Part I Death Receptor

Transcription

1 Part I Death Receptor Apoptosis and Cancer Therapy. Edited by K.-M. Debatin and S. Fulda Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN

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3 1.1 Introduction 3 1 The role of CD95/CD95 Ligand Signaling in Apoptosis and Cancer Karsten Gçlow, Marcin Kamiński and Peter H. Krammer Apoptosis plays a key role in the maintenance of tissue homeostasis. An imbalance between apoptosis and proliferation can result in tumor formation. These imbalances are capable of increasing tumor size and can also render the tumor resistant towards therapy. As death receptor (DR) signaling contributes to efficiency of cancer therapy, insights into the signaling cascades of DRs may provide new strategies for drug development. 1.1 Introduction Cell death is a key event in biology. At least two modes of cell death can be distinguished: necrosis and apoptosis (programmed cell death) [1]. Apoptosis is a strictly regulated process, which plays a key role in development, morphogenesis, tissue remodeling and immune response, mediating the ordered removal of superfluous, aged or damaged cells. In contrast to necrotic cells, which can elicit an inflammatory reaction, apoptotic cells are removed in an inconspicuous fashion, mainly by phagocytosis by neighboring cells or by specialized macrophagelike cells. Throughout adult life, each second several millions of cells in the human body undergo apoptosis to maintain homeostasis in self-renewing organs. All cells of the human body can proceed towards apoptosis, even in the absence of de novo protein synthesis [2], which suggests that all elements required for at least one apoptotic pathway are constantly present in every single cell [3]. Moreover, pathological effects caused by disturbances in apoptosis regulation illustrate the importance of apoptosis. An abnormal resistance to induction of apoptosis frequently correlates with cellular transformation, cancer or autoimmune diseases. In contrast, enhanced apoptotic decay of cells plays an important role in many pathological conditions (infection by toxin-producing microorganisms, ischemia-reperfusion damage or infarct) as well as in chronic diseases (degenerative diseases and AIDS) [4]. Apoptosis is distinguished by typical morphological and biochemical traits, including cell shrinkage, nuclear DNA fragmentation and membrane blebbing [4]. Apoptosis and Cancer Therapy. Edited by K.-M. Debatin and S. Fulda Copyright c 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN

4 4 1 The role of CD95/CD95 Ligand Signaling in Apoptosis and Cancer A characteristic feature of the classical apoptotic process is the activation of proteases called caspases, which act as key effector molecules [5]. The activity of caspases is largely responsible for the observed morphological changes seen in dying apoptotic cells. The caspase family members are synthesized as zymogens and are mostly activated by proteolytic cleavage. Caspases are characterized by the presence of a cysteine residue in their active centre and by the ability to cleave their substrates at aspartate residues. Most known caspases have a function in apoptosis and can be subdivided into two classes: initiator caspases (e.g. caspase-8, -9 and -10) and effector caspases (e.g. caspase-3, - 6 and -7). Activation of initiator caspases leads to proteolytic activation of effector caspases, which in turn results in dismantling of the vital cellular apparatus The Apoptotic Machinery While individual stimuli may initiate apoptosis, pathways transducing these signals are often very distinct. Many environmental stressors are detected within the cell and initiate the signaling cascade leading to apoptosis. Other stimuli act via cell surface receptors. Apoptotic signaling events can thus be roughly divided into two pathways, depending on the mechanism of initiation: the intrinsic pathway, which mainly depends on mitochondrial changes, and the extrinsic pathway, which is activated by extracellular signals that act via death receptors (DRs). Although different molecules participate in the core machinery of both apoptosis signaling pathways, a crosstalk exists at multiple levels The Intrinsic Pathway Mitochondrial Involvement in Apoptosis Progress in the development of fluorescent dyes and their application to confocal microscopy and flow cytometry led Petit et al. [6] and Skulachev et al. [7] to postulate a hypothesis of a crucial role of mitochondria for the initiation and progression of apoptosis. Mitochondrial stress is proposed to be a primary factor which leads to opening of the permeability transition pore (PTP) accompanied by an increase of mitochondrial volume and dissipation of the mitochondrial electrochemical potential. Key regulators of mitochondrial events during initiation of the apoptotic process are proteins of the Bcl-2 family [8], cytochrome c [9] and Smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP-binding protein with low pi) [10]. In addition, several other less well-defined factors were recently reported, among them: apoptosis-inducing factor (AIF) [11], endonuclease G (EndoG) [12, 13] and HtrA/Omi [14]. Bcl-2 family proteins play a crucial role in control of the mitochondrial pathway. In humans, more than 20 members of this family have been identified, including antiapoptotic (Bcl-2, Bcl-X L, Mcl-1, Bfl-1/A1, Bcl-W, Bcl-G) and proapoptotic (Bax,

5 1.1 Introduction 5 Bak, Bok/Mtd, Bad, Bid, Bik, Bim, Hrk, Puma/Bbc3, Noxa, EGL-1, Bcl-B) proteins [8]. Bcl-2 proteins are localized at or translocated to the mitochondrial membrane. They are thought to modulate apoptosis by permeabilization of the inner and/or outer membrane, leading to release of cytochrome c and other proapoptotic proteins, or by stabilizing mitochondrial barrier function, respectively. Most Bcl-2 family proteins can interact with each other, forming homo- or heterodimers [8]. Cytochrome c is an important component of the mitochondrial respiratory chain and the best-characterized proapoptotic protein of mitochondrial origin [9]. The release of cytochrome c into the cytosol results in ATP-dependent oligomerization of the adapter apoptosis-inducing factor 1 (Apaf-1) and recruitment of procaspase-9. Consequently, a supramolecular complex called an apoptosome is formed. Caspase-9 bound to the apoptosome activates downstream effector caspases such as caspase-3 [15]. Another protein released from the mitochondria upon induction of apoptosis is Smac/DIABLO. Smac/DIABLO inactivates direct inhibitors of active caspases the cytoplasmic inhibitors of apoptosis (IAPs) [10]. Mitochondria release a number of other proapoptotic proteins, whose function is not yet clearly defined. One of these factors is AIF, whose role is controversial. AIF was reported to translocate into the nucleus. Upon addition to purified nuclei, AIF induces caspase-independent chromatin condensation and DNA fragmentation into 50-bp fragments. AIF is also proposed to participate in the regulation of apoptotic mitochondrial membrane permeabilization [11]. Another potentially proapoptotic protein released by mitochondria is EndoG. EndoG is a divalent cation-dependent endonuclease, involved in mitochondrial DNA repair and duplication. It was demonstrated that EndoG translocates from the mitochondria to the nucleus during execution of apoptosis, where it degrades nuclear DNA into oligonucleosomal fragments similar to those generated by the caspase target caspase-activated DNase (CAD) [12, 13]. Identified in 2000, Omi/HtrA2 is a mammalian serine protease that shares significant homology with the bacterial endoprotease HtrA [16]. In mammals, Omi/ HtrA2 is a ubiquitously expressed protein with dual function. Upon release from mitochondria, Omi/HtrA2 can bind to IAPs and disrupt their binding to caspases. It can also induce apoptosis in a caspase-independent manner that seems to depend on its serine protease activity [14, 15] The Extrinsic Pathway The Role of Death Receptors and their Ligands The extrinsic pathway is activated by ligation of cell surface DRs through their specific ligands. The subfamily of DRs is part of the tumor necrosis factor (TNF)/nerve growth factor (NGF) superfamily. Six DRs have been identified so far: TNF-R1 (CD120a), CD95 (APO-1, Fas), DR3 (APO-3, LARD, TRAMP, WSL1), TRAIL-R1 (APO-2, DR4), TRAIL-R2 (DR5, KILLER, TRICK2) and DR6 [17, 18]. They are type I transmembrane proteins and harbor two to five cysteine-rich extracellular repeats (Fig. 1.1). Their common feature is a cytoplasmic

6 6 1 The role of CD95/CD95 Ligand Signaling in Apoptosis and Cancer Fig. 1.1 DRs TNF-R1, CD95, DR3, TRAIL-R1, DD, of approximately 70 amino acids. The DD TRAIL-R2, DR6, DcR1, DcR2, DcR3, OPG (osteoprotegrin), EDA-R (ectodysplasin receptor) and NGF-R (nerve growth factor receptor), and death ligands TNF, CD95L, TL1A, TRAIL, EDA and NGF. Death receptors are type I transmembrane proteins and harbor cysteine-rich extracellular repeats. Their common feature is is necessary for induction of apoptosis. The natural ligands of DRs belong to the TNF family. TNF-a, CD95L and TRAIL are the most frequently involved in apoptotic signaling and are synthesized as type II transmembrane proteins. Yellow squares: cysteine-rich motifs; red: DDs. a cytoplasmic protein interaction domain, the protein interaction domain, the death domain (DD), of approximately 70 amino acids, which is both necessary and sufficient for induction of apoptosis [17]. Death receptors are activated by their natural ligands which are members of the TNF family [18]. So far identified are TNF-b (LT-a), LT-b, TWEAK (APO-3L), TNFa, CD95 ligand (CD95L) (APO-1L, FasL) and TRAIL. TNF-a, CD95L and TRAIL are most frequently involved in apoptotic signaling and are synthesized as type II transmembrane proteins (Fig. 1.1). Soluble forms of these ligands have been reported to be generated by cleavage of the N-terminal portion by specific metalloproteinases [17]. The crucial point of DR signaling is the formation of a multimolecular complex of proteins triggered by receptor crosslinking either with agonistic antibodies or death ligands. The complex that is formed is called the death-inducing signaling complex (DISC) [19]. Among the DR complexes, the CD95 DISC is the most extensively investigated.

7 1.1 Introduction The CD95 System CD95 is a glycosylated type I transmembrane cell surface receptor of a relative molecular mass around kda (consisting of 335 amino acids). It has also been proposed that CD95 can exist in soluble form due to alternative splicing [20]. CD95 is widely expressed in normal and neoplastic tissues. CD95 expression can be enhanced in T cells by cytokines such as interferon (INF)-g and TNF [21]. Resting B cells express low levels of CD95 upon induction by CD40L and endotoxins [22]. CD95L, a 40-kDa glycoprotein is present only on few cell types, such as activated T cells [23] or natural killer (NK) cells [24], where it participates in cellmediated cytotoxicity. Its presence on cells of immune-privileged sites, such as the testes, placenta, anterior chamber of the eye and brain, has also been reported [25 27]. The presence of CD95L in these tissues may contribute to elimination of infiltrating lymphocytes. While most TNF receptor-like molecules require trimerization for the recruitment of signaling molecules and activation of the signaling cascade, the CD95 receptor was also suggested to exist in a trimeric (inactive) form pre-assembled via preligand binding assembly domains (PLAD) [28]. In this case, signaling would be induced either by conformational changes of preformed DR trimers or, alternatively, by the formation of multimeric complexes upon ligand binding [29]. Furthermore, a refined but still intensively discussed model of proximal steps of CD95 signaling was proposed, involving (i) formation of CD95 microaggregates, (ii) DISC formation, (iii) formation of large CD95 surface clusters and (iv) internalization of activated CD95 [30]. The transduction of the apoptotic signal starts with the formation of the DISC, within seconds after receptor engagement. The CD95 DISC consists of oligomerized CD95 receptors, the adapter protein FADD (Fas-associated DD, also known as MORT-1), two isoforms of procaspase-8 {procaspase-8/a [FADD-like interleukin (IL)-1b-converting enzyme (FLICE), Mach-a1, Mch5b]} and procaspase-8/b (Mach-a2)] [31], procaspase-10 and c-flip L/S/R (Fig. 1.2). The crucial factor for DISC formation is FADD, which bridges the receptors with caspases by homotypic interactions of the DD and death effector domain (DED). FADD binds via its DD to the DD of the receptor, and via its DED it recruits DED-containing procaspases-8 and -10. Thereafter, procaspase-8 is cleaved at the DISC, which leads to formation of active caspase-8, a major player in the pathway. The active form of caspase-8 is a heterotetramer of two small (p10) subunits and two large (p18) subunits [32, 33]. The prodomain of caspase-8 remains at the DISC, while active caspase-8 dissociates from the DISC to initiate a cascade of caspase activations and the execution phase of apoptosis [32]. One major regulator of CD95-mediated apoptosis at the DISC level is the cellular FLICE-inhibitory protein (c-flip, also called FLAME-1, I-FLICE, Casper, CASH, MRIT, CLARP and Ursupin) [34 42]. Multiple splice variants of c-flip have been reported, but so far only three designated forms, i.e. c-flip L, c-flip S

8 8 1 The role of CD95/CD95 Ligand Signaling in Apoptosis and Cancer Fig. 1.2 The CD95 DISC consists of oligomerized and DED. FADD binds via its DD to the DD of CD95 receptors, the adapter protein FADD, procaspase-8, procaspase-10 and the regulatory protein c-flip. The crucial factor for DISC formation is FADD, which bridges the receptor with caspases and the regulator protein c-flip by homotypic interactions of its DD the receptor, and via the DED it recruits DEDcontaining procaspase-8, procaspase-10 and/ or c-flip. Multiple splice variants of c-flip have been reported, but so far only three, designated c-flip L, c-flip S and c-flip R, could be detected at the protein level. and c-flip R, have been detected at the protein level [43, 44]. c-flip L contains tandem DEDs and a caspase-like domain. However, it lacks amino acid residues that are critical for caspase activity, most notably the cysteine of the catalytic centre. High levels of c-flip L expression were shown to inhibit caspase-8 processing in the DISC [45]. Surprisingly, it has also been shown that c-flip L at low expression levels can act as an activator of caspase-8 by forming a heterodimer with procaspase-8 within the DISC [46]. c-flip S and c-flip R resemble their viral counterparts, and consist only of two DEDs and a short C-terminal part that differs from c-flip L [35]. Recruitment of c-flip S/R to the DISC prevents caspase-8 cleavage completely. In addition to FADD, procaspase-8, procaspase-10 and c-flip, various other proteins have been described to bind to the DISC, but their putative role and importance in the regulation of apoptosis remains to be solved [47] The Two-pathways Model for CD95 Signaling Two pathways of CD95 apoptosis signaling, based on the quantity of production of active caspase-8 at the DISC, were described [48]. In type I cells, a high production of caspase-8 at the DISC leads to direct processing and activation of the effector caspase, caspase-3, and ultimately leads to apoptosis (Fig. 1.3). In type II cells, however, only a small amount of caspase-8 is produced at the DISC (Fig. 1.3). The DISC in type II cells is formed quite poorly and, subsequently, active caspase-8 is generated in lower amounts. Apoptosis in these cells is dependent, at least in part, on the cleavage of Bcl-2 homology domain-3 (BH3) containing

9 1.1 Introduction 9 Fig. 1.3 Two CD95 signaling pathways. In pendent on cleavage of Bid by caspase-8. The type I cells, a high production of caspase-8 at the DISC leads to direct processing and activation of the effector caspase, caspase-3, and to ultimate apoptosis of the cell. In type II cells, only a small amount of caspase-8 is produced at the DISC. The DISC in these cells is formed quite poorly and less active caspasecleavage results in a proapoptotic fragment termed tbid. This fragment induces the proapoptotic functions of the mitochondria, cytochrome c is released and forms the apoptosome with Apaf-1 and caspase-9. Caspase-9 activates caspase-3, resulting in induction of apoptosis. 8isinduced. Apoptosis in these cells is de- Bcl-2 family member Bid [49, 50]. This cleavage results in a proapoptotic fragment termed truncated Bid (tbid). This fragment induces proapoptotic functions of mitochondria by causing aggregation of Bax and Bak [51], and subsequent loss of cytochrome c from the mitochondrial intermembrane space. Apaf-1, cytochrome c and ATP form a large protein complex, the apoptosome, a sort of cytosolic DISC at which caspase-9 as the initiator caspase is activated [52]. Induction of apoptosis is accomplished by activation of effector caspases like caspase-3.

10 10 1 The role of CD95/CD95 Ligand Signaling in Apoptosis and Cancer CD95-mediated apoptosis in type II cells is further affected by the expression of antiapoptotic members of the Bcl-2 family. Expression of either Bcl-2 or Bcl-X L renders type II cells resistant to CD95-mediated apoptosis [48]. Type I cells are not protected from CD95-mediated apoptosis even by the expression of very high levels of Bcl-2 or Bcl-X L [48]. The physiological importance of the two-pathways model has been validated by the description of mice which forcibly express members of the apoptotic pathway or which are deficient in various molecules involved in apoptosis. Mice deficient in Bid have thymocytes that are not protected from CD95-mediated apoptosis, whereas their hepatocytes are resistant towards CD95-induced apoptosis [53]. Bax/Bak double-deficient mice demonstrate resistance to CD95-mediated apoptosis in hepatocytes, whereas their thymocytes and T cells are affected in a way similar to that in control mice [54]. The hepatocytes of mice expressing Bcl-2 as a transgene are also protected from CD95-mediated apoptosis, whereas transgenic thymocytes and T cells are not protected from apoptosis mediated by CD95 [55 57]. Thus, thymocytes and T cells are type I cells, and hepatocytes are type II cells The Death Ligand CD95L In contrast to CD95, expression of CD95L is restricted to a few cell types, such as activated T cells [23], NK cells [58] and a few nonlymphoid tissues, particularly, of immune-privileged sites [59]. Constitutive or inducible expression of CD95L in immune-privileged sites can provide protection of these tissues from inflammatory damage. The best studied site is the eye. To maintain its functions, the eye cannot tolerate destructive inflammatory responses. Minor episodes of inflammation can result in impaired vision or even blindness. Expression of CD95L in the eye limits the number of inflammatory cells that infiltrate into the anterior chamber of the eye in response to infections [60]. CD95L-mediated privilege is also reported in other tissues such as testis, thyroid, brain, lung, kidney and liver [59]. Existence of soluble forms of CD95L has also been reported. CD95L was found on killer cell-derived vesicles [61]. It could be also cleaved from the membrane by metalloproteases [62]. At present, the role of soluble forms of CD95L is unclear, e.g. soluble human CD95L can induce apoptosis, whereas soluble mouse CD95L cannot [62, 63]. In T cells, expression of CD95L is strictly regulated. CD95L is not present on resting T cells, but is highly expressed upon activation of T cells by crosslinking of the T cell receptor (TCR) with an antigen or by reagents that mimic the antigen signal like agonistic anti-cd3 antibodies. The CD95/CD95L system plays a major role in regulation of the fate of T cells. During an immune response, activated T cells undergo rapid proliferation and differentiation. In turn, termination of an immune response requires elimination of activated T lymphocytes in a CD95L-dependent manner, called activation-induced cell death (AICD). Therefore, the pathways leading to CD95L expression after TCR triggering are so far the best char-

11 1.1 Introduction 11 acterized and can serve as a model for regulatory mechanisms. The earliest signaling events following TCR engagement are the sequential activation of tyrosine kinases including Lck and ZAP70. Both Lck and ZAP70 are required for calcium (Ca 2+ ) mobilization in T cells. The increase in cytosolic Ca 2+ causes activation of calcineurin, which dephosphorylates the nuclear factor of activated T cells (NF- AT). Upon translocation into the nucleus activated NF-AT initiates gene transcription and is thus regarded as one of the key participants in CD95L regulation [64]. ZAP-70 is also involved in activation of protein kinase C (PKC). The u isoform of PKC has been shown to be essential for activation-induced CD95L expression [64] and for TCR-induced NF-kB activation [65]. Activated NF-kB was shown to be required for high expression levels of CD95L in T cells [66 69]. Activation of T cells via TCR also leads to a Ras-activated cascade of kinase activity including Raf, Mek, Erk and p38 mitogen-activated tyrosine kinase (MAPK). This pathway is crucial for optimal CD95L induction and involved in the activation of the transcription factor AP-1 (Fos/Jun) [64]. Since the CD95L promoter contains an AP-1 binding site, AP-1 is engaged in the regulation of CD95L in human T cells [64]. However, the CD95L gene is under control of a large array of other cis-acting promoter elements [e.g. c-myc, IFN-regulatory factors (IRFs), SP-1, Nur77 (also called NGFI-B, N10, Nak1, TR3) and Egr], which act in concert to achieve a fine degree of control over the transcriptional activity of this gene (Fig. 1.4) [64]. Additional transcription factors such as apoptosis-linked gene 4 (ALG- 4) and RORgt were also reported to regulate CD95L expression despite the absence of binding sites within the CD95L promoter. It was also reported that expression of class II transactivator (CIITA) in T cells activates MHC class II but inhibits CD95L expression. This repression was proposed to be due to competition between CIITA and NF-AT for binding to the common cofactor CBP/p300 [64]. Reactive oxygen species have also been shown to play an important role in the transcriptional control of CD95L expression. It has been reported that inhibition of oxidative signals interferes with induction of CD95L expression in T cells [70 73]. The transcription factors NF-kB and AP-1 can be directly activated by oxidative signals. Thus, oxidative signals are able to influence the activity of the CD95L promoter [64, 74]. Recently, it has been shown that Ca 2+ and oxidative signals alone are insufficient for induction of CD95L expression, but have to act in concert [73].

12 Hagedor Kommun 12 1 The role of CD95/CD95 Ligand Signaling in Apoptosis and Cancer Fig. 1.4 The CD95L gene is under control of a large array of other cis-acting promoter elements (AP-1, NF-AT, NF-kB, ATF2, c-myc, IRFs, SP-1 and Egr) which act in concert to achieve a fine degree of control over the transcriptional activity of the gene.

13 1.2 Cancer and Apoptosis Cancer and Apoptosis In cells and tissues of multicellular organisms, potent physiological mechanisms govern cell proliferation and homeostasis. Many of these growth control mechanisms are linked to apoptosis: excessive proliferation or growth at inappropriate sites induces apoptosis in the affected cells. Tumors can proliferate beyond those constrains. Therefore, resistance of tumor cells to apoptosis is an essential feature of cancer development. This assumption is confirmed by the finding that deregulated proliferation alone is not sufficient for tumor formation. On the contrary, it leads to cell death [75]. For example, the overexpression of growth-promoting oncogenes such as c-myc, E1A or E2F1 sensitizes cells to apoptosis. In addition to the expression of proteins that promote cell proliferation, tumor progression requires the expression of antiapoptotic proteins or the inactivation of essential proapoptotic proteins [76] Resistance Mechanisms Expression of Antiapoptotic Proteins Some tumors are characterized by the expression of high levels of c-flip, which interferes with apoptosis induction at the level of the DRs. Viral analogs of FLIP (v-flip) are encoded by some tumorigenic viruses including HHV8 [77]. In cells that are latently infected with HHV8, v-flip is expressed at low levels, but its expression is increased in advanced Karposi s sarcomas or on serum withdrawal from lymphoma cells in culture [78]. Therefore, v-flips may contribute to the persistence and oncogenicity of v-flip-encoding viruses. Another mechanism by which tumors interfere with DR-mediated apoptosis may be the expression of soluble receptors that act as decoys for death ligands. To date, two soluble receptors soluble CD95 (scd95) and decoy receptor 3 (DcR3) competitively inhibit CD95 signaling. scd95 is elevated in several autoimmune disorders and in the sera of certain cancer patients. High scd95 serum levels were associated with a poor prognosis in melanoma patients [79, 80]. DcR3 interacts with CD95L and thereby inhibits CD95L-induced apoptosis. DcR3 is genetically amplified in several lung and colon carcinomas, and is overexpressed in adenocarcinomas, glioma cell lines and glioblastomas [81 83]. Expression of the IAP family protein, survivin, is highly tumor specific [84]. It is found in most human tumors, but not in normal adult tissue [85]. In neuroblastoma, expression of survivin correlates with a more aggressive progression of the disease [86]. However, so far it is not clear whether survivin acts directly as an apoptosis inhibitor or not. Survivin might also be necessary for completion of the cell cycle as overexpression of survivin counteracts apoptosis [84]. Another IAP family member, c-iap2, is affected by translocation t(11;18)(p21;p21) that is found in about 50% of marginal cell lymphomas of the mucosa-associated lymphoid tissue (MALT). This is indicative of a role of c-iap in the development of MALT lymphoma [87].

14 14 1 The role of CD95/CD95 Ligand Signaling in Apoptosis and Cancer A common feature of follicular B cell lymphoma is the chromosomal translocation t(14;18), which couples the Bcl-2 gene to the Ig heavy chain locus, leading to enhanced Bcl-2 expression. Bcl-2 cooperates with the protooncogene c-myc or in acute promyelocytic leukaemia with the promyelocytic leukemia retinoic acid receptor-a (PML RARa) fusion protein, thereby contributing to tumorigenesis. Some other studies have shown a correlation between high levels of Bcl-2 expression and the severity of malignancy of human tumors [76]. In addition, other antiapoptotic Bcl-2 family members are reported to be involved in resistance of tumors to apoptosis. Bcl-X L can confer resistance to many apoptosis-inducing pathways in cell lines and is suggested to be upregulated by a constitutively active mutant epidermal growth factor receptor (EGFR) in vitro [88] Resistance Mechanisms Inactivation of Proapoptotic Genes Apart from overexpression of antiapoptotic genes, tumors can acquire resistance by downregulation or mutation of proapoptotic genes. In certain types of cancer, the proapoptotic Bcl-2 family member Bax is mutated [89, 90]. Tumor cell lines with mutations of Bax are more resistant towards apoptosis. Metastatic melanomas have found another way to escape apoptosis. They do not express Apaf-1, which forms an integral part of the apoptosome [91]. Another strategy has been reported for neuroblastomas in which the N-Myc oncogene has been amplified. In these tumors, the gene for the initiator caspase-8 is frequently inactivated by gene deletion or methylation, although mutations of caspase genes in tumors have only been identified with low frequency. In addition to neuroblastoma cells, caspase-8 was found to be inactivated by hypermethylation in malignant brain tumors, Ewing sarcoma, retinoblastoma, rhabdomyosarcoma or small cell lung carcinoma [92]. Furthermore, signaling molecules of DR-dependent apoptotic pathways such as CD95/CD9L system could be downregulated or mutated. 1.3 Cancer and CD95/CD95L System Resistance Mechanisms Mutations and Reduced Expression of CD95/CD95L In general, CD95 or CD95L loss of function may occur due to mutations, promoter methylation, transcriptional repression, histone acetylation and/or regulation of signaling molecules [93]. Expression of the CD95 DR was found to be downregulated in several tumor types, such as hepatocellular carcinoma [94], melanoma [95] cutaneous T cell lymphoma and adenocarcinomas or colon carcinomas [96]. The expression of CD95 can be transcriptionally induced by the tumor-suppressor protein p53 which binds to an intronic enhancer element [97]. In addition, three putative p53-bind-

15 1.3 Cancer and CD95/CD95L System 15 ing sites showing limited homology with the p53 consensus binding site have been identified in the CD95 promoter [97]. Therefore, the downregulation of expression of the CD95 receptor could be partially explained by mutations in the TP53 gene, which is a common feature of cancer cells. For example, the described mechanism was observed in the case of hepatocellular carcinoma cells [98]. Functional impairment of CD95 due to mutations of the CD95 gene has been reported in many tumors, e.g. in T cell leukemia, myeloma and non-hodgkin s lymphoma cells [99 101]. Table 1.1 presents additional examples of CD95 somatic mutations found in patients suffering from various cancers of different origin. In most cases of non-hodgkin s lymphomas [101] or multiple myelomas [100], the described mutations were situated in the cytoplasmic region of CD95 and seemed to confer a dominant-negative function. Similarly, about 20% of B cell lymphomas (Tab. 1.1) derived form postgerminal center B cells were found to carry mutations in the last exon of the CD95 gene which encodes the DD. Tab. 1.1 Somatic CD95 gene mutations in cancer (after [64]) Malignancy Cases with CD95 mutations References Solid tumors bladder cancer 3/ malignant melanoma 3/ nonsmall cell lung cancer 5/ squamous cell cancer 3/ gastric cancer 5/ prostate cancer 4/ T cell leukemia adult T cell leukemia 3/ Non-Hodgkin s lymphoma 16/ Postgerminal center B cell lymphoma B cell chronic lymphocytic leukemia 1/ B cell lymphoma of low-grade MALT type 3/5 101 MALT-type B lymphoma 4/5 165 diffuse large B cell lymphoma 9/ / / follicular center cell lymphoma 2/ multiple myeloma 7/ Hodgkin s disease 2/9 167 Lymphomas thyroid lymphoma 3/ chronic lymphocytic thyroiditis 16/26 168

16 16 1 The role of CD95/CD95 Ligand Signaling in Apoptosis and Cancer The CD95 gene was proposed to play a role as tumor suppressor [93]. During differentiation of the B cell lineage, expression levels of CD95 are highest at the germinal center stage. Therefore, disruption of CD95-mediated apoptosis may lead to prolonged survival of B lymphocytes and subsequent accumulation of additional mutations in oncogene or tumor suppressor genes. Furthermore, in families characterized by germline CD95 mutations leading to autoimmunoproliferative syndrome (ALPS), the incidence of lymphomas is generally increased. Additional data suggesting the importance of the CD95/CD95L system for tumor progression emerged from studies on lpr and gld mouse strains, carrying mutations in CD95 and CD95L, respectively. Davidson et al. [102] observed that both strains develop plasmacytoid tumors as they age. Moreover, when lpr or gld mice were backcrossed to tumor-predisposed mice such as Em-myc transgenic, bcl-2 transgenic or T cell-deficient mice, the incidence of hematopoietic tumors was increased significantly [ ]. Thus, it is likely that a defective CD95/ CD95L signaling allows accumulation of mutations that initiate tumor development. This assumption is further supported by the fact that positive correlation between the occurrence of myeloma and the presence of mutations in the CD95 gene was only observed in adults, but not in pediatric patients [100]. Functional impairment of the CD95/CD95L system in a tumor cell can be of major importance for facilitating tumor escape from an immune response. NK cells and cytotoxic T cells are the most powerful antitumor effectors of the immune system. Both cell types are able to kill tumor cells directly by two basic mechanisms: (i) secretion of a membrane-permeabilizing protein, perforin and proteolytic enzymes known as granzymes, and (ii) by induction of apoptosis via triggering of CD95 on tumor cells by expression of CD95L. Therefore, loss of functional CD95 by a tumor cell confers tumor resistance towards an antitumor immune response Resistance Mechanisms Induction of CD95/CD95L Signaling Hypothesis of Tumor Counterattack CD95L may also act against the immune system when it is produced by tumor cells. A scenario in which CD95L on tumor cells could induce selective apoptosis of activated, antitumor T cells was named tumor counterattack [106]. This hypothesis was supported by data showing that various cancer cells indeed express CD95L [107, 108]. The data also suggested that tumors are able to kill activated lymphocytes [109]. Furthermore, the relevance of a tumor counterattack was implied by (i) delayed growth of CD95L-expressing melanoma cells in lpr mice [110], and (ii) depletion of NK cells [111] and diminished antibody responses [112] by CD95L overexpressing tumor cells. Moreover, the well-known fact that high levels of scd95l in the patient s serum are associated with a poor prognosis further suggests the relevance of a tumor counterattack mechanism. However, attempts to induce CD95L expression on tumor cells resulted in tumor rejection [113, 114]. In addition, rejection of tumors expressing CD95L by chemoattracted neutrophils

17 1.3 Cancer and CD95/CD95L System 17 has also been observed [114]. At present, a number of contradictory studies exist in the literature. Thus, it has to be stated that, despite the doubtless attraction of a tumor counterattack hypothesis, so far no study has conclusively demonstrated its relevance for an in vivo situation. Controversies might have arisen due to additional factors, such as level and time point of CD95L expression, activation status of infiltrating T cells, levels and form of CD95L, and/or presence of immunosuppressive factors, e.g. transforming growth factor (TGF)-b [115] Angiogenesis New blood vessel formation is a prerequisite for malignant tumor progression and metastasis. Several reports demonstrated a potential involvement of the CD95/CD95L system in this process. Administration of anti-cd95 antibody in Matrigel implants induced neovascularization and infiltration of inflammatory cells [116]. On the one hand, this observation could be explained by an activating CD95-mediated signal transduced to endothelial cells and/or inflammatory cells. It is known that under certain conditions, CD95 signaling might costimulate proliferation of T cells and fibroblasts [117, 118] or neuronal regeneration and branching [119]. This so called Janus face of CD95 signaling is currently under intensive investigation. On the other hand, the possibility that angiogenesis is secondary to recruitment of inflammatory cells as a local source of growth factors cannot be excluded. There is evidence that CD95 engagement or enforced CD95L expression could lead to inflammation, IL-1 and IL-8 secretion, and neutrophil infiltration [120]. In contrast to the angiogenic role of CD95/CD95L, Volpert et al. suggested that the CD95/CD95L system may be involved in regulation of the activity of antiangiogenetic proteins, such as pigment epithelium-derived factor (PEDF) and trombospondin-1 (TSP-1) [121]. Both inhibitors were ineffective in the corneal angiogenesis model applied to lpr and gld mice. It seems also possible that antiangiogenetic mechanisms of engiostatin and andostatin are related to CD95/CD95L signaling [93]. In addition, IL-12/IL-2 treatment inhibited neovascularization in a CD95-dependent manner [122] The CD95/CD95LSystem and Cancer Therapy The predominant role of apoptosis has been implicated in tumor-directed cytotoxicity of chemotherapy, g-irradiation, UV or immunotherapy. Thus, being of major importance for the apoptotic machinery, the role of the CD95/CD95L system in these cell death scenarios, has also been addressed.

18 18 1 The role of CD95/CD95 Ligand Signaling in Apoptosis and Cancer Involvement of the CD95/CD95L Pathway in Chemotherapy and Drug-induced Apoptosis Although CD95L appears to be considerably less selective for cancer cells than TRAIL, many studies have implicated the role of the CD95/CD95L system in tumor cell death induced by chemotherapy [123]. Drug treatment is known to lead to enhanced expression of CD95L. CD95L could in turn stimulate the apoptotic pathway of the CD95 receptor by binding in a paracrine or autocrine manner. As mentioned above, enhanced expression of CD95L has been observed on many different tumor cells, e.g. leukemia, melanoma, neuroblastoma, hepatoma, small cell lung carcinoma, malignant brain tumor, prostate, colon or breast cancer cells in vitro and ex vivo [92]. CD95L upregulation was related to the activity of various drugs, with different intracellular targets, i.e. induction of DNA damage (doxorubicin, etoposide, bleomycin, cytarabine) or inhibition of DNA repair or DNA synthesis (cisplatin, methotrexate) [92]. The role of the CD95/CD95L system has also been suggested in thymineless death of colon carcinoma cells following treatment with 5-fluorouracil (5-FU) [124]. Since the CD95L promoter bears binding sites for NF-kB and AP-1 transcription factors, activation of these transcription factors was reported to be responsible for increased CD95L mrna levels upon response to chemotherapy [125, 126]. Furthermore, doxorubicin treatment seemed to mimic CD3 triggering in TCR-positive leukemia cells, resulting in expression of functional CD95L and paracrine cell death [127]. Chemotherapeutic agents have also been shown to upregulate surface expression of the CD95 receptor, particularly in the case of cells harboring wild-type p53. This might be explained by the transcriptional dependence of the CD95 gene on active p53. Moreover, the outcome of chemotherapy may involve activation of the CD95-dependent apoptotic machinery. Drug treatment can also influence the expression and activity of downstream pro- and antiapoptotic elements. Colon carcinoma cells treated with doxorubicin, cisplatin and mitomycin C demonstrated an upregulation of FADD and caspase-8 [128, 129]. Concomitantly, antagonistic anti-cd95l antibodies, soluble antagonistic CD95 receptors or the double-negative form of FADD (DN-FADD) were shown to effectively reduce apoptosis occurring after drug application [92]. These findings are further supported by in vivo studies demonstrating that 5-FU-induced apoptosis of mouse thymocytes could be blocked by neutralizing CD95L antibodies and was also impaired in lpr mice [123]. In addition, the association between resistance to CD95-triggered apoptosis and crossresistance to various anticancer agents has been described in some leukemia and solid tumor cell lines [130, 131]. Thus, it seems reasonable to conclude that in cells that can induce CD95/ CD95L signaling, drug-induced apoptosis may include cell death mechanism analogous to AICD of T cells. Nevertheless, despite the body of evidence suggesting an involvement of the CD95/CD95L system in drug-induced apoptosis, the proposed mechanism has been challenged by a number of other studies [ ]. Using other experimental settings it was shown that antagonistic antibodies against CD95L or CD95 did

19 1.3 Cancer and CD95/CD95L System 19 not confer protection to chemotherapy-induced apoptosis [133, 136, 137]. Cytotoxic drug-induced cell death was not alleviated by enforced expression of FLIP and DN-FADD despite clear inhibition of caspase-8 activity. Drug cytotoxicity was also not influenced after targeted disruption of genes encoding FADD and caspase-8 in experiments with nontransformed cells, such as murine embryonic fibroblasts [138]. Several factors may be responsible for the described discrepancies. The quality of reagents applied to block the CD95/CD95L interaction, such as anti-cd95 antibody, anti-cd95l antibody or soluble decoy CD95-Fc receptors, seems to be of crucial importance [123]. In addition, Hyer et al. [139], using adenoviral transfer of CD95L-Green Fluorescent Protein, suggested that in certain situations CD95/ CD95L neutralizing agents may not gain access to their targets due to a CD95/ CD95L pre-interaction in intracellular compartments. The differences may also stem from the variety of experimental systems applied. The results obtained through the application of nontransformed cells have to be considered with special care, since malignant and nonmalignant cells can vary considerably with respect to their sensitivity towards various death-inducing stimuli. Furthermore, response to chemotherapeutic drugs can differ depending on whether the cells tested depend on type I or II CD95 signaling (Fig. 1.3). Thus, when cells overexpressing FADD-DN or Bcl-X L were studied, drug-induced cell death of type I cells involved both receptor and mitochondrial apoptotic pathways. However, type II cells displayed a protective effect of Bcl-X L and predominant dependence on the mitochondrial pathway. To this end, it is important to note that although considerable evidence supports the role of the CD95/CD95L system in chemotherapy-induced apoptosis, most drugs are considered to predominantly influence the mitochondria-dependent apoptotic pathway [140]. However, CD95-induced signaling could still play a role in the amplification of apoptotic signals induced by anticancer drugs [123]. In addition, studies showing that a functional apoptosome is dispensable for drug-induced apoptosis have challenged the concept that the mitochondria-dependent apoptotic pathway is central for drug-induced apoptosis [141] Importance of CD95/CD95L Signaling for Tumor Progression and Treatment Prognosis As mentioned above, several groups postulated a role for CD95 as a tumor suppressor. It was also proposed that the CD95/CD95L system can play an important role in tumor progression. CD95-sensitive melanomas were shown to metastasize to the lung in gld, but not in wild-type, mice [142]. Experiments on late-passage mouse embryo fibroblasts with epigenetically induced loss of CD95 expression showed that restoration of CD95 significantly inhibited tumor growth [143]. Thus, it seems that CD95/CD95L signaling poses an important barrier for tumor metastasis.

20 20 1 The role of CD95/CD95 Ligand Signaling in Apoptosis and Cancer Foremost, as already mentioned, alterations in the functional CD95 pathway, such as loss of CD95 expression or activity of DISC components, can be directly linked to tumor immune escape. A number of studies demonstrated the existence of a link between defective CD95/CD95L proapoptotic signaling and resistance to chemotherapy. Downregulation of CD95 expression has been described for drug-resistant leukemia and neuroblastoma cells [130, 131]. The influence of FLIP expression levels on conferring apoptosis resistance after chemotherapy may vary between cell types. Overexpression of FLIP did not result in protection against cytotoxicity insult in T cell leukemia cells, but FLIP antisense oligonucleotides sensitized osteosarcoma cells for treatment with cisplatin [144, 145]. Elevated FLIP expression in tissue samples from patients was found in the case of Burkitt s lymphoma, pancreatic carcinoma, melanoma and neuroblastoma, and in tumors resistant to chemotherapy [123]. Caspase-8-deficient neuroblastoma cells were found to be resistant to DR- and doxorubicin-mediated apoptosis [146]. Consequently, restoration of caspase-8 expression by gene transfer or by demethylation treatment sensitized resistant tumor cells for DR or drug-induced apoptosis [123]. Many clinical and preclinical studies tried to assay expression levels of apoptotic regulators, particularly Bcl-2 and Bax proteins, in cancer patients for prognosis and/or treatment responsiveness [123]. However, the present state of research shows that the situation is rather complex and regulators of apoptosis do not seem to play a role as single univariate parameters. Screening of genes involved in DR pathways identified a significant percentage of mutations in hematological malignancies and solid tumors [123]. A high incidence of CD95 mutations led to the idea of a tumor-suppressive function of the CD95 system. This notion was further supported by the finding that increased serum levels of scd95, produced by alternative splicing in adult T cell leukemia patients, correlate with reduced survival outcome [147]. CD95 expression was also associated with response to therapy in acute myeloid leukemia patients [148]. Moreover, c-flip expression was associated with enhanced tumor progression in vivo, pointing to its role as prognosis factor [149, 150]. Limited information is currently available on the prognostic value of caspase-8 deficiencies. However, it was demonstrated that loss of caspase-8 was associated with unfavorable survival outcome in patients with medulloblastoma [151] Therapeutic Attempts to Exploit the CD95/CD95L System for Cancer Treatment Current strategies to influence apoptotic pathways for cancer therapy aim to act on individual molecules that constitute key switches in the control of cell death. Great promise is held by therapeutic procedures targeting Bcl-2, IAPs and TRAIL receptor proteins. Due to the ability of agonistic anti-cd95 antibodies to induce apoptosis in a fast and specific manner, they were initially expected to be useful as antitumor drugs. In 1989, Trauth et al. [152] reported that human B cell lymphoma cells transplanted into nude mice underwent rapid regression

21 References 21 after i.v. administration of human agonistic anti-cd95 antibodies. However, further experiments with the systemic application of mouse agonistic anti- CD95 antibodies (Jo2) into mice demonstrated that such a treatment inevitably leads to hypertension, liver failure and death of all mice within 6 h following the administration of antibodies [153]. Therefore, systemic use of CD95 agonists such as anti-cd95 antibodies or recombinant CD95L is currently impossible without additional means to ensure selectivity. Nevertheless, treatment directed to activate CD95 in tumors may be possible in the case of local administration. For example, it was shown that i.p. injection of supernatant containing CD95L could efficiently kill CD95-expressing murine lymphoma cells implanted in capsules in the peritoneal cavity of mice without systemic toxicity [154]. Other possible antitumor strategies involving CD95/CD95L signaling are based on the use of cytokines such as IFNs and TNF to induce CD95 expression and sensitize tumor cells to CD95-mediated apoptosis. Identification of IFN-sensitive response elements in the caspase-8 promoter was accompanied by findings that IFN-a directly activates caspase-8 transcription and that INF-g overcomes resistance of tumor cells towards drug- and DR-induced apoptosis [ ]. Therefore, treatment with IFNs could provide an additional possibility for the sensitization of tumors to DR-induced apoptosis. In general, the great advantage of DR-induced apoptosis with respect to cancer treatment is its ability to proceed in a p53-independent manner. Thus, death by this apoptotic mechanism can circumvent the loss of p53 activity, which constitutes the major obstacle for tumor-directed genotoxicity observed in many tumors. Acknowledgments We thank Dr. B. Fritzsching, Dr. D. Klemke, Dr. I. Lavrik, A. Golks and A. Hong for critical reading of the manuscript. This work is supported by the Wilhelm Sander Stiftung, the Deutsche Forschungsgemeinschaft, the European Community and the Tumor Center Heidelberg/Mannheim. References 1. Kerr, J. F., A. H. Wyllie and A. R. Currie Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: Ellis, H. M. and H. R. Horvitz Genetic control of programmed cell death in the nematode C. elegans. Cell 44: Kroemer, G., B. Dallaporta and M. Resche-Rigon The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 60: Hengartner, M. O The biochemistry of apoptosis. Nature 407: Thornberry, N. A. and Y. Lazebnik Caspases: enemies within. Science 281: Petit, P. X., S. A. Susin, N. Zamzami, B. Mignotte and G. Kroemer Mitochondria and programmed cell death: back to the future. FEBS Lett 396:7.

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