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The hedgehog (Hh) signaling pathway has been reported to be associated with the growth of pancreatic cancer, but its role in the invasive phenotype is poorly understood. Therefore, we investigated the role of the Hh pathway in pancreatic cancer cell invasiveness using a Matrigel invasion assay. Blockade of the Hh pathway by cyclopamine inhibited pancreatic cancer cell invasion in association with a decreased expression of matrix metalloproteinase (MMP)-9. By contrast, activation of the Hh pathway by the addition of exogenous Sonic hedgehog increased cell invasion and MMP-9 expression. Stable transfection of pancreatic cancer cells with Gli1 increased their invasiveness, which was associated with activation of MMP-9. We also showed that inhibition of MMP-9 by small interfering RNA blocked the increased invasiveness of Gli1-transfected cells. Furthermore, inhibition of Gli1 by small interfering RNA suppressed the invasiveness and MMP-9 expression of pancreatic cancer cells. Taken together, these findings suggest that members of the Hh pathway, especially Gli1, play an important role in the invasiveness of pancreatic cancer cells through the regulation of MMP-9 expression. (Cancer Sci 2008; 99: 1377–1384)
Pancreatic cancer is one of the most lethal malignancies that has yet to be successfully controlled with therapy. Although various therapies such as surgery, chemotherapy, and radiation therapy have been carried out, few patients survive for 5 years.(1,2) One reason for this lethality is its invasive and metastatic character. A better understanding of the mechanisms that underlie the development of pancreatic cancer would help to identify novel molecular targets for treatment.
The Hh signaling pathway is crucial to growth and patterning in a wide variety of tissues, including the pancreas, during embryonic development.(3–6) Of three Hh ligands, Shh, Ihh, Dhh, the former is reported to play an essential role in the development of pancreatic cancer as well as pancreatic organogenesis. The response to Shh is mediated by two transmembrane proteins, Smo and Ptch, and by downstream transcription factors that are members of the Gli family. Three Gli genes have been identified. Gli2 and Gli3 have distinct context-dependent repressor and activator functions.(7–9) By contrast, Gli1 is an activator of target genes, and is itself a transcriptional target of the Hh pathway.(10–12) Recent studies have reported an association between Hh pathway activation and initiation of human tumors.(6) Recent studies have also shown cell autonomous ligand-dependent activation of the Hh pathway in small cell lung cancer and carcinomas of the esophagus, stomach, biliary tract, and pancreas.(13–15) It has been also shown that cyclopamine, a Smo antagonist, suppresses the growth of pancreatic cancer.(14,15) These findings suggest that the Hh pathway could be a viable therapeutic target for the treatment of pancreatic cancer.
In the present study, we analyzed the relationship between Hh pathway activation, especially Gli1, and the invasive ability of pancreatic cancer cells.
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Recent increasing data indicate a contribution of the Hh pathway not only to cell proliferation, but also to the repair of several tissues.(23) For example, it has been shown that, during repair of bronchial epithelium, transient activation of Hh pathway occurs within the normally quiescent bronchial epithelium, and such a process might well occur during adult gut epithelial turnover.(13) As we know, many kinds of molecules, including MMPs, take part in degradation of extracellular matrix are participating in tissue repair. This suggests that the Hh pathway might also contribute to cell invasion by mediating MMPs.
Based on these observations, we hypothesized that the Hh pathway plays an important role in the invasive ability of pancreatic cancer. In the present study, we selected two human cancer cells, AsPC-1 and SUIT-2. Blockade of the Hh pathway suppressed the invasive ability of both cell types (Fig. 1), but activation of the Hh pathway increased their invasive ability (Fig. 2). These findings indicate a close relationship between the Hh pathway and the invasive ability of pancreatic cancer cells.
How does the Hh pathway affect the invasive ability of pancreatic cancer cells? It is noteworthy that Hh pathway inhibition with cyclopamine affects neither the proliferation nor the migration ability of these cells, under the present experimental conditions (Fig. 1 and data not shown). These results indicate that the Hh pathway could play a relatively specific role in Matrigel degradation. Most MMPs probably participate in the process of matrix degradation. The degradation of basement membrane type IV collagen is a critical early event in tumor invasion, suggesting that MMP-2 and MMP-9 might play a particularly important role.(24,25) Therefore, we focused on MMP-2 and MMP-9. Real-time RT-PCR and gelatin zymography showed that cyclopamine and rhShh decreased and increased MMP-9 expression, respectively (Fig. 3). Furthermore, a Gli1-expressing pancreatic cancer clones showed increased MMP-9 mRNA expression and MMP-9 enzymatic activity (Fig. 4b,c), and the increased invasive ability of these cells was abolished by RNAi against MMP-9 (Fig. 4f). Thus, the Hh pathway contributes to the invasive ability of pancreatic cancer cells through MMP-9 induction.
It has been reported that aberrant activation of MMP-2 is more frequent in human pancreatic cancer than that of MMP-9.(26) Although we evaluated MMP-2 expression by gelatin zymography, we could detect only pro-MMP-2, not active-MMP-2. Moreover, the expression of pro-MMP-2 was not significantly correlated with Hh pathway activity. Considering these results we had come to a conclusion that the Hh pathway did not affect MMP-2 activity, at least not in the pancreatic cancer cells we used in this study.
It is important that stably Gli1-expressing pancreatic cancer cell clones (pcDNA-Gli1 cells) showed a marked increase in their invasive ability (Fig. 4a) and increased active-MMP-9 expression (Fig. 4b), and that knockdown of Gli1 resulted in suppression of the invasive abilities (Fig. 5d) and MMP-9 mRNA expression (Fig. 5c). These data indicate that Gli1 might play a crucial role both in the invasive ability and in the MMP-9 expression of these cells.
In our study, it is still unclear how the Hh pathway activates MMP-9 expression. Recently, it has been reported that the Hh pathway interacts with the Ras/MAPK pathway.(27) Because the MAPK pathway has been reported to regulate MMPs,(28) we suggest that one of the possible mechanism of activating MMP-9 is the Ras/MAPK cascade.
Interestingly, Feldmann et al.(29) showed that the Hh pathway might play an important role in pancreatic cancer invasion and metastasis using a xenograft model. They focused on snail and E-cadherin as the key molecule of the Hh-related invasion, and suggested that epithelial-to-mesenchymal transition might participate in the Hh-related invasion in pancreatic cancer. In the present study, we also showed an important role for the Hh pathway in the invasive ability of pancreatic cancer cells. We focused on MMP-9 as the key molecule and suggested that degaradation of the extracellular matrix also participates in the Hh-related invasion. We showed for the first time that MMP-9 plays an important role in the invasive ability mediated by the Hh pathway in pancreatic cancer.
We also showed that the Hh pathway, especially Gli1, contributes to pancreatic cancer invasion through MMP-9 induction. The results of the present study suggest that Gli1 might become a new therapeutic target for inhibiting the invasion of pancreatic cancer cells.