934 Immune Factors Involved in the Promotion of Esophageal Adenocarcinoma As Defined by siRNA Library Screening

Abstract

BACKGROUND: Esophageal adenocarcinoma (EAC) is a classic example of inflammationassociated cancer, which develops through GERD (gastro-esophageal reflux disease)-Barrett's esophagus (BE)-dysplasia-adenocarcinoma sequence. The incidence rate for EAC has increased 4-10% per year among men since 1976 in the USA, more rapidly than for any other type of cancer. Patients with Barrett's esophagus can progress to dysplasia and EAC at 30-60 times that of the general population. NF-kB plays a key role in inflammation process and TNF-α is a pro-inflammatory cytokine and a known NF-kB activator. It has been reported that TNF-α and TNFR1 are significantly increased in BE and EACs. Glutathione Peroxidase 7 (GPX7), a recent member of GPX family which currently has 8 members, is frequently silenced through location-specific hypermethylation during Barrett's tumorigenesis. Its functions remain largely uncharacterized. In this study, we investigated the potential role of GPX7 in regulating NF-kB activity. METHODS and RESULTS: In vitro experiments and de-identified human tissue samples were utilized for quantitative real-time PCR, immunofluorescence, luciferase reporter, immunoblotting and intracellular ROS (reactive oxygen species) assays. Reconstitution of GPX7 in BE and EAC cell models abrogated TNF-αinduced NF-kB reporter activity and nuclear translocation of NF-kB-p65 (P<.01). In addition, we detected marked reduction in phosphorylation levels of components of NF-kB signaling pathway (p-p65, p-IkB-α, and p-IKKα/β) and significant downregulation of NF-kB target genes (TNF-α, IL-1β, IL-6, IL-8, CXCL-1, and CXCL-2) in GPX7 expressing cells as compared to control cells. These effects were validated by knockdown of endogenous GPX7 in HET1A cells. We also found that the GPX7-mediated suppression of TNF-α-induced activation was independent of its capacity to regulate ROS. On the other hand, we demonstrated that GPX7 promotes protein degradation of TNFR1 and TRAF2 which could explain GPX7-dependent regulation of the IKK IkB NF-kB-p65 axis. The relevance of these findings to Barrett's was further confirmed by showing that GPX7 suppressed bile salts-induced activation of NF-kB and expression of cytokines in vitro and the presence of inverse relationship between GPX7 and cytokines' expression in esophageal tissue samples. CONCLUSIONS: The loss of GPX7 expression could unleash the TNF-αand bile salt-induced activation of pro-inflammatory NF-kB signaling that contributes to the GERD-associated BE-dysplasia-EAC sequence.