Regulation of Adenosine Levels in Healthy vs. Malignant Tissue
Extracellular adenosine, a nucleoside and derivative of ATP, is involved in the regulation of diverse physiological processes including vasodilation (4), kidney-exerted water reabsorption (5), pain perception (6), and fine-tuning of the sleep–wake cycle (7). Even though levels of extracellular adenosine within healthy tissues are negligible (8–11), upon injury this nucleoside sharply accumulates at the interstitium where it potently restricts immune responses (12) and directly promotes wound healing (13). Under homeostatic conditions in healthy tissues, the cytosolic concentration of ATP ranges from 1 to 10 mM (14), while its extracellular levels are negligible (15). This sharp gradient can be rapidly disrupted however upon breaches of the plasma membrane induced by necrosis, apoptosis or mechanical stress, as well as by regulated ATP efflux. The latter, induced by a variety of stimuli including hypoxia, ischemia and inflammation, has been shown to extensively occur via exocytosis, transmembrane transfer through ATP-binding cassette (ABC) transporters, as well as by diffusion through a variety of anion channels or non-selective plasma membrane pores formed by connexins, pannexin-1 or the ATP receptor P2X7R (16–18). For instance, stimulated T cells release ATP through pannexin-1 hemi-channels and via exocytosis (19, 20).
Once in the extracellular space, ATP undergoes rapid stepwise dephosphorylation by ecto-nucleotidases (21, 22) including the E-NTPDase CD39, which converts ATP or ADP to ADP or AMP, respectively, and the 5′-nucleotidase CD73, which dephosphorylates AMP to adenosine (18, 23) (Figure 1). Additional enzymes whose ecto-activity contributes toward extracellular adenosine generation are other E-NTPDases, members of the ecto-phosphodiesterase/pyrophosphatase (E-NPP) family, nicotinamide adenine dinucleotide (NAD+) glycohydrolases, the prostatic acid phosphatase (PAP), and the alkaline phosphatase (ALP) (21) (Figure 1). Briefly, the co-enzyme NAD+, another key cellular component whose extracellular concentration significantly rises in injured tissue (24, 25), is converted to adenosine diphosphate ribose (ADPR) by the NAD+ glycohydrolase CD38 (26), while ADPR as well as ATP are metabolized to AMP by the E-NPP CD203a (27). Moreover, PAP, which is predominantly, but non-exclusively, expressed in prostate tissue (28), is capable of converting extracellular AMP to adenosine (29), whereas ALP catalyzes the hydrolysis of ATP, ADP and AMP to adenosine (21). Finally, adenosine can also be produced intracellularly either by S-adenosylhomocysteine hydrolase (SAHH)-exerted hydrolysis of S-Adenosylhomocysteine (SAH), a metabolite of the transmethylation pathway, or due to soluble CD73-mediated catabolism of AMP, a nucleoside participating in multiple cellular processes and whose concentration rises within cells of low energy charge (30) (Figure 1). Intracellularly-generated adenosine can be secreted in a diffusion limited-manner through bidirectional equilibrative nucleoside transporters (ENTs) (31). However, although there is evidence suggesting that hypoxia can boost intracellular adenosine production (32, 33), the contribution of this pathway toward injury-caused interstitial adenosine buildup is considered minor due to concurrent hypoxia-induced downregulation of the aforementioned transporters (34, 35). Given its diverse effects, adenosine presence at the extracellular space is subject to tight spatiotemporal control (12, 13, 36). For instance, extracellular accumulation of adenosine is counteracted by its inward transfer through ENTs or concentrative, sodium gradient-dependent, symporters (31) as well as by the function of intra/extracellular adenosine deaminase (ADA) and of cytosolic adenosine kinase (ADK), which respectively convert adenosine to inosine or AMP (37) (Figure 1).
Figure 1  Regulation of interstitial adenosine levels in injured tissue. Stress-induced, extracellular buildup of ATP or NAD+ fuels catabolic adenosine-generating pathways, such as the one mediated by CD39 and CD73. The activity of other ecto-nucleotidases including CD38, CD203a, ALP, and PAP, also contribute toward extracellular adenosine accumulation. Adenosine can also be produced intracellularly by SAHH-exerted hydrolysis of SAH, as well as by soluble CD73-mediated catabolism of AMP, and it can be exported by ENTs in a diffusion-limited manner. On the flip side, the combination of CD26-bound ADA activity and of adenosine cellular uptake, either through equilibrative ENTs or via concentrative CNTs, limits interstitial adenosine levels. Intracellularly, adenosine can be eliminated via its conversion to SAH by SAHH, to AMP by ADK, or to inosine by ADA. SAHH, S-adenosylhomocysteine hydrolase; SAH, S-Adenosylhomocysteine; ENTs, equilibrative nucleoside transporters; CNTs, concentrative nucleoside transporters; ADK, adenosine kinase; ADA, adenosine deaminase. In contrast to homeostatic conditions, ATP levels are highly elevated in the TME as a result of necrosis, apoptosis, hypoxia, and persistent inflammation (17, 18), and intra-tumoral adenosine levels can reach micromolar concentrations (9, 10, 38). ATP catabolism in tumors is primarily mediated by CD39 and CD73 (39–41), and high expression of these ecto-nucleotidases is strongly associated with poor clinical outcome for patients suffering a variety of cancer-types (3, 42, 43). In particular, CD39 and/or CD73 (over)expression has been detected on the surface of tumor cells (39, 44–51), cancer-associated fibroblasts (CAFs) (52–54), mesenchymal stem cells and stromal cells (55–57), endothelial cells (ECs) (45, 46, 51), myeloid derived suppressor cells (MDSCs) (58–60), tumor associated macrophages (TAMs) (53, 61), Tregs (46, 62–64), Th17 cells (65) and of antigen experienced/exhausted conventional CD4+ and CD8+ T cells (64, 66–68). In addition, CD39/CD73-bearing exosomes (69, 70), released by tumor cells (71), Tregs (72), and MDSCs (57, 73) further contribute to adenosine generation. Currently, hypoxia as well as incessant inflammation are considered to be the main drivers of intra-tumoral CD39 and CD73 overexpression. Namely, hypoxia-induced (74, 75) HIF1α (76–79) and Sp1 (80) activity promotes expression of these ecto-nucleotidases. Along the same lines, signaling pathways initiated by inflammation-associated molecules, such as IL-2 (81), IL-6 (66, 82), IL-1β (83), TNFα (83–85), type I IFNs (86, 87), IL-27 (66, 88), TGFβ (82, 89, 90) as well as by inducers of the Wnt (91, 92) or cAMP (83, 93–95) signaling pathways also boost CD39 (66, 81, 82, 88, 89, 95) and CD73 (81–87, 89–94) levels.
Although CD39 and CD73-mediated catabolism of extracellular ATP is considered to account for the bulk of intra-tumoral adenosine generation, expression levels of ecto-enzymes participating in alternative adenosine production pathways also rise in the advent of cancer. For instance, CD38 is frequently upregulated within neoplastic tissues (26, 96, 97) and sporadic evidence suggests that CD203a levels also increase on TME components (98, 99). Along the same lines, the serum concentration of PAP increases during prostate cancer progression (100) while others suggest it gets upregulated on cancerous tissue as well (28). Finally, several studies have demonstrated elevated levels of ALP on cancer cells (101, 102) as well as a correlation of serum ALP levels and disease stage (103–105). Critically, the relative contribution of these alternative adenosine-producing pathways toward intra-tumoral buildup of this nucleoside remains to be determined. Finally, along with aberrant production, defective uptake resulting from the down-modulation of equilibrative (106, 107) as well as concentrative (108–110) nucleoside transporters, also driven by hypoxia (34, 35, 111), further contributes to adenosine accumulation in the TME.