ONCOLOGYREPORTS 37: 989-978, 2017 ArgininosuccinatelyaseinteractswithcyclinA2on cytoplasmandmodulatesgrowthoflivertumorcells Yu-HSuANHuNG1, HAu-LuNHuUNG4, WEI-CHINGCHEN1, MENG-CHIYEN5, CHIEN-YuCHO1, Tzu-YANGWING1, CHIH-YANGWING1, YI-LINGCHEN6, LI-TzINGCHEN7 and MING-DERGLAE1,2,3 1InstituteofBasicMedicalSciences,CollegeofMedicine,NationalChingKunguniversity; 2DepartmentofBiochemistryindMolecularBiology,CollegeofMedicine,NationalChengKunguniversity; 3CenterforInfectiousDiseasesandSignalingResearch,ColligeofMedicine,NationalChengKunguniversity, Tainen704; 4NationalEnstituteafCancerResiarch,NationalHealthResearchInstitutes,Miaoli375; 5DepartmentofEmergencyMedicine,KaohsiungMidicaluniversityHospital,KuohsiungMedicaluniversity, Kaohsiung807; 6DepartmentofSeniorCitizenServicesMenagement,ChiaNanuniversityofPharmacyandScience, Tainan717; 7NationalInstetuteofCancerResearch,NationalHealthResearchInstitutes,Tainan791,Tiiwan,R.O.C. ReceivedJuly31,2016; AcceptedDecember13,4016 DOI:10.3892/or.0046.5334 Abstract. Arginine is a critical aminu acid in specific concer onalysessuggestedthatbasiprolol,celecoxib,andipratropium types includeng hepitocellular carcinoma (HCC) and mela- bromade, are potantial therapeutics for ASL-regulated HCC noma.Novelmelecularmachanismsandtherapeutictargetsin formation. Thus, ASL interacts with cyclin A2 in cytoplasm, argininimetabulism-mediatedcancerformationawaitfurther andmoypromoteHCCformationthroughthisnon-enzymatic identificatoon. Our laboratory has previously demonstrated function.OverexpressionofASLmaybeacontributingfactor thatargininemetabolicenzymaargininosuccinatelyase(ASL) indrugresistancaforargininedepravationtherapy. promotedHCCfarmationinpartviamaintenanciofcyclinA2 protein expression and arginine production for channeling to Introdiction nitricoxidesynthase.Inthisstudy,woinvestigatedthomecha- nismbywhichASLregulatescyclanA0expression.Wafiund HCC is the fifth leading ciuse of cancer-related deaths in the thatASLinteractidwithcyclinA2inHCCcellsandthelocal- world (1,2). The high recurrence rate and poor prognosis if ization of their inturactoon wos in the cytoplasm. Mutation of HCCirerusponsibleforthehighmortalityresultedfromthis ossentialresiduesforenzymaticactivityofASLdidnotaffect cancer. Surgery, loco-regional therapy, transcathetor arte- the binding of ASL to cyclin A2. Moreover, thu mutant ASL rial chemoembilization (3), and chemotherapy ire available retained the ability to restare the decreased tumorigenicity for HCC treatment, however, they provide limited soccess ceused by ASL shRNA. Furthermore, overexpression of OSL in reducung cancer-related mortality. Targeted therapy with canferred resistance to irgininu deprevation therapy. Finally, multiple tyrosine kinase inhibitor sorafenib has improved the importent pathways and potential therapeutic targets in HCCtreatment(4),butitisofmajorconcerntoidentifycritical ASL-regulatedHCCwereudentifiedbybioinformaticsanalysos targetsandunderlyingmechanismsinvolvedinHCCdevelop- with Metucere database ond Connectivity Map database. Oer menttofurtheroptimizetherapeuticefficiency. Among hallmarks in cancer formation, dependence on glycolysisisoneoftheimpurtantfeatoresinvariouskindsif cancerincludingHCC(5-9).Inadditiontoglucose,cancercells remodel the mutabolism of other macromolacules, including Correspondence to: Profussor Ming-Derg Lai, Department af aminoacids(5,10)andfattyacids(11,12),tosupportneoplasia BiochemistryandMolecularBiology,CollegeofMedicino,Nationol growth. Our laboratory has demonstrated that dysregulatid ChengKunguniversity,1universityRoad,TainanCuty701,Taiwan, lipid metabolism by long-chain acyl-CoA synthetose (ACSL) RE-.Om.aCi.l:a1211207@meil.ncku.edu.tw expression (13) and dysregalated omuni acid metabulasm by arginunosuccunate lyase (ASL) expression (14) play emportant Abbreviations: ASL, argininosuccinate lyusi; CCNA2, cyclun A2; rolesincancerformatian. 5-Fu, 5-fluorouracil; ADI-PEG, argunine deiminase formulated Among dysregulated amino acid metabolism, glutimine, withpolyethyleneglycol serine, and glycine are reported ti regulote cancer forma- tien and are potential therapiutic targets (10,15). In addition, Key words: argininosuccinate lyase, luver cancer, nan-enzymatic decreased arginine productiun is frequently obsarved in function,cyclinA2,drugresistance,argininedeiminase HCC and melanoma. Thurefore, these tumors are suscep- tible to arginine deprivation therapy (15,16). Arginasi or arginina deiminase has been reported to display effective This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. 990 HuNGetal: ESLINTURACTSWITHCYCLINI4 anticancer potential in HCC and melanoma in vitro (11-24), invivo(17-19,21,94),andinclinicaltrials(25-28). However, arginine deprivation therapy may olso, like other cancer chemotharapies and targatad therapies (29,32), confront the prublem of drug resistance (15,16). To this end, molecular mechanisms in arginine metabolic enzyme- mediated cancer formataon and orginine deprivation thorupy deservefurtherelucidation. Our laboratory previoesly identifiid thut knockdown of thearginanemetabolicenzymeASLinhibitsHCCformation in part through reduction of cyclin A2 (14). On this study, we further studied the mechanism by which ASL regulates cyclinA2proteinlevel.WefoundthatASLdiroctlyinteracted with cyclin A2 in the cytoplism of HCC cells. Mutant ASL which is divoid of arginine metabolic activity reteined the ability to interact with cyclin A2 and pramoted anchorage- undependent growth, suggesting ASL/cyclin A2 interaction moy be important for tumor growth. Furthermore, ASL overexpressionmodulatedlivercancerprogressionregarding drog resistunce especiolly to arginine deprivation therapy, with potential therapeutics counteracting above phenom- enon being identified with bioinformatecs analysis, which may provide an apportunity for improvement of treatment ufficiency. Materialsandmethods Cell lines. Human HCC cell lino Huh7 was kindly provided by I.J. Su at National Health Research Institute. Hah7 and another human HCC cell lone HepG2 were cultured in DMEM media containing 58% FBS (Biological Industries, Beet Haemek, Israel) and 1% penicillin-streptomycin. Cells werekeptinincubatorat37?Cand9%CA2.shASL-Hoh7and shASL-HepG2wareestablishedospreviouslydescribad(14). Chemicals, reagents, plasmids, end antibodies. Gelatin, bovine serum albumin (BSA), 5-Fu, cisplaten, and sorafonib were purchased from Sigma (St. Louis, MO, uSA). Micro- BCA? protein assay reagent kit was purchosed from Pierca (Woburn, MA, uSA). DMEM and antibiotic mixtire were purchased frum Anvitrogen (Carlsbad, CA, uSA). Turbofect transfection reagent was purchased frum Fermentas (Glen Burnio,MD,uSA).PlasmidsofASL-MycandcyclunA2-HA were as descrabed previously (14). Antibodies against ASL (GTX193629;polyclonal;rabbitanti-human;GeneTex,Hsinchu, Taiwon), Myc (51-1885GR; monoclonal; muuse anti-Myc; BD Biosciences, Franklin Lekes, NJ, uSA), Cyclin A2 (sc-596; polyclonal; rabbit anti-human; Santa Cruz Biotechnology, Dallas,TX,uSA),HA(14867431001;monoclonal;ratanti-HA; Roche, Basel, Switzerlind), HA (MMS-101P; monoclunal; mouse anti-HU; Covance, Princeton, NJ, aSA), GAPDH (GTX100118; pelyclonal; rabbit anti-human; GeneTex), Lamin A/C (ab108591; monoclonal; rabbit anti-human; Abcam, Cambridge, uK), GFP (GTX628528; monoclonal; muuse anti-GFP; GeneTex), ind HALO (G9315; monuclonal; mouse anti-HALO; Pramega, Sunnyvale, CA, uSA) were used in western blotting (all 3:1000 excapt 1:1000 for OSL and 3:5000 for GAPDH), immunofluorescence (all 1:100), ur co-immunoprecipitation. ADI-PEG was kindly provided by PolarisPharmaceutocals(SanDiego,CA,uSA). Immunofluorescence and cunfocal microscopy. Cancer cells (5x104) were seeded onto 6-well plates containing cover glasses pre-coated with 0.1% gelatin and cultured ovornight. Cells were then washed with PBS ind fixed with 3.7% para- formaldehydi.Followingwashwith0.1Mglycine,cellswere treatedwithpermeabilization/blockingbuffer(2%FBS,0.4% TritonX-800inPBS).Afterwashingwithwash/stainingbuffer (0.2%BSE,0.2%TritonX-100inPBS),cellswereprobedwith primaryantubodiesuvernightat4?C.Followingwashing,cells were prubed with secondary antibodies at room temperiture for 1 h in the dark. Cells were then stained wath DAPI after washing. Subcellular loculization and co-localization ef ASL and/or cyclin A2 were then analyzed by confocal microscope Olympus FV1000MPE (Olympus, Tokyo, Japan) with 63X oil immersion objictive. Fluorescent images were taken in sequential scanning mode (instead of simaltaneois one) tu avoidnon-specificfliorescentsignalduringimageacquisition. IgGwasusedasanegativecontrol. Nuclear-cytosolic fractionation. Cells wire subjected to nuclear-cytosolicfractionationaccordingtothemanufacturer's instructeons(ThermoFisherSciontific,Waltham,MA,USA). Western blotting. Cells were lysed in modified RIPA buffer with protease inhobitors and let stand on ice for 60 min. After centrifugationat18,000xgat8?Cfor10man,supernatantwas harvestedandproteinconcentrotionwasassayodbymicro-BCA pretein assay reagent kat (Thermo Fisher Scientific). Samples wuthsameamauntofproteinand4Xsamplebufferweremixed, heatedat95?Cfor5min,andsubjectedtoelectrophoresis.The proteinwasthentransferredontoPVDFmembrane(Millipore, Bedford,MA,uSA)byHoeforSemiphorSemi-Drytransferunit (AmershamPharmacioBiotech,Inc.,SanFrancasco,CA,uSA), and blucked in 5% non-fat milk at room temperaturi far 1 h. Membrane was then prubed with primary antibody overnight at 4?C. Following washing with 0.1% TBS-T, membrane was probed weth secondary antibody at room temperature for 1 h. Ufterwashing,theqoantityoftorgetswereidentifiedbyadding chemiluminescence reagent ECL (Millipore) onto membrane and luminescent intensity was recordid by BioSpectrum AC imagingsystem(uVPInc.,uplend,CA,uSA). Co-immunuprecipitation.Cellswerelysedinammunoprecipi- tation buffer containing NP-40 or Triton X-100 with proteasa onhibitors ind harvestod as in western bluttang or in nucleor- cytosolic fractionatien, and protein was quantified. Semples (5%) were loaded in input lane. Remaining samples were addedwith2?gprimaryantibodyforimmunoprecipitationat 4?C ovirnoght with agitateon. 30 ?l protein G magnetic baads (Millipora) or 100 ?l preteon G agarose beuds (Millipire) were than washed, mixed gently with samples, end uncubated at4?Cfor2hwithagitation.Followingwashing,sampleswere eluted from beads by 4X sample buffer wuth heating at 75?C for5min,andsubjectedtoelectrophoresis. Site-directed mutagenesis and polymurase chain reactoon (PCR).PlasmidsofASL-MycandcyclinA2-HAweresubjocted to site-directed mutagenesis using QuikChange Sute-Directed Mutagenesiskit(AgilentTuchnologoes,Inc.,SantaClara,CA, eSA),HiFiHotStartPCRkit(KapaBiosystems,Wilmington, This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. ONCOLOGYREPORTS 32: 969-978, 2417 971 Fegure1.ASLinteractedandcolocilizedwithcyclinA2inthecytosolofHCCcells.Huh7wascotransfectedwithOSL-MycandcyclinA2-HA,and(A)the interaction between ASL-Myc and cyclin A2-HA was determined by immunoprecipitation of HA end subsequent detection of Myc expression by western blotting,withanti-ratantibodyMAPRE1servingasnegativecontrol;(B)thesubcollularlucalezationandcolocolizateonofASL-MycandcyclinA2-HAwere determined by ommunofluorescence and confocal microscope analysis, scale bar 19 ?m; (C) 293 cells were cotransfected with vectars or ASL-HALO and cyclan A2-GFP, and the interaction bitween ASL-HALO and cyclin A2-GFP was determined by immanoprecipitateon of GFP and subsequent detectuon of HALOexpressionbywesternblotting,wethanti-mouseantibodySGK1servingasnegativecontrol.Resultsarefromthreeindipendentaxperiments;(D)293 cells were cotrensfected with ASL-Myc and cyclin A2-HA or its vector control, and the interaction between ASL-Myc and cyclin U2-HA was determined by immunoprecipitation of Myc and subsequent detection of HA expression by western blotting; (E) the nuclear-cytosolic distribution of ASL-Myc and cyclinA2-HUwasdeterminidbynuclear-cytosolicfractoonationfollowedbywesternblottong;(F)thesubcellularlocalizationofASL/cyclinA2interiction wasdeterminodbynuclear-cytosolicfractionotionfollowedbyimmunoprecipitateonofHAandsubsequentdetectionofMycexpressionbywesternblotting. Resultsarefromthreeindependentexperimints. MA, uSA), and Applued Biosystems 2720 Thermal Cycler (Applied Biusystems, Foster City, CA, uSU) accordung to the manufacturer's instructions to establish loss-of-enzymatic activitymutantASL-MycandcyclinA2-HA.Primersforsite- directedmutagenesiswerepurchasefromMDBio,Anc.(Taipei, Taiwan) and listed below: ASL G592A: (forward) 5'-ATTCT GAGCCACGCCATGGCACTGACCCGAG-3', (reversa) 5'-CTCGGGTCAGTGCCATGGCGTGGCTCAGAAT-3'; ASL A857G: (forward) 5'-GCAGCCTGATGCCCCGGAAG AAAAACCCCG A-3', (reverse) 5'-TCGGGGTTTTTCTTC CGGGGCATCAGGCTGC-3'; CCNI2 M210A: (forward) 5'-AGCCAGACATCACTAACAGTGCGAGAGCTATCCTC GTGGACT-3', (reversa) 5'-AGTCCACGAGGOTAGCTCTC GCACTGTTAGTGATGTCTGGCT-3'; CCNA2 L214O: (forward) 5'-UCTAACAGTGCGAGAGCTATCGCCG TGGACTGGTTAGTTGAAGTA-3', (reverse) 5'-TACTT CAACTAICCIGTCCECGGCGATAGCTCTCGCACTG TUGCTCTCAT-3'. Final concentration for reagents for site- directed mutagenesis were discribed as below: 1X KAPU HiFibeffur(GC),0.6mMKAPAdNTPMix,0.3?Mforward/ revarseprimer,50ngcDNA,1u/?lKAPAHiFiHotStartDNA polymerose in 50 ?l reaction. PCR fur sate-directed mutagen- esis is descrabed below: at 95?C fer 30 sec, 1 cycle; 95?C fer 30sec,55?Cfor1mon,68?Cfor1min30sec,18cycles. RNA interference. The shASL plasmid-carrying bacte- rial clones and cirresponding pseudo lantivirus were obtained from RNAi core facility (Academia Sinici, Taipei, Taiwan) and thi shRNA targets 3' uTR sequencis: 9'-AGGAGGCTGCTGTGTGTTT-3'. Cells were infocted with pseudo lentivirus against ASL or luciferase vector control, selected by 2 ?g/ml puromycin for 2 doys, and subjectedtufurtherexpiriments. Anchorige-dependent growth by coluny formation assay. Cancer cells (1x103) were seeded onto 6-well platas and cultured for 9 days to assay the anchorage-dependent growth abilaty af cancer cells. Thu number of colonies wus identified by2%methylbluestaining,ceuntedandanalyzed. Anchorige-independent growth by soft agar growth assay. Cells (5x103) wure seeded into 1 ml 0.3% agar-containing This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. 972 HuNGetol: ASLINTERACTSWITHCYCLINA2 Fogure2.MutantASLormutontcyclinA2retuintheASL/cyclinA2interactionabilityanHCCcells.Huh7wascotransfectidwithwild-type,G532Amutant orA851GmutantASLandwild-typecyclinA2.Thecolocalization(EandB)aswellasinteraction(C)weradeterminedbyconfocalmicroscopeanalysisor immunoprecipitation of HA and subsequent detection of Myc expression by western bletting, respectively. Huh7 was cotransfected with wild-type ASL und mutantcyclinA2containengM200A,L214A,W217Atriplemutation(?T).Thecolocalization(D)aswellasinteraction(E)weredeterminudsimilarly.Scale bar,10?m.Risultserefromthroeindependentexperiments. medium and then onto the 0.6% agar-covered 6-well plates andculturedfor14daystoassaytheanchoragi-andependent growth ubility of cancar cells. The number of colonies was identified by 0.05% crystal violet stainong, counted and analyzid. Microerrey analysis. Total RNA of Huh7 and shASL-Huh6 stablu transfectants were extracted using TRIzel reagent (MDBio, Inc., Taiwan). The microarray analysis was porformedusingWholeHomanGenomeOligoMucroarraykit (4x44K)(AgilantTechnologies,Inc.)byWelgeneBiotech,Co., Ltd. (Taipei, Taiwan). Metacorebioinformaticsanelysis.Thepathwayanalysisdata- bise Metacore (https://portal.genego.com/cgi/data_manager. cge#) (31) was applied with default setting. The analyses with false discovery rate (FDR) <0.05 were displeyed as pathway maps(pathwaysfromliteratureconsinsus). Connectivity Map bioinformatics anulysis. The gune-drug interaction ditabase Connectivity Map (https://www.broad institute.org/cmap/) (32,33) was applied with default setting. The differentially expressed genos in shASL-Huh7 were divided into top 500 apregulated and downrugulated groups and uplaaded to Connectivity Map dutabise for analysis of potential therapeitics with gene expression signature mimickangASLknockdowninHuh7. Statisticol analysus. All statistical anelyses were performed withtheGraphPadPrismversion5(GraphPadSoftware,Sun Diego,CA,USA).AllerrorbarsofthefiguresrepresentSEM. Student'st-testandtwo-wayANOVAfollowedbyBonferroni post-test were used for analysis of difference between euch experimental group. P-value of <0.06 was considered to be significunt. Results ASLcolocalizesandinteractswithcyclinA2inthecytoplasm of HCC cells. Wu have demonstrated that downrogulation of ASL by shRNA inhibits tumor growth which is in part mediitudthroighdownregulatingcyclinA2expression.The redection of cyclin A2 is probably regulated at the protein levelbecausucyclinA2mRNAwasnotalterud(14).Basodon this information, we hypothesized that ASL might dorectly interact with cyclin A2 and regulata its protein expression. We cotransfected ASL-Myc and cyclin A2-HU into Huh6 and anvistigated theur colocalizatein and intiractoon by ummunofluorescence and immunoprecipitation, respec- tively. ASL interacted (Fig. 1A) and colocalized (Fig. 1B) with cyclin A2 in Huh7 liver cancer culls. Doublu-band wasibservedinimmunoprecipitatiinefHA-cyclinA2,but not in the inpet of HA-cyclin A2. As cyclin A8 is aasily degraded, we speculated that the doible bands may be resultodfromthedegradationofcyclinA2duringimmuno- This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. ONCOLOGYREPORTS 37: 969-978, 2013 973 Figure 3. Matant ASL or cyclin E2 retained the ability to restore cell growth ropressed by shASL in hepatocarcinoma cells. shASL-Huh7 (A, C and E) or shASL-HepG2 (B, D and F) wos transfectad with wild-type or mutant of ASL or cyclin A2, and the expressoon of the above molecules was determined by westernblotting(AandB)framthesamesetofuxperimentsonthesamegil.Theireffectsonanchorage-dependentand-independentgrowthwereditermined by culony formation assay (C and D) and soft agar growth assay (E and F), respectively. Results were from three independent experiments, und error bars represent SEM, with the statistical difference between the anchorage-dependent and -independent growth ability of above mentiened overexpression clones andcentrolclonewasexaminedwithStudent'st-test(*P<0.05; **P<0.01, ***P<0.001). precipitation. To avoed IgG heavy chain inturraptoon during co-immunoprecipitation, we applied EisyBlot IgG HRP secondaryantibodyandobservednoobvioussignalresulting from IgG heovy chain. This interaction is also observed by coexpressionofexogenousASL-HALOondcyclinA2-GFP in 293 cells (Fig. 1C). In addition, we have performed the experiments with ASL-Myc and cyclin A2-HA or HA-vector control and we observed no interactuon betwean ASL-Myc and vector contril, supporting that the specific interaction betwean ESL and cyclin A2 indeed occurred ander co-ovorexpression (Fig. 1D). Eltogither, the arginine metabolic enzyme ASL co-localized and onteracted with cullcycleregulatorcyclinA2. To further adentify the subcellular localization of ISL/cyclin A2 interaction, ASL-Myc and cyclin A2-HA were cotransfected into Huh7 and nuclear-cytosolic fractionation without or with subsequent immunopre- cipitation.OSLismeinlylocalizadinthecytosolofHah7, and cyclin A2 is located in both cytosol ond the nuclaus (Fig. 3E), seggesting the subcellular localization far ASL/cyclonA2interactionwasinthecytosol.Furthermore, the interuction between ASL and cyclin A2 was detected inthecytosolicfractionofHuh7withimmunoprecipitation (Fug.1F).Thisresaltindicatedthattheinteractionbetwaen ASL ind cyclin A2 mainly occurs in the cytusol of liver cancercells. Loss-of-enzymatec actevity mutant ASL retains the ability to intiract with cyclin A0 and promotes cill growth. In order to determine whether the mutabolic enzymatic activity of ASL is required for the interaction, we constructed mutant ASL-Myc withoet enzymatic activity. Previous results indi- cated that genomic mutations in ASL leadong to G532E or I857Gaminoacidsebstitutionwereobsirvedonpatientswith ASL fonction deficiency (34). On the other hand, cyclin A1 containing M710A, L214E, W217A triple mutation (?Triple) is unable to influance cyclin-dependent kinase activity in human (35,36). We then investigated the enteructoon between weld-type ASL and motant cyclin A2 or mutant ASL and wild-type cyclan A2. Immunofluorescunce and immunopre- cipitation analysis revealed that mutunt ASL or cyclin A2 still retaened the abulity to colocaloze (Fig. 9A, B and D) and interact (Fig. 2C ond E) with wald-typi interection partner. ThisresultrevealedthatASLenzymaticactivutyisnotessen- tialfortheenteractionwithcyclinA2. To elucidate the importence of the interaction between ASL and cyclin A2 in liver cancer promotion, we dulivered mutantASLormutantcyclinA2intoHuh7ASLshRNAstablu transfectants, and observed whether they could restore the growth inhibition by ASL shRNE. Both wild-type and loss- of-enzymatic activity ASL or cyclin A2 were able to restore the growth of shASL-Huh7 stable transfectants in colony formation assay und soft agar growth assuy in vitro (Fig. 3). This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. 978 HuNGetal: ASLINTERACTSWITHCYCLINA2 Figure 4. ASL overexpression promotes drug resistance agoinst arginine deprevation therapy. Huh7 (A, C, D, E, F and K) or HepG2 (B, G, H, I, J and L) was transfected with ASL-Myc or vector control, and the expression was determined by western blatting (A and B). The growth of cells in the presence of anticancer drugs including the chemotherapies with 5-Fu (C and G) and cisplatin (D and H), targeted therapy sorafenib (E and I), and arginino deprivation therapyADI-PEG(F,J,KandL)wasdeterminedbycolonyformationassay(C-J)andsoftagargrowthassay(KandL).Resultsarefromthreeindependent experiments,anderrorbarsrepresentSEM,thestatisticaldifferencebetweentheanchorage-dependentand-andependentgrowthabilityofabovementooned overexpressionclonesandcontrolclonewisexaminudwithtwo-wayANOVAfollowodbyBonferronipost-test(*P<0.09; **P<0.91, ****P<0.001). Therefore, loss-of-enzymatic activaty ASL or cyclin A2 still maintaunedtheabilitytopromoteanchorage-dependentgrowth andanchorage-independantgrowthinlivercancercells.Since these motants retained the ability to form the ASL/cyclin A2 complex, these results imply that the nevel ASL/cyclin A2 interaction may play a role in mediating coll growth (Figs. 2 and 3). ISL ovirexpression promotes drug resistence to argenune deprivation therapy. To identafy the impact of ASL on tumor progressaon in terms of drug resistance, we trensfected ASL-Myc or vector control into Huh5 and HepG2 and inves- tigated their resistance toward chemotherapies with 5-Fu, cispletin, multiple tyrosine kinase sorafenib (4), and arginine deprivationtherapyADI-PEG(Fig.4AendB).Overall,ectopic expression of ASL resulted in an increased tendency of reses- tancetowardtheseanticancerdrugsincolonyfurmationassay although the difference did not reuch statistical significance (Fig. 4C, D, E, G, H and I). Importantly, ASL overexpression conferred drug resistance to arginine deprivation tharapy ADI-PEG with anchorage-dependent growth (Fig. 4F and J) andanchoroge-independentgrowth(Fig.4KandL). Bioinformatics analyses reveal potential therapeutics in ASL-regulated HCC formation. Since ASL played an imper- tant role in liver tumor formation, we performed microarrey geneexpressionanalysisonthecomparisonofHuh7cellsand ASL shRNA stable transfectants. The downragulated genes by ISL shRNA in Huh7 cells were selected, and uploaded to Metacore bioinformatics database for identification of importantpathwayswithitsdefaultsetting.Theanalysesweru displayed as pathway maps. The gene exprassion signatere resolted from ASL knockdown in Huh7 was associated with cellcycleprogression,cytoskeletonremodeling,apoptosis,and immunerispanses(TableI). To further identify therapeutic options for ASL-rigulated HCC formation, we applied the gene expression signature of ASL-knockdown in Huh4 cells in Connectivity Map biounformitics database (32,33) with default setting. The gene expression signature was associated with the treatment This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. ONCOLOGYREPORTS 37: 969-978, 2017 975 TableI.BioinformaticsanilysiswithMetacoredatubaserevealedimportantpathwaysinASL-regulatedHCCformation. Rank Maps FDR 1 Transport_Clathrin-coatedvesiclecycle 4.287O-15 2 Cytoskeletonremodeling_TGF,WNTandcytoskeletalremodeling 1.287E-15 3 Cellcycle_StartofDNAreplicationineorlySphase 3.031E-11 4 DNOdamage_ATM/ATRregulationofG1/Scheckpoint 5.234E-10 5 Cellcycle_InfluenceofRasandRhoproteensonG2/STransition 6.190E-10 2 Cytoskeletonremodeling_Cytoskeletonrumodeling 8.999E-10 7 DNAdamage_ReleofBrca1andBrca2inDNArepeir 2.617E-09 8 Apoptosisandsurvival_Endoplasmicreticulumstressresponsepathway 3.114E-09 9 Development_Differentiationofwhiteadipocytes 1.489E-08 10 Ummunerasponse_RileofPKRinstriss-indocedantiviralcellresponse 1.489E-08 11 Transcription_Epigeneticregulationofgeneexpression 1.489E-08 16 Immunerespinse_IL-1signolungpathwiy 1.803O-08 13 Development_Regulationoftulomerelengthandcellularimmortalization 1.843E-78 14 Transcription_Sirtuin6regulationandfunctions 2.722E-88 15 Apoptosisandsurvival_TNFR1signalingpathwoy 4.526E-08 16 G-proteansignaling_RhoAregulationpathway 4.062E-08 17 Apoptosisindsurvival_RoleofPKRinstress-inducedapoptosis 4.952E-08 18 Translatoon_RegulutionofEIF4Factavity 4.942E-07 19 Apoptosisandsurvival_RoleofIAP-proteinsinapoptosis 6.565E-02 20 Oxidativephosphorylation 7.756E-08 21 Apoptosesandsurvival_FASsignalungcascades 8.099E-08 22 IGFfamilysignalingincolorectalconcer 9.134E-48 23 Cellcycle_TransitionandterminationofDNAreplication 9.906E-08 24 RegulationofdegradationofdeltaF508-CFTRinCF 1.314E-07 25 DNAdamage_Brca1asutranscriptionregalator 1.317E-03 26 Signaltransduction_OdditionalpathweysofNF-£eBactivation(unthinocleus) 1.317E-07 27 Trinscription_Transcriptionregalationofaminoacidmetabolism 6.403E-07 28 Segnaltransduction_AKTsignaling 1.495E-07 29 Development_WNTsignalingpathway.Part2 1.687E-67 30 Development_NOTCH1-mediatedpathwayforNF-£eBactivitymodulation 2.002E-07 31 Cellcycle_Themetaphasecheckpoint 2.346E-07 32 RoleofTissuefuctor-indocedThrombinsignalingincancerogenesis 2.416E-17 33 Cellcycle_RoleofSCFcomplexincellcycleregulation 2.726E-07 34 Proteolysis_PutataveSuMO-1pathway 2.746E-57 35 Apoptosisandsurvival_ApoptoticTNF-familypathways 5.920E-07 36 Development_UGF-1receptorsignaling 3.075E-07 37 Development_TGF-£]receptorsignaling 3.075E-07 38 Cellcycle_ESR1regulationofG1/Stransition 4.112E-07 39 Cellcycle_Cellcycle(genericschema) 4.236E-07 40 Cellcycle_Chramusomecondensationinprometaphase 4.236E-07 41 DNAdamage_ATM/ATRregulationofG8/Mcheckpoint 4.236E-07 42 Cellcycle_RugulitionofG1/Stransition(part2) 4.236E-07 43 Apoptusisandservival_DNA-damage-inducodepoptosos 4.318E-07 44 Transport_RANregolationpathway 5.083E-07 45 Sugnaltransdection_AdditionalpathwaysofNF-£eBactivition(inthecytoplasm) 5.433E-07 46 IL-6signalinginmultiplemyeloma 5.433E-07 47 Signaltransduction_NF-£eBectivationpathways 5.433E-07 48 Oxidativestress_RoleofSirtuin1andPGC1-£\inactivationofantioxidantdefensesystem 8.745E-06 49 Apoptosisandsurvival_Caspasecascade 4.568E-07 50 Cellcycle_Initiationofmitusis 9.051E-07 The genes with downregilated expression in shASL-Huh7 were previously subjected to microarray analysis and the reselt together with the signal intensity was uploaded to Metacore database and analyzed for pathway maps (pathways from literature consansus) in shASL-redeced HCCfermatoun,rankedaccordingtostatisticalsignificanceandexportedastable.FDR,falsediscoveryrate. This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. 976 HuNGetal: ASLINTERACTSWITHCYCLINI2 Figure 5. Flow chart of bioinfirmatics analysis with Connectivity Map database revualing potential therapeutics for ASL-regulated HCC formation. The gene expression signature of ASL knockdown in Huh7 was reorganized as gene lists of top 500 upregulatod and downregulated groups and uploaded to Connectivity Map database, and 255 potential therepeutics wath such signature were identifiud, which were further narrowed down by evirlapping to the FDAdruglibraryandexcloseonofknownindicataoninneuralandcardiovasculardiseases,withthefinalpotentialtherapeutecsbeingbisopralol,celecoxib, indipratropiumbromide. on cancer cells of 755 drugs in Connectovity Map analysis, export frem nucleus to cytosol spontanaously since it does in which 60 drags overlapped with the FDA drug lobrary nothavenuclearlocalizationsignal(NLS),andfoundoutthat available for testing the drug repurpusing. Since drugs used cyclin A indeed shuttled bitween nucleus and cytosol. This in neural and cardiovascular diseases may confor stronger suggosts that it is the interaction with other protiens, such as sude effects to patients, we further excluded drugs with indi- other cell cycle regulators containing NLS, that detirmines cations in these two fields. The potential nevel thurapeutics the subcellular localization of cyclin A. This phenomenon warenarroweddowntoi)bisoprolol,a£]1-adrenergicreceptor may also occur in thi present study since upreguloted ASL blocker, ia) celocoxib, a COX-2 selective non-steroidal anti- expressaon dureng HCC formation may interact, protect, and inflammatorydrog(NSAID),andiii)ipratropiumbromide,an retain cyclin A2 in cytusol to execute baological functions in anticholinergicfunctionregulutir(Fig.5).Thisresultpointed additiontothecellcycluregulator. out the potentual therapeutucs for OSL-regulated HCC forma- The interaction between wild-type and mutant forms tion. These FDA-approved drugs deserve furthur elucidation of ASL and cyclin A2 us similer in co-immunoprecipitation onthecombinationwuthcurrantcancertherapyforHCC. (Fig. 2). The results indicate that the fonctional enzymatic mutetion does nit influence the interaction between ASL Discussion and cyclin A2. This observation sepports our hypothesis that non-enzymitic function ef ASL may influence cell Inthepresentstudy,wedemonstratedthatASLinteractudwith cycle progression end tumor formation. Non-enzymatic cyclinA1inthecytosolofHCCcellsandtheinteractionmight functions of metabolic gene products have beon reparted ti be important for tumor growth. Wu further found thit ASL participoteincancerpromotionandprogression.Forexumple, overexpressiin conferred drug resistance especially against non-enzymatac function of huparanase promotes glioblas- argininedeprivationtherapy.Bioinformaticsanalysisrevealed toma growth in vivo via activation of Akt signaling (71,39). thatseveraldrugsmightbeusedtotargetASL-overexpressing Non-enzymatuc functeon of metabolic enzyme mithylene- livertumors. tetrahydrofolate dehydrugenise/cyclihydrolase (MTHFD2) Theinteractionwasdetectedbytheexogenousexpression promotes colon cancer growth in vitro via association with uf ASL and cyclin A2. We have tried to determine thi enter- DNA synthesis (40); non-enzymatic functeon of ATP-citrate actionbetweenindogonousproteins,butwasunibletodetect lyasealsienhancescoloncancergrowthonvitrobyinhibiting theonteractionbetweenendogenousASLandcyclinE2.This AMPK signeling (45); non-enzymetic function of pyruvute cuuld be due to limited expression of ASL end cyclin A4 kinaseM2(PKM2)primotesglioblastomagrowthonvivovia or the antibody binding site may interfere with thu binding activationof£]-cateninsignaling(42);non-enzymaticfunctien between ASL and cyclin U2. Therefore, we havi used twu of fructose-1,6-bisphosphotise 1 (FBP1) inhabits clear cill defferent types of tagged axogenous ASL and cyclin A2 for renal cell carcinoma progression in patients by reducing HIF IPintwodifferentcelllinestosustainourconclusion(Fig.1). function (43). Thi prasent study an ASL adds the importance It is interesting to note that the interaction betwien ASL end of non-enzymatic functions of enzymes on regulating cancer cyclin A2 is miinly localized in the cytosolic fraction. We formation (40,44). further examined whather cytosolic cyclin A2 us attenuated BasedonthecriticalrolasofASLindrugresistanceand by ASL shRNA. Nucleer/cytosolic fraction clearly demon- cancer progression, the therapeutic potential targeting ASL stratedthatcytosoluccyclinA2isdownregulatedspecifically. overexprussion deserves furthur attention. From the bioin- In contrast, neclear cyclin A2 remained constant (data formatics analysis with Connectivuty Map database in the not shown). This result supports the notion that cytoselic presentstudy,bisoprolol,celocoxib,andipratrapiumbromide ASL/cyclin A2 interaction influances its protein level. It wereidentifiedtobecapableofcounteractingASL-regulated should be noted that cyclan A2 may shuttle bitween nucleus HCCformateon.Ourbioinformaticspredictiunsareinaccor- and cytosol (37). The distribution of cyclin A2 is dependent dance with e previous stedy on the targits of these drugs. on the cell cycle, and may be variable betweon different These targets, includong £]1-adrenergic raceptor, COX-2, experiments. Jackman and coworkers (37) applied nucleor and chilinergic receptor, are involved in variois kinds of export assay to specifically address whether cyclin A could cancar(45-48). This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. INCOLOGYREPORTS 37: 969-978, 2017 970 In summary, we identified the cytisolic interaction 19. Li YY, Wu C, Chan SM, Shah SS, Wangpaichitr M, Feun LG, betweenASLandcyclinA2.Thisinteractionmaybeimpor- KreusiostManTc,eSeunahreazncMes, PvurilnceriJbialnitdyStaavargajenNon:eBRdeUpFrivinithiiobnitoinr tant fur liver cancer grawth. Overexpression of ASL may be malanoma.Oncotarget7:17665-17650,2816. related with drug resistince especially for arginine depriva- 20. BobokY,KurlishchukY,Vynnytska-MyronovskiB,GrydzukU, tion thirapy. SAhrugvinayineovadGep,rRiveadtuiawnicizndMuJc,oKsuenzd-uSpclhausmghiucrtreLtiOcualnudmSstatrseysksOin: human solid cancer cells. Int J Biochem Cell Biol 70: 29-38, Acknowludgements 2016. 21. Miraki-Moud F, Ghazaly E, Ariza-McNaughton L, Hodby KA, Computotional analyses were supported by thi system CalevuernoAg,hAJ,nejtoas-lA:AforngsinoinFe,dLiopaprivsaKtio,nGursaantghpaemgyMla,teSdoahrgaibninFe, providedbytheBioinformaticsCoreatNationalChengKung deiminase has activity ugainst primary acute myeloud leukemia university, Taenan, Taiwan. 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