Abstract 4144: Extracellular nucleotides and purinergic signaling as novel, underappreciated, pro-metastatic factors for human lung cancer cells
Authors: Schneider Gabriela , Glaser Talita , Ismail Ahmed Abdelbaset , Ulrich Henning , Ratajczak Mariusz Z. 
Published: August 2015

Abstract
Background: One of the challenging problems of current radio-chemotherapy is recurrence and metastasis of cancer cells that survive initial treatment. We have proposed that one of the unwanted effects of radio-chemotherapy is release of phospho-sphingolipids in response to treatment and thus induction of pro-metastatic microenvironment in several organs (Mol Cancer Res 2013; 11:793). However, at the same time also intracellular nucleotides such as ATP and UTP are released from damaged (“leaky”) cells exerting chemotactic functions. Hypothesis: Based on these observations that, in addition to phospho-sphingolipids, also nucleotides (e.g., ATP and UTP) are released from damaged cells we hypothesized that these molecules if released into extracellular microenvironment may also direct chemotaxis and metastasis of cancer cells - alone or in synergy with phospho-sphingolipids. Materials and Methods: Several complementary in vitro and in vivo approaches were employed to demonstrate a novel role of extracellular nucleotides (EXN) in a model of metastasis of lung cancer cells. In our studies we employed four large cell and two small cell lung cancer cell lines and a set of nucleotides including ATP, ADP, AMP, UTP, TTP, CTP, and GTP. Real time RT-PCR analysis of the expression of purinergic P1 and P2 receptors as well as chemotaxis, adhesion, proliferation, intracellular calcium flux and cell signaling studies in response to EXN stimulation were performed. Concentrations of ATP and UTP in several organs before and after radio-chemotherapy were measured by colorimetric kits. Purinergic receptor agonists and antagonists, inhibiting all or selected subtypes, were assayed in vitro and in vivo in pro-metastatic assays. Results: We observed that EXN accumulate in in several organs in response to radio-chemotherapy. RT-PCR analysis indicated that most of the P2X, P2Y and adenosine receptor subtypes are expressed in tested lung cancer cell lines. By employing in vitro migration assays, we found that, out of all the nucleotides tested, ATP, AMP and UTP have the strongest chemotactic activity for most of the human lung cancer cell lines correlating with phosphorylation of MAPKp42/44 and AKT. We also observed increased adhesion to fibronectin after stimulation with tested EXN, whereas proliferation was not affected by of EXN. More important, metastasis of lung cancer cells could be inhibited in immune-deficient mice in a presence of specific small molecule inhibitors of nucleotide receptors. Conclusions: Both systemic and local radio-chemotherapy leads to upregulation of EXN release in damaged tissues, and the side effect of such treatment is induction of an unwanted pro-metastatic microenvironment in different organs. Based on this data, EXN are novel pro-metastatic factors and inhibition of their pro-metastatic effects could become an important part of anti-metastatic treatment.
Citation Format: Gabriela Schneider, Talita Glaser, Ahmed Abdelbaset Ismail, Henning Ulrich, Mariusz Z. Ratajczak. Extracellular nucleotides and purinergic signaling as novel, underappreciated, pro-metastatic factors for human lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4144. doi:10.1158/1538-7445.AM2015-4144