The Mechanism Of MMP13 On The Regulation Of Large Cell Lung Cancer Vasculogenic Mimicry

Purposes Lung cancer is one of the fastest growing causes of morbidity and mortality malignant cancer in the world that impact on human health and cause life-threating。Large-cell lung cancer(LCLC) is the most aggressive neoplasm of non-small cell lung cancer(NSCLC), rapid growth and early metastasis of the lymph and blood can lead to poor prognoses. Vasculogenic mimicry(VM) is a new pattern of tumor microcirculation present in aggressive tumors, VM closely related to tumor development, metastasis and poor prognosis. There are fewer research of VM in lung cancer, this study thought to investigate the molecular mechanism role of MMP13 in VM formation of NSCLC and explore the relationship between the regulation of MMP13 and MMP2 in different blood supply modes, to provide a theoretical basis for further define tumor angiogenesis.Methods 1. Immunohistochemical and CD34/PAS double staining to detect and analyze the expression of MMP13, VM and MVD in 51 cases of human LCLC tissues. Analyze the correlation between MMP13 expression levels and clinicopathological parameters, VM and MVD of lung cancer among large cell. There was also analysis of the relationship of MMP13 expression and LCLC patients’ survival and prognosis.2. Large cell lung cancer cell lines H460 and H661 cells were culture in vitro. Using plasmid transfection method to to establish stable MMP13 up or down regulation cell lines by transfecting with pc DNA-3-MMP13 or p RNAT-MMP13 sh RNA. The tube formation, invasion and migration ability changes of up-regulated and down-regulated the MMP13 expression tumor cells were observed by three-dimensional culture in vitro. Recombinant human MMP2 and MMP13 protein were added in cultured H460 and H661 cells to observe its effect on the ability of tumor cells tube formation.3. The use of silver staining technique to detect MMP13 and MMP2 cleaved Ln-5 fragments and using mass spectrometry to detect MMP13 cleaved Ln-5 fragments to determine their amino acid sequence. Western blot and immunofluorescence to observe and analyze the EGFR, F-actin, viment and α-tublin changes after treated with exogenous MMP13 or MMP2 cleaved Ln-5 fragment on large cell lung cancer cell line H460.4. Immunohistochemical staining was used to detect the expression level of VE-cadherin in 51 cases of LCLC tissues. Analyse the correlation between MMP13 and VE-cadherin expression. Western blot and immunofluorescence detected the VE-cadherin expression level after treatment of different concentration exogenous MMP13 in cell lines H460 and H661.5. The human umbilical vein endothelial cellscellscells cells(HUVEC) were also cultured to evaluate the effects of MMP2 and MMP13 on the endothelial cell invasion and tube formation.6. Xenograft model of nude mouse by subcutaneous injection with H460 and MMP13 overexpressed H460 cells was made to investigate the effect of up regulating MMP13 on VM formation ability. The existence of VM, Ln-5 and EGFR expression were detected by immunohistochemistry and endomucin/PAS dual staining. Time depended mouse model was established to observe and analyse the correlation of MMP13 and MMP2 expression and tumor blood supply pattern in the different stages of H460 xenograft growth.Results 1. In 51 cases of LCLC, there were 20 cases showed high MMP13 expression and MMP13 was low expressed in 31 cases. MMP13 expression was positive correlated with tumor stage and tumor size. Kaplan-Meier survival analysis results showed that the overall survival in large cell lung cancer patients with high MMP13 expression was significantly lower than the low expression of MMP13, the difference was statistically significant(P<0.05). Tumor tissues with high MMP13 expression were less in the number of VM, tumors showed a larger number of VM in MMP13 low expression tissues, the difference was statistically significant(P<0.05), and MMP13 high epression tissues showed a higher MVD compared to low MMP13 expression tumor tissue in LCLC, the difference was statistically significant(P <0.05).2. In vitro cell culture models, the ability of tube formation was stronger in low express MMP13 cell line H460 than MMP13 high expression cell line H661 cells in three-dimensional culture. Up regulated MMP13 expression in H460 diminished the tube formation capacity and down regulated H661 MMP13 expression enhancement tumor cell tube formation capacity. In addition, after adding exogenous MMP13 protein the tube number formed by H460 cells was reduction.3. MMP13 degraded Ln-5 into 20 KD fragments, which can inhibit the tumor cells tube formation. The molecular weight 20 KD fragment belonged to Ln-5 protein γ2 subunits peptide analysis by mass spectrometry, its amino acid sequence was Cys540-Arg694.4. Western blot and immunofluorescence showed the H460 cells treated with Ln-5 fragments degraded by MMP2 increased EGFR and F-actin expression, vimentin and α-tublin showed no significant change. After treatment Ln-5 fragments degraded by MMP13 the EGFR and F-actin expression reduction, vimentin and α-tublin showed no significant change.5. In 51 cases of large cell lung cancer tissues, VE-cadherin high expressed in 12 tumor cases and low expressed in 31 cases. There was only one case of VE-cadherin high expressed in the MMP13 positive tissues, 11 cases VE-cadherin high expressed in 31 MMP13 negative expression cases, VE-cadherin was negatively correlated with the expression of MMP13, the difference was statistically significant(P<0.05). Western blot showed that the expression of the VE-cadherin protein of H460 and H661 cells was decreased with adding different concentrations of exogenous MMP13. Immunofluorescence also showed the the cells surface VE-cadherin expression was reduced after MMP13 treatment.6. Training exogenous MMP13 and MMP2 for HUVEC, the tube structure number of endothelial cells significantly increased in the three-dimensional medium. Transwell migration assay results indicate that, MMP13 and MMP2 can promote the migration ability of endothelial cells. Ln-5 neutralizing antibody could decrease the tube gormation and migratin capacity.7. MMP13 overexpression tumor group xenografts growth significantly lower than the H460 control group at the early tumor growth, the difference was statistically significant(P<0.05). Immunohistochemical staining showed VM, Ln-5 and EGFR expression levels in H460 MMP13 overexpression tumor xenografts lower than the H460 control group, consistent with the in vitro results.8. MMP13 expression in large cell lung cancer xenografts showing time-dependent trend. With the tumor growth, MMP13 expression in tumor cells gradually increased, and MMP13 expression was negatively correlated with VM in the xenografts, but it was positively correlated with the number of capillaries, the difference was statistically significant(P<0.05). MMP2 was high expressed during xenografts growth, no expression levels significant changes.Conclusion 1. The higher expression of MMP13 in large cell lung cancer tissues was correlated with tumor aggressive degree and poor prognosis.2. MMP13 negatively regulated Vasculogenic Mimicry formation while positively regulated endothelium dependent vessels.3. MMP13 may be through degradation of laminin-5 and inhibit EGFR/F-actin cytoskeleton changes affect Vasculogenic Mimicry formation4. The effect of MMP-13 in regulating cell migration,invasion and VM formation via degradation VE-cadherin expression.5. MMP13 promoted the migration ability of endothelium cells which resulted in promotion of endothelium dependent angiogenesis.6. MMP13 may play in the replacement of Vasculogenic Mimicry by endothelium dependent vessels to be main blood supply pattern in the late stages of tumor growth.