| Lung cancer is the major cause of cancer-related lethality among human cancer patients globally. Non-small cell lung cancer(NSCLC) accounts for nearly 80% of all lung cancer, including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. There is approximately 70% of NSCLC diagnosed at an advanced stage or with metastasis, and these patients often with low five-year survival rate and less than 15%. The poor prognosis of this cancer is mainly explained by metastasis. Metastasis of cancer cells is a major killer of patients and interacts between cancer cells and host tissues through a complex process. The molecular mechanism underlying metastasis remains unclear. As a result, the identification of functional metastasis genes and their molecular mechanisms underlying the metastatic process will contribute effective treatment of lung cancer and reduce the mortality.The microenvironment regulates the metastasis, and in vivo studies have proved some microenvironment-mediated mechanisms, including the extracellular matrix(ECM). ECM outside is required for balancing cell and tissue homeostasis and cancer cellular migration. Metastatic cancer cells are usually highly mobile and use multiple ECM-degrading proteases including matrix metalloproteinases(MMPs) to enable invasion. These structures have some common components, cell adhesion molecules and regulators of the actin cytoskeleton and so on. A large amount of evidence has testified that MMP, especially membrane type-1 MMP(MT1-MMP) and MMP-2 are important to invasion and metastases of various carcinomas including non-small cell lung carcinomas through degradation of ECM. It has been reported that the metalloproteinase domains of MMP and ADAM has high sequence homology through Catalytic site. ADAM is involved in the invasion and metastases of human lung cancers like MMP.A disintegrin and metalloprotease(ADAM) belongs to the protease family. ADAMs can degrade ECM and shed the membrane-bound precursors that modulate cell-cell and cell-matrix interactions. There are two types of ADAMs: membrane-anchored ADAM and secreted type ADAM. Secreted type ADAM is also called A Disintegrin and Metalloprotease with Thrombospondin Motifs(ADAMTS). It has been proved that ADAMTS can degrade ECM, promoting invasion and metastasis. ADAMTS has been detected in numerous tumors. Some examples such as ADAM-8,-12,-15 and-28 are highly expressed in non-small cell lung cancer, ADAM-9 and-12 in breast cancer, ADAM-9 in liver cancer, ADAMTS4 and ADAMTS5 in glioblastomas.ADAMTS5 is a member of the protease family. It is highly expressed in glioblastomas. Focusing on lung cancer, this article displayed that the expression of ADAMTS5 was high in non-small cell lung cancer with lymph nodes metastases. Meanwhile, in cell level, we found the expression of ADAMTS5 was high in A549 cells, H1299 cells and Spca-1 cells. When we silence ADAMTS5 with sh RNA(short hairpin RNA), the motility of A549 cells reduced in vitro. These findings suggest a novel function for ADAMTS5 in regulating cancer progression by increasing tumor cell migration.Objective:To detect the expression of a disintegrin and metalloprotease with thrombospondinmotifs 5(ADAMTS5) in non-small cell lung cancer(NSCLC) and to investigate the correlation of ADAMTS5 with clinicopathologic variables in NSCLC. To evaluate the particular effect of ADAMTS5 on NSCLC cell migration and clarify the role of ADAMTS5 in tumor metastasis and invasion.Methods:1. Collect eight lung cancer tissue samples and eight normal tissue samples from patients underwent surgery at Department of Pathology, Affiliated Hospital of Nantong University. Western blot(WB) was used to detect the expression level of ADAMTS5 of the 8 pair NSCLC tissues and adjacent non-tumorous tissues. Western blot was used to detect the expression of ADAMTS5 in three NSCLC cell lines, such as A549, H1299 and Spca-1. Immunofluorescence analysis(IF) was used to detect the location of ADAMTS5 in NSCLC cells.2. One hundred and forty Lung cancer sections from patients underwent surgery between 2007 and 2010 at Department of Pathology, Affiliated Hospital of Nantong University. Affiliated Hospital of Nantong University Hospital is good at formalin-fixed and paraffin-embedded for histopathologic diagnosis and immunohistochemical study. Expression of ADAMTS5 was analyzed by immunohistochemistry(IHC) on 140 paraffin-embedded slices. Pearson ?2 test was performed to analyze the associations of ADAMTS5 expression with clinicopathologic. COX regression analysis and Kaplan–Meier analysis were used to evaluate the contribution of ADAMTS5 expression on the outcome of NSCLC patients.3. With liposome transfection method, A549 cell line with high expression of ADAMTS5 transfected with ADAMTS5-sh RNA to establish ADANTS5 gene silencing cell A549-ADAMTS5-KD. Then cell motility was determined by wound healing assays and transwell migration assay.4. Western blot(WB) was used to detect the expression of E-cadherin as well as vimentin. It is aimed to clarify the role of ADAMTS5 in tumor metastasis and invasion.Results:ADAMTS5 was over-expressed in human NSCLC tissues and NSCLC cell linesTo explore a possible role of ADAMTS5 in NSCLC, western blot analysis was performed to exam different expression level of ADAMTS5 in eight paired tumors and adjacent non-tumor tissues of NSCLC, and in three cell lines including NSCLC cell lines A549, H1299 and Spca-1. We found that ADAMTS5 was significantly higher in tumor tissues than the adjacent non-cancerous lung tissues in most cases. Then we examined the basic expression of ADAMTS5 in human NSCLC cells A549, H1299 and Spca-1. We can find in Fig. 1B, the endogenous ADAMTS5 in three NSCLC cell lines expressed remarkably, which consistent with its expression pattern in NSCLC tissues. To further evaluate ADAMTS5's location, IF assay was performed to exam the location of ADAMTS5 in A549 cell line. As show in Fig. 1C, ADAMTS5 located in the cytoplasm significantly.Correlation of ADAMTS5 expression with clinicopathological parameters in NSCLCTo further evaluate the expression of ADAMTS5 and its clinical significance in NSCLC, immunohistochemical staining was performed to determine the expression of ADAMTS5 in 140 NSCLC tissues. We found that ADAMTS5 was seen predominantly in the cytoplasm. Increased expression of ADAMTS5 was found in the sample tissues with lymph node metastasis. Then the expression tendency of ADAMTS5 was associated with tumor pathology grade. Its expression was up-regulated in poor differentiation NSCLC tissues than well differentiation NSCLC tissues.Moreover, we analyzed the association between ADAMTS5 expression and clinicopathologic parameters of the patients, which were summarized. High ADAMTS5 expression shows a significant correlation with gender(p=0.043), lymph node metastasis(p<0.001), clinical stage(p=0.036), pathology grade(p<0.001) and grade(p<0.001), which was no statistical relationship between the presence of ADAMTS5 with smoking status or distal metastasis status.Survival analysis of ADAMTS5 expressionThe Kaplan-Meier survival curve was used to analyze the prognostic significance of ADAMTS5. The result shown, we found that the high expression of ADAMTS5 was significantly related with poor patients' survival(p<0.001). In a word, ADAMTS5 might be a prognostic factor for patients' recurrence status. Besides, univariate survival analysis showed that gender(p=0.011), tumor size(p=0.001), lymph node metastasis(p<0.001), pathology grade(p<0.001), grade(p<0.001) and ADAMTS5 expression(p<0.001) were prognostic factors of overall survival. Additionally, the Cox proportional hazards model was used to demonstrate that gender, tumor size, pathology grade and ADAMTS5 expression were independent prognostic indicators of overall survival.ADAMTS5 was positively correlated with the cancer metastasis and invasionSince ADAMTS5 was associated with lymph node metastasis, pathology grade and grade which were correlated with the migration of lung cancer, we proposed that ADAMTS5 may play a key role in the metastasis and invasion of lung cancer. The expression of ADAMTS5 was high in tissues with lymph nodes metastases, and low in tissues without lymph nodes metastases. We have already proved that the endogenous expression of ADAMTS5 was remarkably high in three NSCLC cells lines. So we were interested in investing the function of ADAMTS5 in cell level. Since the protein level in three NSCLC cell lines was not obvious, but A549 cell was in the shape of spindle, then we chosed A549 cell which had high expression level in three NSCLC cells.Low expression of ADAMTS5 reduced NSCLC cellular metastasis and invasionTo further research the role of ADAMTS5 in NSCLC cellular metastasis and invasion, A549 cells were transfected with control-sh RNA, ADAMTS5-sh RNA#1, ADAMTS5-sh RNA#2, ADAMTS5-sh RNA#3, and ADAMTS5-sh RNA#4 and the Western blot was used to assess the protein level of ADAMTS5. The findings showed that the ADAMTS5 protein levels were remarkably decreased in A549 cells which transfected with ADAMTS5-sh RNA#1, ADAMTS5-sh RNA#2, ADAMTS5-sh RNA#3, and ADAMTS5-sh RNA#4 compared with the cells transfected with control-sh RNA, meanwhile ADAMTS5-sh RNA#1 had the highest knockdown efficiency. As we all know, cell motility is indispensable for cancer metastasis and invasion. We performed wound healing assays and trans-well assays to determine the influence for ADAMTS5 to promote cell migrate. By creating the slower closure of a wound, which is scratched into a confluent epithelial monolayer, the wound healing process could be decreased by low expression of ADAMTS5. Meanwhile, the low expression of ADAMTS5 could decrease the cell migration to the bottom chambers which compared to the control as shown. Therefore,the results show that ADAMTS5 does increase cell motility.The mechanism of ADAMTS5's influence on NSCLC cells' metastasis and invasionThe expression of ADAMTS5 in cells transfected with ADAMTS5-sh RNA#1 was significantly decreased. We also found that the downregulation of ADAMTS5 caused an decrease in the expression of epithelial marker E-cadherin and a increase in the expression of mesenchymal marker vimentin. In summary, the result confirms that the downregulation of ADAMTS5 decrease the motility of NSCLC cells.Conclusions:1. ADAMTS5 might play a negative regulatory role in non-small cell lung cancer, and the over expression of ADAMTS5 may promote the progression and invasion of NSCLC. ADAMTS5 was up-regulated in NSCLC tissue and the expression of ADAMTS5 was positively associated with lymph node, clinical stage and histological differentiation. High expression of ADAMTS5 predicted poor prognosis of NSCLC patients.2. ADAMTS5 was positively correlated with the cancer metastasis and invasion. It may play a key role in the degradation of ECM. Inhibition of ADAMTS5 could restraint the motility of NSCLC cells through reducing the degradation of ECM. Targeting ADAMTS5 and the signal pathway of ADAMTS5 might become a potential treatment of NSCLC. |
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