Expression of collagenase-3 (matrix metalloproteinase-13) in human gastric cancer

Ayman Elnemr 2 3 Yutaka Yonemura 1 2 Etsurou Bandou 1 2 Kazuo Kinoshita 1 2 Taiichi Kawamura 1 2 Shigeru Takahashi 1 2 Shizuka Tochiori 0 2 Yoshio Endou 0 2 Takuma Sasaki 0 2 0 Department of Experimental Therapeutics, Cancer Research Institute, Kanazawa University , Kanazawa, Japan 1 Department of Surgery, Shizuoka Cancer Center , Shizuoka, Japan 2 Offprint requests to: Y. Yonemura Present address: Department of Gastroenterological Surgery , 1007 Shimo-nagakubo, Nagaizumi-machi, Shizuoka 411-8777, Japan Received: May 22, 2002 / Accepted: October 2, 2002 3 Department of Surgery, Faculty of Medicine, Tanta University , Tanta, Egypt Background. Collagenase-3 (matrix metalloproteinase-13; MMP-13) is a recently identified member of the matrix metalloproteinases (MMPs) with broad substrate specificity, and a potential role in tumor metastasis and invasion has been proposed for this enzyme. To date, in gastrointestinal tract tumors, collagenase-3 expression has been reported only in esophageal carcinoma; the presence and possible implications of this enzyme in the progression of gastric cancer are unknown. Methods. In this study, MMP-13 mRNA expression was analyzed in a series of 110 matched gastric adenocarcinomas and the corresponding adjacent normal mucosae as well as in nine gastric cancer cell lines. In addition, the mRNA expression of gelatinase A (MMP-2) and membrane type-1 matrix metalloproteinase (MT1-MMP), two MMPs which have the ability to activate MMP-13 in vitro, was also examined in the same cases and cell lines. The production and localization of MMP-13, MMP-2, and MT1-MMP were investigated by immunohistochemistry, immunofluorescence, Western blot analysis, and zymography. Results. MMP-13 mRNA was expressed in 23 of the 110 carcinomas (21%), and MT1-MMP mRNA was expressed in 45 (40%), but no MMP-13 or MT1-MMP mRNA was detected in any of the normal mucosae. Also, eight of the nine gastric cancer cell lines expressed mRNA of MMP-13, and in each cell line there was coordinate expression with either MT1MMP or MMP-2 mRNA. MMP-13 and MT1-MMP were detected at the bases of invadopodia of the cultured cancer cells as well as in the invasive front of the tumors, as shown by immunofluorescence and immunohistochemistry, respectively. Western blot analysis revealed the presence of MMP13 protein in those cell lines and carcinomas that expressed its mRNA. On zymography, almost all cell lines that expressed MMP-13 showed gelatinolytic bands corresponding to the ac- - tive form of MMP-13 or one of its intermediate forms. Also, zymographic analysis of the tumor specimens revealed strong gelatinolytic bands of MMP-13 and MMP-2, whereas these bands in normal mucosa were weak. There was no significant relationship between MMP-13 mRNA expression and histologic type, lymph node metastasis, wall invasion, or distant metastasis. However, patients with MMP-13 mRNA-positive tumors had a poorer prognosis than those with MMP-13mRNA-negative cancer. Furthermore, patients with simultaneous expression of MMP-13 and MT1-MMP mRNA showed the poorest prognosis, as compared with those having tumors expressing either MMP-13 or MT1-MMP, or neither MMP-13 nor MT1-MMP mRNA. Conclusion. These findings suggest that MMP-13 expression may contribute to the progression of gastric cancer, and its coordinate overexpression with MT1-MMP and/or MMP-2 may have a cooperative effect in the progression of gastric cancer. The incidence of gastric carcinoma is the highest of all carcinomas, and it is the leading cause of death from cancer in Japan. In spite of improvements in surgical treatment and chemotherapy, the prognosis is still poor, owing to local recurrence or metastasis [13]. Degradation of the extracellular matrix during tumor invasion and metastasis is thought to result from a combined action of several proteolytic enzyme systems, including the collagenases and other matrix metalloproteinases (MMPs) [4,5] and serine proteases, such as plasmin generated by the urokinase pathway of plasminogen activation [6]. Human collagenase-3 (MMP-13) is a recently identified member of the matrix MMP family that was originally isolated from breast carcinoma [7]. Expression of collagenase-3 has been detected in squamous carcinomas of the head and neck [8], chondrosarcoma [9], transitional-cell carcinoma of the urinary bladder [10], oral mucosal epithelium of chronic inflammation [11], rheumatoid synovium, and developing bone [12,13], but not in normal adult tissues. Biochemical characterization of collagenase-3 has revealed that it is a very potent enzyme that, after activation through a proteolytic cascade mechanism, displays a broad spectrum of activity against connective tissue components [14,15]. Thus, collagenase-3 degrades very efficiently the native helix of all fibrillar collagens, with preferential activity on type II collagen. In addition to its proteolytic activity on fibrillar collagens, collagenase-3 is also a powerful gelatinase and thus may contribute to further degrade the initial cleavage products of collagenolysis to small fragments suitable for further metabolism [15]. Because fibrillar collagens are the most abundant structural components of human connective tissues, it is conceivable that the ability to degrade collagen extracellular matrix is crucial for the invasion of neoplastic cells [16]. The wide substrate specificity of MMP-13 also makes it a potent proteolytic tool for invading tumor cells [810]. As collagenase-3 widely degrades components of the basement membrane and connective tissue surrounding tumor cells, this collagenase is likely to play crucial roles in modulating extracellular matrix degradation and cellmatrix interactions involved in metastasis. Previous experiments using recombinant human procollagenase-3 showed that MT1-MMP, as well as MMP-2, may be able to activate progelatinase-3, alone or in concert, thereby establishing a new activation cascade consisting of three members of the MMP family; these experiments have also shown that active collagenase-3 can activate gelatinase B (MMP-9) [14,17]. In gastrointestinal tract malignancies, only esophageal cancer was shown to produce collagenase-3 and its activator MT1-MMP [18]; however, to our knowledge, there is no report about collagenase-3 expression in gastric cancer. In the present study, using nine human gastric cancer cell lines and resected specimens of 110 gastric adenocarcinomas, we investigated the mRNA and protein expression, as well as the enzymatic activity, of MMP13, MMP-2, and MT1-MMP. In addition, we studied whether collagenase-3 plays an important role in tumor aggressiveness in association with MT1-MMP. Materials and methods Cell lines and culture conditions The gastric cancer cell lines TMK-1, KATO-III, AZ521, NUGC-3, MKN-28, MKN-45, MKN-45-P, and KMST-6 cells, a human immortalized fibroblast cell line were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA). The gastric cancer ce (...truncated)