SHORT REPORT TRANSGENIC OVEREXPRESSION OF A DOMINANT NEGATIVE MUTANT OF FADD THAT, ALTHOUGH COUNTERSELECTED DURING TUMOR PROGRESSION, COOPERATES IN L-myc-INDUCED TUMORIGENESIS Anne-Odile HUEBER 1 , Susanne B¨ OSSER 2 and Martin Z¨ ORNIG 2 * 1 Institute of Signaling, Developmental Biology and Cancer Research, Centre National de la Recherche Scientifique Unite ´ mixte de Recherche 6543 Centre A. Lacassagne, Nice, France 2 Georg-Speyer-Haus, Frankfurt, Germany Activation of the so-called death receptors, e.g., CD95/Fas/ Apo-1, is a potent stimulus to trigger apoptosis. Overexpres- sion of the C-terminal FADD deletion mutant FADD-DN blocks death receptor–induced apoptosis, but despite this antiapoptotic activity, lck FADD-DN transgenic mice do not develop lymphomas. To analyze whether functional inactiva- tion of FADD cooperates with Myc overexpression in tumor- igenesis, lck FADD-DN transgenic mice were crossed with E L-myc transoncogenic animals. While no tumors were de- tected in single transgenic FADD-DN or L-myc mice within 15 months, 5 of 17 (29%) FADD-DN/L-myc double transgenic animals developed lymphomas with an average latency pe- riod of 47 weeks. Protein analysis of FADD-DN/L-myc tumors showed, however, undetectable levels of FADD-DN protein. FADD-DN protein expression was again lost in 16 of 17 FADD-DN/p53 k.o. T-cell lymphomas, though no significant acceleration of tumorigenesis in P53-deficient lck FADD-DN mice compared to p53 k.o. animals was observed. These data suggest a strong counterselection against the FADD-DN pro- tein during tumor progression, which could be explained by the cell cycle inhibitory activity of FADD-DN. Such counter- selection would have to be compensated for by other anti- apoptotic mutations, and indeed, strong upregulation of the antiapoptotic Bcl-2 family member Bcl-x L was found in one of the tumors. This in vivo mouse model demonstrates that an antiapoptotic protein involved in the onset of tumorigenesis is selected against and consequently lost during tumor pro- gression because of its additional antiproliferative activity. © 2004 Wiley-Liss, Inc. Key words: FADD-DN; death receptor; cell cycle; oncogene coop- eration Apoptosis as a special form of programmed cell death has been recognized as a fundamental process both throughout embryogen- esis and in the adult organism to maintain tissue homeostasis and remove dangerous or surplus cells without inflammation. 1 For a cancer to occur and progress, apoptosis must be inhibited as the tumor cells are constantly exposed to a variety of apoptotic stimuli, e.g., overexpression of apoptosis-inducing oncogenes, hypoxia or lack of survival signals after detachment of metastatic cells from the tumor mass (anoikis). 2 Furthermore, conventional treatment of cancer by chemotherapy and irradiation is based on the selective induction of apoptosis in tumor cells, and tumor resistance to such treatment is a result of the inhibition of drug- or radiation-induced apoptosis. 3 The extrinsic apoptotic pathway is triggered by so-called death receptors, which together with their ligands belong to the TNF/ TNFR superfamily. The death receptor subfamily consists of CD95/Fas/Apo-1, TNFR1, DR3 and the TRAIL receptors DR4/ DR5, DR6, NGFR and EDAR. 4 Death receptors contain in their intracellular part a specific protein-binding motif, the DD, which mediates homophilic interactions with other DD-bearing proteins. FADD is essential for death receptor–induced apoptosis as it is used as an adaptor protein by several DD-containing death recep- tors. 5–15 In the activated CD95 receptor, FADD binds with its N-terminal DD to the CD95 DD. FADD then recruits the apical caspase-8 via another protein-binding domain in its C terminus, the DED. Within this assembled intracellular receptor complex (DISC 16 ), caspase-8 becomes activated (“induced proximity” 17 ) and starts to proteolyse its targets. Death receptor–mediated cell killing is normally induced by the death receptor ligands and plays an important role during physio- logical cell death as well as in different antitumor strategies (e.g., cytotoxic T-cell killing 18 ). Consequently, several mutations have been identified in tumors which lead to impairment of death receptor–induced apoptosis. 1,19 Examples include overexpression of the inhibitory FLIP proteins in melanomas, 20 transcriptional downregulation of caspase-8 mRNA by gene deletion or promoter methylation 21 and CD95 mutations in different cancers. 22–24 Direct proof of the tumor-suppressing proapoptotic activity of the CD95 receptor has been obtained by in vivo cooperation experiments. Tumor development in L-myc transgenic mice is accelerated when mice are bred onto a CD95-deficient lpr background. 25 Interference with FADD functionality leads to the expected impairment of death receptor killing. 26 –30 Surprisingly, however, not only cell death but also T-cell proliferation is strongly inhibited in lck FADD-DN transgenic mice 28 –30 and in rag1 –/– k.o. animals reconstituted with a FADD –/– k.o. T-cell compartment. 26 The influence of such a FADD-dependent signaling pathway on pro- liferation is not restricted to T cells 31,32 and supports further data linking nonapoptotic death receptor signaling to proliferation. ( 33,34 and references therein). While downregulation of apoptosis is clearly an oncogenic event, inhibition of proliferation represents a tumor-suppressing activity. We investigated whether the inhibition of death receptor– induced apoptosis by FADD-DN overexpression can accelerate tumorigenesis and how the antiproliferative influence of FADD-DN would affect tumor progression. As lck FADD-DN transgenic mice do not develop lymphomas, 28 –30 we crossed these Abbreviations: DD, death domain; DED, death effector domain; DISC, death-inducing signaling complex; DN, dominant negative; ECL, enhanced chemiluminescence; FADD, Fas-associated DD protein; k.o., knockout; PVDF, polyvinylidene difluoride; TCR, T-cell receptor; TNF, tumor ne- crosis factor; TNFR, TNF receptor; TRAIL, TNF--related apoptosis- inducing ligand. Grant sponsor: Centre National de la Recherche Scientifiqu; Grant sponsor: Association pour la Recherche contre le Cancer; Grant sponsor: Deutsche Krebshilfe; Grant number: 10-1605. *Correspondence to: Georg-Speyer-Haus, Paul-Ehrlich-Strasse 42-44, 60596 Frankfurt, Germany. Fax: +49-69-63395297. E-mail: zoernig@em.uni-frankfurt.de Received 21 January 2004; Accepted after revision 28 April 2004 DOI 10.1002/ijc.20422 Published online 16 June 2004 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 112, 536 –540 (2004) © 2004 Wiley-Liss, Inc. Publication of the International Union Against Cancer