SDF-1 And CXCR4 Axis Interaction In Tumor Growth And Metastasis Of Non Small Cell Lung Cancer

Backgroud and AimsLung cancer is the most commonly diagnosed malignancy and the leading cause of cancer-related deaths worldwide. This high mortality is probably attributable to early metastasis. The greedy invasiveness of NSCLC cells results in an extremely poor average 5-year survival rate in patients so far. Recent studies indicate that tumor cells express chemokine receptors and may use chemokine-mediated mechanisms during the metastasis process. Among the chemokines and chemokine receptors identified to date, the membranous CXC chemokine receptor 4 (CXCR4) and its ligand, stromal-cell-derived factor 1α(SDF-1αor CXCL12), has been found to play a key role in tumorigenicity, proliferation, metastasis and angiogenesis. Although the mechanisms underlying SDF-1a/CXCR4-mediated tumor invasion have been studied in many cancers, the role of SDF-1a/CXCR4 in the process of lung cancer cells proliferation and migration remains largely unknown.Therefore, the expression of CXCR4 in NSCLC patients was detected by RT-PCR and immunohistochemistry in this study. Based on the results, the role and mechanism of SDF-1/CXCR4 axis in metastasis of lung cancer were further studied by up-regulation expression of CXCR4 in lung cancer cells in vitro and in vivo.Methods(1) Immunohistochemical staining and RT-PCR were used to detect the expressions of CXCR4 in 60 cases of NSCLC tissues. The relationship of CXCR4 experssion in NSCLC was analyzed in the pathologic types, differentiation degrees and TNM stages.(2) CXCR4 stable over expression human lung cancer cell lines (A549) were established by CXCR4 transfection (CXCR4-A549). The migration and invasion assay was performed to detect the migration ability both in CXCR4-A549 and A549 cells. In vivo, CXCR4-A549 and A549 cells were injected subcutaneously into nude mice(each group in 6).Four weeks later the mice were scarified and the tumors in situ and the lungs were taken out to be examined histologically.(3) Th cell cycle distribution was measured by flow cytometry to analyse the effect of SDF-1/CXCR4 in the cell proliferation and atoposis. The changes of the intracellular calcium concentration after SDF-1 activation were aslo measured by flow cytometry. Western blot was used to anlayse the phosphorylation of AKT and ERK, and the expression of MMP-2 and VEGF-C.Results(1) The results of immunohistochemical staining analyzing showed that there were 50 of 60(83.3%) cases with positive staining of CXCR4 protein. Staining of CXCR4 protein was identified in the cell membrane and/or cytoplasm of cancer cells with a rate in 83.3%, but not significantly detected in the normal lung cells taken from non-cancerous regions in 38.3%, adjacent to lung cancer tissue in31.7%. Comparing the expression of CXCR4 protein bewwent adenoid (87.1%) and squamous (79.3%) carcinoma, high, medium and lower differentiation cells, there were no significant differences statistically. However, the expression of CXCR4 was significantly associated with TNM stages, stage I in 72.7%,stage II in 83.9%,stage III in 88.9%(P<0.05). Also the differential expression of CXCR4mRNA was confirmed by semi-quantitative RT-PCR in lung cancer cells and normal lung cells (P<0.05).(2) A stable p-EGFP-C1 expressive plasmid with a CXCR4 sequence was constructed and transfected into A549 cells. With a hogh expression of CXCR4, it demonstrated the CXCR4-A549 cells were able signifcantly to increase expression of the protine by Western blotting analysis. The migration of A549-CXCR4 cells was increased 1.62 times than that of of A549 cells (P<0.05). The chemotactic activity in both of both A549-CXCR4 and A549 cells induced by SDF-1 were improved, but the former much more than the latter in vitro (P<0.05). To assay the tumor formation and metastasis ability induced by over expression of CXCR4 in vivo, twelve nude mice were resepectively inoculated subcutaneously with CXCR4-A549 cells and A549 cells (six mice in each group). The mean weight of the tumors respectively was 4.37±0.96g and 3.24±1.16g in A549-CXCR4 and A549 group (P<0.05). Moreover, the metastatic tumors were affirmatively formed microscopically and pathologically in 6 mice iduced by A549-CXCR4 cells group. In contrast, there were no typical metastatic tumors formed just only 2 mice with invasive tumor cells within the lungs in A549 cells group. It was suggestted the ability of prolifation and invasion of lung cancer cells with CXCR4 over expression.(3) Compared with A549 control cells, CXCR4 over expression increased the proportion of the cells in G2 and S phase in CXCR4-A549 cells and the proliferation index was added 9.72% and proportion of the cells apoptosis was decreased 3.60%. But there was no significant difference between CXCR4-A549 and A549 cells. Subsequently, though intracellular calcium was increased rapidly in a time-dependent mode while both CXCR4-A549 and A549 cells stimulated with SDF-1. However, the mean fluorescence in CXCR4-A549 cells was stronger than that in A549 cells. There was a significant difference in MMP-2 and VEGF-C expression between CXCR4-A549 and A549 cells by Western blot and RT-PCR analysis. These results indicated that regulation of CXCR4 expression was corelated to MMP-2 and VEGF-C activity in lung cancer cells. Phosphorylation of AKT and ERK was strongly increased after stimulation with SDF-1 in 100ng/ml during maximum 30 min. It was suggested that MAPK and PI3K pathway activated by SDF-1 with CXCR4 might be cooperated in metastasis of lung cater cells.Conclusions(1) There were no significant differences in expression of CXCR4 protein between histological types or differentiation degrees of the lung cancer cells. However, it was significantly associated with TNM stages.(2) CXCR4 over expression was functionally actived by SDF-1a. Sequently, migration and invasion of lung cancer cells were promoted the in vivo and in vitro either.(3) The calcium channel might cooperated with MAPK/ERK pathway activated by SDF-1/CXCR4 axis and PI3K/AKT pathway resulting in the activity of downstream mediators, MMP-2 and VEGF-C activity in metastasis of lung cater cells.Based on the results in this study, the role and mechanism of SDF-1/CXCR4 axis in metastasis of lung cancer both in vitro and in vivo were aproached. SDF-1a/CXCR4 axismay be a potential target for NSCLC therapy.