Chronic Stress Promotes The Metastasis Of Hepatocellular Carcinoma Through Activating Sympathetic Nervous System

1.BackgroundHepatocellular carcinoma(HCC)is one of the most common malignant tumors.Metastasis and recurrence of HCC is the main cause of poor prognosis in patients with HCC.It is very important to study the molecular mechanism of HCC metastasis.At present,the medical model has changed to the bio-psychosocial medical model,and the influence of psychosocial factors on malignant tumor has attracted more and more attention.Many studies showed that patients with malignant tumors were under chronic stress.It has been demonstrated that chronic stress was involved in tumor progression by regulating tumor cells,tumor microenvironment,and the immune system.Chronic stress promotes the activation of sympathetic nervous system(SNS)to release stress hormones,catecholamine,which plays an important role in the process of tumor progression.Norepinephrine(NE)has been shown to promote the invasion and migration of tumor cells.But whether chronic stress regulates the malignant progression of HCC through activating SNS has not been reported.Therefore,to explore the impact of chronic stress on tumor progression of HCC patients would be helpful to elucidate the molecular mechanism of HCC metastasis from a view of psych-oneuroendocrinology and reveal a new theory of HCC metastasis.Invasion and migration of tumor cells is a complex process.The changes of tumor cell cytoskeleton remodeling,adhesion and EMT are important factors for tumor cell metastasis.Sympathetic nerve fibers innervated in various tumor tissues.Many studies had demonstrated that the stress hormone,NE promoted the epithelial-mesenchymal transition(EMT)of ovarian cancer cells through activating the human telomerase reverse transcriptase(h TERT).NE promotes the EMT by activating the ?-AR/HIF-1 signaling in gastric cancer and colon cancer cell and regulating the invasion and metastasis of cancer cells.Whether the stress hormone activates the ARs on the HCC cells directly in HCC patients under chronic stress,and it is still unclear that EMT is involved in this process.The purpose of this study is that: 1.To study the effect of chronic stress on HCC metastasis and prognosis of HCC patients.2.To explore the molecular mechanism that chronic stress promotes the EMT of HCC cells through activating the key AR of cells and regulating downstream signaling pathways.2.Methods(1)To evaluate the chronic stress of HCC patients,the change of heart rate variability(HRV),stress index of HCC patients was detected by Body-Checker,and the level of stress hormones(NE and EPI)in patients‘ plasma was detected by ELISA.The relationship among chronic stress with HCC recurrence,distant metastasis and survival time of patients was analyzed.(2)To establish the chronic stress-liver orthotopic model,SMMC-7721-Luc-cherry expressed luciferase gene was build.IVIS Spectrum Pre-clinical In Vivo Imaging System was used to observe the effect of chronic stress on the metastasis of HCC in nude mice,and the role of chronic stress on the survival time of tumor bearing nude mice was analyzed.(3)Based on the chronic stress-liver orthotopic model,6-OHDA was used to chemical denervated sympathetic nerve fibers.The impact of sympathetic denervation on the metastasis of HCC was observed by IVIS Spectrum Pre-clinical In Vivo Imaging System.(4)Immunofluorescence and q RT-PCR were used to detect the expression of ARs on HCC cells.Invasion and migration of HCC cells was studied after treatment with the NE and adrenergic receptor blockers(propranolol etc.)by Transwell invasion and migration assay,Wound-healing assay and Western blot,in vitro.The key AR was found in this part of study.(5)Lentivirus plasmid was used to knockdown the different ?-ARs of HCC cells.To study the key role of the AR in regulating the metastasis of tumor in chronic stress-liver orthotopic model,IVIS Spectrum Pre-clinical In Vivo Imaging System was used to observe the HCC metastasis.(6)Transwell invasion and migration assay,Western blot and q RT-PCR were used to investigate the molecular mechanism of the key AR activation to mediate the invasion and metastasis of HCC cells,and clarify the important role of EMT related genes in HCC metastasis,after knockdown or specific blocker the key AR.(7)Atomic force microscopy(AFM)and immunofluorescence was used to detect cytoskeleton remodeling,morphologic changes,Young's modulus,and EMT of HCC cells after activating key AR.(8)PCR array was used to investigate the changes of 84 genes in process of HCC cells EMT,and the role of the gene in EMT process of HCC cells,after activating AR signaling.(9)Dual-luciferase report gene system and chromatin immunoprecipitation(Ch IP)assay were used to study the effects of key signaling molecule in the process of EMT mediated by stress hormone.(10)Immunohistochemy was used to detect the expression of key signaling molecule in the tumor tissues.The relationship among stress index,HRV index,stress hormones of HCC patients with the clinical pathological characteristics and prognosis of HCC patients were analyzed.3.Results(1)The results of HRV,concentrations of NE and EPI showed that: stress and HRV indexes were obviously related with stress hormones.The combination of the three results can be used to evaluate the chronic stress state of HCC patients.Further analysis found that the level of stress index was significantly related to tumor size,vascular inv asion,intrahepatic metastasis,TNM stage,recurrence,but also correlat ed with disease free survival(DFS)and overall survival(OS).HRV time domain index es of Standard deviation of the NN intervals RR(SDNN),square root of the mean squared differences of successive NN intervals(RMSSD)and frequency domain indexes of low frequency(LF),high frequency(HF)and total power(TP)were positively correlated with DFS and OS.The concentration of NE was significantly correlated with vascular invasion,TNM sta ge,and recurrence.The concentration of EPI was significantly correlated with tumor size,vascular invasion,differentiation,TNM stage and recurrence.Furthermore,concentrations of NE and EPI were negatively related to DFS and OS of HCC patients.(2)IVIS system imaging results indicated that chronic stress promotes HCC metastasis in liver-orthotopic model.Further analysis found that chronic stress could elevate concentrations of NE and EPI in liver tissues.Higher concentrations of NE and EPI were associated with tumor metastasis.Chronic stress reduced the OS in tumor bearing mice.(3)Sympathetic nerve fibers were denervated by 6-OHDA.The metastasis of tumor reduced significantly in stress+6-OHDA group,compared to stress group.The concentrations of NE and EPI were significantly decreased in stress+6-OHDA.Symapathetic denervation extends the OS in tumor bearing mice.(4)The results of immunofluorescence and Western blot showed that a variety of ARs expressed on liver cancer cells.It showed that NE promoted the invasion and migration of HCC cells.The invasion and migration of HCC cells was inhibited by ?-blockers,but the invasion and migration of HCC cells unchanged after treatment with ?-ARs blockers.Activation ?-ARs of HCC cells induced higher level of N-cadherin and Vimentin,and inhibited the expression of E-cadherin.However,the expression of matrix metalloproteinase 2(MMP2),matrix metalloproteinase 9(MMP9)and vascular endothelial growth factor(VEGF)unchanged after blocking ?-ARs.(5)IVIS system imaging results indicated that tumor metastasis was reduced in chronic stress-liver orthotopic model after knockdown ?2-AR of HCC cells,but tumor metastasis unchanged after knockdown ?1-,?3-AR.The survival analysis also found that OS of tumor bearing mice increased after knockdown ?2-AR in chronic stress stimulation nude mice.(6)Transwell invasion and migration assay found that activating ?2-AR of HCC cells,the invasion and migration ability of HCC cells was enhanced,but knockdown or blocking ?2-AR inhibited the invasion and migration ability of HCC cells.Further investigation found that isoproterenol(ISO)inhibited the expression of E-cadherin,and promoted the expression of N-cadherin and Vimentin,but knockdown or blocking ?2-AR inhibited this promotion effect.(7)AFM scan found that activating ?2-AR induced morphologic changes of HCC cells.It is showed that tight junctions between cells became weakened,extended the pseudopodi a,increased granule on cell surface and became rougher.Blocking ?2-AR inhibited the morphological changes of HCC cells.Immunofluorescence found the expression of Vimentin was up-regulated.F-actin was accumulated and abnormal arrangement.After blocking ?2-AR,Vimentin expression and F-actin accumulation decreased.These results demonstrated that cytoskeleton remodeling was inhibited after blocking ?2-AR.The Young's modulus of HCC cells was detected by AFM.It was found that ISO inhibited the hardness of HCC cells(the lower hardness association with the larger deformability),and the hardness of HCC cells increased after blocking ?2-AR.(8)PCR array results showed that 8 genes associated with progression of HCC changed more than 5-fold after activating ?2-AR in HCC cells,compared to the control group.They are including ANGPT2,CCL5,EGF,LEF1,MTDH,STAT3,PIN1,and CDH1(E-cadherin)was a down-regulated gene.Further analysis showed that 3 genes(Myc,CCND1,and LEF1)were down-regulated more than 5-fold,and CDH1 was up-regulated 5-fold in ISO+ICI118551 group,compared to ISO group.These results indicated that the activation of ?2-AR played the key role in the regulation of CDH1 and LEF1.The results of q RT-PCR and Western blot verificated that the expression of LEF1 was promoted after activating ?2-AR of HCC cells and the expression of LEF1 decreased after blocking ?2-AR.(9)According to the results of the PCR array,?2-AR signaling was involved in the regulation of LEF1,Myc,CCND1,and MTDH.Furthermore,these genes were associated with activation of ?-catenin signaling.However,the results of PCR array showed that the expression of ?-catenin unchanged after activating or blocking ?2-AR.It was found that the protein level of ?-catenin was no difference between the two groups.It is reported that the activation of ?-catenin signaling is associated with the nuclear translocation.Immunofluorescence and Western blot detection found that ?-catenin nuclear translocation was promoted after activating ?2-AR of HCC cells,and specific ?2-blockers inhibited the ?-catenin nuclear translocation.It was found that the expression of LEF1,Vimentin and N-cadherin was increased and the expression of E-cadherin was decreased accompanied with the increase of nuclear ?-catenin after the activation of ?2-AR.The effect of ?2-AR simulation on the invasion and migration of HCC cells was inhibited after knockdown ?-catenin.Bio-informatics analysis showed that ?-catenin translocated into the nucleus and combined with LEF1/TCF.The complex of ?-catenin/LEF1 would bind to the promoter of downstream Vimentin and regulation of gene transcription.It was confirmed that ?-catenin could bind to the promoter of Vimentin by Ch IP assay.Further investigation found that the activity of Vimentin promoter was inhibited after knockdown ?-catenin.It was confirmed that ?-catenin translocated into the nucleus to promote the transcription of Vimentin after activating ?2-AR,and mediated cytoskeletal remodeling and EMT of HCC cells.(10)It was found that the expression of ?-catenin located in nucleus was positively correlated with stress index and concentrations of NE and EPI.The expression of ?-catenin located in nucleus NE was significantly correlated with vascular invasion,TNM stage,and recurrence.The expression of ?-catenin was negatively related to OS of HCC patients.4.Conclusion(1)Chronic stress promotes the metastasis of HCC by activating SNS,and leading to the poor prognosis of HCC patients.(2)Chronic stress promotes cytoskeleton remodeling and EMT of HCC cells th rough stimulating ?2-AR of HCC cells by stress hormones.(3)Chronic stress promotes the invasion and metastasis of HCC cells th rough mediating ?2-AR/?-catenin/LEF1/Vimentin pathway.In this study,we found that chronic stress promoted HCC metastasis.Chronic stress promoted the activation of ?2-AR/?-catenin/LEF1/Vimentin pathway to mediate cytoskeleton remodeling and EMT of HCC cells.These results would provide a theoretic al basis and experimental basis for exploring new strategies for prevention HCC metastasis.