USF1 defect drives p53 degradation during Helicobacter pylori infection and accelerates gastric carcinogenesis

Abstract

Objective: Helicobacter pylori (Hp) is a major risk factor for gastric cancer (GC). Hp promotes DNA damage and proteasomal degradation of p53, the guardian of genome stability. Hp reduces the expression of the transcription factor USF1 shown to stabilise p53 in response to genotoxic stress. We investigated whether Hp-mediated USF1 deregulation impacts p53-response and consequently genetic instability. We also explored in vivo the role of USF1 in gastric carcinogenesis.

Design: Human gastric epithelial cell lines were infected with Hp7.13, exposed or not to a DNA-damaging agent camptothecin (CPT), to mimic a genetic instability context. We quantified the expression of USF1, p53 and their target genes, we determined their subcellular localisation by immunofluorescence and examined USF1/p53 interaction. Usf1^-/- and INS-GAS mice were used to strengthen the findings in vivo and patient data examined for clinical relevance.

Results: In vivo we revealed the dominant role of USF1 in protecting gastric cells against Hp-induced carcinogenesis and its impact on p53 levels. In vitro, Hp delocalises USF1 into foci close to cell membranes. Hp prevents USF1/p53 nuclear built up and relocates these complexes in the cytoplasm, thereby impairing their transcriptional function. Hp also inhibits CPT-induced USF1/p53 nuclear complexes, exacerbating CPT-dependent DNA damaging effects.

Conclusion: Our data reveal that the depletion of USF1 and its de-localisation in the vicinity of cell membranes are essential events associated to the genotoxic activity of Hp infection, thus promoting gastric carcinogenesis. These findings are also of clinical relevance, supporting USF1 expression as a potential marker of GC susceptibility.

Keywords: DNA damage; gastric cancer; genetic instability; helicobacter pylori infection; oncogenes.

Figure 1

Correlation of p53 and USF1 loss with gastric carcinogenesis and Hp infection. (A) Expression heatmap depicting mRNA expression of genes distinguishing most significantly GC patients according to their overall survival. Expression data for (low survival: 665 days or high survival: 1095 days) were obtained from TCGA (STAD, n=188) (see online supplementary figure S1A). (B) Survival curve for GC patients according to USF1 and TP53 mRNA levels (low: green, medium: blue or high: red). (C) Expression heatmap depicting median mRNA expression of p53 target genes (Fisher_direct_p53_targets_meta_analysis, GSEA) and putative USF1 target genes (genes having at least one occurrence of transcription factor binding site V$USF_01 (v7.4 TRANSFAC) in the regions spanning up to 4 Kb around their transcription starting sites, gene set enrichment analysis (GSEA),) significantly enriched in both low and high survival GC patients (top 50 genes, see online supplementary figure S1E, F). (D) Expression heatmap depicting p53 and USF1-target genes expression (p53-targets: orange and pink; USF1-targets: blue and green; common: black), previously correlated with low (pink and green) or high survival (orange and blue) using data from Hippo and colleagues comparing non-cancerous and cancerous tissues. (E) Relative USF1 gene expression in gastric biopsies from GC patients (n=34) measured by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) (tumorous vs adjacent tissue); bar: median value. Mann-Whitney test (low (<1) vs high (>1) expression; ****p<0.0001). (F) Log-fold enrichment of p53-target and USF1-target genes expression in Hp-infected gastric cells. Data from GSE55699 (Koeppel and colleagues) and E-GEOD-74577 (Hong and colleagues). GC, gastric cancer; Hp, Helicobacter pylori; TCA, The Cancer Genome Atlas.

Figure 2

Loss of USF1 exacerbates gastric tumourigenesis associated to Hp infection. Usf1^-/- and Usf1^+/+ mice were oro-gastrically infected with HpSS1 for 9 and 12 months as described in online supplementary methods. (A) Representative gastric histological changes on H&E stained tissue section, in Usf1 ^-/- (d, e, f, j, k, l) and Usf1 ^+/+ (a, b, c, g, h, i) mice, Hp-infected (b, c, e, f, h, i, j, k, l) and non-infected (a, d, g, j), after 9 months (a–f) and 12 months (g–l). As early as 9 months, cysts and atypia are observed in Usf1^-/--infected mice (arrows). Dysplasia is only detected in Usf1^-/- infected mice at 9 months pi (arrows). (B) Semiquantitative evaluation of gastric lesions in Hp-infected Usf1^-/- and Usf1^+/+ mice (see online supplementary information). Mann-Whitney test, infected versus non-infected (*p<0.05). (C) p53 if (green) and nuclei (Hoechst, blue) on gastric tissue sections from Hp-infected Usf1^-/- and Usf1^+/+ mice at 12 months pi, showing a depletion of p53 in Hp-infected Usf1^-/- mice. Scale bar 100 µm.

Figure 3

Hp impairs host DNA repair function by downregulating USF1 and p53. MKN45 cells were infected with Hp7.13 (MOI 100:1) for 2 and 24 hours. Control cells were not infected. (A) USF1 (B) Western blot analysis of USF1 (C) TP53 (D) p53 and GADPH (E) paired USF1-TP53 (F) CSA, HR23A and GADD45A mRNA level quantified by quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). Results are relative to the 18SrRNA. Mean±SD, n=3. (B) WB analysis of (B) USF1 (E) p53 and GAPDH (loading control) in protein extracts from infected and non-infected cells. The histogram below corresponds to immunoblot quantification. Error bars: SD, n=3. Student’s t-test, infected versus non-infected (*p<0.05; **p<0.01; ****p<0.0001). Hp, helicobacter pylori.

Figure 4

Hp leads to USF1 foci in the vicinity of cell membranes. (A) Immunofluorescence analysis of USF1 and p53 levels and localisation in MKN45 cells infected as in figure 3. p53 immunostaining (red), USF1 (green) and nuclei (Hoechst, blue). Phalloidin actin staining (grey) indicates the cells shape. Scale bar 5 µm. (B) Quantification of USF1 and p53 nuclear if intensity (n=150–220 cells/condition). Mann-Whitney test, infected versus non-infected (*p<0.05; ****p<0.0001). (C) Maximum intensity projection of representative Hp-infected and non-infected cells at 24 hours. USF1 staining (green) shows foci (yellow arrows) in the cytoplasm and vicinity of cell membranes in infected-cells (left part). Quantification of USF1 spots number per cell according to defined spot criteria as indicated in material and methods (right panel) (n=150–220 cells/condition). Experiments in triplicate with 5–7 microscopic fields analysed. Mann-Whitney test, infected versus non-infected (*p<0.05; ****p<0.0001). Hp, Helicobacter pylori.

Figure 5

USF1 foci are specifically induced by Hp infection. (A) Immunofluorescence analysis of USF1 and p53 levels and localisation, in MKN45 cells treated or not with CPT (50 nm) and infected with Hp 7.13 for 2 and 24 hours. p53 (red), USF1 (green), nuclei (Hoechst, blue) and phalloidin actin staining (grey). The delocalisation and accumulation of USF1 are specifically observed in the cytoplasm and membrane surrounding area of Hp-infected/CPT-treated cells. Scale bar 5 µm. (B) Quantification of USF1 and p53 nuclear if intensity (n=150–220 cells/condition). (C) Quantification of USF1 spots number/cell as in figure 4. USF1 foci are only observed in the presence of Hp (n=150–220 cells/condition). Mann-Whitney test: treated or treated/infected versus control (**p<0.01; ***p<0.001; ****p<0.0001). Experiments in triplicate with 5–7 fields analysed. CPT, camptothecin; Hp, Helicobacter pylori.

Figure 6

Hp inhibits the USF1/p53 complexes in response to a chemical genotoxic stress. (A) Duolink PLA analysis of USF1/p53 complexes (pink foci), in Hp-infected cells either CPT-treated (50 nM) or not as described in methods. nuclei (Hoechst, blue). Experiments in duplicate (5–7 fields analysed). Scale bar: 10 µm for each time-point: right panels zoom: fields delimited in red, scale bar 5 µm. (B) Quantification of USF1/p53 nuclear interaction (5–7 fields analysed). Student’s t-test, CPT-treated and/or infected versus control (*p<0.05; **p<0.01; ***p<0.001). CPT, camptothecin; Hp, Helicobacter pylori; PLA, proximity ligation assay.

Figure 7

Hp infection makes more susceptible gastric cells to a genotoxic stress. (A) Experimental schedule. MKN45 cells were first infected with Hp 7.13 or not, as in figure 3. After 24 hours, cells were washed three times and either treated or not with CPT (50 nM) for 24 hours. (B) Immunofluorescence analysis of USF1 (green) and p53 (red). nuclei (Hoechst, blue) and phalloidin actin staining (grey). Experiments in duplicate (5–7 fields analysed per condition). Scale bar 5 µm. CPT, camptothecin; Hp, Helicobacter pylori.

Figure 8

Schematic representation of the data. (A) Gastric epithelial cells infected with Hp show lower nuclear level of USF1 and p53 and the formation of USF1 foci mainly at the periphery of cells close to membranes. Hp infection inhibits USF1 and CPT-induced USF1/p53 complexes in the nuclei. These data support that, in response to a genotoxic stress, the nuclear localisation of USF1 is important to maintain p53 in the nucleus to carry out its function. (B) Exacerbation of gastric carcinogenesis due to synergistic effects of Hp and environmental DNA damaging factors in chronically infected individuals. According to our data, the progressive nuclear decrease of USF1 and p53 in Hp-positive subjects should lead to further accumulation of DNA damage all lifelong. This supports that Hp increases the sensitivity to DNA damaging effects of genotoxic environmental factors, thus promoting the risk of GC. CPT, camptothecin; GC, gastric cancer; Hp, Helicobacter pylori.