IMPLICATION OF HYPUSINATION IN HELICOBACTER PYLORI-INDUCED GASTRIC CARCINOGENESIS

Introduction: Dysregulated polyamine metabolism during Helicobacter pylori infection can lead to the development of gastric cancer. The polyamine spermine is back-converted to spermidine by spermine oxidase (SMOX). We have shown that H. pylori-infected Smox^–/– mice have a significant reduction in inflammation, dysplasia, and cancer compared to infected wild-type (WT) animals. Deoxyhypusine synthase (DHPS), the rate-limiting enzyme in the hypusination pathway, utilizes spermidine to generate hypusine, an amino acid that causes a post-translational modification on eukaryotic translation initiation factor 5A (EIF5A). Hypusinated EIF5A (EIF5A^Hyp) is important in translation of specific mRNAs and has been implicated in cell proliferation, and thus potentially in cancer. We recently found that infected Smox^–/– mice have decreased gastric levels of EIF5A^Hyp versus infected WT. Our aim was to elucidate the role of the hypusination pathway in gastric epithelial cells in H. pylori infection. Methods: We generated a stomach epithelial cell-specific knockout of Dhps by crossing Foxa3-cre mice with Dhps^fl/fl mice, thus obtaining C57BL/6 Dhps^+/fl; Foxa3^cre (Dhps^Δepi) mice. We validated deficiency of DHPS and EIF5A^Hyp via western blot. Mice were infected with H. pylori PMSS1 for 8 weeks. Colonization was quantified, and histology was analyzed by H&E staining. We infected a gastric adenocarcinoma cell line (AGS cells) with H. pylori PMSS1 at a multiplicity of infection of 100 for 24 h ± the DHPS inhibitor, N¹-guanyl-1,7-diaminoheptane (GC7; 25 μM). Protein lysates were subjected to isobaric tag for relative and absolute quantification proteomic analysis. AGS cells were treated with GC7 (0-50 μM) for 24 or 48 h and total and live cell counts were assessed using trypan blue. Results: There was a marked decrease in levels of EIF5A^Hyp in Dhps^Δepi mice, and a significant decrease in gastric inflammation in the infected Dhps^Δepi mice (1.32 ± 0.17, n=31) compared to infected Dhps^+/fl mice (1.88 ± 0.22, n= 20; P=0.04). Proteomic analysis revealed that of the 153 proteins induced by at least 1.3-fold with H. pylori infection, 99 were reduced with GC7. Proteins including laminin subunit gamma-2, which is overexpressed in H. pylori-infected gastric cancer cells and has been implicated in cell invasion and resistance to apoptosis, and KIF20B, a positive regulator of cell proliferation, were decreased in cells treated with GC7. When we examined cell viability in AGS cells, there was a significant, concentration-dependent reduction in total and live cell counts in cells treated with 5, 10, 25, and 50 mM GC7 after 24 and 48 h. Conclusions: Spermidine generated by SMOX leads to increased EIF5A^Hyp, which can promote translation of pro-inflammatory and pro-carcinogenic proteins. Hypusination may represent a new therapeutic target for gastric cancer prevention and treatment.