Role of Raf kinase inhibitor protein in Helicobacter pylori-mediated signaling in gastric cancer

Abstract

Helicobacter pylori is a helical bacterium that colonizes the stomach in over half of the world's population. Infection with this bacterium has been linked to peptic ulcer disease and gastric cancer. The bacterium has been shown to affect regulatory pathways in its host cells through specific virulence factors that control gene expression. Infection with H. pylori increases levels of phosphorylation of Raf kinase inhibitor protein (pRKIP) in gastric adenocarcinoma (AGS) cells in vitro and in vivo. We investigated the role of H. pylori in the phosphorylation of RKIP as a possible mechanism to downregulate pro-survival signals in gastric adenocarcinoma. pRKIP induces RKIP transcriptional activity, which serves to induce apoptosis of damaged cells to prevent further tumorigenesis. Infection of wild type and RKIP knockout mice with H. pylori for 2 months further confirmed roles of RKIP and pRKIP in the prevention of gastric cancer progression. Loss of RKIP in AGS cells results in increased expression of the Cag A virulence factor after H. pylori infection and RKIP overexpression inhibits H. pylori-mediated STAT3 phosphorylation and STAT3 and NF-κB transcriptional activity. We examined the role of mTOR (mammalian target of rapamycin) after H. pylori infection on the phosphorylation of RKIP. Cells treated with rapamycin, an inhibitor of mTOR, displayed less expression of pRKIP after H. pylori infection. Microarray antibody analysis was conducted on wild-type and RKIP-knockdown AGS cells and showed that in the absence of RKIP, there was increased expression of pro-tumorigenic proteins such as EGFR, Raf-1, and MAPKs. Although further work is needed to confirm the interaction of RKIP and mTOR in AGS cells as a result of H. pylori infection, we hypothesize that H. pylori-mediated induction of pro-survival signaling in gastric epithelial cells induces a feedback response through the activation of RKIP. The phosphorylated, or active, form of RKIP is important in protecting gastric epithelial cells from tumorigenesis after H. pylori infection.

FIG. 1

(A, C) AGS gastric cancer cells were transfected with STAT3 or NF-κB luciferase reporter constructs and in some samples a RKIP expression vector. After 48 h, cells were infected with H. pylori (MOI: 200:1) for 16 h. Cells were harvested and luciferase activity measured with Dual reporter luciferase activity kit (Promega). (B) Western blot analyis of AGS gastric cancer cells transfected with an expression plasmid for RKIP for 48 h and then infected with H. pylori (MOI 200:1) for 3 h. Whole cell lysates were prepared and Western blot analysis performed to examine the levels of the indicated proteins. The upper band in the gel of RKIP transfected cells is ectopic and the lower band is endogenous RKIP.

FIG. 2

AGS cells were treated with rapamycin alone (R), H. pylori (HP), and both H. pylori and rapamycin (R+HP). Control samples are labeled “C.” Cells were pretreated with 10 nM rapamycin (R) for 1 h and then infected with 200 MOI of H. pylori for 0.5 or 3 h. Actin was used as a loading control.

FIG. 3

AGS cells were treated with rapamycin alone (R), H. pylori (H), and both H. pylori and rapamycin (HR). Cells were pretreated with 10 nM rapamycin (R) for 1 h and then infected with 200 MOI of H. pylori for 1 or 2 h. Actin was used as a loading control. Rapamycin reduces expression of pRKIP.

FIG. 4

The results in Figure 4 were adjusted for differences in actin expression. pRKIP pixel density was divided by actin pixel density to find relative pRKIP expression for each sample condition.

FIG. 5

AGS cells were treated with rapamycin alone (R), H. pylori (HP), and both H. pylori and rapamycin (R+HP). Control samples are labeled “C.” Cells were pretreated with 10 nM rapamycin (R) for 1 h and then infected with 200 MOI of H. pylori for 0.5 or 3 h. Actin was used as a loading control.

FIG. 6

Samples were treated with 10 nM rapamycin (R), or infected with H. pylori at 200 or 400 MOI (H200, H400), or pretreated with 10 nM rapamycin 1 h prior to being infected with H. pylori at 200 or 400 MOI (RH 200, RH400).

FIG. 7

Immunohistochemistry of paraffin embedded sections of four representative stomachs of wild-type and RKIP knockout mice [from a total of 17 (seven wild type and 10 knockout)] infected with H. pylori for 2 months and examined for RKIP and pRKIP. Bar is 10 um. Significantly, in RKIP knockout mice there is more inflammation after H. pylori infection and the development of pre-neoplastic gastric pathological changes (Table 2).

FIG. 8

Hematoxylin and eosin stain of infected and uninfected wild-type mice and RKIP K/O mice 2 months postinfection. This figure shows a greater degree of inflammatory cells in the knockout mice when compared to wild type after H. pylori infection.

FIG. 9

Immunohistochemistry of paraffin embedded sections of representative stomachs of wild type and mice infected with H. pylori for 8.5 and 9.5 months and examined for RKIP and pRKIP. Bar is 50 um. There is development of pre-neoplastic gastric pathological changes (Table 3).

FIG. 10

Western blot analyis of AGS wild-type and RKIP KD cgastric cancer cells infected with H. pylori (MOI 200:1) for 3 h. Whole cell lysates were prepared and Western blot analysis performed to examine the levels of the indicated proteins.

FIG. 11

Model depicting the complex signaling after H. pylori infection. The phosphorylation of STAT3 (1) and RKIP (2) occurs after infection. RKIP overexpression (3) abrogates H. pylori-mediated STAT3 phosphorylation. Rapamycin-mediated inhibition (4) of the mTOR pathway (Fig. 2) results in the simultaneous inhibition of RKIP phosphorylation by H. pylori.