The Expression And Significance Of CEP55 In PN0 Stage Esophageal Squamous Cell Carcinoma

BackgroundEsophageal carcinoma is one of the common upper gastrointestinal malignant tumors worldwide with obvious regional distribution differences. Esophageal Squamous Cell Carcinoma(ESCC) is one of the main pathological types of esophageal carcinoma. China is one of the areas showing the highest incidence rate of ESCC while esophageal adenocarcinoma(ECA) is more prevalent in Europe and America. As is widely accepted, surgery is the best therapeutic modality for localized ESCC. Ivor-Lewis esophagectomy is a widely adopted operative type in clinic because that ESCC has the predilection of mid-thoracic esophagus. Prognosis of ESCC patients is tumor-node-metastasis (TNM) staging-specific. However, it is not very accurate and sensitivity to predict the prognosis of ESCC patients just using the TNM stage. According to the latest esophageal cancer treatment guidelines by National Comprehensive Cancer Network (NCCN) recommended for pNO ESCC patients, pTl-2N0M0 patients were not recommendatory candidates for prophylactic postoperative adjuvant therapy, while there was no consensus on whether pT3-4aN0M0 patients need postoperative adjuvant therapy or not. However, our previous study indicated that even pNO ESCC patients underwent lymphatic metastatic recurrence after complete resection. Because of the different main histological type, the foreign experience might not be suitable for China. Therefore it is significant to search for a biological marker to further stratify pNO ESCC patients based on different recurrence risk and survival time. For patients with high risk of recurrence, suitable adjuvant treatment should be taken to reduce recurrence and improve long-term survival. In our previous study, some molecular indictor can be used for predicting lymphatic metastatic recurrence in pN0 esophageal squamous cell carcinoma. But there is no indicator which is widely accepted and adopted in clinic. So it is mandatory to search for new indicators to predict the prognosis of patients with pNO ESCC. There is no report about the correlation between the overpression of CEP55 and prognosis after Ivor-Lewis esophagectomy in pNO ESCC patients.Centrosomal protein 55 (CEP55) is a latest found mitotic phosphoprotein. It plays an important role in cytokinesis, the final stage of cell division during which physical separation of the two daughter cells occurs. CEP55 is required for the establishment and proper function of the midbody structure. And it plays an essential role in abscission involved in membrane fission events.^Overexpression of CEP55 causes cytokinesis defects and an increase in the number of multinucleated cells, which is chromosome instable and prone to tumorigenesis. Notably, CEP55 has been found highly expressed in certain human tumors and various tumor cell lines, while expression was barely detectable in normal tissues except for testis and thymus. Overexpression of CEP55 in mammalian cells is associated with enhancing cell migration and invasion. Furthermore, suppression of CEP55 expression significantly retards the growth of cancer cells, which is associated with increased apoptosis. All the findings indicate that aberrant expression of CEP55 could contribute to tumorigenesis. And it should be a factor that contributes to poor prognosis. But there is no report that announced the correlation between the overexpression of CEP55 and the prognosis in pNO ESCC patients. This study was designed to investigate the correlation between CEP55 overexpression in cancer tissues and prognosis in patients with pNO ESCC after Ivor-Lewis esophagectomy. It was aimed at finding new indicator to stratify the pNO ESCC patients and providing individual therapeutic schedule to improve prognosis. Moreover, Lentivirus carrying small interfering RNA (siRNA) was used to transfected esophageal squamous cancer Eca109 cells to deplete the expression of CEP55. MTT assay and transwell assay was used to identify the role of CEP55 in tumor proliferation and invasion, aiming at screening ideal targets for gene therapy of ESCC.Part I:Clinicopathological Significance of CEP55 Expression in Patients with pNO Esophageal Squamous Cell Carcinoma(ESCC)Objective:In this study we aimed to investigate the clinicopathological significance of CEP55 expression in patients with pNO Esophageal Squamous Cell Carcinoma(ESCC).Methods:In this study,196 eligible patients who suffered from mid-thoracic esophageal squamous cell carcinoma(ESCC) and Ivor-Lewis esophagectomy in January 2007 to December 2009 in Shandong Provincial Hospital affiliated to Shandong University were enrolled. All patients were confirmed to take radical resection and at pN0 staging by postoperative pathology. One pair of samples consisting of ESCC tissue and adjacent normal esophageal mucosa (ANEM) were harvested from surgical specimen of every selected patient. CEP55 expression levels in ESCC tissues and ANEM of all patients were validated by western blot analyses and immunohistochemistry. SPSS 17.0 software was employed to analyze the correlation between CEP55 expression and clinicopathological characteristics of the patients with ESCC. The χ2 test was employed to analyze the correlations between CEP55 overexpression and clinicopathological factors. The measurement data was represented with average±standard deviation. Differences were considered significant when the P-value was less than 0.05.Results:By immunohistochemistry method, the positive expression of CEP55 was detected as yellow or brownish yellow stain in the cytoplasma. The expression level of CEP55 in ESCC was obvious higher than that in ANEM(P<0.001). The result of Western blot corresponded to that of immunohistochemistry method. CEP55 overexpression was detected in 124 patients(63.3%). Clinicopathological factors including tumor stage (P=0.021), Tumor length (P=0.012), TNM stage (P=0.010) were related to CEP55 overexpression.Conclusions:There is CEP55 overexpression in ESCC. CEP55 overexpression was correlated with Tumor stage, Tumor length and TNM staging. Overexpression of CEP55 might plays an important role in carcinogenesis and invasion of ESCC.Part Ⅱ:Prognostic significance of CEP55 in pN0 esophageal squamous cell carcinoma after Ivor-Lewis esophagectomyObjective:This study was to investigate the prognostic significance of CEP55 in pN0 esophageal squamous cell carcinoma after radical resection via Ivor-Lewis esophagectomy.Methods:This study enrolled 196 eligible patients with mid-thoracic ESCC in Shandong Provincial Hospital affiliated to Shandong University from January 2007 to December 2009. All patients suffered from radical resection via Ivor-Lewis esophagectomy and thoracic-abdomen two fields lymph node dissection. And it was confirmed that all patients were at pN0 staging by postoperative pathology. CEP55 expression levels in ESCC tissues of all patients were validated by immunohistochemistry. All patients were followed up. Univariate analysis was performed by modeling Kaplan-Meier survival curves. The log-rank test was used to calculate the survival rate. Multivariate analysis was carried out by the use of the COX proportional hazard model. Differences were considered significant when the P-value was less than 0.05. The statistical data were obtained using an SPSS software package (SPSS 17.0, Chicago, IL, USA)Results:The 1,3, and 5-year overall survival (OS) rate of 196 patients was 96.4%, 62.8%, and 35.2% respectively. Univariate analysis indicated that CEP55 expression level (P=0.001) was significant prognostic factor. The 5-year survival rate of patients without CEP55 overexpression in ESCC tissues was significantly higher than that of patients with CEP55 overexpression. By univariate analysis we found that tumor size (P=0.044), T stage (P=0.000), differentiation degree (P=0.000), TNM stage (P= 0.000) were also significant prognostic factors. Cox multivariate regression analysis revealed that T status, TNM stage, tumor differentiation degree and CEP55 expression level were the independent relevant factors in the prognosis of pNO ESCC.Conclusions:The 5-year survival rate in pNO ESCC patients after radical resection was significantly associated with tumor size, T stage, differentiation degree, TNM staging and CEP55 expression. CEP55 expression was the independent relevant factor and predicts the poor prognosis.Part Ⅲ:Blocking the expression of CEP55 in Esophageal squamous Cancer Eca109 Lines by CEP55siRNA Objective:The present study aimed to investigate the influence of blocking the expression of CEP55 on cell proliferation and invasion by silencing CEP55 gene using CEP55siRNA in Esophageal squamous Cancer Eca109 Lines.Methods:Eca109 cells were transfected by lentivirus with CEP55siRNA to block the expression of CEP55. Western blotting was employed to identify the downregulation of the CEP55 expression in the transfected Eca109 lines. Meanwhile, lentivirus without CEP55siRNA was used to transfect Eca109 lines as the blank control group. Transwell assay and MTT assay was used to investigate the influence of downregulating the expression of CEP55 on cell proliferation and invasion.Results:The recombinant lentiviral vector was transfected into human ESCC cell line Eca109 successfully. After transfected by lentivirus with CEP55siRNA, Eca109 cells were detected with constant downregulation of CEP55 expression by RT-PCR and western blot. We found that downregulating the expression of CEP55 can inhibit cell proliferation and invasion in Eca109 cells.Conclusions:CEP55siRNA can block the expression of CEP55, which is associated with the cell proliferation and invasion in esophageal squamous cancer Eca109 cell lines.