nutritional stress induced by tryptophan- degrading ......tophan, glutamine, arginine, leucine,...

Microenvironment and Immunology

Nutritional Stress Induced by Tryptophan-Degrading Enzymes Results in ATF4-DependentReprogramming of the Amino Acid TransporterProfile in Tumor CellsElina Timosenko1, Hemza Ghadbane1, Jonathan D. Silk1, Dawn Shepherd1, Uzi Gileadi1,Lauren J. Howson1, Robert Laynes2, Qi Zhao3, Robert L. Strausberg3, Lars R. Olsen4,Stephen Taylor5, Francesca M. Buffa6, Richard Boyd2, and Vincenzo Cerundolo1

Abstract

Tryptophan degradation is an immune escape strategyshared by many tumors. However, cancer cells' compensatorymechanisms remain unclear. We demonstrate here that ashortage of tryptophan caused by expression of indoleamine2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase(TDO) resulted in ATF4-dependent upregulation of severalamino acid transporters, including SLC1A5 and its truncatedisoforms, which in turn enhanced tryptophan and glutamine

uptake. Importantly, SLC1A5 failed to be upregulated inresting human T cells kept under low tryptophan conditionsbut was enhanced upon cognate antigen T-cell receptorengagement. Our results highlight key differences in theability of tumor and T cells to adapt to tryptophan starvationand provide important insights into the poor prognosis oftumors coexpressing IDO and SLC1A5. Cancer Res; 76(21);6193–204. �2016 AACR.

IntroductionIndoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-

dioxygenase (TDO) are rate-limiting enzymes of the kynure-nine pathway, which convert the essential amino acid (EAA)tryptophan to kynurenine. IDO-mediated tryptophan degra-dation, long recognized as an antimicrobial resistance mech-anism, has now been firmly established as a key metabolicregulator of the immune response in a number of settings,including maternal/fetal interface, autoimmunity, and cancer.

An increasing number of studies have demonstrated thattryptophan catabolism is an important mechanism of immuneescape exploited by IDO- and TDO-expressing tumors. Manyhuman cancers constitutively express IDO (1, 2) and TDO (3,4), and acquisition of these tryptophan-degrading enzymes byimmunogenic tumors prevents their rejection by tumor-spe-cific T cells (1, 3). Furthermore, high IDO expression correlateswith poor clinical prognosis in several human cancers (5) andplays an immunosuppressive role in the context of immu-notherapies (6).

Starvation of cells from tryptophan and other amino acids issensed through the general control nonderepressible protein 2(GCN2) kinase pathway, which is activated in response toaccumulation of uncharged tRNA. Upon activation, GCN2phosphorylates the translation initiation factor eIF2a, whichpromotes translation of the master regulator of the integratedstress response, activating transcription factor 4 (ATF4; ref. 7).ATF4 regulates transcription of a spectrum of genes required foramino acid synthesis and import, redox balance, and angio-genesis (8, 9).

While it is known that in T cells, IDO-mediated tryptophandepletion induces a GCN2-dependent proliferative arrest (10),inhibiting the G1 to S-phase transition (11, 12), the molecularmechanisms by which IDO- and TDO-expressing cancer cellsremain resistant to local tryptophan depletion remain largelyunknown. To address this question, we performed wholetranscriptome sequencing of HeLa cells expressing IDO andcompared this transcriptome profile to that of IDO� HeLa cellsor of HeLa cells treated with IFNg—a known inducer of endog-enous IDO expression (11, 12). These results have led to thedemonstration that overexpression of tryptophan-degradingenzymes IDO and TDO in tumor cells results in the upregula-tion of several amino acid transporters and in particular of

1MRC Human Immunology Unit, Weatherall Institute of MolecularMedicine,UniversityofOxford, JohnRadcliffeHospital,Oxford,UnitedKingdom. 2Department of Physiology, Anatomy and Genetics, Uni-versity ofOxford,Oxford, UnitedKingdom. 3LudwigCancerResearch,New York, New York. 4Center for Biological Sequence Analysis,Department of Systems Biology, Technical University of Denmark,Lyngby, Denmark. 5The Computational Biology Research Group(CBRG), Weatherall Institute of Molecular Medicine, University ofOxford, John Radcliffe Hospital, Oxford, United Kingdom. 6Depart-ment of Oncology,Old Road Campus Research Building, University ofOxford, Oxford, United Kingdom.

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

Current address for H. Ghadbane: Immunocore Ltd, 101 Park Drive, Milton Park,Abingdon, OX144RY, UK; current address for J.D. Silk: Adaptimmune Ltd, 91 ParkDrive, Milton Park, Abingdon, OX14 4RY, UK; and current address for Q. Zhao:Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY10591.

Corresponding Author: Vincenzo Cerundolo, MRC Human Immunology Unit,Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX39DS, UK. Phone: 44-0-1865222412; Fax: 44-0-1865222502; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-15-3502

�2016 American Association for Cancer Research.

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SLC1A5—a sodium-dependent high-affinity glutamine trans-porter of the solute carrier family (SLC) 1 (13, 14), which isoverexpressed in several human cancers.

In this study, we establish a causative relationship betweenIDO-mediated tryptophan degradation and SLC1A5 upregula-tion and characterize the dichotomy of this phenomenon intumor cells and T lymphocytes.

Materials and MethodsCell lines and culture media

The HeLa, A431, DX3, HPAF, and DU145 tumor cell lineswere obtained from Cancer Research UK. Cell line character-izations were performed by the cell bank using short tandemrepeat (STR) profiling. Cell lines were kept frozen and werepassaged in the user's laboratory for fewer than 6 months afterresuscitation. HeLa cells were cultured in Minimum EssentialMedium supplemented with 10% FBS. A431, DX3, and HPAFcells were maintained in DMEM supplemented with 10% FBS.DU145 cells were cultured in RPMI-1640 medium supple-mented with 10% FBS. Customized DMEM depleted of tryp-tophan, glutamine, arginine, leucine, isoleucine, glycine, andserine (Life Technologies) was supplemented with 10% dia-lyzed FBS (Life Technologies) and the missing amino acidsaccordingly.

Transcriptome sequencing (RNA-seq)RNA-seq was performed as described in the Supplementary

Materials andMethods. The data have been deposited in the GEOdatabase (GEO accession GSE75956).

Measurement of 3H-labeled amino acid uptakeCells were washed twice in PBS, followed by a 20-minute

incubation at 37�C. [3H]L-tryptophan or [3H]L-glutamine(both from PerkinElmer) were added to the cells at 50 and200 pmol/mL, respectively, for a fixed time at 37�C. Uptakewas stopped by removing the solution and adding 20 mmol/LL-lysine in ice-cold PBS. The cells were lysed with 1% NaOH and0.1% SDS, and [3H]L-tryptophan/[3H]L-glutamine uptake wasdetermined by liquid scintillation.

CRISPR-Cas9–mediated gene silencingCRISPR-Cas9 technologywas used to silence SLC1A5 andATF4

by targeting unique sequences in the coding region of exon 4 andexon 1, respectively. Cas9 and the chimeric guide RNA weredelivered to wild-type (WT) HeLa cells using the lentiCRISPRv2construct (Addgene plasmid #52961).

Analysis of mRNA expression in primary human tumorsExpression of mRNA measured by RNA sequencing was

downloaded from The Cancer Genome Atlas data portal forliver hepatocellular carcinoma (LIHC; n ¼ 374), ovarian serouscystadenocarcinoma (OV; n ¼ 309), glioblastoma multiforme(GBM; n ¼ 169), brain lower grade glioma (LGG; n ¼ 534),uterine corpus endometrial carcinoma (UCEC; n ¼ 177),adrenocortical carcinoma (ACC; n ¼ 79), cervical squamouscell carcinoma and endocervical adenocarcinoma (CESC; n ¼306), breast invasive carcinoma (BRCA; n ¼ 1,102), andkidney renal clear cell carcinoma (KIRC; n ¼ 534), and theSpearman's rank correlation coefficient was calculated betweenthe expressions of SLC1A5 and WARS, TDO2, IDO1, and

ATF4 for each tumor type. In addition, clinical data weredownloaded for the LGG samples, and Kaplan–Meier curvescomparing survival of patients with low and high expression ofSLC1A5, IDO1, and the combinations thereof were made.Statistical analysis was performed using log-rank test.

Statistical analysisStatistical analysiswasperformedusingPrism software (Graph-

Pad). Unpaired, 2-tailed Student t test was used to determinestatistical significance, unless indicated otherwise. The thresholdfor rejection of the null hypothesis was P < 0.05.

ResultsTumor cells expressing tryptophan-catabolizing enzymesupregulate the expression of several amino acid transportergenes

To investigate the effect of IDO activity on tumor cell wholetranscriptome profile, we developed an in vitro system whereIDO expression was induced either by transducing HeLa cellswith a lentivirus encoding human IDO (IDOþ HeLa; ref. 12) orby treating HeLa cells with IFNg (IFNg HeLa). We confirmedthat IDO was biologically active by measuring the kynurenine-to-tryptophan ratio in the supernatants obtained from IDOþ

and IFNg HeLa cells, as compared with HeLa cells transducedwith a lentivirus encoding GFP (GFP HeLa) or untreated WTHeLa cells (Supplementary Fig. S1A). Using this system, weidentified the genes modulated in IDOþ HeLa cells underconditions of nutritional stress caused by incubation in limitedamount of culture medium and compared them with the geneprofiling of IFNg HeLa cells by RNA-seq (GSE75956). After 72hours of culture, IDOþ HeLa cells upregulated several genesthat were also upregulated in IFNg HeLa cells (Fig. 1A). Suchshared genetic signature between IDOþ HeLa and IFNg HeLacells was more evident at 72 hours rather than at 48 hours (Fig.1B), which could be accounted for by differences in the kineticsrequired by IDOþ HeLa cells to degrade tryptophan containedin the culture medium. At 72 hours, this shared transcriptionalsignature included the expression of genes involved in theregulation of cellular redox signaling and apoptosis (such asTXNIP; ref. 15), t-RNA amino acylation, glutamine/glutamateand monosaccharide metabolism, and amino acid and carbox-ylic acid biosynthesis and transport, among other functions(Fig. 1A). The most abundantly upregulated gene involved in t-RNA amino acylation was tryptophanyl-tRNA synthetase(WARS), whereas the most abundantly upregulated genes asso-ciated with amino acid transport were SLC7A11 (cystine/glu-tamate exchanger), SLC1A4 (glutamate/neutral amino acidtransporter), SLC1A5 (glutamine/neutral amino acid transport-er) and, to a lesser extent, SLC6A9 (glycine neurotransmittertransporter; Fig. 1A and B). Other major amino acid transpor-ters, including CD98 heavy chain (SLC3A2) and LAT1(SLC7A5), showed a less pronounced differential expressionin HeLa cells expressing IDO (Fig. 1B). Notably, of the top 4upregulated SLC genes, SLC1A5 was the only transporter asso-ciated with EAA transport (16) and tryptophan transport net-work, as determined using the MetaCore pathway analysissoftware (Supplementary Fig. S1B).

Further analysis demonstrated that, in addition to the full-length SLC1A5 transcript, hereafter referred to as SLC1A5 long[SLC1A5(L)], there were truncated splice variants of SLC1A5—

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Figure 1.Remodeling of transcriptomic profiling in IDO-expressing tumor cells. A, clustered RNA-seqexpression data showing the fold change in the expression of genes upregulated in both IDO1-transduced versus GFP-transduced HeLa cells cultured for 72 hours (left circles) and IFNg-treatedversus untreated WT HeLa cells cultured for 72 hours (right circles), as indicated in the legendbox on the right. B, RNA-seq profiles of solute carrier (SLC; top) and amino acid metabolism(bottom) genes in IDO1-transduced versus GFP-transduced and IFNg-treated versus untreatedWT HeLa cells cultured for 48 or 72 hours. C and D, WT, IFNg-treated, GFP-transduced, IDO1-transduced, and TDO2-transduced HeLa cells were cultured for 72 hours. SLC1A5(L), SLC1A5(S),SLC1A5(M), SLC7A11, and SLC1A4mRNA expression levels were analyzed by quantitative PCR andare shown as fold change in expression relative to Actb. Bars represent the mean � SEM (C).Western blotting was performed with indicated antibodies and protein levels were quantified bydensitometry (D). Data are representative of at least two independent experiments.

Reprogramming of Amino Acid Transporters in IDOþ Tumor Cells

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SLC1A5 middle [SLC1A5(M)] and SLC1A5 short [SLC1A5(S);Supplementary Fig. S1C]. We utilized the unique sequences inthe noncoding regions of the SLC1A5(S) and SLC1A5(M) tran-scripts to design aquantitative PCRassay for specific amplificationof SLC1A5(L), SLC1A5(S), and SLC1A5(M)mRNA. IFNg-treatedWT, IDO1-transduced HeLa, as well as HeLa cells overexpressingthe tryptophan-degrading enzyme TDO exhibited increasedexpression of all 3 SLC1A5 variants (Fig. 1C). In agreement withthe RNA-seq data, real-time PCR analysis of transcripts associatedwith SLC7A11 and SLC1A4 confirmed their upregulation in IDO-and TDO-expressing HeLa cells (Fig. 1C).

Next, we assessed SLC1A5 protein expression in tumorcells exhibiting IDO or TDO activity using an antibodyspecific for the N-terminus of the full-length SLC1A5, whichis expected to detect SLC1A5(L) but not the other isoforms.SLC1A5 expression levels increased substantially in tumorcells treated with IFNg and those transduced with IDO orTDO (Fig. 1D).

To validate that tumor cells overexpressing SLC1A5 are ableto upregulate their amino acid uptake, we overexpressedSLC1A5(L) and its most truncated isoform, SLC1A5(S), inHeLa cells. Because SLC1A5 has been reported to have highaffinity for glutamine (13, 14, 17), we first examined the

capacity of transduced cells to transport radiolabeled gluta-mine ([3H]Gln). As expected, we observed higher levels ofradiolabeled glutamine uptake in SLC1A5(L)-transducedtumor cells than in those expressing endogenous levels ofthis transporter (Fig. 2A). Notably, glutamine uptake capacitywas also increased in tumor cells overexpressing SLC1A5(S)(Fig. 2A). Consistently with the reported Naþ dependency ofSLC1A5 (13, 14), SLC1A5(L)- and SLC1A5(S)-transducedtumor cells failed to upregulate glutamine uptake underNaþ-free conditions (data not shown). Furthermore, treatmentof both transduced and control tumor cells with a characterizedpharmacologic inhibitor of SLC1A5 O-benzyl-L-serine (BenSer;refs. 18, 19) reduced glutamine uptake in a dose-dependentmanner (Fig. 2A).

Next, we assessed the action of SLC1A5 on radiolabeledtryptophan ([3H]Trp) uptake. Overexpression of SLC1A5(L),and particularly of SLC1A5(S) in HeLa cells, improved theirtryptophan uptake capacity (Fig. 2B). Conversely, radiolabeledtryptophan transport was reduced in the presence of SLC1A5pharmacologic inhibitor BenSer (Fig. 2C). In line with thesefindings, BenSer treatment also reduced mTOR activationin HeLa cells (Supplementary Fig. S2A). Furthermore, SLC1A5knockdown resulted in a significant impairment of HeLa

Figure 2.

SLC1A5(L)- and SLC1A5(S)-transduced HeLa cells exhibit increased glutamine and tryptophan uptake. A–C, uptake experiments inWT, GFP-transduced, SLC1A5(L)-transduced, and SLC1A5(S)-transduced HeLa cells were performed for [3H]-glutamine (A) or [3H]-tryptophan (B and C) in the presence or absence of SLC1A5inhibitor BenSer over 10 or 3 minutes, respectively. [3H]-Tryptophan uptake was also performed in the presence of excess unlabeled tryptophan (B). Bars representthe mean � SEM. � , P < 0.05; �� , P < 0.01; �� , P < 0.001. Data are representative of at least two independent experiments.

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cell proliferation under amino acid sufficient conditions (Sup-plementary Fig. S2B and S2C). This effect was enhanced evenfurther following tryptophan withdrawal (SupplementaryFig. S2C).

Although not reported to transport tryptophan directly (13,17), SLC1A5 has been proposed to facilitate the activity of thedominant neutral amino acid transporter LAT1 (SLC7A5; ref. 20)through bidirectional transport (16). Consistent with this model,we found that tryptophan uptake in HeLa cells is heavily depen-dent on LAT1 activity, as evidenced by the abolished radiolabeledtryptophan transport into tumor cells lacking LAT1 (Supplemen-tary Fig. S2D and S2E).

Collectively, these findings demonstrate for the first time theassociation between tryptophan catabolism and modulation ofmetabolic and amino acid transporter profiling in tumor cells. Inparticular, we show that increased expression levels of SLC1A5isoforms lead to improved transport of glutamine and tryptophaninto tumor cells.

Tryptophan-starved tumor cells upregulate SLC1A5 expressionBecause IDO activity converts tryptophan into kynurenine, we

speculated that upregulation of SLC1A5 in IDOþ tumor cellscould be accounted for by the reduced concentration of trypto-phan in the culture medium. Alternatively, it may be due toaccumulation of tryptophan metabolite kynurenine, which hasbeen identified as an endogenous ligand of the human arylhydrocarbon receptor (AhR; ref. 4). To distinguish between these2 possibilities, we exposed WT HeLa cells, exhibiting no IDO orTDO activity, either to tryptophan-deficient conditions or toincreasing concentrations of AhR agonists kynurenine and 6-formylindolo[3,2-b]carbazole (FICZ) andmeasured their SLC1A5expression. While AhR agonists had no effect on the levels ofSLC1A5 expression (Supplementary Fig. S3), WT HeLa cellsincubated in the culture medium depleted of tryptophan upre-gulated SLC1A5 (Fig. 3A and B). This effect was observable atconcentrations of tryptophan equal to or below 5 mmol/L(Fig. 3B) and was abolished at the concentration of 10 mmol/L

Figure 3.

Tryptophan depletion upregulatesSLC1A5 expression in a panel of IDO-negative tumor cell lines. A and B, WTHeLa cells were incubated in tissueculture medium containing indicatedconcentrations of tryptophan for 72hours (A) or 48 hours (B). SLC1A5(L) andSLC1A5(S)mRNA expression levels wereanalyzed by quantitative PCR and areshown as fold change in expressionrelative to Gapdh. Bars represent themean � SEM (A). Western blotting wasperformedwith indicated antibodies andprotein levels were quantified bydensitometry (B). C, HeLa, A431, DU145,DX3, and HPAF tumor cell lines wereincubated in tissue culture mediumdepleted of tryptophan for 72 hours.Western blotting was performed withindicated antibodies and protein levelswere quantified by densitometry. D, WTHeLa cells were incubated in tissueculture medium depleted of indicatedamino acids for 48 hours. Westernblotting was performed with indicatedantibodies, and protein levels werequantified by densitometry. Data arerepresentative of at least twoindependent experiments.






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or higher, which we confirmed at both themRNA and the proteinlevels (Fig. 3A and B). Importantly, we extended the resultsobtained in HeLa cells to a panel of other human tumor celllines, including epidermoid carcinoma, prostate, and pancreascancer cell lines (Fig. 3C), demonstrating that the mechanismscontrolling SLC1A5 upregulation are shared by a range of tumorcell lines of different origin.

Having established that shortage of tryptophan can upregulateSLC1A5, we decided to extend these results by assessing whetherthe lack of other amino acids could also have an effect on thistransporter's expression in tumor cells. Incubation of WT HeLacells in the culture medium depleted of glutamine or argininemarkedly increased their SLC1A5 expression, whereas this induc-tion was less profound in the absence of leucine or isoleucine(Fig. 3D). Glycine or serine deprivation, on the other hand,exerted no effect on SLC1A5 expression (Fig. 3D). Interestingly,tryptophan depletion, in addition to upregulation of SLC1A5,also increased expression of SLC7A11 and SLC1A4 (Fig. 4G),demonstrating a more general effect of tryptophan catabolismon the amino acid transporter profiling.

Collectively, these findings show that SLC1A5 expression isregulated by amino acid availability and demonstrate its sensi-tivity to variation in the local tryptophan concentration.

SLC1A5 upregulation in tryptophan-starved tumor cells isdependent on the ATF4 pathway

Wenext assessed the possiblemechanisms thatmay account forthe induction of SLC1A5 during tryptophan starvation. Aminoacid depletion is sensed by the ATF4 pathway, which can beactivated in response to increased levels of uncharged tRNA (7).We interrogated our RNA-seqdata for changes in the expression ofstress response genes in IDOþ tumor cells and observed a strongtranscriptional signature associated with ATF4 pathway activa-tion, as evidenced by the upregulation of ATF4, ATF3, CHOP,GADD34, TRIB3, and HERPUD1 genes in IFNg-treated WT andIDO1-transduced HeLa cells (Fig. 4A and B). In line with thereported role of ATF4 in autophagy regulation (21), tumor cellsexposed to tryptophan deprivation also exhibited increasedautophagy levels, as determined by elevated LC3-II to LC3-I ratio(Supplementary Fig. S4).

To investigate whether ATF4 expression is differentially mod-ulated by the absence of distinct amino acids, we measured ATF4levels in WT HeLa cells cultured under various amino acid–deprived conditions. While ATF4 protein was almost undetect-able in complete medium, its expression increased markedly intumor cells cultured in the absence of tryptophan, glutamine, and,to a lesser extent, arginine and isoleucine (Fig. 4C). Lack of glycineand serine, on the other hand, failed to activate ATF4 inHeLa cells(Fig. 4C). Furthermore, similarly to SLC1A5, ATF4 expressionwasinduced by concentrations of tryptophan lower than 5 mmol/L, as

ATF4 could not be detected after supplementing the culturemedium with 10 mmol/L tryptophan (Fig. 4D). We thus demon-strated that ATF4 expression in starved tumor cells mirrors that ofSLC1A5.

To determine the role of the ATF4 pathway in regulation ofSLC1A5 expression under tryptophan-deficient conditions, weknocked down ATF4 from HeLa cells using a lentiviral CRISPRconstruct (Supplementary Table S1). In contrast to WT tumorcells, no SLC1A5 upregulation could be detected in ATF4knockdown tumor cells under culture conditions lacking tryp-tophan (Fig. 4E). We confirmed these results using ATF4 shRNAknockdown strategy (data not shown). Furthermore, similar toSLC1A5(L), ATF4 knockdown tumor cells failed to upregulatetranscription of truncated SLC1A5 isoforms following trypto-phan withdrawal (Fig. 4F), underscoring that SLC1A5 isoforminduction in tryptophan-starved tumor cells is also controlledby the ATF4 pathway. Similarly, we showed that upregulationof SLC7A11 and SLC1A4 in tryptophan-depleted medium wasabolished in ATF4 knockdown cells (Fig. 4G).

Collectively, these findings demonstrate the role of ATF4in modulating the changes in amino acid transporter pro-filing under culture conditions with reduced concentrationsof tryptophan.

SLC1A5 expression in human T cells is unaffected bytryptophan starvation

Tryptophan degradation by IDO-expressing tumors is acommon mechanism of immune escape, which inhibits effec-tor T cells (1, 3). We (12) and others (10, 11, 22) havedemonstrated that T-cell exposure to tryptophan-depletedmicroenvironment induces their proliferative arrest. To deter-mine whether T lymphocytes are able to alter their SLC1A5expression profile when undergoing tryptophan starvation, weincubated resting or activated human CD4þ and CD8þ T cellsin tryptophan-depleted medium. While resting T cells hardlyexhibited any detectable levels of SLC1A5, treatment withaCD3/CD28 antibodies readily upregulated SLC1A5 expres-sion (Fig. 5A), which is consistent with the increased metabolicdemands of activated T cells (23). In line with these results,pharmacologic inhibition of SLC1A5 reduced mTOR activationin stimulated T cells (Supplementary Fig. S5A).

Surprisingly, in sharp contrast to the upregulation of SLC1A5observed in a panel of tumor cell linesmaintained under identicalconditions, resting CD4þ and CD8þ T cells failed to enhanceSLC1A5 expression in response to tryptophan depletion (Fig. 5Aand B). Moreover, while incubation of T cells with aCD3/CD28antibodies led to increased SLC1A5 expression, tryptophan deple-tion of aCD3/CD28-treated T cells failed to induce further upre-gulation of SLC1A5, in the face of ATF4 activation (Fig. 5A).Human T cells kept in tryptophan-depletedmedium also failed to

Figure 4.ATF4 mediates SLC1A5 upregulation upon tryptophan withdrawal. A, RNA-seq transcriptome profiles of the integrated stress response genes in IDO1-transducedversus GFP-transduced and IFNg-treated versus untreated WT HeLa cells cultured for 48 or 72 hours. B, fold change in the expression of indicated genes in IFNg-treated versus untreated WT HeLa cells cultured for 72 hours (top circles) and in IDO1-transduced versus GFP-transduced HeLa cells cultured for 72 hours(bottom circles), as indicated in the legend box on the right.C andD,WTHeLa cells were incubated in tissue culturemedium depleted of indicated amino acids (C) orin thepresenceof indicated tryptophan concentrations (D) for 48hours.Westernblottingwasperformedwith indicated antibodies andprotein levelswerequantifiedby densitometry. E–G, WT and ATF4 knockdown (ATF4-KD) HeLa cells were incubated in tissue culture medium depleted of tryptophan for 48 hours. Westernblotting was performed with indicated antibodies and protein levels were quantified by densitometry (E). SLC1A5(L), SLC1A5(S), and SLC1A5(M)mRNA expressionlevels (F) and SLC7A11 and SLC1A4 mRNA expression levels (G) were quantified by quantitative PCR and are shown as fold change in expression in tryptophan-depleted versus complete tissue culture medium, relative to Hprt1. Bars represent the mean � SEM. ND, nondetected. Data are representative of at leasttwo independent experiments.






express the alternative SLC1A5 isoform (Fig. 5B). Notably,although T cells increased the levels of SLC1A5 expression uponstimulation, this upregulationwas not sufficient to facilitate levelsof tryptophanuptake comparable to thoseobserved in tumor cells(Supplementary Fig. S5B).

Furthermore, in contrast to tumor cells, tryptophan-starved Tcells also failed to upregulate tryptophanyl-tRNA synthetaseWARS, whereas its expression, similarly to that of SLC1A5, wasenhanced upon T-cell activation (Supplementary Fig. S5C). Thesefindings highlight differential regulation of ATF4-dependentgenes controlling compensatory mechanisms triggered by theshortage of tryptophan in tumor and T cells.

Upregulation of SLC1A5 in aCD3/CD28-treated T cells isconsistent with the increased metabolic demand of activated Tcells for amino acids such as glutamine (24, 25). However, itremains unclear whether upregulation of SLC1A5 upon T-cellreceptor (TCR)-dependent T-cell activation can be modulated bythe strength of the TCR signaling events. To address this question,we stimulated an NY-ESO-1–specific human CD8þ T-cell clonewith increasing concentrations of the cognate NY-ESO-1157–165peptide (26) or with aCD3/CD28 antibodies, as a control. Wethen used the previously characterized SLC1A5-specific stainingreagent (27), which utilizes the binding specificity of the RD114virus envelope glycoprotein for this amino acid transporter (28),to correlate SLC1A5 surface expression with T-cell activation, asdefined by CD25 upregulation. We observed that upregulation ofSLC1A5 directly correlated with the amount of NY-ESO-1157–165peptide added (Fig. 5C) and with CD25 upregulation (data notshown).

Collectively, these findings demonstrate that SLC1A5 expres-sion in human T cells is dependent on the strength of TCRengagement. However, unlike human tumor cell lines, restingand activatedCD4þ andCD8þ T cells fail to upregulate SLC1A5 inresponse to tryptophan starvation. These results highlight a keydifference in the ability of tumor cells and T lymphocytes to adaptto tryptophan starvation.

SLC1A5 and IDO coexpression is observed in several primaryhuman tumors and is associated with poor survival in brainLGG

Having identified the relationship between IDO/TDO-induced tryptophan depletion and SLC1A5 upregulation inhuman tumor cell lines, we next tested whether IDO-expressingtumors upregulate SLC1A5 expression in vivo. To address thisquestion, we injected IDO-expressing or IDO-negative mouseEG7 GFP thymoma cells into B6.SJL-Ptprca mice and assessedSLC1A5 transcription after 5 days of in vivo growth. Althoughwhen kept in normal tissue culture medium, EG7 GFP IDO andEG7 GFP cells had similar transcription levels of SLC1A5 (datanot shown), upon in vivo injection EG7 GFP IDO tumorsexhibited significantly higher levels of SLC1A5 expression thanthe IDO-negative EG7 GFP tumors (Supplementary Fig. S6A).

We then investigated whether the association between trypto-phan catabolism and SLC1A5 expression can be observed inprimary human tumor samples. In line with regulation ofSLC1A5 expression by the ATF4 pathway, our in silico analysis ofavailable RNA-seq data demonstrated that in a large proportionof human tumors, SLC1A5 positively correlates with ATF4

Figure 5.

Human T cells upregulate SLC1A5 expression upon TCR stimulation, but not in response to tryptophanwithdrawal.A,WTHeLa, DU145, HPAF cells, and human CD4þ

andCD8þ T cells, either stimulated (þ) or unstimulated (�) with plate-boundaCD3/CD28 antibodies, were incubated in tissue culturemedium in the presence (þ) orin the absence (�) of tryptophan for 48 hours. Western blotting was performed with indicated antibodies. B, WT HeLa and resting human CD4þ and CD8þ

Tcellswere incubated in tissue culturemediumdepleted of tryptophan for 48hours.SLC1A5(L) andSLC1A5(S)mRNAexpression levelswere analyzedbyquantitativePCR and are shown as fold change in expression in tryptophan-depleted versus complete tissue culture medium, relative to Hprt1. Bars represent themean � SEM. ND, nondetected. C, NY-ESO-1–specific CD8þ T cell clone was stimulated with aCD3/CD28 antibodies or cocultured with antigen-presenting cellsprepulsed with the indicated concentrations of NY-ESO-1157–165 (V) peptide analogue. After 48 hours, SLC1A5 surface expression was assessed by flowcytometry using SLC1A5 cell surface RBD ligand staining. Bars represent the mean � SEM. MFI, mean fluorescence intensity. Data are representativeof at least two independent experiments.

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Figure 6.

Expression of SLC1A5 correlates with the expression of tryptophan-degrading enzymes in primary human tumors and is associated with decreased survival in brainLGG. A, Spearman rank correlation between SLC1A5 and WARS, TDO2, IDO1, or ATF4 gene expression in RNA-seq data of human LIHC, OV, GBM, brain LGG,UCEC, ACC, CESC, BRCA, and KIRC. The size of the circle indicates significance of correlation. B, Kaplan–Meier curves comparing survival of patients with brain LGGtumors expressing high or low levels of SLC1A5 (P¼ 0.00006). C, Kaplan–Meier curves comparing survival of patients with brain LGG tumors expressing high or lowlevels of IDO1 (P ¼ 0.00053). D, Kaplan–Meier curves comparing survival of patients with brain LGG tumors expressing high or low SLC1A5 together with highor low levels of IDO1 (P ¼ 0.00005). High and low expression of SLC1A5 or IDO1 was defined as above and below median expression, respectively.






expression (Fig. 6A). However, as ATF4 activation can occur inresponse to several stresses, including hypoxia/anoxia and nutri-ent deprivation (29), we assessed whether coexpression ofSLC1A5 and ATF4 correlated with the upregulation of genesmorerelevant to catabolism of tryptophan in particular, such as tryp-tophanyl-tRNA synthetase WARS, which we have previouslyshown to be upregulated in IDO-expressing and tryptophan-starved HeLa cells (Fig. 1A and B and Supplementary S5C). Theresults of this analysis demonstrated a strong positive correlationbetween SLC1A5 and WARS expression in several tumors, par-ticularly human LIHC, OV, glioblastoma multiforme, and brainLGG (Fig. 6A). Importantly, in these tumor types, SLC1A5 alsocorrelated significantly with the expression of one or both of thetryptophan-degrading enzyme-encoding genes IDO1 and TDO2(Fig. 6A), hence supporting our in vitro data describing thetryptophan shortage–induced SLC1A5 upregulation in IDOþ andTDOþ tumor cell lines.

We next assessed the significance of SLC1A5 and IDO1 tumorexpression for patient prognosis. We found that, in brain LGG,which exhibited a particularly strong positive correlation betweenSLC1A5 and IDO1 expression (Fig. 6A), high expression ofSLC1A5 was significantly associated with decreased patient sur-vival (HR¼ 0.40526; log-rank test P¼ 0.00006; Fig. 6B). We alsoobserved a similar association between survival and high IDO1expression in this tumor type (HR ¼ 0.46179; log-rank test P ¼0.00053; Fig. 6C). Furthermore, patients with IDOhiSLC1A5lo

LGGs survived significantly longer than thosewith IDOhiSLC1A5hi

tumors (HR ¼ 0.49285; log-rank test P ¼ 0.01564), whereasIDOloSLC1A5lo tumors correlatedwithbest prognosis (Fig. 6DandSupplementary S6B).

These data highlight the clinical importance of IDO andSLC1A5 upregulation by human tumors such as brain LGG andsuggest their potential use as prognostic biomarkers.

DiscussionIn this study, we showed that IDO activity induces extensive

remodeling of tumor cells' amino acid metabolism gene expres-sion and alters the expression of the amino acid transporter–encoding genes SLC7A11, SLC1A4 and SLC1A5, including 2 novelSLC1A5 splice variants. Such changes in the amino acid trans-porter profiling of IDO-expressing tumor cells are induced byshortage of tryptophan, rather than accumulation of kynurenine,and are dependent on the activation of the transcription factorATF4—a master regulator of numerous stress response genes,including tryptophanyl-tRNA synthetase (WARS; refs. 8, 30).

Upregulation of SLC1A5 (and its splice variants), in addition toenhancing the uptake of glutamine, also improves tryptophantransport, which can be blocked by the SLC1A5-specific inhibitorBenSer (18, 19) or by deleting expression of LAT1 (SLC7A5;ref. 20)—a component of the ubiquitous amino acid transporterSystem L, which constitutes the major neutral amino acid trans-port system, importing large hydrophobic amino acids withbranched or aromatic side chains, such as leucine and tryptophan(31). Being an obligate amino acid exchanger, LAT1 activitydepends largely on the exchange of intracellular glutamine forEAAs (16). Our results describing the abolishment of tryptophanuptake in LAT1knockout tumor cells are consistentwith the abovedata demonstrating that SLC1A5-dependent glutamine effluxfacilitates EAA uptake by activating System L bidirectional trans-port mechanisms (16).

Previous results have implicated that SLC1A5, which, simi-larly to SLC7A5, is highly expressed in several human cancers(32), in promoting tumor cell growth and survival. The anti-proliferative effect of targeting SLC1A5 has been demonstratedin various tumor types (18, 33–40). We have extended thesefindings by demonstrating a role for SLC1A5 in tumor cellproliferation under not only amino acid sufficient but alsotryptophan starvation conditions. These results are consistentwith the hypothesis that SLC1A5-mediated amino acid uptakeacts as a compensatory mechanism to facilitate proliferation ofIDOþ cancer cells under low tryptophan conditions. However,we cannot rule out the alternative hypothesis that SLC1A5 co-operates with IDO to facilitate increased influx of tryptophan,thus ensuring its further deprivation from the tumor microen-vironment through IDO-dependent degradation. Additionalexperiments to address this possibility, as well as the involve-ment of other potentially important tryptophan shortage com-pensatory mechanisms, such as WARS-mediated tryptophanyl-tRNA stock repletion, are warranted.

The mechanisms underlying regulation of glutamine transpor-ters such as SLC1A5 in cancer are largely poorly understood.Whilewe have demonstrated that SLC1A5 isoform induction in IDO-and TDO-expressing tumor cells is a direct consequence of tryp-tophan depletion, we have also observed differential modulationof SLC1A5 expression by the withdrawal of distinct amino acidsfrom the culture medium. These results are consistent withSLC1A5 upregulation representing a specific amino acid deple-tion response mechanism. Importantly, we established that con-centrations of tryptophan ranging from 0 to 5 mmol/L enhanceSLC1A5 expression. Because tryptophan concentrations below 5mmol/L induce T-cell proliferative arrest (22), our findings indi-cate a coordinated cancer immune evasion mechanism designedto inhibit T-cell proliferation while simultaneously upregulatingcompensatory signaling events in cancer cells.

ATF4 is a known regulator of the nutritional stress response (7,8), which we showed to be upregulated in IDO-expressing tumorcells and, importantly, to be activated upon exposure of tumorcells to culture conditions with limited amounts of tryptophan.Notably, all 3 top upregulated transporters in IDOþ tumor cellsshowed evidence of regulation by the ATF4 pathway. These dataare in agreementwith thepreviously reported regulationof severalamino acid transporter genes by the ATF4 pathway (8, 37) andwith the role of ATF4 in promoting tumor cells' survival andproliferation under amino acid starvation conditions (41). Ourresults highlight the importance of the ATF4 stress responsepathway for sustaining metabolic homeostasis in tumor cellsexposed to tryptophan deprivation and identify it as a potentialtarget for treatment of IDOþ and TDOþ tumors. Other pathwayshave also been reported to regulate SLC1A5 expression, includingc-Myc (42), N-Myc (37), Rb/E2F (43), EGF signaling (44), andRNF5 (45). These cell intrinsic mechanisms, linked to the neo-plastic process, could synergize with the environmental effect oftryptophan depletion andmay prove to be important therapeutictargets.

The immunosuppressive effects of tryptophan catabolism on Tcells have been well-documented (1, 10, 12, 22), and the involve-ment of the GCN2/ATF4 pathway has been proposed (10).However, very little is known about the effects of ATF4 pathwayactivation on the amino acid transporter expression profile in Tcells. It is clear that stimulated T cells undergo extensivemetabolicreprogramming to meet the increased energetic and biosynthetic

Timosenko et al.





demands of proliferating cells. This is reflected in the switch toanaerobic glycolysis (23) and enhanced ability of activated T cellsto transport amino acids like phenylalanine, leucine, and gluta-mine (24, 25, 46). Furthermore, T-cell activation is associatedwith upregulation of several amino acid transporters, includingSLC7A5 (LAT1), SLC3A2 (CD98hc), as well as glutamine trans-porters SLC38A1 (SNAT1), SLC38A2 (SNAT2), and SLC1A5 (24,46). Consistent with these findings, we observed enhancedexpression of SLC1A5 in human CD4þ and CD8þ T cells treatedwith aCD3/CD28 antibodies and demonstrated the modulationof SLC1A5 upregulation by the strength of TCR engagement.

The dichotomy between tumor cells and T cells in their abilityto upregulate SLC1A5 in a tryptophan-low microenvironment isof importance. Consistent with previous findings (10), wedetected activation of the ATF4 pathway in activated T cells.Strikingly, however, although T cells were able to upregulateSLC1A5 in response to aCD3/CD28 treatment, which has beenpreviously shown to bemediated by CARMA1 (25), they failed toincrease the levels of this transporter following tryptophan with-drawal, irrespectively of their activation status. Tumor cell lines,on the other hand, readily upregulated SLC1A5 under theseconditions, which was coupled to robust ATF4 expression. Ascenario where ATF4 binding to the C/EBP-ATF composite siteof the target gene occurs without stimulation of transcriptionalactivity has been reported for SLC38A2 (SNAT2) transporter (47).The authors describe a repressive signal downstream of ATF4binding to SNAT2 promoter, which inhibits SNAT2 transcriptionin response to the unfolded protein response. We speculate that asimilar mechanismmay account for the lack of SLC1A5 upregula-tion in tryptophan-starved T cells. It would be insightful tocompare the epigenetic landscape of SLC1A5 promoter followingtryptophan deficiency-inducedATF4 activation in tumor cells andT cells.

In a tumor microenvironment where essential nutrients arerestricted, cancer and immune cells' capacity for metabolic repro-grammingmay play a crucial role in the fate of tumor progression.In line with this, we found that the strong positive correlationbetween SLC1A5 and IDO1 expression in brain LGG is signifi-cantly associatedwithdecreased patient survival. This observationis clinically important, as it suggests the potential use of SLC1A5and IDO1 expression as prognostic biomarkers, and providestherapeutic opportunities. Our observations complement previ-ous studies, which have proposed high SLC1A5 expression as amarker of poor prognosis in MYCN-driven neuroblastoma (37),non–small cell lung cancer (35), and breast cancer (45).

In conclusion,wehavedescribed that in tumor cells, shortage oftryptophan induced by the expression of tryptophan-degradingenzymes initiates ATF4-dependent reprogramming of several

amino acid transporter gene expression, including SLC1A5 andits truncated isoforms, which allows improved glutamine andtryptophan import into tumor cells. Notably, this strategy is notshared by resting human T cells, which provides importantinsights into the mechanisms by which cancer cells but nottumor-infiltrating T cells can compensate for the shortage oftryptophan in the local microenvironment. Finally, IDO andSLC1A5 tumor expression can have significant clinical implica-tions, as demonstrated by the decreased survival associated withIDOhiSLC1A5hi brain LGGs. Our studies shed light on the com-pensatory mechanisms employed by tumors to allow survivalunder conditions of nutritional stress and have relevance for theidentification of novel targets for pharmacologic inhibition oftumors expressing amino acid–degrading enzymes IDO andTDO.

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

Authors' ContributionsConception and design: E. Timosenko, H. Ghadbane, J.D. Silk, R.L. Strausberg,V. CerundoloDevelopment of methodology: E. Timosenko, J.D. Silk, U. Gileadi, R. Laynes,R. Boyd, V. CerundoloAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E. Timosenko, H. Ghadbane, J.D. Silk, D. Shepherd,U. Gileadi, L.J. Howson, R. LaynesAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E. Timosenko, H. Ghadbane, J.D. Silk, U. Gileadi,L.J. Howson, R. Laynes,Q. Zhao, R.L. Strausberg, L.R.Olsen, S. Taylor, F.M. BuffaWriting, review, and/or revision of the manuscript: E. Timosenko, H. Ghad-bane, J.D. Silk, R.L. Strausberg, L.R. Olsen, F.M. Buffa, R. Boyd, V. CerundoloAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E. Timosenko, R. LaynesStudy supervision: V. Cerundolo

AcknowledgmentsWe thank Adrian Harris, Ji-Li Chen, Mariolina Salio, Giorgio Napolitani,

Craig Waugh, Kevin Clark, Margarida Rei, Helio Pais, and Joey Riepsaame(WIMM, Oxford) for their help and assistance.

Grant SupportThis work was supported by Cancer Research UK (ProgrammeGrant #C399/

A2291), the Ludwig Institute for Cancer Research, the Harry Mahon CancerResearch Trust, UK, and the Medical Research Council.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 4, 2016; revised August 11, 2016; accepted August 26, 2016;published OnlineFirst September 20, 2016.

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2016;76:6193-6204. Published OnlineFirst September 20, 2016.Cancer Res Elina Timosenko, Hemza Ghadbane, Jonathan D. Silk, et al. Transporter Profile in Tumor CellsResults in ATF4-Dependent Reprogramming of the Amino Acid Nutritional Stress Induced by Tryptophan-Degrading Enzymes

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