The Role And Molecular Mechanism Of CEP55 In The Proliferation,Migration And Invasion Of Esophageal Squamous Cell Carcinoma

BackgroundEsophageal cancer(EC)is one of the most common aerodigestive tract malignant tumors and is the sixth leading cause of mortality in the world.The histological types of EC mainly include esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EA),and in China ESCC is the most common type.In some areas of Henan and Shandong province of China,the incidence rate of ESCC can account for more than 90%.In addition,ESCC is also high lethal,and more than 200,000 patients die each year in China.In recent years,although comprehensive treatment of tumors including surgery,radiotherapy,chemotherapy and immunotherapy has made great progress,the overall survival of ESCC patients has not been significantly improved.According to the National Comprehensive Cancer Network(NCCN)guidelines,ESCC patients without lymph node metastasis(pN0)may not accept postoperative adjuvant treatment after complete tumor resection.However,the 5-year overall survival rate of patients in this stage is only 70%,and about 1/3 of individuals may not survive more than 5 years.Obviously,patients with pNO ESCC showed significantly different prognostic tendencies.Therefore,we believe that patients in this stage should be stratified more effectively based on the different prognosis,and then provide personalized treatment in order to improve the survival of patients.Centrosome protein 55(CEP55),also known as FLJ10540,C10orf3,and URCC6,is the latest identified member of the centrosome-and midbody-associated protein family,and it mainly participates in the process of cell division.But CEP55 overexpression may lead to abnormal cytokinesis,which in turn leads to unstable multinucleated cells and tumorigenesis.CEP55 protein overexpression are rare in normal human tissues(except thymus and testicular tissue),but a large amount of evidence indicates that CEP55 is up-regulated in many solid tumors including lung cancer,breast cancer,head and neck squamous cell carcinoma,and ovarian epithelial tumor.In addition,it was identified as a prognostic signature,and its overexpression was significantly associated with poor prognosis of patients with oral squamous cell carcinoma,ovarian epithelial tumors,hepatocellular carcinoma,prostate cancer,and pancreatic cancer.Also in the process of genetic analysis of 12 tumors including medulloblastoma,lymphoma,breast cancer,glioma,mesothelioma and lung cancer,CEP55 was identified as one of the 70 genes with the highest differential expression.And CEP55 was also found in the TGCA database to be up-regulated in ESCC tissues,and its overexpression was associated with bad survival of patients.In addition,it has been reported that CEP55 overexpression can participate in the malignant behavior of tumor cells and promote the proliferation,migration and invasion of cells.However,the prognostic value of CEP55 in patients with pN0 ESCC and its biological function in ESCC cells are still unclear.All of these unknowns have become the hot topics in the field of thoracic surgeon and oncology.We hope that CEP55 may become a predictor and a treatment target of ESCC.The phosphatidylinositol-3-kinase(PI3K)/Akt pathway is an important signaling pathway that is implicated in multiple oncogenic processes,including cell proliferation,differentiation,apoptosis,epithelial-mesenchymal transition(EMT),migration and invasion of tumors.PI3K can phosphorylate phosphatidylinositol 4,5-diphosphate(PIP2)into 3,4,5-trisphosphate(PIP3),which in turn activates Akt.PIK3CA(p110)is a catalytic subunit of PI3K that maintains the activation of PI3K.It was reported that CEP55 may interact with PI3KCA,and then combine with each other to form the complexes,which can increase the activation of Akt.In lung adenocarcinoma and hepatocellular carcinoma,CEP55 overexpression can promote cell migration and invasion by activating the PI3K/Akt pathway.In addition,the PI3K/Akt pathway was also reported to participate in CEP55-mediated cell cycle in gastric cancer.Although some studies have shown that the PI3K/Akt pathway can regulate the biological behavior of ESCC cells by interacting with other molecules,we don't know whether it is involved in CEP55-mediated malignant behavior of tumor cells.In this study,we want to identify the differential expression of CEP55 protien in ESCC tissues,and then to explore its relationship with the clinicopathological features of pNO ESCC patients and to evaluate of the prognostic value of CEP55.In addition,by silencing and up-regulating the expression of CEP55 in ESCC cells,we want to explore the role of CEP55 in biological behavior of tumor cells.Additionally,the role of PI3K/Akt pathway in CEP55-mediated proliferation,migration and invasion of ESCC cells also needs to be explored by blocking of this signaling pathway with specific inhibitors.This study aims to investigate the prognostic significance of CEP55 in patients with pNO ESCC and the molecular mechanism in the process of proliferation,migration and invasion of tumor cells.Part ?The expression of CEP55 in esophageal squamous cell carcinoma tissues and its prognostic significanceObjective1.To explore the expression of CEP55 protein in pN0 ESCC tissues;2.To analyze the relationship between CEP55 protein expression and the clinicopathological features of pN0 ESCC patients;3.To detect the prognostic significance of CEP55 in pNO ESCC patients.Methods1.30 pairs of frozen ESCC tissues and their corresponding noncancerous tissues(more than 5 cm from the margin of the tumor)were collected from Shandong Provincial Hospital Affiliated to Shandong University from December 2015 to May 2016.In addition,196 formalin-fixed paraffin-embedded(FFPE)tumor specimens were harvested from patients who underwent Ivor-Lewis esophagectomy with two-field lymphadenectomy from January 2005 through December 2007.All patients achieved complete tumor resection(R0),and they were all pathologically diagnosed as pN0 ESCC.Preoperative neoadjuvant chemotherapy and postoperative adjuvant treatment were not imposed2.The expression levels of CEP55 protein in 30 pairs of ESCC tissues and their corresponding noncancerous tissues were detected by immunohistochemistry and western blot analysis.It was demonstrated that CEP55 was overexpressed in ESCC tissues.In addition,immunohistochemistry was used to detect the expression of CEP55 protein in another 196 pNO ESCC FFPE tissues.And all of them were divided into two groups according to immunohistochemical score.3.SPSS 17.0(Chicago,IL,USA)was used to establish a database which based on the patients' clinicopathological and prognostic data.?2 test was used to analyze the correlation between CEP55 expression and the clinicopathological features of ESCC patients.The Kaplan-Meier method was used to calculate the survival curves.Log-rank test was used to examine the effect of CEP55 expression on the 5-year overall survival and 5-year progression-free survival of pN0 ESCC patients.Univariate and multivariate analysis were used to calculate the prognostic factors.A statistically significant difference was represented by a two-tailed P-value<0.05.Results1.The results of immunohistochemistry showed that the positive expression of CEP55 protein was presented by yellow or brownish yellow immunostaining in the cytoplasm of ESCC.Negative expression of CEP55 protein was manifested by no or lighter immunostaining in the cytoplasm of tumor cells.The expression levels of CEP55 protein in ESCC tissues were higher than that of their corresponding noncancerous tissues.Western blot analysis further verified the results of immunohistochemistry.The results showed that CEP55 was significantly overexpressed in ESCC tissues(CEP55/GAPDH:0.71±0.14 vs 0.49±0,09,P<0.001).2.According to the results of immunohistochemical score,196 patients with pNO ESCC were divided into overexpression group(124 cases)and low expression group(72 cases).?2 test showed that CEP55 protein expression was significantly correlated with tumor size(P=0.012)and T stage of tumor(P=0.021).On the contrary,there was no correlation between its expression with other clinicopathological features such as age,gender and tumor differentiation degree(P>0.05).3.Kaplan-Meier calculated the patients' 5-year overall survival curve and 5-year progression-free survival curve.Log-rank test found that the 5-year overall survival(P=0.001)and 5-year progression-free survival(P=0.001)of pNO ESCC patients were significantly decreased with CEP55 overexpression.Univariate analysis showed that tumor T stage(P<0.001),tumor differentiation(P=0.025)and CEP55 protein expression(P=0.001)were prognostic factors of 5-year overall survival;tumor T stage(P<0.001),tumor differentiation(P=0.002),and CEP55 protein expression(P=0.001)were also prognostic factors of 5-year progression-free survival.Cox multivariate analysis found that CEP55 protein expression was an independent prognostic factor of pNO ESCC patients' 5-year overall survival(P=0.012)and 5-year progression-free survival(P=0.028).Similarly,T stage of tumor can also affect the 5-year overall survival and 5-year progression-free survival of pNO ESCC patients,and was also an independent prognostic factor.Conclusions1.Compared with noncancerous tissues,CEP55 protein is significantly overexpressed in pN0 ESCC tissues.2.The expression of CEP55 protein was significantly correlated with tumor size and T stage of tumor in patients with pNO ESCC.3.CEP55 is an important indicator to evaluate the prognosis of patients with pN0 ESCC.Part ?CEP55 promotes proliferation,migration and invasion of esophageal squamous cell carcinoma cells by activating the PI3K/Akt pathwayObjective1.To detect the role of CEP55 in the proliferation,migration and invasion of ESCC cells;2.To identify the role of PI3K/Akt pathway in CEP55-mediated biological behavior of ESCC cells and its molecular mechanism.Methods1.The expression levels of CEP55 in 5 common ESCC cell lines(Eca109,EC9706,KYSE150,KYSE450 and TE-1)and a normal esophageal epithelial cell line(HET-1A)were detected by qRT-PCR and western blot analysis.And higher CEP55 expression cell lines were used to target silence the expression of CEP55,and lower CEP55 expression cell lines were used to up-regulate CEP55 expression.2.Chemically synthesized human CEP55 shRNA and CEP55 overexpression vector were packaged into lentivirus(Genechem,Shanghai,China).Eca109 and KYSE450 cells were transfected with lentivirus-mediated shRNA to silence the expression of CEP55.and EC9706 cells was transfected with the lentivirus-mediated CEP55 overexpression vector to up-regulate CEP55 expression.Cells transfected with shRNA,scramble sequence,CEP55 overexpression vector and negative control vector were labeled as shCEP55,Scramble,CEP55 and Vector.Cells not transfected with the lentivirus were labeled as the Blank control.5 ?/mL of puromycin was used for cell selection.3.Western blot analysis was used to verify the transfection efficiency of tumor cells,and the most effective silencing sequence and up-regulated vector were used for the subsequent experiments.4.CCK-8,clonogenic assay and nude mice assay were used to verify whether CEP55 can promote the proliferation of ESCC cells.Transwell assays were performed to detect whether CEP55 can promote the migration and invasion of tumor cells.In addition,the EMT process of tumor was also detected in this research.5.For CEP55 down-regulated and up-regulated ESCC cells,western blot analysis was used to detect the expression of Akt,phosphorylated AktS473(pAktS473)and phosphorylated AktT308(pAktT308),6.PI3K inhibitors(LY294002 and wortmannin)and Akt inhibitor(MK2206)were used to block the PI3K/Akt pathway.Then CCK-8 and Transwell assays were used to detect the changes of cell proliferation,migration and invasion.The corresponding changes in EMT were also verified by western blot analysis.7.Two-tailed t-test was used to analyze the statistical difference between two groups.And the differences among more than two groups were calculated by one-way ANOVA.A significant statistically difference was represented by P<0.05Results1.The expression of CEP55 in both mRNA and protein levels in five ESCC cell lines and a normal esophageal epithelial cell line(HET-1A)were analyzed through qRT-PCR and western blot analysis.Both mRNA and protein expression levels of CEP55 in tumor cells were significantly higher than those of HET-1A,especially in KYSE450 cells.Compared with the other 4 tumor cells,EC9706 cells exhibited the lowest expression level of CEP55.Therefore,we used Eca109 and KYSE450 cells to silence the expression of CEP55,while EC9706 cells were used to up-regulate the expression of CEP55.2.Compared with the Blank control and Scramble groups,the expression of CEP55 was significantly decreased in tumor cells transfected with shCEP55-1 and shCEP55-2,especially shCEP55-1.Therefore,shCEP55-1 was used to kcockdown the expression of CEP55 in Eca109 and KYSE450 cells for subsequent experiments.Similarly,compared with the Blank control and Vector groups,the expression of CEP55 was significantly increased in EC9706 cells transfected with CEP55 overexpression vector(CEP55 group),so it was used for subsequent experiments.3.CEP55 can promote the proliferation of ESCC cells.CCK-8 showed that the growth rate of tumor cells transfected with shCEP55-1 was significantly slower than that of Scramble group.And for tumor cells transfected with CEP55 overexpression vector,the growth rate of cells was significantly faster than that of Vector group.The clonogenic assay showed that the number of clones was significantly reduced after silencing CEP55 expression.And compared with Vector group,the number of clones in the CEP55 group was significantly increased.In nude mice assay,the growth rate of the xenografted tumors in the shCEP55-1 group was significantly slower than that in the Scramble group,and the tumor weight was also significantly reduced.Similarly,the growth rate of the xenografted tumors in the CEP55 group was significantly faster than that in the Vector group,and tumor weight was also increased significantly.4.CEP55 can promote the migration and invasion of ESCC cells,and mediate the EMT process of tumor.Transwell assays showed that CEP55 can promote the migration and invasion of ESCC cells,which showed that the number of migrated Eca109 and KYSE450 cells was significantly decreased after silencing CEP55 expression.And for EC9706 cells whose CEP55 expression was up-regulated,the number of migrated cells was significantly increased.Similarly,the invasive ability of tumor cells with CEP55 knockdown was significantly decreased;and this ability of tumor cells with CEP55 overexpression was significantly increased.In addition,the expression of EMT markers(E-cadherin,N-cadherin,Vimentin,Snail and Claudin-1)also altered with changes of CEP55 expression.Silencing CEP55 expression mcreased the expression of epimenal markers(E-cadnerm and Claudin-1),whlle the expression of mesenchymal markers(N-cadherin,Vimentin and Snail)was significantly decreeased.In contrast,up-regulation of CEP55 significantly increased the expression of mesenchymal markers(N-cadherin,Vimentin,and Snail),while the expression of epithelial markers(E-cadherin and Claudin-1)was significantly decreased.5.Western blot analysis showed that the expression of pAktS473 and pAktT308 was significantly decreased with CEP55 knockdown in ESCC cells.Correspondingly,for tumor cells with CEP55 overexpression,the expression of pAktS473 and pAktT308 was also significantly increased.This indicates that CEP55 can mediate the activation of PI3 K/Akt pathway in ESCC cells.6.After blockade of the PI3K/Akt pathway with inhibitors,we found that the enhanced biological behaviors of tumor cells were significantly decreased,showing that the increased OD value and the increased migrated and invaded cells were all abrogated.Cadherin switching was also reversed by inhibition of the PI3K/Akt pathway.Conclusions1.CEP55 overexpression can promote the proliferation,migration and invasion of ESCC cells.2.PI3K/Akt pathway is involved in CEP55-mediated proliferation,migration,invasion and EMT of ESCC cells.