The A2A adenosine receptor (A2AR) has been shown to be a critical and nonredundant negative regulator of immune cells in protecting normal tissues from inflammatory damage. We hypothesized that A2AR also protects cancerous tissues by inhibiting incoming antitumor T lymphocytes. Here autoimmunity ͉ cancer ͉ therapy ͉ hypoxia ͉ inflammation T he coexistence of tumors and antitumor immune cells is currently explained by the inhibition of immune cells in a poorly understood ''hostile'' tumor microenvironment (1-3). This unidentified immunosuppressive mechanism limits promising cancer therapies using antitumor T cells (4-14). We hypothesized that cancerous tissues are protected from antitumor T cells because of immunosuppressive signaling via T cell A2A adenosine receptor (A2AR) (15-17) activated by extracellular adenosine produced from hypoxic tumor (Fig. 1a). Indeed, hypoxic cancerous tissues may be protected by the same hypoxia3adenosine3A2AR pathway that was recently shown to be critical and nonredundant in preventing excessive damage of normal tissues by overactive immune cells in vivo (18). It is well established that some areas of solid tumors often have transient or chronic hypoxia (19,20), which is conducive to extracellular adenosine accumulation (21). Hypoxia has been implicated in mechanisms of tumor protection against ionizing radiation and some chemotherapeutic agents (19) and is associated with poor prognosis (20).T cells, including antitumor T cells, do predominantly express cAMP-elevating Gs protein-coupled high-affinity A2AR and͞or low-affinity A2B adenosine receptors (A2BR) (16,17,(22)(23)(24); the number of A2AR per T cell may determine the intensity of maximal T cell response to adenosine (25, 26). Whereas we focused on A2AR, others have discounted A2 receptors and suggested the A3 adenosine receptors as responsible for inhibition of antitumor killer T cells (27,28). Here we report that genetic deletion of A2AR accomplishes the complete rejection of immunogenic tumors by antitumor CD8 ϩ T cells in the majority (Ϸ60%) of mice, whereas the antagonists of A2 receptors facilitate CD8 ϩ T cell-mediated retardation of tumor growth. Results The Gradient of T Cell-Inhibiting Extracellular Adenosine in Tumors.It was important to confirm the presence of elevated extracellular adenosine levels in cancerous tissues using a reliable method (29). The HPLC analysis and the use of equilibrium dialysis probes demonstrated higher levels of extracellular adenosine (Fig. 1b), increased adenosine metabolism, and the concomitant increase in cAMP (29) in a solid tumor microenvironment (Fig. 7, which is published as supporting information on the PNAS web site). We also confirmed that antitumor CD8 ϩ T cells used in this study do express the cAMP-elevating functional A2AR and A2BR (Fig. 1c). To directly test whether A2AR inhibit antitumor T cells in vivo, we studied the effects of A2AR gene deletion or competitive antagonists on tumor growth in mice using different CD8 ϩ T celldependent cancer immunosurveillance and ad...

Short-chain fatty acids, generated in colon by bacterial fermentation of dietary fiber, protect against colorectal cancer and inflammatory bowel disease. Among these bacterial metabolites, butyrate is biologically most relevant. GPR109A is a G-protein-coupled receptor for nicotinate, but recognizes butyrate with low affinity. Millimolar concentrations of butyrate are needed to activate the receptor. Although concentrations of butyrate in colonic lumen are sufficient to activate the receptor maximally, there have been no reports on the expression/function of GPR109A in this tissue. Here we show that GPR109A is expressed in the lumen-facing apical membrane of colonic and intestinal epithelial cells and that the receptor recognizes butyrate as a ligand. The expression of GPR109A is silenced in colon cancer in humans, in a mouse model of intestinal/colon cancer, and in colon cancer cell lines. The tumor-associated silencing of GPR109A involves DNA methylation directly or indirectly. Re-expression of GPR109A in colon cancer cells induces apoptosis, but only in the presence of its ligands butyrate and nicotinate. Butyrate is an inhibitor of histone deacetylases, but apoptosis induced by activation of GPR109A with its ligands in colon cancer cells does not involve inhibition of histone deacetylation. The primary changes in this apoptotic process include downregulation of Bcl-2, Bcl-xL, and cyclin D1, and upregulation of death receptor pathway. In addition, GPR109A/butyrate suppresses NF-κB activation in normal and cancer colon cell lines as well as in normal mouse colon. These studies show that GPR109A mediates the tumor-suppressive effects of the bacterial fermentation product butyrate in colon.