We had previously established a cell line, NPC-BM1, derived from a bone marrow biopsy of a female Taiwanese patient with NPC . Some basic differences in baseline secretion of IL-6, IL-6 receptor α and IFN-γ between NPC-BM1 and other NPC cell lines derived from primary tumors have recently been documented . In the current study, this parental cell line was further separated to non-invasive and highly invasive cells, designed NPC-BM00 and NPC-BM29, respectively. After separation, the metastatic phenotype of these two cell lines was confirmed by cell morphology change suggestive of EMT. While NPC-BM00 cells exhibited typical well-attached polygonal epithelial cell morphology, NPC-BM29 was more heterogeneous and included cells that were spindle-shaped cells and those resembled fibroblasts.
EMT is characterized by the loss of epithelial characteristics and the gain of mesenchymal attributes. It has been associated with physiological and pathological processes requiring epithelial cell migration and invasion. In addition, evidence is mounting suggesting the importance of EMT pathways in the progression of carcinoma to metastasis by providing epithelial tumor cells with the ability to migrate, invade the surrounding stroma and disseminate in secondary organs . It has been reported that EMT is one of the major events during the acquisition of the invasive phenotype in tumors of epithelial origin [8, 35]. This process is often accompanied by expression of mesenchymal markers and loss of epithelial markers, especially E-cadherin, which is one of the most commonly observed changes in invasive and metastatic carcinomas [36, 37].
In the scrape-wound assay, we showed that NPC-BM00 cells exhibit slower migration into the wounded area than its counterpart NPC-BM29. This observation was further supported by the findings demonstrating downregulation of E-cadherin and its associated protein β-catenin in NPC-BM29 cells (Fig. 9). Previously we showed that TPA is a highly potent activator of EBV genome replication [27, 28]. TPA also has been shown to activate some isoforms of the important signaling enzyme, protein kinase C (PKC) , which interferes with both cell proliferation and cell adhesion in a variety of cell types . Various studies have demonstrated the involvement of PKC in disassembly of E-cadherin-dependent cell-cell adhesion [39–42]. In the present study, we analyzed the effect of TPA on cell motility and revealed that both NPC-BM00 and NPC-BM29 showed increased rate of cell migration into the wounded area compared to the sham-treated cells but with differential rate. Activation with TPA also resulted in a decrease in the level of E-cadherin in both cases (our unpublished data). Based on these results, we conclude that PKC activation is involved in TPA-induced cell motility.
E-cadherin and β-catenin are another commonly employed index for the epithelial state. These cell adhesion molecules are localized in the adherence junctions. These cell-cell adhesion-related criteria are almost exclusively absent in mesenchymal cells. The E-cadherin plays a central part in the process of epithelial morphogenesis. Expression of this protein is downregulated during the acquisition of metastatic potential at late stages of epithelial tumor progression. In cancer, the maintenance of epithelia is lost, and dissociation of cells is associated with metastatic dissemination. Dissociation of cells can occur through a decrease in the local expression level of E-cadherin.
Invasion and metastasis are determinative features in the pathogenesis and progression of malignant neoplasms. The pathogenesis of metastasis consists of multiple, sequential, selective and interdependent steps. To establish a metastatic focus, tumor cells must detach from the primary tumor (suppression of cell-to-cell and cell-matrix adhesion), degrade and invade the extracellular matrix (ECM), increase in cell motility and enter the circulation, arrest in a capillary bed, gain entrance into organ parenchyma, proliferate and induce angiogenesis . It is now well established that the processes of invasion and angiogenesis are essential for the growth and metastasis of both primary and metastatic tumors .
Matrix metalloproteinases (MMPs) are a family of structurally related zinc-dependent endopeptidases collectively capable of degrading essentially all components of ECM. Based on their structure and substrate specificity, MMPs are classified into subgroups of collagenases: stromelysins and stromelysin-like MMPs and other MMPs . MMPs play an important role in the physiologic degradation of ECM, e.g., in tissue morphogenesis, tissue repair and in angiogenesis. MMPs also have important functions in pathologic conditions characterized by excessive degradation of ECM, such as rheumatoid arthritis, osteoarthritis, periodontitis, autoimmune blistering disorders of the skin, as well as in tumor invasion and metastasis [44–47]. MMP-1 (collagenase-1) cleaves fibrillar collagens with preference for type III collagen, which denatures into gelatin and is further degraded by other MMPs, such as gelatinases. MMP-2 (gelatinase-A) and MMP-9 (gelatinase-B) can both degrade the type IV collagen of basement membranes, the first barrier to tumor invasion. Thus, high expression levels of certain MMPs facilitate tumor cell invasion and metastasis. Our results revealed that after separation, the metastasis potential of NPC-BM29 was confirmed by increased expression of 92-kDa type IV collagenases/gelatinase B (MMP-2 and MMP-9); both enzymes are involved in tumor cell invasion and metastasis.
Ets1 is a member of Ets transcription factor family and recognizes specific nucleotides sequences with a GGAA/T core specific sequence . Physiologically, Ets1 is expressed in various mesodermal derivatives, such as endothelial cells and mesenchymal cells, but is also induced in dissociating epithelial cells, during EMT process occurring in early development and oncogenic transformation to acquire invasive features [30, 48]. Ets1 expression in tumor cells has been found to correlate with the grade of invasiveness in human tumor tissues  and correlates with a degree of invasiveness in breast cancer cell lines . Ets family members were reportedly involved in the control of cell motility and invasion during normal tubular morphogenesis and cancerous scattering in mammary epithelial cells . Furthermore, it has been reported that MMP9 gene promoter contains a binding site for Ets in addition to AP-1 and NF-kB, and thus Ets1 may positively regulate MMP9 gene expression . Therefore, Ets1 may stimulate transcription of many genes associated with tumor invasion and metastasis, and would be an effective target in preventing invasion of malignant tumors.
Using Western blot analysis, we have identified that the expression of E-cadherin and β-catenin was decreased with the increasing metastatic potential of NPC-BM29 cells. Concomitant with the downregulation of E-cadherin and β-catenin, upregulation of the transcription factor Ets1 was detected in NPC-BM29 cells, in agreement with the central role of these molecules in the transition of EMT.
We observed that NPC-BM1 cells formed compact cell islands in tissue culture plates, remained in tight cell-cell contacts and exhibited the typical morphology of epithelial cells. In sharp contrast, NPC-BM18 and 29 cells were scattered and exhibited the typical morphology of mesenchymal fibroblasts, suggesting that the constitutive activation of β-catenin signaling might induce EMT .
It has been well-documented that the transformed cells do not require high concentration of serum in the culture medium to support the proliferation. In this study, we demonstrated that serum deprivation markedly retarded the growth of NPC-BM00 cells with little or no metastatic potential, but had no significant impact on NPC-BM29 cells likely to have higher metastatic potential (Fig. 5). This phenomenon suggests the possibility that NPC-BM00 cells may convert the cell phenotype toward benign, because serum-independent proliferation generally is accepted as a hallmark of tumor cells but not normal cells. Indeed, this speculation is supported further by the observation that significantly fewer colonies were formed in soft agar by NPC-BM00 than by NPC-BM29 cells (Fig. 6).
The preferential expression of sialyl-Tn by NPC-BM29 cells on one hand and the selective expression of HLA-DR by NPC-BM00 cells on the other in a significant proportion of cell population are interesting. While the implications of these results are not immediately clear, further investigations on both observations are warranted.
Angiogenesis is a key step in tumor growth, invasion and metastasis. Massive formation of blood vessels at the tumor site increases the opportunity for tumor cells to enter the circulation. Thus, microvessel density is considered to influence tumor metastasis and consequently prognosis in various human cancers . VEGF, FGF basic (bFGF) and IL-8 are prominent angiogenic molecules. These molecules have been demonstrated to influence microvessel synthesis in various tumors [53, 54]. The associations of neovascularization to both angiogenic and lymphatic metastases have been examined in many malignant tumors, and the contributions of angiogenic molecules such as VEGF, bFGF, and IL-8 to the metastatic potential of tumors have been well documented. The relevance of angiogenic factors to the angiogenesis of these NPC cell lines described in this study was evaluated by studying the relationships of IL-8 and VEGF expression. Our results clearly indicate that the higher level expression of IL-8 in NPC-BM29 was significantly correlated to its invasive and metastatic potential. In contrast, the upregulation of VEGF was found in NPC-BM00 cells suggesting that it may enhance the growth capability by facilitating neovascularization locally.
The roles of EBV infection in the tumorigenesis of NPC are of interest because of the close association of this virus with NPC. Recent reports indicate that EBV LMP-1 is capable of inducing a wide range of cellular factors associated with metastatic character of NPC [13–22]. However, the critical functions of EBV in NPC development remain poorly understood. It has been noted that the EBV genome is commonly lost during the establishment of NPC cell lines from biopsies or xenografts . However, EBV genomes are not lost from tumors being propagated in nude mice. Such tumors retain characteristics of the original NPC including p53 negativity. The loss of EBV genomes during attempts to culture NPC in vitro is associated with inability to establish cell lines from original or nude-mouse passaged tumors.
It should be noted that both the parental cell line (NPC-BM1) and two sublines (NPC-BM00 and NPC-BM29) are EBV-negative (Fig. 3). This observation suggests that EBV may have other important roles in vivo, which may include, but are not limited to, conferring protection to the carcinoma cells from immune surveillance .
Overall, this study reports our observations on important cellular signaling cascades that are associated with metastasis of NPC in the absence of EBV genome. The link between morphologic changes from epithelial to mesenchymal transition and identification of the molecules associated with these changes opens up new avenues in cancer research, allowing the use of these molecular markers to pave the way for the identification of novel targets for therapeutic intervention of invasion and metastasis.