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APAF-1 signaling in human melanoma
Cancer Letters 238 (2006) 168–179 www.elsevier.com/locate/canlet
Mini-review
APAF-1 signaling in human melanoma Andrea Anichini*, Roberta Mortarini, Marialuisa Sensi, Marina Zanon Unit of Human Tumor Immunobiology, Dept. of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian 1, 20133, Milan, Italy Received 11 June 2005; accepted 18 June 2005
Abstract Acquired resistance to mechanisms of programmed cell death is one of the hallmarks of cancer. Human melanoma, in advanced stage, is hardly curable, due to development of several strategies that impair apoptosis induced by the death receptor and the mitochondrial pathways of apoptosis. Among these apoptosis escape strategies, one is based on inactivation of proapoptotic factors such as Apoptotic Protease Activating Factor-1 (APAF-1). APAF-1 couples cytochrome c release from the mitochondria to caspase-9 activation and has been considered a central adaptor in the intrinsic pathway of programmed cell death. Inactivation of APAF-1 in human melanoma may impair the mitochondrial pathway of apoptosis induced by chemotherapeutic drugs that activate the p53 pathway, thus contributing to the development of chemoresistance. In-vivo, loss of expression of APAF-1 is associated with tumor progression, suggesting that APAF-1 inactivation may provide a selective survival advantage to neoplastic cells. However, recent results have indicated the existence of APAF-1-independent pathways of caspase activation and apoptosis in normal and neoplastic cells. Moreover, it has been found that expression of APAF-1 is not necessary for the apoptotic response of melanoma cells to different pro-apoptotic drugs. The emerging picture from results obtained in melanoma and other human tumors is that the relevance of the APAF-1 pathway in programmed cell death is cellcontext-dependent and related to the specificity of the pro-apoptotic-stimuli. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: APAF-1; Apoptosis; Melanoma
1. Introduction Human melanoma is hardly curable in advanced disease and clinical evolution in AJCC Stage III and IV is frequently disappointing [1–2]. The striking and progressive worsening of the clinical outcome, along
* Corresponding author. Tel.: C39 0223902817; fax: C39 0223902630. E-mail address: [email protected] (A. Anichini).
with clinical stage, reflects the powerful mechanisms of resistance to current therapies that can be developed in advanced stage of this disease. With respect to the action of chemotherapeutic drugs, human melanoma can be considered a paradigm of chemoresistance, given the wide range of drugs that have failed to show improvement of patients’ survival, and in spite of decades of clinical trials. Melanoma has proven resistant, in-vivo, to a large array of drugs acting with different mechanisms and belonging to different classes, including alkylating
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.06.034
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agents, antibiotics, plant-derived products, hormonal analogs and platinum drugs (see ref. [3] for review). One of the common mechanisms underlying such resistance of melanoma to pharmacological therapies is the development of defects in the cell death pathways. Such acquired defects are considered one of the hallmarks of cancer [4]. Several cell death pathways have been identified in recent years and can be broadly classified in apoptotic and non-apoptotic [5]. Apoptosis, or programmed cell death (PCD), is the best understood cell-suicide program that plays a central role in embryonic development, in the function of the immune system and in the maintenance of tissue homeostasis [5]. Non-apoptotic cell death pathways are less understood and can be exemplified by senescence, necrosis, mitotic catastrophe and autophagy [5]. The role of the non-apoptotic pathways of cell death in controlling melanoma response to therapy remains to be fully elucidated. In contrast, significant information has been gathered over the past 10–15 years in the molecular circuitry of apoptotic cell death [5] and in the mechanisms of melanoma resistance to apoptosis promoted by several chemotherapeutic drugs. Although human melanoma cells may show multiple defects, or alterations, at different steps along the PCD signaling cascade, several lines of evidence have pointed to the potential relevance of APAF-1 inactivation as an important mechanism of chemoresistance. Here we will review the evidence on the role of APAF-1 in apoptosis and in melanoma response to pro-apoptotic agents.
2. The basic signaling pathways of PCD PCD can be initiated by two partly interconnected pathways: the first one depends on triggering of death receptors expressed on the cell surface (the ‘extrinsic pathway’), while the other one is mediated by molecules released from the mitochondria (the ‘intrinsic pathway’) [6]. Six distinct death receptors have been identified (TNF-R1, Fas/CD95, DR3, TRAIL-R1, TRAIL-R2 and DR6) whose triggering may initiate the extrinsic pathway (see ref. [7–8] for review). Upon binding by the cognate ligand, the death receptors trimerize and, in their intra-cellular portion, assemble a death-inducing signaling complex
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(DISC), where the FADD/MORT1 adaptor recruits ‘initiator’ caspases, as pro-caspase-8 [8]. In the DISC, pro-caspase-8 undergoes auto-proteolytic activation. This then leads to triggering of the enzymatic activity of downstream effector caspases, as caspase-3 and -7. In response to a different set of stimuli, including hypoxia, growth factor deprivation, cell detachment and stress signals, such as drug-induced DNA damage, the intrinsic pathway is activated [6,8]. To this end, pro-apoptotic Bcl-2 family members, as Bax or Bak, can homooligomerize as multimers inserted into the mitochondria outer membrane [8]. This process is thought to produce discontinuities, and perhaps pores in such membrane, leading to mitochondria outer membrane permeabilization (MOMP) [9]. As result of MOMP, a component of the mitochondrial respiratory chain, cytochrome-c, present in the intermembrane space, is released from mitochondria into the cytosol, where it binds to APAF-1. In the presence of ATP, cytochrome-c and APAF-1 assemble into a multi-molecular complex (apoptosome) that recruits and activates the initiator caspase-9 [6,8]. Active caspase-9 then activates downstream effector caspases. In addition to these two main PCD mechanisms, a further ‘extrinsic’ pathway is activated by cytolytic T cells (CTL) and NK cells. In fact, activated T cells, upon specific recognition of antigens on the target, and NK cells, after triggering of activating NK receptors, can release cytotoxic granules. These granules, contain the poreforming protein perforin and several serine proteases (granzymes) [8]. Granzymes activate apoptosis by several mechanisms. Thus, for example, granzyme B can cleave inhibitors of the DNAse that degrades DNA, and can activate Bid, a pro-apoptotic member of the Bcl-2 family. Cleaved Bid then translocates to the mitochondria and promotes cytochrome-c release in the cytosol, thus activating the mitochondrial pathway [8]. In addition to the mitochondrial pathway, recent evidence indicates that even the endoplasmic reticulum (ER) plays a role in the intrinsic pathway of PCD [8]. The ER can initiate death pathways through mobilization of ER calcium stores, in presence of increasing ER stress, such as after accumulation of misfolded proteins [10]. In such conditions, Bcl-2 family members, as the pro-apoptotic Bax and Bak can translocate to the ER, not only to the mitochondria.
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ER-initiated apoptosis has been shown to depend on activation of caspase-12, which localizes at the cytosolic face of the ER and is capable of activating other downstream caspases [11]. Thus, it appears that the intrinsic pathway has at least two gateways: the mitochondria, through release of cytochrome c, and the ER. Although the basic ‘extrinsic’ and the ‘intrinsic’ pathways of PCD may be viewed as signaling cascades leading to activation of effector caspases, it is to be kept in mind that PCD can proceed even by caspase-independent mechanisms [8,12]. Thus, DNA degradation, one of the key events in apoptosis, can be achieved not only by a caspase-activated DNAse (CAD), but even by a caspase-independent mechanism mediated by mitochondrial proteins endonuclease G and AIF [8,12]. Furthermore, the two basic pathways of PCD (the cellular stress-activated, or ‘intrinsic’ and the death receptor-activated, or ‘extrinsic’ pathway) are not to be considered as fully independent, as these signaling cascades are connected at several points. In fact, for example, in FasL-induced apoptosis, the initiator caspases 8 and 10, can cleave Bid, thus leading to activation of the mitochondrial pathway [13]. On the other hand, in apoptosis initiated by drugs that interact with DNA, stabilization of p53 leads to transcriptional upregulation of the CD95 receptor/ligand system, leading to cell death promoted by the extrinsic pathway, and not only by the intrinsic one [14]. Similarly, apoptosis induced by drugs that affect gene transcription (such as the histone deacetylase inhibitors) has been recently shown to be mediated by activation of ligands and receptors of the extrinsic pathway [15].
3. PCD escape strategies in neoplastic cells Five major strategies of tumor resistance to apoptosis have been identified in neoplastic cells of different histological origin, including melanoma. These strategies include: (1) alterations in the p53 pathway, preventing the apoptotic response after cellular stress; (2) alterations in the PI3K/AKT pathway leading to promotion of cell survival; (3) expression of drug transporters that actively expel drugs from the cells; (4) up-regulation of anti-apoptotic
molecules, such as Bcl-2, and inhibitors of apoptosis (IAP proteins); (5) down-regulation/silencing of pro-apoptotic genes, such as Bax, death receptors, and APAF-1 [6]. The available evidence indicates that all these strategies can be exploited during tumor progression by human melanoma cells. This enables this tumor, as suggested by Soengas et al. [3], to become ‘bullet proof’ against a wide range of chemotherapeutic drugs. On the other hand, it seems clear that the different strategies to inactivate PCD do not have similar relevance or frequency in human melanoma. Thus, p53 mutations between exon 5 and 8 (the region spanning the most frequently mutated exons) have been recently described in 24% of a large panel of short-term melanoma cell lines [16]. However, published data (see Ref. [17] for review) indicate a frequency of p53 mutations in only 5–10% of primary and metastatic tumors, and a frequency of 20–30% for positive p53 staining by immunohistochemistry (consistent with enhanced protein stabilization in vivo) [17]. In contrast, constitutive activation of the PI3K/AKT signaling pathway is frequent in human melanoma. This takes place mainly through inactivation of PTEN, which acts as a negative regulator of AKT [18]. In fact, PTEN degrades phosphatidylinositol 3,4,5-trisphosphate (PIP3), the AKT activator [18], thus promoting the pro-survival and anti-apoptotic function of the PI3K/AKT pathway. In melanoma, up to 25–40% of tumors show loss of heterozigosity at the PTEN locus, or loss of PTEN protein (15–30% of tumors) [19–20]. The third strategy to escape from drug-induced apoptosis, by increased drug efflux, has been recently shown to be frequently activated in human melanoma. In fact, human melanoma cells may express, in-vitro and invivo, the drug transporter ABCB5 belonging to the P-glycoproteins family [21]. This transporter promotes the efflux of drugs as doxorubicin and, interestingly, it has been found expressed on a subset of CD133C ‘stem cell-like’ melanoma cells [21]. The fourth PCD escape strategy, based on constitutive expression or upregulation of inhibitors of apoptosis is frequently exploited by human melanoma cells. Thus, melanoma cells have been shown to upregulate inhibitors of apoptosis (IAP proteins), as XIAP, which can bind to and inhibit caspase-3 activation promoted by TRAIL [22]. Human melanomas, but not
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normal melanocytes, express high levels of another inhibitor of apoptosis, ML-IAP/Livin [23]. Interestingly, the activity of ML-IAP is inhibited by binding of SMAC/DIABLO, a mitochondrial protein that promotes caspase activation in the cytochrome c/ Apaf-1/caspase-9 pathway [24]. However, in melanoma cells, signaling through the the MEK Erk1/2 kinase pathway (which can be constitutively active due to frequent B-Raf or N-Ras mutations) has been shown to inhibit release of SMAC/DIABLO from mitochondria [25]. Additional inhibitors of apoptosis highly expressed in human melanoma cells include c-FLIP [26], whose function inhibits PCD through the death receptor pathway [27], and survivin [28], whose expression in lymph node metastases has been shown to represent a negative prognostic factor [28]. The fifth PCD escape strategy of melanoma cells is based on down-regulation/silencing of positive regulators of apoptosis, or of receptors for pro-apoptotic molecules. This strategy is exemplified by the loss of expression of TRAIL receptors [29] or of FAS/CD95 (see Ref. [7] for review), which blocks the death receptor pathway, and by inactivation of APAF-1, reported by Soengas et al. [30] in 2001, which has been thought to impair the mitochondrial pathway of PCD.
4. Structure and function of APAF-1 and of the apoptosome At the beginning of the past decade, the genetic analysis of the nematode Caenorhabditis elegans (C. elegans) identified three genes (Ced-3, -4 and -9) that controlled the process of programmed cell death [31–32]. Ced-3 (homologous to the pro-apoptotic cysteine proteases known as caspases) and Ced-4 were shown to be required for the execution of the apoptotic program [31]. In contrast, Ced-9, (homologous of mammalian bcl-2 family members) acting upstream of Ced-3 and Ced-4, prevented their activation [32]. In 1997, Zou et al. [33] reported the purification, cDNA cloning and characterization of the human homolog of C. elegans Ced-4 gene, which was named Apoptotic Protease Activating Factor-1 (APAF-1). The APAF-1 mRNA was found to be ubiquitously expressed in human adult and fetal tissues [33] and to yield a 130 kd cytoplasmatic protein of 1194 amino acids able to bind cytochrome-c and contributing
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to caspase-3 activation [33]. At the structural level (Fig. 1 A), the APAF-1 protein was initially shown to consist of a Ced-4 homologous domain that binds dATP/ATP, flanked by a caspase recruitment domain (CARD) and by 12 WD-40 repeats involved in the negative regulation of APAF-1 [34]. Different spliced forms of APAF-1 have been identified over the past years. These include APAF-1L, APAF-1XL, APAF1M, and APAF-1XS. These alternative APAF-1 forms differ from the original APAF-1 molecule in the number of WD40 repeats (12 or 13) and/or for the presence of additional sequences inserted between the CARD and the Ced-4 homologous domains [35–37]. Soon after the initial characterization of APAF-1, it was found that caspase-9 could bind to APAF-1 in a cytochrome-c- and dATP-dependent fashion [38], thus becoming activated [39]. The binding of caspase-9 to APAF-1 was mediated through the respective CARD domains present in both proteins [38]. After binding to APAF-1, and its activation, caspase-9 could then cleave and activate caspase-3 [38]. Interestingly, it was also found that cytochromec, released from mitochondria at the beginning of the apoptotic cascade, formed with APAF-1 a large (O1300 kd) multimeric complex (named apoptosome [40]), containing several APAF-1 subunits. The definition of the three-dimensional structure of ˚ resolution, by electron the apoptosome at 27 A cryomicroscopy [41], has provided significant insight into the mechanism of assembly and function of this macromolecular complex. The results of this investigation have indicated that the APAF-1-cytochrome-c apoptosome is a wheel-like multiprotein particle with 7 spokes and a central hub (Fig. 1B). According to the model proposed by Acehan et al [41], seven APAF-1 molecules contribute, through their CARD domains, and with the N-terminal portion of their Ced-4 homology domain, to the central hub of the apoptosome, where interaction with pro-caspase 9 takes place. The remaining part of each APAF-1 molecule contributes an arm and a Y shape domain (Fig. 1B), ending with two lobes. Each lobe, made of 6 or 7 WD-40 repeats (depending on the APAF-1 isoform) folds as a b propeller, and a cytochrome-c molecule binds between the two b propellers (Fig. 1B). According to this model, in the absence of cytochrome-c, APAF-1 would adopt an autoinhibitory conformation, with the CARD domain
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Fig. 1. Structure of APAF-1 and of the apoptosome. A. Structure of the APAF-1 protein showing the CARD domain, the Ced-4 homology domain and the WD40 repeats. B. Structure of the apoptosome after binding of APAF-1 to cytochrome-c, according to the model proposed by Acehan et al [41]. See text for details on the structural domains identified in the figure. C. Schematic diagram of the three-dimensional structure of APAF-1 (residues 1–591) in an inactive state, bound to ADP (yellow), as described by Riedl et al [42]. The structure was drawn with DS Viewer Pro 5.0 from the crystal structure data file 1Z6T as deposited at http://www.pdb.org. The five APAF-1 structural domains were coloured according to Riedl et al [42]. The N-terminal CARD domain (residues 1–107) is in green. The structure of APAF-1 shown in panel C may contribute to the hub region and to the initial segment of the arm region of the apoptosome, as shown in panel B. However, the APAF-1 structure shown in panel C has to undergo several conformational changes, promoted by binding and hydrolysis of dATP/ATP, to allow the assembly of the apoptosome.
bound to the region of the two b propellers. Assembly of the apoptosome would begin when cytochrome-c displaces the CARD domain from the space between the two b propellers, allowing the Ced-4 homology domain to bind to dATP/ATP and to undergo a conformational change. In turn, this would promote apoptosome assembly by allowing the CARD and Ced-4 homology domain regions of different APAF-1 molecules to interact and build the hub region of the apoptosome. At this stage, 7 pro-caspase-9 molecules, by CARD-CARD interactions with the hub domain, may bind to the apoptosome. The final step is the activation of pro-caspase-9, possibly by an intermediate step requiring the formation of caspase-9 dimers (see ref. [41] for further details on this model). The recent definition of the crystal structure of an
ADP-bound, WD-40-deleted APAF-1 molecule (Fig. 1C) has provided additional elements to understand the mechanism that keeps APAF-1 in an inactive state, before binding of ATP [42]. According to these results, inactive APAF-1 binds ADP, which is buried deeply at the interface between different APAF-1 structural domains (Fig. 1C). As proposed by Riedl et al. [42], ADP acts as an organizing centre that contributes to keep APAF-1 in an inactive state. The binding and hydrolysis of dATP/ATP would then promote extensive conformational changes (see [42] for details) that may lead to the formation of the heptameric apoptosome previously identified by electron cryomicroscopy [41]. Recent results have indicated that the APAF-1 pathway is subjected, in normal and neoplastic cells,
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to positive and negative regulatory interactions with several molecules. At least two positive regulators have been identified. The first one, NAC, is a CARDcontaining protein that associates with APAF-1 and promotes the activation of pro-caspase-9 by the apoptosome [43]. Another positive regulator, Nucling, has been shown to associate with the apoptosome and to promote its functions and traslocation to the nucleus [44]. The APAF-1 pathway has also several negative regulators. IAP proteins, as XIAP, can associate with the apoptosome and inhibit both the activation of caspase-3 and the release of active caspase-3 from the complex [45]. The negative action of IAP proteins, on the activation of the APAF1 pathway is counteracted by proteins as SMAC/ DIABLO [24], released by the mitochondria, that can remove IAP binding to caspase-9 [46], or as the protease OMI/HtrA2 that cleave IAPs [47]. The APAF-1 pathway is also subjected to control by heat shock proteins (HSP). Thus, at least three Hsps (HSp27, Hsp70, and Hsp90) have been implicated in the negative control of cell death by the APAF-1 pathway [48–51]. Hsp27 binds to cytochrome-c preventing its association with APAF-1 [48], while Hsp70 and Hsp-90 bind to APAF-1 and prevent caspase-9 activation [49–51]. More recently, it has been found that even the cAMP pathway contributes to the negative regulation of the APAF-1 pathway. In fact, extracellular signals that increase cAMP levels lead to activation of protein kinase A, which in turn inhibits cytochrome c-dependent recruitment of procaspase-9 to APAF-1 [52].
5. Apoptosome-dependent and -independent pathways of apoptosis Soon after the identification of APAF-1, its role and relevance for cellular homeostasis and development were addressed in gene targeted, APAF1K/K mice [53–54]. In one study [53] APAF1K/K mice exhibited remarkable craniofacial abnormalities, and died at birth, or shortly after birth. Similar abnormalities were found even in another study where APAF1K/K embryos did not survive beyond embryonic day 16 [54]. Moreover, ES cells or embryonic fibroblasts from APAF1K/K mice were resistant to apoptosis induced by UV or chemotherapeutic drugs
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as cisplatin, while the susceptibility to the FASdependent extrinsic pathway was retained in thymocytes and peripheral T cells from these animals [53]. Thus, these data indicated that APAF-1-dependent apoptosis is essential for normal CNS development and that APAF-1 plays a relevant role in stressinduced, but not in death-receptor induced apoptosis. In agreement with these findings, in a recent study by Hao et al. [55], ‘knockin’ mice expressing a mutant cytochrome-c unable to bind APAF-1, but retaining electron transport function (KA/KA mice), have been shown to display embryonic or peri-natal mortality due to extensive CNS defects. Moreover, in agreement with the previous reports on APAF-1K/K mice, no activation of caspase-9 and caspase-3 was achieved after UV irradiation of KA/KA embryonic fibroblasts (MEFs). However, interestingly, and in contrast with the behaviour of thymocytes from APAF1K/K mice, it was found that KA/KA thymocytes were normally susceptible to apoptosis induced by several stimuli, including etoposide, g and UV irradiation. Moreover, in these cells, caspase-9 and caspase-3 could be activated after g-irradiation, in spite of absence of APAF-1 oligomerization [55]. Thus, these data indicate that an apoptosome-independent, caspase activation pathway must exist in thymocytes in response to stress-induced pro-apoptotic stimuli. However, this pathway does not operate in MEFs. These data are not the only evidence that questions the role of APAF-1 in stress-induced apoptosis, in contrast with the early reports. In fact, initial studies had provided evidence for an essential role of caspase9 and APAF-1 in the stress- and oncogene-induced cell death regulated by p53 [56]. Cells from APAF-1deficient mice resisted apoptosis induced by serum deprivation or hypoxia, or even promoted after transduction with c-Myc and oncogenic RAS genes [56]. However, later, it was found that the APAF-1 pathway is not an obligate initiator of caspase activation in stress-induced, or in DNA damagepromoted apoptosis, and that caspase-9 is not the apical, initiator caspase in this process. In fact, caspase-2 activation, and not caspase-9 activation, was found to be the apical event required for activation of the proteolytic cascade after DNA damage in E1A-transformed fibroblasts, and even in some human cancer cell lines [57]. Interestingly, such
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activation of caspase-2 preceded mitochondrial permeabilization, and was required for such process, suggesting that the mitochondrial-APAF-1 pathway could be regarded as an amplifier, rather than an obligate initiator of stress-induced apoptosis. [57]. In addition, in caspase-9K/K and APAF- 1K/K mice, caspase activation and apoptosis could still be achieved [58]. Additional results have indicated that APAF-1K cells can undergo staurosporine-induced apoptosis, although by a caspase-independent mechanism and with a delayed kinetics [59]. More recent results have suggested that the role of the APAF-1 pathway in PCD may be cell-context-dependent. In fact, in response to cytotoxic drugs, processing of caspase-9 and apoptosis could be elicited in APAF1K/K myoblasts, but not in APAF1K/K fibroblasts [60]. In addition, expression of caspase-9, APAF-1, or even caspase-2, was not required for PCD after growth factor withdrawal, while their expression only accelerated the rate of apoptosis [61].
6. Expression and function of APAF-1 in human melanoma As mentioned in previous chapters, in 2001 Soengas et al. [30] described inactivation of APAF1 in human melanoma cells, resulting from loss of one allele and promoter methylation-dependent transcriptional silencing of the other one. This promoted melanoma chemoresistance to Adriamycin, a chemoterapeutic drug that induces p53-dependent apoptosis [30]. In that study the authors also hypothesized that loss of expression of APAF-1 in melanoma might explain the apparent paradox resulting from low frequency of p53 mutations in this tumor, in spite of its known chemoresistance to drugs that activate the p53 pathway. In fact, loss of expression of APAF-1, an important transcriptional target of p53, could contribute to explain the defective apoptotic response of this tumor, even in presence of a functional p53 pathway. Subsequent studies have been aimed at assessing expression of APAF-1 in-vivo, in melanoma lesions, and at correlating loss of APAF-1 with relevant clinical parameters and patients’outcome. Thus, APAF-1 was found expressed at higher levels in nevi compared to melanomas [62]. Moreover, a significant negative correlation was identified
between extent of APAF-1 expression and tumor thickness of the primary lesion, as well as between primary and metastatic melanomas [62]. In a similar study, in 70 primary melanoma biopsies, by immunohistochemistry, it was found that APAF-1 expression was significantly reduced, compared to normal nevi, although no association was found with other relevant parameters, such as tumor thickness, presence of ulceration, patient’s gender, age, or 5 year survival [63]. A significant decrease in APAF-1 expression has been recently confirmed in melanomas compared to nevi, and in primary tumors of increasing thickness [64]. Interestingly, no expression of APAF-1 was found in metastases in such study [64]. In another study, looking at loss of heterozigosity at the APAF-1 locus (12q22-23) by microsatellite probes in a large panel of primary and metastatic melanomas, a higher frequency of LOH was found in metastases compared to primary lesions [65]. Moreover, LOH at the APAF1 locus in metastases correlated with a worse prognosis [65]. Interestingly, in the same study it was found that LOH was frequent in the 12q22-23 region centromeric to APAF-1, suggesting presence of other, yet to be identified, tumor-related genes in the region. In a more recent study, the same authors found that allelic imbalance (AI) at 12q22-23 could be demonstrated in DNA present in serum of AJCC Stage IV patients [66]. Moreover, patients responding to biochemotherapy showed significantly lower frequency of AI at 12q22-23 in DNA from serum, compared to nonresponders. Furthermore, serum AI at 12q22-23 was associated with worse prognosis [66]. Similar results have been obtained even in glioblastomas, were LOH at 12q22-23 was found in 42% of the cases, and this was significantly associated with reduced APAF-1 mRNA and protein levels in the tumors [67]. However, this is not the only example of similarities between melanoma and other tumors. Thus, LOH at 12q22-23 has been documented in colorectal carcinomas and the frequency of LOH increased from 0% in adenomas to 53% in liver metastases [68]. Furthermore, APAF-1 mRNA levels were significantly reduced in tumors with allelic imbalance [68]. Even in cervical cancers significantly less lymph node metastases have been found in tumors with strong or moderate APAF-1 expression, as determined by immunohistochemistry, compared with weakly staining, or APAF-1-negative
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tumors [69]. Moreover, in early stage non-small cell lung cancer, 5-year survival was significantly higher in patients whose tumors showed a nuclear localization of APAF-1, compared to those with a cytoplasmic localization [70]. This suggests that a functional APAF-1 (which is expected to translocate to the nucleus during apoptosis) may be associated with better prognosis. At the functional level, the role of APAF-1 in the apoptosis of melanoma cells has been recently addressed in our laboratory, by evaluating response to different chemotherapeutic drugs in melanoma cell lines expressing or lacking APAF-1. To this end, changes in gene expression, effects on caspase activation and induction of apoptosis were evaluated [71]. We found that drugs acting with different mechanisms (camptothecin, celecoxib and an iNOS inhibitor) did not induce (in an APAF-1-negative tumor), nor upregulated (in an APAF-1-positive tumor) the expression of APAF-1, at either the mRNA or protein levels. In contrast, O30 genes belonging to the intrinsic or the extrinsic pathways of PCD were significantly modulated by one or more of these drugs. Interestingly, APAF-1 has been shown to be a transcriptional target of p53 [72], although in a tissue-specific fashion [73]. However, we found lack of modulation of APAF-1 in tumors with wild type p53, and even in response to drugs that activate the p53 pathway (such as camptothecin) [71]. In addition, we found that caspase-9, the ‘initiator’ caspase of the APAF-1 pathway, could be significantly activated by five different chemotherapeutic agents (celecoxib, cisplatin, camptothecin, betulinic acid and etoposide) even in APAF-1-negative melanoma cells. Accordingly, in these cells, drug treatment led also to activation of caspase-3, the effector caspase that is thought to be activated by caspase-9 [33]. This indicated that expression of APAF-1 is not necessary for activation of caspase-9 in response to cytotoxic drugs, in agreement with the evidence for existence of apoptosome-independent pathways for caspase-9 activation [55,57–60]. Interestingly, we also found that caspase-9 activation, promoted by etoposide or celecoxib, could be significantly reduced by inhibitors of caspase-2, -3 and -8 [71], even in APAF-1-negative melanoma cells. This provided further evidence against a role of APAF-1 as necessary activator of caspase-9 in
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response to these drugs, and suggested that other truly ‘apical’ caspases, can activate caspase-9 in this setting. Finally, in a large panel of melanomas expressing or lacking APAF-1 we did not find significant differences, related to the APAF-1 phenotype, in the extent of apoptosis induced by cisplatin, camptothecin and celecoxib. Only the apoptosis induced by a very high dose of etoposide was significantly higher in APAF-1C melanomas, compared to APAF-1K tumors [71]. Taken together, these results are consistent with the role of the APAF-1 pathway as an amplifier in the apoptotic response of human melanoma cells only to some cytotoxic drugs, such as etoposide. Moreover, it appears that the APAF-1 phenotype of melanoma cells may not represent an immediate predictor of the apoptotic response to several anti-cancer drugs. In agreement with this interpretation, in a screening of 60 cancer cell lines for response to a large set of anti-neoplastic agents, no significant correlation between drug sensitivity and the level of expression of APAF-1 was found [74]. Similarly, no relationship between APAF-1 protein expression and drug resistance was found in acute lymphoblastic leukemias [75]. On the other hand, it is to be pointed out that the relevance of the APAF-1 pathway, in drug induced apoptosis, may be not only drug-specific (as suggested by the differential response of APAF-1C and APAF-1K tumors to etoposide, but not to other drugs), but even cell-context-specific. Therefore, melanoma might represent an instance of a cell type where the APAF-1 pathway plays only a minor role for the response to several pro-apoptotic agents. As mentioned earlier in this review, the cell-contextspecific role of the APAF-1 pathway in apoptosis has been clearly documented by comparing the susceptibility to apoptosis of embryonal fibroblasts and thymocytes from KA/KA mice [55]. The same principle holds true even for the response to cytotoxic drugs by tumors arising from different tissues. In fact, in the model proposed by Fulda et al. [76], the apoptosis induced by anti-cancer drugs is mediated either by death receptors (through CD95 and caspase-8), or the mitochondrial pathway (through APAF-1/caspase-9), depending on the cell type that is responding to treatment. Thus, in lymphoma cells responding to etoposide, a role of caspase-8, the apical caspase in death-receptor-induced
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apoptosis, has been described [77]. In contrast, in glioma cells, overexpression of APAF-1 has been found to increase etoposide-induced apoptosis [78]. Similarly, the APAF-1 pathway has been shown to play a significant role in apoptosis induced in leukemia and lymphoma cells by different stimuli. In fact, in leukemia cell lines, resistance to UVinduced apoptosis has been found to depend on APAF-1 deficiency, and responsiveness can be restored by APAF-1 transfection in these lines [79]. In B lymphoma cells, sequestration of APAF-1 in lipid rafts at the plasma membrane prevents its association with cytochrome c, leading to resistance to etoposide or staurosporine [80]. Enforced expression of APAF-1 increased the concentration of APAF-1 in the cytosol restoring responsiveness to etoposide and staurosporine [80].
7. Why do melanoma cells lose expression of APAF-1? The overall picture, emerging from studies carried out in normal and neoplastic cells, as well as in gene targeted mice, since the human homolog of C. elegans Ced-4 was cloned, is that APAF-1 has a relevant role in the control of apoptosis. However, the specific role and relevance of the APAF-1 pathway for PCD appears to depend to a large extent on the interplay between the cell context where APAF-1 is acting and the nature of the pro-apoptotic stimulus. As discussed in previous chapters, human melanoma is not an exception to this rule, although an apparent paradox needs to be resolved. In fact, the progressive loss of APAF-1 during tumor progression suggests a relevant role of APAF-1 as a tumor suppressor. If the main function of APAF-1 is to act as an adaptor that couples apoptosis initiated by the mitochondria to activation of caspases, then the loss of APAF-1 would be expected to explain, at least in part, the chemoresistance of advanced melanoma. In fact, several chemotherapeutic drugs activate apoptosis through the mitochondrial pathway, even when they do not activate the p53 signaling cascade. However, the prediction that loss of APAF-1 may contribute to explain resistance to several anti-cancer drugs has not been confirmed, although this may be true in tumors other than melanoma. Nevertheless, it
is possible that the true selective advantage for melanoma cells, resulting from inactivation of APAF-1, is not that of acquiring resistance to different drugs currently used in the chemotherapy of this tumor. In support of this hypothesis, progressive loss of APAF-1 has been shown to be an early event in melanoma progression, detected even in primary melanomas of increasing thickness, or in primary tumors compared to nevi [62–64]. At these stages, no chemotherapy has been previously employed. Thus, the initial selective pressure for loss of APAF-1 cannot result from the clinical use of drugs that require a functional APAF-1 pathway. Therefore, it is likely that the loss of expression of APAF-1 might be relevant for promoting survival of neoplastic cells in the presence of forms of cellular stress unrelated to the action of chemotherapeutic drugs. One of such stress signals, that could act as selective pressure favouring early loss/inactivation of APAF-1 in melanoma progression, is hypoxia. Hypoxic regions are common in neoplastic tissues and hypoxia can lead to apoptosis by a p53dependent mitochondrial pathway [81–82]. Interestingly, a new APAF-1 interacting protein (APIP) has been recently identified [83] in skeletal muscle and heart that acts as a negative regulator of ischemic/ hypoxic-induced apoptosis. Moreover, gene profile analysis of neoplastic cells selected for increased apoptosis resistance to hypoxia, has indicated that one of the most upregulated genes is Hsp70 [84]. Among several other functions, Hsp70 is a known inhibitor of the APAF-1 pathway [49–50]. Taken together, these data are consistent with a model where APAF-1 could be a relevant player in hypoxic stress induced apoptosis, providing an early selective pressure for its loss in human melanoma, in spite of a more limited role of the APAF-1 pathway in druginduced apoptosis.
Acknowledgements Results mentioned in this review [71] were obtained thanks to the partial support from grants from the Ministry of Health, Rome and Compagnia di S. Paolo, Turin, Italy.
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Mini-review
APAF-1 signaling in human melanoma Andrea Anichini*, Roberta Mortarini, Marialuisa Sensi, Marina Zanon Unit of Human Tumor Immunobiology, Dept. of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian 1, 20133, Milan, Italy Received 11 June 2005; accepted 18 June 2005
Abstract Acquired resistance to mechanisms of programmed cell death is one of the hallmarks of cancer. Human melanoma, in advanced stage, is hardly curable, due to development of several strategies that impair apoptosis induced by the death receptor and the mitochondrial pathways of apoptosis. Among these apoptosis escape strategies, one is based on inactivation of proapoptotic factors such as Apoptotic Protease Activating Factor-1 (APAF-1). APAF-1 couples cytochrome c release from the mitochondria to caspase-9 activation and has been considered a central adaptor in the intrinsic pathway of programmed cell death. Inactivation of APAF-1 in human melanoma may impair the mitochondrial pathway of apoptosis induced by chemotherapeutic drugs that activate the p53 pathway, thus contributing to the development of chemoresistance. In-vivo, loss of expression of APAF-1 is associated with tumor progression, suggesting that APAF-1 inactivation may provide a selective survival advantage to neoplastic cells. However, recent results have indicated the existence of APAF-1-independent pathways of caspase activation and apoptosis in normal and neoplastic cells. Moreover, it has been found that expression of APAF-1 is not necessary for the apoptotic response of melanoma cells to different pro-apoptotic drugs. The emerging picture from results obtained in melanoma and other human tumors is that the relevance of the APAF-1 pathway in programmed cell death is cellcontext-dependent and related to the specificity of the pro-apoptotic-stimuli. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: APAF-1; Apoptosis; Melanoma
1. Introduction Human melanoma is hardly curable in advanced disease and clinical evolution in AJCC Stage III and IV is frequently disappointing [1–2]. The striking and progressive worsening of the clinical outcome, along
* Corresponding author. Tel.: C39 0223902817; fax: C39 0223902630. E-mail address: [email protected] (A. Anichini).
with clinical stage, reflects the powerful mechanisms of resistance to current therapies that can be developed in advanced stage of this disease. With respect to the action of chemotherapeutic drugs, human melanoma can be considered a paradigm of chemoresistance, given the wide range of drugs that have failed to show improvement of patients’ survival, and in spite of decades of clinical trials. Melanoma has proven resistant, in-vivo, to a large array of drugs acting with different mechanisms and belonging to different classes, including alkylating
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.06.034
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agents, antibiotics, plant-derived products, hormonal analogs and platinum drugs (see ref. [3] for review). One of the common mechanisms underlying such resistance of melanoma to pharmacological therapies is the development of defects in the cell death pathways. Such acquired defects are considered one of the hallmarks of cancer [4]. Several cell death pathways have been identified in recent years and can be broadly classified in apoptotic and non-apoptotic [5]. Apoptosis, or programmed cell death (PCD), is the best understood cell-suicide program that plays a central role in embryonic development, in the function of the immune system and in the maintenance of tissue homeostasis [5]. Non-apoptotic cell death pathways are less understood and can be exemplified by senescence, necrosis, mitotic catastrophe and autophagy [5]. The role of the non-apoptotic pathways of cell death in controlling melanoma response to therapy remains to be fully elucidated. In contrast, significant information has been gathered over the past 10–15 years in the molecular circuitry of apoptotic cell death [5] and in the mechanisms of melanoma resistance to apoptosis promoted by several chemotherapeutic drugs. Although human melanoma cells may show multiple defects, or alterations, at different steps along the PCD signaling cascade, several lines of evidence have pointed to the potential relevance of APAF-1 inactivation as an important mechanism of chemoresistance. Here we will review the evidence on the role of APAF-1 in apoptosis and in melanoma response to pro-apoptotic agents.
2. The basic signaling pathways of PCD PCD can be initiated by two partly interconnected pathways: the first one depends on triggering of death receptors expressed on the cell surface (the ‘extrinsic pathway’), while the other one is mediated by molecules released from the mitochondria (the ‘intrinsic pathway’) [6]. Six distinct death receptors have been identified (TNF-R1, Fas/CD95, DR3, TRAIL-R1, TRAIL-R2 and DR6) whose triggering may initiate the extrinsic pathway (see ref. [7–8] for review). Upon binding by the cognate ligand, the death receptors trimerize and, in their intra-cellular portion, assemble a death-inducing signaling complex
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(DISC), where the FADD/MORT1 adaptor recruits ‘initiator’ caspases, as pro-caspase-8 [8]. In the DISC, pro-caspase-8 undergoes auto-proteolytic activation. This then leads to triggering of the enzymatic activity of downstream effector caspases, as caspase-3 and -7. In response to a different set of stimuli, including hypoxia, growth factor deprivation, cell detachment and stress signals, such as drug-induced DNA damage, the intrinsic pathway is activated [6,8]. To this end, pro-apoptotic Bcl-2 family members, as Bax or Bak, can homooligomerize as multimers inserted into the mitochondria outer membrane [8]. This process is thought to produce discontinuities, and perhaps pores in such membrane, leading to mitochondria outer membrane permeabilization (MOMP) [9]. As result of MOMP, a component of the mitochondrial respiratory chain, cytochrome-c, present in the intermembrane space, is released from mitochondria into the cytosol, where it binds to APAF-1. In the presence of ATP, cytochrome-c and APAF-1 assemble into a multi-molecular complex (apoptosome) that recruits and activates the initiator caspase-9 [6,8]. Active caspase-9 then activates downstream effector caspases. In addition to these two main PCD mechanisms, a further ‘extrinsic’ pathway is activated by cytolytic T cells (CTL) and NK cells. In fact, activated T cells, upon specific recognition of antigens on the target, and NK cells, after triggering of activating NK receptors, can release cytotoxic granules. These granules, contain the poreforming protein perforin and several serine proteases (granzymes) [8]. Granzymes activate apoptosis by several mechanisms. Thus, for example, granzyme B can cleave inhibitors of the DNAse that degrades DNA, and can activate Bid, a pro-apoptotic member of the Bcl-2 family. Cleaved Bid then translocates to the mitochondria and promotes cytochrome-c release in the cytosol, thus activating the mitochondrial pathway [8]. In addition to the mitochondrial pathway, recent evidence indicates that even the endoplasmic reticulum (ER) plays a role in the intrinsic pathway of PCD [8]. The ER can initiate death pathways through mobilization of ER calcium stores, in presence of increasing ER stress, such as after accumulation of misfolded proteins [10]. In such conditions, Bcl-2 family members, as the pro-apoptotic Bax and Bak can translocate to the ER, not only to the mitochondria.
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ER-initiated apoptosis has been shown to depend on activation of caspase-12, which localizes at the cytosolic face of the ER and is capable of activating other downstream caspases [11]. Thus, it appears that the intrinsic pathway has at least two gateways: the mitochondria, through release of cytochrome c, and the ER. Although the basic ‘extrinsic’ and the ‘intrinsic’ pathways of PCD may be viewed as signaling cascades leading to activation of effector caspases, it is to be kept in mind that PCD can proceed even by caspase-independent mechanisms [8,12]. Thus, DNA degradation, one of the key events in apoptosis, can be achieved not only by a caspase-activated DNAse (CAD), but even by a caspase-independent mechanism mediated by mitochondrial proteins endonuclease G and AIF [8,12]. Furthermore, the two basic pathways of PCD (the cellular stress-activated, or ‘intrinsic’ and the death receptor-activated, or ‘extrinsic’ pathway) are not to be considered as fully independent, as these signaling cascades are connected at several points. In fact, for example, in FasL-induced apoptosis, the initiator caspases 8 and 10, can cleave Bid, thus leading to activation of the mitochondrial pathway [13]. On the other hand, in apoptosis initiated by drugs that interact with DNA, stabilization of p53 leads to transcriptional upregulation of the CD95 receptor/ligand system, leading to cell death promoted by the extrinsic pathway, and not only by the intrinsic one [14]. Similarly, apoptosis induced by drugs that affect gene transcription (such as the histone deacetylase inhibitors) has been recently shown to be mediated by activation of ligands and receptors of the extrinsic pathway [15].
3. PCD escape strategies in neoplastic cells Five major strategies of tumor resistance to apoptosis have been identified in neoplastic cells of different histological origin, including melanoma. These strategies include: (1) alterations in the p53 pathway, preventing the apoptotic response after cellular stress; (2) alterations in the PI3K/AKT pathway leading to promotion of cell survival; (3) expression of drug transporters that actively expel drugs from the cells; (4) up-regulation of anti-apoptotic
molecules, such as Bcl-2, and inhibitors of apoptosis (IAP proteins); (5) down-regulation/silencing of pro-apoptotic genes, such as Bax, death receptors, and APAF-1 [6]. The available evidence indicates that all these strategies can be exploited during tumor progression by human melanoma cells. This enables this tumor, as suggested by Soengas et al. [3], to become ‘bullet proof’ against a wide range of chemotherapeutic drugs. On the other hand, it seems clear that the different strategies to inactivate PCD do not have similar relevance or frequency in human melanoma. Thus, p53 mutations between exon 5 and 8 (the region spanning the most frequently mutated exons) have been recently described in 24% of a large panel of short-term melanoma cell lines [16]. However, published data (see Ref. [17] for review) indicate a frequency of p53 mutations in only 5–10% of primary and metastatic tumors, and a frequency of 20–30% for positive p53 staining by immunohistochemistry (consistent with enhanced protein stabilization in vivo) [17]. In contrast, constitutive activation of the PI3K/AKT signaling pathway is frequent in human melanoma. This takes place mainly through inactivation of PTEN, which acts as a negative regulator of AKT [18]. In fact, PTEN degrades phosphatidylinositol 3,4,5-trisphosphate (PIP3), the AKT activator [18], thus promoting the pro-survival and anti-apoptotic function of the PI3K/AKT pathway. In melanoma, up to 25–40% of tumors show loss of heterozigosity at the PTEN locus, or loss of PTEN protein (15–30% of tumors) [19–20]. The third strategy to escape from drug-induced apoptosis, by increased drug efflux, has been recently shown to be frequently activated in human melanoma. In fact, human melanoma cells may express, in-vitro and invivo, the drug transporter ABCB5 belonging to the P-glycoproteins family [21]. This transporter promotes the efflux of drugs as doxorubicin and, interestingly, it has been found expressed on a subset of CD133C ‘stem cell-like’ melanoma cells [21]. The fourth PCD escape strategy, based on constitutive expression or upregulation of inhibitors of apoptosis is frequently exploited by human melanoma cells. Thus, melanoma cells have been shown to upregulate inhibitors of apoptosis (IAP proteins), as XIAP, which can bind to and inhibit caspase-3 activation promoted by TRAIL [22]. Human melanomas, but not
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normal melanocytes, express high levels of another inhibitor of apoptosis, ML-IAP/Livin [23]. Interestingly, the activity of ML-IAP is inhibited by binding of SMAC/DIABLO, a mitochondrial protein that promotes caspase activation in the cytochrome c/ Apaf-1/caspase-9 pathway [24]. However, in melanoma cells, signaling through the the MEK Erk1/2 kinase pathway (which can be constitutively active due to frequent B-Raf or N-Ras mutations) has been shown to inhibit release of SMAC/DIABLO from mitochondria [25]. Additional inhibitors of apoptosis highly expressed in human melanoma cells include c-FLIP [26], whose function inhibits PCD through the death receptor pathway [27], and survivin [28], whose expression in lymph node metastases has been shown to represent a negative prognostic factor [28]. The fifth PCD escape strategy of melanoma cells is based on down-regulation/silencing of positive regulators of apoptosis, or of receptors for pro-apoptotic molecules. This strategy is exemplified by the loss of expression of TRAIL receptors [29] or of FAS/CD95 (see Ref. [7] for review), which blocks the death receptor pathway, and by inactivation of APAF-1, reported by Soengas et al. [30] in 2001, which has been thought to impair the mitochondrial pathway of PCD.
4. Structure and function of APAF-1 and of the apoptosome At the beginning of the past decade, the genetic analysis of the nematode Caenorhabditis elegans (C. elegans) identified three genes (Ced-3, -4 and -9) that controlled the process of programmed cell death [31–32]. Ced-3 (homologous to the pro-apoptotic cysteine proteases known as caspases) and Ced-4 were shown to be required for the execution of the apoptotic program [31]. In contrast, Ced-9, (homologous of mammalian bcl-2 family members) acting upstream of Ced-3 and Ced-4, prevented their activation [32]. In 1997, Zou et al. [33] reported the purification, cDNA cloning and characterization of the human homolog of C. elegans Ced-4 gene, which was named Apoptotic Protease Activating Factor-1 (APAF-1). The APAF-1 mRNA was found to be ubiquitously expressed in human adult and fetal tissues [33] and to yield a 130 kd cytoplasmatic protein of 1194 amino acids able to bind cytochrome-c and contributing
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to caspase-3 activation [33]. At the structural level (Fig. 1 A), the APAF-1 protein was initially shown to consist of a Ced-4 homologous domain that binds dATP/ATP, flanked by a caspase recruitment domain (CARD) and by 12 WD-40 repeats involved in the negative regulation of APAF-1 [34]. Different spliced forms of APAF-1 have been identified over the past years. These include APAF-1L, APAF-1XL, APAF1M, and APAF-1XS. These alternative APAF-1 forms differ from the original APAF-1 molecule in the number of WD40 repeats (12 or 13) and/or for the presence of additional sequences inserted between the CARD and the Ced-4 homologous domains [35–37]. Soon after the initial characterization of APAF-1, it was found that caspase-9 could bind to APAF-1 in a cytochrome-c- and dATP-dependent fashion [38], thus becoming activated [39]. The binding of caspase-9 to APAF-1 was mediated through the respective CARD domains present in both proteins [38]. After binding to APAF-1, and its activation, caspase-9 could then cleave and activate caspase-3 [38]. Interestingly, it was also found that cytochromec, released from mitochondria at the beginning of the apoptotic cascade, formed with APAF-1 a large (O1300 kd) multimeric complex (named apoptosome [40]), containing several APAF-1 subunits. The definition of the three-dimensional structure of ˚ resolution, by electron the apoptosome at 27 A cryomicroscopy [41], has provided significant insight into the mechanism of assembly and function of this macromolecular complex. The results of this investigation have indicated that the APAF-1-cytochrome-c apoptosome is a wheel-like multiprotein particle with 7 spokes and a central hub (Fig. 1B). According to the model proposed by Acehan et al [41], seven APAF-1 molecules contribute, through their CARD domains, and with the N-terminal portion of their Ced-4 homology domain, to the central hub of the apoptosome, where interaction with pro-caspase 9 takes place. The remaining part of each APAF-1 molecule contributes an arm and a Y shape domain (Fig. 1B), ending with two lobes. Each lobe, made of 6 or 7 WD-40 repeats (depending on the APAF-1 isoform) folds as a b propeller, and a cytochrome-c molecule binds between the two b propellers (Fig. 1B). According to this model, in the absence of cytochrome-c, APAF-1 would adopt an autoinhibitory conformation, with the CARD domain
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Fig. 1. Structure of APAF-1 and of the apoptosome. A. Structure of the APAF-1 protein showing the CARD domain, the Ced-4 homology domain and the WD40 repeats. B. Structure of the apoptosome after binding of APAF-1 to cytochrome-c, according to the model proposed by Acehan et al [41]. See text for details on the structural domains identified in the figure. C. Schematic diagram of the three-dimensional structure of APAF-1 (residues 1–591) in an inactive state, bound to ADP (yellow), as described by Riedl et al [42]. The structure was drawn with DS Viewer Pro 5.0 from the crystal structure data file 1Z6T as deposited at http://www.pdb.org. The five APAF-1 structural domains were coloured according to Riedl et al [42]. The N-terminal CARD domain (residues 1–107) is in green. The structure of APAF-1 shown in panel C may contribute to the hub region and to the initial segment of the arm region of the apoptosome, as shown in panel B. However, the APAF-1 structure shown in panel C has to undergo several conformational changes, promoted by binding and hydrolysis of dATP/ATP, to allow the assembly of the apoptosome.
bound to the region of the two b propellers. Assembly of the apoptosome would begin when cytochrome-c displaces the CARD domain from the space between the two b propellers, allowing the Ced-4 homology domain to bind to dATP/ATP and to undergo a conformational change. In turn, this would promote apoptosome assembly by allowing the CARD and Ced-4 homology domain regions of different APAF-1 molecules to interact and build the hub region of the apoptosome. At this stage, 7 pro-caspase-9 molecules, by CARD-CARD interactions with the hub domain, may bind to the apoptosome. The final step is the activation of pro-caspase-9, possibly by an intermediate step requiring the formation of caspase-9 dimers (see ref. [41] for further details on this model). The recent definition of the crystal structure of an
ADP-bound, WD-40-deleted APAF-1 molecule (Fig. 1C) has provided additional elements to understand the mechanism that keeps APAF-1 in an inactive state, before binding of ATP [42]. According to these results, inactive APAF-1 binds ADP, which is buried deeply at the interface between different APAF-1 structural domains (Fig. 1C). As proposed by Riedl et al. [42], ADP acts as an organizing centre that contributes to keep APAF-1 in an inactive state. The binding and hydrolysis of dATP/ATP would then promote extensive conformational changes (see [42] for details) that may lead to the formation of the heptameric apoptosome previously identified by electron cryomicroscopy [41]. Recent results have indicated that the APAF-1 pathway is subjected, in normal and neoplastic cells,
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to positive and negative regulatory interactions with several molecules. At least two positive regulators have been identified. The first one, NAC, is a CARDcontaining protein that associates with APAF-1 and promotes the activation of pro-caspase-9 by the apoptosome [43]. Another positive regulator, Nucling, has been shown to associate with the apoptosome and to promote its functions and traslocation to the nucleus [44]. The APAF-1 pathway has also several negative regulators. IAP proteins, as XIAP, can associate with the apoptosome and inhibit both the activation of caspase-3 and the release of active caspase-3 from the complex [45]. The negative action of IAP proteins, on the activation of the APAF1 pathway is counteracted by proteins as SMAC/ DIABLO [24], released by the mitochondria, that can remove IAP binding to caspase-9 [46], or as the protease OMI/HtrA2 that cleave IAPs [47]. The APAF-1 pathway is also subjected to control by heat shock proteins (HSP). Thus, at least three Hsps (HSp27, Hsp70, and Hsp90) have been implicated in the negative control of cell death by the APAF-1 pathway [48–51]. Hsp27 binds to cytochrome-c preventing its association with APAF-1 [48], while Hsp70 and Hsp-90 bind to APAF-1 and prevent caspase-9 activation [49–51]. More recently, it has been found that even the cAMP pathway contributes to the negative regulation of the APAF-1 pathway. In fact, extracellular signals that increase cAMP levels lead to activation of protein kinase A, which in turn inhibits cytochrome c-dependent recruitment of procaspase-9 to APAF-1 [52].
5. Apoptosome-dependent and -independent pathways of apoptosis Soon after the identification of APAF-1, its role and relevance for cellular homeostasis and development were addressed in gene targeted, APAF1K/K mice [53–54]. In one study [53] APAF1K/K mice exhibited remarkable craniofacial abnormalities, and died at birth, or shortly after birth. Similar abnormalities were found even in another study where APAF1K/K embryos did not survive beyond embryonic day 16 [54]. Moreover, ES cells or embryonic fibroblasts from APAF1K/K mice were resistant to apoptosis induced by UV or chemotherapeutic drugs
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as cisplatin, while the susceptibility to the FASdependent extrinsic pathway was retained in thymocytes and peripheral T cells from these animals [53]. Thus, these data indicated that APAF-1-dependent apoptosis is essential for normal CNS development and that APAF-1 plays a relevant role in stressinduced, but not in death-receptor induced apoptosis. In agreement with these findings, in a recent study by Hao et al. [55], ‘knockin’ mice expressing a mutant cytochrome-c unable to bind APAF-1, but retaining electron transport function (KA/KA mice), have been shown to display embryonic or peri-natal mortality due to extensive CNS defects. Moreover, in agreement with the previous reports on APAF-1K/K mice, no activation of caspase-9 and caspase-3 was achieved after UV irradiation of KA/KA embryonic fibroblasts (MEFs). However, interestingly, and in contrast with the behaviour of thymocytes from APAF1K/K mice, it was found that KA/KA thymocytes were normally susceptible to apoptosis induced by several stimuli, including etoposide, g and UV irradiation. Moreover, in these cells, caspase-9 and caspase-3 could be activated after g-irradiation, in spite of absence of APAF-1 oligomerization [55]. Thus, these data indicate that an apoptosome-independent, caspase activation pathway must exist in thymocytes in response to stress-induced pro-apoptotic stimuli. However, this pathway does not operate in MEFs. These data are not the only evidence that questions the role of APAF-1 in stress-induced apoptosis, in contrast with the early reports. In fact, initial studies had provided evidence for an essential role of caspase9 and APAF-1 in the stress- and oncogene-induced cell death regulated by p53 [56]. Cells from APAF-1deficient mice resisted apoptosis induced by serum deprivation or hypoxia, or even promoted after transduction with c-Myc and oncogenic RAS genes [56]. However, later, it was found that the APAF-1 pathway is not an obligate initiator of caspase activation in stress-induced, or in DNA damagepromoted apoptosis, and that caspase-9 is not the apical, initiator caspase in this process. In fact, caspase-2 activation, and not caspase-9 activation, was found to be the apical event required for activation of the proteolytic cascade after DNA damage in E1A-transformed fibroblasts, and even in some human cancer cell lines [57]. Interestingly, such
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activation of caspase-2 preceded mitochondrial permeabilization, and was required for such process, suggesting that the mitochondrial-APAF-1 pathway could be regarded as an amplifier, rather than an obligate initiator of stress-induced apoptosis. [57]. In addition, in caspase-9K/K and APAF- 1K/K mice, caspase activation and apoptosis could still be achieved [58]. Additional results have indicated that APAF-1K cells can undergo staurosporine-induced apoptosis, although by a caspase-independent mechanism and with a delayed kinetics [59]. More recent results have suggested that the role of the APAF-1 pathway in PCD may be cell-context-dependent. In fact, in response to cytotoxic drugs, processing of caspase-9 and apoptosis could be elicited in APAF1K/K myoblasts, but not in APAF1K/K fibroblasts [60]. In addition, expression of caspase-9, APAF-1, or even caspase-2, was not required for PCD after growth factor withdrawal, while their expression only accelerated the rate of apoptosis [61].
6. Expression and function of APAF-1 in human melanoma As mentioned in previous chapters, in 2001 Soengas et al. [30] described inactivation of APAF1 in human melanoma cells, resulting from loss of one allele and promoter methylation-dependent transcriptional silencing of the other one. This promoted melanoma chemoresistance to Adriamycin, a chemoterapeutic drug that induces p53-dependent apoptosis [30]. In that study the authors also hypothesized that loss of expression of APAF-1 in melanoma might explain the apparent paradox resulting from low frequency of p53 mutations in this tumor, in spite of its known chemoresistance to drugs that activate the p53 pathway. In fact, loss of expression of APAF-1, an important transcriptional target of p53, could contribute to explain the defective apoptotic response of this tumor, even in presence of a functional p53 pathway. Subsequent studies have been aimed at assessing expression of APAF-1 in-vivo, in melanoma lesions, and at correlating loss of APAF-1 with relevant clinical parameters and patients’outcome. Thus, APAF-1 was found expressed at higher levels in nevi compared to melanomas [62]. Moreover, a significant negative correlation was identified
between extent of APAF-1 expression and tumor thickness of the primary lesion, as well as between primary and metastatic melanomas [62]. In a similar study, in 70 primary melanoma biopsies, by immunohistochemistry, it was found that APAF-1 expression was significantly reduced, compared to normal nevi, although no association was found with other relevant parameters, such as tumor thickness, presence of ulceration, patient’s gender, age, or 5 year survival [63]. A significant decrease in APAF-1 expression has been recently confirmed in melanomas compared to nevi, and in primary tumors of increasing thickness [64]. Interestingly, no expression of APAF-1 was found in metastases in such study [64]. In another study, looking at loss of heterozigosity at the APAF-1 locus (12q22-23) by microsatellite probes in a large panel of primary and metastatic melanomas, a higher frequency of LOH was found in metastases compared to primary lesions [65]. Moreover, LOH at the APAF1 locus in metastases correlated with a worse prognosis [65]. Interestingly, in the same study it was found that LOH was frequent in the 12q22-23 region centromeric to APAF-1, suggesting presence of other, yet to be identified, tumor-related genes in the region. In a more recent study, the same authors found that allelic imbalance (AI) at 12q22-23 could be demonstrated in DNA present in serum of AJCC Stage IV patients [66]. Moreover, patients responding to biochemotherapy showed significantly lower frequency of AI at 12q22-23 in DNA from serum, compared to nonresponders. Furthermore, serum AI at 12q22-23 was associated with worse prognosis [66]. Similar results have been obtained even in glioblastomas, were LOH at 12q22-23 was found in 42% of the cases, and this was significantly associated with reduced APAF-1 mRNA and protein levels in the tumors [67]. However, this is not the only example of similarities between melanoma and other tumors. Thus, LOH at 12q22-23 has been documented in colorectal carcinomas and the frequency of LOH increased from 0% in adenomas to 53% in liver metastases [68]. Furthermore, APAF-1 mRNA levels were significantly reduced in tumors with allelic imbalance [68]. Even in cervical cancers significantly less lymph node metastases have been found in tumors with strong or moderate APAF-1 expression, as determined by immunohistochemistry, compared with weakly staining, or APAF-1-negative
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tumors [69]. Moreover, in early stage non-small cell lung cancer, 5-year survival was significantly higher in patients whose tumors showed a nuclear localization of APAF-1, compared to those with a cytoplasmic localization [70]. This suggests that a functional APAF-1 (which is expected to translocate to the nucleus during apoptosis) may be associated with better prognosis. At the functional level, the role of APAF-1 in the apoptosis of melanoma cells has been recently addressed in our laboratory, by evaluating response to different chemotherapeutic drugs in melanoma cell lines expressing or lacking APAF-1. To this end, changes in gene expression, effects on caspase activation and induction of apoptosis were evaluated [71]. We found that drugs acting with different mechanisms (camptothecin, celecoxib and an iNOS inhibitor) did not induce (in an APAF-1-negative tumor), nor upregulated (in an APAF-1-positive tumor) the expression of APAF-1, at either the mRNA or protein levels. In contrast, O30 genes belonging to the intrinsic or the extrinsic pathways of PCD were significantly modulated by one or more of these drugs. Interestingly, APAF-1 has been shown to be a transcriptional target of p53 [72], although in a tissue-specific fashion [73]. However, we found lack of modulation of APAF-1 in tumors with wild type p53, and even in response to drugs that activate the p53 pathway (such as camptothecin) [71]. In addition, we found that caspase-9, the ‘initiator’ caspase of the APAF-1 pathway, could be significantly activated by five different chemotherapeutic agents (celecoxib, cisplatin, camptothecin, betulinic acid and etoposide) even in APAF-1-negative melanoma cells. Accordingly, in these cells, drug treatment led also to activation of caspase-3, the effector caspase that is thought to be activated by caspase-9 [33]. This indicated that expression of APAF-1 is not necessary for activation of caspase-9 in response to cytotoxic drugs, in agreement with the evidence for existence of apoptosome-independent pathways for caspase-9 activation [55,57–60]. Interestingly, we also found that caspase-9 activation, promoted by etoposide or celecoxib, could be significantly reduced by inhibitors of caspase-2, -3 and -8 [71], even in APAF-1-negative melanoma cells. This provided further evidence against a role of APAF-1 as necessary activator of caspase-9 in
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response to these drugs, and suggested that other truly ‘apical’ caspases, can activate caspase-9 in this setting. Finally, in a large panel of melanomas expressing or lacking APAF-1 we did not find significant differences, related to the APAF-1 phenotype, in the extent of apoptosis induced by cisplatin, camptothecin and celecoxib. Only the apoptosis induced by a very high dose of etoposide was significantly higher in APAF-1C melanomas, compared to APAF-1K tumors [71]. Taken together, these results are consistent with the role of the APAF-1 pathway as an amplifier in the apoptotic response of human melanoma cells only to some cytotoxic drugs, such as etoposide. Moreover, it appears that the APAF-1 phenotype of melanoma cells may not represent an immediate predictor of the apoptotic response to several anti-cancer drugs. In agreement with this interpretation, in a screening of 60 cancer cell lines for response to a large set of anti-neoplastic agents, no significant correlation between drug sensitivity and the level of expression of APAF-1 was found [74]. Similarly, no relationship between APAF-1 protein expression and drug resistance was found in acute lymphoblastic leukemias [75]. On the other hand, it is to be pointed out that the relevance of the APAF-1 pathway, in drug induced apoptosis, may be not only drug-specific (as suggested by the differential response of APAF-1C and APAF-1K tumors to etoposide, but not to other drugs), but even cell-context-specific. Therefore, melanoma might represent an instance of a cell type where the APAF-1 pathway plays only a minor role for the response to several pro-apoptotic agents. As mentioned earlier in this review, the cell-contextspecific role of the APAF-1 pathway in apoptosis has been clearly documented by comparing the susceptibility to apoptosis of embryonal fibroblasts and thymocytes from KA/KA mice [55]. The same principle holds true even for the response to cytotoxic drugs by tumors arising from different tissues. In fact, in the model proposed by Fulda et al. [76], the apoptosis induced by anti-cancer drugs is mediated either by death receptors (through CD95 and caspase-8), or the mitochondrial pathway (through APAF-1/caspase-9), depending on the cell type that is responding to treatment. Thus, in lymphoma cells responding to etoposide, a role of caspase-8, the apical caspase in death-receptor-induced
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apoptosis, has been described [77]. In contrast, in glioma cells, overexpression of APAF-1 has been found to increase etoposide-induced apoptosis [78]. Similarly, the APAF-1 pathway has been shown to play a significant role in apoptosis induced in leukemia and lymphoma cells by different stimuli. In fact, in leukemia cell lines, resistance to UVinduced apoptosis has been found to depend on APAF-1 deficiency, and responsiveness can be restored by APAF-1 transfection in these lines [79]. In B lymphoma cells, sequestration of APAF-1 in lipid rafts at the plasma membrane prevents its association with cytochrome c, leading to resistance to etoposide or staurosporine [80]. Enforced expression of APAF-1 increased the concentration of APAF-1 in the cytosol restoring responsiveness to etoposide and staurosporine [80].
7. Why do melanoma cells lose expression of APAF-1? The overall picture, emerging from studies carried out in normal and neoplastic cells, as well as in gene targeted mice, since the human homolog of C. elegans Ced-4 was cloned, is that APAF-1 has a relevant role in the control of apoptosis. However, the specific role and relevance of the APAF-1 pathway for PCD appears to depend to a large extent on the interplay between the cell context where APAF-1 is acting and the nature of the pro-apoptotic stimulus. As discussed in previous chapters, human melanoma is not an exception to this rule, although an apparent paradox needs to be resolved. In fact, the progressive loss of APAF-1 during tumor progression suggests a relevant role of APAF-1 as a tumor suppressor. If the main function of APAF-1 is to act as an adaptor that couples apoptosis initiated by the mitochondria to activation of caspases, then the loss of APAF-1 would be expected to explain, at least in part, the chemoresistance of advanced melanoma. In fact, several chemotherapeutic drugs activate apoptosis through the mitochondrial pathway, even when they do not activate the p53 signaling cascade. However, the prediction that loss of APAF-1 may contribute to explain resistance to several anti-cancer drugs has not been confirmed, although this may be true in tumors other than melanoma. Nevertheless, it
is possible that the true selective advantage for melanoma cells, resulting from inactivation of APAF-1, is not that of acquiring resistance to different drugs currently used in the chemotherapy of this tumor. In support of this hypothesis, progressive loss of APAF-1 has been shown to be an early event in melanoma progression, detected even in primary melanomas of increasing thickness, or in primary tumors compared to nevi [62–64]. At these stages, no chemotherapy has been previously employed. Thus, the initial selective pressure for loss of APAF-1 cannot result from the clinical use of drugs that require a functional APAF-1 pathway. Therefore, it is likely that the loss of expression of APAF-1 might be relevant for promoting survival of neoplastic cells in the presence of forms of cellular stress unrelated to the action of chemotherapeutic drugs. One of such stress signals, that could act as selective pressure favouring early loss/inactivation of APAF-1 in melanoma progression, is hypoxia. Hypoxic regions are common in neoplastic tissues and hypoxia can lead to apoptosis by a p53dependent mitochondrial pathway [81–82]. Interestingly, a new APAF-1 interacting protein (APIP) has been recently identified [83] in skeletal muscle and heart that acts as a negative regulator of ischemic/ hypoxic-induced apoptosis. Moreover, gene profile analysis of neoplastic cells selected for increased apoptosis resistance to hypoxia, has indicated that one of the most upregulated genes is Hsp70 [84]. Among several other functions, Hsp70 is a known inhibitor of the APAF-1 pathway [49–50]. Taken together, these data are consistent with a model where APAF-1 could be a relevant player in hypoxic stress induced apoptosis, providing an early selective pressure for its loss in human melanoma, in spite of a more limited role of the APAF-1 pathway in druginduced apoptosis.
Acknowledgements Results mentioned in this review [71] were obtained thanks to the partial support from grants from the Ministry of Health, Rome and Compagnia di S. Paolo, Turin, Italy.
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