The Clinical Correlation Between Secreted Hsp90α And Esophageal Squamous Cell Carcinoma And The Mechanism Research In Promoting Migration And Invasion

BackgroundEsophageal carcinoma(EC)is one of the most common malignancies in the world.According to the latest global cancer statistics in 2018,the incidence and mortality of EC are seventh and sixth,respectively.The pathological types are classified into esophageal squamous cell carcinoma(ESCC)and adenocarcinal adenocarcinoma(ECA).The proportion of patients with ESCC in China accounts for more than 90%,while in western developed countries,it is mainly adenocarcinoma.In recent years,despite the diagnosis and treatment technology of EC has been continuously improved,the 5-year survival rate of patients with EC is still less than 30%.The reasons of poor prognosis include diagnosed at advanced stage,high degree of malignancy,high rate of recurrence and metastasis after therapy.It is worth noting that patients with EC are generally prone to lymph node(LN)metastasis,which seriously affects their prognosis.Therefore,current efforts have been focused on the development of clinically relevant biomarkers that predicting LN status in ESCC patients.Heat shock protein 90(Hsp90)is a highly expressed molecular chaperone that assists the conformational maturation of client proteins during stress and protects them from degradation.It is exploited by cancer cells to support activated oncoproteins that are essential for oncogenic transformation.Previous study showed that Hsp90 is abundantly expressed in esophageal cancer as well as in esophageal cancer cell lines.Hsp90a is one of the isoforms of Hsp90,which can also be secreted to the extracellular space.It may be found either in a secreted form or on the cell surface,both forms are detected in diverse tumor types.The importance of eHsp90a in promoting tumor invasiveness and metastasis has received more and more attention.Currently,serum Hsp90a has been used as a tumor marker.What’more,it has been demonstrated to be closely related to the degree of malignancy of tumor,especially regional and distant metastasis.Therefore,secreted Hsp90a can be used as a biomarker for assessing disease status and predicting survival outcomes in tumor patients.Secreted Hsp90a interacts with its extracellular clients and surface receptors to promote tumor cell migration and invasion.Matrix metalloproteinase-2(MMP-2)is one of the key proteins in tumor invasiveness and metastasis.Previous studies have confirmed that eHsp90a secreted by tumor cells could improve the activity of MMP-2,thereby promoting the migration and invasion of tumor cells.In addition,epithelial-to-mesenchymal transition(EMT)is one of the most important processes for tumor cells to acquire migration and invasion.Moreover,Hsp90a on the surface of tumor cells has been shown to be closely associated with increased expression of key driver factors during EMT.In conclusion,secreted Hsp90a plays an important role in tumor invasiveness and metastasis,and is clinically related to tumor malignancy.However,there is no relevant research in the ESCC.In this study,we analyzed the association between pretreatment serum Hsp90a levels and ESCC malignancy,as well as lymph node status.The effects of secreted Hsp90a on the migration and invasion of ESCC cells were further studied,and the related mechanisms were preliminarily explored.Part Ⅰ The relationship between pretreatment serum Hsp90a level and esophageal squamous cell carcinoma malignancy in clinicObjective:1.To analyze the differences in clinicopathological features between low and high serum Hsp90a level ESCC patients.2.To analyze the correlation between clinicopathological characteristics as well as serum Hsp90a level and lymph node status.3.Logistic regression analysis was used to evaluate the association between clinical variables and LN status.Methods:193 cases of newly diagnosed ESCC who underwent esophagectomy in Shandong Cancer Hospital from September 2015 to January 2018 were retrospectively analyzed.All enrolled patients had clear pathological diagnosis of ESCC,and serum Hsp90a levels were routinely measured before surgery.The cut-off value of Hsp90a was defined as 82.06 ng/ml,according to the 95%confidence intervals of non-cancer Chinese patients.Serum Hsp90a levels above 82.06 ng/ml were defined as high,while those below 82.06 ng/ml as low.The clinicopathological information of all enrolled patients,including age,gender,tumor length,tumor location,degree of differentiation,T stage,N stage,TNM stage,serum Hsp90a level as well as lymph node status,were recorded and analyzed.Results:1.There were significantly different in T stage(P=0.021),N stage(P=0.011)and TNM stage(P=0.016)between low and high serum Hsp90α groups.The low serum Hsp90a group tended to have earlier T stage and N stage compared with high serum Hsp90a group.2.There were significantly different in tumor length(P=0.012),T stage(P=0.004)and serum Hsp90a level(P=0.021)between LN negative group and LN positive group.The patients with negative lymph node metastasis had earlier T stage and lower serum Hsp90a level.3.By logistic regression analysis,T stage(P=0.037,OR=0.859,95%CI for OR=0.311-2.374)and the preoperative serum Hsp90a level(P=0.027,OR=0.276,95%CI for OR=0.091-0.833)may be served as independent predictors for ESCC LN status.Conclusions:1.High serum Hsp90a level before treatment was positively correlated with ESCC malignancy.2.High serum Hsp90a level before treatment was significantly correlated with positive LN metastasis in ESCC and may be served as an independent predictor for ESCC lymph node status.Part Ⅱ Effect of secreted Hsp90a on esophageal squamous cell carcinoma migration and invasionObjective:1.To detect the secretion of Hsp90a in ECA109 and ECA9706;2.To detect the effect of recombinant Hsp90a protein on migration and invasion of ECA109 and ECA9706;3.To detect the changes in migration and invasion of ECA109 and ECA9706 after the secreted Hsp90a was functionally blocked by Hsp90a neutralizing antibody.Methods:1.ESCC cell cultureECA109 and ECA9706 cell lines were cultured in high glucose DMEM medium containing 10%fetal bovine serum.The incubation conditions were 37℃,5%CO2,95%humidity.2.Western blottingThe total protein in ECA109 and ECA9706 condition media as well as total cell lysate were extracted separately.Western blotting was used to detect the expression level of Hsp90a in cell condition media and total cell lysate.3.Cell migration and invasion assayRecombinant Hsp90αa protein was added to the condition media at a concentration gradient of 0 μg/ml,5 μg/ml,10 μg/ml.The effects of eHsp90a on migration and invasion abilities of the two cell lines were analyzed.The changes in migration and invasion abilities of the two cell lines were also analyzed after secreted Hsp90a was functionally blocked by Hsp90a neutralizing antibody.Results:1.ESCC cell lines ECA109 and ECA9706 secreted a certain amount of Hsp90a continuously.2.The amount of Hsp90a in ECA109 and ECA9706 conditioned medium was decreased after secreted Hsp90a was functionally blocked,while the expression level of Hsp90a in total cell lysate was unchanged.3.Recombinant Hsp90a protein promoted the migration and invasion of ECA109 and ECA9706,and there was significant difference compared with the control group(P<0.05).4.The number of migrated cells and invaded cells treated with Hsp90a neutralizing antibody were significantly smaller than those of the control group(P<0.05).Conclusions:1.ESCC cells secreted Hsp90a and the secreted Hsp90a can be functionally blocked by Hsp90a neutralizing antibody.2.Recombinant Hsp90a protein promoted the migration and invasion of ESCC cells and blockage of secreted Hsp90a inhibited this function.Part Ⅲ Preliminary research on the mechanism of secreted Hsp90a in esophageal squamous cell carcinoma migration and invasionObjective:1.Hsp90a secreted by tumor cells can interact with and activate MMP-2,thereby promoting the migration and invasion of tumor cells.Here we explore whether secreted Hsp90a plays the same role in ESCC cell lines.2.To investigate whether secreted Hsp90a promotes ESCC cell lines migration and invasion through EMT.Methods:ECA109 and ECA9706 were treated with 10 μg/ml recombinant Hsp90a or 10μg/ml of Hsp90a neutralizing antibody for 24 h.MMP-2 is a secreted protein,and the expression level of MMP-2 in ECA109 and ECA9706 cell conditioned medium was detected by Western blotting.E-cadherin and N-cadherin are epithelial and mesenchymal markers in the EMT process,respectively.The expression levels of E-cadherin and N-cadherin in total cell lysate were analyzed by western blotting.Results:1.The expression level of MMP-2 in ECA109 and ECA9706 cells conditioned medium after recombinant Hsp90a treatment were significantly higher than that in the control group.The amount of MMP-2 in cell conditioned medium treated with Hsp90a neutralizing antibody significantly decreased compared with IgG control group.2.The expression levels of E-cadherin were significantly decreased in ECA109 and ECA9706 whole cell lysates treated with recombinant Hsp90a,while the expression level of N-cadherin was significantly increased.In addition,Hsp90a neutralizing antibody reversed this phenomenon.Conclusions:Secreted Hsp90a can enhance the migration and invasion of esophageal squamous carcinoma cells by stabilizing the activity of MMP-2 and promoting EMT progression.