H. pylori infection induces CXCL8 expression and promotes gastric cancer progress through downregulating KLF4

Tumour‐derived CXCL8 facilitates the movement of myeloid‐derived suppressor cells, which are able to restrain antitumour immune responses to the tumour microenvironment. Kruppel‐like factor 4 (KLF4) is a potential tumour suppressor in gastric cancer (GC). However, knowledge regarding correlations between KLF4 and CXCL8 in GC is limited. We use cellular and molecular biological methods to assess whether these two factors interact in GC. Expression CXCL8 and KLF4 was altered in human GC tissues compared to normal gastric tissues in opposite ways. Additionally, cytotoxin‐associated gene A protein (CagA) gene transduction or Helicobacter pylori (H. pylori) infection upregulated CXCL8 expression. Knockdown of KLF4 expression increased CXCL8 protein and RNA expression, whereas its overexpression had the opposite effect. CXCL8‐mediated enhancement of GC cell migration and proliferation was reversed by upregulation of KLF4 expression. Further mechanistic research revealed that KLF4 binds the CXCL8 promoter, suppressing CXCL8 transcription. Moreover, CXCL8 stimulation reduced KLF4 protein expression and promoted GC cell proliferation and migration, eventually promoting neoplasm growth in vivo. Together, our findings demonstrate that CagA promotes CXCL8 and inhibits KLF4. CXCL8 is a decisive downstream target gene of KLF4, and KLF4 negatively regulates CXCL8 in GC. Furthermore, CXCL8's negative regulation of KLF4 in vivo and in vitro, indicates that CagA may downregulate KLF4 by inducing CXCL8 expression, low expression of KLF4 further promotes that of CXCL8, forming a vicious circle in GC. Targeted KLF4 activation might improve the immunosuppressive microenvironment through direct negative regulation of CXCL8, providing a new potential target to strengthen the efficacy of immunotherapy in GC patients.


| INTRODUCTION
Gastric cancer (GC) remains a crucial cancer worldwide, as it ranks as the third leading cause of malignant cancer death and the fifth most frequently diagnosed cancer. 1 GC has a poor prognosis due to its high tendency to be advanced, which is the main cause of the high cancer mortality. 2 In recent years, the rapid development of tumour immunotherapy, especially cancer immune checkpoint therapy has achieved remarkable curative effects in the treatment of malignant melanoma and nonsmall cell lung cancer, 3 bring new hope for the treatment of GC.
The prevalence of Helicobacter pylori (H.pylori), which is a gastric bacterial pathogen aetiologically associated with human gastric carcinoma, is correlated with the incidence of GC.
H. pylori-associated cytotoxin-associated gene A (CagA) protein, which is transferred to gastric epithelial cells via bacterial type IV secretion, is an oncoprotein that can cause malignant neoplasms in mammals. 1,4 Numerous studies have shown that H.
pylori also promotes the progression, metastasis and recurrence of GC, 5,6 which may be closely related to the persistence of H. pylori infection, resulting in a local immunosuppressive microenvironment, 7 that allows GC cells to escape immune surveillance. Among the numerous inflammatory factors, CXCL8 and interferon gamma (IFN-γ), which are accompany chronic H. pylori infection, play a critical role in this process.
Recently, several studies revealed that CXCL8 (also called interleukin-8, ) is a crucial inflammatory chemokine induced by H. pylori infection. It has been reported that CXCL8 was the most significantly upregulated gene in the whole genome after infecting normal gastric epithelial cells with H. pylori for 24 h. 8 CXCL8 is overexpressed in GC and is positively associated with a poor prognosis and tumour metastasis. 9 In a recent study, CXCL8 was identified as a potent chemotactic factor that promotes the recruitment of myeloid-derived suppressor cells (MDSCs) to tumours by binding CXCR1/2, which was discovered on the surface of tumour-derived MDSCs in a tumour engraftment mouse model. This chemotactic activity can be inhibited in vivo with the use of the CXCR1/2 blocking agent reparixin. 10 Several recent studies documented that MDSCs dampen the antitumour immune response mainly by suppressing T cell function via multiple molecular mechanisms. 11 Therefore, effectively inhibiting and blocking the high expression of CXCL8 in GC and disrupting the local poor immunosuppressive microenvironment are important for improving the effect of comprehensive immune treatments for GC.
Krüppel-like factor 4 (KLF4) is a transcription factor containing a zinc finger structure. Its main functions are to inhibit cell proliferation, induce cell differentiation, including the differentiation and maturation of various immune cells, and participate in the body's immune response. 12,13 In our previous study, we demonstrated that KLF4 tends to be absent in gastric carcinoma tissue and that CagA gene transfection of the gastric mucosal epithelium significantly inhibits the expression KLF4. 14,15 Experimental and clinical research has increasingly revealed that KLF4 is a tumour-suppressor gene in GC and that KLF4 inactivation is closely related to the occurrence and development of GC. 16

| Chromatin immunoprecipitation (ChIP)
According to the manufacturer's protocol, GC cells (2 × 10 6 ) were prepared for ChIP with a ChIP assay kit (Catalogue #17-371; Millipore). The resulting precipitated DNA specimens were collected, and fractions of the CXCL8 promoter region were amplified using PCR. The products were electrophoretically resolved on a 1.5% agarose gel and directly viewed using ethidium bromide staining. The following primers were designed for the PCR analysis:

| Immunohistochemical staining
To assess the relationship between the protein expression of KLF4 and CXCL8 in GC patients, a tissue microarray (TMA) of cancer tissues and corresponding adjacent tissues from 75 pairs of GC samples was used to detect KLF4 and CXCL8 expression. Positive brown granular substances were detected and the score was given based on the percentage of positive cells and the dyeing depth of KLF4 and CXCL8. Each section was evaluated by two pathologists who were double-blinded in randomized observations with 10 high-power fields of vision. The positive cell percentage points were as follows: more than 75% for 4 points, 51%-75% for 3 points, 26%-50% for 2 points, 10%-25% for 1 point, and less than 10% for 0 points. The positive staining intensity was defined as follows: colorless meter for 0 points, pale yellow meter for 1 point, tan for 2 points, and tan meter for 3 points. The total score was calculated as the product of positive cell percentage score and the positive staining intensity score, and indicated the expression level as follows: strong positive: 6-12 points; weak positive: 3-6 points; negative: and ≤3 points. The patient characteristics are summarized in Table 1. In addition, The KLF4 and Ki67 protein expression levels in MGC803-control and MGC803-CXCL8 nude mice were detected by immunohistochemistry (IHC 3 | RESULTS

| Expression levels of KLF4 and CXCL8 are inversely altered in GC tissue
To explore the correlation between KLF4 and CXCL8 and their clinical significance in GC, we detected CXCL8 and KLF4 expression in a TMA containing seventy-five paired GC tissues and corresponding adjacent nontumour tissues through IHC. KLF4 expression was reduced in the GC tissue compared with that in the adjacent nontumour tissues ( Figure 1A), while CXCL8 expression was enhanced in the GC tissue compared with that in the adjacent nontumour tissues ( Figure 1B). The cancer tissues showed a relatively high expression of CXCL8 and much lower expression of KLF4. In contrast, the adjacent nontumour tissues showed low CXCL8 expression but high KLF4 expression ( Figure 1C). Furthermore, using The Cancer Genome Atlas data (n = 440), we found a high level of CXCL8 expression in the GC samples with H. pylori infection (p = 0.003, Figure 1D). Taken together, these data strongly support our hypothesis that H. pylori infection leads to upregulation of CXCL8. In addition, CXCL8 expression correlated positively with tumour grade in stomach adenocarcinoma (p < 0.01, Figure 1E).

| H. pylori infection or CagA transfection increases CXCL8 expression
To further study the correlation between KLF4 and CXCL8 expres-

| CXCL8 is a downstream target gene of KLF4
We found that KLF4 overexpression can significantly inhibit the expression of CXCL8 in GC cells, therefore, we sought to study the mechanism. In fact, the bioinformatics analysis showed that the proximal CXCL8 promoter contains two potential KLF4-binding sites.
We constructed vectors containing wild-type and three site-specific mutated proximal CXCL8 reporter sequences with a luciferase reporter gene called WT, Site1, Site2, and Double site ( Figure 4A). The inhibitory effect on the CXCL8 promoter was enhanced when the CagA on CXCL8 promoter activity (Figures 5Ci and 5Cii). These re-   GC cell proliferation and migration. [19][20][21] In addition, KLF4 has been recognised as a potential therapeutic target for GC treatment through the attenuation of its oncogene function; for example, the KLF4 gene has been shown to inhibit STK33 expression by immediately binding the KBS site of its promoter region to control GC progression and metastasis. 22 We found contrasting expression patterns between CXCL8 and KLF4 cin human GC tissues. KLF4 expression in GC tissue was lower than that in adjacent tissue, while the opposite results were observed with CXCL8, indicating a certain link between KLF4 and CXCL8.
CXCL8, which is the most common inflammatory factor, performs various functions in angiogenesis, cell migration, proliferation, and survival. 23,24 CXCL8 is highly expressed in various carcinomas and is associated with a poor prognosis. 25 40,41 In a recent investigation, MDSCs were found to inhibit T cell proliferation and the production of IFN-γ and GrB partially through arginase I production. CXCL8 can induce increased arginase I production in MDSCs via the PI3K-AKT signalling pathway. 42 Given that MDSCs represent an important T cell immunosuppressive component in cancer-bearing hosts, the factors that attract these cells to the tumour microenvironment are considered attractive targets for cancer immunotherapy strategies, which is especially true in the case of CXCL8, which is a central