| Background and significanceInvasion and metastasis are the primary causes of death in patients with malignant tumors, and also are the problems have not yet been solved in the therapeutic field of malignant tumor, therefore, investigate the molecular mechanisms of invasion and metastasis, and corresponding therapeutic strategies is of very important significance. The exact mechanisms of malignant tumor invasion and metastasis are still incompletely clear, but is well known composed of a series of multiple steps with inner links, continuous and multistage extremely complicated dynamic process of interactions of tumor cells with host cells and surrounding microenvironment, involving the bioadhesion reduction between tumor cells, close attachment of tumor cells to the basement membrane, degradation of the extracellular matrix, tumor cells penetrate into blood vessels or lymphatics, arrest at distant sites by adhesion to endothelial cells, invasion outward and induction of angiogenesis, evasion of host antitumor responses and continue to grow at the metastatic sites. Theoretically, blocking any link in the above multistage and continuous process may block the invasion and metastasis of malignant tumor cells.With the continuous progressions in research, it's well known that the growth, invasion and metastasis of tumor cells need a large number of newly born blood vessels, tumor angiogenesis is the process of microvascular growth induced by tumor cells and establishment of blood circulation in tumor microenvironment, plays very important roles in the process of growth, invasion and metastasis of malignant tumors, and then puts forward the new approach to suppress tumor growth-antiangiogenic therapy, has become an important strategy in the modern therapeutic fields of tumors. Tumor angiogenesis is an extremely complex and coordinating process, regulated by a tight balance of various factors at different levels. Antiangiogenic therapy, with new blood vessels for acting targets, by cutting off the nutrients supply and metastatic path to prevent growth, invasion and metastasis of tumor cells, is a kind of important method of targeted therapy. Most angiogenesis regulatory factors are downstream molecules to the present study so far, the role of a single factor is weak, if one of these regulatory factors is blocked, the tumor cells can still regulate angiogenesis through other factors and signaling pathways, therefore, it urgently needs to find a key molecule that can regulate a wide variety of factors involved in tumor angiogenesis.Metastasis-associated gene1(MTA1) is a recently identified gene closely correlated with invasion and metastasis of a variety of human malignant tumors, and it encodes a protein with715amino acid residues. MTA1protein is one of the highly conserved proteins in evolution, located in cell nucleus, as one of transcriptional corepressors component of the nucleosome remodeling and histone deacetylation complex, plays important regulating roles in the signaling pathways of growth, invasion and metastasis of malignant tumor cells, has become a hot molecule in the research field of malignant tumors. Recently, MTA1protein has received more attention as a potent angiogenic factor with regard to its roles involved in tumor angiogenesis. It has been demonstrated that MTA1protein is closely correlated with tumor angiogenesis in bladder cancer, gastric cancer, breast cancer and prostate cancer, and MTA1protein can promote tumor angiogenesis by regulating HIF-1α, VEGF-A and other downstream molecules, suggesting that MTA1protein may be a potential angiogenesis-promoting key molecule of malignant tumors.Tumor angiogenesis plays essential roles in the process of growth, invasion, metastasis and treatment resistance of non-small cell lung cancer and esophageal cancer, however, to the best of our knowledge, the expression of MTA1protein and its angiogenic roles in connection with tumor angiogenesis in non-small cell lung cancer and esophageal cancer has not yet been elucidated so far. In this study, we perform the following three-part research:(1) Detect the expression levels of MTA1protein in patients with stage I non-small cell lung cancer, investigate the relationship of MTA1protein to the clinicopathologic factors, tumor angiogenesis and prognosis;(2) Suppress MTA1protein expression in non-small cell lung cancer cells by RNA interference in vitro, investigate the roles of MTA1protein in regulating migration, invasion and angiogenic ability;(3) Detect the expression levels of MTA1protein in patients with esophageal cancer, evaluate the relevance of MTA1protein expression to the tumor progression, angiogenesis and prognosis.Part I The relationship of MTA1protein to the tumor angiogenesis and prognosis in patients with stage I non-small cell lung cancerObjective:To detect the expression levels of MTA1protein in patients with stage I non-small cell lung cancer, and investigate the relationship of MTA1protein to the clinicopathologic factors, tumor angiogenesis and prognosis.Methods:One hundred and two patients with pathologic stage I non-small cell lung cancer that successfully underwent curative surgical resection plus systematic lymph node dissection at the Department of Thoracic Surgery, Qilu Hospital, Shandong University, were enrolled in this study. Immunohistochemical staining for MTA1and CD34was performed using the streptavidin-peroxidase method, and intratumoral microvessel density (MVD) was recorded by counting CD34-positive immunostained endothelial cells. All statistical analyses were performed with SPSS13.0statistical software. The x2test was performed to examine the association between MTA1protein, MVD and variable clinicopathologic factors. Kaplan-Meier method was used to calculate the survival curves, and log-rank test was used to compare the statistical significance of difference between the survivals of patient subgroups. Multivariate Cox regression analysis was used to identify the significantly independent prognostic factors.Results:MTA1protein overexpression was detected in40.2%cases, no statistically significant correlation was found between MTAl protein expression and any clinicopathologic factors (P>0.05). High MVD was detected in45.1%cases, and significantly associated with histology (P=0.034). The χ2test showed that MTA1protein overexpression was significantly associated with high MVD (P=0.008). MTA1protein overexpression (P=0.004) and high MVD (P=0.007) were significantly associated with tumor relapse, respectively. Univariate survival analysis showed that patients with MTAl protein overexpression and high MVD had a significantly poor disease-free survival (P=0.001and0.004), overall survival (P=0.005and0.043) and disease-specific survival (P=0.006and0.031) at5years after operation, respectively. Multivariate Cox regression analysis showed that high MVD was an independent prognostic factor for unfavorable disease-free survival (P=0.044), while MTA1protein overexpression was an independent prognosticator for unfavorable disease-free (P=0.011), overall (P=0.024) and disease-specific survival (P=0.046), respectively.Conclusions:MTA1protein overexpression is common in stage I non-small cell lung cancer, and significantly associated with tumor angiogenesis and poor survival. These findings suggest that MTA1protein may have clinical potential as a promising predictor to identify individuals with poor prognostic potential and a possible novel target molecular of antiangiogenic therapy for patients with non-small cell lung cancer. Part Ⅱ The roles of MTA1protein in regulating migration, invasion and angiogenic ability of non-small cell lung cancer cells in vitroObjective:To suppress MTA1protein expression in non-small cell lung cancer cells by RNA interference in vitro, and investigate the roles of MTA1protein in regulating migration, invasion and angiogenic ability.Methods:The expression levels of MTA1mRNA and protein were detected in non-small cell lung cancer cell lines A549, H292, H460and95-D by real time PCR and western blot, respectively. MTA1siRNA was transfected into the cell line with the highest expression level of MTA1gene by liposome2000, fluorescently labeled negative control sequence was used to detect the transfection efficiency, real time PCR and western blot were used to detect the inhibition effect of siRNA on the expression of MTA1mRNA and protein. Wound healing assay was performed to detect cell migration ability; Transwell invasion assay was performed to detect cell invasive ability; Zymography assay was performed to detect MMP-9activity; Tube formation assay was performed to detect cell angiogenic ability.Results:Different expression levels of MTA1mRNA and protein were detected in these4non-small cell lung cancer cell lines, of which95-D cell line showed the highest expression level. Forty-eight hours after MTA1siRNA transfection, the results of real time PCR and western blot showed that the expression of MTA1mRNA (P<0.01) and protein (P<0.01) in95-D cells were both inhibited significantly. After MTA1protein expression was decreased by siRNA, wound healing assay showed that the migration ability of95-D cells was significantly inhibited (P<0.01); Transwell invasion assay showed that the invasive ability of95-D cells was significantly inhibited (P<0.01); Zymography assay showed that the MMP-9activity was significantly decreased (P<0.01); Tube formation assay showed that the angiogenic ability of95-D cells was significantly inhibited (P<0.01).Conclusions:Inhibition of MTA1protein expression could effectively down-regulate the migration, invasive and angiogenic abilities of95-D cells in vitro, suggesting that MTA1protein plays important regulating roles in promoting migration, invasive and angiogenic abilities of95-D cells, MTA1protein might be a potential novel target of antiangiogenic therapy for patients with non-small cell lung cancer. Part III The relationship of MTA1protein to the tumor angiogenesis and prognosis in patients with esophageal cancerObjective:To detect the expression levels of MTA1protein in patients with esophageal cancer, and evaluate the relevance of MTA1protein expression to the tumor progression, angiogenesis and prognosis.Methods:One hundred and thirty-one patients with pathologic esophageal squamous cell cancer that successfully underwent subtotal esophagectomy and esophago-gastric anastomosis plus regional lymph node dissection at the Department of Thoracic Surgery, Qilu Hospital, Shandong University, were enrolled in this study. Immunohistochemical staining for MTA1and CD34was performed using the streptavidin-peroxidase method, and MVD was recorded by counting CD34-positive immunostained endothelial cells. All statistical analyses were performed with SPSS13.0statistical software. The χ2test was performed to examine the association between MTA1protein immunoreactivity and patients'clinicopathologic factors. The correlation between intratumoral MVD and patients'clinicopathologic factors and MTA1protein immunoreactivity was analyzed by nonparametric test. Kaplan-Meier method was used to calculate the survival rate, and log-rank test was used to compare the differences between the survival rates of the patient subgroups divided by clinicopathologic factors. Multivariate survival analysis was performed that included the variables significant in the univariate analysis to identify the most significant independent prognostic factors for predicting survival.Results:MTAl protein high expression was detected in42.7%cases and significantly correlated with invasion depth (P=0.041), lymph node metastasis (P=0.021) and pathologic stage (P=0.003). The staining intensity of MVD varied broadly from3.8to93.4/high-power field, and MVD was significantly correlated with differentiation (P=0.032) and invasion depth (P=0.018). Mann-Whitney U test showed that the intratumoral MVD in tumors with MTA1protein high expression was significantly more than that in those with MTA1protein low expression (P=0.044). MTA1protein high expression was significantly associated with tumor relapse (P=0.031). The overall5-year survival rate was37.4%, Univariate survival analysis showed that patients with MTA1protein high expression had significantly poor overall5-year survival (P=0.002), and the factor found on multivariate Cox regression analysis to significantly affect overall survival was only pathologic stage (P=0.040). Further stratified survival analysis split by pathologic stage demonstrated that MTA1protein high expression significantly predicted unfavorable prognosis among patients with pathologic stage Ⅱ disease (P=0.006).Conclusions:MTA1protein high expression is common in esophageal squamous cell cancer, and is closely associated with tumor progression, increased tumor angiogenesis and poor survival. These findings indicate that MTA1protein has clinical potentials as a useful indicator of progressive phenotype, a promising prognostic predictor to identify patients with poor prognosis and a potential novel therapeutic target of antiangiogenesis for patients with esophageal squamous cell cancer.
|
PDF Full Text Request