Abstract 501: CXCR4 induced ROS accumulation through PI3K/AKT

Abstract

Abstract Reactive oxygen species (ROS) and oxidative stress have long been implicated in the onset and progression of many cancers. A mechanism by which ROS contributes to cancer is by relaying signals from the cell surface to important signaling proteins, thereby regulating a variety of cellular processes. Identifying the sources of ROS in cancer cells, and the consequences of oxidative stress, remain areas of intense research. We have previously demonstrated that ROS increased expression and activity of the chemokine (CXC) receptor, type 4 (CXCR4), which resulted in trans-endothelial migration of prostate cancer (PCa) cells. Similarly, a study in human hepatoma cells revealed that CXCR4 and its ligand, stromal cell derived factor 1 alpha (SDF-1α), promoted ROS accumulation. We, and others, have demonstrated that increased ROS generation observed in PCa cell lines was largely contributed by the NADPH oxidase (NOX) family of enzymes. ROS accumulation in cells is linked to survival through activation of PI3K/AKT and ERK1/2 pathways. To that end, we’ve demonstrated that phosphorylated AKT (pAKT) expression increased in response to hydrogen peroxide in a dose-dependent manner. Therefore, we sought to investigate whether SDF-1α-mediated regulation of NOX2 enzymes also involved PI3K/AKT signaling. Our results revealed that treatment with the PI3K inhibitor, LY294002, decreased ROS secretion below basal level in the presence of SDF-1α. In contrast, cells treated with the ERK1/2 inhibitor, PD98059, and SDF-1α exhibited increased ROS secretion, suggesting that SDF1α-mediated ROS production did not depend upon ERK1/2 signaling, but was instead, contributed, in part, by PI3K/AKT signaling. To further delineate the involvement of PI3K/AKT in NOX-mediated signaling, we treated cells with apocynin (NOX inhibitor) and assessed its effect on the phosphorylation of AKT. We observed a concomitant decrease in pAKT, in cells where NOX activity was repressed, compared to untreated cells. Likewise, cells, in which NOX activity was inhibited but stimulated by SDF-1α, expressed little pAKT. To determine whether NOX2 and pAKT physically associated in vitro, NOX2 expression was silenced via siRNA transfection, followed by NOX2 immunoprecipitation, prior to Western blot analysis for pAKT protein expression. When compared to untransfected control, cells transfected with NOX2 siRNA alone exhibited decreased expression of pAKT. Finally, stimulation with SDF-1α did not recover expression of pAKT, further validating coordinated efforts between AKT, NOX2 and the SDF-1α/CXCR4 signaling axis. Results generated from this study will further elucidate the mechanism by which CXCR4 promotes oxidative stress through NOX enzymes. Understanding the relationship between known cancer promoting elements, such as CXCR4, ROS, AKT and NOX, will facilitate the development of more comprehensive therapeutics to enhance current clinical strategies to combat cancer metastasis. Citation Format: Latoya K. Bryant, Kia J. Jones, Cimona V. Hinton. CXCR4 induced ROS accumulation through PI3K/AKT. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 501. doi:10.1158/1538-7445.AM2014-501