Overexpression Of MUC1 Enhances The Development Of NSCLC Through Activation Of AKT And ERK Pathways

ObjectMucin 1(MUC1) is a transmembrane glycoprotein that is aberrantly unregulated in many types of cancer including non-small cell lung cancer(NSCLC). It is known that angiogenesis is an important process required for tumor genesis, growth, invasion and metastasis. Vascular endothelial growth factor(VEGF) in the tumor tissues is the main mechanism of angiogenesis. In this study, we study the expression of MUC1 and VEGF and analyze the correlation between them in NSCLC.We also make a further research on the effect and molecular mechanism of MUC1 over expression in lung cancer angiogenesis.MethodsThe expression of MUC1 and VEGF m RNA was detected by realtime PCR in cancer tissue and adjacent non-cancerous tissues. The expression of MUC1 and VEGF was detected by immunohis-tochemistry(IHC) in 100 cases cancer tissue. The correlation between MUC1 and VEGF expression was analyzed by Spearman’s rank correlation test. Expression of MUC1 was detected in non-small cell lung cancer cell lines by realtime PCR. MUC1 was transfected into A549 and NCI-H460 cells with a low level of endogenous MUC1, two non-small cell lung cancer(NSCLC) cell lines. RT-PCR and western blot analysis was used to examine the expression of MUC1 at both m RNA and protein levels. Transwell migration assay and tube formation assay were used to analyze the effects of MUC1 overexpression on vascular endothelial growth factor(VEGF)-dependent endothelial cell migration and tube formation in A549 and NCI-H460 cells. MTT assay was used to analyze the effects of MUC1 overexpression onproliferation of cells. Clone formation assay was used to analyze the effects of MUC1 overexpression on the colony formation ability of cells.The effect of MUC1 overexpression on the adhesion of NSCLC cells was also examined.Cell cycle distribution was determined by flow cytometric analysis of propidium iodide-stained cells.Enzyme-linked immunosorbent assay(ELISA) was used to measure the production of VEGF m RNA and protein in A549 and NCI-H460 cells, which were transfected with MUC1.A549 cells were stably expressed by the lentiviral vector system.MTT assay and nude mouse tumorigenicity assay were used to analyze the effects of MUC1 over expression on proliferation of cells. Western blot analysis wasused to detect the expression and phosphorylation of Akt,extracellular signal-regulated kinase(ERK), p38,and c-Jun N-terminal kinase(JNK) in A549 and NCI-H460 cells transfected with MUC1 gene. ERK and AKT inhibitor, PD9859 and SH-5, were used to pretreat A549 and NCI-H460 cells individually. Tube formation assay was used to analyze the effects of PD9859 and SH-5 on the overexpression of MUC1-induced tube formation. ResultsThe expression of MUC1 and VEGF m RNA was upregulated in cancer tissues compared with adjacent non-cancerous tissues in NSCLC. The expression levels of MUCl and VEGF in NSCLC had statistically correlation with tumor size, pathological types, differentiation, lymph node metastasis and TNM stage. But the expression of MUCl and VEGF was not related to the age and history of smoking. There was a positive correlation between MUC1 and VEGF in NSCLC(r=0.380, p<0.01). The expression of MUC1 was upregualted in A549 and NCI-H460 cells transfected with MUC1 plasmid using RT-PCR and Western blot.At the same time, the RT-PCR and ELISA showed that overexpression of MUC1 in NCI-H460 and A549 cells significantly enhanced the expression and secretion of VEGF. We showed that enforced expression of MUC1 in A549 and NCI-H460 promoted their ability to induce vascular endothelial growth factor(VEGF)-dependent endothelial cell migration and tube formation. The effect of MUC1 induced endothelial cell migration and angiogenesis could be reversed by VEGF neutralizing antibody. The cell cycle distribution and adhesion to fibronectin was not altered by MUC1 overexpression. The overexpression of MUC1 can significantly improve the cell proliferation and cell cloningability.The results of animal experiments showed that the expression of MUC1 gene could significantly promote the growth of A549 cells, tumor weight and tumor volume were significantly higher than the control group.MUC1 overexpression resulted in a marked elevation in phosphorylated Akt and extracellular signal regulated kinase(ERK) 1/2, indicative of activation of both signaling pathways. Most importantly, inhibition of Akt or ERK signaling using specific chemical inhibitors restrained the pro-angiogenic activity of MUC1-overexpresing NSCLC cells. ConclusionsThe expression of MUC1 and VEGF m RNA was upregulated in NSCLC tissues which may be involved in carcinogenesis and progression in NSCLC,the expression of MUC1 was positively correlated with VEGF.Overexpression of MUC1 promotes expression and secretion of VEGF and induces VEGF-dependent endothelial cell migration and tube formation and facilitates lung cancer cell proliferation and angiogenesis.Overexpression of MUC1 was beneficial for the cloning and proliferation of lung cancer cells.In addition,our present data demonstrate that the aberrant upregulation of MUC1 favors tumor angiogenesis in NSCLC, likely through the activation of both Akt and ERK pathways and elevation of VEGF production. MUC1 may thus be a potential antiangiogenic target in NSCLC.