The Roles And Mechanisms Of Trop2 In Invasion And Metastasis Of Gastric Cancer

BackgroundGC(gastric cancer)is one of the fifth most common tumors and ranks third leading cause of cancer-related mortality in the world.GC is one of the highest incidence of cancer in East Asia,furthmore about 70%of new cases and death cases occurred in East Asia,Eastern Europe and South America each year,while China accounted for 40%.The incidence of GC is the highest in China,which ranks the second of cancer in men,second only to lung cancer,and ranks the fourth in women.With the development of the health condition and diet habit,the incidence of GC has declined in recent years.Due to the high rates of metastasis and recurrence,the prognosis of GC patients is poor:5-year survival rates are less than 20%.The traditional methods toward GC,such as surgery,radiotherapy and chemotherapy,did not significantly improve the prognosis of GC patients,therefore need new method urgently.Nowadays,some genes be reported to have regulated role towards invasion and metastasis of GC and targeted therapy towards the genes have been made some progress,but because of the complexity of the tumor formation and the interaction role of these genes,as well as the limitation of the drug carrier,lead to the poor effects of the targeted therapy.Therefore we need to find newly targets,so as to provide reliable theoratical foundation for medical treatment.Human trophoblast cell surface antigen 2(TACSTD2/Trop2/M1S1/GA733-1)is a membrane surface antigen,located at chromosome 1q32,which have been studied more thoroughly in our Lab.Trop2 is a 36-kDa single-pass transmembrane protein expressed primarily in epithelial cells and was first identified in human trophoblasts[10].Trop2 is be cleaved through TNF-α converting enzyme(TACE)into extracellular domain,intracellular domain,transmembrane domain and a phosphorylation of cytoplasmic tail.Intracellular domain is released from the membrane,and accumulates in the nucleus,so as to play a function.Trop2 is overexpressed in various epithelial tumors,and its expression correlates with aggressive tumor behavior.Our Lab studied of Trop2 since 2009,the results of early study indicated that Trop2 was highly expressed in breast cancer,pancreatic cancer,cervical cancer,but the effects of Trop2 in GC has not yet been reported.Epithelial-mesenchymal transition(EMT)phenomenon,is refer to the epithelial cells transform mesenchymal cells under a normal and specific pathological conditions,which plays an important role in cancer metastasis and make cancer cells have some stem cells s properties through reprogramming.At the beginning of the tumor metastasis,cancer cells need chang in phenotype to adapt the microenvironment.Because multiplayer structure of the normal epithelial tissue adverse to migration and invasion of the malignant tumor cells,tumor cells loss of epithelial phonotype(intercellular adhesion,polarity and immobility)and epithelial cell surface markers(such as E-cadherin),meanwhile gain mesenchymal phenotype(such as fibronectin,vimentin)through EMT phonomenon,so as to acquire athletic ability.EMT phenomenon is very important and absolutely necessary in the beginning of the tumor metastasis.The study is to evaluate whether or not Trop2 could be used as a molecular target for GC target cell therapy,to explore that the functional role of Trop2 in GC,and found Trop2 could activate proliferation,inhibit apoptosis,increase the invation and metastasis of GC cells;in the last part,we explored Trop2 and vimentin expression is positive correlation and Trop2 could mediate β-catenin so as to increase vimentin expression,in order to induce EMT phenomenon and invasion and metastasis of GC in vivo and in vitro.Objectives1.Through stomach clinical specimens,cytological experiments and animal experiments,to detect the expression level of Trop2 and the effects of Trop2 in malignant biological behaviors of gastric cance,so as to evaluate the feasibility of Trop2 as new target for GC.2.To clarify the mechanism of Trop2,which could promote the invasion and metastasis of GC through β-catenin and EMT phenomenon.Methods1.qRT-PCR was performed to determine Trop2 mRNA expression levels in 41 pairs of human GC tissues and matched tumor neighbor tissues.IHC was used to explore Trop2 protein expression level in 830 cases different types of stomach tissue samples.Paired t-test,χ2 test,univariate and multivariate Cox regression,Kaplan-Meier method and a log-rank test were used to analyze whether Trop2 is a prognostic factor in GC.2.OE-Trop2 or shRNA-Trop2 in GC cell lines,to detect the proliferation of GC cells by CCK8 and clone assay,cell apoptosis and cell cycle by FACS,invasion and metastasis of GC by transwell assay.To produce stable cell lines and prepare nude mice tumor model,campare the inhibition of shRNA-Trop2 in vivo.3.IHC detect the EMT phenotype vimentin and Trop2 in 248 cases GC tissues,compared to 86 cases matched tumor neighbor tissues.Paired test,χ2 test,univariate and multivariate Cox regression,Kaplan-Meier method and a log-rank test were used to analyze the correlation of the two genes and GC metastasis and progmosis.4.shRNA-Trop2 in GC cell lines which were examined as higher Trop2 expression or over expression Trop2 in GC cell lines which were examined as lower Trop2 expression,and then detected the invasion and metastasis ability of change in those GC cells by transwell assay.The expression level of EMT phenotype marker(E-cadherin,vimentin and fibronectin)and EMT transcription factors(β-catenin,goocecoid,snail)were detected by western-blot and qRT-PCR.GC cell lines EMT models induced by TGF-β1,and then detected the change of the EMT phenotype marker by morphological observation and immunofluorescence(IF)assay.Next,qRT-PCR was performed to detect the change of Trop2,EMT phenotype marker(β-catenin,goocecoid,snail)expression level in EMT models.IF was used to explore the change of Trop2 and β-catenin expression.Co-IP assay was performed to examine physical interaction between Trop2 and(3-catenin.Western-blot was performed to explore the expression level of Trop2,β-catenin and vimentin in GC cell lines,EMT model cell lines,and shRNA-Trop2-EMT model cell lins.Laser scanning confocal microscope(LSCM)was performed to examined the change of β-catenin and vimentin in OE/shRNA-Trop2-GC cell lines and EMT-shRNA-Trop2 GC cell lines.Analysis of the mechanisms about the Trop2 towards the invasion and metastasis of the GC.5.Mice xenograft transfer model were used to analyze the metastasis ability of GC cell lines,which could be influenced by shRNA-Trop2.IHC was performed to observe the expression change of the Trop2,vimentin and Ki67 in lung tumor tissues.Results1.Trop2 was over expressed in GC tissues.41 pairs fresh frozen GC tissues processed qRT-PCR showed Trop2 mRNA expression was 2.32 ± 1.44 folds higher in GC tissues than that in matched tumor neighbor tissues.IHC carried out on 830 paraffin-embedded GC sections showed that Trop2 expression was higher in GC tissues than in neighboring non-tumor tissues and any other types of gastric tissues.Increased Trop2 protein levels in GC were associated with increased differentiation,tumor node metastasis stage,tumor size,lymph node metastasis,distant metastasis,and H.pylori infection.GC patients with high Trop2 expression also had poor overall survival(OS)rates.These data suggest that Trop2 is a useful prognostic biomarker for GC.2.Over expressed Trop2 induced cell proliferation and clone formation,inhibits cell apoptosis and induces S cell cycle arrest in GC cell lines,meanwhile,knockdown Trop2 inhibited cell proliferation and clone formation,induces cell apoptosis and G1 cell cycle arrest in vitro.Moreover,Trop2 depletion inhibits tumor growth in vivo and the inhibit rate reached 50.40%.3.The expression of Trop2 and vimentin were positively correlated,Trop2 and vimentin were all over expressed in GC and higher than those in matched tumor neighbor tissues.Trop2+/vimentin+ expression in GC were associated with increased differentiation,TNM stage and distant metastasis.GC patients with Trop2+/vimentin+ expression also had poor OS rates.These data suggest Trop2 expression in GC may be associated with EMT phenomenon.4.Trop2 could promote the metastasis and invasion of the GC cells in vitro.Trop2 increased the mesenchymal biomarker(vimentin and fibronectin)and EMT regulator(β-catenin),and meanwhile decreased the epithelial biomarkers(E-cadherin)by western-blot and qRT-PCR.TGF-β1 induced EMT phenomenon of the GC cell lines.After 7d,the shape of GC cell lines changed distinctly,and E-cadherin expression decreased,while vimentin and fibronectin increased by IF and qRT-PCR.These data indicated that the EMT model were induced successfully.Trop2 and β-catenin were all increased in EMT model cell line.The results of Co-IP assay indicated that Trop2 could binding with β-catenin intracellular and Trop2 could up-regulateβ-catenin so as to promote EMT phenomenon in GC cells.LSCM further observation that Trop2 up-regulated vimentin expression in GC cells through increased β-catenin gathered in nulei.These results suggested the mechanism of Trop2,which could promote the invasion and metastasis of GC through β-catenin and EMT phenomenon.5.shRNA-Trop2 could inhibited the metastasis of GC cells in vivo,the IHC results indicated that Trop2,vimentin and Ki67 expression in tumor tissues of the shRNA-Trop2 group is lower than that in shRNA-NC group.Conclusions1.Trop2 highly expressed in GC and were associated with the differetiation,TNM stage,tumor size,lymph node metastasis,distant metastasis and H.pylori infection.GC patients with high Trop2 expression also had poor overall survival rates.These data suggest Trop2 is a useful prognostic biomarker for GC.2.Trop2 highly expressed may facilite proliferation,inhibit apoptosis,increase invasion and metastasis and cell mitosis of GC cells.3.Trop2 and vimentin co-expression in GC tissues and positively related.Trop2+/vimentin+ expression in GC were associated with the differetiation,TNM stage,distant metastasis.GC patients with high Trop2 and vimentin expression also had poor overall survival rates mediate,these data suggest Trop2 expression in GC may related to EMT phenomenon in GC.4.Trop2 could binding β-catenin to increase vimentin expression,so as to induce EMT phenomenon and metastasis of GC.