Study On Epithelial-Mesenchymal Transition Induced By VEGFR-1 Activation Promoting Invasion And Migration In Hepatocellular Carcinoma

BACKGROUNDS: It's estimated that new incidence and fatality of hepatocellular carcinoma(HCC) every year was 564,000, 549,000 in the world and 306,000, 300,000 in China, respectively. It indicated that morbility and mortality was the most high in China. It's been increasing in recent years. Though measures for treating HCC were various, the effect was dissatisfactory. Metastasis and recurrence of HCC was the maximal barrier influencing therapeutic effect. It's unclear that how HCC cells obtain the potential of invasion and metastasis. To explore molecular mechanism about invasion and metastasis of HCC exhibited important theoretical significance and clinically practical value.OBJECTIVES: Although VEGFR-1 initially were believed to be expressed exclusively on endothelial cells (ECs), recent studies had demonstrated the presence of VEGFR-1 on malignant tumour cells from epithelial cells, such as pancreatic cancer, breast cancer, colon carcinoma and prostatic carcinoma cells and so on. It suggested that VEGFR-1 activation could correlate with invasion and migration of malignant tumours. Howover, the molecular mechanism that activated VEGFR-1 how to promote malignant tumour cells invading and migrating were required another investigation. HCC was blood vessel-rich malinant tumours from epithelial cells. It's known to all HCC cells expressed high vascular endothelial growth factor(VEGF), but whether HCC cells expressed VEGFR-1 and what role VEGFR-1 played and how VEGFR-1 played role in invasion and migration of HCC were unclear.METHODS: To confirm whether HCC cells expressed VEGFR-1 and how VEGFR-1 played role in invasion and migration of HCC, we proposed following experiments: Firstly, we immunohistochemically examined the expressing pattern of VEGFR-1, epithelial markers E-cadherin,α-catenin and mesenchymal markers vimentin, N-cadherin in 82 hepatocellular carcinoma specimen and assessed correlation between VEGFR-1 expression and clinicopathological parameters. It's valuated the clinical significance and correlation between VEGFR-1 and cell phenotype markers. Secondly, we screened hepatocellular carcinoma cells lines expressing VEGFR-1. HCC cells lines that expressed VEGFR-1 were used as target cell for following experimental study. Reverse transcription polymerase chain reaction (RT-PCR), western blotting and immunofluorescence were performed to assess changes of epithelial markers E-cadherin,α-catenin and mesenchymal markers N-cadherin, Vimentin after VEGFR-1 activation to investigate whether activated VEGFR-1 could induce epithelial mesenchymal transition in hepatocellular carcinoma cell lines. Lastly, c-Src inhibitor PP2(4-amino-5-(4-chlorophenyl)-7-(tbutyl) pyrazolo [3,4-d]-pyrimidine) and negative control PBS(phosphate buffered solution) were added to investigate the signal transduction pathway of epithelial-mesenchymal transition(EMT) induced by activated VEGFR-1 in HCC cell lines.RESULTS:1. VEGFR-1 was present in 59 detected HCC tissue specimen(59/82, 71.95%); 52 HCC specimen expressed no E-cadherin(52/82, 63.41) and 30 expressed E-cadherin(30/82, 36.59%); 39 HCC specimen expressed noα-catenin(39/82, 47.56%), 43 HCC specimen were positive(43/82, 52.44%); 37 HCC specimen(37/82, 45.12%) expressed N-cadherin,44 HCC specimen expressed Vimentin(44/82, 53.66%). It demonstrated that in hepatocellular carcinoma cells deletion expressing of epithelial markers E-cadherin andα-catenin by statistical analysis, positive expressing of mesenchymal markers N-cadherin and Vimentin correlated significantly with clinically pathological factors such as tumour staging, differentiation, tumour amicula and vein invasion,which showed that the attenuated expressing of epithelial markers and accentuated expressing of mesenchymal markers synergially contributed to invasion and migration of HCC. The expressing of VEGFR-1 correlated strikingly with tumour staging, differentiation, tumour amicula and vein invasion and deleted expressing of E-cadherin,α-catenin and positive expressing of Vimentin, N-cadherin. It suggested that VEGFR-1 promoting invasion and metastasis correlated with phenotypic transition of hepatocellular carcinoma cells in HCC.2. Hepatocellular carcinoma cell line MHCC97-H with high potential of metastasis expressed VEGFR-1 mRNA and protein by RT-PCR and western blotting analysis. The expressing level of VEGFR-1 correlated positively with potential of metastasis of HCC cell lines. HCC cell lines with high potential of metastasis had high expressing of VEGFR-1. In all 4 HCC cell lines detected, MHCC97-H had potential of metastasis at 100% and the expressing level of VEGFR-1 mRNA and protein was the most high. Therefore, we took MHCC97-H cell line as the target cell for following study to identify whether activation of VEGFR-1 could induce EMT in HCC.3. After VEGF-B stimulating MHCC97-H cells for 48h, we observed differences in the gross appearance of VEGF-B- treated cells as compared with untreated cells. The phenotypic changes observed included loss of cell polarity causing a spindle-cell morphology, increased intercellular separation signifying loss of intercellular adhesion, and increased formation of pseudopodia observed emanating from the cell membrane, which preliminarily suggested that activation of VEGFR-1 could induce EMT in HCC cells. A VEGFR-1 neutralizing antibody 18F1 abrogated the above changes of cell morphology and maintained the untreated cell morphology. RT-PCR, western blotting and immunofluorescent staining were performed to assess expressing of epithelial markers E-cadherin,α-catenin and mesenchymal markers Vimentin, N-cadherin after activation of VEGFR-1. Results showed that expressing of epithelial markers E-cadherin,α-catenin decreased in cell membrane and increased in cytoplasm, mesenchymal markers Vimentin, N-cadherin increased in cytoplasm. Results of cell migration and invasion test showed VEGF-B induced at least a fourfold increase in migration compared with the control. The increased migration was blocked by pretreatment with the neutralizing VEGFR-1 antibody 18F1. VEGF-B induced a fourfold increase in invasion compared with the control. Pretreatment with the VEGFR-1 neutralizing antibody 18F1 abrogated this effect, which indicted activation of VEGFR-1 enhanced the capability of migration and invasion. A VEGFR-1 neutralizing antibody 18F1 was added to block VEGFR-1, we could not observe phenomenon in experimental group. It suggested that VEGFR-1 activation could induce EMT in HCC cells.4. Results of interventing experiments showed that VEGF-B could not induce changes that were consistent with EMT after a c-Src kinase specific inhibitor PP2 was added to treat MHCC97-H cell. MHCC97-H cell retained epithelial cell morphology and cell-cell junction. Epithelial markers E-cadherin andα-catenin protein expressed primarily in cell membrane and the quantity of proteinα-catenin and E-cadherin were unchanged; Mesenchymal markers Vimentin and N-cadherin protein expressed principally in cytoplasm and the quantity of mesenchymal proteins were unchanged. Results of cell migration and invasion assay demonstrated VEGFR-1 activation could not enhance the ability of invasion and migration in HCC cells after PP2 were added to block VEGFR-1. After PBS were added to pretreat MHCC97-H cells, VEGFR-1 activation induced morphologic changes consistent with EMT in MHCC97-H cells. It demonstrated that Src kinase signal transduction pathway was one of pathways that mediated EMT of cancer cells and it's confirmed PP2 was a selective tyrosine kinase inhibitor for c-Src and was used widely to study the signal transduction pathway on Src kinase. In view of above experimental results, we thought that EMT induced by VEGFR-1 activation was mediated via Src kinase signal transduction pathway. Src was one of the key factors that regulated EMT induced by VEGFR-1 activation and inhibition targeted to Src could be a effective measure for interventing in invasion and migration of HCC.CONCLUSION:1. VEGFR-1 was present in HCC cells and it's expression correlated with invasion, migration and poor prognosis of HCC. VEGFR-1 was one of hallmarker for predicting the invasion and migration of HCC.2. VEGFR-1 activation could promote MHCC97-H cells invading and migrating by inducing epithelial-mesenchymal transition.3. Epithelial mesenchymal transition induced by VEGFR-1 activation was mediated- Src kinase signal transduction pathway. Src was a key factor that regulated EMT induced by VEGFR-1 activation in HCC cells and could be an effective target for interferencing in invasion and migration of HCC.