Roles And Molecular Mechainsm Of MUC1 In CCL21/CCR7 Induced Lymph Node Metastasis Of Esophageal Squamous Cell Carcinoma

Background:Esophageal cancer(EC) is one of the common malignant tumor of digestive tractwith a variable geographic distribution, it ranks eighth in order of incidence and is the sixth most common cause of death. China is one of the countries with high incidence of esophageal cancer, and the incidence and mortality of esophageal cancer both ranks first in the world.About 210000 patients die due to esophageal cancereach year. The pathological types of esophageal cancercan be divided into mainly two: squamous cell carcinoma and adenocarcinoma, and esophageal squamous cell carcinoma accounts for about 90% of esophageal cancer in china.As a kind of highly aggressive malignant tumor, ESCC is prone to lymph node metastasis.And lymph node metastasis status is the most important factor affecting the prognosis of patients with ESCC. According to the unique anatomical characters, the submucosa of esophagus contains abundant lymphatic vessels, so thetumors could easily achieve lymphatic metastasis via these micro-tunnels. But with the gradual deepening understanding of metastasis, simple anatomical features could not explain the complex mechanism of lymph node metastasis of ESCC.Chemotaxis refers to the directional movement of cells along a concentration gradient, it is achieved by the binding of chemokine and the chemokine receptor onthe cell surface:then intracellular signal pathways will be activated and regulate the expression of certain genes which involve in cell motility. Chemotaxis is essential for lymphatic homing and inflammation and a series of physiological activities.And recent studies have confirmed that chemotaxis is involved in tumor metastasis to specific organs:with the help of chemokine receptor, the tumor cells could achieve migration to target organs which express specific chemokine. CCR7 chemokine receptor, as one of CC chemokine receptor family, which is expressed on surface of mature lymphocytes and dendritic cells, their corresponding specific ligand CCL19/ 21 is widely expressed in secondary lymphatic system, and involve in lymphocyte the homing behavior.Recent studies found aberrant expression of CCR7 invarious tumors such as breast cancer, prostate cancer, lung cancer, and esophageal squamous cell carcinoma, and up-regulation of CCR7 correlated with lymph node metastasis and poor prognosis. These suggests CCL19/21-CCR7 may specificity may play an important role in promoting lymph node metastasis, but the specific molecular mechanisms still need to be explored.Mucin 1 (MUC1) is a kind of transmembrane glycoprotein with relatively high molecular weight. It is formed by the core peptide and sugar chains. MUC1 isnot only involvedindifferentiation, renewal, and integrity of epithelium but also correlated with tumorigenesis and metastasis. MUC1 contains two subunits:MUC1-Cand MUC1-N.MUC1-N is the extracellular subunit containing multi-repeat segment of amino acid and accompanied by extensive glycosylation. And it is located on the cell surface by interacting with MUC-C. MUC1-C is the transmembrane subunit of MUC1, and studies demonstrated that MUC1-C can regulate a variety of biological behavior of cells via influencing multiple signaling pathway. However,recent studies have confirmed the aberrant expression of MUC1 in various tumor cells and correlated with tumor invasion and metastasis.Our former studies have revealed the up-regulation of CCR7/MUC1 in ESCC tissue and confirmed its relationship with lymph node metastasis, recurrence status and poor prognosis of ESCC patients. All these indicated the potential role of CCR7/MUC1 in the progression of metastasis. Although several researches have revealed the mechanism of CCR7 in promoting lymph node metastasis, most focused on the role of CCR7 in inducing directional migration of tumor cells and activating the down-stream signaling pathway and very little was known about the key down-stream molecular that regulate the biological behavior. To our best knowledge, there was no report about the role of MUC1 in CCR7 induced lymph node metastasis of ESCC. In this study, we aimed to detect the expression of CCR7 and MUC1 in ESCC, reveal the correlation between CCR7 and MUC1 and explore their relationship with lymph node metastasis, regional lymphatic recurrence and prognosis of ESCC patients. Based on this, we will further the role of MUC1 in migration and invasion induced by CCL21/CCR7 axis in vitro and reveal the underlying molecular mechanism. And we hope this study would partly reveal the molecular mechanism of lymph node metastasis of ESCC and offer theoretical basis for target therapy of ESCC.PartⅠExpression of CCR7 and MUC1 in ESCC andrelationship with clinical pathological significance.Objective:To explore the expression of CCR7 and MUC1 in ESCC tissue sample, declare their relationship with lymph node metastasis, recurrence and prognosis, and investigate the correlation of CCR7 and MUC1 in ESCC.Methods:153 ESCC patients who underwent esophagectomy in the Department of Thoracic Surgery at the provincial hospital affiliated with Shandong University from January 2006-June 2009 were enrolled in this study. TNM staging was determined by the criteria established by the International Union Against Cancer (UICC) in 2009 and lymph node dissection was undertaken according to the lymph node mapping system for esophageal cancer established by the American Joint Committee on Cancer (AJCC) in 1997.The expression of CCR7 and MUC1 was detected by immunohistochemistry. SPSS 19.0 was used to create databases for data analysis. For continuous variables, Student’s t-test was performed. The correlation between CCR7/MUC1 protein expression and pathological parameters was determined by the chi-square test or Fisher’s exact probability test (two-tailed tests). Survival and recurrence rates were calculated by the Kaplan-Meier method and analyzed by the log-rank test.Spearman correlation analysis was used to evaluate the correlation of CCR7 and MUC1 in ESCC.Results:In 153 ESCC tissue samples, there were 88 cases with CCR7 positive expression and 97 cases with MUC1 positive expression, the expression of CCR7 and MUC1 were both correlated with lymph nodes metastasis(P<0.01, P<0.001). And no significance was found according to age, gender, tumor size, differentiation, depth of invasion. Survival analysis showed that the positive expression of CCR7/MUC1 were both correlated with a higher 3-year recurrence rate(P<0.01, P<0.001) and a lower 5-year survival rate(P<0.01, P<0.01).And the expression of CCR7 was positively correlated with the expression of MUC1 in ESCC tissue (P<0.001).Conclusion:Co-expression of CCR7 and MUC1 was detected in ESCC tissue sample, and both expression of CCR7 and MUC1 were correlated with lymph node metastasis, recurrence and poor prognosis of ESCC patients.Part ⅡEffect of CCL21/CCR7 on MUC1 expression and molecular mechanism.Objective:To explore the effect of CCL21/CCR7 on MUC1 expression in ESCC cell lines and reveal the underlying molecular mechanism.Methods:qRT-PCR, Western-blot and Immunofluorescence staining were used to detect the expression level of CCR7 and MUC1 in ESCC cell lines. Western-blot was used to detect the activation of Akt and Erk pathway in ESCC cell lines. The phosphorylation of Spl was detect by Western-blot. The activity of MUC1 promoter was determined by luciferase reporter system and the binding of Spl to MUC1 promoter at -99/-97 was detected by ChIP.Results:In ESCC cell lines KYSE150,KYSE410,KYSE450 and Eca9706, the lowest expression of CCR7 and MUC1 was detected in KYSE150, and higher expression level of CCR7 and MUC1 was detected in KYSE410 and Eca9706. qRT-PCR showed a remarkable increases in the mRNA level in KYSE410, KYSE450 and Eca9706 cells with a higher expression level of CCR7, while there was no significant change of MUC1 in KYSE150. qRT-PCR and Western-blot confirmed that CCL21 could increase the mRNA level of MUC1 in a dose- and time-dependent manner in KYSE410 and Eca9706 cells. Furthemoreheterologous expression of CCR7 could up regulate the expression of MUC1 in KYSE150 after treated with CCL21. Western-blot confirmed that CCL21/CCR7 could activate the Akt and Erk pathway while inhibiting Erk but not Akt could suppress the up-regulation of MUC1 and the activity of MUC1 promoter in KYSE410 and Eca9706. Then increasing phosphorylation of Spl and binding of Spl to MUC1 promoter were detected by Western-blot and ChIP and inhibiting Erk could suppress the phosphorylation of Spl and binding of Spl to MUC1 promoter.Conclusion:CCL21/CCR7 could up regulate the expression of MUC1 via Erk-Spl pathway in ESCC cell lines.PartⅢRole of MUC1 in CCL21/CCR7 induced migration and invasion of ESCC cell linesObjective:To explore the role of MUC1 invasive biological behavior induced by CCL21/CCR7.Methods:Transwell assay was performed to explore the migration and invasion abilities of ESCC cells. Silencing of MUC1 ESCC cell lines were obtained by transfected of lentivirus containing specific siRNA targeted to MUC1 and ESCC cell lines of heterogeneous CCR7 were obtained by transfected of lentivirus containing f CCR7 cDNA.Results:. Transwell assay confirmed that CCL21 could promote migration and invasion abilities of KYSE410 and Eca9706 compared to the corresponding control group in vitro, and CCL21 could promote migration and invasion abilities of KYSE150 with heterogeneous CCR7 compared to the control group. Silencing MUC1 could significantly suppressed migration and invasion abilities of KYSE410 and Eca9706 induced by CCL21/CCR7.Conclusion:Up-regulation of MUC1 was responsible for the migration and invasion induced by CCL21/CCR7.