Contribution of matrilysin (MMP-7) to the metastatic pathway of human colorectal cancers

Abstract

Background/aim: Matrilysin is one of the matrix metalloproteinases that has a critical role in tumour invasion, and is often expressed in gastrointestinal cancers. The aim of this study was to examine the role of matrilysin in metastasis of human colorectal cancers. PATIENTS (SUBJECTS)/METHODS: The relation between matrilysin expression and Dukes's type was investigated immunohistochemically in 83 surgically resected colorectal cancers, including five with liver metastasis. Moreover, the effects of matrilysin on the in vivo invasive and metastatic potential of colon cancer cells transfected with matrilysin cDNA were examined after subcutaneous injection into SCID mice.

Results: In 46% of primary and all of metastatic liver tumours, over 10% of cancer cells were stained positively for matrilysin. The expression of matrilysin correlated significantly with the presence of nodal or distant metastases (p<0.05). In addition, matrilysin transfectants formed invasive tumours and multiple liver metastases in SCID mice, without producing any significant difference in the subcutaneous tumour growth from mock transfectants. Casein zymography showed that the invading and metastasised tumours showed conspicuous matrilysin activity, which correlated with the number of metastatic lesions (p<0.001).

Conclusions: Matrilysin showed a correlation with metastasis in a cohort of 83 colorectal cancer patients and marked metastatic potentiation in human colorectal cancer xenografts, indicating that it may play a critical role in the metastatic pathway of colorectal cancers.

Figure 1

Immunostaining of human colon cancers with anti-matrilysin monoclonal antibody was performed as described in Materials and methods (original magnification × 200). (A) A well differentiated adenocarcinoma case of Dukes's type C. The monoclonal antibody stained tumour cell cytoplasm and cell membranes, but did not stain the stromal components. (B) Immunostaining with negative control monoclonal antibody of the serial section of (A). Adjacent normal colon epithelium (C) was not stained with anti-matrilysin monoclonal antibody. (D) A well differentiated adenocarcinoma case in Dukes's type D. Metastatic liver nests (E) were stained more strongly than the primary site (D).

Figure 2

(A) Northern blot analysis and (B) casein zymography of parental cells, sublines of matrilysin transfectants, and mock transfectants were performed as described in Materials and methods. The matrilysin transfectants expressed various levels of matrilysin mRNA (A) and secreted promatrilysin (29 kDa) and activated matrilysin (19 kDa; B), but neither was produced by parental cells or mock transfectants: lane 1, CHC-Y1; lane 2, CHC-Mat-1; lane 3, CHC-Mat-2; lane 4, CHC-Mat-3; lane 5, CHC-Mat-4; lane 6, CHC-Mat-5; lane 7, CHC-Mat-6; lane 8, CHC-mock. Clones CHC-Mat-4 (lane 5) and CHC-Mat-6 (lane 7) were used as sublines expressing high and moderate levels of matrilysin respectively.

Figure 3

(A) 3-(4,5-Dimethylthiazol)-2,5-diphenyl- tetrazolium bromide assay showed that there was no significant difference in in vitro growth in CHC-Y1, mock transfectants, and matrilysin transfectants. (B) Gold colloid assay showed that in vitro motility of two matrilysin transfected sublines was equivalent to those of parental cell and mock transfectants.

Figure 4

Tumour volumes of transplanted subcutaneous site in SCID mice were measured and calculated every seven days. Growth of tumours derived from two matrilysin transfected sublines was not significantly different from those derived from mock transfectants or parental cell CHC-Y1.

Figure 5

Each subline of matrilysin transfected cancer cells and mock transfectants formed almost the same sized subcutaneous tumour six weeks after injection into SCID mice. (A) Primary subcutaneous tumours of mock transfectants did not invade the muscle tissues (original magnification × 100). (B) Primary subcutaneous tumours of matrilysin transfectants CHC-Mat-6 invaded the muscle layer (original magnification × 100). (C) The mice injected with matrilysin transfectants showed multiple liver metastases (right), while those injected with mock transfectants did not (left). (D) Microscopic view of liver metastatic nest (C, right) showing colon cancer cells in left upper side of the field (original magnification × 400). Arrowheads indicate the border of tumour cells and hepatocytes.

Figure 6

Primary and metastatic tumours of SCID mice were analysed by zymography as described in Materials and methods. (A) Casein zymography showed that primary subcutaneous tumours derived from the two matrilysin transfectants produced matrilysin, the active form (19k) of which was much more abundant than the latent form (29k). (B) Casein zymography of liver showed that activated matrilysin was effectively expressed in metastatic lesions but neither form of matrilysin was detected in normal murine liver.