Abnormal Expression Of VEGFR-1and Its Mechanism Of Regulation Effect On Snail And MMP-9Associated With Metastasis And Invasion In Hepatocellular Carcinoma

Background and ObjectiveHCC is one of the leading cancers threatening human life and its incidence is increasing.1Despite of some advances in early diagnosis and therapeutic strategies, HCC still shows high postsurgical recurrence, rapid progression and extremely poor prognosis. Understanding of the molecular mechanisms regulating the metastatic and invasive behavior of this malignant tumor is essential for improving the treatments. Moreover, as we know, the prognosis of HCC patients with the same stage and clinico-pathological features often differs in spite of curative surgical resection. Thus, many investigations have focused on the discovery of specific molecular markers that could meet clinical requirements for precise prediction of HCC prognosis. To date, however, the search for valuable molecules remains substantially limited. Activation of growth factor receptors is one of the most important mechanisms for survival and invasion of human cancers. Vascular endothelial growth factor receptor-1(VEGFR-1), one of three typical tyrosine kinase receptors for vascular endothelial growth factor, is a cell membrane-bound tyrosine kinase receptor. It was initially found in endothelial cells and has been demonstrated to play roles in the assembly of endothelial cells and modulation of blood vessel maturation during vasculogenesis and angiogenesis. Emerging evidence, however, suggests that VEGFR-1is also present and functional in cancer cells and promotes invasiveness by a non-angiogenesis-dependent pathway.Intrahepatic spread of HCC often involves degradation of the underlying extracellular matrix barrier through proteolysis to promote invasion and dissemination. MMPs generated by cancer cells appear to contribute to key steps in cancer metastasis. Among MMPs, MMP-9is particularly interesting because it can be induced by Snail or VEGFR-1in the course of cancer invasion. In this regard, the current study focused on investigating expressions of VEGFR-1in both cell lines and liver tissues of patients with HCC, and analyzing the correlation among VEGFR-1, Snail and MMP-9. Furthermore, the levels of these proteins expression were compared with clinico-pathological features and patients'outcomes to evaluate clinical and prognostic value. Based on this data, we address the hypothesis that MMP-9was involved in the activation of VEGFR-1mediated invasion. The present study was also aimed to investigate this possible mechanism of MMP-9regulation and its role in activation of VEGFR-1-mediated invasion. Part IExpression and Clinical value of VEGFR-1, Snail and MMP-9in Hepatocellular CarcinomaObjective1. To detect the location and expression level of the VEGFR-1, Snail and MMP-9in different hepatocellular carcinoma cell lines, and analyze the relation with invasion ability of HCC cell lines.2. To evaluate the correlation between expression level of these three proteins and clinical pathology features, such as TNM stage, tumor multiplicity, tumor tissue encapsulation and so on.3. To analyze the correlation between VEGFR-1, Snail and MMP-9.4. To evaluate the correlation between expression level of these three proteins and recurrence and prognosis of patients with HCC.Methods1. Western blot and immunofluorescence were used to detect expression of VEGFR-1, Snail and MMP-9in4HCC cell lines, respectively.2. The expression of VEGFR-1, Snail and MMP-9was confirmed on the samples from95HCC patients underwent curative resection using immunohistochemistry.3. Receiver operating characteristic (ROC) analysis was used to determine the predictive value of the parameters, such as VEGFR-1, Snail and MMP-9and so on.Results1. Expression of VEGFR-1, Snail and MMP-9proteins in hepatocellular carcinoma cell lines(1) VEGFR-1, a protein band of approximately180kDa, was detected in all4HCC cell lines, with HUVECs used as positive controls. Simultaneously, Snail and MMP-9were also detected. Immuno fluorescent was carried out on the SMMC-7721cell line. VEGFR-1was present in the cytomembrane, Snail in the cell nucleus, and MMP-9mainly in the cytoplasm. While the negative control showed no specific fluorescence.(2) The results of RD by normalizing with P-actin suggested that one cell line with high expression of VEGFR-1also had high expression of Snail and MMP-9. For the MHCC-97H (p<0.001for VEGFR-1, p<0.001for Snail, and p<0.01for MMP-9vs. Hep3B cell line, respectively).2. Expression and relationship of VEGFR-1, Snail and MMP-9in HCC tissues The expression of VEGFR-1, Snail and MMP-9were in accord with the expression in the hepatocellular carcinoma cell lines. High VEGFR-1and Snail expression were observed in43/89(48.3%) and49/92(53.3%), respectively. The scores of VEGFR-1and Snail in cancerous tissues were significantly higher than that in non-cancerous tissues (p<0.001for both), while the level of MMP-9in cancerous tissues was lower than in non-cancerous tissues (p=0.044). Based on the immunohistochemical score, we found that cancerous tissues with high VEGFR-1also showed increased Snail and MMP-9expression with a Pearson correlation coefficient of r=0.418(p<0.001) and r=0.232(p=0.036), respectively. However, there was no significant relationship between expression of Snail and MMP-9in cancerous tissues (p=0.242).3. Correlation with clinical features of patients with HCC Patients with high VEGFR-1or Snail were prone to have a high TNM stage, high rates of intrahepatic metastasis, microvascular invasion, and poor differentiation (p<0.05). Snail expression was also associated with capsular invasion (p<0.05). No features, except intrahepatic metastasis (p<0.05), correlated with high MMP-9expression.4. Univariate Prognostic Analysis of patients with HCC The features that correlated with RFS and OS (p<0.05log-rank test) were tumor size, microvascular invasion, tumor multiplicity, tumor tissue encapsulation, TNM stage. HBsAg positive was also associated with poor OS (p=0.046). Patients with high VEGFR-1expression were statistically less than the median RFS and OS times for patients with low expression (p<0.001and p=0.023, respectively). Patients with high Snail expression tended to have poor RFS and OS (p<0.0001for both). However, there was no difference between high MMP-9expression and low expression in RFS or OS (p>0.05for both, data not shown).5. Multivariate Prognostic Analysis of patients with HCC Both VEGFR-1and Snail were independent risk factor for RFS (Hazard ratio [HR]=1.876,/?=0.003HR=1.759,p=0.032, respectively) and OS (HR=1.72, p=0.044and HR=2.362, p=0.023, respectively).6. Combination of VEGFR-1and Snail expression and ROC analysis The predictive value of the combination VEGFR-1and Snail was the best. Additionally, it can also precisely death. The area under the curve of this combination was0.784(95%CI,0.642to0.927; p=0.002) for recurrence and0.685(95%CI,0.567to0.803; p=0.005) for death.Conclusion1,VEGFR-1distinctively expressed in the cytomembrane of Hepatocelluar carcinoma cells.2,High expression of VEGFR-1was associated with Hepatocelluar carcinoma progression and worse outcome.3,High co-expression of VEGFR-1and Snail may be a novel prognostic marker for prognosis of patients with curative resection of hepatocellular carcinoma, especially in recurrence. Part ⅡEnhance of invasion ability through upregulation of Snail and MMP-9in hepatocellular carcinoma cell line in response to activation of VEGFR-1Objective1. To observe the changes in migration and invasion of HCC cells after activation VEGFR-1.2. To investigate the changes in the expression level of MMP-9after activation VEGFR-1and to observe the changes in invasion of HCC after blocking the proteinase activity using a function-blocking anti-MMP-9antibody (Ab-1).3. To detect the changes of expression level of Snail after activation VEGFR-1.Methods1. Wound healing and invasion assays were used to test the changes in migration and invasion after activation VEGFR-1by VEGF-B167in SMMC-7721cells.2. Western Blot and qRT-PCR were used to observe the change of VEGFR-1and Snail after activation VEGFR-1in SMMC-7721cells and HepG2cells.3. Gelatin zymography assay was used to detect the changes of MMP-9gelatinolytic activity after activation VEGFR-1in SMMC-7721cells and HepG2cells.4. The specific role of MMP-9expression in invasion of HCC cells was determined by blocking the proteinase activity via a function-blocking anti-MMP-9antibody (Ab-1).5. Basing on the result of IHC of TMAs, the relationship between the expression level of VEGFR-1and MMP-9in cancer tissues and prognosis and pathologic features was analyzed.Results1. VEGFR-1activation promotes cell migration and invasion of SMMC-7721cells(l)Wound healing and invasion assays were used to test the changes in migration and invasion after activation VEGFR-1by VEGF-B167in SMMC-7721cells. At the point of12h and24h post-scratch, the relative cell migration was60.5±1.8%and8.4±1.2%, respectively for the untreated group,60.3±1.1%and12.3±1.6%, respectively for the non-specific IgG group, but58.6±1.1%and1.4±1.1%, respectively for the VEGF-B167-treated group. There was no significant increase in the migration among these three groups in initial12h. However, the motility of the VEGF-B167-treated cells was significantly increased compared to the non-specific IgG treated cells or untreated cell after24h (p<0.05).(2) Matrigel Invasive assay was assessed the effect of VEGFR-1on invasion. Invasion of cells treated with VEGF-B167were significantly increased compared to that of the cell treated with non-specific IgG after48h (p<0.05).2. VEGFR-1activation induces MMP-9expression in HCC cell lines(1) The level of MMP-9mRNA expression in the cell treated with VEGF-B167for32h was increased by4.3-fold and2.6-fold in SMMC-7721and HepG2cells, respectively. The expression level was declined at48h post treatment.(2) Western Blot indicated that the level of MMP-9protein, detected as two bands at92kDa and82kDa, was increased in these representative two cell lines. Gelatin zymography assay showed that MMP-9gelatinolytic activity in the culture medium from VEGF-B167-treated SMMC-7721cells was significantly higher than that in the non-specific IgG group or untreated cells. However, MMP-2activity was barely detectable. In contrast, in the VEGF-B167-treated HepG2cells, the activity of both MMP-9and MMP-2were increased in comparison with the controls.(3) Immunofluorescence analysis also showed that the increased MMP-9was mainly localized in the cytoplasm of both SMMC-7721and HepG2cells.3. VEGFR-1activation promotes MMP-9-dependent Matrigel Invasion A broad-spectrum pharmacologic MMP inhibitor (GM6001) and a functional blocking anti-MMP-9antibody (Ab-1) were used to block the proteinase activity. VEGFR-1activation-induced Matrigel invasion was completely blocked after addition of GM6001, indicating that metalloproteinase activity is required for the cellular invasion. To examine if MMP-9is involved in the cellular invasion, functional blocking antibody against MMP-9was used and the results showed that abrogation of MMP-9activity significantly reduced cell invasion by approximately84%when compared with SMMC-7721cells treated with VEGF-B167alone.4. VEGFR-1activation induces Snail expression in HCC cell lines(1) The results showed that mRNA level of Snail in SMMC-7721and HepG2were increased by8.5-fold and2.2-fold after28h and24h of VEGF-B167treatment, respectively.(2) Western blot analysis also indicated the Snail protein level was increased after VEGF-B167treatment.Conclusion1,Activation of VEGFR-1can promote progression and invasion of hepatocellular carcinoma via the proteolytic effect of MMP-9and it may be a novel mechanism of metastasis and invasion of hepatocellular carcinoma.2,Abnormal expression and activation of VEGFR-1in hepatocellular carcinoma can promote invasion and metastasis via upregulating the expression of MMP-9, while Snail maybe participates and mediated this process.