Association of matrilysin mRNA expression with K-ras mutations and progression in pancreatic ductal adenocarcinomas

Hiroshi Fukushima, Hiroyuki Yamamoto,1, Fumio Itoh, Hideaki Nakamura, Yongfen Min, Shina Horiuchi, Shouhei Iku, Shigeru Sasaki and Kohzoh Imai

First Department of Internal Medicine, Sapporo Medical University, South-1, West-16, Chuo-ku, Sapporo 060-8543, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The matrix metalloproteinase matrilysin has been implicated in the progression of gastrointestinal and other cancers. The aim of this study was to examine matrilysin mRNA expression and determine whether it is correlated with K-ras mutations and/or progression of pancreatic carcinoma. Using the semiquantitative reverse transcriptase–polymerase chain reaction (RT–PCR), we analyzed 11 pancreatic cancer cell lines and 70 pancreatic adenocarcinoma tissues for matrilysin mRNA expression. The results were correlated with clinicopathological characteristics and K-ras mutations. Significant amounts of matrilysin mRNA were detected in six of the eight cell lines with K-ras mutations but not in the three cell lines with wild-type K-ras. Matrilysin mRNA was detected in 57 (81.4% ) of the 70 tumor tissues and in all of the eight liver metastases, but not in any of the adjacent non-tumorous tissues. Matrilysin expression was significantly correlated with the size of tumor, tumor spreading, lymph node metastasis, advanced pathologic tumor-node- metastasis stage and K-ras mutations. The relative amounts of matrilysin mRNA in tumor tissues increased with increase in tumor stage and were highest in liver metastatic tumor tissues. Our results suggest that matrilysin, the expression of which is correlated with K-ras mutations, plays a key role in tumor growth and progression of pancreatic carcinoma.

Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMPs, matrix metalloproteinases; pTNM, pathological tumor-node-metastasis; RT–PCR, reverse transcriptase–polymerase chain reaction; TCF, T-cell factor.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Degradation of the extracellular matrix mediated by matrix metalloproteinases (MMPs) is a crucial mechanism during tumor invasion and metastasis. As an MMP, matrilysin is unique in its minimum MMP structure, wide spectrum of substrate specificity, potency to start an activation cascade of MMPs and, most notably, in its production by cancer cells themselves (13). Tumor cell-specific MMP matrilysin has been implicated in tumor invasion and metastasis in colorectal (47), gastric (8,9), esophageal (9,10), hepatic (11) and other cancers (13). Some MMPs, including matrilysin, are involved in not only extracellular matrix degradation but also in cell proliferation, differentiation and angiogenesis (13). Indeed, matrilysin has been shown to play an essential role in tumor initiation or growth in colorectal (12,13) and mammary carcinogenesis (14). Matrilysin has also been detected in colorectal adenomas at lower levels than in cancers, and its expression has been shown to be correlated with the size of adenoma and the degree of dysplasia (1517).

The expression of matrilysin in cancer cells could be advantageous as a biological marker of malignancy. Indeed, associations of matrilysin mRNA expression with tumor progression have been seen in colorectal (18,19), gastric (20,21) and hepatic carcinomas (22). In contrast to these gastrointestinal cancers, there is little information on the significance of matrilysin expression in pancreatic carcinoma. In a previous study, analyzing a small number of tumor samples, matrilysin mRNA was detected in 15 (88%) of 17 pancreatic carcinoma tissues (23). Thus, it seems important to examine matrilysin expression in a large number of pancreatic carcinoma samples.

Despite a number of studies demonstrating the importance of matrilysin in colorectal and other carcinogenesis, the mechanisms underlying the overexpression of matrilysin are poorly understood. Recently, the promoters of mouse and human matrilysin have been shown to contain T-cell factor (TCF) binding sites and to be activated by ß-catenin/TCF-4 (24,25). Moreover, concomitant expression of ß-catenin and matrilysin has been shown in human colorectal cancer tissues (25). These results suggest that matrilysin is an important target gene of APC/ß-catenin/TCF-4 pathways in colorectal carcinogenesis. In contrast to colorectal cancers, intracellular accumulation of ß-catenin was not observed in any of the 40 pancreatic adenocarcinomas (26). Consistently, neither 78 ductal pancreatic adenocarcinomas nor 14 pancreatic cancer cell lines had mutations in exon 3 of the ß-catenin gene (26). Thus, APC/ß-catenin/TCF-4 pathways do not appear to be common targets of genetic alterations in pancreatic carcinogenesis (26, 27).

Another possible mechanism is activation of the K-ras signaling pathway. The human matrilysin promoter contains binding sites for the transcriptional factors AP-1 and PEA3 (28). Both factors are downstream effectors of the ras signaling pathway (25). Indeed, we have previously reported that activated K-ras induces matrilysin expression in colon cancer cell lines (29). Moreover, Ohnami et al. (30) have recently shown that matrilysin was one of the genes down-regulated in the antisense K-ras-transduced pancreatic cancer cell line AsPC-1 and that matrilysin mRNA was overexpressed in four out of five carcinoma tissues. Thus, it seems important to examine the relationship between matrilysin mRNA expression and K-ras mutations in pancreatic carcinoma.

In an attempt to address these issues, we investigated the expression of matrilysin in 70 pancreatic carcinoma tissues, using the semiquantitative reverse transcriptase–polymerase chain reaction (RT–PCR), in relation to K-ras mutations and clinicopathological characteristics.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients and samples
Seventy fresh paired surgical specimens of pancreatic carcinoma and adjacent non-tumorous tissue were obtained from patients receiving surgical treatment. Each tissue specimen was divided into two pieces after resection. For RNA extraction, one sample was immediately frozen in liquid nitrogen at the time of surgery and stored at –80°C until extraction. The other sample was processed for pathologic examination using hematoxylin and eosin staining for the evaluation of tumor cell content. Only specimens containing >75% tumor cells were used for analysis. All of the tumors were adenocarcinomas, and the histopathological and clinical features of the specimens were classified according to the guidelines of the International Union Against Cancer (UICC) (31). Informed consent was obtained from each subject. Human pancreatic adenocarcinoma cell lines AsPC-1, BxPC-3, Capan-1, Capan-2, CFPAC-1, HPAF-II, Hs 700T, Hs 766T, MIA PaCa-2, PANC-1 and SU.86.86 were purchased from the American Type Culture Collection (Rockville, MD). Cells were cultured in DMEM or RPMI 1640 containing 10% fetal bovine serum.

Semiquantitative RT–PCR and K-ras mutations
Total RNA was extracted from specimens using the acid guanidinium thiocyanate–phenol–chloroform extraction method and treated with DNase I. cDNA was synthesized from 1 µg total RNA using SuperScript II reverse transcriptase (Gibco BRL, Gaithersburg, MD) with random hexamers. PCR amplification was performed using primers for matrilysin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes (final concentration 0.5 µM and 0.05 µM, respectively) in duplex PCR reactions (32). GAPDH served as an internal control of the reaction. PCR was carried out with Taq DNA polymerase for one cycle of 94°C for 4 min followed by 28 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 30 s. Primers were 5'-TCTTTGGCCTACCTATAACTGG-3' and 5'-CTAGACTGCTACCATCCGTC-3' for matrilysin and 5'-GTGAAGGTCGGAGTCAACG-3' and 5'-GGTGAAGACGCCAGTGGACTC-3' for GAPDH (32). The primers had been designed to span a large enough intron in the genomic sequences for human matrilysin and GAPDH to completely avoid DNA contamination. PCR products were electrophoresed on 2% agarose gels. Results were analyzed using a multiimage analyzer (BioRad, Richmond, CA). All reactions were controlled without reverse transcriptase. K-ras mutations at codons 12 and 13 were analyzed as described previously (33).

Statistical analysis
Matrilysin expression was assessed for associations with clinicopathological parameters using the following statistical tests: Student's t-test for age, the chi-square two-tailed test for gender; Fisher's exact test for size, lymph node metastasis, distant metastasis and K-ras mutations; the Mann–Whitney test for differentiation, local tumor spread (pathological T factor) (31) and pathological tumor-node-metastasis (pTNM) stage (31). In all tests, the level of significance was set at P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Semiquantitative RT–PCR
To perform semiquantitative analysis for matrilysin mRNA expression using RT–PCR, the range of linear amplification for the matrilysin gene and the control GAPDH gene was examined. The optimal number of PCR cycles and the mixing ratios of primers were determined as described in Materials and methods.

Matrilysin mRNA expression in pancreatic cancer cell lines
Using RT–PCR, matrilysin mRNA was examined in 11 pancreatic carcinoma cell lines (Figure 1Go). Eight of the 11 cell lines showed variable levels of matrilysin mRNA. Among these eight cell lines, Hs700T and Hs766T expressed negligible amounts of matrilysin mRNA. Considering the sensitivity of RT–PCR and the results of the 70 paired tissue specimens, these levels of expression in both cell lines were judged to be negative. Six of the eight K-ras mutated cell lines were matrilysin positive. In contrast, all of the three cell lines with wild-type K-ras were negative for matrilysin expression.



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Fig. 1. RT–PCR analysis of matrilysin mRNA expression in (top) pancreatic carcinoma cell lines and (bottom) pancreatic ductal adenocarcinoma tissues. N and T, matched samples from non-tumoral and tumor tissue, respectively.

 
Matrilysin mRNA expression in pancreatic carcinoma tissues
The expression of matrilysin mRNA was examined in 70 paired specimens of pancreatic carcinoma and non-tumorous tissue. Figure 1Go shows representative results of RT–PCR for matrilysin. Matrilysin mRNA was detected in 57 (81.4%) of 70 carcinoma tissues and in all of the eight liver metastatic tumor tissues, but was not, or only faintly, detected in adjacent non-tumorous tissues. The relationship between matrilysin expression and clinicopathological features is summarized in Table IGo. Matrilysin mRNA expression was significantly correlated with tumor size (P = 0.0264), local tumor spread (P = 0.0254), lymph node metastasis (P = 0.0264) and advanced pTNM stage (P = 0.0112). There was no correlation of matrilysin mRNA expression with age, gender, histological differentiation of the tumor or distant metastasis (Table IGo). Matrilysin mRNA expression was significantly correlated with K-ras mutations (P = 0.0027 and odds ratio = 8.33). Finally, the relative amounts of matrilysin mRNA corrected for that of GAPDH expression in carcinoma tissues were significantly higher in more advanced tumor stages and were highest in the liver metastatic tumor tissues (P = 0.0002, Figure 2Go).


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Table I. Associations between matrilysin mRNA expression and clinicopathological and genotypical characteristics in patients with pancreatic carcinoma
 


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Fig. 2. Relative quantification of matrilysin mRNA expression in pancreatic ductal carcinoma tissues. Band intensities of PCR products were measured using a computerized image analyzer. Higher expression is present in patients with a higher stage (P = 0.0002). Error bars, standard deviation.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
First, we examined the role of matrilysin mRNA expression in the progression of pancreatic carcinoma. Matrilysin mRNA expression was found to be significantly correlated with the extent of tumor spreading, lymph node metastasis and advanced tumor stage. In addition, there was an increase in the relative amounts of matrilysin expression with tumor progression. These results suggest that matrilysin plays an important role in the progression of pancreatic carcinoma. Moreover, matrilysin mRNA was detected in all of the eight liver metastatic tumors, suggesting that matrilysin plays an essential role in liver metastasis in pancreatic carcinoma. Thus, our results suggest that matrilysin contributes to the aggressive behavior of pancreatic carcinoma cells. Analysis of matrilysin expression could therefore be an important part of the management of patients with pancreatic carcinoma.

The significance of matrilysin mRNA expression was further substantiated by its correlation with tumor size. Matrilysin expression has previously been shown to be correlated with the size of colorectal adenomas (17). Although the mechanism by which matrilysin enhances tumor growth is unclear, several lines of evidence suggest a direct role for matrilysin in cellular proliferation (33,34). MMPs, including matrilysin, reportedly activate lumenal or membrane-bound cytokines or growth factors, such as tumor necrosis factor {alpha} and heparin-binding epidermal growth factor, to locally perturb the growth of responsive cells (35,36). Alternatively, matrilysin may enhance tumor growth through the up-regulation of angiogenesis (13).

Second, the correlation between matrilysin mRNA expression and K-ras mutations was investigated. Activating K-ras mutations are one of the most frequent genetic alterations in pancreatic carcinogenesis (37). It is also well-known that K-ras mutations are one of the earliest genetic alterations in pancreatic carcinogenesis. Since matrilysin plays an important role not only in late but also early carcinogenesis, the correlation between matrilysin mRNA expression and K-ras mutations is intriguing. Nevertheless, the correlative evidence presented in this study does not justify the interpretation of a direct cause–effect relationship between K-ras mutation and matrilysin expression. Although Ohnami et al. (30) have recently shown that matrilysin was one of the genes down-regulated in the antisense K-ras-transduced pancreatic cancer cell line, further study is needed to clarify this issue.

An increase in the amount of matrilysin expression with tumor progression cannot be explained by K-ras mutation alone. The results suggest a role for molecular pathways other than the K-ras activating pathway in modulation of matrilysin expression. The human matrilysin promoter has binding sites for various transcriptional factors. Brabletz et al. (25) have shown that maximum activation of the matrilysin promoter is achieved by a combination of TCF-4, ß-catenin, ets-1, c-jun and c-fos expression vectors (25). Therefore, K-ras mutations and other genetic alterations may have an additive effect in the activation of matrilysin gene transcription in the progression of pancreatic carcinoma. Involvement of the Ets-1 gene in overexpression of matrilysin has recently been demonstrated in human hepatocellular carcinoma (38). However, Ets-1 may not play a role in overexpression of matrilysin in pancreatic carcinoma, because there was no correlation between Ets-1 and matrilysin expression (H.Yamamoto and S.Horiuchi, unpublished results). In addition to the stimulatory elements, the matrilysin promoter has TGF-ß inhibitory elements that negatively regulate its transcription (28). It is clear that the inactivation of the TGF-ß signaling pathway is an important genetic alteration in pancreatic carcinogenesis. Goggins et al. (39) have reported that the cumulative incidence of TGF-ß pathway disruption may exceed 80% in pancreatic carcinomas due to the inactivation of SMAD4 (~50%), p15 (~30%), TGF-ß type II receptor (~4%) and ALK-5 (~1%). Overexpression of TGF-ß signaling inhibitors Smad 6 and Smad 7 have also been reported in pancreatic carcinoma (40). Therefore, both constitutive activation of the K-ras pathway and inactivation of the TGF-ß pathway may be necessary for an efficient activation of matrilysin transcription in the progression of pancreatic carcinoma. Further study is required to clarify the complex regulatory mechanisms in matrilysin gene activation in pancreatic carcinoma.


    Notes
 
H.Fukushima and H.Yamamoto contributed equally to this paper

1 To whom correspondence should be addressedEmail: h-yama{at}sapmed.ac.jp Back


    Acknowledgments
 
This work was supported by Grants-in-aid from the Ministry of Education, Science, Sports and Culture (F.I. and K.I.) and from the Ministry of Health and Welfare (F.I. and K.I.), Japan.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Wilson,C.L. and Matrisian,L.M. (1996) Matrilysin: an epithelial matrix metalloproteinase with potentially novel functions. Int. J. Biochem. Cell Biol., 28, 123–136.[ISI][Medline]
  2. Chambers,A.F. and Matrisian,L.M. (1997) Changing views of the role of matrix metalloproteinases in metastasis. J. Natl Cancer Inst., 89, 1260–1270.[Abstract/Free Full Text]
  3. Wilson,C.L. and Matrisian,L.M. (1998) Matrilysin. In Parks,W.C. and Mecham,R.P. (eds) Matrix Metalloproteinases. Academic Press, San Diego, pp. 149–184.
  4. Powell,W.C., Knox,J.D., Navre,M., Grogan,T.M., Kittelson,J., Nagle,R.B., Bowden,G.T. (1993) Expression of the metalloproteinase matrilysin in DU-145 cells increases their invasive potential in severe combined immunodeficient mice. Cancer Res., 53, 417–422.[Abstract]
  5. Yamamoto,H., Itoh,F., Hinoda,Y. and Imai,K. (1995) Suppression of matrilysin inhibits colon cancer cell invasion in vitro. Int. J. Cancer, 61, 218–222.[ISI][Medline]
  6. Itoh,F., Yamamoto,H., Hinoda,Y. and Imai,K. (1996) Enhanced secretion and activation of matrilysin during malignant conversion of human colorectal epithelium and its relationship with invasive potential of colon cancer cells. Cancer, 77, 1717–1721.[ISI][Medline]
  7. Adachi,Y., Yamamoto,H., Itoh,F., Hinoda,Y., Okada,Y. and Imai,K. (1999) Contribution of matrilysin (MMP-7) to the metastatic pathway of human colorectal cancers. Gut, 45, 252–258.[Abstract/Free Full Text]
  8. Yamashita,K., Azumano,I., Mai,M. and Okada,Y. (1998) Expression and tissue localization of matrix metalloproteinase 7 (matrilysin) in human gastric carcinomas. Implications for vessel invasion and metastasis. Int. J. Cancer, 79, 187–194.[ISI][Medline]
  9. Adachi,Y., Itoh,F., Yamamoto,H. et al. (1998) Matrix metalloproteinase matrilysin (MMP-7) participates in the progression of human gastric and esophageal cancers. Int. J. Oncol., 13, 1031–1035.[ISI][Medline]
  10. Yamamoto,H., Adachi,Y., Itoh,F. et al. (1999) Association of matrilysin expression with recurrence and poor prognosis in human esophageal squamous cell carcinoma. Cancer Res., 59, 3313–3316.[Abstract/Free Full Text]
  11. Yamamoto,H., Itoh,F., Adachi,Y., Sakamoto,H., Adachi,M., Hinoda,Y. and Imai,K. (1997) Relation of enhanced secretion of active matrix metalloproteinases with tumor spread in human hepatocellular carcinoma. Gastroenterology, 112, 1271–1277.[ISI][Medline]
  12. Witty,J.P., McDonell,S., Newell,K., Cannon,P., Navre,M., Tressler,R.J. and Matrisian,L.M. (1994) Modulation of matrilysin levels in colon carcinoma cell lines affects tumorigenecity in vivo. Cancer Res., 54, 4805–4812.[Abstract]
  13. Wilson,C.L., Heppner,K.J., Labosky,P.A., Hogan,B.L. and Matrisian,L.M. (1997) Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin. Proc. Natl. Acad. Sci. USA, 94, 1402–1407.[Abstract/Free Full Text]
  14. Rudolph-Owen,L.A., Chan,R., Muller,W.J. and Matrisian,L.M. (1998) The matrix metalloproteinase matrilysin influences early-stage mammary tumorigenesis. Cancer Res., 58, 5500–5506.[Abstract]
  15. Newell,K.J., Witty,J.P., Rodgers,W.H. and Matrisian,L.M. (1994) Expression and localization of matrix-degrading metalloproteinases during colorectal tumorigenesis. Mol. Carcinogen., 10, 199–206.[ISI][Medline]
  16. Yamamoto,H., Itoh,F., Hinoda,Y., Senota,A., Yoshimoto,M., Nakamura,H., Imai,K. and Yachi,A. (1994) Expression of matrilysin mRNA in colorectal adenomas and its induction by truncated fibronectin. Biochem. Biophys. Res. Commun., 201, 657–664.[ISI][Medline]
  17. Takeuchi,N., Ichikawa,Y., Ishikawa,T., Momiyama,N., Hasegawa,S., Nagashima,Y., Miyazaki,K., Koshikawa,N., Mitsuhashi,M. and Shimada,H. (1997) Matrilysin gene expression in sporadic and familial colorectal adenomas. Mol. Carcinogen., 19, 225–229.[ISI][Medline]
  18. Mori,M., Barnard,G.F., Mimori,K., Ueo,H., Akiyoshi,T. and Sugimachi,K. (1995) Overexpression of matrix metalloproteinase-7 mRNA in human colon carcinomas. Cancer, 75, 1516–1519.[ISI][Medline]
  19. Ishikawa,T., Ichikawa,Y., Mitsuhashi,M., Momiyama,N., Chishima,T., Tanaka,K., Yamaoka,H., Miyazaki,K., Nagashima,Y., Akitaya,T. and Shimada,H. (1996) Matrilysin is associated with progression of colorectal tumor. Cancer Lett., 107, 5–10.[ISI][Medline]
  20. Honda,M., Mori,M., Ueo,H., Sugimachi,K. and Akiyoshi,T. (1996) Matrix metalloproteinase-7 expression in gastric carcinoma. Gut, 39, 444–448.[Abstract]
  21. Senota,A., Itoh,F., Yamamoto,H., Adachi,Y., Hinoda,Y. and Imai,K. (1998) Relation of matrilysin messenger RNA expression with invasive activity in human gastric cancer. Clin. Exp. Metastasis, 16, 313–321.[ISI][Medline]
  22. Yamamoto,H., Itoh,F., Adachi,Y., Fukushima,H., Itoh,H., Sasaki,S., Hinoda,Y. and Imai,K. (1999) Messenger RNA expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human hepatocellular carcinoma. Jpn. J. Clin. Oncol., 29, 58–62.[Abstract/Free Full Text]
  23. Bramhall,S.R., Neoptolemos,J.P., Stamp,G.W.H. and Lemoine,N.R. (1997) Imbalance of expression of matrix metalloproteinases (MMPs) and tissue inhibitors of the matrix metalloproteinases (TIMPs) in human pancreatic carcinoma. J. Pathol., 182, 347–355.[ISI][Medline]
  24. Crawford,H.C., Fingleton,B.M., Rudolph-Owen,L.A., Goss,K.J., Rubinfeld,B., Polakis,P. and Matrisian,L.M. (1999) The metalloproteinase matrilysin is a target of ß-catenin transactivation in intestinal tumors. Oncogene, 18, 2883–2891.[ISI][Medline]
  25. Brabletz,T., Jung,A., Dag,S. and Kirchner,T. (1999) ß-Catenin regulates the expression of the matrix metalloproteinase-7 in human colorectal cancer. Am. J. Pathol., 155, 1033–1038.[Abstract/Free Full Text]
  26. Gerdes,B., Ramaswamy,A., Simon,B., Pietsch,T., Bastian,D., Kersting,M., Moll,R. and Bartsch,D. (1999) Analysis of ß-catenin gene mutations in pancreatic tumors. Digestion, 60, 544–548.[ISI][Medline]
  27. Yashima,K., Nakamori,S., Murakami,Y., Yamaguchi,A., Hayashi,K., Ishikawa,O., Konishi,Y. and Sekiya,T. (1994) Mutations of the adenomatous polyposis coli gene in the mutation cluster region: comparison of human pancreatic and colorectal cancers. Int. J. Cancer, 59, 43–47.[ISI][Medline]
  28. Gaire,M., Magbanua,Z., McDonnell,S., McNeil,L., Lovett,D.H. and Matrisian,L.M. (1994) Structure and expression of the human gene for the matrix metalloproteinase matrilysin. J. Biol. Chem., 269, 2032–2040.[Abstract/Free Full Text]
  29. Yamamoto,H., Itoh,F., Senota,A., Adachi,Y., Yoshimoto,M., Endoh,T., Hinoda,Y., Yachi,A. and Imai,K. (1995) Expression of matrix metalloproteinase matrilysin (MMP-7) was induced by activated Ki-ras via AP-1 activation in SW1417 colon cancer cells. J. Clin. Lab. Anal., 9, 297–301.[ISI][Medline]
  30. Ohnami,S., Matsumoto,N., Nakano,M., Aoki,K., Nagasaki,K., Sugimura,T., Terada,M. and Yoshida,T. (1999) Identification of genes showing differential expression in antisense K-ras-transduced pancreatic cancer cells with suppressed tumorigenicity. Cancer Res., 59, 5565–5571.[Abstract/Free Full Text]
  31. Sobin,L.H. and Witterkind,C.H. (1997) TNM classification of malignant tumors. In International Union Against Cancer (UICC), 5th Edn. John Wiley, New York, pp. 87–90.
  32. Kataoka,H., Meng,J.-Y., Uchino,H., Nabeshima,K., Kihira,Y., Matuo,Y. and Koono,M. (1997) Modulation of matrix metalloproteinase-7 (matrilysin) secretion in coculture of human colon carcinoma cells with fibroblasts from orthotopic and ectopic organs. Oncol. Res., 9, 101–109.[ISI][Medline]
  33. Rodgers,W.H., Osteen,K.G., Matrisian,L.M., Navre,M., Giudice,L.C. and Gorstein,F. (1993) Expression and localization of matrilysin, a matrix metalloproteinase, in human endometrium during the reproductive cycle. Am. J. Obstet. Gynecol., 168, 253–260.[ISI][Medline]
  34. Sires,U.L., Griffin,G.L., Broekelmann,T.J., Mecham,R.P., Murphy,G., Chung,A.E., Welgus,H.G. and Senior,R.M. (1993) Degradation of entactin by matrix metalloproteinase. Susceptibility to matrilysin and identification of cleavage sites. J. Biol. Chem., 268, 2069–2074.[Abstract/Free Full Text]
  35. Gearing,A.J.H., Beckett,P., Christodoulou,M. et al. (1994) Processing of tumour necrosis factor-{alpha} precursor by metalloproteinases. Nature, 370, 555–557.[ISI][Medline]
  36. Lanzrein,M., Garred,O., Olsnes,S. and Sandvig,K. (1995) Diphtheria toxin endocytosis and membrane translocation are dependent on the intact membrane-anchored receptor (HB-EGF precursor): studies on the cell-associated receptor cleaved by a metalloprotease in phorbol-ester-treated cells. Biochem. J., 310, 285–289.[ISI][Medline]
  37. Almoguera,C., Shibata,D., Forrester,K., Martin,J., Arnheim,N. and Perucho,M. (1988) Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell, 53, 549–554.[ISI][Medline]
  38. Ozaki,I., Mizuta,T., Zhao,G., Yotsumoto,H., Hara,T., Kajihara,S., Hisatomi,A., Sakai,T. and Yamamoto,K. (2000) Involvement of the Ets-1 gene in overexpression of matrilysin in human hepatocellular carcinoma. Cancer Res., 60, 6519–6525.[Abstract/Free Full Text]
  39. Goggins,M., Shekher,M., Turnacioglu,K., Yeo,C.J., Hruban,R.H. and Kern,S.E. (1998) Genetic alterations of the transforming growth factor ß receptor genes in pancreatic and biliary adenocarcinomas. Cancer Res., 58, 5329–5332.[Abstract]
  40. Kleeff,J., Ishiwata,T., Maruyama,H., Friess,H., Truong,P., Buchler,M.W., Falb,D. and Korc,M. (1999) The TGF-ß signaling inhibitor Smad7 enhances tumorigenicity in pancreatic cancer. Oncogene, 18, 5363–5372.[ISI][Medline]
Received February 2, 2001; revised March 21, 2001; accepted March 22, 2001.