The Role And Underlying Mechanism Of PTEN Phosphatase Inactivation In Gastric Carcinogenesis

Background and objective:Gastric cancer is the fourth most common malignancy in the world.Also,China has a high risk of incidence for gastric cancer.The latest research shows that the incidence and mortality of gastric cancer in China rank at the second among all types of malignancies,which has brought great harm to national health.Therefore,it is of great significance to clarify the mechanism of occurrence and development of gastric cancer and active exploration of the new methods for early diagnosis and gene therapy is the focus of our work.PTEN is an important tumor suppressor gene,which was discovered in 1997.Its encoded protein has the dual activity of protein phosphatase and lipid phosphatase,and can regulate a variety of cell signal transduction pathways or functional molecules to form a complex network system,such as to promote apoptosis,inhibit cell survival,and arrest cells cycle progression and suppress epithelial-mesenchymal transition.PTEN is often inactivated in tumors.Therefore,it is particularly important to explore the role and specific mechanisms of PTEN phosphatase inactivation in the formation,growth and metastasis of gastric cancer.Undoubtedly,the discovery of this problem will be very important for the elucidation of the mechanisms for pathogenesis and development in gastric cancer and will help to provide new therapeutic targets.Hippo signaling pathway was a highly conserved growth control signaling pathway discovered in the Drosophila in 1995.Under normal conditions,the activity of Hippo pathway is strictly controlled,but once the regulation is abnormal,which will cause uncontrolled proliferation and apoptosis inhibition,thus participates in tumorigenesis.However,currently,the regulation mechanism of Hippo pathway is not clear.At present,the most in-depth study of PTEN is its negative regulation on PI3K/Akt pathway through phosphatase-dependent manner,but it can also display its function independent on PI3K/Akt pathway.It suggests that there may be some other important molecular mechanisms.Interestingly,PTEN can affect the expression of TAZ,a core molecule of the Hippo signaling pathway.Moreover,Hippo and PI3K/Akt signaling pathways have synergistic effects in regulating cell proliferation and apoptosis.So form above,we wonder how PTEN phosphatase inactivation leads to the occurrence and development of gastric cancer? Are the Hippo,PI3K/Akt signaling pathway involved in PTEN phosphatase inactivation induced gastric tumorigenesis? What is the relationship between Hippo and PI3K/Akt signaling pathway during this process? Therefore,in view of these unknowns,this study targeted at PTEN phosphatase inactivation,PI3K/Akt signaling pathway and Hippo signaling pathway,and conducted an in-depth studies from the human,animal and cell levels,which will provide new insights into the diagnosis and treatment of gastric cancer.Materials and methods:1.Effect of PTEN phosphatase inactivation on malignant biological phenotype in gastric cancer patients(1)The expression of p-PTEN was detected by immunohistochemistry in 90 cases of gastric cancer and matched nomal tissue.(2)Analysis of the correlation between p-PTEN expression and clinicopathological features of gastric cancer patients and conduct Kaplan-Meier survival analysis.2.Studies on the effect of PTEN phosphatase inactivation on malignant biological phenotype of gastric cancer in vitro and in vivo(1)Establishment of gastric cancer cell lines which stably express the target genes.Application of MTT,Brd U,plate cloning,high-content analysis system and Transwell assay to detect proliferation and migration of BGC-823-Control,BGC-823-WT PTEN,BGC-823-Mut1,BGC-823-Mut2,SGC-7901-Control,SGC-7901-WT PTEN,SGC-7901-Mut1,and SGC-7901-Mut2 cells in vitro.(2)Subcutaneous xenograft model in nude mice was used to observe the effect of PTEN phosphatase inactivation on the growth of gastric cancer in vivo.3.PTEN phosphatase inactivation regulates Hippo signaling pathway in gastric cancer(1)The expression of YAP was detected by immunohistochemistry in 90 cases of gastric cancer and matched nomal tissue.Analysis of the correlation between YAP expression and clinicopathological features of gastric cancer patients and conduct Kaplan-Meier survival analysis and prognosis evaluation.(2)Application of western blot,nuclear plasma separation and immunofluorescence methods to detect the expression of Hippo signaling pathway related molecules and the cellular distribution of YAP in gastric cancer cell lines with highly expressed target genes.(3)Co-immunoprecipitation method was used to detect the binding force of MOB1 and LATS1/2 in gastric cancer cell lines with highly expressed target genes.(4)Different concentrations of verteporfin were used to treat the gastric cancer cell lines with highly expressed target genes,and the changes in cell proliferation and motility were observed.4.PI3K/Akt activation induced by PTEN phosphatase inactivation depends on Hippo signaling pathway(1)Western blot was used to detect the expression of PI3K/Akt signaling pathway-associated molecules in gastric cancer cell lines with highly expressed target genes.(2)The above gastric cancer cell lines were treated with different concentrations of verteporfin and the expression of p-Akt protein was detected.Results:1.Effect of PTEN phosphatase inactivation on malignant biological phenotype in gastric cancer patients(1)The expression of p-PTEN was significantly different in gastric cancer and normal tissues.The expression of p-PTEN in gastric cancer tissues was significantly higher than that in adjacent tissues.(2)The expression of p-PTEN was positively correlated with tumor invasion depth T-grade,lymph node metastasis N-grade and clinical stage.(3)Patients with high expression of p-PTEN had a significantly worse prognosis.2.Studies on the effect of PTEN phosphatase inactivation on malignant biological phenotype of gastric cancer in vitro and in vivo(1)Cell-level results showed that the cell lines with high expression of mutant PTEN C124 S and G129 E had significantly higher cell proliferation and migration capacity than those with empty lentivirus-transfected gastric cancer cell lines,whereas the cell proliferation and migration ability in gastric cancer cell lines with high expression of wild-type PTEN were significantly lower than those of the gastric cancer cell line transfected with the empty lentivirus.(2)Animal-level results showed that tumor cell growth rate,tumor volume,and tumor mass in mutant PTEN C124 S and G129 E groups were higher than control.However,tumor cell growth rate,tumor volume,and tumor mass in wild-type PTEN group were significantly reduced.3.PTEN phosphatase inactivation regulates Hippo signaling pathway in gastric cancer(1)The expression of YAP was significantly different in gastric cancer and matched normal tissues.The expression of YAP in gastric cancer tissues was significantly higher than that in noncancerous tissues.(2)The expression level of YAP total protein and YAP protein in nuclei were positively correlated with tumor size,tumor invasion depth T grade,lymph node metastasis N grade,and clinical stage.The cytoplasmic expression of YAP did not correlate with the patient's gender,age,tumor size,tumor distribution in the stomach,T-grade,N-grade,and clinical stage.(3)High YAP total protein expression and high YAP nuclear protein expression led to a worse prognosis.High YAP total protein expression and high YAP nuclear protein expression were independent prognostic factors for gastric cancer.(4)There was a correlation between p-PTEN and YAP total protein expression or YAP nuclear expression.(5)PTEN phosphatase inactivation decreased the expression of the Hippo signaling pathway molecules,like SAV1,LATS1,LATS2,MOB1,and p-YAP(Ser127)in gastric cancer cell lines,while enhancing the expression of YAP/TAZ;and enhancement of PTEN phosphatase activity could significantly increase the expression of SAV1,LATS1,LATS2,MOB1,and p-YAP(Ser127),while reducing the expression of YAP/TAZ.PTEN phosphatase inactivation promoted the migration of YAP/TAZ into the nucleus in gastric cancer cell lines.(6)High expression of mutant PTEN C124 S and G129 E reduced the binding of MOB1 to LATS1/2 in gastric cancer cells,while high expression of wild-type PTEN increased the binding of MOB1 to LATS1/2 in gastric cancer cells.(7)After inhibition the binding of YAP with the transcription factor TEAD by verteporfin,it was found that the proliferation and migration ability of gastric cancer cell lines that highly expressed target genes were significantly decreased,and the proliferation and migration ability of PTEN phosphatase-inactivated gastric cancer cells were more significantly decreased.4.PI3K/Akt activation induced by PTEN phosphatase inactivation depends on Hippo signaling pathway(1)Compared with the control,the highly expressed mutant PTEN C124 S and G129 E significantly increased the PI3K/Akt signaling pathway molecules,like p-Akt(Ser473),p-Akt(Thr308),p-Fox O1(Ser256),p-GSK-3?(Ser9),?-catenin,and p-Bad(Ser112),also decreased expression of Fox O1 and P27 Kip1;on the contrary,high expression of wild-type PTEN significantly decreased p-Akt(Ser473),p-Akt(Thr308),p-Fox O1(Ser256),p-GSK-3?(Ser9),?-catenin,and p-Bad(Ser112),also enhanced expression of Fox O1 and P27 Kip1.(2)Different concentrations of verteporfin were used to treat gastric cancer cells that highly expressed target genes.As the concentration of verteporfin was increased,expression of YAP and TEAD in the gastric cancer cell lines transfected with empty lentivirus,wild-type PTEN,PTEN C124 S and G129 E were significantly reduced,and the expression of p-Akt was also observed decreased in a concentration-dependent manner.Conclusions1.PTEN phosphatase inactivation plays an important role in regulation on the malignant biological phenotypes of gastric cancer.2.Hippo signaling pathway is involved in PTEN phosphatase inactivation enhanced malignant biological phenotypes in gastric cancer.3.The activation of PI3K/Akt signaling pathway induced by PTEN phosphatase inactivation is dependent on the regulation of key molecules in Hippo signaling pathway in gastric cancer.