Crosstalk Between Insulin And G Protein-coupled Receptor Signaling Stimulates Yap Activity In Pancreatic Cancer Cells

Background: As a perilous digestive tumor,Pancreatic cancer is becoming one of the most important health problems known.Cell signal transduction plays a key role in the process of cancer development,including cell growth,proliferation,apoptosis and migration.Cell membrane receptor including G protein coupled receptor and growth factor receptor.GPCRs and their cognate agonists are increasingly implicated in pancreatic cancer cell lines,and also dramatically synergize with insulin/IGF-1 in inducing mitogenic signaling.The transcriptional co-activators YAP is a major downstream effector of the Hippo pathway and novel sensors of insulin and GPCR signaling.The identification of YAP in the signaling network that mediates crosstalk between GPCRs and insulin/IGF-1 in Pancreatic cancer cells is of importance for identifying novel targets for treatment and/or prevention of this devastating disease.Objective: 1.To explore crosstalk between GPCRs and insulin/IGF-1 affect nuclear localization,dephosphorylation and transcriptional activity of YAP in PDAC cells.2.To study crosstalk between GPCRs and insulin/IGF-1 mediate YAP via PI3 K or mTOR.3.To verify crosstalk between GPCRs and insulin/IGF-1 mediate YAP via PKD.4.To identify statins affect pancreatic cancer cell proliferation and regulate YAP.Methods: 1.Confluent Panc-1 and MiaPaCa-2 cells were stimulated with neurotensin and/or insulin,Immunofluorescence to examine YAP localization;Western blot to examine phosphorylation of YAP Ser127;qRT-PCR to examine YAP transcriptional activity;[3H]-Thymidine Incorporation to examine DNA synthesis;Cell Count and Colony Formation to examine cell growth and proliferation;Knockdown of YAP and TAZ levels via siRNA transfection to examine phosphorylation of YAP,transcriptional activity and cell growth and proliferation.2.Prior stimulate with neurotensin and/or insulin,cells were exposure to A66 and KU63794 to examine phosphorylation of pAktThr308 and p70S6KThr389,YAP transcriptional activity.Pancreatic cells were transiently transfected with AKT-PH-GFP to monitor PIP3 activity.3.Prior stimulate with neurotensin and/or insulin,cells were exposure to CRT0066101 and kb142-70 to examine phosphorylation of PKD pSer916 and the expression of YAP/TEAD-regulated genes;Knockdown of PKD1,PKD2 and PKD3 levels via siRNA transfection to examine phosphorylation of PKD and YAP transcriptional activity.4.Cell Count and Colony Formation to examine Statins effect on cell growth and proliferation;qRT-PCR to examine Statins effect on YAP transcriptional activity.Results: 1.Insulin/IGF-1 and GPCR agonist promote nuclear localization,dephosphorylation of YAP,DNA synthesis and stimulate YAP/TEAD-regulated gene expression in PDAC cells.Transient transfection of pancreatic cells with siRNAs targeting YAP and TAZ prevented the increase in YAP/TEAD-regulated gene mRNA levels and virtually extinguished the detection of YAP protein.The combination of the stimuli promoted a marked increase in both cell number and colony formation,while the knockdown of YAP/TAZ averted the increase.2.Treatment of these cells with A66(gradient concentration)prevented the increase in the expression of CTGF,CYR61 and CXCL5 mRNA levels.Stimulation with insulin and/or neurotensin induced an increase in the ratio of membrane to cytoplasm fluorescence of AKT-PH-GFP,which can be inhibited by A66.3.Treatment with CRT0066101(dose-dependent)and kb NB 142-70 abrogated PKD autophosphorylation at Ser916 and YAP/TEAD gene expression.Transfection of a mixture of siRNAs targeting PKD1,PKD2 and PKD3 abrogated the increase in CTGF,CYR61 and CXCL5 expression.4.Treatment of these cells with statins abrogated cell proliferation and YAP/TEAD-regulated gene mRNA levels.Conclusion: 1.Crosstalk between insulin and GPCR signaling pathways rapidly regulates YAP localization,phosphorylation and transcriptional co-activity in PDAC cells.2.PI3 K mediate YAP activation in response to insulin and neurotensin in pancreatic cancer cells.3.PKD mediate YAP activation in response to insulin and neurotensin in pancreatic cancer cells.4.Statins inhibit YAP activation through mevalonate pathway.