Cleavage of CD95 by matrix metalloproteinase-7 induces apoptosis resistance in tumour cells Susanne Strand* ,1 , Petra Vollmer 1 , Lothar van den Abeelen 1 , Daniela Gottfried 1 , Vijay Alla 1 , Hans Heid 2 , Ju¨rgen Kuball 3 , Matthias Theobald 3 , Peter R Galle 1 and Dennis Strand 1 1 I. Department of Internal Medicine, Johannes Gutenberg University, Obere Zahlbacherstr. 63, 55101 Mainz, Germany; 2 Department of Cell Biology, German Cancer Research Center, 69120 Heidelberg, Germany; 3 Department of Hematology and Oncology, Johannes Gutenberg University, 55101 Mainz, Germany The ability of tumour cells to resist apoptosis-inducing signals by cytotoxic T cells may decide the success or failure of tumour elimination. An important effector of apoptosis is the CD95/CD95 ligand system (APO-1/Fas) that mediates perforin-independent cytotoxic T-cell killing of tumour cells. We propose a new strategy by which tumour cells can resist CD95-induced apoptosis. We identified matrix metalloproteinase-7, MMP-7 (Martily- sin), as the first physiologically relevant protease that can specifically cleave CD95. MMP-7 is of unique importance because it is produced by the tumour cells themselves at early stages of tumour development. Microsequencing of the positions in CD95 cleaved by MMP-7 revealed two sites in the N-terminal extracellular domain of CD95, important for preligand assembly of CD95. MMP-7 cleavage of CD95 results in reduced CD95 surface expression and decreased CD95-mediated apoptosis sensitivity of tumour cells. Treatment of MMP-7-positive HT-29 tumour cells with MMP-7-antisense oligonucleo- tides led to an increase in CD95-mediated apoptosis sensitivity. Finally, specific cytotoxic T-cell killing was reduced in the presence of MMP-7. Thus, MMP-7 expression in tumour cells may contribute to an apoptosis- resistant phenotype, which ultimately promotes immune escape. This activity may account for the well-established role of MMP-7 in early tumour development. Oncogene (2004) 23, 3732–3736. doi:10.1038/sj.onc.1207387 Published online 12 April 2004 Keywords: CD95; MMP-7; apoptosis; immune evasion Alterations in the control of apoptosis mediated through the CD95 system contribute to the pathogenesis of a number of disorders such as cancer, autoimmunity, AIDS and liver diseases (Galle et al., 1995; Strand et al., 1996; Kondo et al., 1997; Strand et al., 1998; Krammer, 2000). Decreased sensitivity to CD95-mediated apopto- sis is a common trait shared by many cancer cells, which provides them with critical survival advantages ulti- mately promoting malignancy (Khong and Restifo, 2002). Tumours are potentially antigenic, so that the immune system is capable of recognizing and destroying these abnormal cells (Dunn et al., 2002). Thus, the ability of tumour cells to resist apoptosis-inducing signals may decide the success or failure of tumour elimination. Cytotoxic T lymphocytes (CTLs) have been consistently implicated in tumour surveillance and regression leading to the development of immunologi- cal-based cancer therapies founded on these principles (Theobald et al., 1995; Rosenberg, 1999). The efficacy of these strategies depends on the apoptosis-inducing function of CTLs that occurs primarily through both the CD95- and perforin systems (Shresta et al., 1998; Medema et al., 1999; Seki et al., 2002). Loss of CD95 expression (Galle and Krammer, 1998) and expression of soluble CD95 (sCD95) contribute to the immune escape of tumour cells and are correlated with poor prognosis (Nonomura et al., 2000; Mizutani et al., 2001; Osorio et al., 2001; Ugurel et al., 2001). Here, we propose a new strategy by which tumour cells can resist CD95-induced apoptosis; namely, by cleavage of CD95 by matrix metalloproteinase-7 (Matrilysin/MMP-7). Matrilysin/MMP-7 is distinguished from other MMP family members as being the smallest member contain- ing only the common catalytic domain and Zn 2 þ binding region, but lacking the haemopexin-like domain common to other MMPs. Along with its prometastatic function, a fundamental role for MMP-7 has also been established in early tumour development (Egeblad and Werb, 2002). Ablation of MMP-7 in a mouse model for intestinal cancer reduced tumour formation by 67% (Wilson et al., 1997). On the other hand, overexpression of MMP-7 in MMTV-neu mice, a mouse model for mammary tumorigenesis, significantly promoted tumour development in mammary glands (Rudolph-Owen et al., 1998). The mechanism by which MMP-7 contributes to early tumour growth is still unknown. We analysed the influence of MMP-7 on CD95-mediated apoptosis, with particular attention to the CD95 receptor and the cytotoxic action of specific T cells. MMP-7 specifically cleaves CD95 We expressed recombinant CD95 protein in Escherichia coli and incubated the purified protein with six different Received 25 July 2003; revised 20 November 2003; accepted 27 November 2003; Published online 12 April 2004 *Correspondence: S Strand; E-mail: sstrand@mail.uni-mainz.de Oncogene (2004) 23, 3732–3736 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc