Extracellular ATP and Purinergic P2Y2 Receptor Signaling Promote Liver Tumorigenesis in Mice by Exacerbating DNA Damage

Abstract

Release of ATP to the extracellular compartment and subsequent activation of purinergic receptors is a conserved mechanism mediating inflammatory responses and cell fate decisions in various organs including the liver. Previous findings suggest that extracellular ATP may promote liver tumorigenesis, however, the underlying mechanisms are poorly understood. Therefore, our aim was to dissect the functions of extracellular ATP and P2Y₂ receptors (P2Y₂R) during hepatocarcinogenesis. Liver tumors were induced in wild-type and P2y₂r ^-/- knockout mice by intraperitoneal diethylnitrosamine (DEN) injection. Tumorigenesis was analyzed after 8 to 10 months and molecular analyses were performed at different stages of tumorigenesis in vivo, as well as in primary mouse hepatocytes in vitro. Liver tumor incidence and tumor numbers were strongly reduced in P2y₂r ^-/- mice, whereas tumor size and morphology were comparable to wild-type controls, suggesting that P2Y₂R contributes to tumor initiation. Mechanistically, hepatocyte proliferation in DEN-treated P2y₂r ^-/- mice was reduced, which correlated with reduced c-JUN and CCND1 but increased p21 expression. Moreover, DNA damage as determined by hepatocellular expression of γH2A.X and of genes related to genotoxic stress, as well as STAT3 phosphorylation, was reduced in the absence of P2y₂r. Administration of genotoxic agents to primary hepatocytes in vitro confirmed that DNA damage was indeed exacerbated by extracellular ATP, subsequent P2Y₂R activation, and downstream intracellular calcium-dependent signal transduction. In conclusion, our data reveal that extracellular ATP and subsequent P2Y₂R function stimulate DNA damage responses and hepatocyte proliferation, thereby promoting hepatocarcinogenesis. Targeting this pathway may be an attractive approach for chemoprevention of hepatocellular carcinoma. SIGNIFICANCE: Extracellular ATP and subsequent P2Y₂ receptor function stimulate DNA damage responses and hepatocyte proliferation, thereby promoting hepatocarcinogenesis in mice. Targeting this pathway may be an attractive approach for chemoprevention of hepatocellular carcinoma. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/4/699/F1.large.jpg.