The Role And Mechanism Of ADAM10 In Invasion And Metastasis Of Esophageal Squamous Cell Carcinoma

BackgroundEsophageal carcinoma is one of the most predominant malignant tumors and is harmful to human health in the world, especially in China. In spite of significant advancement in surgical intervention, radiotherapy, chemotherapy and other comprehensively adjuvant therapies, the five-year survival rate of patients is still very low, and moreover, the majority of cancer deaths are attributable to tumor invasion and metastasis. Therefore, a better understanding for the molecular mechanism of tumor invasion and metastasis and discovery of a more effective target for anticancer therapy help to prolong the survival and improve the life quality of patients with esophageal carcinoma.The ADAMs(A Disintegrin And Metalloprotease domain) are modular type I transmembrane proteins those contain a metalloprotease and a disintegrin-like domain. In fact, ADAM stands for “A Disintegrin And Metalloprotease” and has become the most widespread name to designate this group of proteins. To date, 40 ADAMs have been identified in the human genome. The ADAMs family members are closely related to the degradation of basement, cell migration, cell fusion, signals transduction, etc. Therefore, ADAMs plays an important role in the progression, invasion and metastasis of cancers. ADAM10 is an important member of this family located on 15q22 and encoded proteins with 748 amino acids. ADAM10 could take part in the progression, invasion and metastasis of cancers via degrading a groups of substrate: cell adhesion molecules(CAMs), extracellular matrix(ECM), etc.However, until now there has no related report about the role and mechanism of ADAM10 in the progression, invasion and metastasis of esophageal squamous cell carcinoma at home and abroad.ObjectiveIn order to analyze the role of ADAM10 in the progression, invasion and metastasis of esophageal squamous cell carcinoma, and the relationship between the expression of ADAM10 and E-cadherin, we firstly studied the expression levels of ADAM10 and E-cadherin in esophageal squamous cell carcinoma tissues and analyzed their expression with the progression, invasion and metastasis of esophageal squamous cell carcinoma. Secondly, we inhibited the expression of ADAM10 in esophageal squamous cell carcinoma cell lines and analyzed the effect of down-regulation of ADAM10 on the invasion and metastasis ability of esophageal squamous cell carcinoma cells and the expression levels of E-cadherin in vitro. Finally, we detected the effect of ADAM10 on the growth and E-cadherin expression of xenograft tumors of esophageal squamous cell carcinoma in the nude mice in vivo.PartⅠ: The role of ADAM10 expression in esophageal squamous cell carcinoma tissuesMethods1. The immunohistochemical staining was used to measure the protein expression of ADAM10 and E-cadherin in 122 cases of esophageal squamous cell carcinoma(ESCC) tissues and 60 cases of normal esophageal mucosa tissues.2.In situ hybridization was used to detect the m RNA expression of ADAM10 and E-cadherin in 122 cases of ESCC tissues and 60 cases of normal esophageal mucosa tissues.3.All the data were analysed by using SPSS version 17.0 statistical package. The difference of count data was examined by χ2 test. P value less than 0.05 was defined as statistically significant.Results1.The positive rates of ADAM10 m RNA and protein in the esophageal squamous cell carcinoma tissues were significantly higher than those in normal esophageal mucosa tissues, and the differences were statistically significant(P<0.01). Furthermore, the expression of ADAM10 m RNA and protein in the esophageal squamous cell carcinoma tissues were positively correlated with invasion depth, lymph node metastasis and TNM staging(P<0.05), but not correlated with sex, age and histological grade(P>0.05).2.The positive rates of E-cadherin m RNA and protein in the esophageal squamous cell carcinoma tissues were significantly lower than those in normal esophageal mucosa tissues, and the differences were statistically significant(P<0.01). Furthermore, the expression of E-cadherin m RNA and protein in the esophageal squamous cell carcinoma tissues were negatively correlated with the histological grade, invasion depth, lymph node metastasis and TNM staging(P<0.05), but not correlated with sex and age(P>0.05).3.The relevant analysis showed that the expression level of ADAM10 was negatively correlated with the expression level of E-cadherin in the esophageal squamous cell carcinoma tissues(P<0.01).PartⅡ: The role and mechanism of ADAM10 on the invasion and metastasis ability of esophageal squamous cell carcinoma cell lines in vitroMethods1.The mRNA and protein expression of ADAM10 in three kinds of esophageal squamous cell carcinoma cell lines, Eca109, EC-1 and TE-1, were analyzed by using real-time PCR and western blot methods. At the same time, the primarily cultured normal esophageal epithelial cells(NEECs) were used as control.2.The invasion and metastasis ability of three kinds of esophageal squamous cell carcinoma cell lines, Eca109, EC-1 and TE-1, were analyzed by Boyden chamber invasion assay and wound healing assay, respectively. Moreover, the relationship between the expression of ADAM10 and invasion and metastasis ability in each cell line was also analyzed3.si RNA targeting ADAM10 was chemically synthesized, negative si RNA was also chemically synthesized and used as negative control. ADAM10 si RNA and negative si RNA were transfected into EC-1 cells with high invasiveness by using Lipofectamine 2000, blank EC-1 cells were used as blank control. Real-time PCR and western blot were used to examine the inhibition effect of ADAM10 si RNA on the expression levels of ADAM10 and E-cadherin.4.Boyden chamber invasion assay, wound healing assay and CCK-8 assay were used the analyzed the effects of ADAM10 si RNA on the invasion, metastasis and proliferation ability of EC-1 cells.5.All the data were analyzed by using SPSS version 17.0 statistical package. The difference of count data was examined by χ2 test. Meanwhile, quantitative data were expressed as means ± standard deviations and the difference of quantitative data was examined by t test. P value less than 0.05 was defined as statistically significant.Results1.The m RNA and protein expression of ADAM10 in three kinds of esophageal squamous cell carcinoma cell lines, Eca109, EC-1 and TE-1, were significantly higher than those in normal esophageal epithelial cells(NEECs)(P<0.01). In addition, the expression level of ADAM10 in EC-1 was highest in three kinds of cell lines, while the expression level of ADAM10 in TE-1 was the lowest, and the difference was statistically significant(P<0.05).2.The invading cell numbers and healing ability of EC-1 cells in three kinds of esophageal squamous cell carcinoma cell lines, Eca109, EC-1 and TE-1, were significantly higher than those of Eca109 cells and TE-1 cells(P<0.05), while the invading cell numbers and healing ability of TE-1 cells were significantly lower than those of Eca109 cells and EC-1 cells(P<0.05).3.The m RNA and protein expression of ADAM10 in EC-1 cells transfected with ADAM10 si RNA for 48 h were significantly lower than those in EC-1 cells transfected with negative si RNA or blank EC-1 cells(P<0.01).4.Compared with those in blank EC-1 cells and EC-1 cells transfected with negative si RNA, the m RNA and protein expression of E-cadherin in EC-1 cells transfected with ADAM10 si RNA for 48 h were significantly higher, and the difference was statistically significant(P<0.01).5.The invading cell numbers, healing ability and proliferation ability of EC-1 cells transfected with ADAM10 si RNA for 48 h were also significantly decreased compared to those of blank EC-1 cells and EC-1 cells transfected with negative si RNA(P<0.05).Part Ⅲ: The effect of ADAM10 on the growth and E-cadherin expression of xenograft tumors of esophageal squamous cell carcinoma in nude miceMethods1.In order to establish the xenograft tumor models of esophageal squamous cell carcinoma, EC-1 cells were subcutaneously injected into the right armpit of nude mice.2.The 15 nude mice which had been successfully constructed xenograft tumors were divided into 3 groups randomly, with 5 in each group. The nude mice in each group were injected with ADAM10 si RNA, negative si RNA or PBS, respectively. The volume of xenograft tumors was observed every 2 days, altogether 15 times.3.The weight of xenograft tumors was measured after the nude mice were executed. At the same time, HE staining was used to observe the morphological characteristics of xenograft tumors, real-time PCR and immunohistochemistry methods were used to examine the m RNA and protein levels of ADAM10 and E-cadherin in xenograft tumors.4.All the data were analyzed by using SPSS version 17.0 statistical package.The difference of count data was examined by χ2 test. Meanwhile, quantitative data were expressed as means ± standard deviations and the difference of quantitative data was examined by t test. P value less than 0.05 was defined as statistically significant.Results1.The volume and weight of xenograft tumors of esophageal squamous cell carcinoma in the nude mice treated with ADAM10 si RNA were significantly lower than those in the nude mice treated with negative si RNA or PBS(P<0.01). However, there was no significant difference in the tumor volume and weight between the nude mice treated with negative si RNA and PBS(P>0.05).2.The morphological characteristics of xenograft tumors in nude mice treated with ADAM10 si RNA were changed greatly compared to those treated with negative si RNA or PBS. Nuclear condensation and fragmentation, and visible point necrosis could be found under the microscope.3.Compared to those treated with negative si RNA or PBS, the m RNA and protein expression levels of ADAM10 were decreased in the xenograft tumors of esophageal squamous cell carcinoma in the nude mice treated with ADAM10 si RNA, and the difference was statistically significant(P<0.01). However, there was no significant difference in the m RNA and protein expression levels of ADAM10 between the nude mice treated with negative si RNA and PBS(P>0.05).4.Compared to those treated with negative si RNA or PBS, the m RNA and protein expression levels of E-cadherin was increased in the xenograft tumors of esophageal squamous cell carcinoma in the nude mice treated with ADAM10 si RNA, and the difference was statistically significant(P<0.01). However, there was no significant difference in the m RNA and protein expression levels of E-cadherin between the nude mice treated with negative si RNA and PBS(P>0.05).Conclusions1.ADAM10 was overexpressed in esophageal squamous cell carcinoma tissues and cells and its overexpression was positively correlated with the invasion and metastasis of esophageal squamous cell carcinoma. ADAM10 might play an important role in the invasion and metastasis of esophageal squamous cell carcinoma.2.ADAM10 si RNA could silence the expression of ADAM10 effectively, and moreover, inhibit the invasion, metastasis and proliferation ability of EC-1 cells and regulate the expression of E-cadherin in vitro. ADAM10 could participate in the invasion and metastasis of esophageal squamous cell carcinoma via regulating the expression of E-cadherin.3.ADAM10 siRNA could also decrease ADAM10 expression and increase E-cadherin expression in xenograft tumors of esophageal squamous cell carcinoma in the nude mice and inhibit the growth of xenograft tumors. ADAM10 could be used as a molecular target in molecular targeted therapy.