Helicobacter pylori infection‐induced H3Ser10 phosphorylation in stepwise gastric carcinogenesis and its clinical implications

Abstract Background Our previous works have demonstrated that Helicobacter pylori (Hp) infection can alter histone H3 serine 10 phosphorylation status in gastric epithelial cells. However, whether Helicobacter pylori‐induced histone H3 serine 10 phosphorylation participates in gastric carcinogenesis is unknown. We investigate the expression of histone H3 serine 10 phosphorylation in various stages of gastric disease and explore its clinical implication. Materials and Methods Stomach biopsy samples from 129 patients were collected and stained with histone H3 serine 10 phosphorylation, Ki67, and Helicobacter pylori by immunohistochemistry staining, expressed as labeling index. They were categorized into nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia, low‐grade intraepithelial neoplasia, high‐grade intraepithelial neoplasia, and intestinal‐type gastric cancer groups. Helicobacter pylori infection was determined by either 13C‐urea breath test or immunohistochemistry staining. Results In Helicobacter pylori‐negative patients, labeling index of histone H3 serine 10 phosphorylation was gradually increased in nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia groups, peaked at low‐grade intraepithelial neoplasia, and declined in high‐grade intraepithelial neoplasia and gastric cancer groups. In Helicobacter pylori‐infected patients, labeling index of histone H3 serine 10 phosphorylation followed the similar pattern as above, with increased expression over the corresponding Helicobacter pylori‐negative controls except in nonatrophic gastritis patient whose labeling index was decreased when compared with Helicobacter pylori‐negative control. Labeling index of Ki67 in Helicobacter pylori‐negative groups was higher in gastric cancer than chronic atrophic gastritis and low‐grade intraepithelial neoplasia groups, and higher in intestinal metaplasia group compared with chronic atrophic gastritis group. In Helicobacter pylori‐positive groups, Ki67 labeling index was increased stepwise from nonatrophic gastritis to gastric cancer except slightly decrease in chronic atrophic gastritis group. In addition, we noted that histone H3 serine 10 phosphorylation staining is accompanied with its location changes from gastric gland bottom expanded to whole gland as disease stage progress. Conclusions These results indicate that stepwise gastric carcinogenesis is associated with altered histone H3 serine 10 phosphorylation, Helicobacter pylori infection enhances histone H3 serine 10 phosphorylation expression in these processes; it is also accompanied with histone H3 serine 10 phosphorylation location change from gland bottom staining expand to whole gland expression. The results suggest that epigenetic dysregulation may play important roles in Helicobacter pylori‐induced gastric cancer.


| INTRODUC TI ON
Gastric cancer (GC) is the fifth most common malignancy and the third leading cause of cancer-related death worldwide. Nearly half of the disease burden occurs in Eastern Asia, particularly in China, South Korea, and Japan. 1 GC can be subdivided into intestinal type and diffuse type based on Lauren's classification. 2 The development of noncardia intestinal-type GC follows a well-defined histological sequence of progression in Helicobacter pylori (H. pylori, Hp)-infected gastric mucosa, initiating as chronic gastritis followed by atrophy, intestinal metaplasia, dysplasia, and GC. [3][4][5][6] H. pylori infection is the known single most important factor for GC, and eradication of H. pylori not only cures gastritis but also prevents the progression to long-term complications, such as atrophic gastritis, intestinal metaplasia, recurrence of ulcers and reduces the incidence of GC.
However, how H. pylori infection results in the initiation of GC remains unknown.
Histone modification plays important roles in various cellular functions and cancer; they affect gene expression by changing the state of transcription factors accessing to chromatin. [7][8][9][10][11] The post-translational modification of histone tails also affects different levels of DNA organization, including acetylation, phosphorylation, methylation, ubiquitylation, and ADP ribosylation on it amino acid residues, 8,[11][12][13] the combination of these modifications are important marks of epigenetics, commonly known as "histone code." Increasing evidences have indicated its role in tumor initiation and development. 14 Several reports have indicated that histone H3 serine 10 (H3Ser10) is involved in epithelial carcinogenesis. For example, phosphorylation of H3Ser10 (p-H3Ser10) is an essential regulatory mechanism for epidermal growth factor (EGF)-induced neoplastic cell transformation, which involves the induction of c-fos and c-jun promoter activity. 15,16 Overexpression of phosphorylated histone H3 in gastric tissue has been reported as an indicator of poor prognosis in gastric adenocarcinoma patients. 17 Increased p-H3Ser10 is critical in EB virus-induced carcinogenesis of nasopharyngeal carcinoma; 18 active protein-1, mitogen-and stress-activated kinase 1 (MSK1) kinase activity and phosphorylation also participate in this process. 18 Our previous work has demonstrated that H. pylori infection alters histone modification and host response via cagPAIdependent mechanisms; we showed that wild-type H. pylori induced time-and dose-dependent dephosphorylation of H3Ser10 and decreased acetylation of H3 lysine 23 (H3K23ac), but have no effects on seven other specific modifications. 19 However, whether H. pylori infection affects histone modifications in human stomach and its role in gastric carcinogenesis remains elusive.
In this report, we evaluate the expression pattern of p-H3Ser10 in stomach biopsy samples in chronic gastric disease and patients with GC, and assess its role in the stepwise gastric disease progress.
The results indicate a strong correlation of histone H3Ser10 phosphorylation with chronic gastric disease progression and implicate its role in H. pylori-induced gastric carcinogenesis.

| Reagents and immunohistochemistry staining
Polyclonal rabbit antihistone H3 phospho-specific at serine 10 antibody was purchased from Cell Signaling Technology (Cell Tokyo, Japan) were used to observe the section and take photographs for staining evaluation.

| Evaluation of immunostaining
Immunostaining results were scored as described previously. 20

| Statistical analysis
The data were analyzed using SPSS for Windows ( Table 3). Representative IHS images of p-H3Ser10 in different groups are presented in Figure 1. Both Ki67 and H3Ser10 phosphorylation showed mild to strong nuclear expression.

| Labeling index of p-H3Ser10 and Ki-67 among different stages of gastric disease
In H. pylori-negative patient groups, LI of p-H3Ser10 was increased as disease progress from NAG to CAG, IM, and LGIN groups, and peaked at LGIN; its level declined in HGIN and GC groups ( Figure 2A). In H. pylori-infected groups, LI of p-H3Ser10 followed the similar pattern as above; however, increased LI of p-H3Ser10 was noted in all groups when compared with H. pylorinegative groups, except in NAG group, whose expression level was lower when compared with H. pylori-negative NAG group (P < .05, Figure 2B and Table 3).
The general LI of Ki67 showed a trend of gradually increasing from NAG stage to GC stage groups, except slightly decreased in CAG groups. In H. pylori-negative groups, the LI of Ki67 was significantly higher in GC than that in CAG and LGIN groups (P < .05); and in IM group compared with CAG group (7.79 ± 1.06 vs 5.82 ± 1.50, P < .01) ( Figure 2C and Table 3). In H. pylori-infected patients, Ki67 LI followed the similar pattern as in H. pylori-negative groups and peaked at GC groups (P < .01); higher level of Ki67 LI in HGIN was noticed when compared with CAG group (P < .05) ( Figure 2D and Table 3). To explore the correlation between p-H3Ser10 and Ki-67 among all these disease groups, Spearman's correlation coefficient was used to assess their relationship; the results indicated that there was no significant correlation between p-H3Ser10 and Ki-67 LI among different disease groups (P > .05).

| Location changes in p-H3Ser10-and Ki-67stained cells among various stages of gastric disease
As location of p-H3Ser10-and Ki-67-stained cell in gastric gland may be closely linked to its function, and their distribution in stepwise gastric carcinogenesis has not been explored, we therefore investigated their location changes during disease progress. The results indicated that locations of p-H3Ser10-stained cells were gradually moved from bottom of gastric gland to the whole gland distribution as disease progress from benign stage to GC.
In H. pylori-negative groups, approximately half of the p-H3Ser10stained cells located at bottom of gastric gland in NAG, CAG, and IM groups, and they start to spread to the body, neck, and whole gland as disease progress from LGIN to GC stage of disease (67.86% to 98.08%) ( Figure 3A). In H. pylori-positive groups, similar distribution pattern of p-H3Ser10-stained cells was noticed as in H. pylori-negative groups, but staining level of p-H3Ser10-positive cells located in the bottom of gastric gland was higher in NAG group than that in H. pylori-negative NAG controls (76.5% vs 51.33%, Figure 3B).
For Ki67, the positive stained cells mainly lie in the bottom of benign gastric gland in NAG, CAG, and IM groups; as disease progress, they start to become whole gland distribution ( Figure 3C and D).
There were no differences in the percentages of Ki67-stained cells in each location between H. pylori-infected and noninfected groups ( Figure 3C and D).   In summary, the current works indicate that stepwise gastric carcinogenesis is associated with altered H3Ser10 phosphorylation, H. pylori infection modified this process and resulted in early stage p-H3Ser10 reduction but later stage increased expression; this is also accompanied with p-H3Ser10 location change from bottom of gland staining expand to the whole gland expression as disease progress. The results suggest that p-H3Ser10 may play an important role during neoplastic transformation processes; further investigation would be helpful to understand how H. pylori induce gastric cancer through epigenetic dysregulation.

ACK N OWLED G EM ENTS
The authors are grateful to the technical staffs of The Digestive Endoscopy and Department of Pathology for their valuable assistant in the study.

E TH I C A L A PPROVA L
This study was approved by Ethics Committee of People's Hospital of Zhengzhou University, Zhengzhou, China.

D I S CLOS U R E O F I NTE R E S T S
All the authors declare that they have no conflict of interests.