FLIP overexpression inhibits death receptor-induced apoptosis in malignant mesothelial cells

Oncogene (2004) 23, 7753–7760 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc ORIGINAL PAPERS FLIP overexpression inhibits death receptor-induced apoptosis in malignant mesothelial cells Maria Rita Rippo*,1, Simona Moretti1, Silvia Vescovi1, Marco Tomasetti1, Sara Orecchia3, Giuseppe Amici4, Alfonso Catalano1,2 and Antonio Procopio1,2 1 Department of Molecular Pathology and Innovative Therapies, Polytechnic University of Marche, 60100 Ancona, Italy; 2Laboratory of Cytology, Italian National Research Center on Aging, 8 60124 Ancona, Italy; 3Department of Anatomical Pathology, S Antonio and Biagio Hospital, 15100 Alessandria, Italy; 4Pediatric Hospital G Salesi, 60100 Ancona, Italy Tumors have developed several forms of resistance to receptor-induced cell death. Here, we show that malignant mesothelial (MM) cell lines as well as primary MM cells and normal mesothelial (NM) cells express Fas and TNFrelated apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5. We found that, although Fas expression levels are comparable, only MM cells are resistant to cell death. Furthermore, MM cells show resistance to TRAILinduced apoptosis. Caspase-8 (FLICE) is not activated by death receptors triggering in malignant cells whereas it is well activated by nonreceptor stimuli, such as UV radiation. We found that FLIP (FLICE-Inhibitory Protein) is constitutively expressed in all MM cell lines and is more expressed in primary MM cells than in NM cells. Knockdown of FLIP expression in MM cell lines, by a FLIPsiRNA, re-established the normal response to apoptosis induced by Fas or DR4/DR5, which was blocked by pretreatment with the caspase-8 inhibitor z-IETD-fmk. These results indicate that MM cells develop an intrinsic resistance to apoptosis induced by death receptors upregulating the expression of the antiapoptotic protein c-FLIP. Oncogene (2004) 23, 7753–7760. doi:10.1038/sj.onc.1208051 Published online 30 August 2004 Keywords: malignant mesothelioma; FLIP; caspase-8; apoptosis Fas; TRAIL; Introduction Pleural malignant mesothelioma (MPM) is a fatal tumor, which is linked to asbestos exposure (Attanoos and Gibbs, 1997) and associated to SV40 infection (Carbone et al., 1994). It is expected that the incidence of malignant mesothelioma (MM) will increase in the next decades. The survival of patients with MPM is not more than 6% with a median survival time after diagnosis of 6–12 months. Mesothelioma is a chemoresistant malignancy. Only multimodality regimens of *Correspondence: MR Rippo; E-mail: Received 22 April 2004; revised 22 July 2004; accepted 23 July 2004; published online 30 August 2004 treatment, based on surgery, radiotherapy and chemotherapy, improve the long-term survival for a very small subgroup of patients (Jaklitsch et al., 2001; Tomek et al., 2003). Furthermore, it has been shown that MM patients have impaired immune responsiveness (Manning et al., 1991). In normal cells, death is induced by several immunological, physiological stimuli, among them the triggering of the death receptor Fas (CD95/Apo-1) by its ligand (FasL). TRAIL receptors, DR4 and DR5, are expressed and selectively induce apoptosis in a wide variety of tumors without exhibiting toxicity to normal cells or tissues (French and Tschopp, 1999). Fas and DR4 and DR5 have an intracytoplasmic death domain (DD), which is important for the transduction of the proapoptotic signaling cascade. In fact, death receptor trimerization, through the DD, induces the recruitment of the adaptor protein FADD that, in turn, binds to and activates the caspase-8 proenzyme. The protein complex formed by all these proteins has been called DISC (Death-Inducing Signaling Complex) (Peter and Krammer, 2003). Activated caspase-8, in turn, mediates the dissipation of the mitochondrial transmembrane potential and the activation of the postmitochondrial caspase-9. Mitochondria, however, take part also in nonreceptor proapoptotic processes. Expression of Fas and TRAIL death receptors on normal mesothelial (NM) cells has been recently demonstrated. FasL induces apoptosis in cultured NM cells (Catalan et al., 2003). The sensitivity to Fas-induced cell death of NM cells may play an important role in controlling homeostasis in normal mesothelium, which is not only a protective layer but it is also a dynamic structure regulating serosal responses to injury, disease and infection (Mutsaers, 2002). The alteration of the Fasinduced apoptotic program could therefore play a role in the tumorigenesis of mesothelial cells. Many tumors are resistant to Fas- and TRAIL-induced cell death through different molecular mechanisms (Wright et al., 1994). Some produce decoy receptors (DcRs) (Roth et al., 2001) some, such as colon cancer cells, express FasL to perform the so-called ‘tumor counterattack’ (Hahne et al., 1996; O’Connel et al., 1996), others upregulate the expression of apoptosis inhibitors (French and Tschopp, 2002; Mitsiades et al., 2002; FLIP inhibits Fas and TRAIL-induced apoptosis in MM cells MR Rippo et al 7754 Thomas et al., 2002). The caspase-8 inhibitor FLIP (FLICE-Inhibitory Protein) has a caspase-like domain, but it lacks amino-acid residues that are critical for caspase activity. Two forms of FLIP, long (FLIPL) and short (FLIPS), exist both with inhibiting functions. The mechanism of cell death inhibition by FLIP may reside in its ability to preclude recruitment of caspase-8 to the DISC and thereby its proteolytic cleavage (Krueger et al., 2001). Much information is available about the mechanisms mediating the proliferation and progression of MM, but little is known about MM cells sensitivity to death receptors. We and others have previously demonstrated that MM cell lines show low susceptibility to TRAIL (Liu et al., 2001; Tomasetti et al., 2004) (Vivo et al., 2003) and that this is not due to Bcl-2 (Narasimhan et al., 1998). Here, we show that, although MM primary cells and cell lines express Fas and DR4/DR5, they are not killed by their crosslinking. Furthermore, we demonstrate that the mechanism of resistance to Fas and TRAIL in MM cells systems is due to FLIPL, which is overexpressed in malignant, but not in normal cells. Results MM cells are resistant to Fas- and TRAIL-induced cell death Little information exists on the sensitivity of MM cells to death receptor-induced apoptosis, thus, we have first analysed Fas expression in normal and malignant mesothelial (MM) cells. Seven MM cell lines (Ist-Mes3, MM-B1, MPP-89, Meso-2, H-Meso, Ist-Mes1, Ist-Mes2), five MM primary cultures (MesRF, Mes-SS, Mes-ST, Mes-PR, Mes-DP), and four NM cell cultures (NM-1, NM-8, NM-12, NM-13) were subjected to cytofluorimetric analysis, which revealed that Fas is expressed in all MM primary cell cultures, cell lines and NM cells and its levels appear apparently comparable, as shown in Figure 1a, where a representative cytofluorimetric analysis of MM-B1, Mes-DP and NM-1 of the extended one (Table 1) is indi (...truncated)