Masayuki Koizumi

PURPOSE: Loss of intercellular adhesion and increased cell motility promote tumor cell invasion. In the present study, E- and N-cadherin, members of the classical cadherin family, are investigated as inducers of epithelial-to-mesenchymal transition (EMT) that is thought to play a fundamental role during the early steps of invasion and metastasis of carcinomas. Cell growth factors are known to regulate cell adhesion molecules. The purpose of the study presented here was to investigate whether a gain in N-cadherin in pancreatic cancer is involved in the process of metastasis via EMT and whether its expression is affected by growth factors. EXPERIMENTAL DESIGN: We immunohistochemically examined the expression of N- and E-cadherins and vimentin, a mesenchymal marker, in pancreatic primary and metastatic tumors. Correlations among the expressions of N-cadherin, transforming growth factor (TGF)beta, and fibroblast growth factor 2 was evaluated in both tumors, and the induction of cadherin and vimentin by growth factors was examined in cultured cell lines. RESULTS: N-cadherin expression was observed in 13 of 30 primary tumors and in 8 of 15 metastatic tumors. N-cadherin expression correlated with neural invasion (P = 0.008), histological type (P = 0.043), fibroblast growth factor expression in primary tumors (P = 0.007), and TGF expression (P = 0.004) and vimentin (P = 0.01) in metastatic tumors. Vimentin, a mesenchymal marker, was observed in a few cancer cells of primary tumor but was substantially expressed in liver metastasis. TGF stimulated N-cadherin and vimentin protein expression and decreased E-cadherin expression of Panc-1 cells with morphological change. CONCLUSION: This study provided the morphological evidence of EMT in pancreatic carcinoma and revealed that overexpression of N-cadherin is involved in EMT and is affected by growth factors.

Abstract The stromal cell-derived factor-1 (SDF-1)/CXCR4 system is implicated in various instances of cell migration in mammals, including the migration of lymphocytes and the formation of metastases. We have recently synthesized a potent novel CXCR4 antagonist, TN14003. The purpose of this study was to investigate the role of SDF-1/CXCR4 axis in the pancreatic cancer metastasis via cell migration and invasion, and the inhibitory effect of TN14003 on pancreatic cancer cell metastasis. The expression of CXCR4 was detected in six pancreatic cancer cell lines by Western blotting and immunocytochemistry. In migration and invasion assays, SDF-1 stimulated both migration and invasion of cancer cells in a dose-dependent manner. The maximal effect of SDF-1 was observed at 100 ng/ml. SDF-1-induced migration and invasion of cancer cells were completely blocked by 100 nm TN14003. The stimulatory effect of SDF-1 on cancer migration and the inhibitory effect of TN14003 were mediated via the alteration in phosphorylation of mitogen-activated protein kinases. Treatment of cancer cells with 100 ng/ml SDF-1 resulted in a significant increase of actin polymerization, which was reduced by 100 nm TN14003. SDF-1 enhanced cancer cell adhesion to laminin, which was not reversed by TN14003. Taken together, SDF-1/CXCR4 axis is involved in pancreatic cancer metastasis through migration and invasion. The small molecule antagonists against CXCR4 such as TN14003 might be an effective anti-metastatic agent for pancreatic cancer.

BACKGROUND: The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene was initially isolated as a transformation suppressor gene. The RECK gene is expressed widely in normal organs but is undetectable in many tumor-derived cell lines. When artificially expressed in such cell lines, RECK negatively regulates at least matrix metalloprotease (MMP)-9, MMP-2, and MT1-MMP activation and suppresses the invasive and metastatic potentials of these cells. Clinical relevance of these observations, however, is yet to be established. The aim of this study was to examine RECK expression in pancreatic cancer, where intensive invasiveness and metastasis are frequently observed, and investigate its clinical significance. We also analyzed the correlation between RECK expression and MMP activation. METHODS: (a) RECK expression in surgically resected tissue samples of invasive ductal carcinomas of the pancreas (n = 50) was examined immunohistochemically, and its correlation with clinicopathological factors was analyzed; and (b) gelatin zymography was used for the detection of latent and activated forms of MMP-2 and MMP-9 in some of the tissue samples (n = 33). The gelatinase activity was quantified by densitometory, and the ratio of intensity of the active MMP-2 band to the total intensity of the pro- and active MMP-2 bands was evaluated as an indicator of MMP-2 activation. The MMP-9 activation was also studied. RESULTS: Among the 50 ductal carcinoma samples, 26 (52%) were stained positive for RECK. In the normal pancreas, both acinar and beta cells were stained positive, but ductal cells did not. Tumors with positive RECK staining were significantly less invasive as compared with RECK-negative tumors (P = 0.0438). Importantly, patients who had tumors with high RECK expression showed significantly better prognosis than those who had RECK-negative tumors (P = 0.0463, by Log-rank test). Zymographic analysis indicated significant inverse correlation between the level of RECK expression and extent of MMP-2 activation (P = 0.0374). CONCLUSIONS: Our findings support the hypothesis that the RECK protein has negative effects on the invasiveness of pancreatic cancer by inhibiting MMP-2 activation and suggest the potential value of RECK as a prognostic molecular marker for pancreatic cancer.