Targeting the Apoptosome for Cancer Therapy Elizabeth C

Molecular Pathways

Targeting the Apoptosome for Cancer Therapy Elizabeth C. Ledgerwood and Ian M. Morison

Abstract Apoptosis is a programmed mechanism of cell death that ensures normal development and tissue homeostasis in metazoans. Avoidance of apoptosis is an important contributor to the survival of tumor cells, and the ability to specifically trigger tumor cell apoptosis is a major goal in cancer treatment. In vertebrates, numerous stress signals engage the intrinsic apoptosis pathway to induce the release of cytochrome c from mitochondria. Cytochrome c binds to apoptosis protease activating factor-1, triggering formation of the apoptosome, a multisubunit protein complex that serves as a platform for caspase activation. In this review we summarize the mechanisms of apoptosome assembly and activation, and our current understanding of the regulation of these processes. We detail the evidence that loss-of-function of the apoptosome pathway may contribute to the development of specific cancers. Finally we discuss recent results showing enhanced sensitivity of some tumor cells to cytochrome c ^ induced apoptosis, suggesting that agents able to directly or indirectly trigger apoptosome-catalyzed caspase activation in tumor cells could provide new approaches to cancer treatment.

Background process controlled by Bcl-2 family members. This results in the release of cytochrome c and other proapoptotic factors Apoptosis is a programmed mechanism of cell death that is (e.g. HtrA2/Omi and Smac/Diablo) to the cytosol (3, 6). The essential for normal development and tissue homeostasis (1). identification of cytochrome c in the cytosol during apoptosis, Because dysregulation of apoptosis is a major contributor to and the subsequent characterization of how cytochrome c the survival of tumor cells, there is much interest in identifying induces caspase activation, has led to new opportunities for new ways to activate this process in malignant cells. The aim of therapeutic targeting of this pathway both to block and to the apoptotic program is to activate caspases, the family of trigger apoptosis. proteases that drive the biochemical events leading to the Cytochrome c is the major inducer of caspase activation organized disassembly of the cell. Two well-described pathways downstream of mitochondria. The proapoptotic activity of lead to caspase activation in mammalian cells: the extrinsic cytochrome c requires interaction with apoptosis protease pathway, triggered by an extracellular signal, such as activating factor-1 (Apaf-1). This promotes assembly of the the binding of tumor necrosis factor or Fas ligand to cell apoptosome, a protein complex that serves as a platform for surface receptors, and the intrinsic pathway that responds to activation of the initiator caspase procaspase-9. Subsequently intracellular signals such as DNA damage (1, 2). caspase-9 cleaves and activates executioner caspases such as The intrinsic apoptosis pathway is also referred to as the caspase-3, leading to death of the cell. Cytochrome c, Apaf-1, mitochondrial apoptosis pathway because it requires the release and procaspase-9 form the core of the mitochondria-mediated of proapoptotic factors from the mitochondrial intermembrane intrinsic apoptosis pathway (refs. 7–9; Fig. 1). space, a key player being cytochrome c (2, 3). The discovery in Manipulation of apoptosome assembly and activation has 1996 that this electron carrier also acts as a cytosolic apoptosis significant therapeutic potential, but relies on detailed under- effector (4) was described at the time as "bizarre and standing of the molecules involved in its formation and unexpected" (5). It is now known that multiple pathways regulation. The crucial initiation step for apoptosome assembly activated by intracellular stress signals converge at the is the binding of cytochrome c to the functional Apaf-1 splice mitochondrion causing loss of outer membrane integrity in a variant, Apaf-1XL (10). Apaf-1 has three functional regions: the NH2-terminal caspase recruitment domain that binds procaspase-9, the central nucleotide-binding and oligomerization domain (NOD, also called the NB-ARC or Ced-4 domain), and Authors’ Affiliation: Department of Biochemistry, University of Otago, Dunedin, 13 WD40 repeats in the COOH-terminal half. A 12.8 A˚ cryo- New Zealand Received 9/2/08; revised 9/11/08; accepted 9/11/08. electron microscopy apoptosome structure shows that a single Grant support: Health Research Council of New Zealand, Lottery Grants Board molecule of cytochrome c binds between two h-propellers (New Zealand). formed by the WD40 repeats (11). This interaction is of high Requests for reprints: Elizabeth Ledgerwood, Department of Biochemistry, affinity and primarily electrostatic with contributions from University of Otago, PO Box 56, Dunedin, New Zealand. Phone: 64-3-4795195; lysine residues distributed over the entire surface of Fax: 64-3-4797866; E-mail: [email protected]. F 2009 American Association for Cancer Research. cytochrome c (12–16). Our recent report of mutation of doi:10.1158/1078-0432.CCR-08-1172 glycine 41 to serine in human cytochrome c that increases its

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Fig. 1. Cytochrome c ^ induced apoptosome formation and caspase activation. In healthy cells cytochrome c is sequestered in the mitochondrial intermembrane space and Apaf-1exists as an inactive monomer in the cytosol. In response to stress stimuli (e.g. DNA damage or chemotherapeutic drugs) cytochrome c is released from mitochondria to the cytosol where it binds to Apaf-1, triggering a conformational change and hydrolysis of the Apaf-1-bound dATP/ATP. In a process dependent on exchange of dATP/ATP for dADP/ADP, the Apaf-1-cytochromec heterodimers assemble into the apoptosome, which provides a platform for activation of the initiator caspase procaspase-9. Activated caspase-9 then cleaves and activates executioner caspases such as caspase-3.

caspase-activating activity (17) shows that the electrostatic formation does not occur in response to ‘‘accidental’’ release of interactions may not be the sole binding determinant. small amounts of cytochrome c in healthy cells. Cytochrome c undergoes conformational fluctuations, Beyond these factors, our understanding of how the particularly in the region of residue 44, and it may be that apoptosome is regulated in normal cells and in cancer cells is there is a favored conformational state necessary for its binding in its infancy. In Caenorhabditis elegans, the Bcl-2 homologue to Apaf-1 (18, 19). In healthy cells Apaf-1 exists as an inactive CED-9 binds directly to the Apaf-1 homologue CED-4 to dATP/ATP-bound monomer in which the WD40 domains are inhibit caspase activation. It was therefore expected that folded back on the rest of the protein, preventing oligomer- apoptosome assembly and activity would be regulated by ization and caspase-9 binding (20, 21). The binding of additional protein factors. Early reports suggested that Bcl-XL cytochrome c induces a conformational change in Apaf-1, plays a similar role in vertebrates as CED-3 does in C. elegans, exposing the NH2-terminal regions and triggering hydrolysis binding to Apaf-1 to regulate its activity (29, 30). However, of the Apaf-1–bound dATP/ATP. However, dADP-bound subsequent results have not sustained these observations (31). Apaf-1 remains inactive and only assembles into the apopto- The isolation of endogenous active apoptosomes from cultured some once the exchange of dATP for dADP has occurred cells has enabled characterization of the components by mass (11, 21). Seven cytochrome c –bound Apaf-1 molecules spectrometry (32, 33). Only Apaf-1, caspase-9, caspase-3, and assemble into a wheel-like complex with the caspase recruit- X-linked inhibitor of apoptosis protein (XIAP) are present. ment domains at the hub and the WD40-cytochrome c bound XIAP is a caspase-9 inhibitor, which in healthy cells contributes regions forming the spikes of the wheel (11). In this complex to the prevention of accidental caspase activation. During the Apaf-1 caspase recruitment domains are free to bind apoptosis, the presence of XIAP in the apoptosome is initially procaspase-9, which is then activated. required for binding of caspase-3 (33). Subsequently, the The cell has a number of mechanisms in place to ensure that proapoptotic factors Smac/Diablo and Omi/HtrA2, which are active apoptosomes are only formed in response to apoptotic released from mitochondria alongside cytochrome c, bind to stimuli. Apoptosome assembly is inhibited by physiologic and displace XIAP from the apoptosome (7, 32). Interestingly, concentrations of potassium (22–25), calcium (26), and cytochrome c was only transiently associated with the isolated nucleotides (16, 25). High ionic strength disrupts the apoptosomes, suggesting it is necessary for apoptosome interaction between cytochrome c and Apaf-1, calcium binds assembly but not for apoptosome activity. The failure to detect to Apaf-1 preventing nucleotide exchange, and ATP binds to additional proteins in the apoptosome complex in these studies surface lysine residues on cytochrome c preventing binding to does not, however, rule out regulatory roles for other proteins. Apaf-1. During apoptosis the inhibitory effects are reduced as Indeed a large number of proteins have been reported to the concentration of cytochrome c increases and the cytosolic enhance or inhibit apoptosome assembly and/or activity, concentrations of these factors decrease (16, 27, 28). although only a few of these effects have been independently This combination of mechanisms ensures that apoptosome confirmed. For example, it was initially reported that heat

www.aacrjournals.org 421 Clin Cancer Res 2009;15(2) January 15, 2009 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2009 American Association for Cancer Research. Molecular Pathways shock protein (Hsp) 70 inhibited apoptosome-dependent detecting Apaf-1 protein may lead to incorrect conclusion of caspase activation (34, 35); the inhibitory effect of Hsp70, Apaf-1 loss (56, 57). Loss of Apaf-1 has also been reported in however, has subsequently been shown to be due to high salt ovarian and leukemic cancer cell lines, and in glioblastomas concentrations in Hsp70 preparations (32, 36). Similarly, (58–60), and resistance to cytochrome c –induced caspase Hsp27 and Hsp90 have been reported to bind to cytochrome activation was common in cells isolated from patients with c (37) and Apaf-1 (38, 39), respectively, thereby inhibiting acute myeloid leukemia (61). Sequestration of Apaf-1 away apoptosome activity, although for Hsp27 this result has been from the cytosol has been proposed as an alternative disputed (35). mechanism of apoptosis resistance in B-cell lymphoma (62). There is accumulating evidence that pp32/putative HLA class Analysis of cytochrome c –induced apoptosis in a panel of II-associated protein-1 (PHAP1) is a bone fide apoptosome non–small cell lung cancer cell lines found that primary drug activator. pp32/PHAP1 indirectly increased both procaspase-9 resistance correlated with an inability to activate caspases-9 and association with the apoptosome and caspase-9 activity, caspase-3 although apoptosome formation was normal (63). without altering apoptosome assembly (40). A recent report This inhibition was overcome by the addition of pp32/PHAP1 suggests PHAP1 requires at least Hsp70 and cellular apoptosis both in the cell lines and in a murine tumor model in vivo, but sensitivity protein as Apaf-1-binding cofactors to promote because there was no difference in pp32/PHAP1 levels in the nucleotide exchange on Apaf-1, thereby enhancing apopto- resistant versus sensitive cell lines, the mechanism of resistance some formation (41). Prothymosin-a is a putative negative is unclear. regulator of the apoptosome pathway. Prothymosin-a blocks Investigation of cells isolated from different stages of colon the stimulatory activity of PHAP1 (40), and knockdown cancer showed that the cells became more resistant to treatment of prothymosin-a expression sensitizes HeLa cells to UV with cisplatin, ionizing radiation, or Fas ligation as the disease and staurosporine-induced apoptosis (40, 42). One report progressed. Resistance correlated with the acquisition of genetic suggested prothymosin-a interacts with p8 to inhibit changes in apoptosis effectors, including down-regulation of cytochrome c –induced caspase activation (42); however, Apaf-1 and up-regulation of XIAP (64). However, when the prothymosin-a also binds directly to cytochrome c (43), ability of cytochrome c to induce caspase activation was providing an additional mechanism for inhibition. determined in cytosols prepared from a range of lung, colon, Other proteins reported to regulate apoptosome formation stomach, brain, and breast cancer cell lines, most cells were and/or activation include histone H1.2, HCA66, NAC/CARD7, found to be more sensitive to cytochrome c –induced caspase Nucling, AFP, AChE (enhancers), and Bcr-Abl, Aven, AIP, APIP, activation than normal cells (65). Breast tumors are signifi- JNK1&2,PKA,ERK,Akt,PKCzeta,HBXIP,andTUCAN cantly more sensitive to microinjected cytochrome c than (inhibitors; reviewed in ref. 44). In addition, modification of surrounding normal mammary epithelial cells (66). This cytochrome c itself may alter its ability to bind to Apaf-1 and difference has been attributed to the increased levels of pp32/ induce apoptosis (45, 46). The involvement of many of these PHAP1 in the cancer cells. Similarly, whereas normal brain proteins in multiple cellular processes and the disparity tissue is highly resistant to cytochrome c –induced apoptosis between findings from different laboratories and in different due to low expression of Apaf-1, brain tumors have increased cell types make it difficult to determine physiologic relevance. expression of Apaf-1 (67). This results in increased susceptibil- ity of human glioblastoma and medulloblastoma cell lines, and Clinical-Translational Advances mouse high-grade astrocytomas and medulloblastomas, to apoptosis following cytosolic microinjection of cytochrome c. Because the inhibition of apoptosis is one of the key features High levels of Apaf1 have previously been associated with the of cancer (47), identifying ways of overcoming or bypassing inability of endogenous inhibitors of apoptosis to protect this inhibition to activate caspases has become an important pheochromocytoma cells against cytochrome c –mediated strategy in the development of new cancer treatments. To date apoptosis (68). major efforts to achieve this have involved targeting the death Cell-free apoptosis assays have been used to screen small receptors, Bcl-2 family proteins, inhibitors of apoptosis, and molecule libraries for compounds that induce apoptosome direct caspase activation (48). In contrast, activation of the activation (40, 69). a-(Trichloromethyl)-4-pyridineethanol apoptosome has received relatively little attention (44, 49, 50). (PETCM), which was identified in a high-throughput screen, However, characterization of the apoptosome pathway in enhanced cytochrome c –induced caspase activation in vitro in specific cancers suggests it is a valid target. In particular, in the absence of added nucleotides, and could overcome the tumors that are refractory to treatments aimed upstream of inhibitory effect of prothymosin-a (40). A series of indolene mitochondrial cytochrome c release, but which have intact and carbamate compounds also induced caspase activation by postmitochondrial apoptotic machinery (51), the ability to an unknown mechanism, and one of these (compound 2) had directly cause apoptosome activation could be valuable. The proapoptotic activity in cell lines (69). The activity of PETCM profiling of a number of cancer and normal cell types has been and compound 2 required the presence of cytochrome c. undertaken to determine when the apoptosome would be a One approach that is currently being explored is the valid target and whether it is possible to capitalize on development of small molecule cytochrome c mimetics, which differences between tumor and normal cells (reviewed in refs. will trigger the conformational change in Apaf-1 that is 44, 51, 52). necessary for apoptosome assembly and caspase-9 activation Melanoma, which is generally refractory to chemotherapy, (66, 67). Another way of targeting the apoptosome for cancer was the first cancer in which loss of Apaf-1 was shown treatment is the use of compounds that directly induce (53–55). However, others have not been able to replicate these cytochrome c release from mitochondria. Triacsin c, an findings in melanoma, and have suggested that difficulties in inhibitor of acyl-CoA-synthase, selectively induced cytochrome

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c release and apoptosome-mediated cell death in tumor cells the development of some cancers and to chemoresistance. Based and decreased tumor growth in a xenograft model (65). on our emerging knowledge, however, being able to trigger In cancers where a decrease in apoptosome components cell death via the apoptosome may provide a means to contributes to drug resistance, it may prove beneficial to use specifically kill tumor cells in some malignancies. The ongoing agents that increase expression of apoptosome components. study of the apoptosome will inform the development of drugs For example the ability of the pan–histone deacetylase that can activate the apoptosome and ultimately provide new inhibitor LAQ824 to cause cell death is associated with tools in the treatment of cancer. induction of Apaf-1 and procaspase-9 expression (70). In recent years significant progress has been made in understanding the core components of the apoptosome, Disclosure of Potential Conflicts of Interest but there are still many questions regarding its regulation. Defects in the apoptosome pathway may contribute to No potential conflicts of interest were disclosed.

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Elizabeth C. Ledgerwood and Ian M. Morison

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