Tu1658 Inhibition of Peritoneal Metastasis by Overcoming Death Receptor Resistance by Small Molecule Targeting of C-FLIP

Abstract

Background: Helicobacter pylori (H.pylori) infection plays an important role in gastric carcinogenesis and exemplifies the strong association between inflammation and cancer. DARPP-32 (Dopamine and cAMP-regulated phosphoprotein, Mr 32000) is a novel cancer gene. The aim of this study was to investigate the role of inflammation and H.pylori in regulating DARPP-32 expression. Methods: Immunohistochemistry (IHC) were used to determine protein expression. DARPP-32 promoter activity was measured by a luciferase reporter system. The mRNA and protein levels were evaluated by quantitative real-time PCR and Western blot analysis. Results: The IHC analysis demonstrated overexpression of p-NFκB and DARPP-32 in the early stages of human gastric cancer. Therefore, we examined the relationship between NF-kB and DARPP-32, and found that DARPP-32 does not regulate the transcriptional activity of NF-κB as measured by pNF-κB luciferase reporter and western blot analysis. On the other hand, we found that TNF-α treatment leads to activation of NFκB and mediates upregulation of mRNA and protein levels of DARPP-32 in several gastric cancer cell lines (P 10-fold, P<0.001) and protein levels. In addition, treatment with an NF-κB inhibitor (BAY 11-7082) decreased DARPP-32 mRNA and protein levels. In an attempt to study the transcriptional regulation, we cloned a 2500 bp of the DARPP-32 promoter into the luciferase reporter (DARPP-32-Luc). The TNF-α treatment of AGS and MKN-45 cells led to a significant induction of luciferase activity of DARPP-32 reporter (P<0.01), and treatment of these cells with BAY 11-7082 (5 μM) reversed these effects (P<0.01). Furthermore, transient expression of NF-kB-p65 in AGS cells led to a 3fold induction in the luciferase activity of DARPP-32 reporter (P<0.01). Using deletion constructs, we have found that the -1000bp of promoter area is the most active region. We confirmed that NF-kB-p65 binds to DARPP-32 promoter by ChIP assay. In vitro data using gastric cancer cell lines and H.pylori infection (J166 and 7.13 strains) demonstrated simultaneous increase in the p-NF-kB and increase in the mRNA and protein levels of DARPP-32. Using the Tff1-KO gastric cancer mouse model that is characterized by NF-kB activation, we found that DARPP-32 was overexpressed at the protein and mRNA levels. Treatment of wild-type and Tff1-KO mice with H.pylori (PMSS1 strain) led to significant induction of both p-NF-kB and DARPP-32. Conclusion: Our results demonstrated, for the first time, that H. pylori infection leads to induction of NF-κB-dependent DARPP-32 expression. Given the oncogenic functions of DARPP-32 in regulating the PI3K/AKT and CXCR4 signaling, this novel DARPP-32 regulationmechanism provides a new paradigm in gastric carcinogenesis.