Adrenaline Enhances Pancreatic Cancer Cell Migration Via HuR-TGF? Regulatory Axis

BackgroundPancreatic cancer patients suffer from not only cancer itself,but also the psychological stress.Accumulating evidence showed that catecholamines are involved in the regulation of cancer genesis and progression in many cancer types.Smoking and chronic stress are well-documented risk factors that are associated with?-adrenoceptors in the development of pancreatic cancer.In addition,multiple studies have revealed that pancreatic cancer and cancer cell lines express adrenergic receptors abundantly,indicating a potent role of adrenoceptor signaling in pancreatic cancer progression.Metastasis is the leading cause of cancer associated mortality.Especially,pancreatic ductal adenocarcinomas?PDACs?are highly metastatic with poor prognosis,while whether adrenoceptor signaling promotes pancreatic cancer cell migration is unknown.TGF-?signalling has two opposite roles in tumor progression and metastasis,depending on the stage of carcinogenesis and the responsiveness of tumor cells.It is clear that in the late stages,TGF?promotes cancer development via promoting metastasis.TGF?signal itself is coordinately regulated by epigenetic and transcriptional mechanisms.Nowadays,posttranscriptional regulation,which controls mRNA turnover and translational efficiency,is found to be essential in fine tuning multiple signal pathways,including TGF?signal pathway.The ELAV family member RNA binding protein HuR has been found to stabilize a subset of short-lived mRNAs found to harbor AU-rich elements?AREs?in the 3?untranslated region?UTR?.Previous study has found that HuR could promote fibrogenic response through stabilizing TGF?.In addition,HuR was found to be activated under multiple stress conditions and cancers.Taken together,it is reasonable to hypothesize that adrenaline might activate HuR and then promotes migration via TGF?signal pathway in pancreatic cancer.Materials and MethodsCell culture.The human pancreatic cancer cell lines PANC-1 was obtained from the American Type Culture Collection?Manassas,VA,USA?and grown in 1640 medium?Invitrogen?,supplemented with 2 mM glutamine,200?g/ml penicillin,100?g/ml streptomycin and 10%fetal bovine serum?FBS??Sijiqing,Hangzhou,China?at 37°C in a humidified atmosphere of 5%CO2.Adrenaline?adrenaline bitartrate?,ICI118,551 and SB431542 were obtained from Sigma–Aldrich?Munich,Germany?.Synthesis and transfection of NC and siHuR.Negative control and si HuR were synthesized in Shanghai Genepharm.Transfection of these RNAi duplexes was done using Lipofectamine2000.After recovery for 12 h,cells were grown in the complete medium and subject to further treatment.Migration assay.Cell migration was evaluated using Transwell cell migration plates?Corning Inc.,Corning,NY,USA?.Cells?1.0×10⁴?were seeded in serum-free medium into the upper chamber and allowed to migrate towards 10%FCS in the lower chamber as a chemoattractant.After 8?h,the cells that migrated through the membrane and adhered to the underside of the membrane were stained with crystal violet and counted.Western blot assay.Cells with indicated treatments were washed twice with ice-cold PBS and lysed with whole cell lysis buffer or cytoplasmic and nuclear sub-fraction lysis buffer?Thermo Fisher?.Protein concentration was determined by the BCA protein assay?Thermo Fisher?.Equal amounts of cell lysates were run on SDS polyacrylamide gels and transfered onto nitrocellulose membranes.Membranes were then incubated in blocking solution,followed by incubation with the indicated antibodies?HuR,pSmad2,pSmad3,Smad2/3,lamin B or?-actin?at 4°C overnight.The membranes were then washed in TBS-T and incubated with HRPO-conjugated secondary antibodies for 1 h at room temperature.Antibody detection was performed with an enhanced chemiluminescence reaction.TGF?luciferase activity assay.3×SBE reporter plasmid,which harbors 3 copies of Smad binding elements flanking the firefly luciferase,together with the internal control pRL-TK,is co-transfected into PANC-1 cells.Transfected cells were further treated with control or adrenaline or in combination with siHuR as indicated before lysis using passive lysis buffer.Firefly and Renilla luciferase activities were analyzed using the dual-luciferase reagent assay kit?Promega?according to the manufacturer's instructions.The ratios of firefly to renilla were expressed as means±SEM from at least 5 independent experiments.Immunohistochemistry.Pancreatic cancer tissues and the adjacent normal tissues were fixed in 10%neutralized formalin and embedded in paraffin blocks.Sections?4?m?were prepared for immunohistochemical examination.Briefly,endogenous peroxidases were blocked using 0.75%H2O2 in phosphate-buffered saline?PBS?for 30 minutes,followed by incubation with 5%bovine serum albumin blocking buffer.Incubation with primary antibody anti-HuR?1:300?was performed for 24 hours at 4°C.Immunodetection was performed in a 3-step protocol,using streptavidin-horseradish peroxidase complex,with visualization by 3,3-diaminobenzidine.Statistical analysis.Data are expressed as mean±SD of 3 or more independent experiments.Student t test was used for comparisons between 2 groups,and one-way ANOVA for multi-group comparisons.Significant difference was considered when p<0.05.ResultsAdrenaline increases PANC-1 cell migration via?₂-adrenoceptor signalingConsistent with previous studies,we found both?1 and?2 adrenoceptors were abundantly expressed in PANC-1 cells.As expected,adrenaline dose-dependently increased the cell migration.Consistent with the increased migration ability,adrenaline decreased the E-cadherin expression while increased the vimentin expression in a dose dependent manner.These data indicate that adrenaline promotes PANC-1 migration via Epithelial-Mesenchymal transition.With the blockade of?2 adrenoceptor with ICI118,551,adrenaline induced cell migration was nearly totally inhibited.Consistently,ICI118,551 restored the expression of E-cadherin in PANC-1 cells,while ICI 118,551 alone had no obvious effects on cell migration.?₂-adrenoceptor signaling induces TGF?activation3×SBE luciferase reporter was used for monitoring the TGF?activity.Adrenaline induced an about 3 fold induction of SBE luciferase activity,which was nearly blocked by ICI 118,551.Accordingly,increased phosphorylation of Smad2 and Smad3 was observed upon adrenaline treatment,while ICI 118,551 nearly blocked the induction.No obvious change of the total Smad2/3 was observed.All of these data indicated that adrenaline activated TGF?pathway.?₂-adrenoceptor activates TGF?via cytoplasmic translocation of Hu RAdrenaline induced a robust cytoplasmic translocation of HuR,while ICI 118,551reduced the cytoplasmic translocation.Additionally,knockdown of HuR reduces HuR expression efficiently and efficient HuR knockdown nearly blocked the adrenaline induced TGF?activation,as shown by reduced SBE luciferase activity and phosphorylation of Smad2/3.HuR is essential for adrenaline induced PANC-1 migrationPANC-1 cells transfected with NC or siHuR were additionally treated with adrenaline.Knockdown of HuR mildly reduced cell migration in the control group,and it nearly blocked the adrenaline induced cell migration.The block efficiency was comparable to the effects of ICI 118,551 and SB43152.Clinical sample analysis further revealed increased HuR expression correlates with metastasis in pancreatic cancer samples.Together,these data indicate that HuR might be a key effector of adrenaline on cell migration and a key regulator of TGF?pathwayPlasma adrenaline is negatively associated with patients'overall survivalWe found that adrenaline in pancreatic cancer patients was nearly 3 fold higher,with27.55±8.55 pg/m L in normal group and 93.67±50.89 pg/mL in pancreatic cancer patients.Kaplan–Meier analysis revealed that low adrenaline patients had a median survival of 16.6months as compared with 9.6 months for the patients with high adrenaline.Moreover,plasma adrenaline correlated with the expression of TGF?at mRNA level.All of these data revealed that adrenaline–HuR–TGF?regulatory axis at least partially contributes to clinical pancreatic cancer development.ConclusionHere,we reported that neurotransmitter adrenaline promoted pancreatic cell PANC-1migration in a dose dependent manner.Block of the?2-adrenoreceptor with ICI118,551,significantly reduced cell migration,indicating that?2-adrenoreceptor activation is essential for the migration promoting role of adrenaline.Furthermore,we found that adrenaline induced a cytoplasmic translocation of RNA binding protein HuR,which in turn activated TGF?,as shown by the SBE luciferase assay and phosphorylation of Smad2/3.Either HuR knockdown or TGF?inhibition reduced cell migration induced by adrenaline.Taken together,our study revealed that adrenaline–HuR–TGF?regulatory axis at least partially contributes to the psychological stress induced metastasis in PANC-1cells,shedding light on therapeutic targeting psychological stress in improving the prognosis of pancreatic cancer.