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Hepatocellular carcinoma (HCC) is one of the most deadly human cancers because of its high incidence of metastasis. Despite extensive efforts, therapies against metastasis of HCC remain underdeveloped. Vacuole membrane protein 1 (Vmp1) was recently identified to be involved in cancer-relevant processes; however, its expression, clinical significance and biological function in HCC progression are still unknown. Therefore, we evaluated the expression of Vmp1 in human HCC specimens. To functionally characterize Vmp1 in HCC, we upregulated its expression in HCCLM3 cells using a plasmid transfection approach, following which both in vitro and in vivo models were used to elucidate its role. A significant downregulation of Vmp1 was found in human HCC tissues and closely correlated with multiple tumor nodes, absence of capsular formation, vein invasion and poor prognosis of HCC. Such expression was verified with HCC cell lines including HepG2, MHCC97-L and HCCLM3, and the Vmp1 expression levels negatively correlated with metastatic potential. Interestingly, upregulation of Vmp1 significantly affects proliferation, migration, invasion and adhesion of HCCLM3 cells. Using a mouse model, we demonstrated that upregulation of Vmp1 was associated with suppression of growth and pulmonary metastases of HCC. Therefore, our data suggest Vmp1 is a novel prognostic marker and potential therapeutic target for metastasis of HCC.
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of death from cancer, resulting in more than 600 000 deaths per year.[1, 2] During the past decade, the long-term survival rate remains unsatisfactory because of a high incidence of recurrence and metastasis after hepatic resection, with a 5-year actuarial recurrence rate of 75–100% reported in the literature. To predict recurrence, metastasis and prognosis in patients with HCC after hepatic resection is a clinical issue of great significance. Various prognostic markers of HCC have been identified, such as vascular endothelial growth factor (VEGF), osteopontin (OPN) and transforming growth factor-β (TGF-β). Based on a genomic analysis, we previously reported ras homologous gene C (RhoC), high mobility group A1 (HGMA1) and Wiskott–Aldrich syndrome protein family verprolin-homologous protein 2 (WAVE2) as potential markers for metastasis of HCC.[4-7] All these findings represent significant progress in this field. However, the mechanisms underlying HCC metastasis are still not fully understood, supporting a need for further studies. Thus, exploring new prognostic markers and further inhibition of invasion and metastasis is of great importance in HCC therapies.
It is well known that reduced levels of cell–cell adhesion proteins often correlate with tumor invasion and metastasis. The loss of functional tight junctions would further result in changes of cytoskeletal organization, which in turn might lead to a higher invasive potential of the tumor cells. Vacuole membrane protein 1 (Vmp1) is a conserved putative membrane protein whose function is now beginning to be elucidated. In Drosophila, Vmp1 (known as TANGO5) was identified in a functional genomic screen and found to be required for protein secretion and Golgi organization. It has previously been reported that the overexpressed protein is an inhibitor of cell proliferation, anchorage-independent growth and secretory membrane transport. Moreover, recent studies have demonstrated that Vmp1 is necessary in autophagy and its expression induces the formation of autophagosomes.[14, 15] Interestingly, the expression profiling on a cDNA array and another complementary DNA microarray revealed that VMP1 was downregulated in renal cell carcinoma.[16, 17] Furthermore, Vmp1 is an essential component of initial cell–cell contacts and tight junction formation where it colocalizes with zonula occludens-1 (ZO-1) in spots between neighboring cells. Downregulation of VMP1 by RNAi results in loss of cell adherence and increased invasion capacity.
However, evidence for the function of Vmp1 in human malignancies is still limited, especially in the case of HCC. Therefore, we carried out the present study to determine the expression of Vmp1 in human HCC tissues as well as cell lines. Furthermore, the biological functions of Vmp1 in HCC were also elucidated in vitro and in vivo.
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Alterations in cell–cell and cell–matrix adhesion seem to have a central role in facilitating tumor cell migration, invasion and metastatic dissemination. The overexpressed human Vmp1 was reported to localize in the endoplasmic reticulum (ER) and Golgi complex, where it appeared to be required for protein secretion and Golgi organization, as well as organelle biogenesis and multicellular development. Besides its different functions including membrane traffic, growth and autophagy,[14, 15, 28, 29] interestingly, recent reports showed that Vmp1 was located in the plasma membrane playing a critical role in cell–cell contact.[18, 23, 24] These results suggest a role for Vmp1 in tumor relevant cellular processes, which is further supported by expression profiling data.[16, 17] However, the expression profile and function of Vmp1 in HCC remains unknown, and the correlation between its expression and prognosis of patients has not been documented at present. On this basis, we aimed to define the cancer-relevant processes Vmp1 is involved in and how this protein is associated with HCC characteristics and prognosis.
Our data revealed that both mRNA and protein levels of Vmp1 were significantly decreased in HCC tissues compared with those in the corresponding PCLT and NL tissues, and were even lower in PVTT compared with HCC (primary tumors). The prediction of recurrence, metastasis and prognosis in patients with HCC after hepatic resection is an important clinical issue, which could determine surgical therapeutic regimens. Analysis of the association of Vmp1 expression and the clinicopathological characteristics in 124 HCC patients revealed that Vmp1 downregulation was significantly correlated with multiple tumor nodes, absence of capsular formation and vein invasion of HCC, which are widely accepted factors associated with metastasis and poor prognosis of HCC. Furthermore, the expression level of Vmp1 protein was significantly downregulated in NHCC when compared with SLHCC and SHCC, which correlates with the higher metastasis potential of NHCC than that of SLHCC, supporting the correlation between Vmp1 expression levels and metastasis in HCC. The Kaplan–Meier analysis showed that the HCC patients with low Vmp1 expression in general had worse prognosis than those with high Vmp1 expression. A multivariable Cox regression analysis indicated that low Vmp1 expression is an independent risk factor for the prognosis of HCC patients, suggesting that Vmp1 might serve as a useful prognostic biomarker of HCC.
Of particular interest is the correlation between Vmp1 expression and the ability of HCC cells to metastasize, which was uncovered when we analyzed Vmp1 expression in three HCC cell lines (HepG2, MHCC97-L and HCCLM3) with different spontaneous metastatic potential. HepG2 cells exhibited a moderate metastatic potential whereas HCCLM3 cells were highly invasive, as demonstrated by extensive metastases via both subcutaneous and orthotopic inoculation. In agreement with the difference in metastatic potentials, expression of Vmp1, both in mRNA and protein levels, was markedly lower in the HCCLM3 cell line when compared with the MHCC97-L and HepG2 cell lines, suggesting an association of downregulation of Vmp1 with the metastatic potential of HCC. As a previous study has demonstrated, this system can serve as a useful model for the study of HCC metastasis.
To gain insights into a role for Vmp1 in HCC metastasis, we used a plasmid transfection approach to specifically upregulate the expression of Vmp1 in HCC cells and characterized the changes. Our data show that upregulation of Vmp1 expression resulted in marked inhibition of proliferation, migration and invasion of HCCLM3 cells. The adhesion test confirmed that upregulation of Vmp1 is associated with higher homogeneity adhesion and lower heterogeneity adhesion ability, further suggesting that the expression level of Vmp1 determines the invasion capacity of cancer cells. Together, these results support a critical role for Vmp1 in the invasion and metastasis of HCCLM3 cells. Such a role for Vmp1 is further supported by our in vivo study in which we showed that upregulation of Vmp1 inhibited proliferation of primary tumors of HCC, as well as the metastatic ratio of pulmonary metastases.
Vmp1 localization in mammalian cells has been described to be in the ER, Golgi apparatus, autophagosomes and the plasma membrane. Our immunohistochemical staining showed Vmp1 is mostly distributed in cytoplasm and in a punctate pattern in the plasma membrane. This phenomenon is in accordance with the finding of Sauermann et al., that is:
…Vmp1 precisely colocalized with Calnexin in both ER and vacuoles. However, in cells with low overexpression of Vmp1, Vmp1 was found in the plasma membrane distributed in a punctate pattern, suggesting that localization in the ER was due to accumulation of protein caused by high level of overexpression.
Vmp1 is predicted by Hirokawa et al. to be a seven transmembrane domain protein with major parts of the protein located towards the extracellular space. Proteins destined for the plasma membrane mostly pass through the ER and Golgi network, and it is possible that an overexpressed protein with seven transmembrane helices is not folded correctly, leading to its retention in the endoplasmic reticulum. Furthermore, recent studies indicate that Vmp1 is essential for cell–cell contacts and tight junction formation, and it colocalizes with the tight junction protein ZO-1 in spots between neighboring HEK293 cells.[18, 23, 24] Meanwhile, ZO-1 is important for clustering of claudins and occludin, resulting in the formation of tight junctions.[33, 34] To further confirm the subcellular localization of Vmp1 and its regulating mechanism, we performed co-immunoprecipitations with HCCLM3VMP1+ cell lysates using a ZO-1 capture antibody, confirming the direct interaction of these two proteins in HCC cells. As ZO-1 plays an important role in cancer-related cell biological systems,[35, 36] Vmp1's direct interaction with ZO-1 might provide some evidence on the phenomenon that Vmp1 controls HCC behaviors thorough regulation of cell–cell contacts.
Vmp1 has been associated with different functions including autophagy[14, 15, 28, 29] and cell adhesion,[18, 23, 24] while there are still uncertainties in its underlying mechanism of tumor suppression, whether it is functioning in autophagy (when present in the ER) or in cell adhesion (when located in the plasma membrane). Autophagy has recently emerged as a key regulator of multiple aspects of cancer biology and autophagy deficiency leads to tumorigenesis in the liver.[37, 38] Impairment of autophagy causes accumulation of p62, which might contribute to hepatoma development. In previous published studies, elevated p62 was reported in human HCC, and immunohistochemistry staining of p62 was significantly different between the frequencies of tissues with cancer (62.5%) and paracarcinomatous tissues (0%) in patients with HCC. As it has been reported that p62 colocalizes with upstream autophagy factors such as VMP1, an interesting hypothesis is that downregulation of Vmp1 might cause suppression of autophagy, which is always accompanied by marked accumulation of p62. Also, the overexpression of p62 as a result of autophagy inhibition was shown to be important in the promotion of tumorigenesis through a variety of mechanisms, including deregulation of NF-&kB signaling, accumulation of ROS and increased DNA damage. Additionally, p62 binds to Keap1, leading to the upregulation of NRF2,[43, 44] and persistent NRF2 activation appears to be critical for anchorage independent growth of hepatocellular carcinoma cells in the context of p62 overexpression. Another possible mechanism is that downregulation of VMP1 and the consequent lack of Vmp1 protein (which directly interact with ZO-1) at the plasma membrane lead to failure of cell junction assembly, resulting in loss of cell–cell adhesion and subsequent cell detachment. Disruption or failure in the formation of intercellular junctions is commonly associated with metastasis of cancer cells, and loss of cell–cell adhesion is a prerequisite for tumor cell invasion. All of these possibilities need to be explored further.
In conclusion, the present study has shown for the first time that downregulation of Vmp1 significantly correlates with a poor prognosis of HCC. Furthermore, we have demonstrated that Vmp1 inhibits metastasis of HCC by affecting cell adherence. Collectively, our data suggest Vmp1 as a novel prognostic marker and a potential therapeutic target for metastasis of HCC.