The Role And Mechanism Of Hippo/YAP Signaling Pathway On The Helicobacter Pylori CagA-induced Epithelial-mesenchymal Transition

Background and objective:Although a steady decline of gastric carcinoma morbidity and mortality over the last few decades have been observed,stomach cancer still remains the most malignant disease in eastern Asia including china.According to the latest epidemiological data in China,gastric cancer ranks the second in both incidence and mortality rates among the commonest cancers.The majority of patients with gastric cancer already have advanced disease when diagnosed,which leads to the limited treatment strategies and poor prognosis.Helicobacter pylori(H.pylori)infection is not only the primary cause of chronic gastritis,peptic ulcer,but also is associated with diffuse-type and intestinal-type gastric cancer.H.pylori was classified as class I carcinogen by the world Health Organization(WHO)in 1994.Recent studies have indicated that eradication of H.pylori significantly reduce the risk of gastric carcinoma.Understanding the mechanisms underlying the pathogenesis of H.pylori in promoting gastric carcinogenesis,will contribute to the development of novel stratedies for early diagnosis of gastric cancer and for better gastric cancer prevention.Currently,approximately 50%of the world population is infected with H.pylori,but only 1-3%of those infected with H.pylori develop gastric cancer eventually.In addition to host-related factors and environmental factors,the bacterial virulence factors have been demonstrated to be the predictors of severe clinical outcomes after H.pylori infection.CagA functions as a virulence factor inH.pylori pathogenicity.After delivered into gastric epithelial cells via type IV section system(T4SS),translocated CagA stimulates cellular functions including excessive proliferation,inhibited apoptosis,enhanced migration by physically interacting with intracellular signaling pathways,which eventually contributes to gastric carcinogenesis.It has been confirmed that infection with CagA~+strains of H.pylori could significantly increase the risk of gastric adenocarcinoma.CagA transgenic mice developed gastric polys and adenocarcinoma of stomach.In addition,translocated CagA has been reported to be involved in the epithelial to mesenchymal transition(EMT)including loss of epithelial cell polarity and acquisition of elongated mesenchymal phenotype.However,the underlying molecular mechanism of EMT induced by H.pylori CagA remains unclear.Hippo-YAP signaling pathway plays an important role in regulating-tissue growth and organ size control depending on the balance between cell proliferation and cell death.Recent studies have shown that aberrant activation of YAP,which is a key effector of the Hippo pathway,are involved in tumorigenesis and progression.Deregulation of Hippo pathway activates YAP expression,which induces nuclear translocation of YAP and upregulates downstream genes,thereby promoting cell proliferation,survival and migration.It has been shown that YAP and its target genes 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,it remains unclear whether or not H.pylori infection alone induce deregulation of Hippo/YAP signaling pathway to activate oncoprotein YAP.In addition,it is unclear whether the role of H.pylori CagA play in the regulation of Hippo/YAP pathway.It has been documented that the Hippo/YAP signaling pathway play a major role in regulating the epithelial-mesenchymal transition(EMT),and therebypromoting tumor cell malignant transformation.Inactivation of Hippo signaling pathway results in overexpression of YAP and facilitates its nuclear translocation,which also induces EMT.EMT is a major hallmark of gastric mucosa malignant transformation including tumor cell invasion and metastasis.During EMT,gastric epithelial cells lose cell polarity and cell-cell adhesion,in concert withacquirement of a mesenchymal phenotype with a higher motility.Despite that previous studies showed that infection with CagA~+H.pylori strains induces EMT,the roles of H.pylori CagA~+andHippo/YAP signaling pathway andpromoting EMT remain defined.In this project,we aimed to investigate the effect of Hippo/signaling pathway on H.pylori-induced gastric carcinogenesis.In addition,we aimed to elucidate the role of H.pylori CagA in regulating Hippo/YAP signaling.Finally,we explored how H.pylori infection promotes gastric carcinogenesis via deregulation of the Hippo/YAP signaling in clinical subjects.Materials and methods Ⅰ.In vivo and in vitro characterization of how H.pyloriregulated Hippo/YAP signaling pathway in gastric mucosa epithelial cells1.Gastric tissues of patients with different stages of gastric carcinoma were obtained.Immunohistochemistry staining was performed to detect the major effectors of Hippo/YAP signaling pathway including YAP and its paralog TAZ.Furthermore,the relationship between their expressions and clinical pathological features were statistically analyzed.2.CagA~+H.pylori strains(7.13,PMSS)and CagA~-H.pylori strain(SS1)were cocultured respectively with gastric epithelial AGS cells.Expression of key factores involved in Hippo-YAP signaling pathway were determined by western blot.The mRNA levels of the YAP-regulating genes such as CTGF,CYR61 were quantified by qRT-PCR.Ⅱ.Generation and characterization of PMSS1 DcagA isogenic mutants1.According to the whole genome sequence of PMSS1 cagA gene,a pair of primers cagAupF and cagAL-5’RXS or cagAdnR and cagAF-3’FXS were designed,and overlapping PCR was performed to generate amplicon(namely cagA up-dn)consisting of the upstream and downstream regions of the PMSS1 cagA genes.Subsequently,the cat(chloramphenicol acetyltransferease gene)cassette was ligated in the overlapping site of cagA up-dn.Finally,the recombinant plasmids were generated.2.These recombinant plasmids were introduced into PMSS1 strain by electroporation or natural transformation.Positive transformants were selected and amplified.PMSS1 DcagA isogenic mutants then were obtained.3.A pair of primers were designed to verify the complete deletion of cagA from the genome of these Transformants.Another pair of primers were designed to detect the orietation of cat cassette.Western blot was performed to verify whether cagA has been knocked out in PMSS1 DcagA mutant strains.Ⅲ.Mechanistic studies of the role of virulence factor CagA in H.pylori-induced YAP expression,nuclear translocation and transcriptional modulation of YAP downstream target genes.1.PMSS1 wild-type and DcagA isogenic strains were cocultured with gastric epithelial AGS cells,western blot was performed for CagA translocation and phosphorylation.And morphological changes of these cells were examined under microscope.2.AGS cells were infected with the clinical H.pylori strains isolated from the human subjects with chronic non-atrophic gastritis(CNAG),intestinal metaplasia(IM)and gastric carcinoma(GC).Translocated CagA and its phosphorylated levels were determined by Western blot.3.AGS cells were cocultured with CagA~+H.pylori strains(PMSS1 or 7.13)and their CagA~-isogenic mutants:Western blot assays were performed to evaluate YAP protein expression in Hippo/YAP signaling pathway;Immunofluorescence assay was performed for cellular location of YAP;qRT-PCR assay was performed for determing mRNA levels of YAP and its downstream genes CTGF,CYR61.Ⅳ.Effects of Hippo/YAP signaling transduction in H.pylori CagA-induced EMT.1.AGS cells were cocultured with CagA~+H.pylori strains(PMSS1 or 7.13)and the CagA~-isogenic mutants.Immunofluorescence were performed to detectE-cadherin expression,an epithelial cell marker.2.The plasmid of YAP cDNA were introduced into AGS cells,and how this treatment influencesexpression patterns of EMT-associated markers were determined.3.AGS cells were treated with CagA~+H.pylori strains(PMSS1 or 7.13)in combination with a YAP inhibitor Verteporfin.E-cadherin expression was detected by Immunofluorescence staining,EMT-associated markers were detected by Western blot,and Transwell cell invasion assay was performed.4.Human CNAG,IM,Dys and GC tissues were collected.Immunohistochemistry staining was performed for evaluating YAP and E-cadherin expression.In addition,,influence of H.pylori infection on the expression of gastric YAP and E-cadherin in these gastric tissues with different histopathological stages of human gastric cancer were analyzed.ResultsⅠ.In vivo and in vitro characterization of the role of H.pylori in modulating Hippo/YAP signaling pathway in gastric mucosa epithelial cells1.YAP and its paralog TAZ were distributed in both the cytoplasm and nucleus in the majority of gastric tumors.Compared with the adjacent noncancerous tissue,expression of YAP and TAZ were significantly increased in gastric cancer tissues.Additionally,higher levels of cytoplasmic YAP were noted in the early tumor stages,whereas YAP was predominantly located in the nucleus at the advanced tumor stages.While YAP and TAZ expression were not associated with gender,age of patients and location,increased YAP and TAZ levels in the gastric tissues was positively correlated with invasion depth and lymph node metastasis.2.Short-term infection with H.pylori upregulated the core effector YAP expression of the Hippo signaling,and up-regulated transcription of its downstream genes CTGF,CYR61.However,phosphorylation of YAP and its upstream kinase LATS1 was increased in AGS cells.These findings suggest that the activation of oncoprotein YAP in Hippo signaling pathway andthe increased expression of YAP downstream genes CTGF and CYR61 via CagA~+H.pylori infection does not neccesarily require upstream kinases.Generation and classification of PMSS1 DcagA isogenic mutants1.The recombinant cagA knockout plasmids were constructed successfully.Two recombinant plasmids 54 and 55 were selected because their genomes contain opposite orientations of the cat cassette(upstream and downstream primers were 54,55respectively.).These recombinant plasmids were introduced into recipient PMSS1 strain by electroporation or natural transformation.Transformants were selected on sheep blood agar containing 25μg/ml of chloramphenicol;four Cm~R Transformants,namely 54E,54N,55E and 55N.2.The results of agarose gel electrophoresis have indicated that CagA has been completely deleted in all of DcagA isogenic mutants.The transcriptional orientation of cat was 54→55 in the mutants of 54E and 54N;and the orientation of cat was 55→54 in the mutants of 55E and 55N.The results of Western blot assay have shown that there was no CagA protein expressed in these four DcagA isogenic mutants.Taken together,the data have demonstrated that PMSS1 DcagA isogenic mutants have been constructed successfully.Ⅱ.The mechanistic studies of the role of virulence factor CagA in H.pylori-induced YAP expression,nuclear translocation and downstream transcriptional regulation1.Translocation and phosphorylation of CagA were detected in AGS cells co-cultured with parental strains PMSS1 or 7.13 but not with their respective mutants.In addition,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 54N,was not significantly different from those of the uninfected controls.2.Compared with clinical H.pylori strain isolated from the gastric tissues of patient with CNAG and IM,these clinical H.pylori strains isolated from the patient with GC translocated more CagA into gastric epithelial cells.However,there is no difference between CNAG and IM group.3.Infection with CagA~+H.pylori strains induced higher levels of YAP mRNA and protein and increased YAP nuclear translocation compared with infection with the CagA~-mutants and the uninfected controls.Also,CagA~+H.pylori increased downstream genes CTGF and CYR61 expression.However,knock out of CagA in H.pylori strains abrogated this effect.These results have demonstrated that H.pylori CagA plays an important role in dysregulating Hippo/YAP pathway.Ⅲ.Effects of Hippo/YAP signaling transduction in H.pylori CagA-induced EMT.1.Treatment with CagA~+H.pylori strains(7.13 or PMSS1)led to the reduction of E-cadherin expression in AGS cells compared with the uninfected controls as visualized by immunofluorescence,whereas E-cadherin expression were partially restored in the AGS cells infected with CagA~-H.pylori mutants.2.Transient overexpression of YAP decreased the level of E-cadherin,which indicated that YAP plays an important role in promoting EMT.3.Treatment of the YAP inhibitor Verteporfin partially augmented epithelial marker E-cadherin expression and attenuated mesenchymal markers N-cadherin and Slug in CagA~+H.pylori-infected AGS cells.4.Treatment with YAP inhibitor Verteporfin significantly suppressed CagA~+H.pylori induced gastric cell invasion.5.The expression of YAP was gradually increased during gastric neoplastic progression(CNAG→IM→Dys→GC),whereas there was a gradual decrease in the expression of E-cadherin.Pearson correlation analysis predicted that the levels of YAP expression were negatively correlated with the levels of E-cadherin.Furthermore,we found that H.pylori~+gastric tissues contained significantly higher levels of YAP and lower levels of E-cadherin compared to H.pylori–tissues for CNAG but not for IM,Dys and GC.These finding indicated that dysregulation of Hippo/YAP signalling pathway plays a very important role in CagA~+H.pylori-induced EMT.Moreover,H.pylori infection elevated YAP expression in concert with reduction of E-cadherin in the early stage of the gastric tumorigenesis cascade,which could be one of the key mechanisms underlying H.pylori-induced gastric carcinogenesis.These findings could at least partially explain why H.pylori~+patients have a higher risk for the development of gastric cancer compared with H.pylori~-patients.Conclusion1.H.pylori infection causes aberrant transduction of Hippo/YAP signaling pathway via elevated activation and increased nuclear localization of the core effector YAP that further upregulates its downstream genes.The bacterial protein CagA functions as a key effector during this process.2.CagA~+H.pylori promotes EMT in gastric carcinogenesis via triggering oncogenic YAP pathway.