Person:

Wu, Yan

Background: Extracellular adenosine triphosphate (ATP) functions as a novel danger signal that boosts antitumor immunity and can also directly kill tumor cells. We have previously reported that chronic exposure of tumor cells to ATP provokes P2X7-mediated tumor cell death, by as yet incompletely defined molecular mechanisms. Methodology/Principal Findings Here, we show that acute exposure of tumor cells to ATP results in rapid cytotoxic effects impacting several aspects of cell growth/survival, leading to inhibition of tumor growth in vitro and in vivo. Using agonist and antagonist studies together with generation of P2X7 deficient tumor cell lines by lentiviral shRNA delivery system, we confirm P2X7 to be the central control node transmitting extracellular ATP signals. We identify that downstream intracellular signaling regulatory networks implicate two signaling pathways: the known P2X7-PI3K/AKT axis and remarkably a novel P2X7-AMPK-PRAS40-mTOR axis. When exposed to high levels of extracellular ATP, these two signaling axes perturb the balance between growth and autophagy, thereby promoting tumor cell death. Conclusions: Our study defines novel molecular mechanisms underpinning the antitumor actions of P2X7 and provides a further rationale for purine-based drugs in targeted cancer therapy.

Interferon gamma (IFNγ)-producing CD8+ T cells (Tc1) play important roles in immunological disease. We now report that CD3/CD28-mediated stimulation of CD8+ T cells to generate Tc1 cells, not only increases IFNγ production but also boosts the generation of reactive oxygen species (ROS) and augments expression of CD39. Inhibition of NADPH oxidases or knockdown of gp91phox in CD8+ T cells abrogates ROS generation, which in turn modulates JNK and NFκB signalling with decreases in both IFNγ levels and CD39 expression. CD39+CD8+ T cells substantially inhibit IFNγ production by CD39−CD8+ T cells via the paracrine generation of adenosine, which is operational via adenosine type 2A receptors. Increases in numbers of CD39+CD8+ T cells and associated enhancements in ROS signal transduction are noted in cells from patients with Crohn's disease. Our findings provide insights into Tc1-mediated IFNγ responses and ROS generation and link these pathways to CD39/adenosine-mediated effects in immunological disease.

Extracellular ATP (eATP) accumulation within the tumor microenvironment (TME) has the potential to activate purinergic signaling. The eATP evoked signaling effects bolster antitumor immune responses while exerting direct cytotoxicity on tumor cells and vascular endothelial cells, mediated at least in part through P2X7 receptors. Approaches to augment purinergic signaling in TME e.g. by ectonucleotidase CD39 blockade, and/or boosting P2X7 functional responses, might be used as immunomodulatory therapies in cancer treatment. In this study, we delineated the translatable strategy of hyperthermia to demonstrate impacts on P2X7 responsiveness to eATP. Hyperthermia (40°C) was noted to enhance eATP-mediated cytotoxicity on MCA38 colon cancer cells. Increased membrane fluidity induced by hyperthermia boosted P2X7 functionality, potentiating pore opening and modulating downstream AKT/PRAS40/mTOR signaling events. When combined with cisplatin or mitomycin C, hyperthermia and eATP together markedly potentiate cancer cell death. Our data indicate that clinically tolerable hyperthermia with modulated P2X7-purinergic signaling will boost efficacy of conventional cancer treatments.