TRAIL: apoptosis signaling, biology, and potential for cancer therapy

Cytokine & Growth Factor Reviews 14 (2003) 337–348

Apo2L/TRAIL: apoptosis signaling, biology, and potential for cancer therapy Alexandru Almasan a , Avi Ashkenazi b,∗ a

Department of Cancer Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA b Department of Molecular Oncology, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA

Abstract Apo2 ligand or tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) is one of several members of the TNF gene superfamily that induce apoptosis through engagement of death receptors. Apo2L/TRAIL is unusual as compared to any other cytokine as it interacts with a complex system of receptors: two pro-apoptotic death receptors and three anti-apoptotic decoys. This protein has generated tremendous excitement as a potential tumor-specific cancer therapeutic because, as a stable soluble trimer, it selectively induces apoptosis in many transformed cells but not in normal cells. Transcriptional activation of Apo2L/TRAIL by interferons (IFNs) through specific regulatory elements in its promoter, and possibly by a number of other cytokines, reveals its possible involvement in the activation of natural killer cells, cytotoxic T lymphocytes, and dendritic cells. In this review, we focus on the apoptosis signaling pathways stimulated by Apo2L/TRAIL, summarize what is known to date about the physiological role of this ligand and the potential for its application to cancer therapy. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Apo2L/TRAIL; Death receptors; Apoptosis; Interferons; Caspases

Contents 1. Introduction: Apo2L/TRAIL and its receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Apoptosis signaling by Apo2L/TRAIL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. The Apo2L/TRAIL DISC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Involvement of Bcl-2 family proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Modulation of sensitivity to Apo2L/TRAIL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Decoy receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. FLIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Inhibitor of apoptosis proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Interferons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. NF␬B and other factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Physiological roles of Apo2L/TRAIL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Regulation of Apo2L/TRAIL expression by interferons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Apo2L/TRAIL as a mediator of IFN function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Role in anti-tumor and anti-viral immune surveillance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Cancer therapeutic potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: Apo2L, Apo2 ligand; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; DR, death receptor; IFN, interferon; NK, natural killer; MM, multiple myeloma ∗ Corresponding author. Tel.: +1-650-225-1853; fax: +1-650-225-6443. E-mail addresses: [email protected] (A. Almasan), [email protected] (A. Ashkenazi).

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1. Introduction: Apo2L/TRAIL and its receptors Apo2L/TRAIL was originally identified and cloned based on sequence homology to the Fas/Apo1 ligand (FasL) and TNF [1,2]. Subsequent work led to identification of four

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Table 1 Apo2L/TRAIL and its receptors: nomenclature and chromosomal localization Gene designation

Map (human)

References

Apo2L, TRAIL, TNFSF10 DR4, TRAILR1, TNFRSF10A DR5, Apo2, TRAILR2, KILLER/DR5, TRICK2, TNFRSF10B DcR1, TRAILR3, TRID, TNFRSF10C DcR2, TRAILR4, TRUNDD, NFRSF10D OPG, OCIF, TNFRSF11B

3q26 8p21 8p22-p21

[1,2] [3] [4–11,125]

8p22-p21 8p21 8q24

[3,4,10,12,13] [14–16] [18–20]

novel, closely related cell-associated members of the TNF receptor (TNFR) superfamily that bind this ligand, as well as a fifth, soluble receptor that is more distantly related to the other four. The nomenclature for Apo2L/TRAIL and its receptors is summarized in Table 1. Two of the receptors that bind Apo2L/TRAIL contain cytoplasmic “death domains” and signal apoptosis: death receptor 4 (DR4) [3] and DR5 [4–11]. The other three receptors appear to act as “decoys”. Decoy receptor 1 (DcR1) [3,4,10,12,13] and DcR2 [14–16] have close homology to the extracellular domains of DR4 and DR5. DcR2 has a truncated, non-functional cytoplasmic death domain while DcR1 lacks a cytosolic region and is anchored to the plasma membrane through a glycophospholipid moiety (Fig. 1). Both receptors are therefore incapable of transmitting an apoptosis signal. The soluble TNFR family member osteoprotegerin (OPG) [17,18], was discovered first to bind the TNF superfamily member RANKL, but later found to bind Apo2L/TRAIL. However, a biological connection between OPG and Apo2L/TRAIL remains to be firmly established. OPG has a low affinity for Apo2L/TRAIL at physiological temperature [19]. On the other hand, a recent study suggests that cancer-derived OPG may be an important survival factor in hormone-resistant prostate cancer cells: a strong negative correlation was observed between levels of OPG and the capacity of Apo2L/TRAIL to induce apoptosis in prostate cancer cells that endogenously produced high levels of OPG [20].

Apo2L/TRAIL is expressed as a type 2 transmembrane protein, however, its extracellular domain can be proteolytically cleaved from the cell surface. Like most other TNF family members, Apo2L/TRAIL forms a homotrimer that binds three receptor molecules, each at the interface between two of its subunits [21,22]. A Zn atom bound by cysteines in the trimeric ligand is essential for trimer stability and optimal biological activity [23,24]. Because of this unique structural feature, the method of preparing recombinant soluble ligand may be important for its selectivity against transformed cells. Ligand preparations lacking the Zn have reduced solubility and tend to aggregate, which might explain the reported toxicity of certain recombinant Apo2L/TRAIL versions to hepatocytes [25,26]. In contrast, the untagged, trimeric Apo2L/TRAIL that contains stoichiometric Zn, has no cytotoxic effect on human or non-human primate hepatocytes [26] or keratinocytes [27]. On the other hand, aggregated or tagged, antibody-crosslinked forms of Apo2L/TRAIL can trigger apoptosis in certain normal cell types. Thus some cells, such as Jurkat T cells, hepatocytes, and keratinocytes are resistant to the trimeric ligand but quite sensitive to these higher-order oligomeric forms.

2. Apoptosis signaling by Apo2L/TRAIL 2.1. The Apo2L/TRAIL DISC Similar to FasL, Apo2L/TRAIL initiates apoptosis upon binding to its cognate death receptors by inducing the recruitment of specific cytoplasmic proteins to the intracellular death domain of the receptor, which form the death-inducing signaling complex (DISC; Fig. 2) [28]. In untransfected cells, the Apo2L/TRAIL DISC is similar to that of FasL, with the adaptor protein Fas-associated death domain (FADD, also called Mort-1 (Chapter by Genhong Chen; Reference—CGFR CHAPTER: cell death-death domain adaptors in signaling activated by TNF family)) and the apoptosis initiator caspase-8 being recruited to DR4 and/or DR5 shortly after addition of Apo2L/TRAIL [29–31].

Fig. 1. Apo2L/TRAIL and its receptors. Apo2L/TRAIL is a homotrimeric ligand that interacts with four closely related and chromosomally linked members of the TNFR superfamily: DR4 and DR5 contain a cytoplasmic death domain and signal apoptosis; DcR1 is linked to the plasma membrane by a glycophosphatidylinositol moiety and lacks signalling activity; DcR2 has a truncated, non-functional death domain. OPG, a soluble, more distantly related receptor, is capable of binding to Apo2L/TRAIL although the physiological significance of its interaction with this ligand is unclear.

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Fig. 2. Activation of the cell-extrinsic and cell-intrinsic apoptosis pathways by Apo2L/TRAIL. The apoptosis signaling pathways engaged by death receptors on the one hand and the Bcl-2 gene superfamily on the other are called the cell-extrinsic and cell-intrinsic pathways. Engagement of the extrinsic pathway by Apo2L/TRAIL is sufficient in some cell types to trigger apoptosis, whereas in other cell types amplification of this pathway through engagement of the intrinsic pathway is needed to commit the cell to apoptosis. Crosstalk between the extrinsic and intrinsic pathways requires cleavage of Bid. In mouse cells, Bax and Bak function redundantly downstream of Bid. In human colon cancer cells, Bax is absolutely required to connect the extrinsic and intrinsic pathways; if the cells have DNA mismatch repair deficiency, Bax is readily lost through mutation which renders the cells resistant to Apo2L/TRAIL. This resistance can be circumvented by pre-exposing the cells to chemotherapy, which upregulates the transcription of Bak and DR5.

Apo2L/TRAIL can trigger apoptosis independently through DR4 or DR5 [29,30]. In cells that express both DR4 and DR5, these receptors can form heterocomplexes [29]. Caspases-8 and -10 are closely related initiator caspases, which transduce the signal from Apo2L/TRAIL to the downstream apoptotitc executioner caspases. Once recruited to the DISC, caspase-8 autoactivates by proteolysis, as evidenced by the presence of cleaved prodomain fragments found in the DISC. The role of caspase-8 in receptor-mediated apoptosis has been well-documented (Chapter by Genhong Chen; Reference—CGFR CHAPTER: background on signaling, caspases, etc.). The role of caspase-10, however, is less clear and until recently its participation in the Apo2L/TRAIL and FasL DISC was controversial. Caspase-10 is found in primates but not rodents. Lymphocytes from ALPS II patients bearing a caspase-10 mutation are resistant to Apo2L/TRAIL killing [32]. Nevertheless, several groups were unable to show endogenous caspase-10 in the native Apo2L/TRAIL DISC [30,31]. The discrepancy was resolved by showing that many commercially available polyclonal antibodies raised against a specific isoform of caspase-10 crossreact with the similarly sized Hsp60 protein [33]. Experiments with well-characterized caspase-10-specific antibodies (monoclonal and polyclonal) demonstrated that endogenous caspase-10 is recruited and activated in the Apo2L/TRAIL and FasL DISC, and that it is capable of transmitting an apoptosis signal, albeit attenuated, in the absence of caspase-8 [33]. Similar results were obtained in another study [34]. Consistent with the data from APLS II patients, loss of caspase-10, often post-transcriptionally, is much more common in lung and breast carcinoma cell lines than loss of caspase-8, suggesting that caspase-10 might have a more significant role in resistance to apoptosis in cancer cells [33]. A third re-

port confirmed the observation that caspase-10 is recruited and activated in the Apo2L/TRAIL and FasL DISC, although caspase-10 tranfection of caspase-8-deficient cells did not rescue apoptosis induction except at high levels of caspase-10 expression [35]. 2.2. Involvement of Bcl-2 family proteins Apoptosis initiated by death ligands depends on the cell-extrinsic signaling pathway, which involves death receptor engagement, DISC formation, proteolytic activation of the apical caspases, caspase-8 and -10, and consequently, activation of effector caspases such as caspase-3, -6, and -7 (Fig. 2). In certain types of cells, effector caspase activation requires amplification of DISC signals by engagement of the cell-intrinsic pathway, which is the main apoptosis signaling pathway that is activated by DNA-damaging agents such as chemo- and radiotherapeutic agents. A critical step in the cell-intrinsic pathway is the activation and translocation of the Bcl-2 family member Bax to the mitochondria, leading to dissipation of the mitochondrial transmembrane potential and cytochrome c release to the cytosol. This facilitates assembly of the Apaf-1 apoptosome with recruitment and activation of caspase-9, as an initiator caspase, and subsequently the effector caspases [36,37] (reviewed in [38]). Multi-domain pro-apoptotic members of the Bcl-2 family such as Bax, or its homologue Bak, which contain three Bcl-2 homology domains (BH1–3), are counteracted by the anti-apoptotic family members Bcl-2 or Bcl-XL, which contain an additional BH4 domain [39]. A subset of the Bcl–2 family, such as the proteins Bid, Bik, Bim, NOXA, and PUMA, contain only the BH3 domain. BH3-only proteins interact with pro-apoptotic Bcl-2 family members to augment their activity. Once cleaved by caspase-8, Bid

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translocates to the mitochondria and activates Bax and Bak, thus providing a mechanism for crosstalk between the extrinsic and intrinsic apoptotic pathways [40,41]. Bcl-2 or Bcl-XL overexpression during Fas-induced apoptosis defines two types of cells with a differential dependence on the mitochondrial pathway [42]. In Type I cells, Bcl-2 or Bcl-XL block all the mitochondrial changes associated with cell death without affecting Fas-initiated apoptosis. In these cells, robust DISC association and processing of caspase-8 activates the effector caspases directly, committing the cells to death without participation of the mitochondrial pathway. Thymocytes from mice devoid of Bax and Bak remain sensitive to Fas despite the inactivation of the intrinsic pathway [43]. In Type II cells, such as Jurkat and CEM, overexpression of Bcl-2 or Bcl-XL prevents apoptosis induction through Fas. Hepatocytes provide an example of cells that become resistant to Fas-induced apoptosis in vivo when Bid, or both Bax and Bak, are deleted [44]. Importantly, in the Bcl-2/Bcl-XL overexpression studies, Apo2L/TRAIL-dependent killing does not necessarily recapitulate the Type I/II model described for Fas, since in Type II cell lines ectopic Bcl-2 expression does not block activation of caspase-8 and apoptosis following Apo2L/TRAIL addition [45,46]. In HL60 or HCT116 cells [47,48] and a variety of other cell lines [49–52], however, overexpression of Bcl-2/XL blocks Apo2L/TRAIL-triggered apoptosis. Since the difference between Types I and II cells may be dependent on DISC assembly, it is quite possible that the requirement for the mitochondrial pathway may depend on the particular death receptor initiating the apoptotic signal, in addition to cell type [46]. Gene knockout studies are perhaps a more definitive system than Bcl-2/XL overexpression for exploring the importance of the intrinsic pathway for Apo2L/TRAIL-initiated cell death. Experiments with a colon carcinoma cell line that carries a Bax gene deletion [53–55] or selected for Bax mutation [53] demonstrated the absolute Bax requirement for Apo2L/TRAIL-mediated apoptosis, even though Bak was expressed in these cells [53]. Early events triggered by Apo2L/TRAIL, such as DISC formation, caspase-8 activation, and Bid cleavage were not dependent on Bax, however, mitochondrial depolarization, cytochrome c release and activation of caspase-9 were prevented in Bax-deficient cells [53,54]. Thus, in these cells, the intrinsic pathway was required for Apo2L/TRAIL-mediated apoptosis, with Bax being essential for induction of the mitochondrial events.

3. Modulation of sensitivity to Apo2L/TRAIL Numerous reports indicate that while many human tumor cell lines are sensitive to apoptosis induction by Apo2L/TRAIL, most normal cells are not. It is not completely clear why normal cells and certain tumor cells are resistant to Apo2L/TRAIL. Some of the potential mechanisms are discussed below.

3.1. Decoy receptors The increased Apo2L/TRAIL sensitivity of tumor cells has been initially postulated to result from the lack of DcR expression. Apo2L/TRAIL binds with high affinity to two receptors, DcR1 and DcR2, incapable of transmitting an apoptotic signal due to absent or incomplete death domains. Overexpression of these receptors protects cells from apoptosis induction by Apo2L/TRAIL, suggesting that they act as “decoys”, by sequestering the ligand from the signaling death receptors [4,16]. Many normal adult tissues express at least one of the DcRs [4,14,16]. Examination of cancer cell lines and tumors failed to provide any correlations between DcR expression and Apo2L/TRAIL resistance. However, almost all of these studies relied on detecting mRNA [56–58] rather than looking for cell surface expression of the proteins [59]. It is possible that even detection of receptors by immunoblots [47] may be misleading since the DcRs may localize within the cell rather than at the cell surface [60]. It is not fully clear how widespread is the decoy receptors surface expression in tumor or normal cells, or how these receptors modulate Apo2L/TRAIL signaling. 3.2. FLIP A screen for genes in a human placental cDNA library that could confer Apo2L/TRAIL resistance to a sensitive colon carcinoma cell line [48] recovered only the short form of the cellular FLICE-inhibitory protein (FLIPs) and Bcl-XL. FLIP has homology to caspase-8 and -10, but lacks protease activity [61]. It is therefore thought that FLIP recruitment to the DISC in place of the initiator caspases blocks their activation. Some overexpression studies suggest that FLIP is an inhibitor of caspase-8 activation at the Apo2L/TRAIL DISC [62,63], and others have found correlations between FLIP levels and Apo2L/TRAIL resistance [56,64]. An inducible pathway for degradation of FLIP apparently sensitizes tumor cells to Apo2L/TRAIL-induced apoptosis [65]. Other studies, however, failed to find such a correlation [66,67]. Moreover, recent work suggests that FLIP can actually promote caspase-8 activation in response to Fas engagement [68,69]. Thus it remains unclear whether FLIP plays an important role in the Apo2L/TRAIL resistance. 3.3. Inhibitor of apoptosis proteins In addition to the proteolytic caspase cascade, caspase activity is regulated by the inhibitor of apoptosis proteins (IAP). Of these, the best-characterized is XIAP, which inhibits caspase-9 and -3 through binding to their intermediate and fully cleaved forms [70]. Smac/DIABLO is released from the mitochondria of apoptotic cells and accelerates cell death activation by displacing XIAP from the caspases [71,72]. A comparison of Apo2L/TRAIL-sensitive and -resistant melanoma cell lines showed a strong correlation

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between sensitivity and effector caspase activity [66]. Although levels of XIAP did not differ between cell lines, association between XIAP and cleavage products of the caspase-3 zymogen correlated with Apo2L/TRAIL resistance. Apo2L/TRAIL-sensitive cell lines released more Smac/DIABLO to the cytosol, so one factor contributing to resistance in some cells may be a decreased propensity to release Smac/DIABLO in response to Apo2L/TRAIL, leading to insufficient activation of effector caspases. Why the propensity varies remains to be determined. Smac/DIABLO agonists sensitize human acute leukemia Jurkat T cells for apoptosis induction by Apo2L/TRAIL or cancer chemotherapy agents [73] and induces regression of malignant glioma in vivo [74]. Smac/DIABLO transfection bypasses Bax requirement for Apo2L/TRAIL-induced apoptosis of HCT116 cells [54]. Therefore, it seems that in addition to promoting the activation of caspase-9, the mitochondrial pathway contributes to Apo2L/TRAIL-induced apoptosis by releasing Smac/DIABLO to the cytosol and relieving XIAP inhibition of caspase-3. 3.4. Interferons Interferons (IFNs), particularly IFN␤, can also sensitize cells to Apo2/TRAIL [75]. This sensitization was suggested, at least in melanoma, to be due to the X-linked inhibitor of apoptosis-associated factor-1 (XAF1), which may interact with and thus inhibit XIAP. The degree of sensitization by XAF1 was similar to that provided by IFN pretreatment and was correlated with the level of XAF1 expressed. Expression of a zinc-finger portion of XAF1 blocked IFN-dependent sensitization of melanoma cells to the pro-apoptotic effects of Apo2L/TRAIL. These results suggested that IFN-dependent induction of XAF1 strongly influenced cellular sensitivity to the pro-apoptotic actions of Apo2L/TRAIL [76]. 3.5. NFκB and other factors NF␬B is potently and rapidly activated after TNF binding to TNFR1, generating a prosurvival signal that must be overcome in many cell lines to enable TNF to induce apoptosis (reviewed in [77]). NF␬B has been reported to induce expression of FLIP, Bcl-XL and XIAP, which are considered to be responsible for its ability to protect cells from death. While Apo2L/TRAIL can also activate NF␬B, this stimulation is significantly attenuated and delayed as compared to that of TNF, and requires a high concentration of the ligand. Thus, NF␬B induction by Apo2L/TRAIL may be a secondary, indirect event [4]. Nonetheless, in a cancer cell line with high constitutive NF␬B activity, specific downregulation of NF␬B by inactivation of the I-␬B kinase significantly sensitized the cells to Apo2L/TRAIL [55]. Thus while Apo2L/TRAIL is unlikely to activate NF␬B directly, in some contexts this transcription factor can moderate sensitivity to the ligand.

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Additional cell survival promoting pathways are likely to influence susceptibility to Apo2L/TRAIL-induced apoptosis. The tumor suppressor p53, an important mediator of apoptosis in response to cell damage [36], upregulates DR5, thereby sensitizing cells to Apo2L/TRAIL [11]. Protein kinase C [78,79], MAP kinase [80] and Akt [81,82] activity have also been reported to affect Apo2L/TRAIL action, although the mechanisms are unclear at present. A possible regulation of Apo2L/TRAIL by the FOXO family of forkhead transcription factors, FKHRL1 has been suggested [83]. This finding correlated the decreased activity of FKHRL1 and FKHR in prostate cancers resulting from loss of PTEN, leading to a decrease in Apo2L/TRAIL expression that may contribute to increased survival of the tumor cells. A variety of natural and synthetic ligands of peroxisome proliferator-activated receptor (PPAR-␥) also sensitize to apoptosis induction by Apo2L/TRAIL. PPAR-␥ ligands selectively reduce levels of FLIP. Both PPAR-␥ agonists and antagonists displayed these effects, regardless of the levels of PPAR-␥ expression and even in the presence of a PPAR-␥ dominant negative mutant, indicating a PPAR-independent mechanism [84]. Reductions in FLIP and sensitization to Apo2L/TRAIL-induced apoptosis were also not correlated with NF␬B, further suggesting a novel mechanism. PPAR-␥ modulators induced ubiquitination and proteasome-dependent degradation of FLIP, without concomitant reductions in FLIP mRNA. The findings suggest the existence of a pharmacologically regulated novel target of this class of drugs that controls FLIP protein turnover, and raise the possibility of therapeutic combination of PPAR-␥ modulators with Apo2L/TRAIL [65]. Therefore, DcRs, FLIP, IAPs, and Bcl-2/XL seem to contribute to resistance when overexpressed, however, the exact mechanism of how they exert their biological roles in unmanipulated cells needs to be further explored. NF␬B, p53, and various protein kinase signaling pathways may also modulate Apo2L/TRAIL sensitivity, particularly in the context of transformed cells, where many of these activities are aberrant. The interactions of these signals may be complex and the important determinants of Apo2L/TRAIL sensitivity or resistance remain to be further identified. Many of the combination treatments with Apo2L/TRAIL and chemo- or radiotherapeutic agents are designed to overcome resistance of certain tumors to Apo2L/TRAIL used as a single agent. 4. Physiological roles of Apo2L/TRAIL 4.1. Regulation of Apo2L/TRAIL expression by interferons IFNs are a family of pleiotropic cytokines, which consist of Type I (predominantly ␣ and ␤) and II (␥) IFNs. They play an essential role in host defense, having both anti-viral and anti-tumor effects. Induction of cell death was not initially recognized as a property of IFNs, as only a few

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reports of this activity were available [85,86]. However, recent work demonstrates that IFNs can act as apoptosisinducing cytokines on various cancer cell lines (reviewed in [87]), including multiple myeloma (MM, a plasma B cell malignancy; [88]), melanoma [89], and ovarian carcinoma [90]. A preferential induction of apoptosis by IFN-␤ compared with IFN-␣2 has been noted in MM [88] and melanoma [89,91], with some melanoma cells being completely resistant to IFN-␣. In some resistant cells, IFN-␤ pretreatment sensitizes human melanoma cells to Apo2L/TRAIL-induced apoptosis [75,76]. IFNs bind to their specific receptors to phosphorylate and activate the Janus kinases (JAKs) and the signal transducers and activators of transcription (STATs; [92]). Once activated, STAT proteins dimerize and translocate to the nucleus where they bind to distinct DNA motifs to induce a large number of IFN-responsive genes. Type I IFNs primarily activate STAT 1 and 2, which are then translocated to the nucleus to bind IFN-stimulated regulatory elements (ISRE) to induce genes expression. Of those IFN-stimulated genes, several were recently reported to be associated with apoptosis. Notable amongst them is Apo2L/TRAIL; its transcriptional induction is one of the earliest events following IFN administration in MM [88,93]. The Apo2L/TRAIL gene spans approximately 20 kb and contains five exons (Fig. 3). The 1.2 kb promoter region upstream of the translation initiation codon was cloned and its transcription start site defined. It lacks a recognizable TATA box but contains several putative transcription factor-binding sites. Luciferase reporter constructs, transfected into Jurkat cells, indicated transcriptional regulation by IFNs. Deletion analysis indicated that the Apo2L/TRAIL promoter region controls the expression of the gene following IFN-␤ treatment [93]. Thus, following IFN binding to its receptor, the STAT transcription factors may bind to cis-elements in the human Apo2L/TRAIL promoter (ISRE, Fig. 3) and stimulate its transcriptional activity. Apo2L/TRAIL mRNA levels are also increased following ␥-irradiation of the Jurkat, MOLT-4, and CEM T cell lines, as well as peripheral blood mononuclear cells (PBMC). Increased Apo2L/TRAIL protein levels were found in MOLT-4 and Jurkat cells. The response to radiation in MOLT-4 cells was lost when only 430 bp of 5 -proximal flanking sequence was maintained [94], pointing to possible regulatory elements required for the radiation response.

Finally Apo2L/TRAIL has a 3 -UTR, which may impact on the post-transcriptional regulation of the stability of its mRNA [84]. A recent screening of the Apo2L/TRAIL gene revealed three single nucleotide polymorphisms (SNPs) in the 3 -UTR. Their impact on gene expression has not been fully determined. 4.2. Apo2L/TRAIL as a mediator of IFN function IFNs play an important role in immune surveillance. One mechanism by which IFNs may fulfill their role could be through regulation of other cytokines. IFN-␣/␤, typically activated by virus infection, can induce Apo2L/TRAIL expression in various cell types [95–97]. In addition, several viruses induce the expression of Apo2L/TRAIL in infected cells [98], which may serve to eliminate such cells by autocrine apoptotic suicide (Benedict; Reference—CGFR CHAPTER: viruses and TNF family). Similarly, induction of Apo2L/TRAIL by viruses can mediate apoptosis of neighboring, uninfected effector cells in vitro. Secretion of IFN-␣/␤ from infected cells activates the JAK/STAT pathway in effector cells, thus resulting in the induction of Apo2L/TRAIL. On the other hand, apoptosis of infected cells mediated by death ligands such as Apo2L/TRAIL may be countered by several viral strategies (reviewed in [98]). While Apo2L/TRAIL may be an important mediator for the effects of IFNs, other factors, such as IL6 [99] and PKR [100] also may be involved in regulation of IFN-induced apoptosis. Apo2L/TRAIL expression in response to IFN-␥ is minimal in MM and melanoma, however, it is significant in cytotoxic T cells (CTL) [101]. Apo2L/TRAIL is expressed on different cells of the immune system and plays a role in NK cell mediated tumor surveillance. In allogeneic hematopoietic-cell transplantation, the reactivity of the donor T cell against malignant cells is essential for the graft-versus-tumor (GVT) effect. While the cytolytic activity of T cells is primarily mediated through the Fas–Fas ligand and perforin–granzyme pathways, T cells deficient for both FasL and perforin can still exert GVT activity in vivo in mouse models. A comparison of clinically relevant mouse bone-marrow transplantation models revealed that alloreactive T cells expressed Apo2L/TRAIL, however, the absence of this ligand had no effect on their proliferation and cytokine production in response to alloantigens.

Fig. 3. Organization of the human Apo2L/TRAIL gene and its 5 -regulatory region. A 20 kb fragment contains five exons (kb) and four introns (bp). ATG and TAA designate the initiation and termination codons, with vertical bars representing exons, and open box the promoter. A 1.2 kb region of Apo2L 5 -flanking sequence was analyzed for known cis-acting elements. Of several putative binding sites identified for various transcription factors, ISRE and forkhead (FKHR) elements are likely to be important for IFN and FOXO regulation. A 3 -UTR, which may provide additional regulation, is indicated.

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Apo2L/TRAIL-deficient T cells showed significantly lower GVT activity than did control T cells, while no important differences in graft-versus-host disease were observed. These data suggest that strategies to enhance Apo2L/TRAIL-mediated GVT activity could decrease relapse rates of malignancies after hematopoietic-cell transplantation without exacerbation of graft-versus-host disease [102]. 4.3. Role in anti-tumor and anti-viral immune surveillance The receptor system for Apo2L/TRAIL in mice appears complex than in humans with only one signaling receptor, homologous to both DR4 and DR5 [103]. The biology of Apo2L/TRAIL may therefore differ significantly between the two species. Nonetheless, the phenotype of an Apo2L/TRAIL mouse knockout could be very informative with respect to the normal function of this death ligand in vivo. Apo2L/TRAIL knockout mice are viable, fertile and have no obvious haematological defects [104,105], suggesting this death ligand does not have an essential developmental function. Studies with these mice confirm earlier reports of the importance of Apo2L/TRAIL in immune surveillance. In the mouse, Apo2L/TRAIL is expressed on liver NK cells but not on other lymphocytes isolated from liver or spleen [106]. Blocking Apo2L/TRAIL with neutralizing antibodies partially prevents liver NK cell cytotoxicity in vitro, and dramatically increases liver metastasis of Apo2L/TRAIL-sensitive cell lines in vivo. Both Apo2L/TRAIL expression and its contribution to preventing liver metastases depend on IFN-␥ signaling (see below). Follow-up experiments show Apo2L/TRAIL is important in reducing the incidence of fibrosarcomas induced by low doses of the carcinogen methylcholanthrene [107]. Furthermore, these studies show that tumors are much more likely to be sensitive to Apo2L/TRAIL if they arise in the presence of Apo2L/TRAIL blocking antibodies. Many of these results were confirmed in Apo2L/TRAILdeficient mice [104]. Cytotoxicity of liver but not spleen NK cells is reduced whereas liver metastases, tumor growth of allografts in the mammary fat pad and fibrosarcoma induction by methylcholanthrene are all increased in the absence of Apo2L/TRAIL expression. Where it was directly compared, the magnitude of the effect of Apo2L/TRAIL gene ablation was similar to that achieved by anti-Apo2L/TRAIL blocking antibodies. A more recent study shows that syngeneic renal cell carcinomas grow faster and show increased metastasis to the liver in Apo2L/TRAIL knockout mice as compared with wild type controls [126]. Apo2L/TRAIL therefore appears to play an important role in host defense against tumor initiation and metastasis in mice. As noted above, IFNs are known to have potent anti-tumor and anti-viral effects, and Apo2L/TRAIL is emerging as

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an important effector of these activities. Both the mouse and human Apo2L/TRAIL promoters are regulated by IFN [93,108], and Apo2L/TRAIL is one of the earliest genes induced by IFN. Fibroblasts infected with human CMV and treated with IFN upregulate both Apo2L/TRAIL and its death receptors. IFN potentiates the apoptotic response in infected cells by upregulating Apo2L/TRAIL on neighboring uninfected cells while simultaneously downregulating the death receptors, making CMV-infected cells differentially more sensitive to apoptosis induced by the ligand [95]. Similar differential effects on target and effector cells are seen when monocytes are treated with IFN: Apo2L/TRAIL is rapidly upregulated and DR5 downregulated. The monocytes become resistant to Apo2L/TRAIL and acquire anti-tumor cytotoxicity that is dependent on the ligand [109]. In addition, IL-2 stimulation induces Apo2L/TRAIL in human NK cells, which use the ligand to kill tumor cell targets [110]. Apo2L/TRAIL is not found on the surface of resting peripheral blood T (PBT) cells, but is dramatically induced when PBT cells are stimulated with anti-CD3 antibodies in the presence of IFN [111]. These stimulated PBT cells have enhanced cytotoxicity against transformed cell lines that is Apo2L/TRAIL-dependent. The anti-tumor effects of IFN-␣/␤ on MM is also associated with rapid upregulation of Apo2L/TRAIL, and is blocked by a dominant negative form of DR5 [88], as is the case with ovarian cancer cells [90], demonstrating the functional importance of Apo2L/TRAIL induction in apoptosis activation by IFNs. Type I IFNs induce apoptosis through activation of Apo2/TRAIL not only in MM cell lines but also in malignant plasma cells freshly isolated from the bone marrow of MM patients, with only samples containing malignant (CD38+ /CD45−/dim ) plasma cells undergoing apoptosis upon treatment with IFNs [88]. Soluble Apo2L/TRAIL was more effective than IFNs at inducing apoptosis in these patient-derived cells. In human bone marrow-derived dendritic cells, surfaceexpressed Apo2L/TRAIL cooperated with other death ligands to induce apoptosis in cancer cells [112]. In addition to Apo2L/TRAIL’s contribution to immune surveillance, which is consistent with its known pro-apoptotic activity, Apo2L/TRAIL inhibits autoimmune inflammation in experimentally-induced rheumatoid arthritis [113] and multiple sclerosis [114]. Intriguingly, these effects do not appear to be associated with induction of apoptosis in infiltrating lymphocytes. It will be interesting to investigate whether Apo2L/TRAIL knockout mice have a greater propensity to develop autoimmune disease in response to experimental immunization. Apo2L/TRAIL thus mediates a significant part of the anti-tumor and anti-viral cytotoxicity of dendritic cells, monocytes, NK and T cells, often augmented by IFNregulated induction, suggesting that it may be an important innate effector molecule in immune surveillance as well as a negative regulator of autoimmunity.

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5. Cancer therapeutic potential Apoptosis induction in response to most DNA-damaging drugs usually requires the function of the tumor suppressor p53, which engages primarily the cell-intrinsic apoptotic-signaling pathway [36]. In most human cancers, following tumor progression or as a result of clinical treatments p53 is inactivated, resulting in resistance to further therapy. Death receptors can trigger apoptosis independently of p53, and therefore their targeting might be a useful therapeutic strategy, particularly in cells in which the p53-response pathway has been inactivated, thus helping to circumvent resistance to chemo- and radiotherapy. In tumors that retain some responsiveness to conventional therapy, death receptor engagement in combination with chemotherapy or radiation might lead to synergistic apoptosis activation, as well as reduce the probability that tumor cells resistant to either type of agent will emerge. Recombinant soluble Apo2L/TRAIL induces apoptosis in a variety of cancer cell lines regardless of p53 status. Moreover, recent studies suggest that Apo2L/TRAIL is effective at inducing apoptosis in primary tumor samples from patients with MM [115] or colon carcinoma [116]. In mouse models, Apo2L/TRAIL demonstrated remarkable efficacy against tumor xenografts of colon carcinoma [53,117], breast carcinoma [118], MM [115], or glioma [74,119]. Further, combinations of Apo2L/TRAIL and certain DNA-damaging drugs [90,117] (Ray and Almasan, unpublished data) or radiotherapy [94,120] may exert synergistic anti-tumor xenograft activity. Some variation between the effectiveness of Apo2L/ TRAIL against tumors in published reports is likely based on the use of various recombinant versions of human Apo2L/TRAIL that have been generated. One version contains Apo2L/TRAIL amino acids 114–281 fused to an amino-terminal polyhistidine tag [1]. A second variant contains amino acids 95–281 fused amino terminally to a modified yeast Gal-4 leucine zipper (LZ) that promotes trimerization of the ligand [118]. A third version contains residues 95–281 fused to an amino-terminal ‘Flag’ epitope tag; crosslinking of this tagged protein with anti-Flag antibodies enhances its activity against certain cell lines [88,94]. Currently, a fourth recombinant version of the ligand probably is the most preferred for clinical application: it contains amino acids 114–281 of human Apo2L/TRAIL without any added exogenous sequences. This latter version is therefore the least likely to be immunogenic in human patients. The production of this version has been optimized by the addition of Zn and reducing agent to the cell culture media and extraction buffers, and by formulation of the purified protein at neutral pH [26,117]. Many tumor cell lines are sensitive to this non-tagged, Zn-optimized version of Apo2L/TRAIL [26]. Hepatocytes and keratinocytes are resistant to this optimized ligand version [26,27] but show significant sensitivity to apoptosis induction by non-optimized or antibody-crosslinked variants of the ligand [25].

One potential explanation for this difference is that commitment of these normal cells to apoptosis might require high-order multimerization of the DR4 and DR5 receptors. The versions of the ligand that are tagged and not optimized for Zn content can have a low solubility and tend to aggregate and/or precipitate at high concentrations, as does the antibody-crosslinked ligand. Therefore, these preparations may over-multimerize death receptors, leading to a signal that surpasses the high threshold for apoptosis activation in the normal cells. Initial studies in non-human primates, namely, cynomolgus monkeys and chimpanzees, show that short-term intravenous administration of non-tagged, Zn-bound Apo2L/TRAIL is well tolerated even at high doses [26]. Thus, given Apo2L/TRAIL’s preferential pro-apoptotic activity on cancer cells over normal cells, studies are in progress to enable clinical investigation of this ligand as a potential cancer therapeutic. Besides using the recombinant ligand, one might envision alternative modalities of expression of Apo2L/TRAIL for therapeutic purposes. One approach is to engage the death receptors DR4 or DR5 with agonistic antibodies [121]. A second approach is gene therapy. Anti-tumor activity and prolonged expression from an Apo2L/TRAILexpressing adenoviral vector has been recently shown [122]. Apo2L/TRAIL was also expressed from heterologous promoters. Such an adenoviral vector, expressing the GFP/TRAIL fusion gene from the hTERT promoter (designated Ad/gTRAIL), elicited high levels of transgene expression and apoptosis in a variety of breast cancer cell lines, including those resistant to doxorubicin or soluble Apo2L/TRAIL protein, or their tumor xenograft derivatives [123]. Furthermore, treatment with Ad/gTRAIL effectively elicited apoptosis in malignant cells but not in normal human primary hepatocytes (NHPHs) in vitro and suppressed tumor growth and prolonged duration of survival in vivo [124]. However, as most gene therapy protocols are still in their infancy, the success of applying this modality to Apo2L/TRAIL will depend on demonstration of efficient delivery, safety and lack of immunogenic response of these vectors.

6. Conclusions and future directions Apo2L/TRAIL is a powerful inducer of apoptosis that acts through an unusually complex receptor system. Interferons are important modulators of Apo2L/TRAIL expression, and consistent with this finding, the ligand seems to play an important role in surveillance by cells of the innate immune system against viral-infection and malignant transformation of host cells. Because of the selectivity of soluble, Zn-bound Apo2L/TRAIL toward transformed versus normal cells, this protein bears exciting promise as a potential cancer therapeutic agent, and work is underway to enable its investigation in cancer patients. Much progress has been made on elucidating the endogenous biochemical pathway leading to

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Apo2L/TRAIL-induced apoptosis in cancer cells. Why normal cells and some types of tumor cells resist Apo2L/TRAIL remains the subject of intense investigation, as do the biological roles and evolution of this intriguing ligand–receptor system.

Acknowledgements Supported in part by research grants from the National Cancer Institute CA81504, CA82858 (A. Almasan).

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