CyclinA/CDK2-mediated Phosphorylation Of BAG3 At Ser291 Site Enhances Proliferation And Anti-apoptosis In Gastric Cancer MGC-803 Cells

Object To evaluate the expression of Cyclin A/CDK2/BAG3 in human gastric cancer(GC) tissues and adjacent non-cancerous tissues and correlation among Cyclin A,CDK2 and BAG3. To explored the possible relationship between BAG3 expression and clinicopathological parameters. To study the role of BAG3 and phosphorylation of BAG3 at Ser291 site in the cell proliferation and anti-apoptosis of GC cells.Methods Western Blot assay was employed to examine the expression of Cyclin A/CDK2/BAG3 protein level in 180 resected GC samples and matched adjacent non-cancerous samples from patients who underwent gastrectomy for GC in Fujian Medical University Union Hospital between December 2012 and July 2012.The correlation among Cyclin A,CDK2 and BAG3 and the relationship of BAG3 expression in GC tissue samples with the clinicopathological features were analyzed. We generated wild-type(WT)-, Ser187Ala(S187A)- or Ser187Asp(S187D)-BAG3 stably expressing MGC-803 cells and BAG3 sh RNA stably down-regulating BAG3 level. CCK8 assay and plate clonogenic assay was used to study the effect of BAG3 and phosphorylation of BAG3 at Ser291 site in the cell proliferation.Cell apoptosis was assayed by flow cytometry and cleaved-PARP detection by WB. Transcriptional activity of FOXO1 was subjected to dual luciferase assays. Immunofluorescence staining and nuclear & cytoplasmic extraction were used to study FOXO1 nuclear export.Results 1.BAG3 could be detected in the GC tissues and adjacent non-cancerous tissues. The immunostaining for BAG3 was mainly located in the cytoplasm of tumor cells. The BAG3 expression in GC lesions was significantly lower than that in the non-cancerous gastric mucosa. Subsequent spearman correlation analysis showed that expression levels of BAG3 were inversely correlated with the Cyclin A/CDK2 expression levels. 2. BAG3 was directly phosphorylated by Cyclin A/CDK2 on Ser291 site. 3. Phosphorylation of BAG3 downregulates its protein stability. 4. Overexpressed BAG3 in gastric cancer cells suppresses cell proliferation, sustained phosphorylation sites S291 is more significant. 5. BAG3 overexpression in gastric cancer cell lines are more sensitive to oxaliplatin-induced apoptosis. 6. Overexpression of BAG3 accelerates FOXO1 nuclear export and promotes P27 and Bim transcription.Conclusions 1.The BAG3 expression in GC lesions was significantly lower than that in the non-cancerous gastric mucosa. The expression of Cyclin A/CDK2 in GC lesions was significantly higher than that in the non-cancerous gastric mucosa. 2. Cyclin A/CDK2 directly phosphorylates BAG3 on Ser291 site and downregulates its protein stability. 3. Overexpression of BAG3 accelerates FOXO1 nuclear export and suppresses proliferation and anti-apoptosis in gastric cancer MGC-803 cells.