Colitis-associated cancer (CAC) is a complication of inflammatory bowel disease (IBD). Binding of extracellular ATP to the purinergic receptor P2RX7 has emerged as a critical event in controlling intestinal inflammation, acting to limit elevation of proinflammatory mast cells and cytokines and promote survival of regulatory T cells (Treg) and enteric neurons. In this study, we investigated the effect of P2RX7 blockade in an established mouse model of CAC. Using genetic and pharmacologic tools, we found unexpectedly that while P2RX7 mediated inflammatory responses, it also acted at an early time to suppress CAC development. P2RX7 blockade enhanced proliferation of intestinal epithelial cells and protected them from apoptosis. The proliferative effects of P2RX7 blockade were associated with an increased production of TGFb1 that was sufficient to stimulate the proliferation of intestinal epithelial cells. Finally, P2RX7 blockade also altered immune cell infiltration and promoted Treg accumulation within lesions of the digestive system. Taken together, our findings reveal an unexpected role for P2RX7 in preventing CAC, suggesting cautions in the use of P2RX7 inhibitors to treat IBD given the possibility of increasing risks CAC as a result. Cancer Res; 75(5); 835-45. Ó2015 AACR.

Keywords: autophagy, bacteria, Crohn disease, hypoxia inducible factor, LAP Abbreviations: AIEC, adherent invasive E. coli; AMPK, AMP-activated protein kinase; ATG5, autophagy-related 5; ATG16L1, autophagy-related 16-like 1; BNIP3L, BCL2/adenovirus E1B 19kDa interacting protein 3-like; BECN1, Beclin 1, autophagy-related; CD, Crohn disease; CEACAM6, carcinoembryonic antigen-related cell adhesion molecule 6 (nonspecific cross reacting antigen); CRTC1/TORC1, CREB regulated transcription coactivator 1; EEA1, early endosome antigen 1; GFP, green fluorescent protein; HBSS, Hank's balanced salt solution; HIF1A, hypoxia inducible factor 1, a subunit (basic helix-loop-helix transcription factor); IBD, inflammatory bowel disease; IRGM, immunity-related GTPase family, M; LAP, LC3-associated phagocytosis; MAP1LC3-II (LC3-II), microtubule-associated protein 1 light chain 3-II; MOI, multiplicity of infection; SQSTM1/p62 (SQSTM1), sequestosome 1; TEM, transmission electron microscopy; TLR5, toll-like receptor 5; ULK1, unc-51 like autophagy activating kinase 1; VAV2, vav 2 guanine nucleotide exchange factor.The hypoxia inducible transcription factor HIF1 activates autophagy, a general catabolic pathway involved in the maintenance of cellular homeostasis. Dysfunction in both autophagy and HIF1 has been implicated in an increasing number of human diseases, including inflammatory bowel disease (IBD), such as Crohn disease (CD). Adherent invasive E. coli (AIEC) colonize ileal mucosa of CD patients and strongly promote gastrointestinal inflammatory disorders by activation of HIF-dependent responses. Here, we aim to characterize the contribution of HIF1 in xenophagy, a specialized form of autophagy involved in the degradation of intracellular bacteria. Our results showed that endogenous HIF1A knockdown increased AIEC survival in intestinal epithelial cells. We demonstrate that the increase in survival rate correlates with a dramatic impairment of the autophagic flux at the autolysosomal maturation step. Furthermore, we show that AIEC remained within single-membrane LC3-II-positive vesicles and that they were unable to induce the phosphorylation of ULK1. These results suggested that, in the absence of HIF1A, AIEC were found within LC3-associated phagosomes. Using blocking antibodies against TLR5 and CEACAM6, the 2 well-known AIEC-bound receptors, we showed that downstream receptor signaling was necessary to mediate ULK1 phosphorylation. Finally, we provide evidence that HIF1 mediates CEACAM6 expression and that CEACAM6 is necessary to recruit ULK1 in a bacteriacontaining signaling hub. Collectively, these results identify a new function for HIF1 in AIEC-dedicated xenophagy, and suggest that coactivation of autophagy and HIF1A expression may be a potential new therapy to resolve AIEC infection in CD patients.