Resistance to arginine deiminase treatment in melanoma cells is … · cells was associated with...

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Published OnlineFirst November 24, 2009; DOI: 10.1158/1535-7163.MCT-09-0794

Resistance to arginine deiminase treatment in melanoma cellsis associated with induced argininosuccinate synthetaseexpression involving c-Myc/HIF-1α/Sp4

Wen-Bin Tsai,1 Isamu Aiba,1 Soo-yong Lee,1

Lynn Feun,2 Niramol Savaraj,2 and Macus Tien Kuo1

1Department of Molecular Pathology, The University of TexasM. D.Anderson Cancer Center, Houston, Texas and 2SylvesterComprehensive Cancer Center, University of Miami,Miami, Florida

AbstractArginine deiminase (ADI)–based arginine depletion is anovel strategy under clinical trials for the treatment of ma-lignant melanoma with promising results. The sensitivityof melanoma to ADI treatment is based on its auxotrophyfor arginine due to a lack of argininosuccinate synthetase(AS) expression, the rate-limiting enzyme for the de novobiosynthesis of arginine. We show here that AS expres-sion can be transcriptionally induced by ADI in melanomacell lines A2058 and SK-MEL-2 but not in A375 cells, andthis inducibility was correlated with resistance to ADItreatment. The proximal region of the AS promoter con-tains an E-box that is recognized by c-Myc and HIF-1αand a GC-box by Sp4. Through ChIP assays, we showedthat under noninduced conditions, the E-box was boundby HIF-1α in all the three melanoma cell lines. Under argi-nine depletion conditions, HIF-1αwas replaced by c-Myc inA2058 and SK-MEL-2 cells but not in A375 cells. Sp4 wasconstitutively bound to the GC-box regardless of arginineavailability in all three cell lines. Overexpressing c-Myc bytransfection upregulated AS expression in A2058 andSK-MEL-2 cells, whereas cotransfection with HIF-1α sup-pressed c-Myc–induced AS expression. These resultssuggest that regulation of AS expression involves inter-play among positive transcriptional regulators c-Myc

Received 4/7/09; revised 8/25/09; accepted 10/10/09; publishedOnlineFirst 11/24/09.

Grant support: National Cancer Institute grants 1RO1 CA79085 andCA89541, and M. D. Anderson Institutional CTT/TI-3D grant (M.T. Kuo)and R01 CA109578 (L. Feun), and a Merit research grant fromVA (N. Savaraj).

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 solelyto indicate this fact.

Note: W.-B. Tsai and I. Aiba contributed equally. N. Savaraj and M.T. Kuocontributed equally.

Requests for reprints: Macus T. Kuo, Department of Molecular Pathology,Unit 951, The University of Texas M. D. Anderson Cancer Center,7435 Fannin Street, Houston, Texas 77030. Phone: 713-834-6038;Fax: 713-834-6085. E-mail: [email protected]

Copyright © 2009 American Association for Cancer Research.

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and Sp4, and negative regulator HIF-1α that confers resis-tance toADI treatment in A2058 and SK-MEL-2 cells. Inabil-ity of AS induction in A375 cells under arginine depletionconditions was correlated by the failure of c-Myc to inter-act with the AS promoter. [Mol Cancer Ther 2009;8(12):3223–33]

IntroductionThe incidence of malignant melanoma continues to in-crease in the past decade. Conventional immunotherapy-,chemotherapy-based, or combination of these therapies yieldan overall response rate of <25%. Thus, novel strategies fortreating melanoma have been sought (1, 2). It has been re-ported that majority of melanomas and hepatocellular carci-nomas are auxotrophic for arginine because of their lack ofargininosuccinate synthetase (AS) expression (3–5). Arginineis synthesized from citrulline through two-step reactions cat-alyzed by AS and argininosuccinate lyase, in which AS is therate-limiting enzyme. In light of these unique properties,arginine deiminase (ADI), a bacterial enzyme that convertsarginine to citrulline and ammonium, resulting in argininedeprivation, was developed for treatment ofmelanoma andhepatocellular carcinomas. To suppress its immunogenicityand enhance its stability in circulation, the clinically usedADI (ADI-PEG20) was conjugated with polyethylene glycol(PEG). In the laboratory setting, exposure of melanoma cellsto ADI results in cell death, whereas normal cells that expressAS are able to survive (6–9). Phase II clinical trials using ADI-PEG20 has been conducted in melanoma and hepatocellularcarcinomas. Arginine was successfully degraded from serumby ADI-PEG20, and a response has been seen (6–9). In somepatients, however, insensitivity to ADI treatment was associ-ated with the induced expression of AS (10), suggesting thatinduction of AS expression under ADI treatment may con-tribute to failure ofADI treatment.However, themechanismsby which AS was expressed in ADI-treated patients havenot been elucidated.The current study was initiated to investigate the mecha-

nism by which AS is induced in three melanoma cell linesunder arginine deprivation conditions, either by treatingthese cells with ADI-PEG20 or by culturing them in argi-nine-free medium. We found that the inducibility of AS ex-pression was correlated with ADI resistance and thatexpression of AS was reversibly regulated by arginine avail-ability. Further investigations revealed that induction of ASexpression by arginine depletion involves the positive tran-scription regulator c-Myc and negative regulator HIF-1α,which recognize the E-box in collaboration with Sp4, whichrecognizes the GC-box at the AS promoter in A2058 and SK-MEL-2 cells. Failure of AS induction in A375 melanoma

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Induction of Argininosucinate Synthetase by ADI3224


cells was associated with the inability of c-Myc to interactwith the E-box. Our study reveals the important roles ofc-Myc/HIF-1α/Sp4 in the regulation of AS expression thatconfer ADI-PEG20 resistance in melanoma cells.

Materials and MethodsReagent, Cell Culture, and Antibodies

ADI-PEG20 (specific activity, 5–10 IU/mg) was obtainedfrom Polaris Pharmacologies, Inc. Deferoxamine, cobaltchloride, and sulforhodamine B (SRB) were purchased fromSigma and LY294002 was purchased from Cayman Chemical.A2058, SK-MEL-2, and A375 melanoma cells were pur-

chased from American Type Culture Collection Centerand maintained in DMEM containing 10% fetal bovine se-rum. Arginine-free medium was purchased from Invitrogenand was supplemented with 10% fetal bovine serum thathad been dialyzed against PBS. For arginine depletion, cellswere washed with PBS and maintained in DMEM contain-ing 0.05 μg/mL ADI-PEG20 or in arginine-free medium.For cell growth assay, 100 to 500 cells were seeded in 96-

well plates for various lengths of time. Cells were fixed with10% tri-chloro acetate, washed with distilled water, andstained with 0.4% SRB. The excess SRB dye was washedwith 0.1% acetic acid and the remaining SRB was dissolvedin 10 mmol/L Tris buffer. The relative number of cell wasmeasured by absorbance at 405 nm.Mouse anti–β-actin, anti-HA (Sigma), mouse anti-AS (BD

Bioscience), mouse anti-myc (9E10, Roche), rabbit anti-Sp1(Santa Cruz Biotechnology), rabbit anti-Sp2 (Santa Cruz Bio-technology), rabbit anti-Sp3 (Santa Cruz Biotechnology), rabbitanti-Sp4 (Santa Cruz Biotechnology), rabbit anti–c-Myc (N262,Santa Cruz Biotechnology), mouse anti–c-Myc (C33, SantaCruz Biotechnology), and mouse anti-HIF1α (BD Biosci-ence) were purchased commercially.Plasmid DNA Construction and siRNA Transfection

The expression vector for pCGN-HA-c-Myc was gener-ously provided by Dr. William P. Tansey, Ruttenberg CancerCenter, NewYork, NY (11). The pCMV4-Sp2 vector was gen-erously provided by Dr. Jonathan M. Horowitz, North Car-olina State University, Raleigh, NC (12). The Sp3 expressionvector was described previously (13). The expression vectorfor pCMV-Sp4 was generously provided by Dr. ManoharRatnam,University ofOhio, Toledo,OH (14). The constitutiveactive or dominant negative forms of HA-Akt expressionconstructs were described previously (15).The proximal promoter region of AS gene (from −1822

to+300)was clonedbyPCRusingGC-richPCRsystem (Roche).The used primers are F: 5′-ACGCGTCCTCACTGTCCCT-CATGTCACTGG-3′, R: 5′-CTCGAGCGGGGCGGGC-GTCTTCTAC-3′. The deletion contracts were created by PCRusing same reverse primer and following forward primers;−1334: 5′-ACGCGTCCTGTGAGAGGGCTGAGGGGGCC-3′,−846: 5′-ACGCGTCTTTGGAGCCGGCAGACCAAGCTG-3′,−602: 5′-ACGCGTGGGATGAAAGCCGGGCCTTCCCGC-3′,−358: 5′-ACGCGTGCCACCCTCCGCCCCTGAGTTACA-3′,−85: 5′-ACGCGTGCGCGCGTCCCCGCCACGTGT-3′, and−37: 5′-ACGCGTCCTGTGCTTATAACCTGGGAT-3′. The am-

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plified DNA fragments were cloned using TOPO PCR system(Invitrogen) and sequence integrity was analyzed. The pro-moter region were digested by MluI/XhoI and ligatedwith pGL3 vector. The point mutations were introducedinto E-box and GC-box sequence by using mutagenesiskit (Stratagene).The siRNAs for HIF1α (5′-GAUUAACUCAGUUUGAA-

CU-3′) and AS (5′-GCAAUGACCUGAUGGAGUA-3′)were synthesized by Sigma, c-Myc siRNA was purchasedfrom Cell Signaling, and control siRNA and Sp4 siRNAwere from Santa Cruz Biotech. The siRNA and plasmidDNA were transfected using lipofectamine 2000 (Invitro-gen) according to the manufacturer's instruction.Immunoprecipitation and Immunoblotting

All immunoprecipitation experiments were done as de-scribed previously (16). Briefly, protein samples were incu-bated with an antibody and 25 μL 50% protein Asepharose slurry, and Protein A beads were collected. Im-munoprecipitates were resolved by SDS-PAGE and ana-lyzed by immunoblotting. For immunoblot analysis, theprotein samples were subjected to SDS-PAGE and trans-ferred onto polyvinylidene difluoride membranes. Themembranes were blocked, incubated with primary antibo-dies and then with horseradish peroxidase–conjugated sec-ondary antibodies, and visualized by an enhancedchemiluminescence kit.Reverse Transcription-PCR

Total RNA was isolated with trizole reagent (Invitrogen)followingmanufacturer's instruction. The cDNAwas synthe-sized from 1 μg of total RNAby using a Super Script II system(Invitrogen). The synthesized cDNA was subjected to stan-dard PCR or real-time PCR using following oligonucleotideprimers. c-Myc: forward, 5′-CCTACCCTCTCAACGA-CAGC-3′ and reverse, 5′-ACTCTGACCTTTTGCCAGGA-3′; ASS, forward: 5′-AGGCACCATCCTTTACCATG-3′ andreverse: 5′-CTGCACTTTCCCTTCCACTC-3′; β-actin, for-ward: 5′-GAGGCCCAGAGCAAGAGAG-3′ and reverse:5′-AGAGGCGTACAGGGATAGCA-3′.Chromatin Immunoprecipitation Assay

Chromatin immunoprecipitation ChIP assaywas carried outwith a ChIP assay kit (Upstate Biotechnology) following man-ufacturer's instruction. A GC-rich PCR system (Roche) wasused for PCR analysis using following AS promoter–specificprimers (forward: 5′-TGAGTTACATGGGTCGCAGC-CACTG-3′, reverse: 5′-GCCCATCCCAGGTTATAAGCA-CAGG-3′) or primers for glyceraldehyde-3-phosphatedehydrogenase promoter (forward: 5′-TGTCTGCCCTAATTAT-CAGG-3′, reverse: 5′-GGAAAGTTGGGGAACTTCTC-3′). Thequantitative analyses on enrichment of precipitated DNAwere also done as described previously (17).Luciferase Assay

Luciferase assay was carried out using dual promotergene reporter kit (Promega) following manufacture's in-struction. In brief, cells cultured in 24-well dishes weretransfected with 0.2 μg of pGL3 vector and 2 ng of pRLIIvector using Lipofectamine 2000 (Invitrogen) followingmanufacture's instruction. After incubation in normal me-dium containing 0.05 μg/mLADI or in arginine-freemedium,


© 2009 American Association for Cancer Research.


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cells were lysed with passive lysis buffer. Cell lysates werecleared by centrifugation and supernatantswere used for dualluciferase assay. The obtained luciferase activity was normal-ized with that of empty pGL3 vector.

ResultsInduction of AS Expression by Arginine Depletion Is

Associated with ADI Resistance in Melanoma Cell Lines

We first analyzed the effects of ADI treatment on AS ex-pression in A2058 melanoma cells. A2058 cells were main-tained in ADI-containing or arginine-free medium forvarious lengths of time up to 72 hours. The culture mediumwas then replaced with complete medium. AS expressionlevel was analyzed by Western blotting and real-timePCR. AS expression appeared 48 to 72 hours after ADI treat-



ment and gradually decreased after removal of ADI (Fig. 1A).Similar results were observed when A2058 cells were main-tained in arginine-free medium (data not shown). These re-sults indicated that levels of AS expression are controlledby arginine availability.We then determined the expression of AS in two other mel-

anoma cell lines, SK-MEL-2 and A375. Like A2058 cells, thesecells have intact AS genes in their genomes by sequencing(data not shown). The cells weremaintained in ADI-containingor arginine-free medium for 72 hours. AS expression wasinduced in SK-MEL-2 cells but the level of induction waslower than that in A2058 cells. AS expression was not de-tected inA375 cells even under induction conditions (Fig. 1B).These three melanoma cell lines were subjected to sensi-

tivity test under arginine-deprivation culturing conditions,

Figure 1. Induction of AS expression by arginine depletion contributes to ADI resistance. A, kinetic study of AS induction by arginine depletion. A2058cells were maintained in medium containing 0.05 μg/mL ADI for the time intervals as indicated. Thereafter, cells were cultured in normal medium for anadditional 24, 48, and 72 h. The cells were harvested and AS expression levels were analyzed by Western blotting or real-time PCR. The AS mRNA levelsare represented as the expression level relative to β-actin. B, A2058, SK-MEL-2, and A375 cells were maintained in normal medium, medium containing0.05 μg/mL ADI, or arginine-free medium for 72 h and AS expression levels were analyzed by Western blotting and real-time PCR. The AS mRNA levels arerepresented as expression level relative to β-actin. C, proliferation of A2058, SK-MEL-2, and A375 cells after ADI treatment or being cultured in arginine-free medium. Cells (100 each) were seeded on 96-well plates and cultured in normal medium, medium containing 0.05 μg/mL ADI, or arginine-free me-dium. Cells were fixed by 10% trichloroacetate every 24 h and the growth rate was analyzed by SRB assay. D, A2058 cells were transfected with controlor AS siRNA (100 nmol/L each) and maintained in normal medium, medium containing 0.05 μg/mL ADI, or arginine-free medium and cell growth rate wasanalyzed. Seventy-two hours after transfection of AS siRNA, AS expression levels were examined by Western blotting 72 h after transfection. Points,mean by Student's t test from three independent experiments (*, P < 0.01); bars, SD.





whereas the regular medium (DMEM) we used contained0.48 mmol/L ariginine. A2058, SK-MEL-2, and A375 cellscultured under arginine-free medium showed very littleproliferative activity up to 5 d. ADI treatment completelyinhibited the growth of A375 cells for the same period oftime, but A2058 and Sk-MEL-2 cells showed significantgrowth in the presence of ADI (Fig. 1C). These data indicatethat AS inducibility correlates with ADI resistance. Indeed,we were able to establish ADI-resistant cell lines fromA2058 and from SK-MEL-2 cell lines but not from A375 celllines (data not shown). We also noticed that A2058 and SK-


MEL-2 cells could partially maintain growth under ADItreatment but not in the arginine-free medium, yet both cultur-ing conditions can induce AS expression (Fig. 1B). These resultssuggest that induction of AS alone is not sufficient to supportcell growth under complete arginine-deprivation conditions.To address the role of AS induction in ADI resistance, we

used siRNA to knockdown AS mRNA. As shown in Fig. 1D,transfection with AS siRNA effectively suppressed AS induc-tion in ADI-containing medium or arginine-free medium, andthere was a concomitant reduction in cell growth, support-ing the role of AS expression in ADI resistance.

Figure 2. Analysis of arginine deprivation–responsive cis-elements on the AS promoter. A, deletion analysis of the AS promoter. A2058 cells weretransfected with pGL3-AS recombinants encoding various lengths of AS promoter sequences and pRLII. Seventy-two hours after transfection, the pro-moter activities of each construct were assayed. B, mutation analysis of the AS promoter. The point mutations were introduced on the E-box and/or GC-box of pGL3-AS-85 as indicated. A2058 cells were transfected with mutant AS promoter constructs and promoter activity was assayed. The AS promoteractivity was normalized with that of the empty pGL3 vector. Columns, mean by Student's t test from three independent experiments. (*, P < 0.01); bars,SD. C, E-box–dependent activation of AS promoter activity by c-Myc. A2058 cells were transfected with wild-type, GC-box mutant, E-box mutant, ordouble mutant of pGL3-AS-85 with empty or c-Myc expression vector and maintained in normal medium, medium containing 0.05 μg/mL ADI, or arginine-free medium for 72 h. The AS promoter activity was analyzed and was normalized with that of the pGL3 vector. Columns, mean from three independentexperiments; bars, SD. D, A2058 cells were cotransfected with pGL3-AS-85 and expression vectors for Sp1, Sp2, Sp3, and Sp4. The promoter activityand the expression of transfected vectors were analyzed. Columns, mean from three independent experiments (*, P < 0.01); bars, SD. Expression levelsof the transfected (+) Sp1, Sp2, Sp3, and Sp4 were determined by Western blottings using their respective antibodies. Representative expression levelsof β-actin were used as loading control. E, additive effect of c-Myc and Sp4 in AS promoter activity. A2058 cells were transfected with pGL3-AS-85 andexpression vectors for c-Myc and Sp4 alone, or c-Myc plus Sp4, and AS promoter activity was analyzed. The AS promoter activity was normalized withthat of the empty pGL3 vector. Columns, mean from three independent experiments; bars, SD.






Identification of Arginine Deprivation-Responsive

cis-Elements in the AS Promoter

To identify the cis-elements within the AS promoter thatrespond to arginine availability, we first constructed a re-porter recombinant pGL3-AS-1822 that contains nucleotides−1822 to +300 of the AS promoter linked to the bacterial leu-ciferase reporter gene. We also constructed several AS pro-moter deletion reporters by progressively removing theupstream AS sequences in pGL3-AS-1822. These deletion re-porters were transfected into A2058 cells and their promoteractivity was analyzed. As shown in Fig. 2A, the reporterswith deleted sequences down to −85 nucleotide still re-tained responsiveness to arginine depletion, but further de-letion to −37 nucleotide markedly reduced both basal andarginine depletion-responsive activities, suggesting that−85 to −37 nucleotides contain sequence important for bothbasal and inducible AS expression. By using computationalanalysis described previously (18), we found an E-box and aGC-box sequences between −85 and −37 nucleotides of theAS promoter (Fig. 2B).To determine whether these sequences are involved in AS

induction, we introduced mutations into both the E-box andGC-box in pGL3-AS-85 (Fig. 2B, underscores) and analyzedtheir effects on AS promoter activity. Mutations at either theE-box or the GC-box alone reduced AS promoter activityand compound mutations further reduced the promoter ac-tivity in response to arginine depletion (Fig. 2B, bottom).These results suggest that both E-box and GC-box se-quences play important roles in the induction of AS by ar-ginine depletion.Two basic helix-loop-helix transcription factors, c-Myc

and HIF-1α, were considered as strong candidates for inter-acting with the E-box sequence. We first analyzed whetherc-Myc indeed plays a role in regulating AS expression.Wild-type or mutant pGL3-AS-85 reporter recombinants



were cotransfected with empty or c-Myc expression vector.As shown in Fig. 2C, cotransfection with c-Myc expressionvector upregulated reporter activity of wild-type and GC-box mutated pGL3-AS-85, but not of the mutant E-box ordouble mutant constructs. These results indicate that c-Myc positively regulates AS promoter activity through itsinteraction with the E-box.The Sp1 transcription factor family is well known to reg-

ulate their target genes through interactions with the GC-box. To investigate the impact of Sp1 transcription factorson the AS promoter, we analyzed the effect of cotransfectionof expression vectors encoding Sp1, Sp2, Sp3, and Sp4 onAS promoter activity. Cotransfection with Sp4 recombinantshowed a 4-fold induction of reporter expression levels,whereas cotransfection with Sp3 recombinant reduced>50% of reporter expression (Fig. 2D). Cotransfection withSp1 and Sp2 recombinants increased reporter activity by<1.5-fold (Fig. 2D, left). Expression levels of transfectedSp1, Sp2, Sp3, and Sp4 were comparable in these transfec-tions (Fig. 2D, right). These results suggested that Sp3 andSp4 can modulate AS promoter activity. The role of Sp4 intranscriptional upregulation of AS was also shown by theadditive increases in reporter expression when c-Myc andSp4 expression recombinants were cotransfected (Fig. 2E).Induction of c-Myc Expression and Its Cross-talk with

Sp4 in the Arginine Depletion–Induced AS Expression in

A2058 Cells

To address the roles of c-Myc, Sp4, and HIF-1α (see be-low) in regulating AS expression under the stress of reducedarginine in melanoma cells, we first focused on A2058 cells.We found that levels of c-Myc but not of Sp4 were increasedin cells grown in medium-containing ADI or in arginine-freemedium. Upregulation of c-Myc was also observed at themRNA level (data not shown). We then investigated the ef-fects of c-Myc or Sp4 expression on AS induction. A2058

Figure 3. c-Myc is induced by ar-ginine-depletion and positively regu-lates AS promoter activity throughthe E-box in A2058 cells. A, cellswere transfected with 100 nmol/Lof scramble (control) or c-Myc siRNA.Cells weremaintained in normal, ADI-containing, or arginine-free mediumfor 48 h. Expression levels of AS,c-Myc, and β-actin were analyzed byWestern blotting. B, cells were trans-fected with control or Sp4 siRNA andmaintained in normal medium, mediumcontaining0.05μg/mLADI, or arginine-freemedium for 72 h. Expression levelsof AS, Sp4, and β-actin were analyzedby Western blotting. C, the interactionbetween c-Myc and Sp4 was analyzedby coimmunoprecipitation using anti–c-Myc antibody followed by Westernblotting using anti-Sp4 and anti–c–Myc antibodies. D, the interactionbetween Sp4 and c-Myc was ana-lyzed by coimmunoprecipitation us-ing anti-Sp4 antibody followed byWestern blotting using anti–c-Mycand anti-Sp4 antibodies.





cells were transfected with c-Myc siRNA, Sp4 siRNA, orscramble siRNA. Cells were maintained in normal, ADI,or arginine-free medium thereafter for 48 hours. As ex-pected, in the presence of control siRNA, c-Myc but not ofSp4 (Fig. 3B) protein levels were upregulated by argininedepletion. Knockdown of c-Myc by siRNA to <50% in theADI-treated or arginine-free medium-cultured cells resultedin >80% suppression of AS induction, suggesting that c-Myc


is a positive regulator for AS upregulation (Fig. 3A). Knock-down Sp4 to ∼30% of the control level by siRNA also inhib-ited AS induction (Fig. 3B), suggesting that Sp4 is alsoinvolved in AS induction. The extent of AS suppressionby Sp4 siRNA seemed to be much reduced in comparisonwith the effect of c-Myc knockdown (Fig. 3A). However,knockdown of Sp3 levels to ∼50% of control by siRNAdid not significantly altered AS expression levels (data not

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Figure 4. Roles of HIF-1α in theregulation of AS expression. A, argi-nine deprivation suppresses HIF-1αexpression. A2058 cells were main-tained in normal medium, mediumcontaining 0.05 μg/mL ADI, or arginine-freemediumwithorwithout100μmol/Ldeferoxamine (DFX) or 100 μmol/LCoCl2. Seventy-two hours aftertreatment,cellswereharvestedandsub-jected for Western blotting. B, A2058cells were transfected with 100 nmol/Lscramble (control) or HIF-1α siRNAand maintained in normal medium,medium containing 0.05 μg/mLADI, or arginine-free medium with100 μmol/L deferoxamine. Untrans-fected A2058 cells maintained innormal medium, medium containing0.05 μg/mLADI, or arginine-freeme-diumwere used for control. Levels ofAS, HIF-1α, c-Myc, Sp4, and β-actinwere measured by Western blot-tings. C, A2058, SK-MEL-2, andA375 cells were transfected withc-Myc or cotransfected with HIF-1αexpression vector, and pCGN (con-trol) vector, and cell lystates wereanalyzed by Western blot. As an ex-pression control, the level of totalexpression of each protein was ex-amined by immunoblotting withantibodies against whole protein asindicated or β-actin (loading con-trol). D and E, kinetics of HIF-1α,AS, c-Myc, and Sp4 expression inA2058 cells grown in arginine-freemedium (D) or 0.05 μg/mL ADI-containing medium (E) for the timecourses as indicated.

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shown). We repeatedly observed that the c-Myc siRNAprobe we used was less effective in suppressing its targetRNA in A2058 cells grown under regular conditions thanin those grown under induced conditions. This was proba-bly due to the low abundance of c-MycmRNA in cells grownunder noninduced conditions, thereby providing reducedtarget size for the attack by siRNA. Nonetheless, this differ-ence should not affect the result that knockdown of c-Myc inthe ADI- or arginine-free medium-treated cells was associ-ated with downregulation of AS expression.We then explored the possibility of physical interactions

between c-Myc and Sp4. c-Myc protein was immunopreci-pitated from A2058 cell lysate by anti–c-Myc antibody andthe precipitate was subjected to Western blotting using anti–c-Myc and anti-Sp4 antibodies. Figure 3C shows that Sp4was coimmunoprecipitated with c-Myc from the lysate ofcells treated with ADI or cultured in arginine-free medium,but not from cells maintained in normal medium. The inter-actions between c-Myc and Sp4 were also shown by usinganti-Sp4 antibody in reciprocal immunoprecipitation, whichshowed that c-Myc was coimmunoprecipitated with Sp4from the lysate of cells treated with ADI or cultured inarginine-free medium but not from cells maintained in theregular medium (Fig. 3D). These results showed the physi-cal interactions between c-Myc and Sp4 in A2058 cells cul-tured under arginine-depleted conditions.



HIF-1α Is a Negative Regulator for Arginine Depletion-

Induced AS Expression in A2058 Cells

Arnt/HIF-1β is another potential interacting transcrip-tion for the E-box sequence. HIF-1β forms heterodimer withHIF-1α andmodulates target gene expression under hypoxiccondition. Given that the physiologic role of HIF1α is oftenopposite to that of c-Myc (19–21), we explored the possibleinvolvement of HIF-1α in AS gene expression. Figure 4Ashows that treating A2058 cells with ADI or growing themin arginine-free medium resulted in downregulation ofHIF-1α (compared among lanes 1–3; results were clearly ob-served with prolonged exposure of the autoradiograph; datanot shown). Induction of HIF-1α using hypoxia mimicsCoCl2 or deferoxamine resulted in accumulated HIF-1α le-vels, and elevated HIF-1α was associated with downregula-tion of AS expression. Strikingly, downregulation of HIF-1αin the hypoxia mimics–treated A2058 cells by siRNA re-versed the inhibitory effects of HIF-1α on AS expression inthe arginine-depleted conditions (Fig. 4B). These resultsshowed that HIF-1α plays a negative role in the regulationof AS expression under arginine-depleting conditions. More-over, we found that in the CoCl2− and deferoxamine-treatedA2058 cells, accumulation of HIF-1α in the ADI-treated or ar-ginine deficiency–cultured A2058 cells was associated withdownregulation of c-Myc (Fig. 4A, compare lanes 5, 6, 8,and 9with 2 and 3). The downregulation of HIF-1α by siRNA

Figure 5. Mechanistic investigation of c-Myc, HIF-1α, and Sp4 in the regulation of AS expression in melanoma cells under arginine-available and arginine-deficient conditions. A, Western blotting analyses of expression levels of AS, HIF-1α, c-Myc, and Sp4 in A2058, SK-MEL-2, and A375 cells grown in theregular, ADI-containing (0.05 μg/mL), and arginine-free medium for 72 h. B to D, ChIP assay of the interactive transcriptional regulators with AS promotersunder arginine depletion conditions.B andC, c-Myc displaces HIF-1α binding fromAS promoter in response to arginine depletion in A2058 and SK-MEL-2cells, respectively.D, dissociation of HIF-1α, but no concomitant association of c-Myc, to theAS promoter in response to arginine depletion in A375 cells.E, stabilization of HIF-1α by the hypoxia mimic CoCl2 resulted in persistent association between HIF-1α and the AS promoter and in the prevention ofc-Myc to interact with the promoter in A2058 cells cultured under arginine-deficient conditions. Top, the antibodies used for ChIP and control IgG. Input,genomic DNA prior to immunoprecipitation. AS promoter sequences from ChIP were quantified by PCR assay.





abolished the inhibitory effect of deferoxamine on AS induc-tion through the recovery of c-Myc induction (Fig. 4B, com-paring lanes 8 and 9with 5 and 6). These results suggest thatHIF-1α also exerts a suppressive role in the regulation of ASinduction through downregulation of the c-Myc pathway inA2058 cells.We next investigated the effects of overexpression of c-Myc

and HIF-1α on the expression of AS. We transfected HA-tagged c-Myc recombinant DNA into A2058, SK-MEL-2, andA375 cells. Overexpression of c-Myc inducedAS expression inA2058 and SK-MEL-2 cells, but not in A375 cells (Fig. 4C, left).Cotransfection of Myc-tagged HIF-1αwith HA-c-Myc recom-binants suppressed c-Myc–induced AS expression (Fig. 4C,right). These results support the positive role of c-Myc andnegative role of HIF-1α in regulation of AS expression.To determine the expression kinetics of transcription reg-

ulators and the induction of AS, we analyzed the timecourse of HIF-1α, c-Myc, and Sp4 expression in referenceto AS expression in A2058 cells after treatment with ADIor under arginine-free culture conditions. We chose A2058cells because they express comparably higher steady-statelevels of HIF-1α than do SK-MEL-2 and A375 cells (see below;Fig. 5A). With prolonged exposure of autoradiographs, HIF-1α signals in theWestern blots could be clearly visualized. Fig-ure 4D and E show that reduction of HIF-1α was seen within10 minutes after the treatment, whereas induction of c-Mycstarted4hours after the treatment, and rapid surgeofAS levelswas seen 48 to 72 hours thereafter. Levels of Sp4did not changethroughout the entire time course analysis. These results indi-cate that induction of AS expression by arginine deprivationin A2058 cells is a delayed effect, initiated by the downregula-tion of HIF-1α and followed by the upregulation of c-Myc.Mechanistic Analyses of c-Myc, HIF-1α, and Sp4 in the

Regulation of AS Expression in Melanoma Cell Lines in

Response to Arginine Deprivation

We then investigated the mechanistic aspects of c-Myc,HIF-1α, and Sp4 in the induced AS expression in A2058,SK-MEL-2, and A375 cells. Expression of HIF-1α, c-Myc, andSp4 in these three cell lines were determined byWestern blot-tings.Our results showed that: (a) comparingwithA2058 cells,the steady-state levels of HIF-1αwere much reduced in SK-MEL-2 and A375 cells, regardless of arginine availability(Fig. 5A); (b) like A2058 cells, levels of c-Myc were low inSK-MEL-2 cells under noninduced conditions but were in-duced in the ADI- and arginine-free medium-treated cells.Furthermore, when induced levels of c-Myc between A2058cells and SK-MEL-2 cells were compared, it appears that le-vels of c-Myc were correlated with levels of AS induction.These results are consistent with the positive role of c-Myc inthe regulation ofAS expression in these two cell lines; (c) levelsof c-Mycwere high inA375 cells grown in the regularmediumbut were lower under arginine-depleting conditions; and(4) levels of Sp4 were constitutively expressed in all thethree melanoma cell lines, regardless of arginine availability.To gain mechanistic insights into how these transcription

factors regulate AS expression in response to arginine deple-tion, we carried out ChIP analysis. A2058 cells grown in theregular medium, arginine-free medium, or treated with


ADI-PEG20 were fixed with formaldehyde, sonicated, andthe sheared chromatin was immunoprecipitated by anti-bodies against c-Myc, HIF-1α, or Sp4. The precipitated DNAfragments were renatured, purified, and subjected to PCRanalyses. Significant enrichment of AS promoter sequencewas observed in the chromatin fractions of ADI-treatedand arginine-free–cultured cells pull-down by anti–c-Mycantibody. In contrast, reduction of AS promoter sequencewas observed in the samples when anti–HIF-1α antibodywas used. No significant difference of AS sequence whenSp4 antibody was used (Fig. 5B). These results suggest thatwhen A2058 cells were cultured under the noninduced con-ditions, the AS promoter was mainly bound by HIF-1α butnot by c-Myc. When these cells were switched to ADI-containing or arginine-free medium, downregulation ofHIF-1α resulted in the replacement of c-Myc occupancy at theAS promoter. Similar results were observed in SK-MEL-2cells (Fig. 5C).We hypothesized that AS expression in A375 cells could

not be induced by ADI treatment because c-Myc was unableto participate in transcriptional activation at theAS promoterlevel. To test this hypothesis, we performed ChIP analyses.As shown in Fig. 5D, although dissociation of HIF-1α fromthe AS promoter was seen in A375 cells cultured under argi-nine-depleted conditions, no engagement of c-Myc to the ASpromoter was found under these conditions. These resultssupport the hypothesis that failure of induced AS expressionin A375 cells under arginine-depleting conditions is becauseof the inability of c-Myc to interact with AS promoter cul-tured under arginine-deficient conditions.We further showed that accumulation of HIF-1α in the

CoCl2-treated A2058 cells persists the binding of HIF-1αat the AS promoter and prevents the displacement by c-Myc (Fig. 5E). Alternatively, these results may be explainedby that the absence of c-Myc in the engagementAS promoteris due to reduced levels of c-Myc in the CoCl2-treated cellsresulting from stabilization of HIF-1α (Fig. 4A). Nonetheless,the present results, at the very least, support that persistentoccupancy of AS promoter by HIF-1α is associated with thesuppressive effect of AS induction by arginine deprivation,thus supporting the negative role of HIF-1α in the regulationof AS expression.

DiscussionThe finding that human malignant melanoma is auxotro-phic for arginine provides a molecular basis for the develop-ment of targeted therapy of this disease based on argininedeprivation using recombinant ADI (6). In this study, wefound that in A2058, SK-MEL-2, and A375 melanoma celllines, AS expression levels were very low. The expressionof AS could be induced in the former two cells lines butnot in the last one by ADI. The inducibility of AS expressionis correlated with the resistance to ADI. These results have aclinical reminiscence that some melanoma patients do notresponse to ADI treatment in clinical trials, underscoringthe importance of elucidating the mechanism of inducedexpression of AS by ADI treatment.






The association between AS expression levels and argi-nine availability was first noted >40 years ago (22). Multiplemechanisms have been reported for the regulation of ASexpression, including transcriptional regulation (23–27)and epigenetic regulation (28, 29). However, many of thesestudies used nonmelanoma cells that express high basallevels of AS, so the results are not applicable to cancer che-motherapy using ADI because these cells used were notsensitive to ADI treatment. In the present study, we iden-tified an E-box and a GC-box as arginine deprivation–responsive elements that controlAS expression and elucidatedthat c-Myc/HIF-1α are the E-box transcription regulatorsand Sp4 is the major GC-box factor. Importantly, we de-scribe that c-Myc and HIF-1α function as a positive and anegative regulator, respectively, and that a switch fromHIF-1α to c-Myc binding to the E-box is accompanied withthe induction of AS expression by arginine deprivation inA2058 and SK-MEL-2 cells (Fig. 6A). We further showedthat failure of AS induction in A375 cells by ADI was asso-ciated with the inability of c-Myc to interact with the ASpromoter (Fig. 6B). To our knowledge, this is the first mo-lecular description of howAS gene expression is induced insome but not all melanoma cells that bears clinical rele-vance to cancer chemotherapy using ADI-PEG20 (10).c-Myc and HIF-1α are among the most important onco-

proteins in cancer biology. c-Myc plays a central role in atranscriptional regulation network that regulates cellgrowth, differentiation, apoptosis, and metabolism signal-ing (30, 31). Likewise, HIF-1α plays a critical role in tumorangiogenesis, glycolysis, metastasis, redox signaling, and re-sponse to radiation and chemotherapy (32–35). All of these



pathways are critically involved in the growth, prolifera-tion, invasiveness, and responsiveness to therapy of cancercells. For these reasons, regulation mechanisms of c-Mycand HIF-1α have been extensively studied. However, themechanisms by which arginine starvation modulatesc-Myc and HIF-1α, which in turn regulate AS expres-sion, are currently unknown. It is plausible that phosphoi-nositide 3-kinase/Akt signaling may be involved. Indeed,several studies have documented the cross-regulatorymechanisms among c-Myc and HIF-1α and phosphoinosi-tide 3-kinase/Akt signaling. (a) It has been described pre-viously that constitutively activated Akt upregulates HIF-1αexpression in melanoma in vivo (36), suggesting a feedbackmechanism of Akt activity through HIF-1α level that can beabrogated by hyperactivation of Akt. (b) It has been shownthat phosphoinositide 3-kinase/Akt signaling regulatesc-Myc promoter activity, mRNA stability, and protein sta-bility through its downstream genes, GSK3β and β-catenin(37, 38). (c) Akt phosphorylates and sequesters the tran-scription factors FOXO3a, Mad-1, andMiz-1, which inhibitthe transactivation of c-Myc target genes (39–41). (d) More-over, it has been shown that HIF-1α can inhibit c-Myc ac-tivity by direct interactionwith c-Myc, transactivation of c-Myccorepressor Mxi1, or promoting proteosome-dependentdegradation of c-Myc (31, 42). (e) Energy starvation bymeans of amino acid deprivation has profound effects onthe function of HIF-1α and c-Myc (43) relevant tomitochon-drial physiology. Expression of HIF-1α has been shown toaffect two mitochondrial enzymes, pyruvate dehydroge-nase, and cytochrome C oxidase (44–46). In contrast, c-Mycpromotes mitochondrial respiration through mitochondrial

Figure 6. Models depicting induc-tion of AS expression in A2058 andSK-MEL-2 cells by arginine depletionthrough HIF-1α displacement by c-Myc(A). a, under the regular culture con-ditions, HIF-1α acts as a repressor bybinding to the E-box of AS gene. b, un-der arginine-depletingconditions,HIF-1αis degraded and c-Myc is upregulatedand directly binds to the E-box thatdrives transcription through interac-tion with the transcription factorSp4, which is bound to the GC-boxof AS promoter. c, in the presenceof hypoxia mimics, CoCl2, HIF-1α isstabilized and remains bound at theE-box, thereby preventing the in-duced AS expression by arginine de-pletion. B, failure of c-Myc bindingtoAS promoter in A375 cells is asso-ciated with inability of AS expres-s ion under a rg in ine -dep le t ingconditions. a, under the regular cul-ture conditions, HIF-1α acts as a re-pressor by binding to the promoter ofAS gene.b, under arginine-depletingconditions, HIF-1α is degraded butc-Myc does not bind to the E-box.





biogenesis (47). Moreover, it has been reported that disrup-tion of HIF-1α activity using RNAi resulted in increased mi-tochondrial biogenesis and O2 consumption. Conversely,targeted disruption of c-Myc diminished mitochondrialDNA and O2 consumption (31). These results suggest thatHIF-1α and c-Myc are oppositely regulated through mito-chondrial metabolism. These observations, together withour finding that HIF-1α can also regulate c-Myc levels, illus-trate a complex inter-regulatory network involving HIF-1αand c-Myc through phosphoinositide 3-kinase/Akt signalingin the regulation of AS expression in response to arginineavailability. This complex mechanism may explain, at leastin part, the observed delayed induction of AS expressionby arginine deprivation.The complex regulatory mechanism of AS expression by

arginine deprivation is compounded by the observationsthat c-Myc–interacting transcription factor Sp4 is alsoinvolved. We showed that Sp4 and Sp3 are the GC-box–interacting factors. Previous studies on regulation of ASgene expression have showed that Sp1 interaction withthe GC-box is important for transactivation of the AS pro-moter (23, 24, 48). Our present results show that Sp1 showsonly a minor stimulatory function on the AS promoter com-pared with Sp4 (Fig. 2D). Further investigation using Sp4siRNA strategy supports the functional role of Sp4 in theregulation of AS expression. Moreover, coimmunoprecipita-tion results showed a physical interaction between Sp4 andc-Myc. It is important to note that roles of other membersof Sp1 family were not examined in these previous studies(23, 24), norwere siRNAand cotransfection assays performed.This study also raised several important issues that have

yet-to-be resolved: (a) Induction of AS expression by argi-nine deprivation in some melanoma cells is apparently ini-tiated by the rapid disappearance of HIF-1α, becauseaccumulation of HIF-1α by hypoxia mimics blocked thesubsequent events leading to suppression of AS induction.It is likely that arginine deprivation induces HIF-1α degra-dation because HIF-1α mRNA levels were not correspond-ingly reduced.3 HIF-1α is a highly unstable protein (t1/2,∼10 minutes under normaxia conditions). The underlyingmechanisms that accelerate HIF-1α degradation under argi-nine-depleted conditions remain to be determined. Alterna-tively, arginine deprivation may also suppress thetranslation of HIF-1α. (b) Our results showed that apparent-ly, dissociation of HIF-1α from the E-box is not sufficientand that c-Myc has to be engaged in transcriptional activa-tion of AS expression as shown in A375 cells. The precisemechanism(s) by which c-Myc fails to interact with the ASpromoter in A375 cells under arginine depletion conditionsare currently unknown. It is plausible that the E-box in A375chromatin may not be in open configuration due to an epi-genetic mechanism such as DNA methylation (29) ormasked by yet-to-be determined transcriptional repressors.Alternatively, c-Myc in A375 cells may be sequestered, ren-dering it unable to transcriptionally activate AS expression

Unpublished results.
3

under arginine deprivation conditions. (c) The regulatorymechanisms underlying the delayed AS induction by argi-nine deprivation remain to be critically investigated.(d) Finally, the roles of HIF-1α, c-Myc, and Sp4 in the reg-ulation of AS expression that associated with ADI sensitiv-ity in malignant melanoma chemotherapy remain to bedetermined. Studies aimed at addressing these issues arecurrently under way.In summary, we have revealed the roles of three transcrip-

tional regulators, c-Myc, HIF-1α, and Sp4, in the regulationof AS expression in response to arginine deprivation inthree melanoma cell lines. Among which, the accessibilityof c-Myc to interact with the AS promoter is critical forthe induction of AS expression. Lack of AS expression hasbeen considered a biomarker for sensitivity to ADI treat-ment in melanomas. Our present study shows that induc-ibility of AS expression is associated with sensitivity toADI treatment. These results suggest that mere screeningof AS expression levels in melanoma patients before ADItreatment is not sufficient. Our present study has goneone step further and elucidated the mechanisms of AS in-duction. The identification of positive and negative regula-tors should provide an important rationale for thedevelopment of effective treatment modalities for malignantmelanoma through modulation of AS expression.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Polaris, Inc., for providing ADI-PEG20.

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2009;8:3223-3233. Published OnlineFirst November 24, 2009.Mol Cancer Ther Wen-Bin Tsai, Isamu Aiba, Soo-yong Lee, et al.

/Sp4αsynthetase expression involving c-Myc/HIF-1cells is associated with induced argininosuccinate Resistance to arginine deiminase treatment in melanoma

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