Mutual amplification of HNF4α and IL-1R1 composes an inflammatory circuit in Helicobacter pylori associated gastric carcinogenesis

Abstract

Helicobacter pylori (Hp) is an environmental inducer of gastritis and gastric cancer (GC). The immune response to Hp and the associated changes in somatic gene expression are key determinants governing the transition from gastritis to GC. We show that hepatocyte nuclear factor 4α (HNF4α) is upregulated by Hp infection via NF-κB signaling and that its protein and mRNA levels are elevated in GC. HNF4α in turn stimulates expression of interleukin-1 receptor 1(IL-1R1), which amplifies the inflammatory response evoked by its ligand IL-1β. IL-1β/IL-1R1 activates NF-κB signaling, thereby increasing HNF4α expression and forming a feedback loop that sustains activation of the NF-κB pathway and drives the inflammation towards GC. Examination of clinical samples revealed that HNF4α and IL-1R1 levels increase with increasing severity of Hp-induced gastritis and reach their highest levels in GC. Co-expression of HNF4α and IL-1R1 was a crucial indicator of malignant transformation from gastritis to GC, and was associated with a poorer prognosis in GC patients. Disruption of the HNF4α/IL-1R1/IL-1β/NF-κB circuit during Hp infection maybe an effective means of preventing the associated GC.

Keywords: HNF4α; Hp; IL-1R1; NF-κB; gastric carcinogenesis.

Figure 1. Expression ofHNF4α in clinical gastric tissues and its role in regulating gastric cell proliferation

A. IHC staining of HNF4α in atrophic gastritis and gastric cancer samples. Representative images are shown here (magnification 100x,400x; Scale bars:200μm,50μm). B. The mRNA levels of HNF4α in 30 atrophic gastritis samples and 30 gastric cancers were measured by real-time PCR. The horizontal bars indicate the mean value of each sample group. Mann-Whitney U-test was used to calculate the P value. C. HNF4α mRNA expression levels in indicated gastric cancer cell lines and immortalized gastric cells GES-1. ***p<0.001 by Student's t-test. D. and E. HNF4α mRNA and protein levels in indicated cells transfected with HNF4α siRNA or HNF4α over-expression plasmid. *p<0.05, **p<0.01, ***p<0.001 by Student's t-test. F. Foci formation after cells transfected HNF4α siRNA or HNF4α over-expression plasmid. *p<0.05, **p<0.01 by Student's t-test. Representative images are shown here from three independent biological replicates. G. Western blot shows loss of HNF4α in BGC-823 cells transfected with lenti-HNF4α shRNA; Tumors formed in nude mice induced by BGC-823 cells transfected with lenti-negative control group(NC)and lenti-HNF4α shRNA group(Hsh). H.Tumor growth curve and tumor weight of NC group and Hsh group. *p<0.05 by Student's t-test.

Figure 2. HNF4α expression level was up-regulated by Hpinfection in vitro and in vivo

A. Real-time PCR shows mRNA levels of HNF4α in GES-1, AGS, and BGC-823 cell lines when cells were treated with PBS or Hp26695 for 4 hours respectively,*p<0.05,**p<0.01 by Student's t-test. B. and C. Western blot shows protein levels of HNF4α in AGS and BGC-823 cells incubated with Hp strains(26695 or SS1) for 4, 6, and 8 hours (multiplicity of infection (MOI) 1:100 (cell:Hp)) D. AGS cells were infected by Hp26695 with MOI at 1:25, 1:50, and 1:100 for 4 hours. Western blot indicates a gradual increasing trend in HNF4α. E. AGS and BGC-823 cells were transfected with cagA over-expression plasmid for 48 hours. Western blot shows increased protein level of HNF4α with cagA transfection. F. Hematoxylin and eosin (HE) staining and IHC assay of HNF4α and Ki-67 in mucosal epithelial tissue of control group and MNU-Hp infection group. Representative images are shown here (magnification: 100x, 400x; Scale bars:200μm, 50μm).

Figure 3. HNF4α induced by Hp is p65 dependent

A. p65 binding sequence in HNF4αP1 and P2 promoter. B. Western blot shows HNF4α expression in AGS infected by Hp26695 with or without administration of BAY 11-7082(1.0 mM) C. Western blot shows HNF4α expression changes in AGSand BGC-823 cells infected by Hp26695 with or without transfection of p65 siRNA. D. RT-PCR and Western blot analysis of mRNA and protein levels of HNF4α expression in AGS and BGC-823 cells transfected with p65 over-expression plasmid. *p<0.05,**p<0.01 by Student's t-test. E. HNF4α P1 and P2 promoter activity increased with transfection of p65 over-expression plasmid in AGS cells, and only P1 promoter increased in BGC-823 cells. Mutation of p65 binding sequence reduced the induction effect of p65 on luciferase activity. F. ChIP assay confirmed the direct binding of p65 and HNF4αP1 and P2 promoter. Data are mean±SD of 3 biological replicates. **p<0.01,***p<0.001 by Student's t-test. G. Western blot indicates protein level of p65 and HNF4α in AGS and BGC-823 cells transfected with p65 plasmid and HNF4α siRNA solely or jointly. H. Attenuation of pro-foci formation effects of p65 by knockdown of HNF4α expression in AGS and BGC-823 cells,*p<0.05,**p<0.01 by Student's t-test.

Figure 4. IL-1R1 is up-regulated in gastric cancer tissues and it is the direct target of HNF4α

A. The mRNA levels of IL-1R1 in 30 atrophic gastritis samples and 30 gastric cancers were measured by real-time PCR. The horizontal bar indicates the mean value of each sample group. Mann-Whitney U-test is used to calculate the P value. B. Positive correlation between HNF4α and IL-1R1 expression in the above tissues. Each data point represents an individual gastric tissue sample, and a correlation coefficient r is shown. C. RT-PCR indicates IL-R1 mRNA levels in indicated cells transfected with HNF4α siRNA and HNF4α over-expression plasmid. D. Luciferase reporter assay confirmed the direct interaction between HNF4α and IL-1R1 promoter. Data are mean±SD of 3 biological replicates. *p<0.05,**p<0.01,***p<0.001 by Student's t-test. E. and G. IL-R1 protein level changes in indicated cells transfected with HNF4α siRNA and HNF4α over-expression plasmid using western blot and immunofluorescence. F. Western blot shows HNF4αand IL-1R1 protein expression in tissues from nude mice in NC group and Hsh group.

Figure 5. HNF4α and IL-1R1 is essential for IL-1β-induced expression and phosphorylation of p65

A. and B. Western blot shows pre-treatment with HNF4α siRNA in AGS and BGC-823 cells decreased p65 expression and phosphorylation induced by IL-1β(10ng/ml). Foci formation assay showed knock down of HNF4α attenuates the pro-foci formation effect of IL-1β in AGS and BGC-823 cells. *p<0.05,**p<0.01 by Student's t-test. C. Western blot and foci formation assay shows HNF4α over-expression markedly increased p65 phosphorylation and foci formation induced by IL-1β in GES-1 cell lines. D. Western blot sand foci formation assay shows HNF4α over-expression in GES-1 cells retrieved IL-1β transduction when IL-1R1 was blockaded by IL-1RA (500ng/ml). E. p65, HNF4α, IL-1R1, and ki67 expression in serial sections from atrophic gastritis and gastric cancer. Representative images are shown here (magnification 400x;Scale bars:50μm). F. Schematic representation of the proposed HNF4α and IL-1R1 feedback circuit in Hp associated gastric oncogenesis.

Figure 6. Expression of HNF4α and IL-1R1 in clinical gastric samples

A. Box plots indicate the IHC score of HNF4α and IL-1R1 in different stages of gastritis and gastric cancer. The line in each box represents the median. *p<0.05, **p<0.01,***p<0.001 by Mann-Whitney U-test. B. IHC staining showed HNF4α and IL-1R1 were both up-regulated in the progression of gastric cancer. Representative images are shown here(magnification 40x, 400x). C. Kaplan-Meier survival curves for survival (months) of 90 gastric cancer patients stratified by HNF4α and IL-1R1 expression, including HNF4α^HighIL-1R1^High(n=45), HNF4α^LowIL-1R1^Low(n=15), HNF4α^HighIL-1R1^Low(n=13), and HNF4α^LowIl-1R1^High(n=17). The HNF4α^HighIL-1R1^High group had poorer outcomes than the HNF4α^LowIL-1R1^Low group (p=0.0011) D. Representative cases of different types of gastric cancer in HNF4α^HighIL-1R1^High group and HNF4α^LowIL-1R1^Low group. Magnification 40x, 400x.