The importance of Slug, a transcription factor and E-cadherin repressor, to allow cancer cells to downregulate epithelial markers and upregulate mesenchymal markers in order to become motile and invasive has recently been reported.16, 30
Moreover, Slug has been shown to modulate expression of a variety of adhesion structures, including desmosomes and adherens junctions.31
Recent studies have shown that Slug is upregulated in many types of epithelial cancers, including breast,9
esophageal squamous cell,10
and cholangiocarcinoma. Slug seems to have an important role in tumor progression and invasion as well as in the downregulation of the adhesion molecule E-cadherin.
In our study, we reported that immunohistochemical analysis using clinical samples revealed the expression of Slug in pancreatic cancer, suggesting that Slug might have a critical role regarding tumor node metastasis in pancreatic cancer, as Slug reportedly does in other types of malignancies.9, 10, 34, 35
A significant relationship was observed between Slug upregulation and tumor node metastasis status; Slug expression was increased in node-positive tumors. Slug may serve as a marker for the prediction of the malignancy of pancreatic cancer after operation. However, in a previous study a correlation between the expression level of Slug and tumor node metastasis status was not obvious in human pancreatic cancer specimens.15
The difference may be because of the limited number of patients or the evaluation the immunohistochemistry results.
E-cadherin is an important mediator of epithelial adhesion and signaling transduction. Loss of E-cadherin expression has been described in invasion and metastasis of many cancers, including pancreatic cancer.34, 35, 36
In the present study, we found that reduced E-cadherin was significantly associated with histological grade, TNM stage and tumor-node-metastasis, but expression of Slug in pancreatic cancer cells showed no significant relation to a decreased expression of E-cadherin.These results are consistent with recent data indicating that Slug was not associated with E-cadherin in hepatocellular carcinoma.8
A previous study found that there was no relationship between the expression of E-cadherin or MMP-9 with stage, histology stage, or perineural invasion, but a significant relationship was found between the expression of E-cadherin/MMP-9 ratio and perineural invasion.37
In this study, we found that expression of MMP-9 was significantly associated with nodal metastasis and nervous metastasis in pancreatic cancer tissue; results were consistent with previous study.19
We also found that the pancreatic cancer tissue with high Slug expression had strong MMP-9 expression, and moreover the expression of Slug was significantly associated with MMP-9. This suggests that Slug promotes migration and invasion, and may correlate with the upregulation of matrix metalloproteinase-9, but not with E-cadherin expression.
To test the significance of Slug expression in pancreatic cancer, we transfected the Slug cDNA into pancreatic cancer cells PANC-1. After transfection, the invasive ability of PANC-1 cells increased dramatically. Slug-transfected cells implanted into nude mice metastasized widely compared with parental untransfected cells. This corroborated the role of Slug in pancreatic cancer progression, and indicates that Slug has an important role in pancreatic cancer invasion and metastasis.
We also found that Slug cDNA promoted metastasis in pancreatic cancer, but it did not inhibit the expression of E-cadherin in pancreatic cancer cells. We suggested that Slug accelerates pancreatic cancer progression, but not through E-cadherin repression; results that are consistent with a previous study.16
Thus, it would be unlikely that E-cadherin
would be the only target gene for Slug proteins, and similarly unlikely that it drives the whole process. These apparent discrepancies in relation to E-cadherin regulation could be a reflection of the specific contribution of various cellular contexts.
It is reasonable to theorize that Slug is largely involved in migration, ie, cell motility of tumor cells. However, it has been largely unclear how Slug proteins exhibit the invasive activity, and the exact mechanism of Slug-dependent invasion is almost unknown. The present study for the first time has explored the relationship between Slug and MMP-9 in pancreatic cancer and clarified the novel mechanism.
In the present study, we found that MMP-9 is mainly located in the perinuclear cisterna in PANC-1 cells, but in Slug-transfected clones, MMP-9 is mainly located in the anterior extremity of the filopodia, allowing MMP-9 to digest the extracellular matrix (ECM). In vitro and in vivo studies showed that Slug apparently upregulated both the expression levels and activities of MMP-9, leading to enhancement of the invasive activity. MMP-9-specific BB94 blocked Slug-induced Matrigel invasion in the malignant PANC-1 cells. These findings indicate that Slug may regulate the metastatic nature of PANC-1 by directly upregulating MMP-9, through enhancement of the invasive activity, ie, the degradation of the ECM. Such Slug-dependent transcriptional change in MMP-9 genes is a new and interesting finding, and the detailed mechanisms, especially the transcriptional factors linking to Slug, may be clarified in future studies.
A key cellular process associated with the invasive or metastatic program in many cancers is the transformation of epithelial cells toward a mesenchymal state, a process called EMT. Actin-dependent protrusion of cell pseudopodia is a critical element of mesenchymal cell migration and, therefore, of cancer metastasis. However, whether EMT occurs in human cancers and, in particular, whether it is a prerequisite for tumor cell invasion and metastasis, remains a subject of debate.38
F-ascin is an actin-bundling protein involved in filopodia assembly and cancer invasion and metastasis of multiple epithelial cancer types. Fascin forms stable actin bundles with slow dissociation kinetics in vitro
and is regulated by phosphorylation of serine 39 by protein kinase C (PKC). Cancer cells use invasive finger-like protrusions termed invadopodia to invade into and degrade the ECM.39
In the present study, we initially analyzed the intracellular F-actin in Slug-transfected clones and FCM showed that the intracellular F-actin in Slug-transfected clones increased 2.1-fold. We then studied the expression of F-actin using immunofluorescence staining and determined that, in non-treated PANC-1 cells, F-actin is mainly located in the perinuclear cisterna; however, in Slug-transfected clones, F-actin is principally located in the anterior extremity of the filopodia. The results suggested the invadopodia in PANC-1 cells was formed by intracellular F-actin polymerization induced by Slug. We also found that MMP-9 and F-actin together were mainly located in the anterior extremity of the filopodia in Slug-transfected clones, suggesting Slug might transport MMP-9 via F-actin networks. These findings indicate that Slug may regulate the metastatic nature of PANC-1 by stimulating cell motility (probably by modulation of the cytoskeletal structure that is well established).
Taken together, there is a possibility that Slug may not only transcriptionally increase MMP-9 expression, but is also responsible for the remodeling of the cytoskeleton network. Acting in concert, they may together mediate invasion and metastasis in PANC-1 cells; further study is warranted to elucidate the exact mechanisms involved, and also to evaluate the potential scope of application in additional human carcinoma cell types.