Helicobacter pylori CagA promotes epithelial mesenchymal transition in gastric carcinogenesis via triggering oncogenic YAP pathway
Helicobacter pylori (H. pylori) delivers oncoprotein CagA into gastric epithelial cells via the T4SS and drives activation of multiple oncogenic signalling pathways. YAP, a core effector of the Hippo tumour suppressor pathway, is frequently overexpressed in human cancers, suggesting its potential tumor-promoting role. Although CagA is a casual factor in H. pylori induced gastric carcinogenesis, the link between CagA and YAP pathway has not been identified. In this work, we investigated the regulation of oncogenic YAP pathway by H. pylori CagA.
Expression of YAP and E-cadherin protein in human gastric biopsies were assessed by immunohistochemistry. H. pylori PMSS1 cagA− isogenic mutant strains were generated. Gastric epithelial cells were co-cultured with H. pylori wild-type cagA+ strains or isogenic mutants and were also treated by recombinant CagA expression. Immunofluorescence was performed for YAP localization. Immunoblot and quantitative PCR were performed for examining levels of YAP, downstream effectors and markers of epithelial-mesenchymal transition. Verteporfin and siRNA silencing were used to inhibit YAP activity.
YAP is significantly upregulated in human gastric carcinogenesis. We generated PMSS1 CagA isogenic mutant strains with chloramphenicol resistance successfully. Our analysis indicated that H. pylori infection induced YAP and downstream effectors in gastric epithelial cells. Importantly, knockout of CagA in 7.13 and PMSS1 strains reduced the expression of YAP by H. pylori infection. Moreover, Inhibition of YAP suppressed H. pylori infection-induced Epithelial-mesenchymal transition (EMT).
Our results indicated that H. pylori CagA as a pathogenic protein promotes oncogenic YAP pathway, which contributes to EMT and gastric tumorigenesis. This study provided a novel mechanistic insight into why cagA+ H. pylori infection is associated with a higher risk for the development of gastric cancer.
KeywordsH. pylori CagA YAP Epithelial-mesenchymal transition Gastric carcinogenesis
Cytotoxin-associated gene A
Chronic non-atrophic gastritis
Connective tissue growth factor
Cysteine-rich angiogenic inducer 61
- H. pylori
Administration of 1-methyl-3-nitro-1-nitrosoguanidine
Multiplicity of infection
The gram-negative microaerophilic bacterium, Helicobacter pylori (H. pylori), infects approximately half of the world’s population and is mostly acquired in childhood [1, 2]. It has been estimated that 80–90% of H. pylori infection is asymptomatic; 10–15% and 1–3% of H. pylori-infected individuals develop gastric ulcer and gastric adenocarcinoma respectively . H. pylori infection can lead to gastric carcinogenesis through the histopathological Correa cascade of steps which include atrophic chronic gastritis, intestinal metaplasia and dysplasia culminating in gastric cancer . The clinical outcome of H. pylori infection is determined by multiple factors including pathogenicity of individual H. pylori strains, host susceptibility and environmental stimuli [5, 6]. Epidemiological data indicate that infection with cagA+ (cytotoxin-associated gene A) H. pylori strains is associated with more severe gastric inflammation and a higher risk for the development of pre-neoplastic lesions including intestinal metaplasia and dysplasia in comparison with cagA- H. pylori strains . It has been also documented that CagA plays an important role in H. pylori-induced gastric tumorigenesis in rodent models [8, 9]. CagA is encoded on the cag pathogenicity island and can be delivered into gastric epithelial cells through Type IV secretion system . Upon delivery into the target cells, CagA promotes various pro-oncogenic signalling pathways . Notably, CagA in the host cells is tyrosine-phosphorylated and interacts with SHP-2, leading to cellular morphological changes associated with increased cell motility and scattering, termed the “hummingbird phenotype” [12, 13].
The mammalian Hippo tumor suppressor signalling pathway is crucial in maintaining developmental organ size and tissue homeostasis . The central components of the Hippo pathway comprise MOB1 (Mps One Binder kinase activator), Sav1 (also known as WW45), MST1/2 (STE20-like protein kinase 1), LATS1/2 (large tumor suppressor 1) and two major downstream effectors YAP (Yes-Associated Protein), and transcriptional co-activator TAZ (PDZ-binding motif) . Canonically, when this signalling is on, MST1/2 phosphorylates LATS1/2 at Thr1079/Thr1041 sites, stimulated by SAV1 and MOB1. Then phosphorylated LATS1 directly phosphorylates YAP at Ser127, resulting in cytoplasmic sequestration via binding to 14–3-3 proteins. By contrast, when Hippo signalling is off, YAP is activated and translocated from cytoplasm into nucleus through interaction with transcriptional factors TEADs [16, 17]. This process leads to expression of downstream oncoproteins, such as connective tissue growth factor (CTGF), cysteine-rich angiogenic inducer 61 (CYR61) and MYC. Increased expression of YAP is positively associated with progression of different human cancers . Inducible overexpression of YAP in mouse liver can lead to expansion of liver size and eventual hepatocellular carcinoma . Activation of YAP is functionally important for proliferative and pro-survival activity in colon cancer cell lines . Therefore, YAP is considered an oncogenic protein.
Epithelial mesenchymal transition (EMT), a hallmark of tumorigenic transformation, is a cellular program by which epithelial cells lose cell-cell adhesion and acquire mesenchymal traits [. During this process, epithelial cells undergo marked biochemical changes, including enhanced cell elongation, loss of polarity, and migratory capacity. Expression of some common biomarkers involving cellular proliferation, including suppression of epithelial markers (e.g. E-cadherin, ZO-1) and upregulation of mesenchymal markers (e.g. N-cadherin, Vimentin, Snail and Slug), can promote EMT . It has been experimentally documented that overexpression of YAP results in reduction of the epithelial marker E-cadherin and phenotypic alteration that is associated with EMT, promoting cancer cell invasion and metastasis [23, 24].
Additionally, it has been reported that intracellular CagA can disrupt cell-cell junctions and cause loss of epithelial adhesion, thereby directly activating EMT . Moreover, a recent study showed that YAP mRNA levels were significantly elevated by H. pylori SS1 infection in C57BL/6 mice in combination with administration of 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) . However, the role of CagA in H. pylori-induced activation of YAP signalling pathway is poorly understood. In this study, by using CagA+ H. pylori strains PMSS1 and 7.13 as well as their ΔcagA mutants, we characterized effects of CagA on activation of the YAP signalling pathway in AGS cells. Also, we examined how H. pylori infection in humans influenced expression of YAP and an epithelial marker E-cadherin during progression of a cascade of gastric cancer from chronic non-atrophic gastritis (CNAG), intestinal metaplasia (IM), dysplasia (Dys) to cancer (GC).
Materials and methods
Antibodies, siRNA and plasmids
Antibodies and their sources were as follows: YAP inhibitor verteporfin from Sigma-Aldrich (St. Louis, MO, USA) for western blot assay, Anti-GAPDH (#2118), Anti-YAP (#4912), Anti-Phospho-YAP Ser127 (#4911), and Anti-Slug (#9585), from Cell Signaling Technology (Beverly, MA, USA); Anti-TAZ (HPA007415) from Sigma (St. Louis, MO, USA) (Anti-E-cadherin (#610405), Anti-N-cadherin (#610921) from BD Biosciences (San Jose, CA USA); Anti-CagA (sc-28,368) and Anti-phospho-tyrosine (sc-7020) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-H. pylori urease B (ab127916) from Abcam (Cambridge, MA, USA). For immunohistochemistry assay, Anti-YAP (#4912) from Cell Signaling Technology, Anti-TAZ (HPA007415) from Sigma, Anti-E-cadherin (#610405) from BD Biosciences For immunofluorescence assay, Anti-YAP (#4912) from Cell Signaling Technology, 4′,6-diamidino-2-phenylindole (DAPI), fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit antibodies from Invitrogen (Thermo Fisher Scientific, Suwanee, GA, USA). The recombinant plasmid of YAP CDNA, CagA were constructed and purchased from GeneChem, Shanghai, China. YAP siRNA was purchase from Santa Cruz Biotechnology.
H. pylori strains and generation of PMSS1 ΔcagA isogenic mutants
CagA+ H. pylori strain PMSS1 (pre-mouse Sydney strain 1) and its mouse-adapted SS1 which is deficient in CagA function due to a mutation in cagY were used in this study. H. pylori strain 7.13 and an isogenic cagA mutant were also included in this study, which were kindly provided by Dr. Richard Peek at Vanderbilt University Medical Center, Nashville, TN, USA. All H. pylori strains were cultured on trypticase soy agar with 5% sheep blood agar plates (Thermo Fisher Scientific) for in vitro passage.
For constructing PMSS1 ΔcagA isogenic mutants, an overlapping PCR amplicon (namely cagAdel) consisting of the upstream and downstream regions of the PMSS1 cagA gene(s) were produced using a pair of primers cagAupF and cagAL-5’RXS or cagAdnR and cagAF-3’FXS respectively as described previously (Additional file 3: Fig. S3A). Subsequently, a 0.7-kb cat (chloramphenicol acetyltransferease) cassette was ligated into a SmaI site in the overlapping site of cagAdel; two recombinant plasmids 54 and 55 which had the opposite orientations in reference to that of cagA were selected (Additional file 3: Fig. S3B and 4C). Then these recombinant plasmids were introduced into PMSS1 strain by electroporation or natural transformation as described previously . Transformants were screened on sheep blood agar containing 25 μg/ml of chloramphenicol; four CmR transformants, namely 54E, 54 N, 55E and 55 N, were obtained. Complete deletion of cagA from the genomes of these transformants were verified using PCR with four primer sets F1/R1 and F2/R2 for targeting cagA, and Fup/54 and Fup/55 for detecting the locus containing the sequence upstream of cagA and the cat cassette as well as direct sequencing. The sequences of all primers were listed in Additional file 6: Table S3.
Cell culture and cell transfection
Human gastric epithelial cells AGS and MKN-45 (CRL-1739; ATCC, Manassas, VA, USA) were cultured in RPMI 1640 (Gibco, CA, USA) containing 10% fetal bovine serum (Sigma Aldrich, MO, USA) and 1% penicillin/streptomycin (Gibco) at 37 °C in 5% CO2 atmosphere. AGS cells were transiently transfected with the YAP CDNA plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
When the cells reached 70% confluence, they were serum-starved overnight prior to H. pylori infection. All H. pylori strains were grown in Brucella broth supplemented with 5% fetal bovine serum at 37 °C for 24 h under microaerobic conditions; OD600nm of bacterial suspensions was then adjusted with 1% FBS DMEM media to concentrations corresponding to a multiplicity of infection (MOI; the number of bacteria per cell at the onset of infection) of 50, 100 and 200. After co-culture with H. pylori strains for 6 or 24 h, AGS cells were collected for qPCR, Western blotting, or immunofluorescent staining.
Cells were treated with H. pylori strains or in combination with verteporfin. Cells were washed three times in cold PBS before adding cell lysis buffer (Cell signaling, Beverly, MA, USA) with protease inhibitor cocktail (Roche, Amherst, CA, USA). Protein concentration was determined using a BCA assays. The cell lysates with an equal amount of protein (25 μg) was separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in Odyssey blocking buffer (Li-COR, Lincoln, NE, USA) at room temperature for 1 h and then incubated with primary antibodies in 5% BSA-TBST at 4 °C overnight. The membranes were then incubated with IRDye-conjugated anti-mouse and anti-rabbit secondary antibodies (1:10000, Li-COR) in Odyssey blocking buffer containing 0.2% Tween-20 at room temperature for 1 h. The protein bands on the nitrocellulose blots were imaged and quantified by the Odyssey imaging system through the analyze module (Li-COR). Band intensity of proteins of interest was normalized to GAPDH.
Real-time quantitative PCR analysis
For measuring mRNA levels of target genes, total RNA was extracted using Trizol Reagents (Invitrogen) and converted to cDNA using the High Capacity cDNA Archive kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Levels of YAP, CTGF and CYR61 mRNA were measured by qPCR using commercial probe mixtures ((Thermo Fisher Scientific) in the 7500 Fast Real-Time PCR system (Life Technologies). Taqman Fast Universal PCR Master Mix (Thermo Fisher Scientific) was used in this assay. Transcript levels were normalized to the endogenous control glyceraldehyde-3-phosphate dehydrogenase mRNA (GAPDH) and expressed as fold change compared with sham-dosed control mice using the Comparative CT method (Applied Biosystems User Bulletin no. 2).
Cell elongation assay
AGS cells were seeded onto 12-well cell culture plates at a density of 1× 105 cells per well. After incubating for 3~ 4 h, H. pylori cells were added at AGS cells at MOI of 200. At 24 h post infection, cells were stained by a three-step staining set (Thermo Fisher Scientific). Images were captured under a contrast microscope (Zeiss Axioskop 2). The length and breadth for at least 30 elongated cells each group were measured using ImageJ software, and then the length-to-breadth ratios were statistically analyzed.
Cells were washed three times with iced-cold PBS and incubated for 15 min at room temperature with 4% formaldehyde in PBS. The cells were permeabilized with 0.25% Triton X-100 for 15 min and then blocked for 1 h in PBS with 3% bovine serum albumin (BSA). The cells were incubated with primary antibody of YAP or E-cadherin overnight at 4 °C, and then incubation with anti-rabbit-FITC or Alex-Fluor-568-conjugated anti-mouse secondary antibody. Cell nuclei were counter-stained with DAPI. All slides were examined, and images were captured using a fluorescent microscope (Zeiss Axioskop 2).
Cell migration and invasion assay
For Boyden chamber assay, AGS cells were suspended in 200 μl serum-free DMEM and seeded into a 24-well Boyden chamber (8 μm pore size, Corning, NY, USA) with Matrigel-pre-coated inserts (BD, Franklin Lakes, NJ, USA). The chambers were incubated in DMEM medium with 10% FBS. After a 24 h incubation, cells attached to the chambers’ lower surface were fixed with 4% paraformaldehyde, and then stained with 0.1% crystal violet, and counted under a microscope (Nikon Ti-S). Wound healing assay and transwell assay for cell migration and invasion were performed as previously described .
Gastric specimens and immunohistochemistry
A total of 199 paraffin-embedded human gastric adenocarcinoma and corresponding non-cancerous specimens were obtained from surgical samples without adjuvant therapy. The clinical characteristics of all patients are listed in Additional file 5: Table S2. Additional 230 paraffin-embedded specimens were collected from endoscopic patients diagnosed with chronic non-atrophic gastritis (80 cases), intestinal metaplasia (50 cases), dysplasia (60 cases) and gastric cancers (40 cases). All specimens were provided by The First Affiliated Hospital of Nanchang University. The study protocol and exemption of informed consent were approved by the Ethics Committee of The First Affiliated Hospital of Nanchang University. Status of H. pylori infection for these clinical specimens was determined with a rapid urease test and Giemsa staining. Immunohistochemical staining was performed to examine expression profiles of YAP, E-cadherin and YAP/TAZ on these samples as described previously , which were evaluated and scored for intensity (scaled 0–3) and frequency (scaled 0–4) by two pathologists blinded to sample identity. For statistical analysis, expression levels of YAP and E-cadherin proteins were illustrated by an expression score in range of 0 to 12 using the formula intensity×frequency .
All the statistical analysis was performed using SPSS 20.0 software. Data were presented as mean ± standard deviation (SD) of three independent experiments. Statistical significance of the in vitro studies for continuous variables were determined by one-way Analysis of variance (ANOVA) and Student’s t-test. All comparison protein expression and clinicopathological parameters were performed with Kruskal-Wallis (> 2 groups) or Mann-Whitney tests (2 groups). Pearson correlation analysis was performed for the correlation between YAP and E-cadherin expression. P value ≤0.5 was considered significant (***, P < 0.001, **, P < 0.01, *, P < 0.05).
Expression of YAP was upregulated in human gastric cancer tissues and was correlated with tumour sizes and metastatic status
Activation of YAP and suppression of E-cadherin expression was positively correlated with progression of chronic non-atrophic gastritis to gastric cancer and H. pylori infection
Epidemiological data indicate that H. pylori is a major risk factor for the development of gastric cancer . To characterize how H. pylori infection influenced expression of gastric YAP and E-cadherin, clinical samples were grouped into H. pylori + and H. pylori– subjects. We found that H. pylori+ gastric tissues contained significantly higher levels of YAP (Fig. 2f, P < 0.01) and lower levels of E-cadherin (Fig. 2g, P < 0.01) compared to H. pylori− tissues for CNAG but not for IM, Dys and GC (Fig. 2f, g). These results suggest that H. pylori infection elevated YAP expression in concert with reduction of E-cadherin in the early stage of the gastric tumorigenesis cascade, which could promote EMT and the possibility of eventual gastric cancer via induction of the YAP signalling pathway.
CagA+ H. pylori infection elevated YAP expression in gastric epithelial cells
Construction and characterization of PMSS1 isogenic ΔcagA mutants
The loss of CagA in the PMSS1 ΔcagA mutants was further characterized by examining CagA expression and the ability of these mutants to translocate CagA into AGS cells and cause a cytopathic “hummingbird” phenotype. CagA was present in cell lysates from PMSS1 and 7.13, but was absent from all 4 PMSS1 ΔcagA mutants and the 7.13 cagA- mutant. In addition, CagA translocation and phosphorylation was detected in the AGS cells co-cultured with parental strains PMSS1 and 7.13 but not with their respective mutants (Fig. 4d and Additional file 3: Fig. S3D). After delivery into gastric epithelial cells, CagA induced the “hummingbird” phenotype . Using this assay, AGS cells infected with CagA+H. pylori strains PMSS1 and 7.13 exhibited significant cell elongation compared to uninfected controls, whereas the morphology of cells infected with CagA− H. pylori mutants, 7.13 cagA− or 54 N, was not significantly different from those of the uninfected controls (Fig. 4e). In addition, there was no “hummingbird” phenotype on AGS cell co-cultured with CagA- H. pylori SS1. These results collectively demonstrated that PMSS1 ΔcagA mutants lost functional CagA.
H. pylori CagA promoted the YAP pathway signalling
CagA+ H. pylori infection enhanced EMT through the activation of the YAP pathway
To further verify the role of YAP in promoting EMT, we treated AGS cells with verteporfin, a YAP inhibitor through disrupting YAP interaction with TEADs and promoting trypsin cleavage of YAP . YAP expression was decreased by treatment with verteporfin in a concentration-dependent manner (Additional file 3: Fig. S3E). Treatment of verteporfin attenuated partially augmented E-cadherin expression (Fig. 6c) and H. pylori-induced mRNA levels of CTGF and CYR61 (Fig. 6d) and in CagA+ H. pylori-infected AGS cells. In contrast, the levels of Slug (a repressor of E-cadherin expression) and N-cadherin (a mesenchymal marker of EMT) were decreased in the cells treated with both H. pylori and verteporfin compared with the cells treated with H. pylori alone (Fig. 6e). Furthermore, knockdown of YAP by YAP siRNA in vitro upregulated the epithelial marker E-cadherin which was inhibited by H. pylori CagA, and downregulated the mesenchymal marker N-cadherin which was induced by H. pylori CagA (Fig. 6f). These findings indicate that activation of YAP played an important role in H. pylori infection-induced EMT.
A previous study showed that there were elevated mRNA levels of YAP and its downstream targets CTGF, CYR61 and CDX2 in the gastric tissues of C57BL/6 mice treated with a carcinogenic agent MNNG in combination with CagA− H. pylori SS1 infection when compared with uninfected controls . This finding appears to be in disagreement with our results that H. pylori SS1 and CagA− mutants of H. pylori strains PMSS1 and 7.13 did not enhance expression of YAP and its downstream genes CTGF and CYR61 in AGS cells. At least two factors could contribute to this discrepancy. First, experimental systems used in these two studies are fundamentally different. The AGS cell line used in our study were originally derived from a gastric cancer tissue . The direct interaction between AGS cells and H. pylori allowed analysis under a more defined condition. In contrast, the responses of C57BL/6 mice to H. pylori infection are modulated by a more complicated environment full of cytokines and immune cells involved in pro-inflammatory and anti-inflammatory pathways. Second, in the study of Jiao et al. , it is unclear how treatment with MNNG alone or H. pylori alone influenced Yap expression in C57BL/6 mice, because these groups were not described in their study.
EMT is frequently activated in cancer invasion and metastasis, and it also contributes to the initiation of gastric adenocarcinoma. During this process, activated EMT reduces gastric epithelial features and confers mesenchymal characteristics . Our results showed that infection with CagA+ H. pylori strain PMSS1 or 7.13 led to downregulation of the epithelial marker E-cadherin expression in concert with increased expression of a mesenchymal marker N-cadherin, further promoting invasion and migration of gastric epithelial cancer cell lines AGS. Importantly, CagA deficiency in H. pylori strains PMSS1 and 7.13 or treatment of verteporfin suppressed YAP expression and partially restored E-cadherin expression and inhibited cell migration. The partial restoration of E-cadherin in the CagA− H. pylori-infected AGS cells suggested that additional H. pylori factors could also contribute to EMT. Our results are also consistent with a previous study reporting that induction of EMT on AGS cells by H. pylori strain 60190 was a CagA-dependent process . Mechanistically, it has been reported that the EPIYA motif of CagA can bind to GSK-3, resulting in depletion of GSK-3 and abnormal expression of various cancer-associated genes including AMPK, β-catenin. Intriguingly, recent study has indicated that YAP enhances the transcriptional activity of β-catenin via GSK-3 activity in glioma progression . Also, the cellular energy sensor AMPK exerts an inhibitory role on the YAP activity via promotion of its phosphorylation . Therefore, these studies suggest that enhanced YAP pathway due to H. pylori infection is probably associated with GSK3/β-catenin or AMPK pathway regulated by CagA. Of note, it has been reported that the Hippo signaling pathway suppresses tumorigenesis by phosphorylation of YAP on Ser127, which can restrict YAP to the cytoplasm where phosphorylated YAP undergoes sequestration via binding to 14–3-3 protein. However, in our study CagA+H. pylori infection elevated the levels of total YAP, but did not alter the ratio of phospho (Ser 127)-YAP to the total YAP. It is likely that the increase of YAP nuclear translocation and upregulation of YAP target genes in the CagA+ H. pylori-infected AGS cells result from the elevation of unphosphorylated YAP.
It has been proposed that gastric cancer is gradually developed over many years through a multistep histopathological cascade . In this study, we showed that gastric YAP expression also increased, which was correlated with the decrease of E-cadherin, from the non-atrophic gastritis, dysplasia to gastric cancer, further suggesting that the YAP signaling pathway plays a pivotal role in promoting gastric tumorigenesis. More importantly, higher YAP levels and lower E-cadherin levels were noted in the chronic gastritis tissues from H. pylori-positive patients compared to those from H. pylori-negative patients, suggesting that activation of the YAP signalling pathway at the stage of CNAG is one of the major molecular mechanisms H. pylori utilizes to promote the cascade of gastric carcinogenesis. This hypothesis is supported by the results that the promotion effect of CagA+ H. pylori on YAP expression was diminished at the stages of IM/Dys/GC and also is consistent with our data that enhancement of YAP expression in the CagA+H. pylori-infected AGS cells occurred at 6 HPI and diminished at 24 HPI. Future investigations into the correlation between H. pylori CagA+ status and YAP expression in clinical samples will further strengthen the role of CagA in increasing a GC risk via activation of the YAP signaling in the early stage of the development of gastric cancer.
In summary, we have demonstrated that H. pylori CagA promotes gastric tumorigenesis via activation of the YAP signalling pathway in AGS cells during the initiation of H. pylori infection. These in vitro results are further linked to the clinical observation that H. pylori infection elevated activation of the YAP expression in concert with the suppression of E-cadherin in the chronic gastritis tissues of H. pylori + patients compared with those of H. pylori- patients. These data have highlighted an important role of YAP in CagA virulence potential in particular and gastric tumorigenesis in general. Thus, the findings from this study may have provided a mechanistic insight into why cagA+ H. pylori infection is associated with a higher risk for the development of gastric cancer. In addition, further investigations into the correlation among H. pylori cagA status, YAP expression and chronic gastritis could potentially develop novel strategies for eradicating H. pylori and prevent the development of gastric cancer.
Our data reveal that H. pylori infection activates the YAP signalling pathway to promote EMT in gastric carcinogenesis via CagA, a defined H. pylori virulence factor. These findings not only highlight a new mechanistic insight into the role of CagA in promoting H. pylori-induced gastric carcinogenesis, but also provide a novel molecular target for developing effective strategies to eradicate H. pylori and prevent the development of gastric cancer.
The funding body had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
This work was supported by China Scholarship Council, The Graduate Innovation Fund of Jiangxi Province (YC2016-B023), National Natural Science Foundation of China (81270479, 81470832, and 81670507), Grants from the National Science and Technology Major Projects for “Major New Drugs Innovation and Development” of China (2011ZX09302–007-03).
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and additional files.
NSL, ZJL, ZMG, NHL conceived and designed the study. NSL and YF performed in vitro experiments and analysed the data in AGS cells; NSL, YBOY, DQH collected human specimens and analysed immunohistochemical data. YH, CH, CX, SCA, YZ provided assistance with analyses of the data and technical issues. NSL, YH, ZMG interpreted the data and drafted the manuscript. NHL, ZMG supervised and oversaw the study. All the authors critically revised the manuscript and provided intellectual content. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was approved by the ethics committee of The First Affiliated Hospital of Nanchang University (2016034), and written informed consent was obtained from all patients.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 20.Zhou D, Zhang Y, Wu H, Barry E, Yin Y, Lawrence E, Dawson D, Willis JE, Markowitz SD, Camargo FD, et al. Mst1 and Mst2 protein kinases restrain intestinal stem cell proliferation and colonic tumorigenesis by inhibition of yes-associated protein (yap) overabundance. Proc Natl Acad Sci U S A. 2011;108(49):E1312–20.CrossRefGoogle Scholar
- 30.Yang Z, Xie C, Xu W, Liu G, Cao X, Li W, Chen J, Zhu Y, Luo S, Luo Z, et al. Phosphorylation and inactivation of PTEN at residues Ser380/Thr382/383 induced by helicobacter pylori promotes gastric epithelial cell survival through PI3K/Akt pathway. Oncotarget. 2015;6(31):31916–26.CrossRefGoogle Scholar
- 35.Jang S, Su H, Blum FC, Bae S, Choi YH, Kim A, Hong YA, Kim J, Kim JH, Gunawardhana N, et al. Dynamic Expansion and Contraction of cagA Copy Number in Helicobacter pylori Impact Development of Gastric Disease. MBio. 2017;8(1).Google Scholar
- 37.Loh JT, Shaffer CL, Piazuelo MB, Bravo LE, McClain MS, Correa P, Cover TL. Analysis of cagA in helicobacter pylori strains from Colombian populations with contrasting gastric cancer risk reveals a biomarker for disease severity. Cancer Epidemiol Biomark Prev. 2011;20(10):2237–49.CrossRefGoogle Scholar
- 41.Wang Y, Pan P, Wang Z, Zhang Y, Xie P, Geng D, Jiang Y, Yu R, Zhou X. beta-catenin-mediated YAP signaling promotes human glioma growth, 2017. J Exp Clin Cancer Res. (36, 1):136.Google Scholar
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