CLINICAL ROLE OF MMP-2/TIMP-2 IMBALANCE IN HEPATOCELLULAR CARCINOMA Gianluigi GIANNELLI 1 * , Carlo BERGAMINI 1 , Felice MARINOSCI 1 , Emilia FRANSVEA 1 , Michele QUARANTA 2 , Luigi LUPO 3 , Oronzo SCHIRALDI 1 and Salvatore ANTONACI 1 1 Department of Internal Medicine, Immunology and Infectious Diseases, Section of Internal Medicine, University of Bari Medical School, Bari, Italy 2 Laboratory of Clinical Diagnostic, Oncology Institute, Bari, Italy 3 Department of Emergency and Organ Transplantation, University of Bari Medical School, Bari, Italy An imbalance between the proteolytic activity of matrix metalloproteinase-2 (MMP-2) and the tissue inhibitor of MMP-2 (TIMP-2) is responsible for degradation of extracellular matrix (ECM) components and plays a critical role in tumor invasion and in metastasis formation. The occurrence of intra-hepatic metastasis, which severely affects prognosis and long-term sur- vival, is commonly observed in the course of hepatocellular carcinoma (HCC). We investigated the expression of MT1- MMP in tissues, whereas both MMP-2 and TIMP-2 were evalu- ated in the sera and tissues (primary and metastatic nodules) of HCC patients with and without metastasis, whose clinical out- come was followed over a 2-year period. MT1-MMP expression was similar among primary nodule tissues of patients with and without metastasis. Serum and tissue levels of MMP-2 were not statistically different between patients with and without metas- tasis, but MMP-2 was concentrated at the invasive edge of the metastatic tissue. On the contrary, serum and tissue levels of TIMP-2 were significantly higher in HCC patients without me- tastasis than in those with. This situation was not only observed in the primary HCC tissues, but also in the metastatic nodules. These results correlate with the clinical outcome, because more than 90% of the patients with high levels of TIMP-2 were still alive after 2 years, whereas less than 30% with low levels of TIMP-2 had survived. Furthermore, we found a strict correla- tion between tissue and serum levels of TIMP-2, this suggesting that a MMP-2/TIMP-2 imbalance and in particular TIMP-2 lev- els, could represent an important prognostic factor in patients with HCC. © 2002 Wiley-Liss, Inc. Key words: MMP-2; TIMP-2; hepatocellular carcinoma; metastasis; tumor invasion Matrix metalloproteinase 2 (MMP-2) is a member of the MMPs family, a class of enzymes with proteolytic activity, secreted in a latent form and activated at the cellular surface by the complex membrane type 1-MMP (MT1-MMP) and tissue inhibitor of MMPs (TIMP-2). 1,2 When activated, MMP-2 degrades the extra- cellular matrix (ECM) components of the basement membrane (BM) removing tissue boundaries and facilitating epithelial cell motility. 3–6 Its activity, however, is inhibited by TIMP-2 so that a too extensive degradation is avoided. 7,8 For these reasons, the role of TIMP-2 is intriguing, not only in view of its involvement in the MMP-2 activation process, but also because of its inhibitory func- tion of MMP-2 proteolytic activity. In the human body, the ex- pression of MMP-2 and TIMP-2 overlaps in the various different tissues and proteolysis occurs as a result of an imbalance between MMP-2 and TIMP-2 activity. 9,10 MMP-2 has been implicated in a number of situations such as development, morphogenesis and tumor progression, where re- modeling of the tissue architecture and alteration of ECM turn- over occur. 11–14 Several studies have reported that MMP-2 is up-regulated in the course of different malignancies and is in- volved in tumor metastasis. 15,16 Other studies have described a downregulation of TIMP-2 in cancer tissues together with essen- tially normal levels of MMP-2. 17 Metastatic dissemination, how- ever, is facilitated by increased levels of MMP-2 or decreased levels of TIMP-2, either of which can shift the balance in favor of proteolysis and cause BM degradation. 4,18 This is the key point for the beginning of tumor spread and metastasis formation, which have a positive correlation with MMP-2 proteolytic activity. The occurrence of metastasis is one of the most serious factors in the course of cancers such as hepatocellular carcinoma (HCC). HCC is the seventh most frequent malignancy in the world and its fre- quency is constantly increasing in the Mediterranean area because of the spread of HCV infection. 19 Although many improvements have been made in terms of diagnosis and treatment, the long-term survival of HCC patients remains unsatisfactory. Tumor recurrence, as well as intra-hepatic and vessel metastasis, are the worst problems in patients with HCC, even in those undergoing surgical therapy, because these severely affect prognosis and survival. 19,20 Furthermore, no therapy is currently available to prevent or block HCC cell invasion, mainly due to the poor current knowledge of the mechanisms that regulate cancer cell motility and invasion. Recently, we have reported that human HCC cell lines with a motile and invasive phenotype produce and activate high levels of MMP-2, that are used to migrate on the ECM substrate and to invade through a reconstituted BM. 21 A synthetic inhibitor of MMPs blocks migration and invasion in vitro and the same inhibitor showed a cytostatic therapeutic effect in an orthotopic metastatic HCC model in nude mice. 22 In the Mediterranean area as well as in North American coun- tries, HCC develops only in chronically injured livers, where altered turn-over and increased deposition of ECM proteins has been reported. In such an environment, upregulation of MMPs and TIMP-2 has been reported and it is possible they have a role in the rearrangement of the liver tissue architecture. 23,24 There is no BM in the liver, but HCC cancer cells grow surrounded by ECM proteins secreted as a consequence of cirrhosis and therefore proteolytic activity is required to allow HCC cells to penetrate and cross over such tissue boundaries. 21 The goal of our study is to investigate the expression and the clinical role of MMP-2, TIMP-2 and MT1-MMP in patients with HCC. MATERIAL AND METHODS Patients and specimens We have studied serum and tissue samples of 40 Italian patients with HCC (35 men and 5 women age 40 – 82 years) that underwent Abbreviations: BM, basement membrane; HCC, hepatocellular carci- noma; LC, liver cirrhosis; MMP-2, matrix metalloproteinase-2; MT1- MMP, membrane type 1-MMP; TIMP-2, tissue inhibitor of MMP-2. Grant sponsor: Ministry for University Technological and Scientific Research (MURST); Grant sponsor: Ministry for Public Health *Correspondence to: Dipartimento di Clinica Medica, Immunologia, e Malattie Infettive. Clinica Medica II, Policlinico, Piazza G. Cesare 11, 70124, Bari, Italy. Fax: +39-080-5478-670. E-mail: g.giannelli@uniba.intmed.it Received 7 June 2001; Revised 23 July 2001; Accepted 30 July 2001 Published online 22 October 2001; DOI 10.1002/ijc.1635 Int. J. Cancer: 97, 425– 431 (2002) © 2002 Wiley-Liss, Inc. Publication of the International Union Against Cancer