The Role And Mechanism Of Pigment Epithelium-derived Factor In Esophageal Squamous Cell Carcinoma Metastasis

BackgroundEsophageal carcinoma ranked sixth among the causes of cancer-related death globally in2020,and its incidence has rapidly increased in recent decades.The prevalence and mortality rate of esophageal carcinoma in China rank fifth worldwide,and nearly half of all new cases and deaths occur in China every year.Pathologically,there are two main forms of esophageal carcinoma: adenocarcinoma and squamous cell carcinoma.However,there are also regional differences between these two histological types.Esophageal squamous cell carcinoma(ESCC)is the most common histologic type of esophageal cancer worldwide.ESCC patients usually have distant metastasis at the time of diagnosis.The prognosis of ESCC patients is generally poor and is dependent on the existence of metastases.Thus,therapeutic approaches are most likely dependent on the existence of tumor metastases,the genetic composition and the tumor subtype.Hence,more detailed works in the future need to effectively explore the mechanism of ESCC at the molecular level and develop improved diagnostic and therapeutic agents.Epithelial-mesenchynal transition(EMT)is a series of cellular transformation,which is mainly reflected in the lose of epithelial characteristics and gaining mesenchymal characteristics.EMT is related to a variety of tumor cell functions,including tumor initiation,malignant progression,tumor stemness,migration,invasion,tumor angiogenesis,and drug resistance.The previous studies have shown that EMT has a strong correlation with tumor metastasis and invasion.For a long time,EMT has been considered to have two different cell types(epithelial and mesenchymal).Which are usually characterized by the absence of the epithelial marker E-cadherin and the increased expression of the mesenchymal marker Vimentin,as well as conferring cell migration.Similarly,EMT is also closely related to the invasion and metastasis of esophageal squamous cell carcinoma.Promoting EMT of esophageal squamous cell carcinoma can also significantly promote metastatic ability of esophageal squamous cell carcinoma.Pigment epithelium-derived factor(PEDF)has been proven to be the most active endogenous angiogenesis inhibitor,and it plays a series of biological roles in many physiological and pathophysiological processes,including neuroprotection,fiber formation and inflammation.Previous studies have shown that PEDF plays a complex and inconsistent role in angiogenesis,tumor growth and metastasis in different cancers.The previous research of our research group found that PEDF inhibits the metastasis of nasopharyngeal carcinoma,but the expression level of PEDF will increase in some types of cancer,and PEDF promotes the stemness and self-renewal of glioma stem cells.The expression of PEDF in different tumors is controversial.However,the role of PEDF in the metastasis of esophageal squamous cell carcinoma has not been reported yet.Extracellular signal-regulated kinase 1/2(ERK)is a member of the mitogen-activated protein kinase(MAPK)family that contributes in the transmission of extracellular signals to intracellular proteins.The MAPK/ERK cascade has a critical role in the regulation of several fundamental processes such as proliferation,differentiation and cell response to diverse extrinsic stresses.The MAPK/ERK signaling pathway has emerged as one of the most commonly dysregulated signaling pathways in cancer and has attracted extensive attention.The MAPK/ERK signaling pathway has emerged as one of the most commonly dysregulated signaling pathways in cancer and has attracted extensive attention.Previous studies have suggested that the MAPK/ERK signaling pathway is also involved in the migration and metastasis of cancer cells.Additionally,MAPK/ERK signaling inhibitors can suppress the migration and invasion of glioma cell.A large body of evidence indicates that the MAPK/ERK signaling pathway is overactivated in the induction of EMT and the initiation of carcinogenesis.However,the potential upstream mechanism of ERK in esophageal cancer has only been demonstrated in a few papers.Therefore,we hypothesized that PEDF may promote the occurrence and development of esophageal squamous cell carcinoma through the MAPK/ERK signaling pathway.ObjectiveTo explore the relationship between PEDF and the clinical prognosis of esophageal squamous cell carcinoma,to search the relationship between PEDF and the metastasis and invasion of esophageal squamous cell carcinoma cells,to clarify whether PEDF promotes EMT of esophageal squamous cell carcinoma cells,and to clarify that PEDF promotes EMT of esophageal squamous cell carcinoma cells signaling pathways and key molecules.The above studies provide a theoretical basis for PEDF as a candidate drug target for the treatment of esophageal squamous cell carcinoma,and the research of key molecules also provides clues for the discovery of new intervention targets in the same time.Experimental design and results1.PEDF mRNA Expression Is Upregulated in Metastatic Esophageal Carcinoma Cell Lines and TissuesUsing the GEO database mRNA sequencing array,the esophageal squamous cell carcinoma sequencing data in the TCGA database and the data in the Oncomine database,18 genes that may be associated with the metastasis of esophageal squamous cell carcinoma were finally determined.Finally,after screening with the literature,we found two genes most likely to be associated with the metastasis of esophageal squamous cell carcinoma.We found that PEDF expression is higher in esophageal squamous cell carcinoma,and this phenomenon is opposite to the expression of PEDF in other tumors,and the expression of PEDF in precancerous lesions of ESCC samples was lower than that in esophageal carcinoma samples.In addition,the expression of PEDF was significantly positively correlated with the clinical stage and the number of lymph node metastases in patients with esophageal squamous cell carcinoma.These observations indicate that the abnormally high expression of PEDF may play a key role in the metastasis of esophageal squamous cell carcinoma.2.Abnormal High Expression of PEDF of PEDF Is Correlated with an Unfavorable Prognosis in ESCC PatientThe immunohistochemical staining was used to detect the expression of PEDF in esophageal squamous cell carcinoma and adjacent tissues.The results showed that the expression of PEDF was significantly higher in esophageal squamous cell carcinoma than that in adjacent tissues.Further statistics found that there was a significant positive correlation between PEDF expression and N stage,CD8 and TNM stage.However,other clinicopathological factors,including T stage,age,gender,tumor grade,and PD-L1,were not correlated with PEDF expression.Kaplan–Meier survival curves showed that female patients,patients with low expression of PEDF,patients with N0–1 stage disease and patients with TNM stage Ⅰ/Ⅱ disease had significantly longer survival times.Cox proportional hazard regression analyses revealed that high PEDF expression was an independent predictor of unfavorable prognosis in ESCC patients.These results suggest that PEDF could be an ESCC metastasis promoter and that overexpression of PEDF is associated with a poor prognosis in patients with ESCC.We further used multivariate regression analysis to establish a clinical prognostic model for esophageal squamous cell carcinoma.The calibration plots of the nomogram showed good agreement between the actual and nomogram-predicted survival rates.The calibration plots esophageal squamous cell carcinoma.3.Overexpression of PEDF Promotes ESCC Cell Migration,Invasion,and EMTThe results of wound healing assays,Transwell migration assays,and Boyden chamber assays show that overexpression of PEDF can significantly promote the migration and invasion of esophageal squamous cell carcinoma cells.On the contrary,by inhibiting the expression of PEDF in esophageal squamous cell carcinoma cells,the migration and invasion ability of esophageal squamous cell carcinoma cells is reduced.Western blotting experiments showed that PEDF can downregulate the epithelial markers E-cadherin and α-catenin,and upregulate the mesenchymal marker N-cadherin.Interference with PEDF expression produces the opposite phenomenon.These results suggest that PEDF may promotes cell motility and invasiveness by driving EMT in ESCC cells.4.MAPK/ERK Signaling Is Required for PEDF Mediated EMTWestern blotting experiments show that PEDF overexpression did not significantly influence AKT expression or phosphorylated AKT expression but upregulated the expression of phosphorylated ERK,suggesting that the activation of MAPK/ERK signaling by ERK phosphorylation plays a special role in promoting tumor migration and invasion induced by PEDF.We further used ERK inhibitors PD98059 and U0126 to treat overexpressing PEDF esophageal squamous cell carcinoma cells.Western blotting and Transwell experiments showed that ERK inhibition PD98059 and U0126 impaired the PEDF-induced migration,invasion,and EMT of KYSE140 and KYSE510 cells.These results indicate that PEDF promotes EMT of esophageal squamous cell carcinoma by activating the MAPK/ERK signaling pathway.ConclusionOur study found for the first time that PEDF expression was significantly increased in highly aggressive esophageal squamous cell carcinoma cells,and PEDF was positively correlated with the poor prognosis of patients with esophageal squamous cell carcinoma.PEDF promotes the epithelial-mesenchymal transition metastasis and invasion of esophageal squamous cell carcinoma cells by regulating the MAPK/ERK signaling pathway.Phosphorylated ERK may be the key molecule of PEDF’s effect on the MAPK/ERK signaling pathway.The above results suggest that PEDF may be a new molecular target for prognosis and treatment of esophageal squamous cell carcinoma.