| BackgroundAs one of the most common malignancies of the digestive system,gastric cancer is the fifth most common cancer,leading to cancer associated death at the top three position worldwide.Depression is one of the common and yet easily overlooked complications and indicates advanced cancer clinical stage,serving as an independent risk factor of poor prognosis for GC patients.However,the precise mechanism and biological pathways underlying depression-associated adverse clinical outcomes remains poorly understood.Unfortunately,there is no specific treatment recommendation for GC patients with depression in the current NCCN guidelines.Hence,intensive studies of the molecular mechanism and effects of depression on gastric cancer have important clinical significance for improving the prognosis of gastric cancer patients.Previous researches have shown that cancer patients with depression are under chronic psychological stress that potentially affects the progression of cancer.Among these patients,the responses to chronic stress lead to the secretion of several stress-associated neurotransmitters,including catecholamines,via the activation of HPA axis and/or the SNS.On one hand,catecholamines are comprised of three principal components,including Epi,NA,and DA,and the former two are major secreted hormones during chronic stress-induced neuroendocrine response,partly protecting from adverse stimuli.On the other hand,however,catecholamines in tumor TME,which bind to ADRs of cancer cell membranes,induce abnormal expression of oncogenes or tumor suppressor genes,facilitating growth and metastasis of several tumors.In gastric carcinoma,previous researchers have proved that catecholamines could promote EMT via c-Jun activated signaling.So,it is obviously that catecholamines play an important role in the development and progression of gastric cancer with depression and exploring its specific mechanism is rewarding.Recently,more and more researchers have reported that catecholamines could affect tumors’ malignant biological properties,such as phenotype,proliferation,drug resistance and so on,in which the neuroendocrine-liked phenotypic transition has attracted more and more attention.Marked by the increased expression of neuroendocrine markers,SYP,CHGA and CD44 with down-regulated expression of epithelial marker KRT8,the presence of neuroendocrine-like dense-cored secretory vesicles adjacent to the Golgi apparatus,and the ability which triggers activation of Ca2+-dependent secretory pathway,this process is newly defined in this study as epithelial-neuroendocrine transition(ENT).Based on the former cancer researches,neuroendocrine phenotypic acquisition of cancer cells indicated an association with neoplasm metastasis,drug resistance.However,due to the insufficient evidence by now,further studies are still needed to find whether neurotransmitter catecholamines can induce ENT and the direct and persuasive supports between ENT and malignant biological behaviors in GC.According to a previous report,the activation of hepatocyte growth factor(HGF)receptor(c-Met)regulated neuroendocrine features with an increased expression of neuroendocrine markers(SYP,CD44,and CHGA)and neuroendocrine differentiation,promoting tumor metastasis.Interestingly,depend on our long-term study and other researches,the expression of c-Met is controlled by metastasis associated in colon cancer 1(MACC1),an oncogene identified in GC and its transcriptional expression is regulated by c-Jun through promoter region binding.Moreover,our previous study has demonstrated that the upregulated expression of MACC1 could promote EMT in GC.Hence,based on the ability of MACC1 on regulating cellular phenotype plasticity and transcriptional activity of c-Met,we suspected that MACC1 might also play a role in the occurrence of so-called ENT in tumors.However,whether depression-associated catecholamines can regulate oncogenic MACC1 activity and the function behind the MACC1-mediated phenotypic reprogramming of GC still need to be explored.In this study,we found that high depression state was notably correlated with elevated plasma catecholamines,MACC1 levels and metastasis features in gastric cancer patients.Furtherly,we firstly proposed that catecholamines induced ENT in GC via β2-ADR/MACCI/SYP signaling pathway.In detail,catecholamines upregulated MACC1 expression through β2-ADR/c-Jun and the increased MACC1 directly integrated with SYP to promote ENT,which finally accelerated the invasion and metastasis of GC.Moreover,we demonstrated that inhibition of depressive chronic stress-activated β2-ADR signal pathway by the β2-ADR antagonist ICI-118,551 blocked ENT and inhibited lung metastasis of GC in vivo.Hopefully,MACC1 is expected to be a new marker of neuroendocrine-liked phenotype for tumors,and β2-ADR antagonist would be beneficial for clinical treatments of GC,particularly for those GC patients in depressive state.Hypothesis1.Depression may be associated with plasma catecholamines,MACC1 levels and metastatic characteristics in patients with GC;2.Catecholamines may up-regulate MACC1 expression by activating the β2-ADR/c-Jun signaling pathway in GC cells;3.Catecholamines may induce epithelial-neuroendocrine transformation(ENT)through MACC1/SYP signal transduction in GC cells;4.β2-ADR antagonists may block catecholamines-promoted ENT,thereby inhibiting invasion of GC cells;5.ICI-118,551 may blocks depression-promoted ENT and lung metastasis of GC through inactivation of β2-ADR/MACC1/SYP signal axis in CUMS modelsMethods and materials1.Depression is closely associated with plasma catecholamines,MACC1 levels and metastatic characteristics in patients with gastric cancerThe clinical datas and blood samples of gastric cancer patients from Nanfang Hospital in 2017.1-2017.10 were retrospectively analyzed.A total of 51 patients with gastric cancer and another 20 healthy volunteers,serving as the control group,were recruited in this part of the study.Clinical indicators included:gender,age,TNM stage(According to AJCC2012 7th Edition),plasma catecholamine epinephrine(Epi)level,noradrenaline(NA)level,and MACC1 level.51 patients with gastrnc cancer group and 20 cases of healthy controls were assessed with PHQ-9(Patient Health Questionnaire-9)method for their depression level.Meanwhile,we collected all participants’blood samples,and their plasma concentrations of epinephrine and noradrenaline were detected by HPLC-MS/MS with MACC1 determined by ELISA(See experimental method for details).51 cases of gastric patients were divided into two groups of patients with high and low depression by median of their PHQ-9 scores,and the levels of epinephrine,noradrenaline,MACC1,TNM stage,lymph nodes and/or distant metastasis were compared between the two groups.To explore the relationship between catecholamines and MACC1 levels in plasma of gastric cancer patients,the correlation between epinephrine,noradrenaline,and MACC1 was analyzed in plasma samples of gastric cancer patients.Finally,to examine possible mechanisms behind the impacts of depression on GC,we delved into the GEO databases,searching for clinical database which contained samples of cancer patients categorized as low and high depressive status.Furtherly,gene set enrichment analysis(GSEA)to explore highly enriched genes,establishing a preliminary foundation for subsequent study on mechanism.2.Catecholamines up-regulate MACC1 expression by activating the β2-ADR/c-Jun signaling pathway in GC cells.Catecholamines:Epi and NA were used to stimulate gastric cancer cells,and MACC1 expression was detected by RT-PCR,western blot and immunofluorescence imaging(See the experimental method for details).To test whether the c-Jun signaling pathway activated by catecholamine plays a key role for MACC1 expression,western blot assay was used to observe whether c-Jun signaling pathway inhibitors could reverse the MACC1 expression up-regulated by catecholamines.To further clarify the binding site of catecholamine in gastric cancer cells,gastric cancer cells were pretreated with a-adrenergic receptor blocker or β-adrenergic receptor blocker,and then cells was exposured to catecholamines.RT-PCR and western blot were used to observe MACC1 expressed levels.β-adrenergic receptors are classified into three main types:β1-ADR,β2-ADR,β3-ADR,which were detected by RT-PCR in GES-1 and BGC823,MKN45 gastric cancer cells.Dobutamine(β1-ADR agonist),salbutamol(β2-ADR agonist),and Isoprenaline(Iso,β3-ADR agonist)were used to stimulate gastric cancer cells.ICI-118,551(β2-ADR antagonist)was applied to testify whether catecholamine-activated MACC1 expression could be retarded by β2-ADR blockade.3.Catecholamines may induce epithelial-neuroendocrine transformation(ENT)through MACC1/SYP signal transduction in GC cellsFirstly,neurotransmitter catecholamines were used to stimulate gastric cancer cells for observing epithelial-neuroendocrine phenotypic transition(ENT),as marked by:(1)Altered expression of neuroendocrine markers SYP,CHGA and CD44,and epithelial marker KRT8 were detected by RT-PCR,western blot and immunofluorescence assay;(2)Neuroendocrine secretory vesicles around Golgi apparatus in gastric cancer cells were observed by transmission electron microscopy and the diameters and numbers were recorded;(3)Fluo-4 AM labeled Ca2+ was used to detect intracellular Ca2+ by immunofluorescence and flow cytometry,patch-clamp recordings were applied.To examine the role of MACC1 in catecholamine-promoted ENT of gastric cancer cells,we used siRNA for MACC1 gene silencing and observed neuroendocrine phenotypic switch of gastric cancer cells.Further,we examined the effects of siMACC1 on catecholamines-induced ENT and observed whether siMACC1 could reverse catecholamines-induced ENT,highlighting the role of MACC1 in catecholamine-induced neuroendocrine phenotypic transition of gastric cancer.Moreover,for exploring the underling mechanisms in neuroendocrine phenotype transformation induced by catecholamine/MACC1,the correlativity of synaptic protein SYP and MACC1 in gastric cancer cells,gastric cancer tissue and normal tissue adjacent to carcinoma was detected for seeking co-localization and co-expression relation.Immunohistochemistry score of SYP and MACC1 in gastric cancer tissue were statisticed for investigating linear correlation relationship between scores,and calculate the correlation index.Following the above,the co-immunoprccipitation(Co-IP)method was used to explore the relationship between MACC1 and SYPR Finally,to explore the role of MACC1/SYP signal axis in neuroendocrine phenotype transformation,we used SYP over-expressive plasmid(oeSYP)transfection to observe three classic phenotypic transformation of neuroendocrine phenotype indicators for probing whether oeSYP could restores the weakened epithelial-neuroendocrine phenotype induced by siMACC1.4.P2-ADR antagonist blocks catecholamines-promoted ENT,thereby inhibiting invasion of GC cellsTo explore whether ENT affects GC cells invasion and the role of MACCI/SYP signaling axis in invasion of GC cells effected by depression,the gastric cancer cells were pretreated with siMACC1 or oeSYP and then stimulated with catecholamines to detect the changes of invasion and ENT in gastric cancer cells.On account of the upstream and downstream signal connection between β-ADR and MACC1/SYP,we observed the invasion of gastric cancer cells after β-ADR blockade and examine whether β-ADR could delay the invasion promoted by catecholamines.Specifically,gastric cancer cells were pretreated with PPL or ICI-118,551 and then treated with catecholamine to detect invasion.In combination with the above results from GEO database,we explore the two molecular mechanisms of cell adhesion and ECM remodeling.On the one hand,FITC-Phalloidine was used to mark the cytoskeleton of gastric cancer cells,and we detected the changes of fluorescence intensity of FITC-Phalloidine after the similar treatment as described preiously.On the other hand,MMP9 was used as a marker for extracellular matrix(ECM)remodeling to explore the effect of β-ADR/MACC1/SYP axis on ECM reshape.5.ICI-118,551 blocks depression-promoted ENT and lung metastasis of GC through inactivation of β2-ADR/MACC1/SYP signal axis in CUMS modelsChronic mild unpredictable stress was used to intervene nude mice for continuous 7 weeks for establishing the depression model of mice.The weight changes of nude mice were recorded weekly and the weight change curve was drawn.At the end of 3 week-CUMS,MKN45 gastric cancer cells transfected with Lv-GFP-Luc were injected into tail vein of nude mice and ICI-118,551(5 mg/kg)was injected intraperitoneally every day for 4 weeks.After CUMS procedures,behavioral tests:sucrose preference test,tail suspension test and forced swimming test were performed in nude mice in order to determine whether CMUS method can successfully construct a depression model.After blood was taken from eyeball,plasma epinephrine and noradrenaline were measured by HPLC-MS/MS.To explore whether β2-ADR blocker can block depression-induced ENT and lung metastasis of gastric cancer,the growth of lung metastases from gastric cancer was detected by in vivo bioluminescence in PBS control group,CUMS group,ICI-118,551 group,CUMS+ICI-118,551 group.After sacrificing,metastatic lung tumors were taken,and the expression of MACC1,KRT8 and SYP were observed by RT-PCR and western blot assay for exploring MACC1/SYP signal axis activation.To further explore the role of extracellular matrix remodeling and cell adhesion mechanism in depression-promoted lung metastasis of gastric cancer,masson trichrome staining and H&E staining were used to observe whether metastatic tumors of the lung break through the tumor envelope,releasing collagen fibers,invading extracellular matrix and surrounding blood vessels.StatisticsSPSS Statistics 19(IL,USA)was applied to the analysis of data in this study.Data were recorded as mean±SEM,unless otherwise stated.Either Student’s t-test or one-way ANOVA was used for comparing continuous variables.The Chi-square was used to compare categorical variables.Spearman correlation were used to analyze the R2 between SYP and MACC1 for staining serial section.The values of P<0.05 were considered to be a statistically significant difference.Statistical analyses were showed under the figure legends and all results representing multiple independent experiments were performed in triplicate.Results:1.Depression correlates with high level of plasma catecholamines,MACC1 expression and enhanced metastatic features in GC patients1.1 To explore the correlation between depression and GC progression,we found PHQ-9 scores,plasma Epi and NA were elevated in GC patients compared with health control.From analyzing depression and clinicopathological features in 51 GC patients,high depressive degrees significantly correlated with TNM stage(P= 0.0026),lymph node and/or distant metastasis(P= 0.0423),plasma Epi(P= 0.0175)and NA levels(P= 0.0050).MACC1 level was also positively related to high depressive degrees(P= 0.0370).Furthermore,there existed positive interrelated relationship between MACC1 and Epi,NA levels,respectively(Pearson r=0.6615,P<0.0001;Pearson r=0.5575,P<0.0001).However,no relevance to depression for age and gender.These data suggested that depression was closely related to GC invasion and metastasis,and several molecular mediators,catecholamines and/or MACC1,might be also involved.1.2 To examine possible mechanisms behind the impacts of depression on GC invasion and metastasis,we found a clinical database(GEO;GSE9116)which contained samples of cancer patients categorized as low and high depressive status.Furtherly,highly enriched genes were relevant to cell adhesion molecules,calcium signaling pathway,and ECM receptor interaction by using GSEA2.Depression-associated catecholamines promote MACC1 expression mediated by P2-ADR/c-Jun signaling pathway in GC cells1.1 Firstly,we observed Epi and NA remarkably upregulated mRNA and protein levels of MACC1 in BGC823 and MKN45 GC cells.However,the similar effect of catecholamines on gastric epithelial cell(GES-1)was minimal.In addition,a significant dose-dependent increase in MACC1 gene expression was observed in both GC cells af’ter 12 hours treated with different concentrations of Epi at 0,0.1,1.0,and 10μM.Moreover,with the concentration of 10μM,Epi also promoted MACC1 expression in a time-dependent manner.1.2 Next,we inquired the mechanistic effect of catecholamines on MACC1 activation in GC.SP600125 and J-NK-IN-8,two different inhibitors of c-Jun,both could block Epi-induced MACC1 upregulation showed by western-blot analyses.To further confirm the binding site of catecholamines on GC cells in the process of catecholamines-induced c-Jun/MACC 1 improvement,we incubated BGC823 and MKN45 cells with phentolamine(Phen.;an α-ADR inhibitor)or propranolol(PPL;a 3-ADR inhibitor)for 6 hours before Epi stimulation and found that propranolol but not phentolamine blocked Epi-induced MACC1 elevation.Elevated MACC1 production in mRNA,protein levels was also seen in BGC823 and MKN45 cells after treated with isoprenaline(Iso),a type of β-ADR agonist.These results indicated that(3-ADR but not a-ADR played a key role in catecholamines functioning.In addition,to determine which kind of(3-ADRs,β1-ADR,β2-ADR and β3-ADR,really worked,we detected the expression of these three kinds of β-ADRs in GC cells and GES-1 by RT-PCR analysis,and found that the mRNA levels of β1 and β2-ADRs in GC cells were significantly higher than GES-1,in which β2-ADR had the highest expression in GC cells,while the expression of β3-ADR had almost no difference between GC cells and GES-T.Then,we noticed that salbutamol(a β2-ADR agonist)but not dobutamine(a β1-ADR agonist)treatment induced MACC1 expression in GC cells,which was analogous to the effect of Epi treatment.In turn,we pre-treated GC cells with ICI-118,551(a specific β2-ADR blockader)for 6 hours and observed that Epi-promoted MACC1 production was hindered.All these results defined that the activation of β2-ADR-oriented signaling made a significant contribution to catecholamines-induced MACC1 production in GC.3.Catecholamines induce epithelial-neuroendocrine transformation(ENT)through MACC1/SYP signal transduction in GC cells1.1 To investigate whether depression-induced catecholamines have such neuroendocrine-like effect on tumors,we treated BGC823 and MKN45 GC cells with 10μM Epi for 12 hours,and found GC cells showed neuroendocrine phenotypic transformation,as supported by:(i)Epi treatment upregulated neuroendocrine markers(SYP,CD44,CHGA)expression and downregulated epithelial markers KRT8 at mRNA and protein levels;(ii)The numbers of neuroendocrine-like secretory vesicles under Epi stimulation(n = 27,from eight fields in seven cells;mean diameter:207.61±22.1 nm)increased sharply compared with control group(n = 10,from 11 fields in 10 cells;mean diameter:218.6±14.5 nm)by transmission electron microscope observation;(iii)Epi activated Ca2+-dependent secretory pathway showing by flow cytoletry and the patch clamp technique detected the increased intracellular Caa-currents.It was indicated that these GC cells undergone a ENT process.However,when using short interfering RNA(siRNA)to knock down MACC1 expression 24 hours before Epi treatment,we found siRNA-mediated MACC1 silencing completely abrogated the ENT effect of Epi on both GC cells.To be specific,(i)siMACC1 blocked the above Epi-regulated expression of neuroendocrine markers(SYP,CD44)and epithelial marker KRT8;(ii)Quantity of neuroendocrine vesicles of siMACCl(n=4,from 28 fields in 17 cells;mean diameter:228.7±20.9 nm)blocked Epi-enhanced the neuroendocrine vesicles’ numbers(siMACC1+Epi group,n=11,from 11 fields in 9 cells;mean diameter:236.0±15.2 nm);(iii)siMACC1 treatment inactivated Epi-induced increasing in calcium influx in GC cells.1.2 For probing into the molecular mechanisms underlying MACC1-regulated neuroendocrine phenotype of GC,we found that among the regulation of neuroendocrine markers induced by MACC1 activity,SYP showed the most significant change both in western blotting and qPCR assays.Co-expression of MACC1 and neuroendocrine marker SYP was observed by immunohistochemistry and their immunohistochemical scores were positively correlated(Pearson r = 0.7658,P<0.001)in 53 gastric tumor tissues.Co-expression of MACC1 and SYP was also observed by immunofluorescence assay and the direct interactions between MACC1 and SYP were detected with immune co-precipitation Moreover,siMACC1 treatment weakened neuroendocrine phenotype of GC cells while oeSYP strengthened neuroendocrine phenotype when used alone and also could rescue siMACCl-reduced neuroendocrine phenotype.Hence,we supposed that MACC1 could integrate with SYP and MACC1/SYP axis might be a positive influencing factor on ENT of GC.4.β2-ADR antagonist blocks catecholamines-promoted ENT,thereby inhibiting invasion of GC cells1.1 To further confirm whether catecholamines can affect invasion of gastric cancer cells through ENT,we found siMACC1 completely abolished the effects of Epi on the invasion of GC cell lines.At the same time,we found oeSYP regained Epi-promoted invasion of GC cells although silencing MACC1 before consistent with oeSYP regained Epi-induced ENT of GC cells although treated with siMACC1 before.In particular,we found treatment with oeSYP and Epi respectively showed equal effects on GC invasion and no additive effects occurred with combined application.Likewise,we found that oeSYP could rekindled the Epi-promoted ability on MMP9 production and FITC-Phalloidin fluorescence intensity of GC cells,although there existed an siMACC1-mediated negative effects.1.2 To further survey the aberrant activation of MACC1/SYP signaling,we found that β-ADR inactivation by propranolol or ICI-118,551 completely blocked Epi-promoted invasion of GC cells along with down-regulation of neuroendocrine markers SYP,CD44.PPL or ICI-115,881 completely strangled Epi-enhanced MMP9 expression by western blotting assay and Epi-elevated FITC-Phalloidin fluorescence intensity of GC cells.In conclusion,β2-ADR/MACC1/SYP signal axis initiated by catecholamines was a core promoter for GC cells invasion,which could be attributed to ENT-associated ECM remodeling.5.ICI-118,551 blocks depression-promoted ENT and lung metastasis of GC through inactivation of β2-ADR/MACC1/SYP signal axis in CUMS models1.1 To identify whether the depression nude mouse model was successfully stimulated by CUMS,we firstly measured the changes in mouse weight and evaluated their behaviors at the end of 7-week CUMS exposure.Compared with non-stressed groups,it was observed that body weight was increased slowly in both two stressed groups and the nude mice of this two stressed groups exhibited depressive symptoms shown as fewer sucrose preference,increased time of motionless states in forced swimming test and tail suspension test.Further,plasma catecholamines from eyeball blood extracting were measured by HPLC-MS/MS and we found Epi and NA in depressed groups were apparently higher than non-stressed groups.Using bioluminescence imaging,we found that ICI-118,551 injection(i.p.,5 mg/kg,daily)completely eliminated the effect of depression-promoted GC invasion and lung metastasis 4 weeks after tumor transplantation by tail intravenous injection.Moreover,ICI-118,551 treatment blocked ENT of metastatic tumors in depressive mice reflected at ENT markers alteration.1.2 To explore the mechanism under invasion and metastasis of GC induced by ENT,we observed an increase of MMP9 expression in pulmonary metastatic tumors of depressive mice compared with non-depressive mice.As expected,these effects were annihilated in mice with ICI-118,551 injection.Moreover,H&E staining and masson-trichrome staining demonstrated pulmonary metastatic tumors of depressed mice penetrated through the capsule and invaded into stroma,resulting in the ECM remodeling with increased collagens.However,these effects were reversed in depressive mice treated with ICI-118,551.Overall,β2-ADR activation was critical for depression-promoted ENT,invasion and metastasis of GC.Conclusion1.Depression is closely associated with plasma catecholamines,MACC1 levels and metastatic characteristics in patients with gastric cancer;2.Catecholamines up-regulate MACC1 expression by activating the β2-ADR/c-Jun signaling pathway in GC cells;3.Catecholamines induce epithelial-neuroendocrine transition(ENT)through MACC1/SYP signal axis in GC cells;4.β2-ADR antagonists block catecholamines-promoted ENT,thereby inhibiting invasion of GC cells;5.ICI-118,551 blocks depression-promoted ENT and lung metastasis of GC through blocking of β2-ADR/MACC1/SYP signal axis in CUMS models. |
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