Overexpression Of Vmp1 Inhibits Metastasis Of Hepatocellular Carcinoma Via Regulating ZO-1

Hepatocellular carcinoma (HCC)is one of the most common malignancies in the world.Because of the high incidence, high recurrence and high mortality, HCC has already become the important risk for the people's health in China and all over the world. Thus,exploring further inhibition of invasion and metastasis is of great importance in the HCC therapies.Vacuole membrane protein 1 (Vmp1)is a recently identified conserved putative membrane protein, whose function is now beginning to be elucidated. Recent studies have demonstrated that Vmp1 is required for protein secretion, organelle organization, as well as organelle biogenesis and multicellular development. Vmp1 is recently identified to be importantly involved in cancer-relevant processes, including membrane traffic, proliferation, growth and autophagy. These results suggest a critical role of Vmp1 in tumor relevant cellular processes.More importantly and interestingly, recent reports showed that Vmp1 is an essential component of initial cell-cell contacts and tight junction formation, which playing a critical role in cell-cell adhesion. However, the function of this protein and its mode of action in tumor progression are still unknown. Therefore, we carried out the present study to determine the expression of Vmp1 in human HCC tissues as well as cell lines and attempted to elucidate the function of Vmp1 in the metastasis of HCC by characterizing its role in cell adherence via using both in vitro and in vivo models.1.Semi-quantitative PCR, real-time quantitative PCR and Western blot were employed to detect Vmpl mRNA and protein in 38 cases of HCC fresh tissues,paracarcinomatous liver tissues (PCLTs), and 5 portal vein tumor thromboses (PVTTs). Our results showed that both mRNA and protein levels of Vmpl were significantly decreased in HCC tissues than those in the corresponding PCLTs and NL tissues, even lower in PVTT compared to HCC.Immunohistochemical staining showed a cytoplasmic and membranic distribution of Vmpl,and the positive expression rate of Vmpl was significantly lower in HCC (81 of 124, 65.3%)than that in PCLT(108 of 124,87.1%). The Vmp1 expression levels were found significantly lower in HCCs with multiple nodules, without capsule formation and with vein invasion. Furthermore, the expression level of Vmpl protein was significantly down-regulated in nodular HCC (NHCC)when compared with solitary large HCC (SLHCC) and small HCC(SHCC).Moreover, HCC patients of the low Vmp1 expression group had both poorer disease free survival and poorer overall survival than those of the high Vmp1 expression group.By multivariable Cox regression analysis,low Vmp1 expression was found to be an independent prognostic factor for overall survival.2.We further confirm the Vmp1 expression in three HCC cell lines: HepG2, MHCC97-L and HCCLM3,with a liver cell line CCL13 as a control.The results of RT-PCR and Western blot showed a significantly lower expression of the Vmp1 mRNA and protein in HCCLM3 and MHCC97-L cells than those in HepG2 and CCL13 cells.Among the three HCC cell lines analyzed, HCCLM3 cells have the lowest Vmp1 expression, followed by MHCC97-L and HepG2.Of note is that the reduced expression of Vmp1 were in agreement with the metastatic potential of these HCC cell lines, suggesting a potential role for Vmp1 in HCC metastasis.3.To further define the correlation of Vmp1 expression and HCC metastasis,we employed plasmid transfection approach to up-regulate the expression of Vmp1 in HCCLM3 cells.We successfully constructed recombinant plasmid pCMVtag2C-VMP1.After FuGENE 6 transfection and three-week selection in media supplied with G418,HCCLM3 cell lines, stably transfected with the VMP1-expressing vector or control vector, were obtained and named as HCCLM3VMP1+ and HCCLM3vector respectively. The expression of Vmp1 mRNA and protein were markedly increased in HCCLM3VMP1+ compared with HCCLM3vector, which showed a satisfactory transfection efficiency. By MTT method, an obvious decrease of proliferation was observed in HCCLM3VMP1+ cells compared with HCCLM3vector cell.The wound-healing assay showed that the closure of HCCLM3VMP1+was significantly slower than that of HCCLM3vector. The results of Transwell invasion assay showed the number of HCCLM3VMP1+ cells that passed through matrigel was only 34% as compared with HCCLM3vector cells.Considering the close relation of Vmp1 with cellular adhesion, the adhesion test confirmed HCCLM3VMP1+ cells had significantly higher homogeneity adhesion and lower heterogeneity adhesion ability than HCCLM3vector cells.Together, these results support a critical role for Vmp1 in the adherence and metastasis of HCCLM3 cell.4.We further examined the in vivo relevance of the potential role for Vmp1 in HCC tumorigenesis and metastasis by using a nude mice implantation model.Primary subcutaneous tumor of HCCLM3 was measured every five days and mice were sacrificed 35 days after implantation. We found that the average size of primary tumors in HCCLM3VMP1+ group was dramatically smaller than that of HCCLM3vector group,which suggested that up-regulation of Vmp1 in vivo could inhibit growth of HCC.The expression of Vmp1 in HCCLM3 tumor was determined to ensure the difference of Vmp1 expression in vivo.The results showed that the expression of Vmp1 was significantly increased in HCCLM3VMP1+ tumor than that in HCCLM3vector tumor. The pulmonary metastasis was observed in the lung tissue sections of only two mice in HCCLM3VMP1+ group (two of seven,28.6%),significantly less than the ratio of pulmonary metastasis in HCCLM3vector group(six of seven,85.7%).Together, these data support an important role for Vmp1 in HCC metastasis.5.Since Vmp1 is essential for cell-cell contacts and tight junction formation, where it colocalizes with ZO-1 in spots between neighboring cells, we further explore Vmp1's regulating effect on ZO-1.Firstly, co-immunoprecipitations confirmed the direct interaction of Vmp1 and ZO-1 proteins in HCC cells.Next, the corresponding ZO-1 protein expression of HCCLM3VMP1+ and HCCLM3vector cells were detected, but revealed no significant change at total protein level.Further immunoprecipitation and Western blotting of HCCLM3 MP1+ and HCCLM3vector cells showed ZO-1 was highly tyrosine-phosphorylated in HCCLM3vector, but was dephosphorylated in HCCLM3VMP1+,confirming the hypothesis that up-regulation of Vmp1 decreases the tyrosine phosphorylation of ZO-1,which leads to up-regulation of the function of ZO-1,and these phenomena contribute to inhibition of HCC metastasis.In conclusion, our study has shown for the first time that down-regulation of Vmp1 significantly correlates with poor prognosis of HCC.Furthermore, we have demonstrated Vmpl inhibits metastasis of HCC by affecting cell adherence through reduction of ZO-1 tyrosine phosphorylation. Collectively, our data suggests Vmp1 as a novel prognostic marker and a potential therapeutic target for metastasis of HCC.