Role of Helicobacter pylori infection in cancer‐associated fibroblast‐induced epithelial‐mesenchymal transition in vitro

Abstract Background Major human gastrointestinal pathogen Helicobacter pylori (H. pylori) colonizes the gastric mucosa causing inflammation and severe complications including cancer, but the involvement of fibroblasts in the pathogenesis of these disorders in H. pylori‐infected stomach has been little studied. Normal stroma contains few fibroblasts, especially myofibroblasts. Their number rapidly increases in the reactive stroma surrounding inflammatory region and neoplastic tissue; however, the interaction between H. pylori and fibroblasts remains unknown. We determined the effect of coincubation of normal rat gastric fibroblasts with alive H. pylori (cagA+vacA+) and H. pylori (cagA−vacA−) strains on the differentiation of these fibroblasts into cells possessing characteristics of cancer‐associated fibroblasts (CAFs) able to induce epithelial‐mesenchymal transition (EMT) of normal rat gastric epithelial cells (RGM‐1). Materials and Methods The panel of CAFs markers mRNA was analyzed in H. pylori (cagA+vacA+)‐infected fibroblasts by RT‐PCR. After insert coculture of differentiated fibroblasts with RGM‐1 cells from 24 up to 48, 72, and 96 hours, the mRNA expression for EMT‐associated genes was analyzed by RT‐PCR. Results The mRNA expression for CAFs markers was significantly increased after 72 hours of infection with H. pylori (cagA+vacA+) but not H. pylori (cagA−vacA−) strain. Following coculture with CAFs, RGM‐1 cells showed significant decrease in E‐cadherin mRNA, and the parallel increase in the expression of Twist and Snail transcription factors mRNA was observed along with the overexpression of mRNAs for TGFβR, HGFR, FGFR, N‐cadherin, vimentin, α‐SMA, VEGF, and integrin‐β1. Conclusion Helicobacter pylori (cagA+vacA+) strain induces differentiation of normal fibroblasts into CAFs, likely to initiate the EMT process in RGM‐1 epithelial cell line.


| INTRODUC TI ON
Despite the incidence and mortality of gastric cancer (GC) have been decreasing, this disorder still remains one of the leading causes of cancer-related death rate worldwide. [1][2][3][4] Despite the fact that the adjuvant chemotherapy and surgical resection are the only curative therapies nowadays, most patients are diagnosed with an advanced stage of disease due to lack of specific early symptoms. Furthermore, some patients lose the opportunity of curative resection resulting from the aggressive nature of GC. Although chemoradiotherapy and targeted therapy have confirmed an improvement in host response rates, the cancer recurrences and metastases are frequently observed. [2][3][4][5][6] The bacteria Helicobacter pylori (H. pylori) is one of the major risk factors for GC development. Epidemiology of H. pylori indicates that this bug colonizes the human stomach of about 50% of the world's population. Although all H. pylori-infected subjects develop gastritis, approximately 80% of these individuals remain asymptomatic. 7,8 Besides GC, H. pylori can also induce the gastric and duodenal ulcers and the mucosa-associated lymphoid tissue (MALT) lymphomas affecting about 1%, 15%, and 0.1% of the population, respectively. 7,8 H. pylori colonizes mainly gastric epithelium but may also penetrate the mucus layer reaching pits of gastric glands. 9 We have previously shown that fibroblasts may constitute a direct target for H. pylori 10 next to epithelial cells. 11,12 H. pylori colonization may directly and indirectly interact with fibroblasts, connective tissue, and other extracellular matrix components. Necchi et al 13  Recently, the direct interaction between this bacterial pathogen and fibroblasts has been proposed 16 suggesting that H. pylori can interact with several components of connective tissue components including fibroblasts. The most virulent H. pylori strains have been shown to harbor the cag pathogenicity island encoding the type IV secretion system, 3,17 allowing the delivery of bacterial cytotoxins into gastric epithelial cells, inducing phenotypic alterations reminiscent of an epithelial to mesenchymal transition (EMT). 3,[17][18][19] The EMT is a biological process in which polarized epithelial cells lose the adherence and tight cell-cell junction, enhance their migratory capacity, and become resistant to apoptosis. 20 Moreover, the EMT increased the production of components of extracellular matrix (ECM) and gained the invasive properties to become mesenchymal cells known to play an essential role in cancer progression and metastasis. [21][22][23][24] EMT allows the tumor cells to acquire invasive properties and to develop metastatic growth characteristics. 21,23 These events are facilitated by the reduction in cell-cell adhesion molecule E-cadherin, the upregulation of more plastic mesenchymal proteins such as vimentin, N-cadherin, and α-SMA and deregulation of the Wnt pathway. 23,24 Many EMT-inducing transcription factors (EMT-TFs) such as Twist1, Snail1, Snail2, Zeb1, and Zeb2 can repress E-cadherin both directly or indirectly. [23][24][25][26] Interestingly, the eradication of H. pylori leads to the reduction in the expression of TGF-β1, Twist, Snail, Slug, and vimentin mRNAs, while enhancing the expression of E-cadherin. This suggests that H. pylori infection may trigger the TGF-β1-induced EMT pathway and that H. pylori eradication may inhibit the GC progression by attenuation of this pathway. 27,28 The activated myofibroblasts accompanying tumors known as cancer-associated fibroblasts (CAFs) belong to the principal constituents of the tumor stroma, playing important role in the tumor microenvironment. 29 The CAFs were shown to mediate cancer-related inflammation by expressing proinflammatory and tumor-promoting factors and promotion of the cancer cell invasion and ECM remodeling. 30,31 Moreover, under the control of a variety of stroma-modulating factors, the cancer cells themselves generate a permissive microenvironment favoring further tumor development and invasion. [32][33][34] The proinflammatory factors released by CAFs, such as IL-6, COX-2 and CXCL1, FSP1, CXCL9, CXCL10 (IP-10), and CXCL12 (SDF-1 stromal cell-derived factor 1), were implicated in the mechanism of tumor growth and neoplastic cell invasion. [35][36][37][38][39] The CAFs secrete proangiogenic factors, such as IL-8, SDF-1, vascular endothelial factor (VEGF), and fibroblast growth factor (FGF), into an environment of other stromal cells including endothelial cells to promote tumor angiogenesis. 30,35,38,39 CAFs may enhance invasion of the cancer cells through expression of TGF β, potent EMT inducer, and HGF, which has been shown to promote breast tumorigenesis. 39,40 Since fibroblasts may alter the mRNA expression of structural and cell cycle-associated genes in the presence of H. pylori, 9,41 we have attempted to determine whether H. pylori can interact with fibroblasts by changing them not only into myofibroblasts, but also into CAFs, further being capable of inducing EMT program in normal RGM-1 epithelial cell line.
The cultures of bacteria were grown on Columbia agar with 5% fresh horse blood (BioMerieux, Warsaw, Poland). The plates were incubated under microaerophilic conditions at 37°C for 3-5 days.

| Technique of rat gastric fibroblast isolation and the infection of isolated fibroblasts with H. pylori
Gastric samples were harvested from 8-week-old Sprague-Dawley rats and extensively washed with sterile PBS to remove contaminating debris. Primary fibroblast culture was established by mincing gastric biopsy into 1-to 2-mm 3 pieces with scissors and placing it in tissue culture flasks under sterile conditions. Growth medium DMEM containing 10% FBS and antibiotics were added and gently mixed with minced tissue. The flasks were maintained in a humidified atmosphere of 5% CO 2 at 37°C, and the medium was changed every 2 days. When the cells grew up to 70% of confluence, they were passaged using standard trypsinization techniques to establish a secondary cell culture as reported before. 9,43,44 Before the coincubation with fibroblasts, H. pylori strains were first suspended in sterile PBS and immediately transferred to the dishes containing fibroblasts. The 70% confluent fibroblasts were infected with 1 × 10 9 of live H. pylori per dish and incubated in humidified atmosphere for 72 hours.

| Determination of mRNA expression by RT-PCR
After incubation period, total cellular RNA was isolated according to  with fibroblasts, the cells were harvested and total cellular RNA was isolated as mentioned above. Expression of 18s, α-SMA, collagen I, collagen III, tenascin C (TNC), FAP, FSP, IL-6, IL-8, SDF-1, HGF, TGFβ, and COX-2 transcripts in the rat gastric fibroblasts were determined by RT-PCR using specific primers (Sigma-Aldrich; Table A1, Appendix I).

| Coculture of epithelial cells (RGM-1) with fibroblasts after 72 hours of their incubation with H. pylori (cagA+vacA+)
After 72 hours of fibroblast coculture with H. pylori (cagA+vacA+), the H. pylori was washed out from fibroblasts and the medium was changed into DMEM with 10% FBS and antibiotics. The culture dish was maintained in a humidified atmosphere of 5% CO 2 at 37°C for 4 hours, and then, the incubatory fluid was again replaced with fresh portion f the medium. On the layer of fibroblasts and medium, 0.4μm pore size cell culture inserts (Becton Dickinson) were placed ( Figure 1A,B).
Trypsinized RGM-1 cells were then seeded on the inserts surface, the medium was filled up to the volume of 5 mL, and the cells were coincubated in humidified atmosphere for 24, 48, 72, and 96 hours ( Figure 1B). After incubation period, the total cellular RNA from RGM-1 cells was isolated as described above. 45
F I G U R E 1 The insert coculture of fibroblasts with epithelial RGM-1 cells. After 72 h of fibroblast coculture with Helicobacter pylori (cagA+vacA+) (A), the H. pylori was washed out and medium changed into RPMI with 10% FBS and antibiotics. The 0.4μm pore size cell culture inserts (Becton Dickinson) were placed on the layer of fibroblasts and medium. Trypsinized RGM-1 cells were then seeded on the inserts surface, the medium was filled up to 5 mL and the epithelial cells were subsequently coincubated with fibroblasts in humidified atmosphere for 24, 48, 72, and 96 h (B)

| Contrast-phase microscopy
For microscopic examination, the trypsinized fibroblasts were seeded on cover glasses and cocultured with and without presence of H. pylori (cagA+vacA+) for 72 hours in antibiotic-free DMEM. Then, the medium with H. pylori was removed and the fresh DMEM with 10% FBS and antibiotics was added. After

| Statistical analyses
Statistical analysis of the data was performed with the use of Excel Software. Results are expressed as means ± SEM from six samples per each group. Statistical significance of difference was determined using analysis of variance (one-way ANOVA) test (Statistica Software, StatSoft, Cracow, Poland). Further statistical analysis for post hoc comparisons was carried out with Newman-Keuls test. Differences were considered statistically at P < 0.05. Therefore, for further H. pylori influence examination, only H. pylori (cagA+vacA+) was selected.

| Proinflammatory factors and angiogenic potential markers expressed by rat gastric fibroblasts
One of the CAFs potential characteristics is their ability to secrete proinflammatory cytokines, known to facilitate the recruitment of granulocytes and lymphocytes and to promote tumor growth and metastasis. Therefore, we determined the expression of IL-6, IL-8, COX-2, and SDF-1 mRNA in fibroblasts infected with H. pylori (cagA+vacA+). After 72 hours of coincubation with bacteria, an induction of IL-6 and SDF-1 mRNA expression as compared with control fibroblasts was observed (Figure 2A). We have also noticed a highly significant increase in mRNA expression of COX-2 and IL-8 in cells infected with H. pylori (cagA+vacA+) strain (Figure 2A).

| EMT inducers, ECM components, and proinvasive signals
The mRNA expression for TGFβ and scattering factor HGF was significantly increased at 72 hours of coincubation with H. pylori

| Expression of mesenchymal markers
The expression of α-SMA was significantly increased at 24 hours, with further progression in time up to 96 hours in fibroblasts cocultured with H. pylori ( Figure 4A,B). The N-cadherin mRNA expression showed a significant increase already after 24 hours of coculture, the effect which persisted up to 96 hours of incubation ( Figure 4A,B).

| Expression of promigrative and proangiogenic factors
The statistically significant transcriptional suppression of the ad-  (Figure 4A,B). In addition, the mRNA expression for COX-2, the factor considered as an accelerator of invasion, metastasis, and angiogenesis, was significantly increased at 24 hours following coculture with H. pylori-infected fibroblasts compared with control ( Figure 4A,B). The increase in COX-2 mRNA expression was sustained up to 96 hours of incubation ( Figure 4A,B).
The angiogenic potential was evaluated by determination of VEGF mRNA expression. Consistently, the VEGF mRNA showed a significant increase at 24 hours with further increase with time as compared with control coculture without H. pylori infection ( Figure 4A,B).

| Expression of EMT triggering signals
The expression of the mRNAs for growth factors receptors for TGFβ, HGF, and FGF known as the EMT triggering signals was determined.
The TGFβR and FGFR mRNA expressions were strongly upregulated from 24 hours up to 96 hours of incubation with H. pylori-infected fibroblasts compared with control coculture (Figure 5A,B). The significant increase in mRNA expression for HGFR was also observed after 24 hours with further increase within time up to 96 hours of incubation compared with control coculture (Figure 5A,B).

| Expression of transcription factors specific for induction of EMT process, proliferation, and apoptosis
The expression of mRNA for transcription factors Twist and Snail  Figure 5A,B).  Figure 6A,B), with the most prominent effect being observed at 96 hours of incubation ( Figure 6F).  induced in gastric cancer. 35,38,64 The secretion of IL-6, FAP, and IL-8

| D ISCUSS I ON
by CAFs plays pivotal role in macrophage differentiation or M2 polarization resulting in an immunosuppressive microenvironment. 36 We have previously reported that mRNA for HIF-1α, the another potent tumor activator, 65 is upregulated in fibroblasts infected by H. pylori (cagA+vacA+) strain. 10  In consequence, the alteration such as reduction in cell-to-cell adhesion molecule E-cadherin, the upregulation of more "plastic" mesenchymal proteins such as vimentin, N-cadherin, and α-SMA were facilitated. The E-cadherin to N-cadherin "switch" exerts critical role in cancer progression being essential for enhanced cell motility and migration. 26 Our results are in keeping with these observations as Twist has been shown to act as another potent E-cadherin repressor and N-cadherin activator. 23 changes. Our next goal will be to confirm the observed changes on EMT induced by H. pylori at the level of protein expression.

ACK N OWLED G EM ENTS
This article was supported by Jagiellonian University Medical College grant: K/ZDS/005736.

D I S CLOS U R E S O F I NTE R E S T S
There is no actual or potential conflict of interest including financial, personal, or other relationship with other people or organizations associated with article.