17 Regulation of TRAIL-Induced

From: Cancer Drug Discovery and Development: Death Receptors in Cancer Therapy Edited by: W. S. El-Deiry © Humana Press Inc., Totowa, NJ

297

17 Regulation of TRAIL-Induced

Apoptosis by Transcriptional Factors

Rüdiger Göke and Youhai H. Chen, ^MD , ^P h D

TRAIL AND ITS RECEPTORS

In 1995, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) was identified based on its sequence homology to other TNF family members (1). Among members of the TNF family, TRAIL shares the highest sequence homology with Fas ligand (FasL, CD95L). However, unlike FasL, TRAIL appears to induce apoptosis of tumor cells but not most normal cells (2). To date, five receptors for TRAIL have been cloned: TRAIL-R1 (DR4, Apo2A) (3), TRAIL-R2 (DR5, TRICK, Killer) (3–8), TRAIL- R3 (DcR1, TRID, LIT) (6–9), TRAIL-R4 (DcR2, TRUNDD) (10), and osteoprotegerin (OPG) (11). Unlike other TRAIL receptors, which bind only to TRAIL, osteoprotegerin also binds to osteoprotegerin ligand (OPGL), TRANCE, and RANK ligand (RANKL).

TRAIL-R1 and TRAIL-R2 contain intracellular death domains and induce, via coupling

with intracellular adaptor proteins, the proteolytic cleavage of caspase-8 (12). Caspase-

8 activation initiates the extrinsic and intrinsic apoptotic pathways, resulting in caspase-

3 cleavage, which is an irreversible step in a cell’s commitment to apoptosis. Other

TRAIL receptors do not generate death signals because TRAIL-R3 does not contain a

death domain and is attached to the membrane by a glycolipid anchor (6,9), whereas the

death domain of TRAIL-R4 is not functional (10) and OPG exists only in a soluble form

(11). Therefore, TRAIL-R3 and TRAIL-R4 might act as decoy receptors by competing

with other TRAIL receptors for TRAIL. The expression pattern of TRAIL receptors on

certain cell lines might determine their sensitivity to TRAIL. However, the expression of

TRAIL decoy receptors is not always related to a cell’s resistance to TRAIL-induced

apoptosis (13). Other factors may, therefore, play more decisive roles in determining a

cell’s sensitivity to TRAIL. In this review, we will focus on NF-gB and PPAR-a, two

transcription factors that were recently found to play important roles in TRAIL-induced

apoptosis.

ROLES OF NF-gB IN TRAIL-INDUCED APOPTOSIS

The Rel/NF-gB family of transcription factors regulates a number of biological pro- cesses, including cell proliferation and differentiation, apoptosis, immune response, and inflammation (14–16). Rel/NF-gB are normally present in the cytoplasm in association with a family of inhibitors (IgBs) that mask their nuclear localization sequences (17,18).

Activation of IgB kinase (IKK) leads to phosphorylation and degradation of IgBs. As a result, Rel/NF-gB are released and translocated to the nucleus, where they bind to DNA and induce the transcription of target genes (15).

Rel/NF-gB induce the expression of a number of anti-apoptotic genes, including cellular inhibitors of apoptosis (cIAPs), mitochondrial proteins of the Bcl

family such as Bfl-1/A1 and Bcl-X

, A20, manganese superoxide dismutase (MnSOD), IEX-1L, caspase 8/FADD-like IL-1`-converting enzyme (FLICE)-inhibitory protein (c-FLIP), TNF receptor-associated factor 1 (TRAF1) and 2 (TRAF2), and TRAIL receptor 3 (16,19–

30). Numerous studies exist which demonstrate that Rel/NF-gB inhibit programmed cell death induced by TNF-_, anticancer drugs, and ionizing radiation (16,31–33).

Recently, it has also become clear that Rel/NF-gB regulate TRAIL-induced apoptosis.

Thus, treatment of TRAIL-resistant pancreatic cancer cell line L3.6 with TRAIL and the NF-gB inhibitor NBD (NEMO-binding domain) peptide significantly decreased cell viability and increased apoptosis (34). This effect was most likely due to decreased FLIP levels in L3.6 cells as a result of NF-gB inhibition. Prostate cancer cell lines PC3AR and PC3Neo show different TRAIL sensitivities, which are associated with a difference in NF-gB levels in these cells (35). Blocking NF-gB function by adenoviral transfer of mutated I gB increased apoptotic responses, suggesting a direct role for NF-gB in this system.

Multiple myeloma (MM) is an incurable disease. TRAIL might represent a new treatment option, since it kills most MM cell lines and MM cells freshly isolated from patients. Treatment with the NF- gB inhibitor SN50 enhanced TRAIL-induced apoptosis in sensitive cells and reversed resistance of ARH-77 and IM-9 MM cells (36). Interest- ingly, treatment with SN50 did not sensitize normal B-lymphocytes towards TRAIL- induced apoptosis. Insulin-like growth factor-1 (IGF-1) promotes proliferation of MM cells and protects them against TRAIL-induced apoptosis. In a recent study, IGF-1 was shown to activate NF-gB and upregulate the expression of survival factors FLIP, survivin, cIAP-2, A1/Bfl-1, and XIAP (37). Overexpression of Akt decreased TRAIL sensitivity of MM cells. Interestingly, treatment of cells with an Akt inhibitor abrogated NF-gB activation and prevented the protective effect. These data show that besides NF- gB, the PI-3K/Akt pathway is also involved in the regulation of TRAIL sensitivity. An important role for NF-gB in TRAIL-induced apoptosis has also been demonstrated in lymphoid cell lines. Thus, acute T-cell leukemia cells (CEM, Jurkat) and BJAB cells (Burkitt lymphoma) treated with NF-gB inhibitors showed a significantly increased sensitivity towards TRAIL (38,39).

It has been previously reported that TRAIL, but not other TNF family members,

induces apoptosis in the majority of melanoma cell lines (40–44). The mechanisms of

TRAIL resistance of some melanoma cells are not well understood. Lack of response to

TRAIL was partially due to a loss of TRAIL receptor expression (41). However, a clear

correlation between expression of TRAIL decoy receptors and TRAIL resistance could

not be established (13). In a recent study, pretreatment of melanoma cell lines with the proteasome inhibitor -acetyl-

-leucinyl-

-leucinyl-

-norleucinal (LLnL), which also inhibits NF-gB, sensitized 10 of 12 cell lines to TRAIL-induced apoptosis (45). Further- more, TRAIL resistant melanoma cells generated by intermittent TRAIL exposure exhib- ited elevated levels of activated NF-gB. Resistance in these cell lines could be reversed by LLnL and by a degradation-resistant form of IgB_(45). These results suggest that NF- gB significantly influences TRAIL sensitivity in melanoma cells. Reports on renal cancer cells provide contradictory results. Whereas Pawlowski et al. reported that neither acti- vation nor inhibition of NF-gB signal transduction pathway protected renal carcinoma cells from TRAIL-induced apoptosis (46), a recent report shows that constitutive activa- tion of NF-gB prevents TRAIL-induced apoptosis in these cells (47).

Platelet-activating factor (PAF) is a proinflammatory lipid mediator that acts via a G protein-coupled receptor. Activation of epidermal PAF receptor results in protection against TRAIL-induced apoptosis. Recently, this effect was shown to be NF-gB-dependent since it could be antagonized by a super-repressor form of IgB (48). Therefore, it seems that G protein-coupled receptors like the PAF receptor are able to block TRAIL-in- duced apoptosis in a NF-gB-dependent manner.

Various subunits of NF-gB can have different effects on the cellular response to TRAIL. Thus, the c-Rel subunit promotes TRAIL-induced cell death by increasing the expression of TRAIL receptors 1 and 2; the Rel-A subunit blocks TRAIL-induced apoptosis by increasing Bcl-X

expression (49). However, further investigation is needed to establish the specific roles of each NF-gB subunit in TRAIL-induced apoptosis.

ROLES OF PPAR-a IN TRAIL-INDUCED APOPTOSIS

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors. PPARs form a subfamily consisting of PPAR- _, PPAR-6, and PPAR-a(50). PPAR-a is highly expressed in adipose tissues but also present in intestine, mammary gland, endothelial and smooth muscle cells, monocytes, macrophages, and a number of other cell types (51–58). PPAR- a exists in two isoforms, PPAR-a1 and PPAR-a2, which are produced from a single gene as a result of the utilization of different promoters and alternative splicing (51). PPAR- a forms a dimer with the retinoid X receptor. This complex binds to the promoter region of specific target genes. Binding of PPAR-a ligands leads to conformational changes resulting in activation of the target genes. PPAR-a plays an important role in cell growth and differentiation, lipid metabolism, and inflammation (59,60).

PPAR-a is highly expressed in several cancer cell lines, including breast cancer (53,61–

63), prostate cancer (64,65), pancreatic cancer (66), gastric cancer (67), colon cancer (68,69), liposarcoma (70), bladder cancer (71), lung cancer (72), and related cancer tissues from patients (64,68,70). Activation of PPAR-a resulted in suppression of cell growth, induction of apoptosis, promotion of terminal differentiation, and morphological changes to a well differentiated and less malignant state (50,60,73).

The mechanisms by which PPAR-a ligands promote apoptosis are not well under-

stood. PPAR-a ligands were shown to up-regulate Bax and Bad protein levels and

downregulate Bcl

(61,74). However, in other studies PPAR-a ligands had no effect on

Bax and Bcl

mRNA expressions (75–77). While troglitazone and pioglitazone were

shown to increase the expression of cyclin-dependent kinase inhibitors p21

and p16

(71,78), troglitazone did not change p21

levels in smooth muscle cells (75). Furthermore, PPAR-a ligand-induced inhibition of NF-gB-dependent transcription might be mediated by PPAR-a-independent mechanisms (58,79). Thus, further investi- gations are needed to elucidate the proapoptotic activities of PPAR-a ligands.

TRAIL holds tremendous promise for cancer therapy because of its ability to selec- tively kill transformed cells but not normal cells. However, not all tumor cells are equally sensitive to TRAIL-induced apoptosis. Using Jurkat cells, we showed that pioglitazone inhibits cell growth and sensitizes them for TRAIL-induced apoptosis (39). Similar results were obtained in carcinoid cells that are normally resistant to TRAIL-induced apoptosis (78). In carcinoid cells, pioglitazone treatment upregulated p21

ex- pression. Because overexpression of p21

by adenoviral gene transfer sensitized cells to TRAIL-induced apoptosis, the proapoptotic effect of pioglitazone might be mediated by p21

in carcinoid cells. Recently, the sensitizing effect of PPAR-a ligands on TRAIL-induced apoptosis was also observed in prostate and ovarian cancer cell lines (80). In these cells, PPAR-a ligands induced ubiquitination and proteasome- dependent degradation of FLIP without affecting FLIP mRNA levels. Interestingly, this effect was not related to NF-gB, independent of PPAR-a expression, and could be ob- served even in the presence of a PPAR-a dominant-negative mutant, indicating a novel PPAR-a-independent mechanism. This assertion is supported by a recent study in which the PPAR-a agonist bisphenol A diglycidyl ether (BADGE) induces apoptosis in Jurkat cells independent of PPAR-a, in both caspase-dependent and -independent manners (81).

In summary, transcription factors NF-gB and PPAR-a play important roles in the regulation of programmed cell death. Recent studies strongly suggest that while PPAR- a promotes TRAIL-induced apoptosis, NF-gB inhibits it. Therefore, NF-gB inhibitors and PPAR-a agonists may be used as enhancers for TRAIL-based cancer therapy.

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