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Cancer Therapeutics Insights

Attenuation of Argininosuccinate Lyase Inhibits CancerGrowth via Cyclin A2 and Nitric Oxide

Hau-Lun Huang1,2, Hui-Ping Hsu3, Shu-Chu Shieh4, Yung-Sheng Chang1,2, Wei-Ching Chen1,2,Chien-Yu Cho1,2, Chiao-Fang Teng1, Ih-Jen Su6, Wen-Chun Hung7, and Ming-Derg Lai1,2,5

AbstractArginine biosynthesis and nitric oxide (NO) production are important for cancer homeostasis. Degradation

of argininemay be used to inhibit liver tumorswith low argininosuccinate synthetase (ASS) expression. In this

report, we investigated an alternative therapeutic approach by targeting argininosuccinate lyase (ASL). ASL is

transcriptionally induced by endoplasmic reticulum stress and is overexpressed in some human liver tumors.

Knockdown of ASL expression by short hairpin RNA (shRNA) in three liver cancer cell lines, ML-1, HuH-7,

andHepG2, decreased colony formation in vitro and tumor growth in vivo. Furthermore, lentiviral infection of

ASL shRNA inhibited tumor growth in a therapeutic animal tumormodel. Analysis ofASL shRNA on the cell-

cycle progression revealed a G2–M delay. Among cell-cycle regulatory molecules, cyclin A2 expression was

reduced. Reintroduction of exogenous cyclin A2 restored the cell growth inASL-knockdown cells. Autophagy

was observed in the cells treated with ASL shRNA, as shown by an increase in LC3-II levels and autophago-

some formation. The total cellular arginine level was not altered significantly. Inhibition of autophagy further

attenuated cell growth, suggesting that autophagy induced by ASL shRNA plays a feedback prosurvival

function. Knockdown of ASL reduced NO content, and addition of NO donor partially recovered the growth

inhibition by ASL shRNA. In summary, downregulation of ASL attenuated tumor growth and the inhibition

was mainly mediated by a decrease of cyclin A2 and NO. Mol Cancer Ther; 12(11); 1–12. �2013 AACR.

IntroductionArginine is classified as a nonessential amino acid for

mature and healthy animals but regarded as an essentialamino acid for young and growing animals (1). Argininebiosynthesis is initiated from glutamate or proline via animportant intermediate, citrulline. The endogenous syn-thesis of arginine from citrulline may not be sufficient forhumans in growth or diseased condition. (2). Argininealso plays a major role in producing nitric oxide (NO;refs. 3, 4). Arginine is also the precursor for many biologicmolecules, including polyamines, urea, glutamate, pro-line, and agmatine (2). Many of these biologic productshave been implicated in tumor development, and there-fore, feeding mice with arginine enhances tumor growth

(5). In contrast, depletion of dietary arginine inhibits livermetastasis (6).

The conversion from citrulline to arginine involves twoenzymes, argininosuccinate synthetase (ASS) and arginino-succinate lyase (ASL).ASS seems to be the rate-limiting stepin the production of arginine (7), but citrulline availabilitycontrols theendogenous levelsof arginineandNOthrough-out the body (8). An analysis of ASS expression in tumortissues revealed thatASSexpression is low inmesothelioma(9), renal cell carcinoma (10), melanoma, and liver cancer(11, 12). In contrast to the low ASS expression observed inhepatocarcinoma and melanomas, the overexpression ofASS is observed in several other types of cancers, includingovarian, stomach, lung, and colorectal cancer (8). Tumorswith low ASS expression are dependent on extracellulararginine for cell growth and generally called "arginineauxotrophs" (11). Degradation of plasma arginine by argi-nine deiminase (ADI) provides an alternative therapy forthe treatment of liver cancer and melanoma. Depletion ofarginine by pregylatedADI causes autophagy and caspase-independent apoptosis in prostate cancer cells (13). Treat-mentwith pregylatedADI showed a 25% response rate in aphase I/II study for advanced metastatic melanoma (14).However, a 47% response rate was observed in advancedhepatocarcinoma (15). In a randomized phase II trial ofpregylated ADI for Asian patients with advanced hepato-carcinoma, patients with sustained depletion of plasmaarginine for 4 weeks had a better median survival (16).Another alternative is to usepregylatedarginase todegrade

Authors'Affiliations: 1Institute ofBasicMedical Sciences;Departments of2Biochemistry and Molecular Biology, 3Surgery, and 4Medical LaboratoryScience and Biotechnology; 5Center for Infectious Diseases and SignalingResearch, College of Medicine, National Cheng Kung University; 6NationalInstitute of Infectious Diseases and Vaccinology; and 7National Institute ofCancer Research, National Health Research Institute, Tainan, Taiwan

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

CorrespondingAuthor:Ming-Derg Lai, Center for InfectiousDiseases andSignaling Research, College of Medicine, National Cheng Kung University,University Road, No. 1, Tainan 701, Taiwan. Phone: 886-6-2353535#5549;Fax: 886-6-2741694; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-0863

�2013 American Association for Cancer Research.

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Published OnlineFirst August 26, 2013; DOI: 10.1158/1535-7163.MCT-12-0863

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circulating arginine (17). In addition to hepatocarcinoma,pregylated arginase has been proposed as a potentialtherapy for acute lymphoblastic T-cell leukemia (18). Thesensitivity of tumor cells to arginase orADIdepends on theexpression of other enzymes involved in the regenerationof arginine, including ornithine transcabamylase and ASL(19).ASLcatalyzes the conversionofargininosuccinate intoarginine and fumarate. The enzyme complex of ASL isusually localized in the vicinity of ASS for the efficientchanneling of substrate and product (20, 21). The expres-sion of ASL and ASS are coordinately induced by aminoacid starvation (2). Recently, multiorgan dysfunction andNO deficiency were observed in an ASL-deficient mousemodel. ASL has a structural role in amultiprotein complexthat regulates the production of NO (22).

Because endoplasmic reticulum (ER) is responsible forsynthesis of membrane and secretory proteins, it is plau-sible that amino acid metabolism is regulated by ERhomeostasis. For example, amino acid starvation leads tophosphorylation of eIF2a at Ser51 to inhibit translation.Similarly, ER stress induces PKR-like endoplasmic reticu-lumeIF2akinase (PERK),whichalso inhibits translationbyphosphorylating eIF2a. In addition to PERK, ER stressinduces a set of chaperones that aid in protein folding,including Bip/GRP78 and GRP94 (23, 24). ER stress signaltransduction can be studied with drugs that perturb ERfunction, including the glycosylation inhibitor, tunicamy-cin. ER stress is involved in many types of disease, includ-ing liver disease, cardiac hypertrophy, and diabetic kidneydisease (25, 26). ER-resident molecular machinery is fre-quently deregulated and is involved in the pathology ofmany types of cancers (27–29).

On the basis of the close relationship between aminoacids andprotein synthesis, it is possible that ER stressmayaffect amultitude of amino acidmetabolic genes, includingargininemetabolic enzymes. Because ER stress is frequent-ly activated in cancer, we hypothesized that the expressionof ASLmay also be elevated by ER stress in human cancer.Although ASL may not be the rate-limiting enzyme inarginine biosynthesis, we further hypothesized that down-regulating the expression of this enzyme may alter thearginine–NO enzyme complex and decrease cell growth.

In this report, ASL is upregulated by ER stress and isoverexpressed in hepatocellular carcinoma. Downregula-tion of ASL by short hairpin RNA (shRNA) attenuatedtumor cell growth both in vitro and in vivo. Decrease ofcyclin A2 and a G2–M arrest was observed. Cell growthwas rescuedby the reintroduction of exogenous cyclinA2.Depletion of ASL decreased NO content and causedautophagy, which is not correlated with the total cellularcontent of arginine. Downregulation of ASL is a potentialtherapeutic approach for liver cancer in the presence ofnormal arginine content.

Materials and MethodsCell culture

Mouse liver ML-1 cell line was obtained from Dr. C.P.Hu (TungHai University, Taichung City, Taiwan). HuH-7

humanhepatoma cell linewasprovidedby I.J. Su (Nation-al Health Research Institute, Tainan, Taiwan), HepG2human hepatoma cell line was authenticated by DNA(short tandem repeat) profiling at Bioresource Collectionand Research Center in 2012. All cell lines and theirshASL-stable transfectants were cultured in Dulbecco’sModified Eagle Media (DMEM) containing 10% FBS (Bio-logical Industries), 0.4 mmol/L arginine, 100 mg/mLstreptomycin, and 100 U/mL penicillin at 37�C and 5%CO2.

Chemicals, reagents, plasmids, and antibodiesEthidiumbromide, SDS, G418 sulfates,MTTpowder, 2-

amino purine, and actinomycin D, 3-MA, bafilomycin A1,sodium nitrite, and the amino acid, arginine, were pro-ducts of Sigma-Aldrich. The Micro BCA Protein AssayReagent Kit was from Pierce. Lipofectamine 2000, TRIzolreagent, DMEM, and antibiotic mixture were products ofInvitrogen. Turbofect transfection reagent was from Fer-mentas. Myc-tagged ASL (RC201568) was purchasedfrom the OriGene Technologies Inc.. The expression plas-mid of cyclin A2 (NM_001237.3) was kindly obtainedfrom Dr. Ih-Jen Su.

Reverse transcription PCR analysisTotal RNAwas isolated fromcells usingTRIzol reagent,

and the cDNA was reverse-transcribed using oligo (dT)primers and Moloney murine leukemia-virus transcrip-tase. The PCR reactions were carried out with the cDNAby Pro Taq polymerase (PROtech Technology EnterpriseCo.) using a thermocycler (ABI). The PCR products weremade visible with ethidium bromide staining. The inten-sity of PCR fragments was determined by NIH ImageJsoftware (NIH, Bethesda, MD.). Primer sequences arelisted in (Supplementary Table S1).

Western blot analysisCells were lysed in modified radioimmunoprecipita-

tion assay (RIPA) buffer supplemented with proteaseinhibitors. Total cell lysates were separated using SDS-PAGE, and the proteins were transferred onto polyviny-lidene difluoride membranes (Millipore) using a HoeferSemiphor Semi-Dry transfer unit (Amershampharmacia).The membranes were incubated with the indicated pri-mary antibody and followed by a horseradish peroxi-dase–conjugated antibody. The blots were developedusing enhanced chemiluminescence (ECL) Western blot-ting detection reagents (Millipore) and detected using aBioSpectrum AC imaging system (UVP) according to themanufacturer’s instructions.Antibodies are listed in (Sup-plementary Table S2).

Tissue samplesTumor specimens from 14 patients with hepatocellular

carcinoma were obtained from National Cheng KungUniversity Hospital (Tainan, Taiwan) with the approvalof the Institutional Review Board. The pathology of alltumor parts and adjacent nontumor parts was previously

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assessed by a pathologist in National Cheng Kung Uni-versity Hospital. Total proteins from tumor tissues andadjacent, unaffected tissues were homogenized for a fewseconds in modified RIPA buffer using an Omni TH-115tissuehomogenizer (Omni Instruments), and 30mgof totalprotein was analyzed by Western blot analysis.

RNA interferenceThe following target sequences were used: (i) the 30UTR

of human ASL was 50-AGGAGGCTGCTGTGTGTTT-30

(shASL1669), (ii) the coding sequences of ASL were 50-GCCTATTACCT GGTCCGCAAA-30 (shASL10), (iii)coding sequence 50-CACCTTCAAACTGAACTCCAA-30

(shASL11), (iv) the coding sequences of mouse Asl were50-CAAGTGGCCACTGGAGTCATCTCTA-30 (shAsl1111),and (v) 50-CCATCACTCTCAACAGCAT-30 (shAsl752).The shRNA targeting human ASL and mouse Asl wereconstructed in the pHsU6 vector as described previously(30) or were obtained from the RNA interference (RNAi)Core Facility (Academia Sinica, Taipei, Taiwan). Cellsweretransfected with the target shRNA or control vector andselected for stable transfectants with G418 sulfates orpuromycin (Sigma-Aldrich).

Colony formation assayCells were seeded in 6-well plates. After 7 to 10 days of

growth in complete culture medium, the colonies weredetected by Methyl Blue staining, and the number ofcolonies was counted for statistical analysis.

Soft agar assayOne milliliter of 0.3% agarose in complete growth

medium containing 5,000 cells (ML-1, HuH-7, HepG2,control transfectants, and ASL shRNA transfectants) wasseeded onto 1.5 mL of 0.6% agarose in 6-well plates. After14 days, cells were stained with 0.05% crystal violet, andthe colonies were photographed and scored for statisticalanalysis.

Production and titration of lentivirusPlasmid containing shASL and vector control were

purchased from RNAi Core Facility (Academia Sinica).Lentiviruses were generated in 293T cells by cotransfec-tion of pLKO.1-shASL (target) or pLKO_AS1 (vector con-trol), with psPAX2 and pMD2.G packaging plasmids. Theproduction and tittering procedure was according to theprotocol from RNAi Core Facility.

Animals and tumor modelsSeven- to 8-week-old male nonobese diabetic/severe

combined immunodeficient (NOD/SCID) and BALB/cmicewere obtained from the LaboratoryAnimalCenter atNational Cheng Kung University. All study protocolsinvolving mice were approved by the Animal WelfareCommittee at National Cheng Kung University. BALB/cmice were inoculated subcutaneously in the flank regionwith 1.5 � 106 ML-1 cells or transfectants in 0.3 mL PBS.NOD/SCID mice were implanted subcutaneously in the

flank region with 5 � 106 human tumor cells or theirtransfectants. Tumor size was measured using a calipertwice a week. Tumor volume was calculated using thefollowing formula: volume¼ (d1

2� d2� 0.5236), in whichd1 and d2 represent the shortest and longest diameter,respectively. Mice were sacrificed when the tumor vol-umewas larger than 2,500mm3 orwhen themousewas inpoor condition. For therapeuticmodelwith lentiviral ASLshRNA, 10 days after implantation themice were injectedintratumorally with LV-pLKO_AS1 or LV-shASL at a titerof 2 � 107 transduction units (TU) in 100 mL PBS.

Monodansylcadaverine staining of autophagyAll experiments were carried out using a Cayman

autophagy/cytotoxicity dual staining kit (Item no.600140; Cayman chemical company) according to themanufacturer’s instructions. In brief, cells were incubatedwith propidium iodide solution for 2 minutes at roomtemperature and then in the monodansylcadaverine(MDC) solution for 10 minutes. A 6-hour treatment withtamoxifen (20 mmol/L) served as a positive control. MDCstaining was detected by fluorescence microscope witha UV filter to detect 40,6-diamidino-2-phenylindole(DAPI) staining. These experiments were carried out intriplicate.

Measurement of intracellular arginine contentFor intracellular arginine analysis, 3 � 106 cells were

collected and suspended in 200 mL 1� PBS. The suspen-sions were then sonicated, centrifuged to removedebris, and the supernatants were stored overnight at�80�C before lyophilization. The freeze-dried sampleswere then reconstituted with 20 mL 1� PBS and sub-jected to HPLC analysis using Agilent ZORBAX EclipseAAA column (Agilent PN 993400-902), borate buffer(Agilent PN 5061-3339), and the Agilent derivatizationreagent, OPA (Agilent PN 5061-3335; Agilent Technol-ogies Inc.). The required lamp was UV with a Bio-Radmodel 1801 UV monitor (338 nm for detection of OPAderived amino acid; Bio-Rad Laboratories). The columntemperature for detection was held at 40�C with aheating device (ENSHINE SUPER CO-150). The intra-cellular arginine concentration was quantitated byinterpolation at intermediate points on the standardarginine curve (Sigma-Aldrich).

Intracellular NO detectionAll experiments were carried out using a Cayman

nitrate/nitrite colorimetric assay kit (Item no. 780001)according to the manufacturer’s instructions. Cells weregrown in 24-well plates for 48 hours and subjected toassay. The 80-mL volume of the conditioned medium wascollected from the 1 mL conditioned medium and thenanalyzed. The color development was read at an absor-bance of 540 nm using a plate reader (molecular devicesVERSAmax microplate reader; Molecular Devices Inc.)and the NO value was counted. The determined valuemultiplied by 2.5 was the total NO level.

ASL Regulates Cyclin A2 and NO

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Reporter gene assayTheASLpromoterwas amplified fromgenomicDNAof

Hela cell with the following primers: 50-accatctcagctcactg-caa-30 and 50-ggccgcacggatagtgtc-30. The PCRproductwascloned into TA-Cloning vector (YeasternBiotechCo.). TheASL promoter in TA cloning vector was further amplifiedwith the following primers: 50-gcaatcggtaccctcccgggttcaa-gagattct-30 and 50-gcaatc ctcgagactggccagggcttttctg-30, andsubcloned into pGL3-basic plasmid (Promega) usingKpnI and XhoI restriction enzymes (Takara Bio Inc.).The ASL reporter in pGL3-basic (�976 to þ24 bp) wassequence verified. To analyze the promoter activity ofASL reporter gene, HuH-7 cells were transiently trans-fected with these plasmids as indicated by Lipofecta-mine 2000 according to the manufacturer’s instructionswith slight modification. The total DNA amount foreach experiment was matched equally with controlempty vectors. After 6 hours of incubation with trans-fection mixtures and cells, the transfectants in DMEMwere changed to complete medium and incubated forfurther 12 hours. Luciferase activities in cell lysates oftransfectants were measured by the Luciferase AssaySystem according to the manufacturer’s instructions(Promega).

Statistical analysisGraphPad Prism version 4.00 for Windows (GraphPad

Software) was used for analyses of all numerical data andgraphs. All analyses were conducted three times or more.All of the error bars of the figures represent SEM. Student ttest was used for analysis of the difference betweenexperimental groups. The survival time of mice was sub-jected to Kaplan–Meier analysis.

ResultsASL expression is induced by ER stress andoverexpressed in liver cancerTo test whether ASL is induced by ER stress, the

human hepatoma cell line HuH-7 and mouse hepatomacell line ML-1 were incubated in the absence or presenceof tunicamycin, an artificial ER stress inducer. Theexpression of ASL mRNA was elevated by tunicamycintreatment in both HuH-7 andML-1 cells (Fig. 1A and B).ASL protein expression levels were increased followingtunicamycin treatment in both cell lines too (Fig. 1C andD). ASL expression was induced in the livers of C57BL/6 mice following intraperitoneal injection with tunica-mycin (Fig. 1E). The overexpression of hepatitis B virus

large surface protein has been shown to induce ERstress (31) and can be used as a natural ER stress-stimulant. Upregulation of ASL was also observed inHuH-7 cells stably expressing Hepatitis B virus largesurface protein (Fig. 1F). Altogether, ASL proteinexpression was induced by ER stress. We next examinedwhether ASL expression is elevated in human hepato-cellular carcinoma. Fourteen pairs of hepatocellularcarcinomawith adjacent nontumorous tissue were ana-lyzed by Western blot analysis. Overexpression is indi-cated by more than 1.5-fold expression in tumor partcompared with the adjacent nontumor part. Amongthese, 10 pairs showed concurrent high expression levelof ASL and GRP78, an ER stress marker, in liver tumors(Fig. 1G and Supplementary Fig. S1).

Induction of ASL by ER stress occurs throughtranscriptional regulation

To study whether ASL induction under ER stress ismediated transcriptionally, actinomycin D was used toblock transcription. ML-1 cells were cotreated withtunicamycin and actinomycin D (5 mg/mL), and the ERstress–mediated induction of Asl mRNA was abolished(Supplementary Fig. S2A). To further examine tran-scriptional induction of ASL, a reporter vector carryingthe ASL promoter (�976 toþ24 bp) was constructed andused in luciferase assays. Under ER stress conditions,ASL promoter activity was increased approximately 2-fold compared with unstimulated samples (Supplemen-tary Fig. S2B). These results showed that ER stress–induced ASL expression is mainly mediated throughtranscriptional regulation. To investigate the role ofeIF2a in the induction of ASL expression, HuH-7 cellswere cotreated with tunicamycin and 2-aminopurine,an inhibitor of eIF2a phosphorylation, and examinedfor ASL expression. The induction of ASL under ERstress was attenuated by 2-aminopurine treatment (Sup-plementary Fig. S2C).

Knockdown of ASL expression by ASL shRNAinhibits growth in vitro

To study whether ASL is a potential therapeutic targetfor cancer, three liver cancer cell lines (mouse liver ML-1cells, human HuH-7, and HepG2 cells) were transfectedwith ASL shRNA, and stable transfectants were obt-ained. The shRNAs were designed to target the exon or30-untranslated regions of ASL respectively. All threesets of ASL shRNAs attenuated mouse or human ASL

Figure 1. ASL is inducedbyERstress andoverexpressed in liver cancer.HuH-7 (A) andML-1 (B) cellswere treatedwith tunicamycin (2.5mg/mL) for the indicatedtime points. Total RNA was isolated and subjected to reverse transcription PCR (RT-PCR) analysis using primers specific for ASL, GRP78, and GAPDH.HuH-7 (C) and ML-1 (D) cells were incubated with tunicamycin for different time points, and lysates were subjected to Western blot analysis with specificantibodies for ASL, GRP78, glyceraldehyde-3-;phosphate dehydrogenase (GAPDH), and actin. E, male C57BL/6 were administered tunicamycin(1.5mg/kgbodyweight) intraperitoneally and sacrificed at the indicated timepoints. Liverswere collected andASL,GRP78, andGAPDH levelswere examinedby Western blot analysis. F, cell lysates from stable transfectants expressing human influenza hemagglutinin (HA)-tagged HBV wild-type, pre-S2 deletionmutant large surface protein, or vector control were collected and subjected to Western blot analysis with antibodies for ASL, HA, GRP78, and GAPDH.G, Western blotting analysis of surgical biopsies from patients with hepatocellular carcinoma (14 pairs); N and T denote the adjacent unaffected tissues andtumor tissues, respectively, of the same patients. Quantitative analyses of the Western blot analyses were also conducted. TM, tunicamycin.






expression in three liver cell lines (Fig. 2A and Supple-mentary Fig. S3). The three liver parental cancer celllines and their ASL shRNA stable transfectants were

subjected to colony formation assay to determine theirproliferative potential. Decreased cell growth wasobserved in the ASL shRNA stable transfectants of

Figure 2. Knockdown of ASL inhibitscell growth in vitro and tumor growth invivo. A, reduction of ASL expression.Lysates from shASL stabletransfectants of ML-1, HuH-7, andHepG2 were analyzed by Westernblotting using indicated antibodies orreverse transcription PCR (RT-PCR),using primers specific for ASL andGAPDH. B, the proliferation rates ofML-1, HuH-7, HepG2, and their shASLstable transfectants were determinedby colony formation assay. Onethousand cells were seeded in 6-wellplates and allowed to grow for 7 to10days. The colonieswere detected bymethyl blue staining. C, a soft agarassay was conducted with ML-1,HuH-7, and HepG2 cells andtransfectants. Fourteen days afterseeding on 0.3% top agar, cells werestained with 0.05% crystal violet, andthe colonies were photographed andcounted for statistical analysis(colonies with sizes > 100 mm). D–F,tumor growth andmice survival curves.D, BALB/c mice bearing ML-1 cellsstably expressing shAsl and its U6vector. E, NOD/SCID mice bearingHuH-7 cells stably expressing shAsland its U6 vector. F, NOD/SCID micewere implanted with HuH-7 cells andinjected intratumorally with LV-pLKO_AS1 or LV-shASL at a titer of2 � 107 after tumors were palpable. Allsurvival data were subjected toKaplan–Meier analyses. The number inparentheses is the number of mice inthe experiment. �, P < 0.05,��; P < 0.01; and ��, P < 0.001compared with the parental cell.

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ML-1, HuH-7, and HepG2 cells. These results showedthat ASL knockdown inhibited the growth rate of threeliver cell lines (Fig. 2B and Supplementary Fig. S3).Anchorage-independent growth is an important char-acteristic of cancer cells. Therefore, three liver cancercell lines and their ASL shRNA stable transfectants wereexamined in soft agar assay. Anchorage-independentgrowth was decreased following ASL knockdown inmouse and human liver cell lines (Fig. 2C and Supple-mentary Fig. S3). These results indicated that ASLshRNA decreased both cell growth and anchorage-inde-pendent growth.

Downregulation of ASL inhibits tumor formationin BALB/c and NOD/SCID mouse tumor models

To investigate the effect of ASL downregulation ontumor growth, ML-1 cells stably expressing shAsl (752-1 and 752-2) or its U6 vector were implanted subcuta-neously in immune-competent BALB/c mice. Tumorgrowth was decreased by Asl shRNA, and a Kaplan–Meier survival analysis revealed an increase of mousesurvival time (Fig. 2D). We then compared the tumor-igenicity of HuH-7 cells, control transfectants, and fourASL shRNA stable transfectants (1669-1, 1669-2, 10-1,and 11-1), which were targeted by different shRNAs

Figure 3. ASL knockdown reducescyclin A2 expression and causes aG2–M cell-cycle delay. A, ML-1,HuH-7, HepG2, and their shASLstable transfectants were seededin 6-well plates or 3.5 cm dishes ata density of 3 � 105 cells per well(ML-1: 2 � 105 cells per well). After24 hours, cell lysates wereharvested forWestern blot analysiswith the indicated antibodies. B,total RNA was also collected forreverse transcription PCR (RT-PCR) analysis with the indicatedprimers. C, the three liver cancercell lines and their correspondingASL shRNA stable transfectantswere seeded. After 24 hours,cells were treated with MG132(5 mmol/L), a proteasome inhibitor,for an additional 12 hours, and celllysateswere harvested forWesternblot analysis using antibodiesspecific for ASL, CCNA2, GAPDH,and ACTIN. D, cell-cycle analysiswas conducted on the three livercancer cell lines (HuH-7,ML-1, andHepG2) and their correspondingASL shRNA stable transfectants.Cells were seeded at the density of3�105 cells perwell (ML-1: 2�105

cells/well). After incubation for 48hours, cells were collected forstaining with propidium iodidesolution and then subjected to flowcytometry analyses.






to avoid off-target effects. Downregulation of ASLinhibited tumor growth and extended the time of sur-vival of immune-deficient mice (Fig. 2E). The tumorgrowth of 10-1 transfectant is lower than that of 11-1transfectant, which is correlated with the expression ofASL in the transfectants as shown by Western blotting(data not shown). To mimic therapeutic model, weintratumorally injected recombinant lentiviruses con-taining shASL or vector control. A single injection oflentivirus-shASL slowed down growth rate of HuH-7tumor and extended the survival rate of mice whencompared with the control lentiviral group (Fig. 2F).Taken together, inhibition of ASL expression attenuatedcolony formation, anchorage-independent growth, andtumor growth.

Knockdown of ASL in cancer cells reduces cyclin A2expression

Because downregulation of ASL affects cell growthand tumor formation, we analyzed the expression ofproteins involved in the cell-cycle progression. Amongthe cyclins examined, cyclin A2 expression decreasedsignificantly in ML-1, HuH-7, and HepG2 cells. In con-trast, the expression of other cyclins, including cyclinD1, cyclin E1, and cyclin B1 were not significantlydifferent in three liver parental cells and their ASLshRNA stable transfectants (Fig. 3A). Downregulation

of cyclin A2 by ASL shRNA is probably regulated at theprotein level because cyclin A2 mRNA was not altered(Fig. 3B). Furthermore, treatment with a proteasomeinhibitor, MG-132, restored cyclin A2 expression (Fig.3C). The regulation of cyclin A2 is probably mainly atposttranslational level. Analysis of cell-cycle progres-sion reveals a delay of G2–M progression, which par-tially explain the growth delay (Fig. 3D).

Ectopic expression of cyclin A2 restored the cellgrowth in ASL-knockdown cells

Because knockdown of ASL reduced both the expres-sion of cyclin A2 and cell growth, we investigated therole of cyclin A2 in the cell growth by ectopically exp-ressing cyclin A2 in cell bearing shASL. In the colonyformation assay, ectopic expression of cyclin A2could restore the growth inhibition by ASL shRNA inHuH-7 (Fig. 4A) and HepG2 (Fig. 4B). Ectopic expres-sion of ASL, which is served as a positive control, canrestore the cell growth in ASL-knockdown cells (Fig.4A and B). Furthermore, ectopic overexpression ofcyclin A2 also promoted cell proliferation in parentalHuH-7 cells (Fig. 4A). Taken together, exogenouscyclin A2 could reverse the growth inhibition causedby ASL shRNA. Therefore, reduction of cyclin A2plays a major role in inhibition of cell growth by ASLshRNA.

Figure 4. Ectopic expression ofcyclin A2 or ASL rescues thegrowth inhibition in shASL stabletransfectants. A, HuH-7 and shASLtransfectants. B, HepG2 shASLtransfectants were furthertransiently transfected withexogenous cyclin A2 or ASL andexamined with colony formationassay. Parental cells, shASLtransfectants, shASL/cyclin A2double transfectants, and shASL/ASL double transfectants weregrown for 7 to 10 days, and thecolonies were detected by methylblue staining, photographed, andcounted for statistical analysis. Allexperiments were carried out intriplicates. �, P < 0.05; ��, P < 0.01;and ��, P < 0.001 comparisonbetween the indicated groups.

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KnockdownofASL in cancer cells induces autophagyWe tested whether downregulation of ASL induced

autophagy. ASL shRNA induced autophagy in ML-1,HuH-7, and HepG2 cells, as shown by the presence ofLC3B molecules in Western blotting (Fig. 5A) and theoccurrence of MDC staining autophagosome by fluo-rescence microscopy (Fig. 5B). To examine the relation-ship of autophagy and cell growth, liver cancer cellswere treated with 3-MA or bafilomycin A1, whichare inhibitors of autophagy. Inhibition of autophagyresulted in a further decrease of cell growth (Fig. 5C),suggesting a prosurvival role of autophagy in ASL-knockdown cells. However, the total cellular argininelevel was not significantly altered in the ASL shRNAtranfectants established from the HuH-7 and HepG2(Fig. 5D). Autophagy is induced independent of totalcellular arginine content.

Knockdown of ASL in cancer cells attenuates NOcontent

ASL andNO synthase form as a complex in vivo (22), weexamined NO content in ASL-knockdown transfectants.The NO content was significantly decreased by down-regulation of ASL (Fig. 6A). Furthermore, the growth ratewas attenuated by the addition of an inducible nitric oxidesynthase (iNOS) inhibitor in all liver cancer cell lines (Fig.6B). Treatment of HuH-7 and HepG2 cells with NO donorsodium nitrite enhanced cell growth and partially restoredthe growth inhibition byASL shRNA (Fig. 6C). Altogether,reduction of bothNOand cyclinA2 aremainly responsiblefor the inhibition mediated by ASL shRNA.

DiscussionIn this report, we have shown that ER stress induced

ASL expression and that overexpression of ASL is

Figure 5. Downregulation of ASLinduces autophagy. A, oneautophagy marker, LC3-II, wasexamined by Western blotanalyses in the following cell lines:ML-1, HuH-7, and HepG2, andtheir corresponding shASL stabletransfectants. Tamoxifen, a knowninducer of autophagy, wasincluded as a positive control (top).B, autophagosome formation inshASL stable transfectants. MDCstaining, a fluorescent dye used todetect autophagy, was conductedin these cells (bottom).C, three livercancer cells (ML-1, HuH-7, andHepG2) and their correspondingASL shRNA stable transfectantswere seeded at the density of2�104 cells perwell (ML-1: 1�104

cells/well) in 24-well plates for MTTassay. These cells were treatedwith 3-Methyladenine (3-MA) andbafilomycin A1 for 24 hours. They-axis is the fold of absorbancevalues of cells (with inhibitor/without inhibitor). The value of cellswithout inhibitor is 1. D, intracellulararginine levels in ML-1, HuH-7,HepG2, and their shASL stabletransfectants were detected byHPLC. ��, P < 0.01 compared withparental ML-1 cells.






observed in human liver cancer. Downregulation ofASL expression by shRNA attenuated cell proliferation,anchorage-independent growth, and tumor growth. Theeffects were unlikely due to the off-target effects as fourdifferent ASL shRNAs targeting on various sites on ASLachieved similar effects. Reintroduction of exogenousASL restored the cell growth. Analysis of the cellularpathways reveals that reduction of NO and cyclin A2wasobserved in ASL shRNA transfectants. Reintroduction ofcyclin A2 or addition of NO donor reverses the growthinhibition by ASL shRNA. A schematic drawing is illus-trated in Fig. 6D. To the best of our knowledge, this is thefirst report indicating the link betweenASL and cyclin A2in cell growth.

ASL is essential for systemic NO production as shownby a hypomorphic ASL mouse (22). Thus, ASL is a targetfor manipulating NO production and treatment of NO-related diseases. Our results support the notion thatdysregulation of an enzyme in a metabolic complex leads

to physiologic or pathologic consequences. Overexpres-sion of ASLwas detected in human cancers andmay playa prooncogenic role. Downregulation of ASL by shRNAalso attenuated the stable content of NO in cancer cells. Inaddition, exogenous overexpression ofASL enhancedNOproduction (Fig. 6A, middle). NO production has beenimplicated in cancer initiation andprogression (32). In thisreport, iNOS inhibitor inhibited the growth rate of all celllines we used, suggesting a prooncogenic role for NO. Incontrast to its tumor-promoting effects, NO derived frommacrophages and natural killer cells may participate inantitumor activity (33). The final effects of NO on tumorgrowth will be determined by these opposing factors.

DownregulationofASL leads to agrowthdelay, and thedoubling time of ASL shRNA transfectant is longer thanthe parental cells. For example, the doubling time of theHepG2 is 23 hours and the doubling time of the HepG2ASLshRNAtransfectants is 32hours. The longerdoublingtime is reflected by the shorter S-phase; however, a longer

Figure 6. Nitric oxide is partiallyresponsible for the growthattenuation by ASL shRNA. A, totalnitrate and nitrite content, the finalproduct of nitric oxide in vivo, weremeasured with a nitrate/nitritecolorimetric assay kit. B, fociformation assay was conducted inthe presence of iNOS inhibitor,L-NMMA for these cell lines.�, P < 0.05; ��, P < 0.01; and��, P < 0.001 compared with theparental cell. C, HuH-7, HepG2,and shASL stable transfectantswere seeded at 1,000 cells per wellin 6-well plates. After 24 hours,cells were treated with sodiumnitrite (1.0 mmol/L) and allowed togrow for 7 to 10 days. The colonieswere detected by methyl bluestaining. �,P<0.05 and ��,P<0.01,comparison between the indicatedgroups. D, schematic drawing ofthe pathways through whichdownregulation of ASL attenuatestumor growth. Cyclin A and NO aremainly responsible for theinhibitory effects.

Huang et al.





G2–M phase was observed in all transfectants. In analyz-ing the effects of ASL attenuation on the cell-cycle pro-gression, cyclin A2 was specifically reduced. Cyclin A2 isan important cell-cycle regulator on both G1–S and G2–Mphase (34). In addition, cyclinA2 controls cell invasion viaRhoA signaling (35). The downregulation of ASL alsoleads to a lower migration ability in ASL shRNA trans-fectants (Supplementary Fig. S4). Nitric oxide increaseslysine 48–linked ubiquitination after arterial injury andalters the expression of cyclin A and B (36). In our exper-imental system,NOmaynot be involved in the expressionof cyclin A2 because cyclin B was not affected by ASLshRNA. The ASS–ASL–NO synthase contains a HSP (22).HSP may directly or indirectly regulate the stability ofcyclin A2. Many cellular proteins interacting with cyclinA2have a commonRXLmotif (X is variable; refs. 37–39).Arecent report also indicated that HCVNS5B protein inter-acted with cyclin A through RXL motif (40). ASL proteincontains a RXL motif, which may direct interact withcyclin A2 and regulate its stability or function. Theinteraction between ASL and cyclin A2 warrants furtherinvestigation.Arginine deprivation by pregylated ADI (ADI-PEG20)

is a novel therapy for prostate cancer (13). Autophagy andcaspase-independent cell death were induced by ADI-PEG20, and autophagy was suggested to be a protectiveresponse during the early-treatment stages. Downregula-tion of ASL also leads to autophagy, and the inhibition ofautophagy further decreases cell growth. Therefore,autophagy may function as a protective response duringASL attenuation, which is similar to the autophagy thatoccurs in endocrine therapy (41). We also analyzed theapoptosis fraction by annexin V staining and the expres-sion of PARP, but did not find significant differencebetween the parental cells and transfectant cells (data notshown). It is very interesting to note that the overallcontent of arginine was not decreased in shASL transfec-tants, which is likely due to the sufficient supply ofarginine present in culture medium. The difference ofintracellular arginine content between several ASL trans-fectants may result from the variable efficiency of aminoacid transporter. Because we did not add arginase inhib-itor during lysis of cells, we cannot completely excludethe influence of cellular arginase on the measured argi-nine content. More importantly, our results indicate thatautophagy may be induced independent of the totalarginine concentration; however, we cannot exclude theexistence of variable arginine concentration in specificcellular compartment. The induction of autophagy is

probably due to the signal elicited by the imbalance ofthe ASS–ASL–NOS complex. Alternatively, NO is able toinhibit autophagy (42), thereby, the decrease of NOinduces autophagy during ASL downregulation.

ASL is reported to be downregulated in certain patientswith liver cancer at later stages (43). The differencemaybein part due to the analysis method (Western blot analysisvs. immunohistochemistry). Our report shows the upre-gulation of ASL by ER stress in vitro, and occurrence ofER stress marker GRP78 and ASL expression in tumorsamples. Furthermore, downregulation of ASL leads totumor regression, supporting a cause–effect relationshipbetween ASL and cancer. The lost of ASL in certainpatients with liver cancer in later stages may be betterdefined as diagnostic marker (43). We showed here thatASL plays a proto-oncogenic role in cancer.

In conclusion, ASL is overexpressed in liver cancer, andattenuation of ASL inhibits tumor growth in vivo. Thedecrease of cell growth ismainlydue to reduction of cyclinA2 andNO.Local treatment of tumorswithASL shRNA iscapable of delaying tumor progression. ASL shRNA mayfunction as adjuvant therapy for treating liver cancer inthe future.

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

Authors' ContributionsConception and design: H.-L. Huang, Y.-S. Chang, C.-Y. Cho, I.-J. Su,W.-C. Hung, M.-D. LaiDevelopment of methodology: H.-L. Huang, S.-C. Shieh, Y.-S. ChangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):H.-L. Huang, H.-P. Hsu, W.-C. Chen, C.-F. TengAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): H.-L. Huang, H.-P. Hsu, S.-C. Shieh, C.-Y.Cho, M.-D. LaiWriting, review, and/or revision of the manuscript: H.-L. Huang, C.-Y.Cho, W.-C. Hung, M.-D. LaiAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases):H.-L.Huang,H.-P. Hsu, S.-C. Shieh,I.-J. Su, W.-C. Hung, M.-D. LaiStudy supervision: H.-L. Huang, C.-Y. Cho, M.-D. Lai

Grant SupportThis study was supported by the grant NSC-100-2325-B-006-008 from

National ScienceCouncil, Taiwan andNHRI-EX100-9927B1 fromNationalHealth Research Institute (M.D. Lai), and from Establish Centers ofExcellence forCancerResearch inTaiwan,DOH101-TD-C-111-003Depart-ment of Health, Executive Yuan, Taiwan.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 4, 2012; revised August 2, 2013; accepted August 7,2013; published OnlineFirst August 26, 2013.

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Huang et al.





Published OnlineFirst August 26, 2013.Mol Cancer Ther Hau-Lun Huang, Hui-Ping Hsu, Shu-Chu Shieh, et al. Growth via Cyclin A2 and Nitric OxideAttenuation of Argininosuccinate Lyase Inhibits Cancer

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