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Oncogene (2010) 29, 5346–5358 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc REVIEW Extracellular triphosphate and adenosine in

J Stagg and MJ Smyth

Cancer Program, Sir Donald and Lady Trescowthick Laboratories, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia

Adenosine triphosphate (ATP) is actively released in the mulated the hypothesis of purinergic extracellular environment in response to tissue damage (Burnstock, 1972). Burnstock’s hypothesis that ATP and cellular stress. Through the activation of P2X and could be released by cells to perform intercellular P2Y receptors, extracellular ATP enhances tissue repair, signaling was initially met with skepticism, as it seemed promotes the recruitment of immune and unlikely that a that acts as an intracellular dendritic cells, and acts as a co-activator of NLR family, source of energy would also as an extracellular pyrin domain-containing 3 (NLRP3) inflammasomes. messenger. Nevertheless, Burnstock pursued his work The conversion of extracellular ATP to adenosine, in and, together with Che Su and John Bevan, reported contrast, essentially through the enzymatic activity of the that ATP was also released from sympathetic nerves ecto-nucleotidases CD39 and CD73, acts as a negative- during stimulation (Su et al., 1971). Three decades later, mechanism to prevent excessive immune responses. following the cloning and characterization of ATP and Here we review the effects of extracellular ATP and adenosine adenosine surface receptors, purinergic signaling is a on tumorigenesis. First, we summarize the functions of well-established concept and constitutes an expanding extracellular ATP and adenosine in the context of tumor field of research in health and disease, including cancer . Second, we present an overview of the immuno- (Burnstock, 2007). Although early studies focused on suppressive and pro-angiogenic effects of extracellular the role of purinergic receptors in neurotransmission, it adenosine. Third, we present experimental evidence that soon became obvious that extracellular ATP and extracellular ATP and adenosine receptors are expressed adenosine have important roles in another system by tumor cells and enhance tumor growth. Finally, we discuss requiring efficient cell-to-cell : the recent studies, including our own work, which suggest that . Over the last decade, there has been therapeutic approaches that promote ATP-mediated activa- increasing interest not only in the effects of extracellular tion of inflammasomes, or inhibit the accumulation of tumor- and on inflammatory responses, derived extracellular adenosine, may constitute effective new but also on general biological pathways, such as cell means to induce anticancer activity. survival, proliferation, differentiation and motility. Oncogene (2010) 29, 5346–5358; doi:10.1038/onc.2010.292; It is becoming increasingly clear that the release of published online 26 July 2010 extracellular purines and pyrimidines represents a ubiquitous means of intercellular communication Keywords: immunosuppression; adenosine; used by different cell types, involved in various ecto-nucleotidases; inflammasome; ATP biological processes and conserved throughout evolu- tion, as evidenced by the discovery of ATP receptors in invertebrates (Fountain et al., 2007) and plants (Kim et al., 2006). Introduction Extracellular ATP in immunity For many scientists in the 1960s, cells could not possibly release a molecule as fundamental as adenosine tripho- ATP receptors sphate (ATP). Owing to its established role in the Krebs In 1978, Burnstock proposed two types of purinergic cycle, there was considerable skepticism to the notion receptors: P1 receptors selective for adenosine and that ATP—and purines in general—could exert extra- P2 receptors selective for ATP and ADP. Some P2 cellular function. In 1970, Burnstock et al. described the receptors additionally bind UTP or UDP (Burnstock, release of extracellular ATP as a transmitter substance by 2006). In 1985, a pharmacological approach was non-adrenergic inhibitory nerves, and later in 1972, for- proposed to distinguish between two types of P2 receptors: ionotropic P2X and metabotropic P2Y Correspondence: Dr J Stagg or Professor MJ Smyth, Cancer receptors. Currently, four subtypes of P1 receptors Immunology Program, Sir Donald and Lady Trescowthick Labora- (A1, A2A, A2B and A3), seven subtypes of P2X tories, Peter MacCallum Cancer Centre, Locked Bag 1, A’Beckett receptors and eight subtypes of P2Y receptors have Street, East Melbourne, Victoria 8006, Australia. E-mails: [email protected] or [email protected] been identified (Figure 1). P2Y receptors are subdivided Received 12 May 2010; revised 11 June 2010; accepted 13 June 2010; into five Gq/G11-coupled subtypes (P2Y1, P2Y2, P2Y4, published online 26 July 2010 P2Y6 and P2Y11) and three Gi/o-coupled subtypes Extracellular and adenosine J Stagg and MJ Smyth 5347

Figure 1 Adenosine triphosphate (ATP) and adenosine signaling. Extracellular ATP binds G--coupled P2Y and trimeric channel P2X receptors. P2Y receptors are subdivided into Gq/G11-coupled subtypes that activate the C and triphosphate pathways, and Gi/o-coupled subtypes that inhibit . P2X7 is an atypical ATP receptor, forming ion channels at low concentrations of ATP, activating NLRP3 inflammasomes by K þ efflux and the recruitment of the -1 hemichannel, and inducing cell death at high concentrations of ATP. Extracellular adenosine binds Gi/o-coupled A1 and A3 receptors and Gs-coupled A2A and A2B receptors. In contrast to A1 and A3 receptors, A2A and A2B receptors increase intracellular cyclic AMP levels.

(, P2Y13 and P2Y14). Gq/G11-coupled P2Y recep- 2006) and the processing and of the tors generally activate the and inositol -1b (IL-1b) and IL-18, important activators triphosphate pathways, whereas Gi/o-coupled P2Y of innate and adaptive immune responses. The NLRP3 receptors generally inhibit adenylyl cyclase and mod- inflammasome is the best-characterized inflammasome ulate ion channels (Abbracchio et al., 2009). and belongs to the intracellular NOD-like receptor In contrast to G-protein-coupled P2Y receptors, P2X (NLR) family. Together with Toll-like receptors (TLRs) receptors are trimeric cationic channels permeable to and C-type lectins, NLRs scan the extracellular and Na þ ,Kþ and Ca2 þ upon activation. Six homomeric intracellular environment for pathogen-associated mo- (P2X1À5 and P2X7) and six heteromeric (P2X1/2, P2X1/4, lecular patterns and host-derived danger-associated P2X1/5, P2X2/3, P2X2/6 and P2X4/6) receptors have been molecular patterns to alert the immune system (Schro- described. The homomeric P2X7 receptor is an atypical der and Tschopp, 2010). The NLRP3 inflammasome is with a longer carboxy-terminal activated in response to various danger , such as tail and a number of polymorphisms or spliced variants co-activation of P2X7 and TLRs, increased cytosolic (Gunosewoyo et al., 2007; Wu et al., 2009). The DNA levels, monosodium urate crystals, fibrillar activation of P2X7 receptors is highly regulated by amyloid-b and high extracellular glucose levels extracellular levels of ATP. Whereas low ATP concen- (Schroder and Tschopp, 2010). The NLRP3 inflamma- trations activate P2X7 ion channels to become perme- some can also be activated independently of TLR able to small , high ATP concentrations result in signaling (Kanneganti et al., 2007). Schroder and pore formation permeable to as large as Tschopp (2010) recently proposed that a unifying 900 kDa, which ultimately causes cell death (Khakh and element in NLRP3 activation might be the generation North, 2006). of . The activation of P2X7 receptors and the recruitment of the pannexin-1 membrane pore may also allow NLRP3 to Extracellular ATP and the NLR family, pyrin enter the cells and directly activate the inflammasome domain-containing 3 inflammasome (Kanneganti et al., 2007). In the context of inflammation, the release of ATP by activated monocytes, dying, injured or stressed cells, degranulating platelets or secreted by them- Extracellular ATP and T helper type 17 cell immune selves, acts as a co-activator of the NLR family, pyrin responses domain-containing 3 (NLRP3) (cryopirin/NALP3) T-helper type 17 cell immune responses are required for inflammasome (Piccini et al., 2008; Netea et al., 2009). the control of infectious agents (Cho et al., 2010) and are The NLRP3 inflammasome is a multiprotein complex involved in the pathogenesis of various autoimmune that triggers caspase-1 activation (Mariathasan et al., diseases, such as multiple sclerosis (Axtell et al., 2010)

Oncogene Extracellular adenosine triphosphate and adenosine J Stagg and MJ Smyth 5348 and inflammatory bowel disease (Cho and Weaver, responses. Extracellular ATP, by activation of P2Y 2007). The generation of Th17 cells is controlled by the receptors, has been identified as a potent chemotactic cytokines IL-6, tumor (TGF)-b and IL-23 for immature DCs (Idzko et al., 2002). In (Bettelli et al., 2007). ‘Naturally occurring’ IL-17- contrast, mature DCs exposed to ATP have decreased producing CD4 þ T cells are also present in the gut. migratory capacity (Schnurr et al., 2003). Thus, the Atarashi et al. (2008) recently investigated the contribu- accumulation of ATP at sites of infection may allow the tion of commensal bacteria in the development of Th17 recruitment of immature DCs. The transient inhibition cells. Surprisingly, the study showed that extracellular of DCs’ migration after maturation might then prolong ATP is constitutively released from commensal bacteria antigen encounter. However, extensive exposure to and drives the differentiation of Th17 CD4 þ cells. This extracellular ATP can also inhibit DC functions. ATP- Th17-skewing effect of extracellular ATP has also been stimulated DCs produce less pro-inflammatory shown in monocyte-derived dendritic cells (DCs) cytokines, more IL-10 and synergize with (Schnurr et al., 2005). Thus, excessive accumulation of (IFN)-g in upregulating indoleamine 2,3-dioxygenase extracellular ATP may have an important role in the levels (Marteau et al., 2005). pathogenesis of autoimmune diseases. In support of this hypothesis, mice deficient for the ecto- triphos- phate diphosphohydrolase, CD39, which hydrolyzes ATP, develop exacerbated experimental colitis (Fried- Extracellular adenosine in immunity man et al., 2009). Moreover, patients with low levels of Adenosine receptors CD39 activity have an increased susceptibility for The conversion of extracellular ATP to adenosine, developing Crohn’s disease (Friedman et al., 2009). essentially through the enzymatic activity of the Taken together, these studies are consistent with a membrane-bound nucleotidases CD39 and CD73, acts role for extracellular ATP in driving Th17 immune responses. as a negative-feedback mechanism that prevents ex- cessive immune reactions. The immunosuppressive effects of adenosine are summarized in Figure 2. Extracellular ATP and Extracellular adenosine activates four distinct G-pro- Activation of the ATP receptor P2Y2 has been recently tein-coupled receptors: Gi/o-coupled A1 and A3 recep- implicated in the directed migration (that is, chemotaxis) tors that decrease intracellular cyclic AMP (cAMP) of immune phagocytes, such as macrophages and levels, and Gs-coupled A2A and A2B receptors that neutrophils. However, the means by which P2Y2 increase cAMP levels through the activation of adenylyl regulate the chemotaxis of macrophages and neutrophils cyclase (Hasko and Cronstein, 2004). In contrast to the are markedly different. In monocytes/macrophages, other adenosine receptors, which are all activated extracellular ATP released from an extrinsic source, at physiological concentrations of adenosine (that is, such as apoptotic cells, directly acts as a ‘find-me’ 30–300 nM in interstitial fluid), the A2B receptor requires (Elliott et al., 2009). In contrast, migrating neutrophils high levels of adenosine generated in response to do not migrate toward gradients of ATP, but release pathological conditions (Fredholm et al., 2001). ATP at their leading edge in response to a chemo- attractant, which, through the feedback activation of signaling P2Y and A3 receptors, provides chemotaxis amplifica- 2 A1 and A3 adenosine receptors inhibit adenylyl cyclase tion (Chen et al., 2006). Thus, whereas both macro- and protein A activity, thus decreasing intracel- phages and neutrophils exploit P2Y signaling, only in 2 lular cAMP levels, with studies revealing an important macrophages does it stimulate a non-redundant chemo- function in cardioprotection (Liang and Jacobson, tactic signal. Extracellular ‘find-me’ signals, such as 1998; Reichelt et al., 2005; Hochhauser et al., 2007), ATP, act in concert with ‘keep-out’ signals to induce (Clark et al., 2007) and protection macrophage recruitment. For instance, the release of against septic shock (Gallos et al., 2005). A1 and A3 lactoferrin selectively inhibits the migration of granulo- adenosine receptors have also been linked to phospha- cytes, but not macrophages (Bournazou et al., 2009). tidylinositol 3-kinase, mitogen-activated These recent studies offer a new level of complexity to and protein kinase C pathways (Hasko and Cronstein, the mechanisms governing cellular chemotaxis. The 2004; Gessi et al., 2008). In contrast to other P1 generally conserved functions of purinergic receptors receptors, A3 adenosine receptor presents multiple sites and their ubiquitous expression in different tissues of that are important for receptor suggest that ATP and adenosine receptors may be desensitization after activation (Palmer and Stiles, implicated in the motility of other cell types, including 2000). tumor cells. A2A and A2B adenosine receptors activate adenylyl cyclase and protein kinase A, thereby increasing Extracellular ATP and dendritic cells intracellular cAMP levels (Fredholm et al., 2007). A2A Although monocytes and macrophages are generally and A2B receptors can further activate mitogen- responsible for the non-immunogenic removal of activated protein kinase and protein kinase C. Owing apoptotic cells, DCs are specialized antigen-presenting to its high affinity for adenosine, the A2A adenosine cells whose role is to initiate or regulate immune receptor has a non-redundant immunosuppressive role

Oncogene Extracellular adenosine triphosphate and adenosine J Stagg and MJ Smyth 5349

Figure 2 Immunosuppressive effects of extracellular adenosine. The phosphohydrolysis of extracellular adenosine triphosphate (ATP) to adenosine, essentially through the activity of the ecto-nucleotidases CD39 and CD73, acts as a potent negative-feedback mechanism to suppress the functions of innate and adaptive immune cells.

(Ohta and Sitkovsky, 2001; Odashima et al., 2005). The (Cronstein et al., 1990; Firestein et al., 1995; Bouma A2A adenosine receptor is expressed on monocytes/ et al., 1997). macrophages, mast cells, granulocytes, lymphocytes, DCs, natural killer (NK) cells, natural killer T (NKT) Effect of adenosine on NK cells. In contrast to cells, endothelial cells and airway epithelial cells macrophages and neutrophils, the effect of extracellular (Fredholm et al., 2007). Importantly, the regulation of adenosine on NK cells is not as clearly defined. The A2A receptor expression is driven by at least four activation of A3 adenosine receptors have been shown independent promoters (Yu et al., 2004), which most to enhance NK cell functions, although this could be the likely accounts for tissue-specific functions. A2A recep- result of chronic exposure to A3 agonists that induce tor expression is induced by hypoxia, as well as by receptor desensitization (Priebe et al., 1990; Harish (TNF)-a and IL-1a by nuclear et al., 2003). In contrast, activation of A2A adenosine factor-kB (Khoa et al., 2001). The A2B adenosine receptor has been shown to inhibit IFN-g production receptor differs from the other adenosine receptors by its (Lappas et al., 2005). The inhibitory effect of the A2A relative low affinity for adenosine (Feoktistov and receptor on NK cells most likely reflects its ability to Biaggioni, 1997). A2B is transcriptionally induced by increase intracellular cAMP levels, which has been hypoxia-inducible factor-1a activation (Eltzschig et al., shown to suppress NK cell functions (Goto et al., 1983). 2003; Feoktistov et al., 2004), TNF-a,IL-1b, IFN-g and extracellular adenosine (Xaus et al., 1999; Kolachala et al., 2005; St Hilaire et al., 2008). Effect of adenosine on NKT cells. Natural killer T cells are a unique population of T cells involved in the regulation of , infection, immune tole- Adenosine-mediated immunosuppression rance and tumor immunosurveillance (Godfrey and Effect of adenosine on immune phagocytes. The accu- Kronenberg, 2004; Swann et al., 2009). NKT cells mulation of extracellular adenosine profoundly inhibits recognize glycolipids presented in the context of the the function of phagocytes. In macrophages, adenosine major histocompatibility complex class I-like molecule inhibits phagocytosis, production of superoxide and CD1d and produce large amounts of cytokines upon (Eppell et al., 1989) (Hasko et al., 1996), activation. NKT cells deficient in A2A adenosine TNF-a release, major histocompatibility complex class receptor were found to produce significantly less IL-4, II expression and IL-12 production (Hasko et al., 2000). IL-10 and TGF-b, but more IFN-g upon stimulation In neutrophils, adenosine inhibits degranulation, adhe- with alpha-galactosylceramide (Nowak et al., 2010). sion to endothelial cells and superoxide production Consistent with a role for adenosine in regulating NKT

Oncogene Extracellular adenosine triphosphate and adenosine J Stagg and MJ Smyth 5350 cell function, CD39-deficient NKT cells have been receptor , T cells fail to proliferate and to shown to produce less IL-4 upon activation (Beldi produce IFN-g upon reactivation, thus essentially et al., 2008). promoting T-cell anergy (Zarek et al., 2008). In vivo activation of A2A adenosine receptors drives CD4 þ þ Effect of adenosine on DCs. Extracellular adenosine T-cell differentiation toward Foxp3 regulatory T cells increases the chemotaxis of immature monocyte-derived (Tregs), most likely owing to a concomitant increase in DCs, but inhibits IL-12 production following activation TGF-b and decrease in IL-6 production following A2A (Panther et al., 2001, 2003). Similarly, immature receptor activation (Zarek et al., 2008). In addition, plasmacytoid DCs also migrate toward a gradient of activation of A2A receptors was found to promote the þ adenosine (Schnurr et al., 2004). Upon maturation, DCs generation of LAG-3 Tregs. alter the expression profile of adenosine receptors, switching from Gi- to Gs-coupled receptors, resulting Adenosine and Tregs. Foxp3 þ Tregs are crucial for the in a loss of chemotactic response and an inhibition of control of autoimmune responses and tumor immunity -producing function (Schnurr et al., 2004). and rely on multiple mechanisms for their immunosup- Recently, Novitskiy et al. (2008) investigated the effect pressive function. Recent studies have shown that the of extracellular adenosine on the differentiation of ecto- CD39 and CD73 are important contribu- human DCs from blood monocytes. The activation of tors to the regulatory function of Foxp3 þ Tregs A2B adenosine receptors on monocytes was shown to (Deaglio et al., 2007). In mice, Foxp3 þ Tregs express divert DC differentiation toward a population that high levels of both CD39 and CD73, upregulate CD73 produced high levels of vascular endothelial growth upon activation, and partly rely on ecto-nucleotidase factor, IL-8, IL-6, IL-10, -2, TGF-b and activity and adenosine for immunosuppression (Ring indoleamine 2, 3-dioxygenase. Remarkably, when adop- et al., 2010). Human Foxp3 þ Tregs also express CD39, tively transferred to tumor-bearing mice, adenosine- which is further upregulated upon Treg activation differentiated DCs significantly enhanced tumor growth. (Miyara et al., 2009), whereas CD73 expression remains unclear. The analysis of CD73 expression may be Effect of adenosine on B cells. The activation of A2A affected by -mediated shedding (Airas et al., adenosine receptors, by increased protein kinase A 1997). Nevertheless, inhibition of CD73 has been shown activity, has been shown to inhibit B-cell receptor- to reduce human Treg-mediated suppression in vitro induced NF-kB activation (Minguet et al., 2005). (Mandapathil et al., 2010). A recent study has further Consistent with a role for adenosine in regulating identified extracellular ATP as an activating molecule of B-cell functions, isotype-switched B cells, and more Tregs (Ring et al., 2010). Thus, extracellular release of specifically a subpopulation of memory B cells, upregu- ATP and its hydrolysis to adenosine by CD39 and CD73 late the expression of CD73, an essential ecto-nucleo- have a crucial role in Treg function. tidase for the generation of extracellular adenosine (Anderson et al., 2007). Further in support of this Production of extracellular adenosine. The extracellular model, a recent study revealed that memory B cells have concentration of adenosine is generally constant in most increased expression of involved in adenosine tissues, but can rapidly increase 100-fold in hypoxic signaling, including A2A adenosine receptor, compared tissue and in response to inflammation (Schulte and with naive B cells (Tomayko et al., 2008). Taken Fredholm, 2003). The dominant pathway leading to together, these studies strongly suggest that isotype- high extracellular adenosine levels is the extracellular switched B cells, especially memory B cells, use phosphohydrolysis of ATP by ecto-nucleotidases. adenosine receptors to control antigenic re-stimulation. These include ecto-nucleoside triphosphate diphospho- hydrolases, ecto- pyrophosphatase and Effect of adenosine on T cells. Extracellular adenosine phosphodiesterases, alkaline and CD73 is also an important inhibitor of T-cell functions. Similar (ecto-50-nucleotidase). Ecto-nucleoside triphosphate to its effect on B cells, adenosine inhibits T-cell receptor- diphosphohydrolases (predominantly CD39) and ecto- induced NF-kB activation (Majumdar and Aggarwal, nucleotide pyrophosphatase and phosphodiesterases 2003). The A2A receptor dominantly regulates T-cell hydrolyze ATP and ADP to AMP, which is further functions and is significantly upregulated upon hydrolyzed to adenosine by CD73. Thus, the membrane- T-cell activation. Activation of A2A receptors during bound nucleotidases CD39 and CD73 are key regulators T-cell activation significantly inhibits cytotoxicity and of the phosphohydrolysis of ATP into adenosine. cytokine production (Ohta et al., 2009) and has been CD39 (NTPDase1) is expressed on endothelial cells reported to inhibit T- (Zhang et al., and subsets of leukocytes, where it hydrolyzes ATP and 2004; Deaglio et al., 2007). T cells activated in the ADP released by activated platelets, thereby inhibiting presence of an A2A receptor agonist maintain a platelet recruitment and thrombus formation (Robson suppressed phenotype after removal of the agonist et al., 2006). CD39 expression on leukocytes also (Ohta et al., 2009). A2A receptor activation in T cells regulates leukocyte migration across the suppresses the development of both Th1 and Th2 into ischemic tissue (Hyman et al., 2009). CD39 also has immune responses, in vitro and in vivo (Csoka et al., important immunosuppressive functions through the 2008). When activated in the presence of an A2A phosphohydrolysis of pro-inflammatory extracellular

Oncogene Extracellular adenosine triphosphate and adenosine J Stagg and MJ Smyth 5351 ATP and the generation of extracellular adenosine when et al., 2008), glioma (Bavaresco et al., 2008), glioblas- co-expressed together with CD73 (Dwyer et al., 2007). toma (Ludwig et al., 1999), melanoma (Sadej et al., 2006), ovarian cancer (Jin et al., 2010), cancer CD73 (ecto-50-nucleotidase) (Kondo et al., 2006), esophageal cancer (Fukuda et al., The membrane-bound CD73 (or ecto-50-nucleotidase) is 2004), gastric cancer (Durak et al., 1994), colon cancer considered as the rate-limiting in the generation (Eroglu et al., 2000), prostate cancer and breast cancer of extracellular adenosine (Resta et al., 1998). CD73 (Spychala et al., 2004). Notably, CD73 expression has catalyzes the of and pyrimi- been associated with a pro-metastatic phenotype in dine ribo- and deoxyribonucleoside monophosphates to melanoma and breast cancer (Lee et al., 2003; Leth- the corresponding nucleoside. CD73 expression is Larsen et al., 2009). In breast cancer cells, CD73 upregulated by hypoxia-inducible factor-1a activation expression was regulated by receptor-a, where- and exposure to type I IFNs, and has been associated by loss of -a significantly enhanced with Wnt signaling (Synnestvedt et al., 2002; Niemela CD73 expression (Spychala et al., 2004). In addition, et al., 2004; Spychala and Kitajewski, 2004) and protein CD73 expression was increased in breast tumor cells kinase C activation (Kitakaze et al., 1996). In addition, resistant to doxorubicin (Ujhazy et al., 1996). CD73 also IL-1b,TNF-a and E2 have been shown to increased the resistance of Jurkat leukemic cells to enhance CD73 activity, whereas IFN-g and IL-4 have tumor necrosis factor-related -inducing - been shown to downregulate CD73 (Savic et al., 1990; mediated apoptosis (Mikhailov et al., 2008). Christensen et al., 1992). The structure of CD73 has been reviewed elsewhere (Strater, 2006). Purinergic signaling in cancer CD73 functions. CD73 is expressed on various cell types, including subsets of lymphocytes, endothelial cells Tumor-suppressive effects of extracellular ATP and epithelial cells (Colgan et al., 2006). The expression Owing to its pro-inflammatory role and its direct of CD73 is critical for the regulation of hypoxia-induced cytotoxic function, the ATP receptor P2X7 might act vascular leakage and for the control of inflammatory as a tumor suppressor. Consistent with this hypothesis, responses (Thompson et al., 2004). CD73 upregulation P2X7 levels are significantly lower in various types of on endothelial cells decreases endothelial vascular cell cancer cells compared with adjacent normal tissues of adhesion molecule-1 expression and prevents leukocyte the same origin (Li et al., 2006). In addition, P2X7 attachment. CD73 expression on high endothelial receptors have been found to be cytolytically non- venules also negatively regulates the migration of functional in tumor cells (Slater et al., 2004). Several T and B cells from the blood into draining lymph nodes mechanisms regulate P2X7-induced cell death, including in response to TLR4 activation (Takedachi et al., 2008). glycosylation (Feng et al., 2005), trafficking (Guerra In contrast to its role in lymph nodes, CD73 promotes et al., 2003), oligomerization and internalization (Feng T-cell extravasation in peripheral tissue. Accordingly, et al., 2006). In support of a selective advantage for decreased extravasation of pathogenic T cells was tumor cells with decreased P2X7 levels, a recent study recently suggested as the cause of resistance of CD73- identified putative target sites for micro-RNA-186 and deficient mice to experimental autoimmune encephalo- micro-RNA À150 within the P2X7 , and showed myelitis (Mills et al., 2008). Through the production of that micro-RNA À186 and micro-RNA À150 levels are adenosine and the activation of adenosine receptors on significantly higher in cancer cells than in normal cells endothelial cells, it was suggested that CD73 triggers (Zhou et al., 2008). More evidence that P2X7 may act as adhesion molecules, such as intercellular adhesion a tumor suppressor comes from the analysis of breast molecule 1 on endothelial cells. cancer patients with a genetically dysfunctional P2X7. In addition to its enzymatic function, CD73 has been Breast cancer patients harboring a P2X7 gene associated suggested to have a role in T- (Resta et al., with decreased P2X7 activity have a significantly greater 1998) and . Human T cells treated with a risk of progression to metastatic disease (Ghiringhelli plate-bound anti-CD73 monoclonal antibody and sub- et al., 2009). Taken together, these studies support a mitogenic concentrations of anti-CD3 monoclonal anti- tumor-suppressive role for P2X7. body proliferate more readily and produce more IL-2 (Massaia et al., 1990). CD73 has been shown to regulate Pro-tumorigenic effects of extracellular ATP T-cell adhesion in a mechanism dependent on lympho- Other studies, in contrast, suggest that extracellular cyte function-associated antigen-1 (Airas et al., 2000). ATP can promote tumor growth, either directly by CD73 has also been shown to interact directly with activation of P2 receptors on tumor cells (including , such as , fibronec- P2X7) or indirectly via non-transformed cells. During tin and tenascin C (Dieckhoff et al., 1986; Sadej et al., skin wound healing, the release of extracellular ATP has 2006). been shown to be critical for efficient epithelial repair, revealing the role of purinergic signaling in general cell CD73 on cancer cells. CD73 has been found to be functions. Indeed, activation of ATP receptors stimu- overexpressed in several types of cancer, including lates the proliferation of epidermal keratinocytes (Greig bladder cancer (Stella et al., 2010), leukemia (Mikhailov et al., 2003) and cells (Wilden et al.,

Oncogene Extracellular adenosine triphosphate and adenosine J Stagg and MJ Smyth 5352 1998), and initiates migration of epithelial cells (Boucher against trastuzumab (Herceptin) therapy, which relies et al., 2007). Likewise, extracellular ATP released from on p27 upregulation (Lane et al., 2001). A1 adenosine hepatocytes and Kupffer cells immediately after hepa- receptor has also recently been shown to act as a target tectomy has been shown to promote liver regeneration and regulator of estrogen receptor-a in breast cancer (Gonzales et al., 2010). One mechanism by which cells (Lin et al., 2010). extracellular ATP enhances cell function is through the transactivation of receptor A3 receptors in cancer. A3 receptor is also upregulated (EGFR). G-protein-coupled receptors are important in transformed versus adjacent non-transformed cells transactivators of EGFR, thereby promoting cell (Madi et al., 2004). In the case of A3 adenosine survival and proliferation. A recent study has identified receptors, however, synthetic agonists have been shown a P2Y-dependent pathway (most likely P2Y2) that leads to directly suppress tumor cell survival and/or prolifera- to the release of the EGFR ligand TGF-a and tion (Fishman et al., 2004). The antiproliferative effects transactivation of EGFR, through a mechanism invol- of A3 agonists most likely rely on the downregulation of ving mitochondrial production of reactive oxygen A3 adenosine receptors following chronic activation. A3 species (Myers et al., 2009). Interestingly, other groups agonists have been shown to decrease the activity of have also reported the ability of P2Y2 to induce reactive protein kinase A and Akt, resulting in a suppression of oxygen species, including in prostate cancer cells, cyclin D1, c- and cell proliferation (Fishman et al., resulting in increased tumor growth (Sauer et al., 2004). A3 receptor agonists have also been shown to 2001). Although transactivation of EGFR can trigger enhance the antitumor activity of NK cells by IL-12 kinase-dependent pathways, it may further promote cell upregulation (Harish et al., 2003). Finally, in some survival independently of its kinase activity. Indeed, circumstances, the pro-apoptotic effects of A3 receptor recent studies have shown that EGFR maintains basal agonists can be independent of A3 receptor activation intracellular glucose levels in a kinase-independent (Morello et al., 2008). manner (Weihua et al., 2008). Whether extracellular ATP can maintain intracellular glucose levels by EGFR transactivation remains unknown. However, it has been A2A receptors in cancer. The activation of A2A observed that activation of purinergic receptors in- adenosine receptors on endothelial cells significantly creases intracellular ATP content (Di Virgilio et al., enhances tumor angiogenesis (Ahmad et al., 2009). This is consistent with the fact that endothelial cells 2009). The activation of P2X7 has also been shown to increase tumor cell survival. Accordingly, overexpres- associated with tumors have significantly higher levels sion of P2X increased cell survival in serum-free of A2A adenosine receptors. Analysis of A2A receptor 7 expression in samples and matched normal conditions, whereas blocking P2X7 suppressed proli- feration of lymphocytic leukemia cells (Adinolfi et al., lung tissue revealed that there was a significant increased 2002). Taken together, these studies suggest that expression of A2A in early-stage cancer samples accumulation of extracellular ATP can induce impor- (Ahmad et al., 2009). Other studies have shown that tant pro-tumorigenic effects. activation of A2A adenosine receptors directly promotes In addition to its direct effect on tumor cells, tumor cell survival. Indeed, activation of A2A adenosine extracellular ATP may support tumor growth and receptors was found to significantly enhance the metastasis by indirect means by activation of purinergic proliferation of the -dependent breast cancer receptors on non-transformed cells. Chronic exposure of cell line MCF-7 (Etique et al., 2009). C6 glioma cells to ATP, for instance, enhances the In addition to its pro-angiogenic and proliferative release of the pro-inflammatory factors MCP-1, IL-8 effects, the A2A adenosine receptor is a potent and vascular endothelial growth factor (Jantaratnotai suppressor of endogenous tumor immunosurveillance. Accordingly, Ohta et al. (2006) showed that A2A et al., 2009). In thyroid papillary carcinoma cells, P2X7 activation induces IL-6 production (Solini et al., 2008). receptor-deficient mice mount spontaneous antitumor T-cell responses able to induce T-cell-dependent P2X7 has also been shown to trigger the release of the growth factor substance P from neuroblastoma cells tumor rejection. This study suggested that blocking A2A receptors enhanced existing antitumor immune (Raffaghello et al., 2006). Finally, activation of P2X7 in C6 glioma cells has been shown to increase tumor cell responses and increased IFN-g production from anti- þ migration (Wei et al., 2008). tumor CD8 T cells was suggested as the main mechanism of action.

Adenosine receptors in cancer A2B receptors in cancer. A2B adenosine receptors also A1 receptors in cancer. A1 adenosine receptors have have an important role in angiogenesis (Feoktistov been found to be overexpressed in primary breast tumor et al., 2002). The pro-angiogenic effect of A2B receptors tissues and to enhance breast cancer cell proliferation is due to its expression on both endothelial and hemato- (Mirza et al., 2005). RNA interference knockdown poietic cells. Accordingly, studies in A2B-deficient studies suggested that activation of A1 adenosine mice have shown that tumor-infiltrating CD45 þ cells, receptors decreases p27 expression, thereby upregulating most likely CD11b þ myeloid cells, produce up to CDK4 activity (Mirza et al., 2005). The activation of A1 fivefold more vascular endothelial growth factor when adenosine receptors may thus contribute to resistance proficient in A2B adenosine receptors (Ryzhov et al.,

Oncogene Extracellular adenosine triphosphate and adenosine J Stagg and MJ Smyth 5353 2008). The expression of A2B adenosine receptors on et al., 2007). However, the addition of recombinant mast cells may also significantly enhance tumor angio- CRT and high-mobility group box 1 to live tumor cells genesis. The stimulation of A2B adenosine receptors in is not sufficient to induce antitumor immune responses, mast cells has been shown to induce the production of implying another level of regulation. As activation of IL-4, IL-13, IL-8 and vascular endothelial growth factor inflammasomes is central to the instigation of immune (Ryzhov et al., 2004). responses, Zitvogel and colleagues hypothesized that the NLRP3 inflammasome may constitute a third level of regulation of immunogenic chemotherapy (Ghiringhelli et al., 2009). Zitvogel and colleagues recently showed Modulating purinergic signaling for cancer therapy that activation of the NLRP3 inflammasome in DCs is indeed a critical event in the induction of antitumor ATP release and immunogenic chemotherapy immune responses following treatment with doxorubicin, Specific classes of chemotherapeutic , such as mitoxanthrone and oxaliplatin (Ghiringhelli et al., 2009). anthracyclines, have recently been shown to be potent Ghiringhelli et al. (2009) further tested whether the release of inducers of immunogenic cancer cell death, thereby ATP from dying tumor cells was involved in the activation triggering antitumor immune responses (Ghiringhelli of the NLRP3 inflammasome. Numerous chemotherapeu- et al., 2009) (Figure 3). Most remarkably, the antitumor tic drugs—that is, cadmium, etoposide, mitomycin C, immune responses induced by these specific chemother- oxaliplatin, cisplatin, staurosporine, thapsigargin, mi- apeutic drugs were shown to be essential for therapeutic toxanthrone and doxorubicin—could indeed trigger activity in mice. Intriguingly, drugs such as anthra- the release of extracellular ATP from tumor cells before cyclines and oxaliplatin induce potent immunogenic cell and during apoptosis (Martins et al., 2009). Remark- death, whereas other drugs, such as etoposide and ably, mice deficient in the ATP receptor P2X7 bearing mitomycin C, fail to do so. In an attempt to elucidate EL4 or EG7 tumors failed to respond to oxaliplatin the mechanism of immunogenic chemotherapy, Obeid treatment and failed to mount tumor-specific CD8 þ et al. (2007) showed that pro-inflammatory apoptosis of T-cell responses, a phenotype that could be rescued by tumor cells was directly linked to the rapid translocation the adoptive transfer of wild-type DCs. Taken together, of (CRT) to the cell surface upon exposure these studies strongly suggest that anthracyclines and to the . Dying tumor cells must thus translocate the oxaliplatin induce immunogenic cell death by the endoplasmic reticulum-resident CRT and disulfide iso- translocation of CRT on tumor cells and the activation, merase ERp57 to the plasma membrane to be efficiently on DCs, of TLR4 by high-mobility group box 1 and phagocytosed by DCs. In addition to CRT transloca- P2X7 by ATP release. These events in turn activate the tion, the release of chromatin-binding high-mobility NLRP3 inflammasome in DCs, stimulate antigen group box 1 protein by dying tumor cells and its presentation and IL-1b release, thereby generating activation of TLR4 on DCs are also required (Apetoh IFN-g-producing tumor-specific CD8 þ T cells.

Figure 3 Modulating purinergic signaling for cancer therapy. Altering the balance between extracellular adenosine triphosphate (ATP) and adenosine can induce anticancer activity. Anthracyclines and oxaliplatin trigger ATP release, calreticulin (CRT) translocation and high-mobility group box 1 (HMGB1) release from tumor cells, thereby activating NLRP3 inflammasome and inducing antitumor immune responses that are essential for therapeutic activity (Ghiringhelli et al., 2009). However, tumor cells often overexpress ecto-nucleotidases, such as CD39 and CD73, which hydrolyze extracellular ATP to adenosine. Two recent studies, including our own work, have identified tumor-derived CD73 as an important mechanism of tumor immune escape (Stagg et al., 2010). Antibody-based therapy against CD73 can significantly inhibit tumor growth, chemotaxis and metastasis, suggesting that CD73 may constitute a potential cancer target.

Oncogene Extracellular adenosine triphosphate and adenosine J Stagg and MJ Smyth 5354 Blocking A2A adenosine receptors involving co-activation of the dependence receptor As discussed above, Ohta et al. (2006) showed that mice DCC (Rodrigues et al., 2007). genetically deficient in A2A receptors are significantly protected against CL8-1 and RMA tumor challenges, most likely by enhanced antitumor CD8 þ T-cell Conclusion response. The authors of that study also tested whether pharmacological inhibition of A2A receptors could One of the most important consequences of tumorigen- induce antitumor activity in wild-type mice. Indeed, esis is the disruption of tissue architecture. Among other injection of A2A antagonists significantly enhanced the pathways, tissue damage triggers the release of extra- therapeutic activity of adoptively transferred tumor- cellular ATP, which activates P2X and P2Y receptors to specific CD8 þ T cells. No therapeutic effect was promote tissue repair. The activation of ATP receptors observed against non-immunogenic B16 tumors, or in enhances cell survival, proliferation and migration, and nude mice with CL8-1 tumors, suggesting a T-cell- acts as a chemotactic molecule for the recruitment of dependent mechanism. Interestingly, pharmacological immune phagocytes and DCs. In the presence of danger- inhibition of A2A receptors was associated with the associated molecules, extracellular ATP further development of self-resolving autoimmunity as evi- activates NLRP3 inflammasomes, thereby triggering a denced by transient hair loss. As pointed out by the pro-inflammatory cascade leading to the activation of authors, ‘it is important to carefully consider the known innate and adaptive immune responses. Concurrently, cardiovascular and neurological effects of A2A and A2B regulatory mechanisms are in place to prevent excessive antagonists’. Potential side effects of A2A receptor tissue damage that might be caused by unresolved antagonists might include increased blood and inflammation. Tissue hypoxia and the accumulation of inflammation. However, based on the results of recent specific cytokines, such as type I IFNs and TNF-a, are clinical trials with A2A receptor antagonists against important activators of these regulatory mechanisms. Parkinson’s disease, A2A antagonists seem to be well- One of the most important immunosuppressive regula- tolerated (LeWitt et al., 2008). tory pathways is the phosphohydrolysis of extracellular ATP to adenosine. In the context of cancer, tumor cells can directly Targeting CD73 exploit ATP and adenosine receptors for survival, Although pharmacological antagonists to adenosine proliferation and motility. In addition, tumor cells have receptors may hold therapeutic potential, an alternative been found to express high levels of ecto-nucleotidases, is to target the ecto-nucleotidase CD73. The observation such as CD73, that catabolize the formation of that CD73 is expressed at high levels in various types of extracellular adenosine. Extracellular adenosine gener- cancer supports this approach. Using RNA-interference ated from the activity of CD73 promotes tumor knockdown of CD73, we have recently shown that angiogenesis, immune escape and metastasis. The CD73 expression by breast tumor cells significantly accumulation of extracellular adenosine thus constitutes impairs adaptive antitumor immune responses (Stagg an important regulatory mechanism in the generation of et al., 2010). Another independent group recently antitumor immunity. Our data and that of others corroborated our findings, revealing that tumor-derived strongly suggest that the ecto-nucleotidase CD73 may CD73 inhibited T-cell immunosurveillance of murine constitute a previously unrecognized target for cancer ovarian tumors (Jin et al., 2010). The latter study therapy. showed that CD73 on tumor cells inhibited T-cell It has become increasingly evident that antitumor function and induced T-cell apoptosis. immune responses are essential not only for the We have investigated whether CD73 could be a potential endogenous control of tumorigenesis, but also for the target for antibody-based therapy (clone TY/23). Tumors therapeutic activity of standard treatment, such as of treated mice were significantly reduced in size chemotherapy. However, several regulatory mechanisms, compared with control-treated mice (Stagg et al., or ‘immune-checkpoints’, limit the generation of anti- 2010). As 4T1.2 tumors can spontaneously metastasize cancer immune responses. Various strategies targeting after injection in the mammary fat pad, we also specific immune-checkpoints can be deployed to enhance observed that anti-CD73 monoclonal antibody treat- antitumor immunity. Understanding the relative impor- ment significantly reduced the number of spontaneous tance of each of these immune-checkpoints in specific lung metastases. Our data also revealed that CD73- cancer types in the context of patients’ individual derived adenosine enhanced tumor cell chemotaxis. polymorphism will be of primary importance. Thorough Others had previously shown that CD73 expression in translational studies will thus be essential for the breast cancer cells was associated with increased development of safe and effective new cancer treatments migration (Zhi et al., 2007). Our data further suggested aiming to harness antitumor immune responses. a role for A2B adenosine receptors in tumor cell metastasis. A2B receptors could potentially enhance receptor expression (Richard et al., 2006) or act as an important co-receptor. Accordingly, A2B Conflict of interest adenosine receptors have been shown to promote human colon cancer cell motility by a mechanism The authors declare no conflict of interest.

Oncogene Extracellular adenosine triphosphate and adenosine J Stagg and MJ Smyth 5355 Acknowledgements Breast Cancer Foundation, the Victorian Breast Consortium, the Victorian Cancer Agency, and by JS is supported by a Canadian Institutes of Health Research a National Health and Medical Research Council of Australia (CIHR) Fellowship. MJS is supported by the Susan G Komen (NH&MRC) Australia Fellowship and Program Grant.

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