CDK1 bridges NF-κB and β-catenin signaling in response to H. pylori infection in gastric tumorigenesis

Abstract

Infection with Helicobacter pylori (H. pylori) is the main risk factor for gastric cancer, a leading cause of cancer-related death worldwide. The oncogenic functions of cyclin-dependent kinase 1 (CDK1) are not fully understood in gastric tumorigenesis. Using public datasets, quantitative real-time PCR, western blot, and immunohistochemical (IHC) analyses, we detect high levels of CDK1 in human and mouse gastric tumors. H. pylori infection induces activation of nuclear factor κB (NF-κB) with a significant increase in CDK1 in in vitro and in vivo models (p < 0.01). We confirm active NF-κB binding sites on the CDK1 promoter sequence. CDK1 phosphorylates and inhibits GSK-3β activity through direct binding with subsequent accumulation and activation of β-catenin. CDK1 silencing or pharmacologic inhibition reverses these effects and impairs tumor organoids and spheroid formation. IHC analysis demonstrates a positive correlation between CDK1 and β-catenin. The results demonstrate a mechanistic link between infection, inflammation, and gastric tumorigenesis where CDK1 plays a critical role.

Keywords: CDK1; CP: Cancer; H. pylori; NF-κB; gastric cancer; β-catenin.

Figure 1.. CDK1 is overexpressed in gastric cancer

(A) Analyses of two Gene Expression Omnibus (GEO) datasets from tumor versus normal (TN) and H. pylori (HP) cohorts. Hierarchical clustering heatmap and volcano plots of significant differentially expressed genes (DEGs) are shown in DEG analysis. Overlapping TN cohorts and HP cohorts initially identified 60 DEGs. (B) The mRNA expression levels of CDK1 were examined in the GEO datasets and our local cohort with H. pylori infection. (C) Immunofluorescence staining for CDK1 (red) was performed in mice orogastrically challenged with Brucella broth or PMSS1, and representative images are shown. H&E staining of representative histological features of gastric mucosa mice. (D and E) Western blot and quantitative real-time analysis of CDK1 in GES1, AGS, and MKN28 cells following H. pylori infection. Experiments were performed in triplicates. *p < 0.05 and **p < 0.01.

Figure 2.. Activation of NF-κB-P65 upregulates CDK1 expression

(A) The transcription factor binding sites were predicted by the PROMO website using a 2,000-bp conserved segment of the CDK1 promoter. (B) A list of putative transcriptional factors with higher JASPAR scores in human and mouse CDK1 promoters. (C and D) Western blot analyses of p-P65 (S536), P65, and CDK1 in GES1, AGS, and MKN28 cells following H. pylori infection (7.13 and J166). (E) The western blot analyses of p-P65 (S536), P65, and CDK1 were performed in mice orogastrically challenged with Brucella broth or with PMSS1. (F) The immunoblot analyses and quantitative real-time PCR were performed to determine CDK1 expression in AGS and MKN28 cells transiently transfected with P65 or empty vector. (G and H) The immunoblot analyses and quantitative real-time PCR of p-P65 (S536), P65, and CDK1 were performed in AGS and MKN28 cells following TNF-α and Bay 11-7082 treatment. Experiments were performed in triplicates. *p < 0.05.

Figure 3.. NF-κB transcriptionally upregulates CDK1 expression

(A and B) A table and a scheme showing putative NF-κB transcription factor binding sites on the CDK1 promoter and the CHIP primers designed in the CDK1 promoter (P1–P5). (C) Chromatin immunoprecipitation (ChIP) assay using a specific antibody against P65 to immunoprecipitate formaldehyde-fixed chromatin in AGS cells, followed by quantitative real-time PCR with primers designed for the NF-κB-P65 binding site of the CDK1 promoter region with H. pylori infection or P65 plasmid transfection. (D) Luciferase reporter assay for CDK1-Luc in AGS cells infected with H. pylori alone or combined with Bay 11-7082 treatment. (E and F) Luciferase reporter assay for CDK1-Luc or mutCDK1-Luc in AGS and MKN28 cells treated with TNF-α alone or in combination with Bay 11-7082. (G and H) Luciferase reporter assay for CDK1-Luc or mutCDK1-Luc constructs in AGS and MKN28 cells expressing P65 or empty vector. Experiments were performed in triplicates. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4.. CDK1 regulates β-catenin protein levels and enhances β-catenin transcriptional activity

(A) Gene set enrichment analysis (GSEA) in a mouse model with H. pylori infection was performed by comparing infection cases with non-infection cases. Canonical Wnt signaling pathway was significantly enriched in H. pylori infection samples (p < 0.01). (B) Pearson’s correlation test revealed strong correlations between CDK1 expression and CTNNB1 level in the GEO: GSE84433 cohort (R = 0.39, p < 0.01). (C) The immunoblot analyses were performed to determine β-catenin and p-GSK-3β (S9) expression in GES1 and AGS cells with overexpression of CDK1 or its knockdown by CDK1-specific siRNA in MKN45 cells. (D) Representative immunofluorescent images of β-catenin (green) and CDK1 (red) in AGS cells’ overexpression of CDK1; nuclei were stained with DAPI (blue). (E and F) β-catenin luciferase reporter assays. TOP-flash contains wild-type TCF binding sites. FOP-flash, containing mutated TCF binding sites, is served as a negative control; *p < 0.05 and **p < 0.01. (E) The gastric cancer (GC) cells were transfected with indicated amounts (0.5 or 1.0 μg) of CDK1 expression vector or empty vector control (Ctrl). (F) The MKN45 cells were transfected with CDK1 siRNAs or scrambled siRNA (Ctrl). (G and H) AGS or MKN28 cells were transfected with CDK1 expression plasmid or empty vector (Ctrl), and MKN45 cells were transfected with CDK1 siRNA or scramble siRNA control (Ctrl). Quantitative real-time PCR analysis of β-catenin targets, including AXIN2 and CCND1. Experiments were performed in triplicates. *p < 0.05 and **p < 0.01.

Figure 5.. H. pylori infection increases β-catenin transcriptional activity through a CDK1-dependent manner

(A) Western blot analysis of p-β-catenin (S552), β-catenin, p-GSK-3β (S9), GSK-3β, and CDK1 in GES1, AGS, and MKN28 cells with H. pylori infection (7.13) at different time points (3, 6, and 24 h). (B) The western blot analysis of p-β-catenin (S552), β-catenin, and CDK1 were performed in mice orogastrically challenged with Brucella broth or with PMSS1. (C and D) Western blot analysis of p-β-catenin (S552), β-catenin, p-GSK-3β (S9), GSK-3β, and CDK1 with H. pylori infection and CDK1 siRNA knockdown in GES1 and AGS cells. (E and F) Luciferase reporter assay for TOP or FOP in GES1 and AGS cells with H. pylori infection and CDK1 siRNA knockdown. (G and H) Quantitative real-time PCR analysis of β-catenin targets, including AXIN2 and CCND1, were performed in GES1 or AGS cells with H. pylori infection (7.13). (I) Representative immunofluorescent images of β-catenin (green) and CDK1 (red) in AGS cells with H. pylori infection; nuclei were stained with DAPI (blue). Experiments were performed in triplicates. *p < 0.05 and **p < 0.01.

Figure 6.. CDK1 depletion represses GC cell expansion

(A) GSEA in samples with high-CDK1 expression across the TCGA and GEO datasets. (B) A weak to moderate correlation of the ssGSEA scores with β-catenin and its targets was observed. (C) Representative immunofluorescent images of β-catenin (green) and CDK1 (red) in spheroids from MKN28 cells with stable knockdown CDK1; nuclei were stained with DAPI (blue). (D) Spheroids (scale bars, 100 μm) derived from MKN28 cells with stable knockdown of CDK1 (CDK1 shRNA 01, CDK1 shRNA 02) displayed significantly smaller spheroids compared with the scrambled shRNA cells (Ctrl). (E) The quantification of sphere size and the number was expressed as the mean ± SD of 3 independent fields: **p < 0.01 and ***p < 0.001. (F) Western blot analysis of p-β-catenin (S552), β-catenin, p-GSK-3β (S9), GSK-3β, and CDK1 in spheroids derived from MKN28 cells with CDK1 shRNA knockdown. (G and H) At the age of 8 weeks, K19^creCDK1^fl/fl (control) or K19^creCDK1^fl/fl mice received tamoxifen (50 mg/kg, intraperitoneally [i.p.]) for 10 days. At 10 weeks of age, the mice were infected with H. pylori (PMSS1 strain) for 1 month. The size of gastric organoids established from these mice was measured. Experiments were performed in triplicate. **p < 0.01 and ***p < 0.001.

Figure 7.. In vivo studies of CDK1/β-catenin axis in gastric tumorigenesis

(A) At the age of 8 weeks, K19^creCDK1^fl/fl (control) or K19^creCDK1^fl/fl mice received tamoxifen (50 mg/kg, i.p.) for 10 days. At 10 weeks of age, the mice were infected with H. pylori (PMSS1 strain) for 1 month. Immunofluorescence analyses of β-catenin (green) and CDK1 (red) from these mice were performed. H&E staining of representative histological features of gastric mucosa from mice. (B) Immunofluorescence analyses of β-catenin (green) and CDK1 (red) were performed in gastric organoids established from K19^creCDK1^fl/fl (control) or K19^creCDK1^fl/fl mice receiving tamoxifen treatment and/or H. pylori infection. Nuclei were stained with DAPI (blue). H&E staining of representative histological features of gastric organoids. (C) Representative immunohistochemistry staining (scale bars, 100 μm) of CDK1 (n = 216) and β-catenin (n = 117) in human gastric tumors and adjacent non-tumor tissues. (D) Composite expression score of CDK1 (n = 216) and β-catenin (n = 117) in tumor and non-tumor tissues. (E) Spearman’s correlation between IHC staining scores of CDK1 and β-catenin in tumor slides of 117 patients. TAM, tamoxifen. (F) Illustration of the study model.