CELL DEATH BY MURDER

Death Receptor Pathways

In some cases, apoptosis is initiated through ligation of specific cell-surface receptors. These death receptors belong to the tumor necrosis factor receptor (TNFR) family, and the best studied members include Fas and TNFR1.

Ligand binding to TNFR can also activate adapter proteins that recruit components of the mitogen-activated protein kinase (MAPK) cascade. This leads to activation of Jun-V-terminal kinase (JNK) and c-jun, which participate in receptor-mediated apoptosis by controversial mechanisms.¹ Other adapter proteins recruit signaling components of the NF-κB pathway, which prevents apop­tosis by inducing expression of survival molecules and proinflammatory cytokines. TNFR ligation primarily stimulates inflammation but can induce apoptosis when NF-κB signaling is blocked.

Another ligand-receptor pair that is increasingly understood to play a role in immune regulation and cancer is the TNF-related apoptosis-inducing ligand (TRAIL) and its receptors. TRAIL-induced signaling pathways are similar to those activated by TNFR1. However, TRAIL can also bind to decoy receptors that lack intracellular signaling capability.² The decoy receptors sequester TRAIL from competent signaling receptors and, therefore, block apoptosis. Expression of decoy receptors provides one mechanism by which normal cells escape TRAIL-induced death. On the other hand,

TABLE 27.2

Strategies to Prevent Cell Death

tumor cells exhibit lower expression of decoy receptors due to promoter methylation.³ This may be one explanation for increased sensitivity of tumor cells to TRAIL-induced apoptosis.

Proposed physiological functions for endogenous TRAIL involve immune surveillance against oncogene-transformed and virus-infected cells. A role in immune surveillance against tumors is supported by the increased incidence of metastases in mice with mutagen-induced tumors when endogenous TRAIL is eliminated by gene knockout⁴ or neutralizing antibody.⁵ TRAIL was also proposed to play a role in the interferon (IFN)-dependent host defense against viral infection. IFNs prevent viral replication by inducing apoptosis in infected cells, and they regulate TRAIL expres- sion.6 For example, in response to IFN, cytomegalovirus-infected human fibroblasts become more sensitive to TRAIL, whereas neighboring uninfected cells increase expression of the TRAIL ligand and downregulate death receptors. In vivo studies have shown that TRAIL expression by natural killer cells is critical for limiting viral replication and, furthermore, that TRAIL induces apoptosis of infected cells in a mouse model of adenoviral hepatitis.⁷ Some viruses directly target the TRAIL pathway to overcome immune surveillance and protect infected cells from apoptotic death, as adenoviruses were shown to induce receptor internalization.⁸

Therapeutic Approaches

Modulation of the death receptor pathway occurs at several levels. Decoy receptors compete with death receptors for ligand binding and were described for both TRAIL and Fas.⁹ Downstream of receptor ligation, cellular-FLICE inhibitory protein (c-FLIP) competes with caspase-8 and -10 for DISC binding, and inhibitors of apoptosis proteins (IAPs) block effector caspase activation. Each of these steps can be deregulated in tumor cells to promote survival, and each can be pharmaco­logically targeted to induce cell death.

Whereas attempts at developing death-receptor-targeted chemotherapeutics, such as anti-Fas antibodies or recombinant TNF ligand, were hindered by toxicity,¹⁰ strategies using TRAIL hold more promise.

Inhibitor of Apoptosis Proteins (IAPs)

IAP family proteins were originally discovered in baculovirus-infected cells and are characterized by one or more 70-80 amino acid baculoviral IAP repeat (BIR) domains.¹⁸ Subsequent studies identified cellular homologues¹⁹,²⁰ and showed that IAPs inhibit caspase activation via two mechanisms. IAPs can block self-cleavage of initiator caspases by direct binding of the BIR domain, and they can also target activated effector caspases for proteasomal degradation with the E3 ubiquitin ligase activity of their RING domains. IAPs are inhibited by the proapoptotic molecules Smac/DIABLO and Omi/ HtrA2, which are released from the mitochondria upon the loss of mitochondrial membrane integrity during apoptosis.

Inhibition of IAPs in Cancer Therapy

IAP family members are highly expressed in many types of cancer.²¹ Beyond overexpression correlations, direct genetic evidence for an oncogenic role of IAP members is found in mucosa- associated lymphoid tissue (MALT) B cell lymphomas, in which translocation events result in fusion of the BIR domains of cIAP-2 to the MALT protein.²² In many cancers, downregulation of the overexpressed IAP protein alone might be sufficient to induce apoptosis or at least sensitize cells to other chemotherapeutics.

More recent developments include the identification of small molecule antagonists of IAPs, which target either caspase binding or ubiquitin ligase activity. Compounds targeting XIAP were shown to sensitize multiple cancer cell lines to TRAIL-induced apoptosis and to be effective as single agents in tumor xenograft models, although their precise mechanism of action remains unclear.²⁷ One concern with pharmacologic strategies that specifically target ligase activity is that IAPs might still be able to bind caspases and prevent their activation. Therefore, ligase inhibitors would work better in combination with specific inhibitors of IAP-caspase binding²⁸ or with agents that induce Smac release. Along these lines, injection of Smac peptides in combination with TRAIL ligand was shown to induce complete tumor regression in human glioma cells in nude mice.^29,30 Although IAP family proteins clearly represent an important target for modulation of cell death, current therapeutic options are still in the experimental stages of development. Strategies to inhibit specific IAPs with small molecules will rapidly advance with more detailed structural studies.

Activation of IAPs to Prevent Neuronal Loss

In contrast to cancer, overexpression of IAP family members can be a protective therapeutic approach to prevent apoptosis in neurodegenerative diseases. Loss of function studies in animal models provided the first evidence that neuronal apoptosis inhibitory protein (NAIP) expression is critical to prevent cell loss.³¹ Attempts to therapeutically increase IAP expression showed that injection of adenoviral vectors expressing NAIP or XIAP into the rat hippocampus provide pro­tective effects in a global ischemia model.³² Adenoviral vectors encoding IAP family members were also shown to suppress apoptosis in a sciatic axotomy model.³³

Caspases

Development of therapeutics that modulate apoptosis downstream of mitochondrial permeability primarily focused on the identification of small molecule inhibitors of caspases.

The therapeutic benefit of global caspase inhibition also depends on the specific mechanism contributing to a given disease and can actually exacerbate cell death in disorders involving caspase­independent mechanisms. For example, caspase inhibition was shown to increase toxicity of TNF- α-induced shock in vivo by enhancing oxidative stress and mitochondrial damage.³⁸ In this situation, caspase activity might actually serve a protective function by cleaving phospholipase A₂ and preventing further generation of reactive oxygen species.

In contrast, small molecules that induce apoptosis downstream of mitochondrial permeability were identified through cell-free assays for caspase activation. This strategy identified chemicals that specifically induce oligomerization of apoptotic protease-activating factor 1 (Apaf-1) into the mature apoptosome, causing activation of caspase-9 and -3 without the need for cytochrome c release.³⁹,⁴⁰ Compounds identified in these screens showed cytotoxic activity against multiple cancer cell lines without affecting normal cells. However, further studies in animal models are needed to determine the efficacy of this strategy for future drug development.

Furthermore, controversy remains over the importance of caspase activation following mito­chondrial disruption in determining cell death vs. orchestrating cellular disposal.