in each mouse model. Lung biopsywas performed with mice that showed the pathologic symptoms such as dyspnea. Thus, average age (AveAge) of each group was different and representsapproximate onset time. Note that this result contained 4-month-old analysis (Fig. 4A), and some of DX2 single mice were scarified without symptoms. SCLCwas always detected with other kinds of cancers. F, DX2 expression in SCLC cell lines. Two SCLC cell lines had strong DX2 expression like K-Ras–mutatedH460 (red arrowhead). However, AIMP2 expression levels did not differ betweenNSCLC and SCLC cells.G, analysis of DX2 andAIMP2 transcription, with GAPDH as acontrol. H, detection of autoantibody against DX2 in sera from SCLC patients. Immobilized DX2 was recognized by antibody in 9 of 10 SCLC sera and 8 of 20NSCLC sera (indicated in red) but not in sera of healthy individuals. Lamin A and Snail were used as positive controls.






commonly isolated chemicals from candidate (Supplementary Fig.S5AandS5D).Next,wedirectly checkedspecific inhibition throughGST pull-down assay using DX2-p14/ARF and p53-p14/ARF (Sup-plementary Fig. S5E and S5F). Finally, we obtained five chemicalsthat were selectively inhibited DX2-p14/ARF. Because we did notobtain enough amounts of four chemicals for further experimentinstantly, we went to next step with SLCB050 (Fig. 6B) that couldblock the interaction of DX2 and p14/ARF more obviously thanSLC24, randomproteinbinding inhibitor (Supplementary Fig. S5Dand S5G). Indeed, SLCB050 inhibited DX2-p14/ARF binding inin vitroGST pull-down assay (Fig. 6C and Supplementary Fig. S6A)as well as IP experiment (Fig. 6D) in a dose-dependent manner(Supplementary Fig. S6B). SLCB050 also blocked the interaction ofDX2 and AIMP2 (Fig. 6E), but not on p53-AIMP2 or DX2 binding(Supplementary Fig. S6C) and p14/ARF (Supplementary Fig. S6D),indicating that SLCB050 would be interacted with DX2-specificregion. To verify that SLCB050 is a specific inhibitor of DX2-p14/

ARF, we performed the GST pull-down assay with SLCB050 and itsvery similar chemicals (HJH141204, HJH141206; stereoisomer)and related chemical (SLCB036: ribose ring is replacedbymodifiedbenzene ring; Supplementary Fig. S6E). We also checked the newlysynthesized SLCB050 to exclude the synthetic error. Obviousinhibition effect on p14/ARF-DX2 binding was observed inSLCB050, HJH141204, and HJH141206 (Supplementary Fig.S6F). However, these chemicals did not alter the interaction ofp53-p14/ARF (Supplementary Fig. S6G), and SLCB036 did notshowthe inhibitory effect onbothbinding (Supplementary Fig. S6Fand S6G). These results indicate that unique chemical structure (inparticular, ribose ring) is required for the binding inhibition.

Antitumor effect of the chemical in small-cell lung cancercell lines

So, we next examined the effect of SLCB050 in cell system.Treatment of SLCB050 could block the interaction (Fig. 6F) and

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DX2 suppresses p14/ARF expression. A, reduction of p19/ARF in DX2 and DK MEF. p19/ARF expression was reduced to undetectable levels in DX2 and DK MEF.B, si-DX2 induces p14/ARF expression in lung cancer cell line H1299. Cells were transfectedwith indicated concentration of si-DX2 for 24 hours. Induction of p14/ARFwas detected following dosage of si-DX2. C, si-DX2 increases ectopic expression of p14/ARF. In p14/ARF-transfected A549 and H322 cells, si-DX2 inducedexogenous p14/ARF expression. D, elimination of DX2 increases p14/ARF stability. H1299, transfected with p14/ARF or DX2, was cotransfected with si-DX2 for24 hours. Protein half-life was measured after treatment of cycloheximide (CHX). E, increase of p14/ARF in nucleoplasm by si-DX2. In GFP-p14/ARF-transfectedA549, elimination of DX2 could increase p14/ARF in nucleoplasm. F, DX2 promotes the ubiquitylation of p14/ARF. HEK293 cells were transfected with the indicatedvectors for 24 hours. After incubation with MG132, cell lysates were analyzed by IP with p14/ARF antibody. Ubiquitin (Ub)-conjugated p14/ARF was detectedat high molecular weight (arrowheads). G, the proteasome inhibitor MG132 (10 mmol/L, 6 hours) reverses DX2-induced p14/ARF suppression and inhibits DX2.

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colocalization of DX2 and p14/ARF in nucleus (Fig. 6G). Indeed,this chemical could suppress DX2 expression itself through rapiddegradation (Supplementary Fig. S7A) and relocalization fromnucleolus to cytosol (Supplementary Fig. S7B). Thus, we couldobserve the increase of p14/ARF in SLCB050-treated H1299, H69(Fig. 6H and Supplementary Fig. S7C), as well as reduction of DX2in p14/ARF-deficient cell lines (H322, H460, and A549; Fig. 6I;

Supplementary Fig. S7C). We next measured the cell viability inseveral lung cancer cell lines and found that p14/ARF-deficient celllines were resistant to SLCB050, whereas SCLC cell lines weresensitive to it (Fig. 6J). In fact, SLCB050 completely suppressedthe H128 cell growth (Fig. 6K and Supplementary Fig. S7D).Moreover, SLCB050 derivatives, HJH141204 and 1206, but notSLCB036, also suppressed cell viability (Supplementary Fig. S7E),

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Direct interaction of p14/ARF and DX2. A, colocalization of p14/ARF and DX2 in nucleolus-like structures. HEK293 cells were transfected with GFP-p14/ARF(top) or cotransfected with GFP-p14/ARF and DX2 (bottom) for 24 hours. Cells were incubated with anti-Myc antibody and Rhodamine-conjugatedsecondary antibody (red). GFP-p14/ARF in the nucleoplasm was reduced by DX2 transfection. B, IP analysis using His-DX2. Cells, transfected with indicatedvectors, were incubated with His-DX2 recombinant protein. After IP with His Ab, coprecipitated materials were analyzed by Western blot analysis.Specific interaction between p14/ARF and DX2 was observed. Sup, supernatant after IP analysis. Despite strong expression, GFP-nucleolin did not show theinteraction with DX2, indicating that DX2-p14/ARF binding was a specific event. C, specific interaction of p14/ARF with DX2 but not with AIMP2. A GSTpull-down assay was performed using bead-GST-p14/ARF and His-DX2 or His-AIMP2. A pull-down assay with GST-p53 was performed as a controlexperiment. D, in vitro binding assay. Recombinant His-DX2 comigrated with bead-conjugated recombinant GST-p14/ARF. E, the N-terminal regionof p14/ARF serves as the binding domain for DX2. Full-length p14/ARF (p14/ARF-F; AA 2–132) and the N-terminal p14-ARF fragment (p14-N; AA 2–29) weretested in a binding assay. His-DX2 was strongly associated with p14/ARF-N. F, p14/ARF binding region. A region of DX2 generated by the joining of exons 1 and3 binds to the N-terminal region of p14/ARF.






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indicating that tumor cell growth suppression is achieved bychemical-DX2 binding.

The combinational effect of SLCB050 with antitumor drugsWe previously showed that DX2 provided the drug resistance

(Fig. 2B; Supplementary Fig. S2A and S2B), and others reportedthat p14/ARF is critical for drug sensitivity (39). So, we tested thatSLCB050 could restore the sensitivity to anticancer drug. Resis-tance to GN25 inDX2 andDKMEFwas abolished by cotreatmentof SLCB050 (Fig. 7A). We also observed the significant enhancedeffect of SLCB050 with Adr in DX2 and DK MEF (Fig. 7B) andSCLC cell line H69 that was partially responded to SLCB050 (Fig.7C). However, p14/ARF-deficient H322 did not show enhancedresponse to combinational treatment (Fig. 7C). To extend this, wegenerate tumor xenograft model using H446 that was resistant toAdr and partially responded to SLCB050 (Supplementary Fig.S7F). Exponentially increased tumor volume was moderatelysuppressed by SLCB050 injection (10 mg/kg, 3 times/week;Fig. 7D and Supplementary Fig. S7G). Although we also observedthe tumor repression effect by injection of 5 mg/kg Adr (3 times/week), it evoked rapid weight loss and death (Fig. 7D andSupplementary Fig. S7H). In contrast, combinational treatmentshowedmore obvious antitumor effect despite low dosage of Adr(Fig. 7D). Indeed, combinational treatment of Adr and SLCB050could obviously induce p53 expression in primary tumor cellsobtained from DX2 or DK mouse (Fig. 7E). Next, we tested theeffect of SLCB050 on nontransformed lung cell (L132). However,this chemical did not alter the cell viability or drug sensitivityon these cell lines (Supplementary Fig. S7I). Next, we checked theeffect of combination treatment of SLCB050 with other commer-cial anticancer drugs such as Taxol and Carboplatin. However,these chemicals did not show significant enhanced effect withSLCB050 (Supplementary Fig. S7J). These results indicate thatinhibition of DX2 could enhance drug sensitivity through reac-tivation of p14/ARF in DX2-overexpressed cancer cells.

In vivo antitumor effect of SLCB050To explore the effect of SLCB050 in mouse model, we inject-

ed it into DK mice following our experimental schedule (Sup-plementary Fig. S8A). Combinational treatment of SLCB050and low-dose Adr strikingly suppressed tumor progression(Fig. 7F and G) without significant weight loss (SupplementaryFig. S8B). However, nontoxic dose of Adr did not show anti-

tumor effect on this mouse model (Fig. 7F and G; Supplemen-tary Fig. S8B). More detailed histologic analysis showed thatSCLC region was more obviously erased by combinationaltreatment (Supplementary Fig. S8C). In DX2-reduced condition(Fig. 7H), apoptotic tumor cells were obviously increased(Fig. 7I and Supplementary Fig. S8D). Our results indicate thatDX2, produced by aberrant splicing of AIMP2, promotes tu-mor progression, in particular small cell lung cancer, via directinteraction and inhibition of p14/ARF (supplementary Fig. S9).Thus, under DX2-expressed conditions, oncogene-induced tu-morigenesis would be easily progressed because oncogene-induced cell death or senescence is abolished.

DiscussionAlthough DX2 is elevated in human lung cancer (16, 18),

physiopathological role of DX2 has not been revealed. In thisstudy, we found that DX2 is stabilized by oncogenic stressessuch as oncogenic K-Ras or Her2/Neu-AKT signaling activation(Fig. 1). Concerning this, siah-1 is speculated as responsibleE3-ligase (Fig. 1H and Supplementary Fig. S1F and S1G). How-ever, we did not exclude the involvement of other mechanismon DX2 destabilization, because despite Siah-1 knockdown,AKT can induce DX2 expression (Supplementary Fig. S1E) andphosphatase inhibitor can extend DX2 half-life (Supplemen-tary Fig. S1D). Thus, more intensive investigation on DX2regulation should be performed, including how DX2 is tran-scriptionally induced.

Based on differential sensitivity of DX2 transgenic MEF to p53activators and cellular distribution, we assumed that oncogenicrole of DX2 would be related to nuclear proteins (Fig. 2D) anddrug resistance (Fig. 2A and B). Moreover, the effect of DX2knockdown is fully dependent on p14/ARF status but not onp53 status (Fig. 2C). Indeed, si-DX2 can suppress the viability ofH1299, p53-null lung cancer cell line but not that of A549, despiteintact p53 (Fig. 2C). In addition, DX2 blocks the oncogene-induced p14/ARF expression (Fig. 2D). These results suggest thatoncogenic property ofDX2 is tightly relatedwith p14/ARF. In fact,DX2 suppresses p14/ARF expression (Fig. 4) through direct inter-action (Fig. 5).

In general, oncogenic stimulations activate p53 pathway viap14/ARF and resulted in senescence or apoptosis. Thus, cancercells should escape from oncogene-induced cell death or

Figure 6.Isolation of SLCB050 as an inhibitor of DX2-p14/ARF binding. A, ELISA-based screening method. To confirm the validity of the ELISA system, we measuredincreases in ELISA values with increasing concentrations (Conc) of p14/ARF (left). One chemical, SLCB050, reduced the ELISA reaction considerably (arrow in theright panel). B, chemical structure of SLCB050. C, inhibition of the binding of p14/ARF to DX2. A GST pull-down assay showed complete blocking ofDX2-p14/ARF binding by SLCB050. His-DX2 (red arrow) was detected in a higher molecular weight range than AIMP2 (black arrow) was, because His-DX2recombinant protein was fused with thioredoxin. D, SLCB050 disrupts the interaction of p14/ARF and DX2 in cells. Cotransfected HEK293 cells wereincubatedwithMG132 before IP analysis to prevent the reduction of both proteins. After incubationwith SLCB050 (10 mmol/L, 6 hours), cellswere used for IP analysiswith Myc antibody (DX2). IgGH, immunoglobulin G heavy chain. E, inhibitory effect of SLCB050 on the binding between DX2 and AIMP2. In protein bindingassay using His-DX2 protein, binding between DX2 and AIMP2 was reduced by SLCB050 treatment. F, dissociation of DX2 and p14/ARF. Through IP analysis,dissociation of DX2 and p14/ARF was detected. HEK293 cells were transfected with indicated vectors for 24 hours and incubated with additional 6 hourswith MG132 and SLCB050. G, differences in localization of DX2 and p14/ARF after SLCB050 treatment. Colocalization of DX2 and p14/ARF (top panels) wasdisrupted by SLCB050 (10 mmol/L, 6 hours). DX2 was decreased and p14/ARF was increased in the nucleoplasm. H, SLCB050 treatment increases p14/ARF in theNSCLC cell line H1299 and the SCLC cell line H69. SLCB050 treatment at the indicated concentrations for 6 hours reduced DX2 and increased p14/ARF.I, dose-dependent reduction of DX2. DX2 levels decreased considerably in the p14/ARF-null H322 cells in response to SLCB050 treatment (6 hours). J, effect ofSLCB050 on viability of human lung cancer cells incubated with the indicated concentrations of SLCB050 for 24 hours. Cell viability was determined by MTT assay.SCLC cells were very sensitive to SLCB050. K, soft-agar colony formation assay. H128 cells (SCLC cell line) were seeded in soft-agar plates and incubatedwith the indicated concentrations of SLCB050 for 48 hours. Cells were visualized by trypan blue staining after fixation. Colonies were counted (bottom).






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senescence. In previous, we revealed that induction of Snail inresponse to oncogenic K-Ras suppresses p53 in pancreatic cancer(27). Similarly, Her2/Neu-AKT pathway inhibits activation ofp14/ARF-p53 network through DX2 induction. However, elimi-nation of DX2 via siRNA or SLCB050 could suppress cell viabilityin p53-mutated cells (Figs. 2C and 6J), suggesting that p14/ARFexecutes additional tumor-suppressive functions. Indeed, it hasbeen reported that p14/ARF regulates cell proliferation throughribosomal RNA processing (40). Thus, we are now investigatingthe effect of DX2 on this.

More interesting feature is that DX2 transgenic mice, includ-ing DK mice, generate SCLC (Fig. 3 and SupplementaryFig. S3). In previous, SCLC mouse model is p53/pRb doubleknockout system (41). Although p53 and pRB are most fre-quently deleted genes in SCLC, they are also important formost types of cancers, including NSCLC, colon cancer, pan-creatic cancer, and even in sarcoma (42). Thus, this model hassome limitation for study of SCLC-specific carcinogenesis.Although our double transgenic model (DK mice) can induceSCLC with NSCLC, it would be useful for investigation ofearly SCLC carcinogenesis.

For the human study, we were looking for human specimen.However, unfortunately, we did not obtain the fresh SCLCtissues, because standard protocol of SCLC does not includesurgery. Instead, we obtained the human serum of SCLC. In ourprevious study, some kinds of proteins that are elevated inhuman cancer could work as autoantigen (27). Thus, we alsocheck the DX2 autoantibody in this study. Indeed, we coulddetect DX2 autoantibody from SCLC patient's sera (Fig. 3H).Although it is a prototype at present, more development wouldprovide a new rapid detection method for SCLC through DX2autoantibody. Concerning this, we are examining the physio-pathological relevance of SCLC and DX2 expression. Becausep14/ARF is inactivated in SCLC without genetic mutation (43),DX2 may contribute to this.

Because we provide the evidence about interaction of DX2and p14/ARF, our next step is screening of binding inhibitorthat is required for proof of our concept. Indeed, though ELISA-based chemical screening, we found small chemical inhibitorsagainst p14/ARF-DX2 binding. This chemical (Fig. 6B) and itsresembling derivatives (Supplementary Fig. S6E) could sup-press the interaction of DX2 and p14/ARF (Fig. 6C–G; Supple-mentary Fig. S6) and cell viability in a p14/ARF-dependentmanner (Fig. 6J). Moreover, these chemicals can induce p14/ARF expression and suppress DX2 (Fig. 6H and I).

In fact, SLCB050 show enough anticancer effect in small celllung cancer cell lines as p14/ARF-dependent manner (Fig. 6J).We also observed the significant enhanced effect with GN25,activator of p53 through inhibition of Snail-p53 binding (Fig.7A) or Adriamycin (Fig. 7B–E). However, SLCB050 did notshow even additive effect with EGFR inhibitors in NSCLC(data not shown) or TAXOL in SCLC (Fig. S7J). These resultssuggest that SLCB050 would be related with p53-p14/ARF-mediated anticancer signaling. In fact, combinational treat-ment of SLCB050 and low dosage of Adr could obviouslysuppress tumor progression and induce cell death in ourmouse model. This result indicates that inhibition of DX2 isvery attractive target for therapy for SCLC as well as NSCLC.However, until now, we do not completely understand howSLCB050 can induce cell death in p53-deficient SCLC (Fig.7C). To reveal this, we are now checking several possibilitiesincluding p73 activation or blocking of ribosomal RNAprocessing.

In conclusion, our results have demonstrated that DX2promotes SCLC progression by inhibiting p14/ARF and thatSLCB050 can suppress tumor progression by blocking DX2.SLCB050 may therefore be effective in treatment of lungcancers.

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

Authors' ContributionsConception and design: A.-Y. Oh, J. Kim, J.-H. Lee, G.-Y. Song, B.-J. ParkDevelopment of methodology: J. Kim, J.-H. Lee, G.-Y. Song, B.-J. ParkAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.-Y. Oh, Y.S. Jung, J.-H. Lee, J.-H. Cho, S. Park,N.-C. Ha, J.S. Park, C.-S. Park, G.-Y. Song, B.-J. ParkAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.-Y. Oh, H. Park, S. Lim, G.-Y. Song, B.-J. ParkWriting, review, and/or revision of the manuscript: A.-Y. Oh, J. Kim,G.-Y. Song, B.-J. ParkAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.-Y. Oh, J.-H. Lee, H.-Y. Chun, G.-Y. SongStudy supervision: B.-J. ParkOther (designed and synthesized specific inhibitor againstDX2 and p14/ARFbinding such as SLCB050): G.-Y. Song

Grant SupportThis research was supported by Basic Science Research Program through the

National Research Foundation of Korea (NRF) funded by the Ministry of

Figure 7.Antitumoral effect of SLCB050.A, significant enhanced effect of SLCB050with GN25, p53 activator in DX2 and DKMEFs. Resistance to GN25 in DX2 and DK cells wasabolished by SLCB050 treatment. Cells were incubated with 5 mmol/L of GN25 and SLCB050 for 6 hours. The viability was determined by MTT assay. ##, significantenhanced effect by combination treatment. B, significant enhanced effect of Adr and SLCB050 on viability of primary tumor cells from DX2 and DKmice. Cell viabilitywas determined by MTT assay after the cells were incubated with 0.2 mg/mL of Adr and/or 10 mmol/L SLCB050 for 24 hours. ##, significant enhanced effect bycombination treatment. C, sensitivity of SCLC cell lines to combination treatment with Adr and SLCB050, compared with the sensitivity of the p14/ARF-null H322 cells.Only H69 showed the significant enhanced effect by combination treatment with SLCB050 andAdr (##).D, tumor-suppressive effect of SLCB050. In xenograftmodelusing SCLCH446, 10mg/kgof SLCB050 suppressed tumorgrowth.AlthoughAdr (5mg/kg)blocked the tumorgrowth,within 4weeks, all of themwere ceasedbyhightoxicity (�). In contrast, lowdoseofAdr (1mg/kg)withSLCB050obviously suppressed tumorgrowthwithout severe toxicity. Chemicalsweredeliveredvia i.p injection3times per week. E, in primary tumor cells, combinational treatment could induce p53 synergistically. p53 expression was determined by Western blotting.F, hematoxylin–eosin staining of lung tumor tissues from SLCB050-treated DK mice. Combination treatment with SLCB050 and Adr induced tumor regression.G, reductionof tumorvolumebycombination treatment. Comparedwith tumorareas in age-matcheduntreated (DMSO)or single chemical-treatedDKmice (SLCB050:5 mg/kg and Adr: 1 mg/kg), tumor areas in mice treated with the combination of SLCB050 and Adr (AdrþSLCB050) were considerably smaller. H, reduction of DX2expression in mouse lung tumor tissues in response to SLCB050 treatment. I, TUNEL staining in combined treated DK mice lung tissues. Specific staining ontumor cells was detected in AdrþSLCB050-treated mice.






Science, ICT & Future Planning (NRF-2013R1A1A2010008; B.-J. Park), GlobalFrontier Project (2012M3A6A4054952; B.-J. Park), and by the Ministry ofEducation (NRF-2013R1A1A2062764; G.-Y. Song).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 21, 2015; revised April 21, 2016; accepted May 3, 2016;published OnlineFirst June 14, 2016.

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Oh et al.




2016;76:4791-4804. Published OnlineFirst June 14, 2016.Cancer Res Ah-Young Oh, Youn Sang Jung, Jiseon Kim, et al. Lung Cancer and Delays Tumor ProgressionInhibiting DX2-p14/ARF Interaction Exerts Antitumor Effects in

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