Effects Of PTEN And Its Mutants On AKT Phosphorylation In Gastric Cancer Cells

Research BackgroundGastric cancer is the fifth most common cancer in the world,ranking the third leading mortality of cancer.In China,about 404,000 new cases of gastric cancer have been diagnosed each year,accounting for 42%of the global total cases.Approximately 80%patients in the discovery of gastric cancer has been exacerbated,so that no effective treatment can be found.In vivo,AKT phosphorylation signal is an important pathway of regulating cell proliferation,survival and apoptosis and glucose metabolism.Its abnormal activation can induce constant proliferation of cells and lead to the formation of tumors.It was reported that AKT phosphorylation signal is frequently activated in 40-82%gastric cancer cases,with associating to lymph node metastasis.Sasaki T et al.found in gastric cancer that the survival rate of patients with high p-AKT was significantly lower than that of others.Therefore,AKT may be a therapeutic target for gastric cancer and a good marker for diagnosis and prognosis.The tumor suppressor gene PTEN,a negative regulator of AKT phosphorylation pathway,is one of the most mutated gene associated with tumor in vivo.PTEN mutations can occur in any encoding region of the gene,but most common in the phosphatase domain encoding region(exon 5).PTEN mutant C124S(the N terminal 124th cysteine substituted by serine)results in completed loss of both lipid and protein phosphatase activity,and G129E(the 129th glycine replaced by glutamic acid)results in only loss of lipid phosphatase activity.On the relationship between gastric cancer and PTEN,there are many domestic and foreign researches focused on the change of PTEN expression leading to gastric carcinogenisis due to a variety of factors.It is lacking detailed study about the effect of PTEN phosphatase domain mutations on gastric cancer.In the present study,we explore the effect of PTEN phosphatase domain mutations on AKT phosphorylation in gastric cancer cells in vitro.Part one:Effects of overexpressed wtPTEN on AKT phosphorylation in gastric cancer cellsObjectTo study the effect of overexpressed wild-type PTEN(wtPTEN)on AKT phosphorylation induced by insulin or recombinant human epidermal growth factor(rhEGF)in gastric cancer cell lines AGS and BGC-823.MethodsAGS and BGC-823 cells were cultured in vitro,and then transfected with wtPTEN expression plasmid and stimulated with insulin or rhEGF for 10 min after starvation overnight.Finally,Western blot was used to detect the level of AKT phosphorylation.Results1.wtPTEN inhibited AKT phosphorylation in AGS cells.AKT phosphor ylation was activated after insulin stimulation for 10 minutes.The expression of p-AKT in thransfected group(0.43±0.35)was lower than that in the control(1.40±0.48),with statistically significance(P= 0.049),the corresponding PTEN expression trend was opposite,with statistically significance(P= 0.037).rhEGF can activate AKT phosphorylation in gastric cancer cells as well as.The expression of p-AKT in thransfected group(0.54±0.12)was also lower than that in the control(0.95±0.09),with statistically significance(P= 0.010),the corresponding PTEN expression trend was opposite,with statistically significance(P= 0.024).2.wtPTEN inhibited AKT phosphorylation in BGC-823 cells.After insulin stimulation for 10 minutes,the expression of p-AKT in thransfected group(0.50±0.17)was lower than that in the control(1.08±0.11),with statistically significance(P= 0.008),the corresponding PTEN expression trend was opposite,with statistically significance(P<0.001).Conclusions1.Insulin or rhEGF stimulation can activate AKT phosphorylation signaling pathway in gastric cancer cells.2.Wild-type PTEN plasmid can be transfected into gastric cancer cells and expressed.3.In AGS and BGC-823 cells,PTEN can inhibit AKT signaling pathway induced by insulin or rhEGF stimulation.Part two:Effects of PTEN mutants on AKT phosphorylation in gastric cancer cellsObjectTo study the effect of PTEN C124S and G129E mutant on AKT phosphorylation in gastric cancer cells.MethodsWe cultured cells as before,and then respectively thransfected with PTEN-C124S and PTEN-G129E mutant expression plasmids according to the propocol.After serum starvation overnight,cells were stimulated with insulin or rhEGF for 10 min.At last,Western blot was used to detect the level of AKT phosphorylation.Results1.PTEN-C124S or G129E was no longer inhibited AKT phosphorylation in AGS cells.Compared with the expression of phosphorylated AKT in the control group,no significant difference was found in the transfected C124S group after insulin or rhEGF stimulation(P>0.05).When transfected with rhEGF,we can get the same results(P>0.05).2.In BGC-823 cells,PTEN-C124S or G129E mutant expression plasmids invalid the inhibitive function.After insulin stimulation,there was no significant difference between the expression of phosphorylated AKT in the control group and the transfected group(P>0.05)·ConclusionsThe activation of AKT phosphorylation signal may involve in the pathogenesis of gastric cancer through the way PTEN phosphatase domain mutation in N terminal 124th or 129th amino acids.