Cancer Therapy Vol 4, 47-72, 2006

Sensitivity and resistance of human cancer cells to TRAIL: mechanisms and therapeutical perspectives

Review Article

Luca Pasquini, Eleonora Petrucci, Roberta Riccioni, Alessia Petronelli and Ugo Testa*

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy

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*Correspondence: Dr Ugo Testa, Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy; e-mail: u.testa@iss.it

Key words: apoptosis, cancer, TRAIL, receptors, monoclonal antibodies, resistance, sensitivity

Abbreviations: 2-ciano-3,12-dioxooleana-1,9(11)-dien-28-oic acid, (CDDO); acute lymphoblastic leukaemia, (ALL); Acute Myeloid Leukaemia, (AML); Apoptosis-Inducing-Factor, (AIF); Apoptotic Protease Activating Factor 1, (APAF-1); B-Chronic Lymphocytic Leukemia,, (B-CLL); cellular FLICE inhibitory proteins, (c-FLIP); Chronic Myeloid Leukemia, (CML); c-Jun kinase, (JNK); death domains, (DD); death-induced signalling complex, (DISC); endoplasmic reticulum, (ER); hepatocarcinoma cancer cells, (HCC); Histone acetyltransferases and histone deacetylases, (HDAC); histone deacetylase inhibitors, (HDACi); imidazole derivative of CDDO, (CDDO-Im); inhibitor of apoptosis proteins, (IAPs); interferon-g, (IFN-g); interferon-alpha, (IFN-a); Multiple Myeloma, (MM); non-small cell lung cancer, (NSCLC); osteoprotegerin, (OPG); renal cell carcinoma, (RCC); Tumor necrosis factor, (TNF); Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand, (TRAIL); ultraviolet B radiations, (UVB)

Received: 22 September 2005; Accepted: 3 February 2006; electronically published: February 2006

Summary

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines, able to induce apoptotic cell death in a variety of tumor cells by engaging the death receptors TRAIL-R1 and TRAIL-R2, while sparing most normal cells. Endogenously expressed TRAIL plays an important role in immunosurveillance of developing and metastatic tumors. The majority of studies aiming to evaluate the pro-apoptotic effects of TRAIL on tumor cells have been carried out on cancer cell lines and showed variable levels of sensitivity of these cells to TRAIL-induced apoptosis, while the few studies carried out on primary tumor cells have almost invariably shown low sensitivity or resistance of tumor cells to TRAIL-induced apoptosis. The mechanisms of resistance of tumor cells to TRAIL are related to increased expression or activity in these cells of anti-apoptotic molecules, mainly c-FLIP or one of the IAPs, or to the defective expression of molecules such as FADD or caspases-8, involved in the transduction of the apoptotic signal originated from TRAIL-Rs. However, many studies have shown several strategies able to induce a high sensitivity of tumor cells to TRAIL, based on the combined administration with TRAIL of different drugs, including triterpenoids, proteasome inhibitors, histone deacetylase inhibitors, interferon-g or anti-cancer cytotoxic drugs: these various agents either decrease the expression of anti-apoptotic molecules or increase the expression of molecules involved in TRAIL-Rs signalling, thus restoring a high level of sensitivity of tumor cells to TRAIL. On the other hand, preclinical studies in mice and non-human primates have shown the potential utility of recombinant soluble TRAIL and, mostly, of agonistic anti-TRAIL-R1 or -R2 antibodies for cancer therapy. Two anti-TRAIL-R antibodies are under evaluation in phase I/II clinical studies.

I. Introduction

Chemotherapy, surgical resection and radiotherapy represent the standard therapeutic approaches in the treatment of cancer. These therapeutic strategies, however, are often non curative and associated with considerable secondary and toxic effects, limiting the treatment and result frequently in the selection of highly malignant treatment-resistant tumor cells. Given these limitations there is the absolute need for the development of alternative therapeutic strategies based on new drugs endowed with a different mechanism of action, specifically targeting to the tumor cells and with more acceptable toxicity profiles. In this context, particularly promising is the area of biologic therapies, based on agents able to selectively kill tumor cells, sparing normal cells, display limited in vivo toxicity, and able to bypass or circumvent acquired tumor resistance against conventional treatments.

Among new biologic agents, the death receptor ligand Apo2 Ligand/Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (Apo2L/TRAIL) seems to offer a promising anti-tumor therapeutic potential, based on its capacity to selectively induce the killing of a wide variety of cancer cells, sparing normal cells. Importantly, unlike conventional anti-cancer drugs, death receptor ligands kill tumor cells through a molecular mechanism completely independent on the p53 tumor suppressor gene, very frequently mutated in human tumors.

In this article we provide an overview of the Apo2L/TRAIL molecule and its receptors, of the sensitivity and resistance of the tumor cells to this death receptor ligand and of the possible therapeutic use of Apo2L/TRAIL for the treatment of cancer used alone or in combination with other drugs.

A. Apoptosis and cancer

Defects in DNA repair lead to genomic instability and predispose to cancer development. This genetic instability represents the mechanism through which normal cells can accumulate a sufficient number of mutations to become malignant (Hanahn and Weimberg, 2000). The cells, however, possess important mechanisms of protection against this genomic instability, mainly orchestrated by the tumor suppressor protein p53, which acts as “a guardian of the genome” protecting cells against cancer (i.e. by inducing the death of cells that have accumulated genetic defects) (Brown and Attardi, 2005). The p53 is the most frequently mutated gene in human cancers and its inactivation by mutation certainly plays an important role in tumor development (Brown and Attardi, 2005). There is now evidence that in addition to a p53 inactivation tumor cells exhibit multiple defects in cell death pathways. Resistance to cell death and particularly to apoptotic cell death plays a key role both in tumor development and in the mechanism of resistance to anticancer drugs (Okada and Mak, 2004). The resistance to programmed cell death plays a major role in several pathogenetic mechanisms of tumors, allowing tumor cells to abnormally survive beyond their normal life, reducing the need for exogenous growth factors, providing protection from hypoxia and oxidative stress, increasing the time and therefore the opportunity for the development of additional genetics abnormalities altering cell proliferation, interfering with cell differentiation, promoting angiogenesis and increasing cell motility and invasiveness during tumor progression.

Furthermore, standard anticancer treatments, chemotherapy and radiotherapy, must be administered in relatively small doses to allow the normal tissues to receive from sub-lethal radiation or cytotoxic damage between treatments. However, surviving cancer cells proliferate during the intervals between treatments and this process of repopulation is an important cause of treatment failure (Kim and Tannock, 2005).

The understanding of the mechanisms responsible for the resistance of tumor cells to programmed cell death has a fundamental importance not only for a better understanding of cancer biology, but also for the development of new cancer therapies focused on devising ways to overcome this resistance and to induce apoptosis of cancer cells.

B. Apoptotic pathways

The most common and well defined form of programmed cell death is represented by apoptosis, a physiological process of cellular suicides required for the maintenance of cell homeostasis, embryonic development and for the differentiation and function of haematopoietic and lymphoid cells.

A common feature of the apoptotic process is the constant involvement of caspases, a family of intracellular cysteine proteases (Cysteine Aspartyl-specific Proteases).

These enzymes are present as inactive zymogens in all animal cells, but can be triggered to assume an active state, usually trough their proteolytic processing at conserved aspartic acids residues. Pro-caspases contain three domains: a NH₂-terminal domain, a large subunit (about 20 kDa) and a small subunit (about 10 kDa). During activation the pro-caspase is cleaved to generate large and small subunits; the active enzymes are heterotetramers composed of two large and two small subunits.

It is important to note that active caspases cleave their substrates at the Asp residues and are themselves also activated by proteolytic cleavage at Asp residues.

This unique property implies that these enzymes make part of proteolytic cascades, where caspases activated themselves and each other.

Based on their level of action caspases are distinguished in “initiatior” caspases and “effector” caspases: the first ones act at the origin of the apoptotic process, while the latter ones at the late steps of the apoptotic process. Initiator caspases possess long N-terminal prodomains that contain recognizable protein-protein interaction motifs, while effector caspases have short or no prodomains.

The activation of an effector caspase is mediated by an initiator caspase through cleavage at internal Asp residues that separate the large (p20) and small (p10) subunits. p10 and p20 subunits associate to form a monomer. In contrast, initiator caspases are auto-activated. The process of activation of initiator caspases is tightly controlled (Reidel et al, 2004).

There are two pathways by which caspases activation is induced, the extrinsic and the intrinsic apoptotic pathways. Both the pathways converge on activation of effector caspases, but require different initiator caspases to start the process. The extrinsic pathway is activated by engagement of death receptors on the cell membrane. Binding of ligands, such as FasL, TNF and TRAIL to their respective membrane receptors Fas, TNF-R and TRAIL-Rs induces the formation of the death-induced signalling complex (DISC). DISC in turn promotes caspases-8 recruitment and promotes a cascade of caspases activation that culminates with cell death.

The activation of caspase-8 is antagonized by cellular FLICE inhibitory proteins (c-FLIP), an enzymatically-inactive relative of caspase-8 and -10 that binds to DISC. The knockdown of c-FLIP augments DISC recruitment, activation and processing of caspases-8, thereby enhancing effector-caspase stimulation and apoptosis.

The intrinsic pathway is triggered by various extracellular and intracellular stresses, including growth factor deprivation, DNA damage, oncogene induction, hypoxia and cytotoxic drugs. Cellular signals originated by various mechanisms by these different stresses converge on a cellular target represented by mitochondria. A series of biochemical events is then induced leading to damage of the outer mitochondrial membrane, the release of cytochrome c and other pro-apoptotic molecules, with consequent formation of the apoptosome, a large molecular complex formed by cytochrome C, Apoptotic Protease Activating Factor 1 (APAF-1) and caspase-9, and caspase activation. The release of cytochrome c is essential for caspase-9 activation (Hao et al 2005). Activated caspase-9 in turn activates the downstream effector caspase-3 and -7, which rapidly cleave intracellular substrates. Other released mitochondrial proteins facilitate caspase activation through inactivation of endogenous inhibitors, the inhibitor of apoptosis proteins (IAPs). The permeabilization of the outer mitochondrial membrane is inhibited by antiapoptotic molecules pertaining to the Bcl-2 family.

Activated executioner caspases kill cells via apoptosis; however, inhibition of these caspases only transiently protect cells since, once the mitochondrial permeability is achieved, death will proceed regardless of caspase activation, either due to toxic mediators released from the mitochondria or eventual loss of essential mitochondrial functions (Chipuk and Green, 2005).

The apoptotic intrinsic pathway is also controlled by other mitochondrial proteins. Thus, Apoptosis-Inducing-Factor (AIF) and endonuclease G may induce cell death independently of caspases activation. Furthermore, Smac/DIABLO and HTRA2 promote caspases activation by counteracting the activity of Inhibitors of Apoptosis (IAP): these proteins exert an inhibitory activity on caspases activation.

A third apoptotic pathway originated by stress occurring at the level of the endoplasmic reticulum (ER), the cellular structure involved in the assembly and routing of proteins. In this pathway, caspases-12, localized in outer membrane of the ER acts as an initiator caspase.

In addition to caspase-dependent cell death, some cell death processes occur in a caspase-independent manner. Basically, four caspase-independent cell death processes have been described: autophagy, paraptosis, slow cell death and mitotic catastrophe (Broken et al, 2005). The autophagy is a cell death process characterized by sequestration of a part of cell cytoplasm, including organelles, in autophagic vesicles and their delivery to and subsequent degradation following fusion with the cellular lysosomes. Paraptosis involves cytoplasmic vacuolation and mitochondrial swelling in the absence of caspase activation and nuclear changes typical of apoptosis. Mitotic catastrophe is a caspase-independent cell death process originated by mitotic failure due to defective cell cycle checkpoints and the generation of aneuploid cells. Finally, slow cell death corresponds to a process od a delayed programmed cell death occurring when caspases are asent or inhibited.

C. TRAIL and its receptors

Death receptors are members of the TNF receptor gene superfamily, composed by more than 20 proteins exhibiting a broad range of biological activities. Particularly, some of them play a key role in the control of cell death and survival. Members of the TNF receptors superfamily share some common, structural features consisting in a cysteyne-rich extracellular domain and in a cytoplasmic domain of about 80 aminoacids, called death domain which plays a key role in the transmission of the death signal triggered by the interaction of a death ligand with its receptors. The typical examples of this receptor superfamily are represented by CD95 (also known as Fas), TNF-R1, TRAIL-R1 and TRAIL-R2.

The corresponding ligands of the death receptors are members of the TNF superfamily, which comprise ligands such as FasL, TNF, TRAIL and TWEAK.

These ligands are type II transmembrane proteins, which exists also as soluble proteins released after their cleavage by metalloproteases present in the microenvironment.

TRAIL is a type II membrane bound ligand of the TNF family of about 33-35 KD, displaying 28% aminoacid sequence identity with FasL and 23% identity with TNF (Kimberly and Screaton 2004; Kelley and Screaton, 2004). The native form of the ligand is present as a transmembrane protein with the C-terminal located at the extracellular side and the N-terminal at the cytoplasmic side. The molecule is released in the microenvironment in a vesicle-associated form, or is cleaved and exists as a soluble homotrimeric ligand stabilized by a zinc moiety. The extracellular domain of the molecule can be cleaved proteolytycally to release a soluble ligand. Crystallographic analysis has shown that TRAIL, like other TNF ligands, exists as a homotrimeric molecule, stabilized by an internal Zn²⁺ atom that is coordinated at the level of the single impaired cysteine residue (Cys 230) of each monomer. This zinc atom is essential for structural integrity of TRAIL. At variance with other death receptor ligands, TRAIL expression is constitutively observed in many tissues, with the exception of liver, testis and brain.

The receptor system responsible for the binding and transduction of a cell signalling of TRAIL is complex in that it involves five specific membrane receptors. (Figure 1) Two of these receptors, TRAIL-R1 (also known as DR4) and TRAIL-R2 (also known as DR5) are true death receptors in that they possess death domains in their cytoplasmic tail and are able to transduce a death signal and to engage the apoptosis machinery (Figure 1).

In contrast TRAIL-R3 to TRAIL-R5 are antagonistic decoy receptors, able to bind TRAIL, but not to transmit a death signal (Figure 1); therefore, they compete with TRAIL-R1 and -R2 for TRAIL binding.

TRAIL-R3 is a glycolphosphatidylinositol GPI-anchored cell surface protein, which lacks a cytoplasmic tail; TRAIL-R4 is a transmembrane cell receptor harbouring a truncated cytoplasmic death domain. As a consequence of these structural features TRAIL-R3 and TRAIL-R4 upon binding of TRAIL are unable to induce a death signal. In addition to these four canonical receptors, there is also TRAIL-R5, known as Osteoprotegerin, a soluble decoy receptor exhibiting low affinity for TRAIL, which is involved in the regulation of osteoclastogenesis.

TRAIL-R2 is a membrane protein non-glycosylated exhibiting 58% homology to TRAIL-R1. TRAIL-R2 is a type I trans-membrane protein, of 411 amino acids with a very large signal sequence (51 amino acids), a 132 amino acid extra-cellular region, a 22 amino acid transmembrane domain and a 206 amino acid cytoplasmic domain (Figure 1). This receptor possesses two cysteine rich domains in its extracellular domain. Two different isoforms of TRAIL-R2 are generated (a long and a short, differing for 23 amino acids at the level of the extracellular domain) by a differential RNA splicing event. The two isoforms are equally active in mediating cell death. Recent studies indicate that the TRAIL-R2 is more active than the TRAIL-R1 in mediating TRAIL-induced cell death (Kelley et al, 2005). TRAIL-R2 is widely expressed in human tissues and its expression is upregulated in activated T lymphocytes and in interferon-treated monocytes/ macrophages.

Figure 1. Human TRAIL receptors. A trimeric human TRAIL molecule is able to induce apoptosis through binding to TRAIL-R1 or TRAIL-R2. In fact, both these receptors possess a death domain in their cytoplasmic tail (shown as a black box). In the cytoplasmic region of TRAIL-R2 is present also a TRAF binding motif (shown as red box) responsible for the capacity of this receptor to activate the NF-kB transcription factor. TRAIL-R3 and TRAIL-R4 do not signal apoptosis and act as decoy receptors: TRAIL-R3 is anchored to the cell membrane through a GPI anchor (shown as a red circle) and TRAIL-R4 possesses a truncated death domain in its cytoplasmic tail (shown as a black box) and TRAF binding motifs (shown as red boxes). OPG is a soluble decoy receptor.

The structure of TRAIL-R1 is highly comparable to that of the TRAIL-R2, as well as its pattern of expression and regulation. TRAIL-R1 is a 468 amino acid type I transmembrane protein that contains a 23 amino acid signal sequence, a 226 amino acid extra-cellular region, a 19 amino acid cytoplasmic region (Figure 1). The cytoplasmic domain of TRAIL-R1 contains a death domain. Although the TRAIL-R1 is able, as well as the TRAIL-R2, to induce a death signal, studies with TRAIL mutants suggest a major role of TRAIL-R2 and not TRAIL-R1 in mediating TRAIL-mediated anti-tumor activity (Kelley et al, 2005).

TRAIL-R3 (also known as DcR1) is a 299 amino acid protein with a 23 amino acid signal sequence, a 217 amino acid extracellular region and a 19 amino acid trans-membrane domain (Figure 1). Lacking a cytoplasmic domain, TRAIL-R3 is linked to the cell membrane through a GPI linker. TRAIL-R3 seems to act as an antagonizing decoy receptor: its overexpression induces resistance to TRAIL, while its removal from the cell surface by phosphatidylinositol phospholipase C augments the sensitivity of the cells to the apoptotic effect of TRAIL. However, the impact of TRAIL-R3 in the normal TRAIL/TRAIL-Rs system is limited due to scarce expression of this receptor in normal tissues.

TRAIL-R4 (also known as DcR2) contains a truncated death domain and is therefore unable to convey an apoptotic signal. TRAIL-R4 acts, like TRAIL-R3, as an inhibitor of TRAIL-induced apoptosis acting through mechanisms similar to those reported for TRAIL-R3 and probably also activating the anti-apoptotic NF-kB pathway. TRAIL-R4 may have a greater importance than TRAIL-R3 in the physiology of the TRAIL/TRAIL-R system in that it is widely expressed in normal tissues.

The fifth TRAIL receptor is represented by osteoprotegerin (OPG). The most characterized function of this soluble receptor consists in the inhibition of RANKL-stimulated osteoclast formation. OPG binds to RANKL, preventing its interaction with RANK; however, it is also able to bind with low affinity to TRAIL. The role of OPG in the normal physiology of the TRAIL/TRAIL-R system is unclear. However, in some pathologic conditions, such as prostate cancer and multiple myeloma, OPG could act in a paracrine/autocrine way by binding TRAIL and promoting tumor growth.

The TRAIL-R1 and TRAIL-R2 activate two overlapping signalling pathways: the extrinsic cell death pathway and a cell survival pathway mainly involving the activation of NF-kB transcription factor (Figure 2).

The TRAIL homotrimer induces the trimerization of TRAIL-R1 or TRAIL-R2 on the surface of target cells, which determines the formation of DISC. Briefly, the trimerization of TRAIL-R1 or TRAIL-R2 results in the recruitment of an adaptor molecule FADD through the interaction of their respective death domains (DD). Next, FADD recruits DED-containing initiator pro-caspase-8 through DED/DED (death effector domain) interactions, thus forming the DISC. Control of FADD recruitment to the DISC is mediated by DED-containing c-FLIPs: these proteins exert their inhibitory activity on TRAIL-Rs signalling through their binding to the DED of FADD, thus blocking pro-caspase-8 activation (Figure 2).

In some cells (type I cells) the activation of caspase-8 is sufficient to trigger the activation of the effector caspase-3 and to execute cellular apoptosis (Figure 2).

In other cell types (type II cells), the amplification of the apoptotic cascade through the mitochondrial pathway is required for the induction of cellular apoptosis: in this pathway Bid is cleaved by caspase-8 and, in turn, the truncated Bid activates Bax- and Bak-mediated release of cytochrome C and Smac/DIABLO from mitochondria with subsequent induction of cell apoptosis (Figure 2).

In type I cells, death receptorinduced death cannot be blocked by overexpression of anti-apoptotic Bcl-2 family members. In type II cells, overexpression of antiapoptotic Bcl-2 family members blocks death receptor-induced cell death. Interestingly, the mode and efficiency of death receptor signalling was correlated with the submembrane localization of these receptors: in type I cells a portion of death receptors resides constitutively in lipid rafts (membrane liquid-ordered microdomains enriched in sphingolipids and cholesterol that constitute a distinct biophysical compartment, serving a signalling platform for membrane receptors), while in type II cells, these receptors are excluded from lipid rafts during early signalling(Muppidi and Siegel, 2004). The presence of a death receptor in lipid rafts allow type I cells to undergo apoptosis in response to stimulation with the corresponding death ligand, which cannot induce apoptosis in type II cells (Muppidi et al, 2004). Disruption of lipid raft structure by cholesterol depletion reduces death receptor signalling efficiency in type I cells, but not in type II cells (Legembre et al, 2006).

This signalling pathway for TRAIL-R1 and TRAIL-R2 was now firmly established (Yagita et al, 2004).

In addition to the death signalling pathway, TRAIL-R1 and TRAIL-R2 are also able to activate survival signals via the transcription factor NF-kB (Figure 2), which can up-modulate antiapoptotic genes (Schneider et al, 1997). However, it is important to note that NF-kB activation via TRAIL-Rs is much weaker than that via TNF-Rs and that the activation of NF-kB alone is not sufficient to inhibit apoptosis triggered via TRAIL-R2. Inhibition of NF-kB activation attenuates TRAIL resistance of tumor cells (Jeremias et al, 1998). TRAIL-R1/TRAIL-R2-induced NF-kB activation is mediated by a TRAF2-NIK-IκB kinase alpha/beta signalling cascade (Hu et al, 1999). TRAIL induces NF-kB in two phases: an early caspases independent phase and a late caspases-8, but not caspases-3, dependent phase; this last phase leads to the caspases-mediated cleavage of IkBα (Rathore et al, 2004).

TRAIL has been shown to activate also c-Jun NH₂-terminal kinase (JNK) and the induction of the JNK pathway was shown to have an amplifying effect on TRAIL-induced apoptosis.

Several studies suggest that TRAIL and its receptors may play an important role in the control of development of tumors, acting at the level of several important steps in the development of cancers.

Figure 2. Outline of the signalling cascade triggered by TRAIL-R2. The binding of one homotrimeric TRAIL molecule to the TRAIL-R2 determines its trimerization with consequent binding of a FADD molecule to its cytoplasmic tail through a DD/DD interaction. FADD through its DED domain recruits pro-caspase-8. Caspase-8 activation is controlled by DED-containing FLICE inhibitory proteins (c-FLIPs) that exert an inhibitory activity on this process. In type I cells the activation of caspase-8 is sufficient to induce the activation of caspase-3 with subsequent induction of apoptosis. In type II cells, the amplification of the apoptotic signal through the mitochondrial pathway is required for induction of apoptosis: in this pathway Bid is cleaved by caspase-8; in turn, truncated Bid activates Bax and Bak-mediated release of cytochrome C and SMAC/DIABLO from mitochondria with consequent induction of apoptosis. In addition to death signalling, TRAIL-R2 is also able to activate survival signals via the TRADD-mediated activation of the NF-kB transcription factor. However, it is important to note that usually the TRAIL-R2-mediated activation of NF-kB is weak and not able to counteract the death signalling activity induced by the activation of this receptor.

The role of TRAIL in cancer development is supported by several lines of evidence:

·TRAIL-R1 and TRAIL-R2 are mutated in some tumors, including breast cancer, ovarian cancer, non-Hodgkin lymphomas, non small cell lung cancer and neck cancer.

·Tumor cells may express TRAIL allowing them to kill T cells and thus to suppress the immune system.

·Mice made deficient in TRAIL expression by gene targeting have a clearly increased tendency to develop tumors thus suggesting a physiologic role for TRAIL in immune surveillance against tumor development (Cretney et al, 2002, Takeda et al, 2002). On the other hand, TRAIL-R2 knockout mice exhibit a clear compromission in radiation-induced apoptosis (Finnberg et al, 2005).

TRAIL-R1 or TRAIL-R2 are silenced in some tumors through epigenetic mechanisms, mainly related to gene hypermethylation (Horak et al, 2005).

The analysis of TRAIL and TRAIL-Rs expression in normal tissues at protein level showed that: (i) TRAIL expression was found to be limited mostly to smooth muscle in lung and spleen as well as glial cells in the cerebellum and follicular cells in the thyroid gland; (ii) TRAIL decoy receptors are rarely expressed in normal tissues; (iii) TRAIL-R1 and TRAIL-R2 are expressed in many tissues and particularly on smooth muscle cells in all tissues, around blood vessels, on neuronal cells in cerebellum, on hepatocytes, follicular cells in the thyroid, monocytes and macrophages present in various tissues (Daniles et al, 2005).

The level of TRAIL-Rs may be modulated by various agents.

Agents that induce DNA damage, such as anti-tumor chemotherapies, UV irradiation or g ray radiation have been shown to up-regulate TRAIL-R1 and TRAIL-R2 through p53- and p63- dependent mechanisms (Liu et al, 2004; Gressner et al, 2005).

For TRAIL-R2 a direct p53-dependent trans-activation mechanism via a p53 DNA binding motif within intron-1, has been shown.

In contrast, the upregulation of TRAIL-R2 by interferon-g and by glucocorticoids is independent of p53 activation.

II. Sensitivity of human tumors to TRAIL

Many studies have been carried out to investigate the sensitivity of human tumors to TRAIL. We focused our attention to the literature concerning the brain, lung, prostate, gynaecologic, gastro-enterologic, skin and hematologic tumors. A major limitation derives from the fact that the majority of these studies have been performed on tumor cell lines, that do not reflect entirely the properties of primary tumor cells. In spite these limitations, these studies clearly indicate that in the majority of cases tumor cells are resistant to the pro-apoptotic effects of TRAIL. The analysis of the molecular mechanisms responsible for TRAIL resistance of tumor cells has lead to the identification of some important defects in the apoptotic machinery of cancer cells. The main steps of TRAIL resistance are outlined in Figure 3 and are represented by a single or by a combination of these mechanisms: defective TRAIL-R1 and/or TRAIL-R2 expression, predominant expression of decoy TRAIL-Rs, defective expression of caspases-8 or of FADD, increased expression of c-FLIP or of IAPs (Figure 4).

A. Brain tumors

The sensitivity of neuroblastoma cells to TRAIL was investigated in detail. Initial studies showed that neuroblastoma non-invasive stromal-adherent cell lines were highly sensitive to TRAIL, while invasive cell lines are resistant (Hopkins-Donaldson et al, 2000). The absence of TRAIL-sensitivity correlated with loss of caspase-8 expression. This last finding was correlated with TRAIL resistance in a second study carried out in 18 neuroblastoma cell lines (Eggert at al, 2001). The caspase-8 gene is silenced in neuroblastomas with amplification of the oncogene MYCN through DNA methylation (frequently) as well as through gene deletion (rarely) (Teitz et al, 2000). Analysis of the pattern of TRAIL-R expression in primary tumors showed that the large majority of them do not express TRAIL-R1 and TRAIL-R2, thus suggesting that loss of TRAIL-R expression may represent an additional mechanism of TRAIL resistance in neuroblastoma (Yang et al, 2003).

TRAIL efficiently triggers apoptosis in some medulloblastoma cell lines (Nakamura et al, 2000). Analysis of the pattern of TRAIL-Rs expression by RT-PCR showed that primary medulloblastoma cells expressed TRAIL-R2; however, a loss of caspase-8 expression was frequently (52% of cases) observed (Zuzak et al, 2002). Primary medulloblastoma cells did not express TRAIL, while TRAIL expression was present in reactive peri-tumoral astrocytes (Nakamura et al, 2000). Similarly, the resistance to TRAIL-induced apoptosis in primitive neuroectodermal brain tumors cell lines correlates with a loss of caspase-8 expression (Grotzer et al, 2000). Importantly, the loss of caspase-8 expression and the lack of TRAIL sensitivity in these tumors correlate with unfavourable survival outcome (Pingoud-Meyer et al, 2003).

The majority of glioma cell lines are resistant or are scarcely sensitive to TRAIL-mediated apoptosis (Chunhai et al, 2001). These preliminary observations have been confirmed through the analysis of primary tumors derived from 21 glioblastoma patients, showing that only 4/21 of them are sensitive to TRAIL-induced apoptosis (Jeremias et al, 2004). The analysis of the mechanisms of TRAIL-resistance in glioma cells suggest a possible role of caspase inhibitors, like XIAP, that are constitutively expressed in these cells (Wagenknecht et al, 1999). Analysis of a panel of glioma cell lines showed that 1 glioma line, U373, was resistant to death ligand-induced apoptosis because it expressed very low levels of caspase-8 (Knight et al, 2001). Analysis of primary glioma cells showed that a high proportion of them have low or undetectable caspase-8 levels (Ashley et al, 2005).

In line with this finding, Smac agonists (that counteracts the antiapoptotic effects of IAPs) sensitize for TRAIL and induce regression of malignant glioma in tumor xenograft models (Fulda et al, 2002).

B. Prostate cancer

No data are available about the sensitivity of primary prostate cancer cells to TRAIL. However, several studies have been performed on a panel of prostate cancer cell lines. The response of prostate cancer cells lines to TRAIL depends on the cell type. For example the PC-3, PC-3M, DU-145, ALVA-31 cell lines were sensitive or semi-sensitive to TRAIL (Zhang et al, 2004; Sonneman et al, 2004; Holen et al, 2002) while LNCaP, PC-3-TR, CL-1 cell lines were resistant (Zhang et al, 2004; Ng & Bonavida, 2002).

The mechanism of prostate cancer resistance to TRAIL was explored only in cancer cell lines. The majority of these studies have been carried out in PC-3, DU-145 and LNCaP cell lines.

The TRAIL-apoptotic signalling pathway is subjected to several levels of inhibitory regulation. It was shown that TRAIL-resistance in prostate cancer cells can be associated with: (i) a surface expression of decoy receptors (i.e. TRAIL-R3/DcR1, TRAIL-R4/DcR2 and OPG); (ii) the elevated expression of c-FLIP, a dominant negative form of caspase-8 that lacks the caspase catalytic site (Zhang et al, 2004); (iii) increased level of Bcl-2 antiapoptotic members (Sonneman et al, 2004).

Figure 3. Cell death apoptotic signalling and mechanisms of resistance to TRAIL-induced apoptosis. The cell death signalling apoptotic pathway induced by TRAIL-R1/-R2 is outlined. This cell death pathway may be inhibited at various levels: at receptor level by the decoy TRAIL receptors that act by competing with TRAIL-R1 and TRAIL-R2 for TRAIL binding; at post-receptor level, at the step of DISC formation by c-FLIP that acts by preventing the recruitment of caspase-8; at mitochondrial level by Bcl-2 and Bcl-X_L that act by suppressing the Bax/Bak-mediated release of cytochrome-C and SMAC/DIABLO from mitochondria; at the level of caspase-3 and -9 activity by IAPs attenuating the activity of these caspases (the inhibitory activity of IAPs is counteracted by SMAC/DIABLO). Many of these inhibitory mechanisms are activated in cancer cells.

Figure 4. Schematic representation of the structure of FADD and FLIP proteins. Left, top: the structure of FADD protein, with two boxes indicating one DED domain and one DD domain, is shown. The numbers indicate the amino acid residue. Within the DED domain, nuclear export sequence (NES) and nuclear localization sequence (NLS) have been identified: they determine the nuclear localization of FADD either in the nucleus or in the cytoplasm. In the COOH terminal site two serine residues (Ser 191 and Ser 194), essential for FADD function, are indicated. Right, top: the structure of the three c-FLIP isoforms, FLIP_L, FLIP_S and FLIP_R with their structural domains is shown. Left, bottom: signalling along the extrinsic pathway based on FADD and caspase-8 activation with consequent induction of the effector apoptotic machinery. Right, bottom: block of the apoptotic signalling along the extrinsic pathway when c-FLIP interacts with FADD, hampering the subsequent caspase-8 recruitment and activation.

C. Bladder cancer

Bladder tumor cells show a spread resistance to TRAIL-mediated apoptosis.

This conclusion is based on studies carried out on bladder cancer cell lines (Mizutani et al, 2001; Jonsson et al, 2003; Papageorgiou et al, 2004). Some bladder cancer cell lines displayed a significant sensitivity to TRAIL-induced apoptosis (Steele et al, 2006). The induction of apoptosis in these cells is dependent on both TRAIL-R1 or TRAIL-R2-induced signalling (Steele et al, 2006). The level of TRAIL sensitivity of bladder cancer cells did not depend on the level of TRAIL-R1 or TRAIL-R2 expression. Normal urothelial cells exhibited a moderate sensitivity to TRAIL-mediated apoptosis (Steele et al, 2006).

The resistance to TRAIL-induced apoptosis in bladder cancer seems to be mediated by a high c-FLIP expression. In fact the ratio between the c-FLIP and caspase-8 was directly correlated to resistance to anti-CD95 or TRAIL-mediated apoptosis. Since overexpression of c-FLIP might shift the responsiveness towards the resistant status, c-FLIP could be a prognostic marker for death receptor-sensitivity in immune therapy (Jonsson et al, 2003). Interestingly, TRAIL resistance in some bladder cancer cells was related to a rapid receptor downregulation (Steele et al, 2006).

Histone deacetylase inhibitors clearly enhanced the sensitivity to TRAIL in bladder cancer cells resistant to this death ligand, via a mechanism involving both TRAIL-R2 upmodulation and loss of mitochondrial membrane potential (Earel et al, 2006).

D Breast cancer

The pro-apoptotic activity of TRAIL on breast cancer has been evaluated only in tumor cell lines and not in primary tumor cells. The majority of breast cancer cell lines are resistant or semisensitive (T47D, MCF-7, MDA-MB-453, MDA-MB-468, MDA-MB-157, SKBr-3, ZR75); only one nontransformed (MCF10A) and one breast cancer cell line (MDA-MB-231) are significantly sensitive to TRAIL-induced apoptosis (Keane et al, 1999; Singh et al, 2003). No difference in sensitivity was found between normal and malignant (resistant) cell lines. A large part of breast cancer cell lines express TRAIL-R1 and TRAIL-R2 and at least one of the two decoy receptors (Singh et al, 2003).

Recently, TRAIL-R1 and TRAIL-R2 expression was explored by immunohistochemistry and quantitated by automated image quantitative analysis, providing evidence about an heterogeneous expression of these two receptors on breast cancer cells (McCarthy et al, 2005). TRAIL-R1 expression was not associated with survival, while high TRAIL-R2 was clearly associated with decreased survival (McCarthy et al, 2005). To explain the association between high TRAIL-R2 expression and poor survival it was suggested that high TRAIL-R2 expression may be a marker of tumor cells that have increased activation of NF-kB (Biswas et al, 2004).

Several studies were performed on a panel of cell lines (MCF-7, MDA-MB-453, MDA-MB-231, MDA-MB-468, MDA-MB-361, T-47D, CAMA-1, AU565) to explore the mechanisms which confer TRAIL-resistance to breast cancer. One of the putative mechanisms of TRAIL-resistance is related to the expression of decoy receptors, in particular TRAIL-R4 (Sanlioglu et al, 2005) and OPG (Neville-Webbe et al, 2004). OPG produced by bone marrow stromal cells protects breast cancer cells from TRAIL-induced apoptosis (Neville-Webbe et al, 2004). It was shown that expression at high level of Bcl-2 and Survivin and XIAP, two members of the IAP protein family, can render breast cancer cells resistant to TRAIL-induced apoptosis (Ruiz de Almod½var et al, 2001; Yang et al, 2003). In line with this observation, Smac-mimic compounds act to induce apoptosis alone, as well as sensitize breast cancer cells to TRAIL or etoposide-induced apoptosis via caspase-3 activation (Backbrader et al, 2005). Recent studies show that TRAIL-resistance is correlated with the action of the Small Heat Shock Protein aB-crystallin, a caspase-3 inhibitor (Kamradt et al, 2005), and by the high expression of FLIP (Hyer et al, 2005).

E. Gastric cancer

Few studies were performed on primary gastric cancer cells and no data are available about TRAIL-sensitivity of primary cells. The majority of these works had shown that TRAIL and TRAIL receptors are highly coexpressed in primary and metastatic gastric carcinoma cells (Sheikh et al, 1999; Koyama et al, 2002). Similarly, the large majority of esophageal adenocarcinomas express functional TRAIL-R1 and TRAIL-R2 (Younes et al, 2006).

TRAIL cytotoxicity was examined in a set of tumor cell lines: SNU-668 and MKN28, highly sensitive to TRAIL-induced cell death; SNU-601, SNU-719, SNU-1, SNU-5, moderately sensitive and SNU-216, MKN45, AGS and SGC-7901 almost completely resistant (Nam et al, 2003; Yang et al, 2004).

All gastric cancer cell lines expressed DR4, DR5 and DcR2 indicating that TRAIL-sensitivity is regulated at the intracellular level: in fact, the resistance to TRAIL can be associated with the Akt activity that upregulates c-FLIP_s and by the antiapoptotic effect of NFkB, Survivin and Bcl-2 molecules (Nam et al, 2003; Yang et al, 2004). One additional mechanism of TRAIL resistance could be represented by OPG, whose elevated expression is associated with poor outcome in gastric carcinoma (Ito et al, 2003).

The ensemble of these observations suggest that targeting of TRAIL-R1 and TRAIL-R2 by agonistic monoclonal antibodies may be of value in the treatment of the highly chemoresistant gastro-esophageal carcinomas.

F. Pancreas cancer

The evaluation of TRAIL’s effects has been carried out for the majority in pancreatic cancer cell lines; only one work performed on patient pancreatic adenocarcinomas grown in SCID mice model show heterogeneity of tumor response to TRAIL (Hylander et al, 2005).

Contrasting data are available in pancreatic cancer cell lines about TRAIL-sensitivity. Recent studies performed on a large panel of pancreatic cancer cell lines showed no apoptosis upon stimulation by TRAIL (Matsuzaki et al, 2001; Ozawa et al, 2001; Bai et al, 2005).

An high expression of DR4 and DR5 was found in TRAIL-sensitive pancreatic cancer cell lines (Ozawa et al, 2001), while an overexpression of decoy receptors DcR1 and DcR2 was detected specially in resistant cells (Bai et al, 2005).

The mechanisms through which pancreas cancer cells evade TRAIL-mediated apoptosis is related to the action of antiapoptotic molecules like NFkB, Bcl-X_L and c-FLIP (Thomas et al, 2002; Bai et al, 2005).

G. Colon cancer

Several studies have investigated the effects of TRAIL mainly on colon cancer cell lines and rarely in primary cells.

Using patient colon carcinoma-SCID mouse model has been possible to show a high heterogeneity in TRAIL sensitivity that, probably, reflects inherent patient-to-patient differences (Naka et al, 2002).

Recent studies performed in colon cancer cell lines have demonstrated a broad range in sensitivity of cell lines to TRAIL, in particular an increased TRAIL-sensitivity was found in colon carcinoma more than colon adenoma cells. (Hargue et al, 2005; Vasilevskaya & O’Dwyer, 2005).

The mechanisms responsible for TRAIL resistance of colon cancer cells have been investigated in detail (reviewed by Van Geelen et al, 2005). Loss of function of TRAIL receptor genes by mutations or epigenetic changes is not frequently observed in colon cancer (Arai et al, 1998). Alterations of caspase-8 either due to inactivating mutations (Kim et al, 2003) or to decreased expression due to increased degradation (McDonald and El Deiry, 2004) are involved in TRAIL resistance of colon cancer cells.

The majority of colon carcinoma and adenoma cell lines express TRAIL-R1 and TRAIL-R2, but the expression pattern of TRAIL receptors did not correlate with TRAIL sensitivity of these cell lines (Hargue et al, 2005).

The expression of antiapoptotic molecules (Bcl-2, XIAP, c-FLIP) can be correlated with TRAIL-resistance (Sinicrope et al, 2004; Vasilevskaya & O’Dwyer, 2005).

Hepatocarcinoma cells could be sensitized to TRAIL with proteasome inhibitors (MG132 or PS-341), while this treatment did not modify TRAIL sensitivity of normal hepatocytes (Ganten et al, 2005).

H. Liver cancer

Few data on primary cells reveal that hepatocarcinoma cancer cells (HCC) show lower levels of TRAIL-R1 and -R2 expressions compared to the non-malignant liver tissues.

Moreover, Fas expression was found to be lower in the HCC tissues than in the normal ones (Shin et al, 2002). The expression of survivin was investigated in 38 cases of HCC tissues and survivin protein was detected in 23 (60,5%) of 38 HCCs. The expression of survivin seems to be related to the metastasis of HCC. Survivin might be considered then a very useful marker for evaluation of metastatic potential and prognosis of HCC (Zhu et al, 2005).

Studies performed on cell lines show a broad spectrum of responses. HepG2 cells undergo TRAIL-induce apoptosis in a dose-dependent manner. In contrast, the treatment of HepG2TR (TRAIL resistant variant of HepG2) and Hep3b cells with TRAIL did not result in a significant level of apoptosis induction (Ganten et al, 2004).

7 of 10 HCC cell lines showed resistance to TRAIL-induced apoptosis and 5 of 7 TRAIL resistant cell lines become sensitive to TRAIL by co-treatment with cycloheximide and cisplatin (Shin et al, 2002).

A recent study showed that human hepatocarcinoma tissues usually express membrane-bound TRAIL that enables these tumor cells to evade immune surveillance by inducing apoptosis of activated lymphocytes (Shiraki et al, 2005). TRAIL expression in hepatocarcinoma cells is enhanced by some chemotherapeutic agents, such as doxorubicin (Shiraki et al, 2005).

Since upregulation of TRAILRs or downregulation of DcR1 and DcR2 (decoy receptors) could be a way to overcome resistance studies performed with TRAIL and 5FU have been performed. TRAIL-R1 upregulation potentially contributes to increase sensitivity of HepG2-TR cells to TRAIL-induced apoptosis upon 5 FU treatment.

I. Kidney cancer

Treatments of freshly-derived renal cell carcinoma (RCC) cells with TRAIL as well as treatments of Caki-1 cell line with TRAIL showed resistance. Only combination with 5-FU resulted in a synergistic cytotoxic effect (Mizutani et al, 2002).

RCC cell lines displayed a great heterogeneity in their sensitivity to TRAIL-mediated apoptosis (Brooks and Sayers, 2005, Griffith et al, 2002, Asakuma et al, 2003). Their resistance to TRAIL was related either to c-FLIP expression (Brooks and Sayers, 2005) or to the low level of TRAIL-R1/ TRAIL-R2 expression in combination with high survivin expression (Griffith et al, 2002) or to the constitutive Akt phosphorylation (Asakuma et al, 2003) or the constitutive NF-kB activation (Oya et al, 2001).

J. Lung cancer

Several in vitro studies have shown that some non-small cell lung cancer (NSCLC) cell lines are sensitive to apoptosis induction by recombinant TRAIL (Sun et al, 2000; Kagawa et al, 2001). No data are available about the sensitivity of primary NSCLC to TRAIL-mediated apoptosis.

Analysis of the expression of TRAIL and TRAIL-Rs in NSCLC patients showed that TRAIL-R1, TRAIL-R2 and TRAIL were expressed in 99%, 82% and 91% of the tumors, respectively (Spierings et al, 2003).

The level of TRAIL-R2 positivity was associated with increased risk of death (Spierings et al, 2003).

Mutations of the ectodomain of TRAIL-R1 (Fisher et al, 2001) and of the death domain of TRAIL-R2 (Lee et al, 1999) have been observed in 35% and 11% of NSCLC patients.

Some NSCLC cell lines are resistant to TRAIL through mechanisms related to Bcl-2 overexpression in these cells (Jiang et al, 1995; Ziegler et al, 1997), to mutations at the level of TRAIL-R1 and/or TRAIL-R2 (Fischer et al, 2001; Spierings et al, 2003), to decreased caspase-8 expression, to low DAP-kinase activity to promoter hypermethylation (Tang et al, 2004) due to promoter hypermethylation (Hopkins-Donaldson et al, 2003), and to spontaneous Akt activation (Kandasamy and Srivastava, 2002).

K. Ovarian cancer

TRAIL represents a potentially interesting molecule for ovarian cancer therapy. In several studies, the action of TRAIL was evaluated in primary tumor cells and in ovarian cancer cell lines. Only few studies were carried out on primary tumor cells, showing a variable sensibility to TRAIL-induced cytotoxicity. For example an analysis done on four primary samples showed that two of them were highly resistant to TRAIL-induced cell death, whereas the other two were sensitive (Lane et al, 2004).

There were no more data that could show the effects of TRAIL on primary tumor cells.

Anyway, in another work the expression of TRAIL in ovarian cancer cells was analyzed: ovarian cancer cells exhibit 10-fold higher mean TRAIL expression than normal ovarian epithelial samples. This high TRAIL expression measured by RT-PCR was associated with prolonged survival (Lancaster et al, 2003).

Similar studies on TRAIL resistance were performed on ovarian cancer cell lines. Upon treatment with TRAIL, cell lines were distinguished in TRAIL-sensitive (OVCAR3, CAOV3, MZ-26, ES-2, IGROV-1) and TRAIL-resistant cell lines (SKOV3, UCI-101, OV-4, A2780, A2780ADR, MZ-15) (Vignati et al, 2002; Lane et al, 2004; Tomek et al, 2004,)

The expression of c-FLIP represents one of the most important mechanisms of resistance to TRAIL in ovarian cancer cells. TRAIL-resistant ovarian cancer cell lines express elevated levels of c-FLIP (Tomek et al, 2004). One study has demonstrated that cisplatin-induced apoptosis in ovarian cancer cells is associated with decreased FLIP protein content and with activation of caspase-8 and caspase-3. Moreover, these results have been observed in sensitive but not in resistant cell lines. In fact, the hypothesis was that the overexpression of FLIP induces resistance of ovarian cancer cells to cisplatin and that the downregulation of FLIP sensitizes chemoresistant ovarian cancer cells to cisplatin. These findings suggest that FLIP may play an important role in regulating the sensitivity of ovarian cancer cells to cisplatin treatment (Abedini et al, 2004). Another study suggest that the inhibitory protein cFLIP is involved also in resistance to CD95-mediated apoptosis in ovarian carcinoma cells with wild type p53 (Mezzanzanica et al, 2004).

It is unclear whether the expression of TRAIL decoy receptor could be involved in the genesis of TRAIL resistance in ovarian cancer cells.

L. Melanoma

Melanoma is a cancer characterized by a high metastatic potential as well as by a great apoptosis resistance, both highly contributing to immune escape mechanisms and resistance to chemotherapy.

Melanoma cell lines displayed a differential sensitivity to TRAIL-mediated apoptosis (Griffith et al, 1998; Zhang et al, 1999). The sensitivity of these cell lines to TRAIL was correlated with the level of TRAIL-R1 and, particularly, of TRAIL-R2, while the expression of TRAIL decoy receptors does not correlate with TRAIL resistance (Griffith et al, 1998; Zhang et al, 1999). However, fresh isolates of melanoma are usually resistant to TRAIL-mediated apoptosis, and this phenomenon is associated with low TRAIL-R2 expression (Nguyen et al, 2001).

The mechanisms of TRAIL resistance by melanoma cells are complex and involve the activation of several anti-apoptotic mechanisms, mainly represented by c-FLIP, survivin and Bcl-2 overexpression (reviewed in Hersey and Zhang, 2001). Downregulation of the expression of these anti-apoptotic proteins considerably potentiates the sensitivity of melanoma cells to TRAIL (Chawla-Sarkar et al, 2004).

However, a recent study re-evaluated TRAIL-R expression in melanoma sections showing TRAIL-R1 and TRAIL-R2 in the majority of cases (Kurbanov et al, 2005). Furtehrmore, it was shown that both in melanoma cell lines and primary tumors both TRAIL-R1 and TRAIL-R2 are very frequently expressed and TRAIL-R1 signaling was able to induce apoptois of melanoma cells (Kurbanov et al, 2005). These observations suggest a therapeutic potential of TRAIL-R1 targeting in melanoma.

M. Haematological tumors

1. CML (Chronic Myeloid Leukemia)

Given the IFN-a inducibile expression of TRAIL on human T cells, TRAIL may participate in the process of antileukemic effects against Ph1-positive leukaemias. The apoptotic effect of TRAIL was first investigated in Ph1 positive leukaemia cell lines. Effectively, TRAIL induced apoptosis in Ph1-positive leukaemia cells and it was mostly correlated with the cell-surface expression levels of DR4 and DR5. Notably, TRAIL was also effective against leukaemia cells that were refractary to the BCR-ABL kinase inhibitor imatinib mesylate (STI571) (Kanako Unok et al, 2003).

Regarding TRAIL sensitivity in leukaemic blasts from CML-BC, only one case was evaluated and showed resistance. FADD and caspase-8, component of DISC, as well as c-FLIP a negative regulator of caspase-8, are expressed ubiquitously in Ph1-positive leukaemia cell lines irrespectively of their differential sensitivities to TRAIL.

2. ALL (Acute Lymphoblastic Leukemia)

The activity of TRAIL was investigated in 29 primary precursor B-cell acute lymphoblastic leukaemia (ALL) samples. TRAIL was found to have a modest activity as it killed a maximum of 29% of ALL cells within 18h compared with a high rate of killing (75%) of Jurkat cells (T-cell lymphoblastic origin). This differential effect may indicate that malignant precursor T-cells (Jurkat) may be more sensitive to TRAIL than B-cells. However preliminary data in three primary precursor T-lymphoblastic leukaemia cases do not support this hypotesis as all T-cell ALL cases were also resistant to TRAIL. TRAIL insensitivity of ALL is not related to the overexpression of the decoy R3 or R4 receptors, nor to the overexpression of the antiapoptotic protein c-FLIP. It is possible that the lack of sensitivity is simply related to the low levels of functional R1 and R2 receptors observed in ALL cells (Clodi et al, 2000). Primary malignant cells of haematological origin frequently express TRAIL transcripts and proteins which induce cell death of target cell lines that are known to be sensitive to TRAIL (Jurkat and HL60). This killing effect is dose-dependent and it is TRAIL-specific because anti-TRAIL antibody reversed this effect. Based on these observation, it was suggested that the functional expression of TRAIL by tumor cells could protect them from cytotoxic T cells (Zhao et al, 1999).

3. B-CLL (B-Chronic Lymphocytic Leukemia)

Primary B-CLL cells from patients are generally not sensitive to either TRAIL or anti-CD95, whereas three tumor cell lines, Jurkat, SKW, MC116 are sensitive. Even at high TRAIL concentration (200 ng) B-CLL are resistant to apoptosis. Lower levels of TRAIL death receceptors expression (TRAIL-R1, TRAIL-R2) are generally observed compared to the cell lines, with no detectable expression of TRAIL-R3 or TRAIL-R4. Thus, in agreement with other studies the resistance of the B-CLL cells to TRAIL-induced apoptosis is not probably due to increased cell surfarce expression of ‘decoy’ receptors (Griffith et al, 1998; Leverkus et al, 2000). The resistance of B-CLL cells to TRAIL may be due partly to low surface expression of the death receptors resulting in low levels of DISC formation and also to the high ratio of c-FLIP L (long form) to caspase-8 within the DISC, which would prevent further activation of caspase-8 (MacFarlane et al, 2002). More recently it has been shown that low concentrations of histone deacetylase inhibitors (HDACi) sensitize CLL cells to TRAIL-induced apoptosis by facilitating increased formation of the TRAIL DISC. Peripheral blood lymphocytes from 28 patients with CLL at different clinical stages were exposed to different forms of TRAIL plus HDACi. No increase in the spontaneous level of apoptosis was observed in cells from these different patients exposed to TRAIL alone. Moreover, pretretment with depsipeptide (VPA 2mM) at low concentrations sensitized CLL cells to TRAIL-induced apoptosis by facilitating increased formation of the TRAIL DISC via TRAIL-R1, although most studies suggested that TRAIL-R2 is the primary TRAIL receptor leading to cell death (MacFarlan et al, 2005).

It is also known that B-CLL express at higher level TRAIL ligand respect to unfractioned lymphocytes, and the addiction in culture of recombinant TRAIL increased leukaemic cell survival. Thus an aberrant expression of TRAIL might contribute to the phatogenesis of B-CLL by promoting the survival in a subset of B-CLL cells (Secchiero et al, 2005).

Primary lymphoma cells are scarcely moderately sensitive to the pro-apoptotic effects elicited either by recombinant TRAIL or by agonistic monoclonal antibodies to TRAIL-R1 (HGS-ETR-1) and TRAIL-R2 (HGS-ETR2) (Georgakis et al 2005).

A defective TRAIL-R1 and TRAIL-R2 expression is frequently observed also in non-Hodgkin lymphomas, due to 8p21.3 chromosomal deletions (Rubio-Mascardo et al, 2005). The chromosomal deletions result in deletion of one or the two alleles encoding TRAIL-R1 and TRAIL-R2, determining a reduced expression of these receptors on lymphoma cells and a reduced sensitivity to TRAIL of these tumor cells (Rubio-Moscardo et al, 2005).

Burkitt’s lymphomas are resistant to TRAIL-mediated apoptosis due to a high c-FLIP expression (Djerbi et al, 1999). The high c-FLIP expression was found highly related to a poor prognosis, characterized by a chemoresistant disease, resulting in a high death rate within the first year of diagnosis (Valnet-Rabier et al, 2005). Interestingly, all c-FLIP positive cases exhibit the presence of an active nuclear factor NF-kB.

4. AML (Acute Myeloid Leukaemia)

Studies based on the analysis of primary cells derived from a large set of AML patients pertaining to different FAB subtypes, provided evidence that they are invariably resistant to TRAIL-mediated apoptosis (Riccioni et al, 2005, Jones et al, 2003). Similarly, pediatric acute leukaemias are frequently resistant or scarcely sensitive to TRAIL (Baader et al, 2005).

The mechanisms of TRAIL-resistance of AML blasts have been only in part explored. In this context the frequent expression of TRAIL-R3, and TRAIL-R4 on the surface of AML blasts has lead to suggest that these decoy receptors may be responsible for TRAIL resistance of these cells (Riccioni et al, 2005). Furthermore, a recent study showed that a high proportion of AML blasts exhibit low or absent levels of FADD (Tourneur et al, 2004).

Some reports have suggested a sensitivity of acute promyelocytic cells to TRAIL induced by retinoic acid treatment (Altucci et al, 2001). These findings, however, were not confirmed by other authors (Riccioni et al, 2001). The resistance of AML blasts to TRAIL may be bypassed by co-treatment with histone-deacetylase inhibitors (Insinga et al, 2005).

Paradoxically, leukaemia cells resistant to TRAIL may be stimulated to proliferate by this death ligand, via a mechanism involving NF-kB activation (Baader et al, 2005).

5. MM (Multiple Myeloma)

Primary myeloma cells display a low sensitivity to pro-apoptotic effects of TRAIL (Lincz et al, 2001). In parallel studies on myeloma cell lines showed a variable sensitivity to TRAIL (Lincz et al, 2001). TRAIL resistance of myeloma multiple cells was related to high expression in these cells of the anti-apoptotic proteins c-FLIP and c- IAP-2 (Mitsiades et al, 2002). Interestingly, interferon-alpha (IFN-a) sensitize myeloma cells to both TRAIL (Crowder et al, 2005) and Fas L (Dimberg et al, 2005) via induction of the promyelocytic gene PML that plays an important role in the control of apoptosis.

III. Therapy based on TRAIL and monoclonal antibodies anti-TRAIL receptors

The selective cytotoxic effect of TRAIL against cancer cells, sparing normal cells, stimulated many studies focused to evaluate the potential use of this death receptor ligand, as well as of agonistic anti TRAIL-R1 antibodies as anticancer drugs.

Previous studies on two other death receptor ligands, TNF-a and Fas Ligand, were considerably hampered either by the pro-inflammatory properties (TNF-a or by the induction of fulminant hepatic necrosis (FasL) after in vivo administration. Both these ligands are therefore considered unsuitable as potential anti-cancer drugs, at least when administered by a systemic way.

In contrast, the studies on TRAIL and agonistic anti-TRAIL-R monoclonal antibodies indicate their potential use as anti-cancer drugs.

A part of these studies were focused to evaluate the anti-tumor properties and the toxicity profile of recombinant preparations of human TRAIL.

Although many concerns have been raised about a possible toxicity of TRAIL, carefull studies have shown that some toxicities against normal cells attributed to this death receptor ligand are in reality related to aberrant structural and biochemical properties of the recombinant variants of the protein.

Four different recombinant versions of the human have been generated and molecularly characterized.

A first molecular form contains TRAIL amino acids 114-281 fused to an amino-terminal polyhistidine tag (Pitti et al, 1996). A second variant contains TRAIL amino acids 95-281 fused to an amino-terminal leucine zipper, promoting the trimerization of the ligand (Walczak et al, 1999). A third version contains TRAIL amino acids 95-281 fused to an amino-terminal Flag: the crosslinking of this tagged protein with anti-flag antibodies enhanced anti-tumor activity (Bodmer et al, 2000). A fourth version of the molecule is composed by TRAIL amino acids 114-281 without any additional exogenous sequences (Ashkenazi 2002). The preparation of this lost form of recombinant human TRAIL is improved by addition of zinc and reducing agents to the cell culture medium and extraction buffers and maintaining neutral pH in the buffers (Kelley 2001, Lawrence et al, 2001).

Soluble untagged TRAIL displayed in vivo no toxicity and, particularly, no hepatic toxicity (Ashkenazi et al, 1999), while the His-tagged TRAIL preparation displayed a marked hepatotoxicity (Jo et al, 2000). The membrane bound form of TRAIL induced hepatic toxicity in mice (Ichikawa et al, 2001). The analysis of the toxicity of the different TRAIL recombinants versions confirmed the observations made on hepatocytes. Thus, His-tagged TRAIL 114-281 as well as L2-TRAIL, induced apoptosis on normal keratinocytic cells (Leverkus et al, 2000; Quin et al, 2001), the non-tagged TRAIL showed non apoptotic effect on these cells (Quin et al, 2001). Similar observations have been raised for normal epithelial prostate cells (Nesterov et al, 2002).

The problem of a potential hepatotoxicity of TRAIL for normal hepatocytes was recently reinvestigated using in vitro studies on human hepatocytes in culture or in vivo in chimeric mice harbouring human hepatocytes, providing definitive evidence that non-tagged soluble TRAIL is not toxic for normal human hepatocytes (Hao et al, 2004). Normal human hepatocytes do not express TRAIL and possess very low levels of TRAIL-R2 (Hao et al, 2004).

The analysis of some structural properties of these different versions of human TRAIL may provide an explanation for their differential in vivo toxicity. In fact, the tagged version of recombinant TRAIL are not optimized for zinc content and due to this limitation they have lower solubility levels and spontaneously aggregate: this TRAIL aggregation could induce TRAIL-R multimerization inducing strong receptor signalling bypassing the high threshold for apoptosis activation existing in normal cells (Kelley and Ashkenazi 2004). In contrast, the non-tagged recombinant human TRAIL is highly stable trimer inducing only the formation of trimeric TRAIL-Rs in normal cells, not sufficient to induce apoptosis of these cells (Kelley and Ashkenazi 2004).

In vivo studies in animal models have provided that non-tagged TRAIL/Apo-2L exhibits potent anti-tumor activity and induces little toxic effects in immunodeficient mice xenograft models implanted with several human tumor cell lines (Ashkenazi et al, 2004). However, the in vivo half-life of the recombinant non-tagged TRAIL was very short (< 4minutes), thus suggesting that agonistic anti-TRAIL-R1 or -TRAIL-R2 could have a better pharmacologic impact (Kelley 2001). Initial studies have shown that TRA-8 an agonistic anti-human TRAIL-R2 monoclonal antibody exert in vivo a potent anti-tumor activity against a wide spectrum of human tumors, without affecting the viability of normal cells, and particularly of hepatocytes (Ichikawa et al, 2001).

Agonistic TRAIL-R1 or TRAIL-R2 antibodies may have enhanced therapeutic potential due to a prolonged half-life in vivo compared to TRAIL ligand (Kelley 2001). Another additional advantage of agonistic TRAIL-R1 or TRAIL-R2 antibodies is that they, at variance with TRAIL ligand, do not bind to TRAIL decoy receptors, TRAIL-R3 and TRAIL-R4 often present on the membrane of tumor cells.

Two anti-TRAIL-Rs have been developed for clinical use. One of these two antibodies is called HGS-ETR1 and is a fully human agonistic antibody with high affinity and specificity for TRAIL-R1 (Pulac et al, 2005). This antibody induce cell killing of tumor cell lines through activation of both extrinsic and intrinsic apoptotic pathway. Importantly, HGS-ETR1 was shown to have in vivo a long half-life (7-9 days in mouse) and suppressed the growth of several tumors in xenografts models in athymic mice (Pulac et al, 2005). Finally, EGS-ETR1 potentiated the anti-tumor efficacy of several chemotherapeutic drugs (Pulac et al, 2005). These observations have clearly indicated that HGS-ETR1 has significant potential as a cancer therapeutic agent. The HGS-ETR1 antibody (Human Genome Sciences) was evaluated in Phase I/II clinical trial in patients with advanced solid or haematological tumors, revealing little toxicity (Pulac et al, 2005).

A second fully human antibody to TRAIL-R2, KTMTR2, was recently reported (Motoki et al, 2005). This antibody through its binding to the TRAIL R2 induces the death of tumor cells: no crosslinking is required for induction of cell apoptosis (Motoki et al, 2005).

Phase I and phase II trials in patients with advanced solid tumors, non-small-cell lung cancer, colon cancer and NHL are in progress. The antibodies are being studied as single agents or in combination with cytotoxic chemotherapy (De Bono et al, 2004, Hotte et al, 2004, Cohen et al, 2004, Tolcher et al, 2004).

The use of fusion proteins composed by recombinant human TRAIL fused to a monoclonal antibody against a membrane antigen can be used to induce target antigen-restricted apoptosis. An example of this fusion protein is given by scFv CD7:sTRAIL, composed by the TRAIL genetically linked to an scFv antibody fragment specific for the T-cell surface antigen CD7: this fusion protein induces cytotoxicity of primary acute T lymphoblastic leukemia cells and potentiates the cytotoxic effect of the antitumor drug vincristine (Bremer et al, 2005). Additional examples of these fusion proteins are given by scFv425:sTRAIL (composed by the EGFR-blocking antibody fragment scFv425 genetically fused to soluble TRAIL) (Bremer et al, 2005) or by scFvEGP2:sTRAIL fusion protein (composed by the anti-pancarcinoma-associated antigen EGP2 genetically fused to soluble TRAIL) (Bremer et al, 2004).

IV. Strategies to overcome TRAIL resistance

As outlined in the previous chapters, very frequently primary cancer cells are resistant to TRAIL-mediated apoptosis. This observation had stimulated many studies focused to develop strategies to circumvent TRAIL resistance by cancer cells. The philosophy of these various strategies is based on the combination of TRAIL with another drug: the role of the other drug consists in sensitizing tumor cells to the apoptotic effects of TRAIL.

A. TRAIL and proteasome inhibitors

The 26S is a multicatalytic enzyme present in the cytoplasm and in the nucleus of virtually all eukaryotic cells, involved in the degradation of proteins targeted by ubiquitin conjugation. The proteasome plays a key role in the control of cell homeostasis in that it regulates the half-life of cellular proteins essential for the life of the cell, such as transcription factors, tumor suppressors, oncogenes and proteins involved in cell cycle control. These observations have suggested that the proteasome may represent an important therapeutic target in cancer.

Several proteasome inhibitors have been synthesized and one of them, Bortezomib (known also as VELCADE or PS-341), was approved by the Food and Drug Aministration for cancer treatment (Rajikumar et al, 2005). Bortezomib, a peptide boronate, selectively inhibits the chymotrypsic-like activity of the proteasome at nanomolar concentrations (Rajikumar et al, 2005). In vitro studies have shown that the effect of Bortezomib on cell lines mainly consists on cell cycle inhibition and induction of apoptosis (Rajikumar et al, 2005).

Treatment of tumor cells with Bortezomib results in multiple biological effects, including inhibition of cell cycling, inhibition of NF-kB acivity, changes in cell adherence and increased apoptosis. Among these various effects it was initially observed that Bortezomib treatment may considerably increase the sensitivity of myeloma cells to TRAIL (Mitsiades et al, 2001). In the absence of the proteasome inhibitor, myeloma cells are resistant to TRAIL-mediated apoptosis. Subsequent studies have confirmed these initial observations in other tumor cell types and have explored the potential molecular mechanisms responsible for this phenomenon. Thus, Bortezomib was found to sensitize mouse myeloid leukaemia C1498 cells to TRAIL-mediated apoptosis through a mechanism related to a decreased expression on the anti-apoptotic protein c-FLIP, without significant decrease of Bcl-2 anti-apoptotic members or of the various IAP members (Sayers et al, 2003). In contrast, other studies carried out on different tumor cells, like hepatocellular carcinoma cells, have shown that the proteasome inhibitor MG-132 clearly increased c-FLIP levels; in spite the c-FLIP increase, MG-132-pretreated cells became more sensitive to TRAIL-mediated apoptosis (Ganten et al, 2005).

Other studies have shown that Bortezomib (Johnson et al, 2003) or MG-132, another proteasome inhibitor, (He et al, 2004) induce a marked increase of TRAIL-R1 and TRAIL-R2 expression in prostate, colon and bladder cancer cell lines and, through this mechanism, sensitize these cells to the pro-apoptotic effects of TRAIL. Experiments on a battery of renal carcinoma cell lines have shown that Bortezomib may either increase TRAIL-R1 and/or TRAIL-R2 expression or decrease c-FLIP levels, but in all istances it sensitize these cells to the apoptogenic effects of TRAIL (Sayers and Murphy, 2005).

The increased TRAIL-R2 expression induced by proteasome inhibitors is related to a transcriptional mechanism dependent upon an enhanced promoter activity, mediated by the binding of the CHOP transcription factor (Yoshida et al, 2005).

Since TRAIL binding to its receptors induces NF-kB activation, that can induce several anti-apoptotic genes, it was suggested that proteasome inhibitors may sensitize the cells to TRAIL-mediated apoptosis by blocking NF-kB activity. However, several studies have shown that NF-kB blocking by proteasome inhibition is not essential for the sensitization of several tumor cell types to TRAIL (Sayers et al, 2003). In some tumor cells, NF-kB inhibition may represent an important event contributing to the sensitization to TRAIL (Luo et al, 2004).

Interestingly, the combined addition of proteasome inhibitors and TRAIL resulted in a cytotoxic effect in chemoresistant Bcl-2-overexpressing cells that are otherwise resistant to TRAIL or cytotoxic drugs or proteasome inhibitors alone (Nencioni et al, 2005). The combination of TRAIL and antiblastic drugs was unable to induce the apoptosis of these cells (Nencioni et al, 2005). These observations indicate that proteasome inhibitors plus TRAIL induce mitochondrial dysfunction irrespective of upregulated Bcl-2.

The molecular mechanisms through which proteasome inhibitors induce a decrease of c-FLIP levels and upmodulate TRAIL-R1 and TRAIL-R2 are largely unknown. In this context, it is important to note that several components of the apoptotic machinery, including some members of the Bcl-2 family, IAP family proteins, IkB and p53, are ubiquitinated (Zhang et al, 2004). The decrease in c-FLIP observed in cells treated with proteasome inhibitors is surprising in that c-FLIP was reported to be degraded by proteasome in some cells (Sayers et al, 2003). One explanation could be related to the effect of the proteasome inhibitor on cell cycle: cell cycle arrest in S-G2/M phase could result in c-FLIP decrease because during the normal cell cycle peak levels of this protein are observed in G1, with a marked decrease in G2/S phase. In contrast, the increase of c-FLIP levels induced by proteasome inhibitors observed in other tumor cells may be related to a reduced degradation of c-FLIP protein (Ganten et al, 2005).

Recent studies carried out in bladder and prostate cancer cells have shown that the cell cycle regulatory protein p21, whose levels are greatly enhanced by Bortezomib+TRAIL, plays a key role in the mechanism through which the proteasome inhibitor allows to bypass TRAIL resistance (Lashinger et al, 2005). The increased p21 levels are required for optimal caspase-8 processing (Lashinger et al, 2005).

B. Triterpenoids enhance the sensitivity of tumor cells to TRAIL

Derivatives of naturally occurring triterpenoids have pronounced antiproliferative and anticarcinogenic activities (Kim et al, 2002). Particularly, two compounds of this chemical family, 2-ciano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its imidazole derivative (CDDO-Im) exert a pronounced anti-tumor activity. In vitro studies have shown that these compounds are able to induce differentiation, to inhibit proliferation and to stimulate apoptosis of different types of cancer cells (Place et al, 2003).

The apoptogenic effects of these compounds were relatively weak. However, both CDDO and CDDO-Im, the latter one being more potent than the former one, were able to sensitize leukemic cells to the pro-apoptotic effects of TRAIL (Konopleva et al, 2002). This effect seems to be largely related to the induction of a reduced c-FLIP expression (Konopleva et al, 2002). This last effect seems to be due to the stimulation of a proteolytic pathway involved in c-FLIP degradation (Kim et al, 2002).

Studies carried out in breast cancer cell lines confirmed the findings obtained in leukemic lines showing that CDDO and CDDO-Im markedly enhanced the sensitivity of these cells to the pro-apoptotic effects of TRAIL, via a mechanism involving both a decrease of c-FLIP and an enhanced expression of both TRAIL-R1 and TRAIL-R2 (Hyer et al, 2005). These effects were also observed in vivo in a xenograft model based on the implantation of breast cancer cells to nude mice (Hyer et al, 2005). The addition of CDDO or CDDO-Im to lung cancer cell lines resulted only in a weak pro-apoptotic effect, greatly enhanced by the concomitant addition of TRAIL: the sensitization to TRAIL was related to a selective upmodulation of TRAIL-R2 expression, mediated by JNK, whose activity is clearly stimulated by triterpenoids (Zou et al, 2004).

In addition to triterpenoids, also some flavonoids and particularly luteolin, a compound found in many fruits, vegetables and medicinal plants, sensitizes TRAIL-induced apoptosis in various human cancer cells, through a mechanism involving XIAP downmodulation mediated via protein kinase C inhibition (Shi et al, 2005).

C. Histone deacetylase inhibitors act as modulators of the sensitivity of tumor cells to TRAIL

Histone acetyltransferases and histone deacetylases (HDAC) catalyze the acetylation and deacetylation of lysine residues in the core nucleosomal histone tails, respectively, thus regulating the affinity of the nonhistone proteins transcriptional complexes with DNA and then controls the rate of transcription (Dockmanovic and Marks, 2005). Recently, HDACs have been shown to be involved in leukemogenesis for their capacity to complex with a variety of oncoproteins found in leukaemia and, through this mechanism, to aberrantly suppress the expression of genes required for cell differentiation and growth control. Furthermore, altered histone acetyltransferase or HDAC has been observed in many tumors in addition to hematologic malignancies. Several inhibitors of HDAC, which inhibit tumor growth both in vivo and in vitro have entered clinical trials. HDAC inhibitors exert their anti-tumor effects due to their ability to induce growth arrest, differentiation and apoptosis. Initial studies describing the induction of apoptosis by HDAC inhibitors have suggested that they induce apoptosis via the intrinsic pathway (Insinga et al, 2005). However, many recent observations, carried out in different tumor models, including primary tumor cells, clearly indicate that HDAC inhibitors potentiate death-receptor induced apoptosis. These observations have been performed in leukemic cell lines and primary leukemic cells (McFarlane et al, 2005; Nebbioso et al, 2005), in chronic B lymphocytic leukaemia cells (Inoue et al, 2004; McFarlane et al, 2005), melanoma cell lines (Facchetti et al, 2004). The molecular mechanisms underlying this synergism between TRAIL and HDAC inhibitors is mainly related to a decreased expression of anti-apoptotic proteins (c-FLIP, XIAP, survivin) and an increased expression of TRAIL-R1 and/or TRAIL-R2 (Rosato et al, 2003; Guo et al, 2004; Inoue et al, 2004; Nakata et al, 2004; McFarlane et al, 2005). Particularly, TRAIL-resistant glioma cell lines became sensitive to TRAIL-mediated apoptosis when this cytokine is added together with sodium butyrate, a HDAC inhibitor, that induces a marked downregulation of surviving and XIAP (Kim HS et al, 2005).

D. Ionizing radiations, UV radiations and TRAIL

Part of the signalling cascade of death receptor signalling is also utilized during irradiation-induced apoptosis. Ionizing radiations were shown to trigger death receptor-independent processing and activation of pro-caspase-8 that occurs through a caspase-3 dependent mechanism.

Several studies indicate that ionizing radiations and TRAIL may cooperate in inducing the killing of tumor cells. Initial studies have shown that the combined administration of ionizing radiations and TRAIL was able to induce the death of lymphoma (Belka et al, 2001), breast cancer (Chinnayan et al, 2000) and renal cell carcinoma (Ramp et al, 2003). In animal models of non-small cell lung cancer the combination of TRAIL and radiotherapy significantly increased apoptosis in vivo, inhibited tumor growth, and significantly prolonged survival in mice bearing human tumors (Zhang et al, 2005). The analysis of the possible mechanisms underlying this synergism showed that: (i) the most active schedule in eliciting tumor cell killing consisted in the sequential administration first of ionizing radiations and then of TRAIL (Marini et al, 2005); (ii) pre-irradiation did not sensitize normal tissues to TRAIL; (iii) in the majority of tumor cell lines, ionizing radiations induced a clear up-modulation of TRAIL-R2 expression; (iv) the sequential treatment with ionizing radiations and then with TRAIL inhibits tumor growth in vivo and induces apoptosis of breast cancer xenografts in nude mice (Shankar et al, 2004). An intact Bax activity is strictly required to mediate synergism between ionizing radiations and TRAIL (Wendt et al, 2005).

The ensemble of these observations clearly indicate that in several neoplasias the sequential treatment with irradiation followed by TRAIL provides an approach to enhance therapeutic potential of TRAIL. Interestingly, a possible role for TRAIL and its receptors in the mechanism of cytotoxicity mediated by ionizing radiations is suggested by the phenotypic analysis of TRAIL-R2 knockout mice (Finnberg et al, 2005). These animals, in fact, exhibit reduced levels of apoptosis when exposed to ionizing radiations, compared to the corresponding tissues of normal animals (Finnberg et al, 2005).

Studies on TRAIL-resistant melanoma cells showed that these tumor cells acquire the sensitivity to this death ligand after exposure to ultraviolet B radiations (UVB), through a molecular mechanism mainly involving c-FLIP downregulation (Zeise et al, 2004).

E. Interferon-g and other agents enhancing caspase-8 expression

As mentioned in Section II, several types of cancers, including some brain tumors, colon cancer, retinoblastoma and Ewing sarcoma, display a reduced sensitivity to TRAIL due to a decreased or absent expression of caspase-8. Given this defect in the apoptotic response, attempts have been made in these tumors to reconstitute a sensitivity to the pro-apoptotic effects of TRAIL by pre-treatment or co-treatment of tumor cells with agents that enhance caspase-8 expression, such as interferon-g (IFN-g).

The addition of IFN-g together with TRAIL is based on previous studies in mice showing that TRAIL plays a key role in IFN-g-dependent tumor surveillance (Takeda et al, 2002). Furthermore, IFN-g is known to enhance caspase-8 (Fulda et al, 2002), to facilitate DISC-mediated caspase-8 processing (Siegmund et al, 2005) and activate Sta-1 (Fulda et al, 2002) and IRF-1 (Park et al, 2004) and, through these mechanisms it greatly enhances the sensitivity of tumor cells to TRAIL, including those tumors with low caspase-8 expression due to promoter hypermethylation. Thus, the simultaneous treatment with IFN-g and TRAIL resulted in a consistent pro-apoptotic effect in Ewing’s sarcoma (Kotny et al, 2001; Marchant et al, 2004) hepatoma (Shin et al, 2001) colon carcinoma (Langaas et al, 2001; Van Gleen et al, 2004) and neuroblastoma (Yang et al, 2003) cell lines. Interestingly, the co-administration of IFN-g and TRAIL was efficacious only in a part of TRAIL-resistant neuroblastoma cell lines; in the remaining lines a significant level of apoptosis was achieved only when chemotherapic drugs were added to TRAIL and IFN-g (Yang et al, 2003). IFN-g stimulates TRAIL-induced apoptosis also in thyroid cancer cells, but through a peculiar mechanism involving BAK upmodulation (Wang et al, 2004).

F. Chemotherapeutic agents and TRAIL

Killing of tumor cells by cytotoxic therapies such as chemotherapy, is predominantly mediated by triggering apoptosis. The relative contribution of the receptor and mitochondrial pathway to drug-induced apoptosis was matter of controversy and there is evidence that both ways, extrinsic and intrinsic, are involved (Debatin and Krammer, 2004). Studies on several types of cancer cell lines and primary tumors cells have shown that treatment with anticancer drugs determines an increase of FasL expression, which acts as a triggering stimulus for the death receptors pathway in an autocrine or paracrine manner by binding to its receptor Fas (reviewed in Debatin and Krammer 2004).

Many studies have clearly shown that several anticancer drugs, including etoposide, CPT-11, doxorubicin, 5-FU, carboplatin, topotecan, taxol and fludarabine greatly augment TRAIL-induced apoptosis of epithelial cancer cells (Gliniak and Le 1999; Kean et al, 1999; Kim et al, 2000; Nagane et al, 2000; Komdeur et al, 2004; Tomek et al, 2004; Schmeltz et al, 2004), acute myeloid leukaemia (Johnston et al, 2003) and chronic lymphocytic leukaemia (Wen et al, 2000). These findings were confirmed also in vivo animal models using xenografts of primary tumor cells (Hylander et al, 2005). In this context, particularly interesting are the results obtained in non-small-lung cancer cells that in xenografts models are scarcely sensitive to anticancer drugs (taxol and carboplatin) or to TRAIL. However, the combined administration of TRAIL and taxol or carboplatin resulted in a curative effect in the large proportion of tumor-bearing mice (Jim et al, 2004). Importantly, this treatment was associated with low toxicity. Interestingly, similar observations have been made for prostate cancer xenograft models using a treatment strategy based on the sequential administration first of cytotoxic chemotherapeutic drugs and then of TRAIL (Shamker et al, 2005). Finally, the administration of irinotecan and TRAIL resulted in the elimination of hepatic metastases of colon cancer cells via a p53-indipendent mechanisms (Ravi et al, 2004).

Because chemotherapeutic agents and TRAIL use different BH3-domain-containing proteins to activate BAX and BAK, the simultaneous delivery of both death signals may converge to promote apoptosis of tumor cells. The mechanisms of synergic effect between TRAIL and the chemotherapeutic drugs consists either in the upregulation of pro-apoptotic molecules or the downregulation of anti-apoptotic molecules.

Among the effects on the upregulation of pro-apoptotic molecules a key event is represented by the upmodulation of TRAIL-R1 and TRAIL-R2 exerted by many anticancer drugs (Wen et al, 2000; Jhonston et al, 2003; Komdeur et al, 2004; Schmelz et al, 2004; Tomek et al, 2004). However, the acquisition of an increased TRAIL sensitivity by cancer cells seems to be related to multiple mechanism, involving both TRAIL-R1/ R2 upmodulation and a decrease expression of anti-apoptotic molecules. Interestingly, recent studies have shown that also anticancer drugs, such like cyclooxygenase 2 inhibitors, that do not act like the cytotoxic anticancer drugs, are able to induce apoptosis of tumor cells through upmodulation of TRAIL-R1 and TRAIL-R2 and greatly potentiate the pro-apoptotic effect of TRAIL of non-small-lung cancer cells (Liu et al, 2005).

V. Conclusions

The induction of apoptosis in response to cell damage generally requires the function of the tumor suppressor p53, which engages the intrinsic signalling pathway of apoptosis. Conventional anticancer treatment likely selects for tumor cells displaying inactivated p53, however, resulting in chemoresistance. Since death receptor activation can induce cell death of malignant cells through a mechanism independent of p53, targeting the TRAIL receptors with TRAIL-targeting agents (either recombinant TRAIL, or fully human agonistic monoclonal antibodies anti-TRAIL-R1 or anti-TRAIL-R2) is a rational therapeutic strategy to treat cancer.

TRAIL may represent an ideal therapeutic agent for cancer treatment because it has been shown to be a potent apoptosis inducer in a wide variety of cancer and transformed cell lines without damaging most normal cells. However, the potential application of TRAIL in cancer therapy is limited since the majority of primary cancer cells are found to be resistant to TRAIL-induced apoptosis. The resitance may be due to mutations of TRAIL-R1 or TRAIL-R2, or peferential expression of the TRAIL decoy receptors, TRAIL-R3 and/or TRAIL-R4, low expression of pro-apoptotic molecules involved in TRAIL signalling (caspase-8 or FADD) or high expression of anti-apoptotic molecules FLIP, IAP, Survivin, Bcl-2. Thus, combination of TRAIL with other agents has been a promising strategy to potentiate the citotoxicity of TRAIL and its therapeutic applications. In this context, particularly promising seems to be the use of newly developped agonistic monoclonal antibodies anti-TRAIL-R1 or anti-TRAIL-R2, able to induce apoptisis, like recombinant TRAIL, but exhibiting an in vivo much longer half-life than the death ligand.

The design of specific phase II and III clinical studies aiming to evaluate whether or nor either agonistic monoclonal antibodies to TRAIL-R1 and TRAIL-R2 or recombinant TRAIL and other pharmaceutical agents targeting TRAIL may represent an important step in the treatment of any specific malignancy and will represent one of the main objectives in the future developments in this area. The identification of the ideal tumor types to select for early proof of activity will represent one immediate goal. In this context, particularly useful will be the observations obtained in vitro on the corresponding primary tumor cells to select the tumor to be treated and the pharmaceutic association to be made. However, one of the major limitations could derive from the inadequacy of of these preclinical models to really evaluate the overall complexity of genetic alterations occurring in human tumors.

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