Molecular Cancer BioMed Central Research Open Access Human agonistic TRAIL receptor antibodies Mapatumumab and Lexatumumab induce apoptosis in malignant mesothelioma and act synergistically with cisplatin Larisa L Belyanskaya†1,2, Thomas M Marti*†1, Sally Hopkins-Donaldson1, Stefanie Kurtz1, Emanuela Felley-Bosco1 and Rolf A Stahel1 Address: 1Laboratory of Molecular Oncology, Clinic and Policlinic of Oncology, University Hospital of Zürich, Häldeliweg 4, 8044 Zürich, Switzerland and 2Materials-Biology Interactions Laboratory, Materials Science and Technology (Empa), Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland Email: Larisa L Belyanskaya - [email protected]; Thomas M Marti* - [email protected]; Sally Hopkins- Donaldson - [email protected]; Stefanie Kurtz - [email protected]; Emanuela Felley-Bosco - emanuela.felley- [email protected]; Rolf A Stahel - [email protected] * Corresponding author †Equal contributors Published: 22 October 2007 Received: 3 May 2007 Accepted: 22 October 2007 Molecular Cancer 2007, 6:66 doi:10.1186/1476-4598-6-66 This article is available from: http://www.molecular-cancer.com/content/6/1/66 © 2007 Belyanskaya et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: The incidence of malignant pleural mesothelioma (MPM) is associated with exposure to asbestos, and projections suggest that the yearly number of deaths in Western Europe due to MPM will increase until 2020. Despite progress in chemo- and in multimodality therapy, MPM remains a disease with a poor prognosis. Inducing apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or agonistic monoclonal antibodies which target TRAIL-receptor 1 (TRAIL-R1) or TRAIL-R2 has been thought to be a promising cancer therapy. Results: We have compared the sensitivity of 13 MPM cell lines or primary cultures to TRAIL and two fully human agonistic monoclonal antibodies directed to TRAIL-R1 (Mapatumumab) and TRAIL-R2 (Lexatumumab) and examined sensitization of the MPM cell lines to cisplatin-induced by the TRAIL-receptor antibodies. We found that sensitivity of MPM cells to TRAIL, Mapatumumab and Lexatumumab varies largely and is independent of TRAIL-receptor expression. TRAIL-R2 contributes more than TRAIL-R1 to death-receptor mediated apoptosis in MPM cells that express both receptors. The combination of cisplatin with Mapatumumab or Lexatumumab synergistically inhibited the cell growth and enhanced apoptotic death. Furthermore, pre-treatment with cisplatin followed by Mapatumumab or Lexatumumab resulted in significant higher cytotoxic effects as compared to the reverse sequence. Combination-induced cell growth inhibition was significantly abrogated by pre-treatment of the cells with the antioxidant N-acetylcysteine. Conclusion: Our results suggest that the sequential administration of cisplatin followed by Mapatumumab or Lexatumumab deserves investigation in the treatment of patients with MPM. Page 1 of 13 (page number not for citation purposes) Molecular Cancer 2007, 6:66 http://www.molecular-cancer.com/content/6/1/66 Background (FADD) with the cytoplasmic tail of the receptor. FADD Malignant pleural mesothelioma (MPM) is a generally then recruits procaspase-8, which undergoes spontaneous fatal thoracic neoplasia that arises from the pleural lining. autoactivation. Activated caspase-8, in turn, cleaves and In the majority of the patients, a history of occupational activates the effector caspases-3, -6 and -7 which cleave exposure to asbestos can be elicited [1]. Taking into cellular substrates to execute cell death [7,8]. Recent data account a latency period of 20–50 years and a decline in suggest the existence of considerable cross-talk between workplace exposure to asbestos in Europe since the 1970s, the extrinsic and intrinsic death signalling pathways. Cas- it is estimated that the number of men dying from MPM pase-8, a key player of this communication platform, can in Europe will double each year until a peak is reached in proteolytically activate the BH3 only family member Bid, about between 2015 and 2020 [2,3]. which induces Bax- and Bak-mediated release of cyto- chrome c and Smac/DIABLO from mitochondria [14]. No chemotherapy regimen for mesothelioma has proven Resistance to TRAIL can occur by different mechanisms, curative, although several treatments are valuable for pal- including lack of TRAIL apoptosis receptors, death recep- liation. The clinically best documented chemotherapy is a tor mutations [15], and enhanced expression of TRAIL- combination of cisplatin with an antifolate. A large phase decoy receptors [16]. FLIP, which bears structural similar- III study comparing the combination of cisplatin and ity to caspase-8, but lacks caspase-8 activity, can inhibit pemetrexed with cisplatin alone demonstrated a superior death receptor-mediated signalling by binding to FADD response, survival and a better quality of life for the com- [17]. Both forms of FLIP, the long form c-FLIPL and the bination [4,5]. For earlier stages of disease, specialized short form c-FLIPS can compete for apical caspase recruit- centers offer multimodality therapy with adjuvant or neo- ment to the DISC, whereas FLIPL can also inhibit the full adjuvant chemotherapy, radical surgery with or without processing of caspase-8 [18]. radiotherapy [6]. However, despite such aggressive treat- ment most patients have disease recurrence within 2 MPM cells have been found by others to be resistant or to years. Therefore, new therapeutic options are needed for have a low susceptibility to TRAIL-induced apoptosis, and more effective treatment of this malignancy. As demon- require either FLIPL siRNA, chemotherapeutic drugs, α- strated by our in vitro investigations, the combination of tocopheryl succinate or cycloheximide to be combined cisplatin-based chemotherapy with agonistic TRAIL recep- with TRAIL for apoptosis to occur [19-22]. However, these tor antibodies might be a promising option. studies were performed with a small number of estab- lished human MPM cell lines only and it remains Tumor necrosis factor (TNF)-related apoptosis-inducing unknown whether the majority of MPM cell lines and pri- ligand (TRAIL) is a type II transmembrane protein belong- mary cultures are indeed resistant to TRAIL combined ing to the TNF family of death ligands. Four TRAIL recep- with chemotherapy. In addition, no information exists on tors have been identified of which two, TRAIL-R1/DR4 the sensitivity of MPM cells to two fully human agonistic and TRAIL-R2/DR5, are capable of transducing an apop- monoclonal antibodies which target TRAIL-R1 (Mapatu- totic signal whereas the other two receptors (TRAIL-R3/ mumab) and TRAIL-R2 (Lexatumumab) although they DcR1, TRAIL-R4/DcR2) act as antagonists since they lack have the advantage over TRAIL of a longer plasma half-life death domains and thus cannot engage the apoptotic and a higher specificity [23]. machinery [7,8]. An additional receptor, osteoprotegrin, has been identified but its activity is still matter of debate In the present study, we compared the sensitivity of 13 because of its low affinity for TRAIL at 37°C [9]. TRAIL MPM cell lines or primary cultures to TRAIL and to two can preferentially induce apoptosis in a variety of tumor fully human agonistic monoclonal antibodies which tar- cell types, whereas normal cells do not appear to be sensi- get TRAIL-R1 and TRAIL-R2, and examined the apoptosis tive [10]. This property suggests TRAIL-R targeting is an sensitization of the MPM cell lines with different sensitiv- excellent strategy for selective cancer therapy and oncol- ity to Mapatumumab or Lexatumumab by the cytotoxic ogy trials with TRAIL and TRAIL-R human agonistic anti- drug cisplatin. bodies have been initiated [11,12]. Results Apoptosis-inducing mechanisms by human agonistic Expression of TRAIL receptors in MPM cell lines or TRAIL-R antibodies Mapatumumab and Lexatumumab primary cultures are thought to be similar to TRAIL-mediated apoptosis The expression of the four membrane-bound TRAIL- [13]. TRAIL-induced cell death is triggered by the interac- receptors was analyzed in a large panel of commercially tion of the ligand with TRAIL-R1 or TRAIL-R2 to assemble available and well-established MPM cells lines obtained the death-inducing signaling complex. The latter forms from human biopsies or pleural effusions [24,25]. Flow when death receptor ligation triggers association of the cytometry analyses performed on non-permeabilized cells intracellular adaptor, Fas-associated death domain with monoclonal antibodies raised against the extracellu- Page 2 of 13 (page number not for citation purposes) Molecular Cancer 2007, 6:66 http://www.molecular-cancer.com/content/6/1/66 lar domains of TRAIL-R1 and -R2, respectively, demon- MPM cells expressing both receptors is unknown. To strated expression of both receptors at the cell surface of investigate the relative contribution of TRAIL-R1 and -R2 MPM cells, with higher TRAIL-R2 than TRAIL-R1 expres- in apoptosis induction, MPM cell lines and primary cul- sion in the majority of cells analysed (Table 1). In con- tures were incubated with increasing concentrations of trast, the two decoy receptors TRAIL-R3 and -R4 were specific human selective agonistic monoclonal antibodies either