Expression Of FOG2 In Pancreatic Cancer And Its Role In Modulation Of Cross-talk Between MAPM And PI3K Signaling

Part I. Cross-talk between MAPK and PI3K signaling in pancreatic cancerObjective:To investigate crosstalk between MAPK and PI3-K signalings in pancreatic cancer.Methods:MAPK inhibitor PD98059 or PI3K inhibitor LY294002 was respectively applied to incubate BxPC3 and PANC1 cells for 1 h. Western blot was performed to determine expressions of p-ERK, ERK, p-Akt, and Akt. Then, siRNA was used to transiently knockdown the expression of Rafl gene, after the expression of p-ERK was reduced, western blot was performed to determine the protein levels of p-Akt in BxPC3 and PANC1 cells.Results:MAPK inhibitor inhibited ERK activity, and decreased p-ERK increased levels of p-Akt protein in BxPC3 and PANC1 cells. Level of p-Akt was also increased by decreased expression of p-ERK in siRNA anti-Rafl transfected BxPC3 and PANC1 cells.Conclusion:Cross-talk exists between MAPK and PI3K signaling in pancreatic cancer. ERK inhibits activation of PI3K signaling.Part II. Expression of FOG2 in pancreatic cancer and its role in cross-talk between MAPK and PI3K signalingObjective:To research the expression of FOG2 in pancreatic cancer, and explore its role in cross-talk between MAPK and PI3K signaling.Methods:Protein level of FOG2 was determined in 108 pancreatic cancer tissues by IHC, and the expression in 4 pancreatic cancer cell lines was detected by western blot. shRNA lentiviral vectors targeting FOG2 were transfected into PANC1and BxPC3 cells to knock down gene expression. Scrambled shRNA lentiviral particle was used as a control. Then, MAPK inhibitor and/or PI3K inhibitor was applied to incubate BxPC3 and PANC1cells for 1 h. Western blot was performed to determine expressions of p-ERK, ERK, p-Akt, and Akt. Co-immunoprecipitation was performed to detect the binding between ERK and FOG2.Results:Sixty-six of 108 pancreatic cancer tissues showed a higher FOG2 expression than corresponding adjacent normal tissues. FOG2 is detected in four PC cells, and PANC 1 cell line has the strongest expression. In control group, inhibition of ERK significantly increased p-Akt level, and silencing of FOG2 blocked ERK inhibition on Akt activation. Co-immunoprecipitation showed ERK was combined with FOG2.Conclusion:FOG2 is highly expressed in pancreatic cancer tissues. ERK can inhibit the activation of Akt by binding with FOG2. Silencing of FOG2 disrupted ERK-mediated crosstalk between ERK and AKT signaling. FOG2 is an important regulatory molecule in cross-talk between MAPK and PI3K signaling in pancreatic cancer.Part Ⅲ. Effect of silencing FOG2 on MAPK inhibitor inhibiting biological behavior of pancreatic cancer cellsObjective:To investigate the effect of silencing FOG2 on MAPK inhibitor inhibiting biological behavior of pancreatic cancer cells.Methods:MAPK inhibitor was applied to incubate shRNA-FOG2 transfected PANC1 and BxPC3 cells for 1 h. Transwell migration assay, Colony formation assay, Annexin V/PI apoptosis assay, and MTT assay was respectively implemented to evaluate MAPK inhibition on migration, tumorigenicity, apoptosis, and proliferation in shRNA-FOG2 transfected PANC1 and BxPC3 cells.Results:Transwell assay showed that silencing FOG2 enhanced inhibition of MAPK inhibitor on cell migration. Flow cytometric analysis indicated that after MAPK inhibitor was applied in silencing FOG2 cells, apoptosis rate were increased. Besides, colony formation assay and MTT assay showed that silence FOG2 enhanced MAPK inhibitor’s anti-tumorigenic and anti-proliferative effects respectively.Conclusion:Silencing of FOG2 enhanced inhibition of MAPK inhibitor on cell migration, tumorigenicity, proliferation and cell survival of PANC1 and BxPC3 cells.