Vacuole membrane protein 1 (Vmp1) is described as a cancer-relevant cell cycle modulator, but the function of this protein and its mode of action in tumor progression are still unknown. In this study, we show that the VMP1 mRNA level is significantly reduced in kidney cancer metastases as compared to primary tumors. Further, VMP1 expression is also decreased in the invasive breast cancer cell lines HCC1954 and MDA-MB-231 as compared to the non-invasive cell lines MCF-12A, T-47D and MCF-7. We show for the first time that Vmp1 is a plasma membrane protein and an essential component of initial cell–cell contacts and tight junction formation. It interacts with the tight junction protein Zonula Occludens-1 and colocalizes in spots between neighboring HEK293 cells. Downregulation of VMP1 by RNAi results in loss of cell adherence, and increases the invasion capacity of the non-invasive kidney cancer cell line Caki-2. In conclusion, our findings establish Vmp1 to be a novel cell–cell adhesion protein and that its expression level determines the invasion and metastatic potential of cancer cells.
Vacuole membrane protein 1 (Vmp1) has been shown in several studies to be involved in cancer-relevant processes. We previously reported that the overexpressed protein is an inhibitor of cell proliferation, anchorage-independent growth (Arlt et al., 2005) and secretory membrane transport (Starkuviene et al., 2004). In addition, VMP1 was found to be differentially regulated in various cancers (Boer et al., 2001; Chen et al., 2002; Higgins et al., 2003). Dusetti et al. (2002) found VMP1 mRNA to be highly expressed in acinar cells of rats with acute pancreatitis and in rat kidney after ischemia. They described the rat ortholog as a stress-induced endoplasmic reticulum (ER) protein that promotes vacuole formation and causes cell death when overexpressed. We also found the overexpressed human Vmp1 to induce apoptosis (Sauermann et al., 2007, Supplementary Figure 1), and that the protein is mostly located in the ER and vacuoles. Here, we show for the first time that endogenously expressed Vmp1 is essentially a cell membrane protein and that it is involved in cell–cell adhesion, invasion and metastasis.
We quantified the mRNA levels of VMP1 in breast cancer patient samples (invasive ductal carcinoma, n=45) that had been classified in tumor grades 1 (n=2), 2 (n=22) and 3 (n=21), and detected a decreasing trend in expression level with increasing tumor grade (Figure 1a). Next, we compared the VMP1 expression in primary kidney tumors (n=37) and kidney cancer metastases (n=19). A significant reduction in VMP1 mRNA was observed in the metastases (t-test: P=0.035, Figure 1b). These results suggest that reduced expression of VMP1 correlates with malignancy. Then we examined whether VMP1 expression level influences the invasion potential of breast cancer cell lines. In an initial approach, we determined the invasion capacity of five cell lines, and found that the cell line MCF-12A, derived from normal breast epithelium, as well as the breast cancer cell lines T-47D and MCF-7 cells were not invasive (Figure 1c). In contrast, HCC1954 and MDA-MB-231 had a high invasion rate. Quantification of VMP1 mRNA with RT–PCR showed that cell lines with high invasion rates had significantly lower VMP1 mRNA levels as compared to the non-invading cell lines (Figure 1d). Although the VMP1 mRNA level in T-47D cells was lowest among the non-invasive cells, the expression level was still 4.5-fold higher than in the invasive HCC1954 and MDA-MB-231 cell lines. This demonstrates that apart from the tumor grades of breast cancer patient samples, VMP1 expression level also correlates with invasiveness of breast cancer cell lines. The results from patient samples and breast epithelial cell lines raised the question, if Vmp1 is involved in metastasis-relevant processes in epithelial cells.
Vacuole membrane protein 1 has been predicted to be a seven transmembrane protein (Hirokawa et al., 1998; Figure 2a) and the overexpressed human protein was reported to localize to the ER and Golgi apparatus (Starkuviene et al., 2004; Mehrle et al., 2006). The overexpressed rat protein was shown to induce intracellular vacuole formation with Vmp1 integrated into the membrane of these vacuoles (Dusetti et al., 2002). We confirmed this effect for the overexpressed human Vmp1 protein, and explored the origin of these vacuoles by staining Vmp1-yellow fluorescent protein (YFP)-transfected HEK293 (embryonic kidney epithelial) cells with antibodies against Golgin-97 and Calnexin, marker proteins for Golgi apparatus and ER, respectively (Figure 2b). Vmp1 precisely colocalized with Calnexin in both ER and vacuoles, showing that the vacuoles originated from the ER. However, in cells with low overexpression of Vmp1, no vacuole formation was noticed. Instead, Vmp1 was found in the plasma membrane distributed in a punctate pattern (Figure 2c), suggesting that localization in the ER was due to accumulation of protein caused by high level of overexpression. To verify if Vmp1 is actually a plasma membrane protein, we stained HEK293 cells without permeabilization, using a Vmp1 antibody directed against a predicted extracellular domain. Similar to cells with low overexpression of YFP-fused Vmp1, the endogenous protein was found scattered all over the membrane in single cells (Figure 2c). However, in subconfluent colonies of cells, Vmp1 was seen as brightly stained puncta at sites of initial cell–cell contacts and at cell–cell borders (Figure 2c), suggesting that it might be involved in the assembly of intercellular junctions. To examine if Vmp1 is a cell–cell contact protein, we stained HEK293 cells with antibodies against cell junction markers and examined their possible colocalization with Vmp1 (Figure 3a). Desmoglein-2, N-Cadherin, Zonula Occludens-1 (ZO-1) and Connexin 43 were used as marker proteins for desmosomes, adherens junctions, tight junctions and gap junctions, respectively. No colocalization of Vmp1 was detected either with Desmoglein-2 or N-Cadherin. Although Connexin 43 staining was seen along the edges of Vmp1 plaques, we never observed exact colocalization. However, a partial overlap of Vmp1 signal with that of ZO-1 was observed in spot-like structures. To test the direct interaction between Vmp1 and ZO-1, co-immunoprecipitations with HEK293 cell lysates were performed using a ZO-1 capture antibody (Figure 3b). ZO-1 was visible at 220 kDa in the cell lysate and eluate. A band at 47 kDa corresponding to Vmp1 was detected with a Vmp1-specific antibody in the same lysate and eluate fractions of ZO-1 precipitates, confirming the direct interaction of these two proteins.
The spot-like junctions where Vmp1 and ZO-1 colocalize consist of two structures, each contributed by one cell (Figure 4a). This suggests that similar to E-Cadherin, Vmp1 molecules present on adjacent cells might form complexes in the intercellular space to initiate the formation of cell junctions. The Z-sectional view of HEK293 cell clusters demonstrates that ZO-1 and Vmp1 colocalize in centers, and ZO-1 scatters to the periphery (Figure 4b). However, in areas where the tight junctions are almost complete (shown with an arrow in Figure 4c), no colocalization of Vmp1 with ZO-1 was detected. This shows that the interaction of Vmp1 with ZO-1 is transient, and the interaction ends once ZO-1 is incorporated into the tight junctions. It further hints that Vmp1 functions in primordial junctions, which are formed at points of initial cell–cell contacts, and initiate maturation of tight junctions and adherens junctions (Ando-Akatsuka et al., 1999). Our assumption that Vmp1 plays an essential role in the formation of cell junctions is further corroborated by the results of a calcium switch assay, which we performed using the normal breast epithelial cell line MCF-10A and the non-invasive breast cancer cell line MCF-7. In both cell lines, we observed induction of Vmp1 expression during adherens junction formation by re-adding calcium to Ca2+-depleted cells (Supplementary Figures 2 and 3). Twenty-four hours after induction, no Vmp1 expression was observed, whereas adherens junction formation was almost completed as indicated by E-Cadherin staining.
We then performed RNAi experiments to examine if Vmp1 is essential for the assembly of cell junctions. To this end, HEK293 cells were cultured at low density to avoid cell–cell contacts, and then transfected with either VMP1 or green fluorescent protein (GFP) control siRNA. Downregulation of the endogenous VMP1 mRNA and the protein levels was confirmed by quantitative TaqMan analysis and western blot, respectively (Figure 4e). After 3 days, cells transfected with GFP siRNA were confluent, but cells transfected with VMP1 siRNA rounded up and showed loss of adherence (Figure 4d). Cell detachment was then measured by counting the floating cells by flow cytometry, and was found to be more than twofold higher in cells transfected with VMP1 siRNA as compared to the GFP control (Figure 4e). We also demonstrated that detachment of cells transfected with VMP1 siRNAs was not caused due to apoptosis or cytoskeletal changes (Supplementary Figures 4 and 5). These results suggest that downregulation of VMP1 and the consequent lack of Vmp1 protein 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 epithelial cancer cells (Martin and Jiang, 2001), and loss of cell–cell adhesion is a prerequisite for tumor cell invasion (Benoliel et al., 2003). We presumed that downregulation of Vmp1 might also affect the invasion potential of cancer cells, and therefore investigated this in an in vitro invasion assay (Sahin et al., 2007). The non-invasive kidney cancer cell line Caki-2 was transfected with VMP1 siRNA, and the number of invading cells was compared with that of cells transfected with GFP control siRNA. The reduction in VMP1 mRNA level was measured by quantitative TaqMan analysis and the effect on Vmp1 protein expression was confirmed via western blot (Figure 4f). Downregulation of VMP1 resulted in a more than threefold increase in invading cells as compared to the control (Figure 4f). Thus, our data indicate that the expression level of VMP1 influences the invasion capacity of kidney cancer cells.
Here, we show that reduced VMP1 expression correlates with the invasion capacity of cancer cells as well as with metastasis in kidney cancer. We demonstrate that Vmp1 is a plasma membrane protein located at primordial junctions, and that it interacts with ZO-1 within these structures. Downregulation of Vmp1 results in cell detachment and increased invasiveness. At the molecular level, a comparable mechanism has been described for E-Cadherin, a primordial junction and adherens junction protein, which also functions as an invasion suppressor (Vleminckx et al., 1991). Thus, we have for the first time identified the function and cellular context Vmp1 is involved in, and emphasized its critical role in cancer cell invasion.
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We thank Ute Ernst, Christina Bootz, Sara Burmester, Heike Wilhelm, Esther Backes, Nina Claudino, Stefanie Krauth and Jens Mattern for excellent technical assistance, and Markus Ruschhaupt, Holger Fröhlich and Olga Freidekind for help in TaqMan data analysis. Further we thank Alberto Calabro for providing us with mRNAs. This work was supported by the Grants 01GR0420 (AP and SW) and 01GR0418/9 (AP and HS) of the National Genome Research Network, funded by the Federal Ministry for Education and Research (Bundesministerium für Bildung und Forschung, BMBF) and by the Austrian Genome Program GEN-AU.
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Sauermann, M., Sahin, Ö., Sültmann, H. et al. Reduced expression of vacuole membrane protein 1 affects the invasion capacity of tumor cells. Oncogene 27, 1320–1326 (2008). https://doi.org/10.1038/sj.onc.1210743
- initial cell–cell contact
- cell junctions
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