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Adenosine Inhibits Tumor Cell Invasion viaReceptor-Independent Mechanisms

SannaS.Virtanen1,2, AnuKukkonen-Macchi3,MinnaVainio4, Kati Elima3, PirkkoL.H€ark€onen2, Sirpa Jalkanen3,and Gennady G. Yegutkin3

AbstractExtracellular adenosinemediates diverse anti-inflammatory, angiogenic, and other signaling effects via binding to

adenosine receptors, and it also regulates cell proliferation and death via activation of the intrinsic signalingpathways. Given the emerging role of adenosine and other purines in tumor growth and metastasis, this studyevaluated the effects of adenosine on the invasion of metastatic prostate and breast cancer cells. Treatment with lowmicromolar concentrations of adenosine, but not other nucleosides or adenosine receptor agonists, inhibitedsubsequent cell invasion and migration through Matrigel- and laminin-coated inserts. These inhibitory effectsoccurred via intrinsic receptor-independent mechanisms, despite the abundant expression of A2B adenosinereceptors (ADORA2B). Extracellular nucleotides and adenosine were shown to be rapidly metabolized on tumorcell surfaces via sequential ecto-50-nucleotidase (CD73/NT5E) and adenosine deaminase reactions with subsequentcellular uptake of nucleoside metabolites and their intracellular interconversion into ADP/ATP. This wasaccompanied by concurrent inhibition of AMP-activated protein kinase and other signaling pathways. Nodifferences in the proliferation rates, cytoskeleton assembly, expression of major adhesion molecules [integrin-1b (ITGB1), CD44, focal adhesion kinase], and secretion of matrix metalloproteinases were detected between thecontrol and treated cells, thus excluding the contribution of these components of invasion cascade to the inhibitoryeffects of adenosine. These data provide a novel insight into the ability of adenosine to dampen immune responsesand prevent tumor invasion via two different, adenosine receptor–dependent and –independent mechanisms.

Implications: This study suggests that the combined targeting of adenosine receptors and modulationof intracellular purine levels can affect tumor growth and metastasis phenotypes. Mol Cancer Res; 12(12);1863–74. �2014 AACR.

IntroductionDespite significant progress in cancer biology, the exact

mechanisms underlying tumor growth and metastasis arenot fully understood. Reasons for this, among others,include the variety of tumor types, their high plasticity andability to adapt to environmental challenges, the redundancyof signaling pathways involved, histopathological diversity ofthe tumor microenvironment, and complex cross-talkbetween the tumor and supporting stroma as well as otheradjacent host cells (1, 2). Currently, intense research effortshave focused on selective targeting of metabolic and signal-

ing pathways that may be altered during tumorigenesis, withparticular emphasis on the extracellular purinergic signalingcascade (3–5). The anticancer effects of ATP have beendescribed 25 years ago (6) and since then, evidence isaccumulating on potent tumor-suppressing or -promotingroles for ATP, adenosine, and other purines (4, 7).Most models of purinergic signaling depend on functional

interactions between distinct processes. These include (i)release of endogenous ATP and other agonists; (ii) triggeringsignaling events via a series of ligand-gated P2X and meta-botropic P2Y receptors; (iii) ectoenzymatic inactivation ofnucleotides; (iii) binding of generated adenosine to its ownG-protein-coupled receptors; and finally, (iv) metabolismand re-uptake of adenosine and other nucleosides by thecells (8). In view of diverse, often counteracting effects ofATP and adenosine, substantial attention is given to thetumorigenic role of key ectoenzymes mediating stepwiseATP hydrolysis to adenosine: nucleoside triphosphatediphosphohydrolase-1 (NTPDase1, otherwise known asCD39; ref. 9) and ecto-50-nucleotidase/CD73 (4, 10–12).High activities of ecto-50-nucleotidase/CD73 and othernucleotidases may contribute to the clearance of proinflam-matory and proangiogenic ATP in the tumor environment

1Turku University of Applied Sciences, University of Turku, Turku, Finland.2Department of Cell Biology and Anatomy, University of Turku, Turku,Finland. 3Department of Medical Microbiology, University of Turku, Turku,Finland. 4Department of Biology, University of Turku, Turku, Finland.

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

Corresponding Author: Gennady G. Yegutkin, MediCity Research Lab-oratory, University of Turku, Tykist€okatu 6A, 20520 Turku, Finland. Phone:358-2-3337022; Fax: 358-2-3337000; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-14-0302-T

�2014 American Association for Cancer Research.

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(4, 7, 9), simultaneously leading to A2A adenosine receptor–mediated inhibition of effector immune cells, includingcytotoxic T-lymphocytes, natural killer cells, dendritic cells,and macrophages (3, 13, 14).Along with "classical" receptor-mediated pathways, aden-

osine exerts cytotoxic and other biologic effects via intrinsicmechanisms (5, 15, 16). Most of these effects occur at highmicromolar and even millimolar nucleoside concentrations,which are at least two orders of magnitude higher thanadenosine receptor affinities and generally imply the uptakeof extracellular adenosine and/or its metabolites into the cellsthrough adenosine transporters and their ensuing intercon-version into AMP and further to ADP/ATP. As a conse-quence of these metabolic shifts, adenosine may induceapoptosis of different malignant cells via upregulation ofp53 (17) or AMP-activated protein kinase (AMPK; ref. 18)activities, translocation of apoptosis-inducing factor AMIDfrom the cytosol into the nucleus (19), and other mitochon-drial apoptotic pathways (15). Likewise, high concentrationsof adenosine receptor agonists, antagonists, and other nucle-oside analogues also trigger antiproliferative and cytotoxiceffects on normal and leukemic cells via receptor-indepen-dent mechanisms (3, 20, 21), presumably acting as anti-metabolites competing with natural nucleosides and inhibit-ing key enzymes of purine homeostasis.By using androgen-independent prostate carcinoma cells

PC-3 and triple-negative breast cancer cells MDA-MB-231lacking three major biomarkers of breast cancer subtypes(estrogen and progesterone receptors and the EGFR2,HER2), as appropriate in vitro models for studying tumormetastasis, this study was undertaken to identify a linkbetween extracellular purines and invasion. The resultsdemonstrate that pretreatment of tumor cells with lowmicromolar concentrations of adenosine inhibited theirinvasion and migration. Strikingly, these effects mainlyoccurred through the metabolism and uptake of exogenousadenosine with subsequent deregulation of intracellularpurine homeostasis and related signaling pathways, thusproviding a novel insight into the ability of adenosine todampen immune responses and inhibit tumor metastasis viatwo different, adenosine receptor–dependent and –indepen-dent mechanisms.

Materials and MethodsReagents and antibodiesThe following antibodies have been used in the study:

mouse mAbs against CD44 (Hermes-3), integrin-b1(ITGB1) and isotype-specific 3G6 mAb against chickenT cells (22); rabbit polyclonal anti-phospho-AMPKa1/2(Thr172) and mouse anti-AMPKa1/2 antibody (SantaCruz Biotechnology); rabbit polyclonal anti-phospho-AMPK1 (Thr174) and mouse anti-phosphotyrosine mAb(4G10; Millipore); mouse mAbs against focal adhesionkinase (FAK) and phospho-FAK (pY397; Becton DickinsonBiosciences); mouse monoclonal anti-b-tubulin (TUB2.1)and FITC-conjugated anti-mouse Ig (Sigma-Aldrich); hor-seradish peroxidase (HRP)-conjugated rabbit a-mouse and

swine a-rabbit Ig (DAKO A/S); Alexa488-conjugated goata-mouse and a-rabbit Ig, and Alexa546-conjugated phalloi-din (Invitrogen, Life Technologies). Radiolabeled com-pounds [methyl-3H]thymidine and [3H]-cAMP were fromPerkinElmer Inc, [2-3H]AMP from Quotient Bioresearch(GE Healthcare), and [2-3H]adenosine from AmershamBiosciences.Matrigel and lamininwere fromBDBiosciences.Compound C (also known as dorsomorphin) was fromTocris Biosciences (R&D Systems). 50-(N-cyclopropyl)-car-boxamido-adenosine (CPCA, also known as C103_SIG-MA), 50-N-ethyl-carboxamide-adenosine (NECA), lipopoly-saccharide (LPS) from Escherichia coli O111:B4, adenosine,and other nucleosides and chemicals were purchased fromSigma-Aldrich.

Cell culture and treatmentsThe PC-3 human prostate carcinoma cell line and the

MDA-MB-231 human breast cancer cell line were fromATCC. The cells were maintained at 37�C in a humidifiedatmosphere of 5% CO2 in DMEM containing 10% FBS,0.03 mg/mL penicillin, and 0.05 mg/mL streptomycin. Fortreatment experiments, the cells were harvested with trypsin/EDTA and seeded onto tissue culture flasks (25 cm2) or 6-and 24-well plates in complete media for 24 to 48 hours toreach confluence. The medium was replaced by DMEMcontaining 1% BSA (DMEM-BSA). Adenosine, inosine,guanosine, and uridine diluted in DMEM, as well as stockDMSO solutions of NECA, CPCA, and compound C werethen applied onto the cells at 1:100 to 1:500 dilutions.Vehicle-treated cells containedDMEM-BSAwithout or with0.02%DMSO.The cells were treated for 20hours at 37�C ina humidified 5% CO2/95% air, detached by trypsin/EDTA,and used in subsequent assays, as specified below.Cell cultureplates and invasion inserts (8-mm pore size; BD Biosciences)were coated overnight at 4�C before the experiment withMatrigel (100 mg/cm2) or laminin (5 mg/cm2).

Invasion and migration assaysThe invasion and migration of PC-3 and MDA-MB-231

cells were assayed as described previously (23). Briefly, theinvasionwas started by applying a cell suspension (5� 103 in300 mL DMEM-BSA) to the upper chamber of Matrigel-coated cell culture invasion inserts, followed by a 48-hourincubation. Likewise, the migration assay was performed byincubating the cells for 5 hours on the inserts coated withlaminin. In both assays, 300 mL of conditioned media fromthe MG-63 osteosarcoma cell line was placed in the lowerchamber as a bone cell-derived chemoattractant to induceinvasion and migration. The insert membranes were thenfixed with paraformaldehyde, stained with Mayer's hema-toxylin, and cut off from the inserts. The number of cells onthe lower surface was counted with a microscope (10�objective) from 10 consecutive fields representing 40% ofthe total area of the membrane.

Measurement of intracellular cAMPPC3 cells grown on 24-well plates were pretreated for 30

minutes with the inhibitor of ADA erythro-9-(2-hydroxyl-3-

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nonyl)adenine (EHNA, 10 mmol/L) and the phosphodies-terase inhibitor (IBMX, 250 mmol/L) and subsequentlyincubated for one hour (37�C and 5% CO2) in DMEM-BSA containing adenosine (0–500 mmol/L). A2B receptorantagonist alloxazine was added 5 minutes before adenosinetreatment. Adenosine receptor agonists NECA and CPCAwere also tested in the assay, but without EHNA pretreat-ment. The cells were then lysed with perchloric acid andneutralized by KOH. cAMP content in the supernatants wasdetermined by a protein-binding method using [3H]cAMP(�10,000 cpm per tube) and MAFBMillipore MultiScreenfiltration plates, as described earlier (24).

Proliferation assayPC3 were cultured in 96-well plates at a density of 3,000

cells per well. On the next day, cells were rinsed with PBSand incubated for 20 hours in 200mLDMEM-BSA contain-ing [methyl-3H]thymidine (�7� 105 cpm/well) and aden-osine or other compounds. LPS (20 ng/mL) and NaN3 (1mmol/L) were used as positive and negative assay controls,respectively. Cells were harvested using a semiautomatedplate harvester (Tomtech MACH III; Fisher Scientific) andthe incorporated radioactivity was determined by scintilla-tion b-counting.

Quantitative PCRTotal RNA was extracted from PC3 and MDA-MB-231

cells using theNucleo-Spin RNAII Total RNA IsolationKit(Macherey-Nagel) and reverse-transcribed with iScriptcDNA Synthesis Kit (Bio-Rad) according to the manufac-turer's instructions. TaqMan Gene Expression Assays forADORA1, ADORA2A, ADORA2B, ADORA3, and NT5Ewere used as primer/probe sets, and the PCR reactions werecarried out using the 7900HT Fast Real-Time PCR System(Applied Biosystems) in the Finnish Microarray andSequencing Center, Turku Center of Biotechnology(Turku, Finland). All samples were run as triplicates andthe expression values were normalized using human b-actinas an endogenous control.

Flow cytometryControl and adenosine-treated PC3 cells were either

used directly in the assays or additionally seeded for 2 hoursonto a Matrigel-coated 24-well plate. The cells were resus-pended in PBS supplemented with 2% FCS and 0.01%NaN3 and stained with mouse monoclonal antibodies (100mL,�10 mg/mL each) against CD44, integrin-b1, as well asisotype-specific mAb 3G6. The cells were then incubatedwith second-stage FITC-conjugated anti-mouse Ig andanalyzed using a FACScan with Cell Quest software (BectonDickinson).

Immunofluorescence stainingControl and adenosine-treated PC3 cells were plated for 2

hours on a 24-well plate containing Matrigel-coated cover-slips. The adherent cells were stained with antibodiesagainst FAK, phospho-FAK (pY397), AMPKa1/2, phos-pho-AMPKa1/2 (Thr172), and tyrosine phosphorylated

proteins (4G10), and subsequently incubated with appro-priate second-stage Alexa488-conjugated immunoglobulins.For actin cytoskeleton costaining, the cells were additionallyincubated with Alexa546-Phalloidin. The slides weremounted with ProLong Gold Antifade medium containingDAPI (Invitrogen) and examined with a fluorescent micro-scope (Olympus BX60). For more experimental details, seeSupplementary Data.

Western blottingControl and adenosine-treated PC-3 cells were seeded

for 2 hours onto Matrigel-coated 6-well plates. The cellswere lysed and subjected to 8% SDS-PAGE electrophoresis(�30 mg protein/lane), followed by transfer of separatedproteins to nitrocellulose membranes. The amounts of totaland phospho-AMPK1 (Thr174), total and phospho-FAK(pY397), integrin-b1, CD44, b-tubulin, and phosphotyr-osine kinases were determined by using appropriate primaryand secondary HRP-conjugated antibodies as described inSupplementary Data.

Thin-layer chromatographic analysis of purine-converting pathwaysFor ecto-50-nucleotidase and adenosine deaminase (ADA)

assays, PC3 and MDA-MB-231 cells were seeded overnightonto 96-well clear plates (8,000 cells per well), and incubatedat 37�C in a final volume of 100 mL DMEM containing 4mmol/L b-glycerophosphate and 10mmol/L of [3H]AMP or[3H]adenosine. Aliquots of the mixture were applied ontoAlugram SIL G/UV254 thin-layer chromatographic (TLC)sheets (Macherey-Nagel). 3H-labeled AMP and nucleosideswere separated using appropriate solvent systems and quan-tified by scintillation b-counting as described earlier (25).Intracellular purine-converting pathways were evaluated byincubation of PC-3 cells grown on 24-well plates for 20hours in 500 mL DMEM containing 0, 2% BSA, 4 mmol/Lb-glycerophosphate, and 10 mmol/L [3H]adenosine. Thecells were washed to remove non-bound radioactivity andlysed in mammalian lysis buffer (PerkinElmer) with subse-quent TLC analysis of the relative amounts of cell-incorpo-rated [3H]adenosine and its metabolites.

Quantification of ATP and other purines and basesPC-3 cells were incubated in 6-well plates in a starting

volume of 1.2 mL DMEM-BSA without (control) and with10 mmol/L adenosine. Aliquots of the media (100 mL) werecollected at the beginning (zero point) and after 2 and 20hours of incubation. Extracellular nucleotides/nucleosideswere extracted by adding perchloric acid, adjusted to neutralpH by KOH, and stored at �70�C. At the end of exper-iment, control and adenosine-treated cells were washed andlysed for assaying intracellular content. Nucleotides andnucleosides were separated by reverse-phase high-perfor-mance liquid chromatography (HPLC), monitored byabsorption at 258 nm, and quantified from the areasunder the corresponding peaks, as described previously(25). For more experimental details, see SupplementaryData. Intracellular ATP was additionally determined in the

Adenosine Inhibits Invasion via Nonreceptor Mechanisms

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obtained cell lysates by using luciferin-luciferase kit ATPLite(PerkinElmer).

Human phospho-kinase arrayControl and adenosine-treated PC3 and MDA-MB-231

cells were plated for 2 hours onto 6-well plates coated withMatrigel. The phosphorylation profile of major kinases wasdetermined by using the human Phospho-Kinase Array Kitwith a cocktail of biotinylated antibodies (R&D Systems)according to the manufacturer's instructions. The proteinconcentration in cell lysates was determined using the BCAProtein Assay Kit (Pierce). After exposing the membranes toa streptavidin-HRP reagent, the obtained images wereacquired to the Gel Doc-2000 (Bio-Rad Laboratories) andthe intensities of the spots were quantified using the accom-panying Quantity One software after subtracting the back-ground signal.

Statistical analysesThe experiments were repeated at least three times and

each treatment was done as duplicate or triplicate. Data fromkinetic experiments were subjected to computer analysis bynonlinear least-squares curve fitting (GraphPad Prism), andqPCR results were analyzed with the SDS 2.3 software(SABiosciences). Statistical comparisons were made usingStudent t test, and P values < 0.05 were taken as significant.

ResultsTreatment of PC3 cells with adenosine, but not withother nucleosides and purinergic agonists, diminishestheir invasion and migrationTo evaluate the putative in vitro effects of adenosine,

other nucleosides, and purinergic agonists on tumorinvasion and metastasis, cultured PC3 cells were pre-treated for 20 hours with different concentrations of testcompounds followed by incubation in invasion assays for asubsequent 48 hours in the absence of treatments. Asshown in Fig. 1A, treatment of PC-3 cells with increasingconcentrations of adenosine, but not inosine, guanosine,and uridine, progressively diminished their invasionthrough Matrigel-coated inserts with IC50 of 0.19 �0.04 mmol/L. Pretreatment of PC-3 cells with a nonse-lective agonist of adenosine receptors NECA (21, 26)diminished their invasion by approximately 40%, whereasa general A2 receptor agonist CPCA (capable of stimulat-ing both A2A and A2B adenosine receptors; ref. 27) did notaffect the numbers of invaded cells (Fig. 1B). Adenosine,but not other adenosinergic agonists, also diminished themigration of PC-3 cells through laminin, although theseinhibitory effects occurred only at relatively high (50mmol/L) concentrations of the nucleoside (Fig. 1B).

Inhibitory effects of adenosine are mediated vianoncytotoxic and presumably receptor-independentmechanisms, despite expression of A2B receptors onPC-3 cellsThemost commonpathway implicated in adenosine effects

includes the activation of certain adenosine receptor(s). In

fact, the incubation of PC-3 cells with increasing concentra-tions of adenosine triggered a concentration-dependentincrease in the cAMP level with EC50 values of approximately100mmol/L (Fig. 2A),which could bemarkedly attenuated inthe presence of the A2B-selective antagonist alloxazine (Fig.2B).No changes in cAMP levels were observed in the presenceof other nucleosides, uridine, guanosine, or inosine. In com-parison with adenosine, adenosine receptor agonists served asmore potent activators of adenylyl cyclase (Fig. 2B). Thesedata are consistent with previous reports showing the ability ofsubmicromolar and lowmicromolar concentrations ofNECA(26, 28) and CPCA (27) to trigger cAMP accumulation viaactivation of low-affinity A2B receptors on PC-3 and othercells. The presence of A2B receptors is further supported byqPCR data showing the abundant mRNA expression for A2B

(ADORA2B), but not for other adenosine receptor subtypes inPC-3 cells (Fig. 2C). Nevertheless, relatively moderate (incomparison with adenosine) inhibition of tumor invasion byNECA, and the inability of another adenosine A2 receptoragonist CPCA to impair PC-3 cell invasion (see Fig. 1B)implies the potential involvement of nonreceptor-mediatedmechanisms in the revealed inhibitory effects. To rule outdirect cytotoxic effects of adenosine and other compounds,the proliferation rate of the treated cells was evaluated bymeasuring [methyl-3H]thymidine incorporation. NECA,

Figure 1. The effect of adenosine and other nucleosides and purinergicagonists on PC-3 cell invasion andmigration. PC-3 cells were pretreatedwith the indicated concentrations of adenosine (Ado), inosine (Ino), andguanosine (Guan), as well as nonselective (NECA) and A2-selective(CPCA) agonists of adenosine receptors, followed by incubation ininvasion or migration assays in the absence of tested compounds. Thenumbers of invaded and migrated cells were determined as described inMaterials and Methods and expressed either as actual cell counts (A) oras a percentage of controls (B; mean� SEM; n¼ 4–8). Note, because nodifferences were observed between vehicle-treated cells incubatedwithout and with 0.02% DMSO, these results were pooled andconsidered as a common "Control" (Co). �, P < 0.05, as compared withcontrol.

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adenosine, and other nucleosides had no effects on PC-3 cellgrowth, whereas a known stimulator (LPS) or inhibitor(NaN3) of cell proliferation caused the expected increase ordecrease in [3H]thymidine incorporation, respectively (Fig.2D). Furthermore, staining of control and adenosine-treatedPC-3 cells with a caspase-3 substrate NucView 488 andcontinuous imaging of the cells during the subsequent 48hours did not reveal any effect of adenosine (10 mmol/L)on the percentage of fluorescent apoptotic cells (data notshown).

Adenosine does not affect the expression of adhesionmolecules, pattern of cell–matrix interaction, and MMPsecretion by PC3 cellsTo further evaluate the effects of adenosine on the initial

binding steps, the expression levels of major adhesion mole-cules potentially implicated in tumor cell adhesion andmetastasis were determined. Flow-cytometric and immuno-blotting stainings with antibodies against integrin-b1 andCD44 revealed an abundant cell surface expression of thesemolecules, with no differences in expression levels beingdetected between the control and adenosine-treated PC-3cells (Fig. 3). Adenosine also did not affect the expression

(Fig. 3C) and staining patterns (Supplementary Fig. S1) ofneither total nor phosphorylated (Y397) forms of yet anotherimportant adhesion-associated kinase FAK. Consistent withthese staining results, direct kinetic analysis of binding offluorescently labeled PC-3 cells to Matrigel-coated wellsshowed that adenosine treatment does not affect the max-imal binding capacity nor time-course of cell adhesion toextracellular matrix (Supplementary Fig. S2A). Further-more, pretreatment of PC-3 cells with adenosine had noeffects on the levels of matrix metalloproteinase (MMP)-2 orMMP-9 accumulated in the inserts during the invasion assay(Supplementary Fig. S2B). Taken together, these resultsexclude the possibility that adenosine-dependent inhibitionof PC-3 invasion is mediated via the downregulation ofadhesion molecules, impaired cell–matrix interaction, orchanges in gelatinase secretion.

Evidence for rapid metabolism of adenosine and otherpurines by PC-3 cells, followed by uptake of nucleosidesand their resynthesis into intracellular ATPThe following studies were aimed to characterize major

pathways involved in adenosine homeostasis. Ecto-50-nucle-otidase/CD73 is known to be a key enzyme responsible for

Figure 2. The effect of adenosine on adenylyl cyclase activity and proliferation of PC-3 cells. Adenylyl cyclase was assayed by measuring cAMP level afterincubation of PC-3 cells with increasing concentrations of adenosine (0–500 mmol/L; A) or with the indicated concentrations of adenosine (Ado), othernucleosides, and adenosinergic agonists (B). Cells were also pretreated with A2B antagonist alloxazine (Allox) before the addition of adenosine. �,P < 0.05, ascompared with corresponding controls. C, a qPCR analysis shows the abundant mRNA expression for ecto-50-nucleotidase/CD73 (NT5E) and for A2B

(ADORA2B), but not A1 (ADORA1), A2A (ADORA2A) and A3 (ADORA3) adenosine receptors in PC-3 cells. The average mRNA expression of eachgene was presented as a percentage of b-actin mRNA expression measured from the same sample. D, proliferation of PC-3 cells was determined aftera 20-hour incubation with [methyl-3H]thymidine in the absence (Control) and presence of the indicated concentrations of adenosine and other compounds(mmol/L, except for LPS expressed as ng/mL). The bars show the amount of cell-incorporated radioactivity (mean � SEM) from three independentexperiments. �, P < 0.05, as compared with control.






phosphohydrolysis of ATP/ADP-derived extracellular AMPto adenosine (8). Our qPCR data (Fig. 2C) together withflow-cytometric staining with anti-CD73 mAb 4G4 (datanot shown) confirm the abundant expression of this enzymeon the PC-3 cell surface both at mRNA and protein levels.The ability of PC-3 cells to dephosphorylate [3H]AMP into[3H]adenosine has been further ascertained by using TLCassays (Fig. 4A, top). Furthermore, incubation of PC-3 cellswith [3H]adenosine as an initial substrate revealed its sub-sequent conversion into 3H-labeled inosine/hypoxanthine(Fig. 4A, bottom), which can be prevented by a specific ADAinhibitor EHNA (10 mmol/L; data not shown).Reverse-phase HPLC analysis provides an independent

line of evidence for the rapid metabolism of exogenousadenosine by cultured PC-3 cells with practically all aden-osine being inactivated within 20 hours of incubation (Fig.4B). Concurrent measurement of the whole spectrum ofintracellular nucleotides and related compounds demon-strated that, along with ATP as a predominant nucleotideconstituent, PC-3 cells contain other nucleotides (ADP,UTP, GTP, ITP, and AMP), whereas adenosine and othernucleosides are maintained at very low, nearly undetectable,levels (Supplementary Fig. S3). Strikingly, the exposure ofPC-3 cells to exogenous adenosine significantly increased therelative amount of intracellular adenosine, accompanied by aconcurrent drop in the level of AMP (Fig. 4C). Additionalbioluminescent analysis did not reveal any changes in ATPconcentrations between the control and adenosine-treated

cells (4.5 � 0.5 vs. 5.1 � 0.3 nmol/mg protein; mean �SEM, n ¼ 5).To elucidate the link between extracellular and intracellular

purine homeostasis, PC-3 cells were incubated for 20 hourswith 10mmol/L [3H]adenosine, lysed and assayed byTLC forthe relative amounts of cell-incorporated [3H]adenosine andits 3H-metabolites. As shown in Fig. 4D, the majority of theuptaken [3H]adenosine was converted into [3H]ATP and, tosome extent, into [3H]ADP/AMP, with the total amountof 3H-labeled nucleosides accounting only for less than 5%of total radioactivity incorporated (Inset). Noteworthy, pre-treatment of the cells with an inhibitor of equilibrativenucleoside transporter S-(4-nitrobenzyl)-6-thioinosine(NBTI; 1 mmol/L) or with 1 mmol/L ABT-702 (the inhibitorof adenosine kinase mediating intracellular phosphorylationof adenosine into AMP; ref. 16), did not affect the amount ofcell-incorporated 3H-nucleosides and the pattern of theirsequential interconversion into [3H]ATP (data not shown).Collectively, incubating PC-3 cells with exogenous adenosinewas shown to affect the balance of intracellular purines, mostlikely via the transport of adenosine and/or its nucleosidemetabolites and their subsequent multistep interconversionand transphosphorylation inside the cell.

Adenosine inhibits phospho-AMPK1a and othersignaling pathways in PC-3 cellsAt this point, the most evident mechanism(s) underly-

ing the inhibitory effects of adenosine on PC-3 invasion

Figure 3. Treating PC-3 cells with adenosine does not affect the expression of major adhesion molecules. A, representative histograms for PC-3 cellspretreated without (Control; Co) and with 10 mmol/L adenosine (Ado) and subsequently stained with antibodies against integrin-b1, CD44, and alsowith isotype-matched control antibody (shown in grey). B,mean fluorescence intensities of integrin-b1- andCD44-positive PC-3 cells were determined eitherimmediately after the treatment with adenosine or after an additional 2-hour incubation on Matrigel-coated wells (mean � SEM; n ¼ 3). C, Westernblot analyses of indicated adhesionmolecules in control and adenosine-treated PC-3 cells whichwere activated by seeding themontoMatrigel-coatedwells.For each experiment, the blots were probed with an anti-b-tubulin mAb to normalize for protein loading. The arrows indicate the position of molecularmass markers (kDa).

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may include its cellular uptake and deregulation of intra-cellular purine homeostasis and signaling pathways. Toaddress this hypothesis experimentally, we compared thephosphorylation levels of key protein kinases in controland adenosine-treated cells by using an antibody-basedarray (Fig. 5A and B). The subsequent quantitative anal-ysis of the array images revealed the ability of adenosine todecrease the phosphorylation levels of certain kinases,including TOR, JNK pan, MEK1/2, AMPK1a, andp38a (Fig. 5C), without affecting the phosphorylationprofiles of many other protein kinases (SupplementaryTable). Interestingly, the adenosine-mediated inhibitionof phospho-AMPK1a is basically consistent with theabove HPLC data on decreased AMP/ATP ratio in aden-osine-treated PC-3 cells (see Fig. 4C). Western blottinganalyses further confirmed that, in comparison withuntreated controls, adenosine-treated PC-3 cells are char-acterized by the downregulation of phospho-AMPK1a(Fig. 6A). Additional immunofluorescence stainings withanti-phospho-AMPKa1/2 (Thr172) antibodies also dem-onstrated that along with predominant nuclear fluores-cence, substantial punctate cytosolic green staining wasobserved in the control and, to lesser extent, adenosine-treated PC-3 cells (Fig. 6B, top). Staining patterns with

antibodies against total AMPKa1/2 were fairly compara-ble between the control and treated cells (Fig. 6B, middle).We also examined an overall pattern of phosphotyrosine

signaling by using 4G10 mAb to tyrosine kinase substrates.Both control and adenosine-treated PC-3 cells displayedsimilar phosphorylation profiles, with an abundant tyrosinephosphorylation of several protein bands being detectedwithin a molecular weight range of approximately 50 to140 kDa (Fig. 6A; bottom). These immunoblotting data areconsistent with the data on PC3 stainings with 4G10antibody showing strong green fluorescence in the corticalregions beneath the plasma membranes of both control andtreated cells (Fig. 6B, bottom). Noteworthy, control andadenosine-treated PC-3 cells also displayed similar patternsof Alexa-546-Phalloidin (Fig. 6B, red), as well as tubulin andvimentin (data not shown) stainings, thus excluding thepotential changes of F-actin filaments and the whole cyto-skeleton assembly in the treated cells.

Treatment of MDA-MB-231 cells with adenosine alsodiminishes their invasion and affects phosphorylationprofiles of AMPK1a and other kinasesFinally, we evaluated the effects of adenosine on the

invasiveness and phospho-kinase profile of another highly

Figure 4. Chromatographicanalyses of the extra- andintracellular purine-convertingpathways in PC-3 cells. A, TLCanalyses of the time courses ofmetabolism of 10 mmol/L [3H]AMP(top) and [3H]adenosine (bottom)and the formation of theirmetabolites by cultured PC-3 cells.B, aliquots of conditioned mediawere collected 0, 2, and 20 hoursafter the incubation of PC-3 cellswith 10 mmol/L of adenosine,followed by the separation ofnucleosides by reverse-phaseHPLC. The peaks correspondingto adenosine (Ado), inosine (Ino),and hypoxanthine (Hyp) weremonitored by absorption at 258 nmand indicated in thechromatograms shown. C, PC-3cells were incubated without(control) and with 10 mmol/Ladenosine for 20 hours.Intracellular nucleotides andadenosine (Ado) were determinedinPC-3 cells lysates andexpressedas percentages of intracellular ATPconcentration (mean � SEM; n ¼3). �,P<0.05, as comparedwith thecontrol. D, TLC analyses of cell-incorporated [3H]adenosine and its3H-metabolites determined after20-hour incubation of PC-3 cellswith 10 mmol/L [3H]adenosine.The results are expressed aspercentages of total radioactivityuptaken by the cells (mean� SEM;n ¼ 3).






invasive cell line, MDA-MB-231 breast cancer cells. Thesecells also express A2B receptors (ADORA2B) and CD73(NT5E) at mRNA levels (Fig. 7A), possess high ecto-50-nucleotidase/CD73 activity (Fig. 7B), and, in addition, arecapable of further deaminating adenosine (29), with subse-quent uptake of generated nucleosides and their intercon-version into intracellular [3H]ADP/ATP (data not shown).As in the case of PC-3 cells, pretreatment of MDA-MB-231cells with micromolar concentrations of NECA and aden-

osine, but not with inosine, inhibited their invasion throughMatrigel-coated inserts (Fig. 7C). Importantly, the Phos-pho-Kinase Array also revealed significant inhibitory effectsof adenosine on phosphorylation profiles of certain kinases inMDA-MB-231 cells, including phospho-AMPK1a (Fig.7D and E and Supplementary Table). Finally, we used asmall-molecule AMPK inhibitor compound C capable ofinhibiting phospho-AMPK activity in PC-3 and othercancer cells in a concentration-dependent fashion (30,31). Similarly to adenosine-pretreated cells, compound Csignificantly diminished the number of invaded MDA-MB-231 cells (Fig. 7C), with a similar inhibitory pattern beingalso detected in PC-3 cells (data not shown), thus providingan independent line of evidence for the potential implicationof AMPK-dependentmechanisms in the inhibitory effects ofadenosine.

DiscussionGiven the emerging role of extracellular purines in tumor

growth andmetastasis, this study was undertaken to evaluatethe in vitro effects of adenosine, other nucleosides, andrelated compounds on the invasive and migration patternsof highly metastatic tumor cell lines. Major findings of thiswork are summarized as follows: (i) pretreatment of humanPC-3 prostate carcinoma cells and MDA-MB-231 breastcancer cells with low micromolar concentrations of adeno-sine and with nonselective adenosine receptor agonistNECA or small-molecule AMPK inhibitor compound Cinhibited subsequent cell invasion and migration throughthe Matrigel- and laminin-coated inserts. (ii) The inhibitoryeffects of adenosine presumably involve cellular uptake ofadenosine and/or its nucleoside metabolites, their intracel-lular interconversion into ADP/ATP and eventually, theinhibition of phospho-AMPK1a and other signaling path-ways. (iii) Strikingly, no differences in the proliferation rates,cytoskeleton assembly, expression of major adhesion mole-cules (integrin-1b, CD44, FAK), and MMP secretion weredetected between the control and treated cells, thus exclud-ing the contribution of these components of the invasioncascade to the inhibitory effects of adenosine.At first sight, the inhibitory effects of adenosine are

unexpected because high ecto-50-nucleotidase activity isoften considered as a poor prognostic factor, which allowsthe tumors to avoid immune destruction via the generationof immunosuppressive adenosine and deactivation of tumor-infiltrating inflammatory cells (5, 10, 12, 32). This apparentdiscrepancy could be resolved on the basis of the followingconsiderations. From the standpoint of adenosine homeo-stasis and its pharmacologic activity, it might be morerelevant to examine the ratio of the adenosine-producingcapability of ecto-50-nucleotidase/CD73 to the combinedadenosine-utilizing activities (33). In fact, despite the abun-dant CD73 expression on tumor cells studied, the generatedadenosine was shown to be rapidly deaminated and trans-ported into the cells (see Fig. 4), suggesting a rather transientappearance of adenosine in the external milieu as theintermediate product of the purine-inactivating chain.

Figure 5. The effect of adenosine on the kinase phosphorylation profile inPC-3 cells. Control (Co) and adenosine-treated (Ado) PC-3 cells wereseeded for 2 hours onto Matrigel-coated wells, lysed and assayed for apanel of 46 phospho-kinases using an antibody-based array. A, arepresentative array from the control cells shows a phosphorylationprofile of the kinases studied. The phosphorylation level of each kinase ispresented as duplicate spots and positive control spots are located in thecorners of the membranes (a1–2, g1–2, and a17–18). B, relativephosphorylation levels of the indicated protein kinases. The array signalsfrom scanned X-ray film images from two independent experiments areshown. C, spot pixel densities were quantified after data normalization tothe internal controls and background intensity. The results showmean�SEM from three independent experiments, where each samplerepresents the pooled lysates from two different assays. �, P < 0.05, ascompared with control. For complete densitometric analyses ofphospho-kinases in control and adenosine-treated PC-3, cells seeSupplementary Table.

Virtanen et al.





Therefore, local and transient generation of nanomolaradenosine by tumor-derived CD73 would indeed be suffi-cient for the failure of mounting effective immune responsesvia activation of the A2A receptors on cytotoxic T lympho-cytes and other inflammatory cells (14, 32). On the otherhand, the overall pattern of purine homeostasis is markedlyderegulated in the tumor environment by hypoxia andmetabolic stress, as ascertained by data on constitutivelyelevated levels of intratumoral ATP (7, 34), the acceleratedrate of nucleotide turnover (4, 9, 33), and the ability of solidtumors to maintain adenosine gradient, with the highestnucleoside concentrations being reported in the center (14).Therefore, it may be speculated that besides dampeningantitumor immunity, the sustained exposure of tumor cellsto the elevated adenosine concurrently impairs their inva-siveness (present study) and at higher micromolar concen-trations also retards tumor growth and proliferation (15, 21).This scenario might provide a partial explanation for thewell-known phenomena of the high proliferative and inva-sive capacities of peripheral tumor cells located in theparenchyma and stroma, whereas the core environment ofthe tumor is maintained in a relatively stable, "semiquies-cent," state (2).The salient finding of this study is that the effects of

adenosine on tumor invasion may extend beyond its recep-tor-mediated activity. Potential mechanisms of this inhibi-tion involve adenosinemetabolism via ecto-adenosine deam-inase activity, subsequent cellular uptake, and interconver-sion of transported nucleosides, accompanied by inhibitionof several protein kinases, including AMPK1a. AMPK is anenergy-sensing serine/threonine kinase consisting of ana-catalytic subunit and regulatory b- and g-subunits. It is

activated under conditions of metabolic stress, such asglucose deprivation, hypoxia, and strenuous exercise thatdeplete intracellular ATP and increase AMP (35). A numberof findings suggest that AMPK-mediated reduction in ana-bolic pathways may ultimately lead to reduced tumorgrowth. Hence, pharmacologic activation of AMPK wouldbe advantageous in treating different endocrine-relatedtumors, including prostate and breast cancers (35–37).However, challenging this view, other studies have emergedthat lead to the opposite conclusion, stating that inhibitionof AMPK reduces cancer growth (35, 36). In particular, themalignant human prostate tissue was shown to display ahigher AMPK activity than normal tissue cells. In addition,the downregulation of AMPK in prostate cancer cell lines,but not in nontumorigenic prostate epithelial cells, reducedcell proliferation and induced apoptosis (30, 31). Theseconflicting results presumably depend on the tumor type andmay also reflect a fine-tuned biphasic regulation of AMPKfunction and/or changing role of AMPK at different diseasestates.Interestingly, short-term (1–10 minutes) incubation of

human endothelial cells (38) as well as rat liver and intestinalepithelial cells (39) with micromolar concentrations ofadenosine or its precursor nucleotides triggered a transientactivation of AMPK. However, the mechanisms of theseacute effects may differ substantially from the chronic effectsof adenosine on AMPK inhibition seen in our experimentalsettings. In addition, the modulation of AMPK in normaland transformed cells often triggers the opposing effects dueto differential deletion of downstream tumor suppressors,thus allowing AMPK activationwhilemitigating the growth-limiting effects of the enzyme (31, 35). Under conditions of

Figure 6. The effect of adenosine onphospho-AMPK activity andoverall pattern of phosphotyrosinesignaling in PC-3 cells. PC-3 cellspretreated without (Control; Co)and with 10 mmol/L adenosine(Ado) were activated by seedingonto Matrigel-coated wells. A,Western blotting analysis of PC-3cell lysates stained with antibodiesagainst total and phosphorylatedforms of AMPK, phosphotyrosinekinases (4G10), and b-tubulin as acontrol for the protein loading. Thearrows indicate the positions of themolecular mass markers (kDa). B,AMPK, phospho-AMPK, andphosphotyrosine (4G10) stainingsof PC-3 cells grown on Matrigel-coated coverslips. The sectionswere incubated with the indicatedantibodies and subsequentlystained with appropriatesecondary Alexa488-conjugatedantibodies (green). For the actincytoskeleton staining, the cellswere costained with Alexa546-Phalloidin (shown in red). Nucleiwere stained with blue-fluorescentDAPI. Scale bar, 50 mm.






energetic stress and the demand of continuous cell prolif-eration, AMPK bestows tolerance of cancer cells to nutrientdeprivation without restricting cell growth and proliferation(31). In fact, the decreased AMPK activity in prostate cancercells challenged to certain energy-starved conditionsenhances apoptotic cell death during androgen deprivationand hypoxia (30) and also diminishes their invasive capa-bility when incubated in serum-free media containing aden-osine (present study).Current research on the role of adenosine in tumor

pathogenesis and inflammation is particularly focused ontargeting the releasing pathways of the precursor nucleotideATP (7, 34), directional regulation of ecto-50-nucleotidase/CD73 activity by using selective enzyme inhibitors, siRNAgene silencing and anti-CD73 mAb therapies (10, 12, 32),synthesis of phosphorylated adenosine derivatives acting assite-specific CD73-activated prodrugs of A2A receptors (40),as well as development of nonmetabolizable nucleosideanalogues acting either as selective adenosine receptor ago-nists and antagonists (5, 13) or as metabolic inhibitors ofintracellular purine homeostasis (16, 20). Yet anotherapproach includes tumor treatment with adenosine as a"kindred" metabolite; however, most of the proapoptoticand antiproliferative effects of adenosine reported in studieswith different malignant cells required continuous presence

of supra-physiological nucleoside concentrations (0.1–20mmol/L) in the assay mixture (15, 17–19).Here, we have shown that adenosine is capable of pre-

venting tumor cell invasion at relatively low concentrations(1–10 mmol/L) without any adverse cytotoxic effects, andthat these effects persisted for at least 48 ensuing hours evenin the absence of stimuli applied. Although this study out-lines the potential implication of phospho-AMPK1a intoinhibitory effects of adenosine, the diminished phosphory-lation profiles of other key tumorigenic kinases in adenosine-treated cells (such as p27, p38a, JNK pan, and TOR in caseof PC-3 and STAT1, STAT4 Akt, eNOS, and JNK pan inMDA-MB-231 cells; see the Supplementary Table) suggestthe existence of a rather complex and probably tumor type-specific interplay between the extracellular and intracellularpurine homeostasis, signaling pathways, and tumor behav-ior. Moreover, significant inhibition of invasion in studieswith nonselective agonist NECA (see Figs. 1B and 7C),together with data on predominant expression of the A2Breceptors on PC-3 and MDA-MB-231 cells (refs. 26, 28;also present study), and diverse pro- and antitumoral roles ofA2B receptors in different tumors (13, 26), may also indicatethe involvement of A2B-receptor–mediated signaling incontrol of tumor invasion, growth and proliferation, espe-cially at high micromolar concentrations of agonist.

Figure 7. The effect of adenosine on the invasion pattern and phospho-kinase profile of breast cancer cells. A, a qPCR analysis of MDA-MB-231 celllysates shows the abundant mRNA expression for A2B (ADORA2B), but not other adenosine receptor subtypes (ADORA1, ADORA2A, and ADORA3),and also for ecto-50-nucleotidase/CD73 (NT5E; n ¼ 3). The average mRNA expression for each gene was presented as a percentage of b-actin mRNAmeasured from the same sample. B, TLC analysis of the time course of [3H]AMP (10 mmol/L) hydrolysis and formation of 3H-nucleoside metabolitesby MDA-MB-231 cells. C, invasion of MDA-MB-231 cells after their pretreatment with indicated concentrations (mmol/L) of adenosine (Ado), inosine (Ino),NECA, and compound C (Comp C). The bars show the numbers of invaded cells (mean � SEM; n ¼ 3–5). �, P < 0.05, as compared with control vehicle–treated cells. D, lysates from control (Co) and adenosine-treated (Ado) MDA-MB-231 cells were assayed for phosphorylation levels of protein kinases.The array signals from scanned X-ray film images from two independent experiments are shown. E, the results from D were analyzed using the imageanalysis software, normalized for background intensity and expressed as mean � SEM (n ¼ 3). �, P < 0.05, as compared with the control. For completequantitative densitometric analyses of major kinases, see Supplementary Table.

Virtanen et al.





In conclusion, the results presented here confirm theimportant role of adenosine in tumor pathogenesis andfurther expand the concepts presented above by hypoth-esizing the coexistence of dynamic networks coordinatelyregulating purine homeostasis and signal transductionpathways in cancer cells. Intratumoral adenosine presum-ably exerts dual functions serving as "immunologic barrier"responsible for the failure of mounting an effective immuneresponse toward malignant cells and at higher nucleosideconcentrations, impairing invasion and retarding cellgrowth and proliferation. Noteworthy, these conclusionsmainly rest on comprehensive analyses performed withhighly invasive androgen-independent PC-3 prostate car-cinoma cells, and also with MDA-MB-231 breast cancercells representing a form of triple-negative breast cancerwith poor prognosis. Therefore, additional in vitro and invivo studies with other tumor cell lines and tissues would berequired to elucidate the role of adenosine in prostate,breast, and other endocrine-related cancers. The compleximplication of both extrinsic and intrinsic mechanisms intoa tuned adenosine-dependent control of tumor growth andmetastasis may open up further research for development ofcombined therapeutic approaches selectively targetingadenosine receptors on the cell surface and at the sametime, modulating intracellular purine homeostasis andrelated signaling pathways.

Disclosure of Potential Conflicts of InterestS. Jalkanen has ownership interest (including patents) in an entity. No potential

conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: S.S. Virtanen, S. Jalkanen, G.G. YegutkinDevelopment of methodology: S.S. Virtanen, P.L. Harkonen, G.G. YegutkinAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): S.S. Virtanen, M. Vainio, P.L. Harkonen, G.G. YegutkinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, com-putational analysis): S.S. Virtanen, A. Kukkonen-Macchi, M. Vainio, K. Elima,G.G. YegutkinWriting, review, and/or revision of the manuscript: S.S. Virtanen, P.L. Harkonen,S. Jalkanen, G.G. YegutkinAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): M. VainioStudy supervision: P.L. Harkonen, S. Jalkanen, G.G. Yegutkin

AcknowledgmentsThe authors thank Sergei Samburski for help in performance of reverse-phase

HLPC assays and data analysis, Ruth Fair-M€akel€a for the revision of the text, SariM€akifor technical assistance, and Anne Sovikoski-Georgieva for secretarial help.

Grant SupportThis work was supported by grants from the Academy of Finland and the Sigrid

Juselius Foundation.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received May 27, 2014; revised July 2, 2014; accepted July 10, 2014;published OnlineFirst July 30, 2014.

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2014;12:1863-1874. Published OnlineFirst July 30, 2014.Mol Cancer Res Sanna S. Virtanen, Anu Kukkonen-Macchi, Minna Vainio, et al. MechanismsAdenosine Inhibits Tumor Cell Invasion via Receptor-Independent

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