| Backgroud:A recent epidemiological study has demonstrated that pancreatic ductal adenocarcinoma(PDAC)is associated with a very poor prognosis,highlighted by the close parallel between disease incidence and mortality[1].The overall 5-year survival rate is less than 6%,and less than 20% of patients are eligible for initial resection[2].The low survival rate is attributed to several factors,the most important of which appears to be late stage at diagnosis.More than 85% of PDAC cases are diagnosed when the tumor has already invaded adjacent organs or metastasized to distant organs[3].Even after potential curative resection,the 5-year survival rate is only approximately 25%[1].Autopsy series have shown that approximately 90% of PDAC cases are complicated by distant metastasis[4].Therefore,there is a critical and urgent need to investigate the molecular mechanisms of PDAC growth and metastasis.Furthermore,identifying biomarkers with prognostic value for PDAC patients after surgery may help with selecting suitable follow-up intervals and subsequent therapies.Semaphorins and the semaphorin receptors(neuropilins and plexins)are secretory transmembrane-bound molecules that act as axon guidance cues in the nervous system[5,6].Among semaphorins,those in class 3 are not only involved in nervous system development but also highly expressed in various types of tumor cells,wherein they modulate tumor growth,metastasis,vascular development,and immune cell activation[7-10].A majority of class 3 semaphorins(e.g.,sema3 a,sema3b,sema3 e,and sema3f)inhibit cell migration and seem to exhibit antitumor properties[11-15].In contrast,sema3 c is highly expressed and promotes cell invasion[16]and tumor growth in gastric cancer[17],hepatocellular cancer[18],breast cancer[19] and oral neoplasia[20].Sema3 c expression correlates with poor survival in gastric cancer[17] and breast cancer[20].Furthermore,Sema3 c promotes endothelial cell proliferation,migration and tube formation in vitro and might be involved in angiogenesis [21].A previous study reported that full-length sema3 c inhibits tumor lymphangiogenesis and metastasis[10,22,23].Thus,sema3 c functions as both an oncogene and a tumor suppressor.However,the mechanisms by which sema3 c regulates cancer progression and the clinical significance of sema3 c have yet to be fully elucidated,and whether an d how sema3 c is involved in PDAC pathogenesis have not been investigated.EMT is particularly important in the migration and invasion of many types of tumor cells,including pancreatic cancer cells[24,25].In addition to being a well-characterized embryological process,EMT has been reported to be crucial for the invasion and metastasis of many types of cancer,including pancreatic cancer[26].ERK1/2 has gained widespread attention as a potential target for the prevention and treatment of metastatic tumors associated with EMT[27-29].Herman reported that prostate cancer cells transfected with sema3 c showed increased invasive and adhesive features and downregulation of E-cadherin and α-catenin,two important epithelial markers.The ERK1/2 signaling pathway plays a pivotal role during EMT-induced tumor cell migration and invasion[28,30-32].Multiple signaling pathways,including the Wnt/β-catenin,JAK/STAT3,NF-κB and ERK1/2 pathways,have been reported to regulate EMT in tumor progression[32-34].ERK1/2 is one of the most important pathways contributing to cell proliferation,metastasis and EMT during tumorigenesis[35,36].Increased p ERK1/2 levels have been reported in many types of cancer,including pancreatic cancer[37-39].Furthermore,Javle et al.observed EMT and activated p-ERK in surgically resected pancreatic cancer specimens,and EMT was associated with poor survival in surgically resected pancreatic adenocarcinoma[40].Objectives:1.we first investigated sema3 c expression in pancreatic cancer andsema3 c mRNA expression in five pancreatic cancer cell lines.2.We investigated the clinical significance of semaphorin 3c and its role in growth and metastasisi of pancreatic ductal adenocarcinoma(PDAC).3.we studied whether EMT in pancreatic cancer cells is affected by sema3 c statu s to further understand the molecular mechanism that mediates the aggressive effects of sema3 c.4.We researched the potential involvement of ERK1/2 to better understand the signaling pathways related to the tumor-promoting features of sema3 c in pancreatic cancer.5.To ascertain the effect of sema3 c on tumorigenicity in vivo.Methods:1.we first investigated sema3 c expression in pancreatic cancer andsema3 c mRNA expression in five pancreatic cancer cell lines by RT-PCR and Quantitative real-time PCR analysis of sema3 c in a panel of 26 pairs of tissues.otal protein was extracted from tumor and normal pancreatic tissues and analyzed by Western blotting.Next,we assessed sema3 c protein levels using IHC and examined whether sema3 c expression is related to clinicopathological features.Kaplan-Meier analysis was performed to examine 118 pancreatic cancer patients after surgical resection.2.Sema3 c expression was knocked down in Bx PC-3 cells by sema3c-sh RNA lentivirus transfection(Bx PC-3-shsema3c),and sema3 c was overexpressed in CFPAC-1 cells by sema3 c plasmid transfection(CFPAC-1-exsema3c).Then,Pancreatic cancer cell proliferation was then examined by CCK8.Cell apoptosis was then examined by flow cytometry.3.The migration and invasion abilities of tumor cells were assessed using transwell and wound healing assays.Next,we assessed the expression levels of epithelial markers(α-catenin and E-cadherin)and reduced the expression levels of mesenchymal markers(N-cadherin and ZEB2)by Western blot.4.we investigated sema3 c and p ERK1/2(ERK phosphorylated at Thr202/Tyr204)levels in pancreatic cancer and matched normal pancreatic tissues from patients by IHC.To confirm the involvement and functional effects of ERK1/2 activation in tumor promotion by sema3 c in pancreatic cancer.In vitro,wound-healing and transwell invsion and migration assay were employed to detect the GC cells invasion and migration after PC cells transfected sema3c-shRNA lentivirus transfection(Bx PC-3-shsema3c)and sema3 c plasmid transfection(CFPAC-1-exsema3c).EMT related molecules were detected by Western blot.5.To ascertain the effect of sema3 c on tumorigenicity in vivo,we subcutaneously transplanted Bx PC-3-shsema3 c cells and CFPAC-1-exsema3 c cells into the right lower abdomen of NOD/SCID mice.Results:(1)Sema3c was significantly augmented in PDAC compared with normal pancreatic tissues.Furthermore,Sema3 c mRNA levels were higher in the five pancreatic cancer cell lines than in HPC-Y5 cells.Among the cell lines analyzed,Bx PC-3 cells showed the highest level of sema3 c expression,and CFPAC-1 cells showed the lowest.(2)Sema3c expression was higher in stage IV pancreatic cancer tissues compared with stage I tissues.Moreover,we found a significant correlation between the sema3 c expres sion level and several clinicopathological features.Sema3 c expression is positively associated with tumor stage and inversely associated with patient survival.(3)Sema3c knockdown significantly attenuated pancreatic cancer cell proliferation compared with negative controls.Moreover,the percentage of apoptotic cells was significantly higher among Bx PC-3-shsema3 c cells.Overexpression of sema3 c had the opposite effects.(4)Sema3c promotes pancreatic cancer cell migration and invasion.As we know,EMT is particularly important in the migration and invasion of pancreatic cancer cells.We found that Sema3 c knockdown reverses EMT in pancreatic cancer cells.Conversely,sema3 c overexpression strongly promoted the mesenchymal phenotype of CFPAC-1 cells.(5)The ERK1/2 signaling pathway plays a pivotal role during EMT-induced tumor cell migration and invasion.We confirmed that the tumor-promoting effects of sema3 c in pancreatic cancer are mediated by activation of the ERK1/2 pathway.(6)Sema3c expression also promotes pancreatic cancer growth in vivo.Conclusion:Aberrant expression of sema3 c is correlated with the poor prognosis of PDAC patients and promotes tumor growth and metastasis by activating the ERK1/2 signaling pathway. |
