Oncogene (2003) 22, 2795–2804 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Loss of FADD protein expression results in a biased Fas-signaling pathway and correlates with the development of tumoral status in thyroid follicular cells Le´ a Tourneur1, Sylvie Mistou1, Francine-Marie Michiels2, Vale´ rie Devauchelle1, Laurent Renia1, Jean Feunteun2 and Gilles Chiocchia*,1 1De´partement d’Immunologie, Institut Cochin, INSERM U567, CNRS UMR 8104, IFR 116, Universite´ Rene´ Descartes, 27 rue du fbg St-Jacques, 75014 Paris, France; 2Laboratoire d’Oncologie Mole´culaire, CNRS Unite´ de Recherche Associe´e 1158, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, France Downregulation of proapoptotic molecules like Fas or (Djerbi et al., 1999; Medema et al., 1999) has been caspase 8, or upregulation of antiapoptotic molecules like reported during tumor progression. FLICE inhibitory protein has been suggested to be a Activation of Fas by its ligand leads to recruitment of regulatory mechanism set up by tumor cells to block the the adapter molecule Fas-associated protein with death death signal received via death receptors. In an in-depth domain (FADD), which, in turn, activates FLICE/ study of the Fas/FasL-signaling pathway in thyroid tumor caspase 8, a process leading to apoptosis (Nagata, 1997). development, we have demonstrated that tumor cells FLIP, an antiapoptotic protein, can protect cells from specifically downregulate the multideath receptor adapter apoptosis by preventing FADD–caspase 8 association Fas-associated death domain (FADD). The regulation of (Irmler et al., 1997). Fas death domain-associated FADD expression occurred only at the protein level. protein (Daxx), a second adapter molecule, defines a Furthermore, in the absence of FADD, Fas-signaling different signaling pathway downstream of Fas (Yang resulted in accelerated growth of thyrocytes. Since et al., 1997). It can independently activate both the Jun thyrocytes also acquired FasL expression during tumor N-terminal kinases (JNK) and p38-mitogen-activated development, the absence of FADD protein could lead to protein kinases (p38-MAPK) pathways through the greater resistance to numerous death receptor-mediated apoptosis signal-regulating kinase 1 (ASK1) intermedi- apoptosis, stimulation of their own proliferation through ate (Ichijo et al., 1997; Chang et al., 1998). It seems that Fas/FasL interaction, and the capacity to counter-attack the JNK pathway generally leads to apoptosis, whereas the infiltrating lymphocytes. p38 kinase can have pro- or antiapoptotic effects, the Oncogene (2003) 22, 2795–2804. doi:10.1038/sj.onc.1206399 final result depending on multiple factors such as the type of cell and its state of differentiation. Recently, the Keywords: FADD; Fas ligand; thyroid; tumor kinase receptor-interacting protein (RIP) was reported to be a third adapter for Fas (Holler et al., 2000). It can transduce Fas-mediated caspase-dependent or -indepen- Introduction dent cell death. The thyroid is of particular interest regarding the role Dysregulation of the Fas pathway is responsible for of Fas/FasL since Fas is constitutively expressed by hypo- or hyperproliferative disorders that are as a result of excess or faulty apoptosis, respectively. FasL expres- normal thyroid follicular cells (TFC), and FasL expres- sion can occur in numerous pathological thyroid sion on tumor cells allows the cells to escape the immune conditions (Baker, 1999), including cancer (Mitsiades system by killing infiltrating lymphocytes that express et al. et al. the receptor Fas, although this point is still controversial , 1999), Hashimoto’s thyroiditis (Giordano , 1997) and Grave’s disease (Hiromatsu et al., 1999). (Nagata, 1996; Strand et al., 1996; Favre-Felix et al., In the thyroid gland, guanine nucleotide stimulatory 2000; Restifo, 2000). Usually, when tumor cells coex- factor (Gs) activates adenylate cyclase in response to press Fas and FasL molecules, they turn out to be thyrotropin stimulation, leading to cyclic AMP level resistant to Fas-mediated cell death. Among the elevation and subsequent thyroid cell proliferation regulatory mechanisms that may take place to block (Dumont et al., 1992). Mutations of the a subunit of the death signal, downregulation of proapoptotic the Gs factor (gsp mutations), resulting in constitutive molecules such as Fas (Moller et al., 1994) or caspase activation of adenylate cyclase, are found in approxi- 8 (Teitz et al., 2000), or upregulation of antiapoptotic mately 30% of human thyroid toxic adenomas and 10% molecules such as FLIP (FLICE inhibitory protein) of human thyroid carcinomas (Suarez et al., 1991; Said et al., 1994). Transgenic mice expressing a mutant form *Correspondence: G Chiocchia; E-mail: [email protected] of the Gsa subunit directed to TFC (gsp mice) have Received 17 July 2002; revised 14 January 2003; accepted 16 January turned out to be a powerful model for studying human 2003 thyroid tumors. These mice have inherited with the gsp FADD protein in development of thyroid tumor L Tourneur et al 2796 transgene a highly penetrant predisposition to develop molecule in mouse thyroid AAC. We analysed thyroids thyroid hyperplasia that eventually transforms into at various stages of tumor development by semiquanti- hyperfunctioning thyroid adenomas at the age of 8 tative reverse transcriptase-polymerase chain reaction months (Michiels et al., 1994). Interestingly, adenocar- (RT–PCR) and Western blot. Neither FasL mRNA nor – cinomas of the thyroid have been observed in the FasL protein could be detected in normal thyroid tissue progeny of animal breed of a specific genetic back- from NTG mice (not shown). In nonpathological (NP) ground, suggesting that the gsp-linked phenotype is thyroid glands from gsp mice, FasL mRNA and protein sensitive to the genetic modifiers (F-M Michiels, were absent in all mice tested (Figure 1a, b, respectively), unpublished observations). whereas they could be detected in hyperplastic (HP) We investigated the presence of a wide array of thyroids, and at higher levels in thyroid AAC (Figure signaling molecules of the Fas cascade in these mice. 1a, b). Moreover, we detected both the membrane- Our results show that FasL expression was acquired bound (40 kDa) and soluble (26 kDa) forms of FasL in during tumor development, with a stronger expression AAC. By contrast, RT–PCR and Western blot analysis in adenoma/adenocarcinoma (AAC) than in hyperpla- showed that Fas mRNA and protein were expressed sia, but not in normal thyroid cells. Among the adapters both in NP and pathological thyroids (Figure 1a, b). for Fas, only FADD was regulated, in that FADD protein expression was weak or completely lost in AAC, although FADD mRNA was present. Furthermore, in FADD protein, but not Daxx and RIP, is lost during an in vitro model of thyrocytes culture, the absence of tumor development FADD resulted in a Fas signaling leading to faster Since Fas is known to act through the recruitment of growth of the thyrocytes. adapter molecules, we first investigated the modulation of FADD, Daxx and RIP (Figure 2). mRNAs of these Fas adapters were detectable in thyroids from NTG and Results transgenic mice, and their respective levels of expression of mRNAs were increased during the course of tumor Fas ligand expression is acquired during tumor development, with a maximal expression observed in development AAC (Figure 2a, b). Western blot analysis showed that high levels of FADD protein were detected in all NP Since FasL expression has been reported in human (3/3) and HP (4/4) glands from gsp mice. Interestingly, thyroid carcinoma, we studied the presence of this FADD protein expression was weak (2/5) or completely l a a c a i m g o lo a n o i i h s rc t la a a a p m c p r o o n e n n o p e e y d d N H A A 425 bp FasL 366 bp Fas NP HP b AAC L1 L2 Membrane-bound 40 kDa FasL Soluble 26 kDa NP HP AAC L1 L2 45-48 kDa Fas Figure 1 Fas ligand expression is acquired during tumor development. (a) Semiquantitative RT–PCR of Fas and FasL mRNAs and (b) Western blot analysis of Fas and FasL proteins expression by thyroid glands from gsp transgenic mice at various stages of tumor development. Clone H11 antibody was used for FasL protein detection. Amplified fragments length and proteins size are indicated. L1 and L2 are for differential protein expression by the two lobes of the same adenoma Oncogene FADD protein in development of thyroid tumor L Tourneur et al 2797 Figure 2 Loss of the adapter protein FADD during tumor development. (a) Semiquantitative RT–PCR analysis of FADD, Daxx and RIP mRNAs expression by thyroid glands from gsp transgenic mice at various stages of tumor development. Amplified fragments size is indicated. b-Actin is used as control. FADD expression was evaluated by using two sets of primers covering the entire coding sequence (not shown). (b) FADD, Daxx, RIP and b-actin transcripts were analysed by semiquantitative RT–PCR as in (a), and bands were quantified by densitometry. Results are the mean of four NP, five HP and nine AAC for FADD; three NP, five HP and nine AAC for Daxx; three NP, two HP and six AAC for RIP. Data are expressed as the percentage of b-actin values. (c) Western blot analysis of the three adapter molecules expression by thyroid glands from gsp mice at various stages of tumor development. L1 and L2 are for differential protein expression by the two lobes of the same adenoma. Proteins size is indicated. We detected an approximately 70 kDa form of Daxx. Whatever the tumoral status of the gland, we could not detect the 120 kDa form of Daxx even in the nucleus (not shown). We used two different antibodies to confirm those results. Immunohistochemistry confirmed Daxx and RIP expression and showed that FADD protein expression was limited to restricted areas or completely lost in AAC (not shown).