Alterations in the Apoptotic Machinery and Their Potential Role in Anticancer Drug Resistance

Alterations in the apoptotic machinery and their potential role in anticancer drug resistance

Scott H Kaufmann*,1,2 and David L Vaux3

1Division of Oncology Research, Mayo Clinic, Rochester, MN, 55905, USA; 2Department of Molecular Pharmacology, Mayo Graduate School, Rochester, MN 55905, USA; 3The Walter and Eliza Hall Institute, Victoria 3050, Australia

Anticancer drugs can potentially kill cells in two might be acceptable in some settings, the current fundamentally different ways, by interfering with cellular regulatory climate favors the development of cytotoxic processes that are essential for maintenance of viability or agents. Despite the approval of B100 such agents for by triggering an endogenous physiological cell death the treatment of cancer, however, the age-adjusted death mechanism. Apoptosis is a form of physiological cell rate from most common epithelial neoplasms has death mediated by caspases, a unique family of intracel- scarcely changed in the past four decades (Greenlee lular cysteine proteases. Zymogen forms of these pro- et al., 2001). These observations suggest that cytotoxic teases are found in virtually all somatic cells, but remain agents often fail to selectively eradicate neoplastic cells latent until their activation is induced by ligation of and provide the impetus for asking whether there is specific cell surface receptors (the so-called ‘death something wrong with the cell death process in cancer receptors’), by mitochondrial alterations that allow cells. release of cytochrome c and other intermembrane components, or possibly by other mechanisms. Most anticancer drugs activate the mitochondrial pathway. This All cells are mortal apoptotic pathway is regulated by pro- and antiapoptotic members of the Bcl-2 family of proteins. Once activated, As a starting point, it is important to emphasize that all certain caspases might also be controlled by the inhibitor cells, whether normal or neoplastic, can be killed. If a of apoptosis (IAP) proteins. Alterations in apoptotic drug or toxin blocks a metabolic process that is essential pathway components or their regulators have been for cell survival, the cell will die. Given at high enough detected in a variety of cancers, suggesting that loss of doses, any agent that inhibits an important process, such the ability of cells to undergo apoptosis might contribute as protein synthesis, ATP production, or the ability to to carcinogenesis. Because cancer therapies such as maintain plasma membrane integrity, will be able to kill radiation, glucocorticoids, and chemotherapeutic drugs all cancer cells. The obvious problem is that such a exert their beneficial effects, at least in part, by inducing treatment would probably kill all normal cells as well, apoptosis of cancer cells, the same alterations in apoptotic leading to death of the patient. pathways would be predicted to contribute to resistance. A key issue is whether the direct toxic activity of these treatments is of benefit when neoplastic cells contain changes that diminish their ability to undergo apoptosis. Most cells can be suicidal Oncogene (2003) 22, 7414–7430. doi:10.1038/sj.onc.1206945 A second point to emphasize at the outset is that Keywords: apoptosis; caspases; carcinogenesis; Bcl-2; chemotherapeutic agents can have two distinct but IAP; drug resistance; stress response related effects on cells. As outlined in Figure 1, they can directly block a vital cellular process, and they can stress a cell, indirectly prompting it to kill itself. Either or both of these effects can contribute to cell death. Introduction The first effect can be adequately described by a ‘lock and key’ model. For example, paclitaxel alters b-tubulin The goal of most current cancer therapies, including function, methotrexate blocks dihydrofolate reductase, radiation, chemotherapy, immunotherapy, and gene and camptothecin inhibits the religation step of therapy, is the reduction or elimination of cancer cells. DNA topoisomerase I. For these types of agents, the Although cytostatic effects, if complete and durable, drug–target interaction has been shown to cause cellular changes that could result in cell death. Paclitaxel, for *Correspondence: SH Kaufmann, Guggenheim 1342C, Mayo Clinic, example, causes mitotic arrest followed by exit into an 200 First St., SW, Rochester, MN 55905, USA; aberrant G1-like tetraploid state characterized by E-mail: [email protected] markedly abnormal nuclear : cytoplasmic communica- Apoptosis and anticancer drug resistance SH Kaufmann and DL Vaux 7415 Chemotherapeutic Agent synthesis of a series of polypeptides that can block the cell cycle and repair the damage or cause the cell to kill itself (Zhou and Elledge, 2000; Abraham, 2001). The recognition that chemotherapeutic agents can kill Cellular Damage cancer cells in these two fundamentally different ways raises a number of important questions: Which mechanism, direct killing or induction of apoptosis, is more important therapeutically? Do alterations of apoptotic Damage incompatible Damage pathway components contribute to resistance? Would with survival Sensors novel therapies that enhance the apoptotic mechanism be clinically useful, either alone or in combination with conventional chemotherapeutic agents? In order to Signal Cell death Transduction address these questions, it is first important to under- stand the nature of the apoptotic machinery and its regulation.

Protective responses, e.g., Apoptotic . heatshock response, cell death . metallothionine induction, Apoptosis mechanisms . DNA repair . cell cycle checkpoint activation Studies in the nematode Caenorhabditis elegans, the fly Figure 1 Two types of responses to anticancer therapy. Most toxins, radiation, and chemotherapeutic agents can directly Drosophila melanogaster, and mammalian cells have damage both normal and cancer cells. If the damage is severe demonstrated that the biochemical mechanisms leading enough and sustained for a sufficient time, it will kill a cell directly to apoptosis are highly conserved among metazoans. as illustrated by examples in the text. If the damage is not as great, These mechanisms have been extensively reviewed or it has not had enough time to kill the cell directly, the cell might detect the damage and respond. Some responses allow the cell to (Adams and Cory, 1998; Metzstein et al., 1998; repair the damage, thereby helping protect the cell. Apoptosis is a Hengartner, 2000; Zimmermann et al., 2001; Martin, common stress response in which the cell kills itself. Because of 2002) and are only briefly summarized here. differences in some of the properties described in other reviews in In all of these organisms, the key effector proteins of this monograph (e.g. drug uptake, xenobiotic metabolism, and apoptosis are a family of cysteine proteases termed target abundance), normal cells and cancer cells might have different thresholds for direct toxicity. Likewise, because of caspases that, once activated, cleave their substrates differences in signals that regulate apoptotic pathways, normal after certain aspartic acid residues (Earnshaw et al., cell and cancer cells can have differences in apoptotic thresholds 1999; Nicholson, 1999). Over 200 caspase substrates, some of which are required for the cell to maintain viability, have been identified in mammalian cells. For tion (Theodoropoulos et al., 1999; Blajeski and example, when inhibitor of caspase-activated DNase Kaufmann, 2001). Camptothecin-induced trapping of (ICAD) is cleaved by caspases, it releases the constitu- covalent topoisomerase I–DNA complexes, on the tively expressed endonuclease caspase-activated DNase other hand, leads to DNA double-strand breaks and (CAD), which then cleaves the nuclear DNA (Liu et al., mutations that, if they occur in survival-critical genes, 1997; Enari et al., 1998). Although DNA degradation is could result in death once the corresponding polypep- incompatible with long-term cell survival, and has tides turn over (Ryan et al., 1991; Hashimoto et al., provided a convenient marker allowing detection of 1995). apoptotic mammalian cells, it is important to emphasize The second effect of chemotherapeutic agents results that proteolysis, rather than DNA degradation, is the in a response in which cells kill themselves by activating key regulatory event in apoptosis. In addition to an endogenous cell suicide process (Kaufmann and nuclease activation, proteolysis results in diminished Earnshaw, 2000). This form of cell death, often DNA repair, disruption of cell-cycle progression, recognizable by its characteristic appearance known as inhibition of protein synthesis, cleavage of major ‘apoptosis,’ uses effector mechanisms that are also structural proteins in the cytoplasm and nucleus, and employed for the removal of unwanted cells during disruption of signal transduction required for cellular development and to maintain constant cell number by homeostasis (Earnshaw et al., 1999). balancing mitosis (Arends and Wyllie, 1991). Stress The caspases responsible for this proteolysis are responses, including cell suicide, are much more synthesized as inactive precursors (zymogens) that must complicated than the ‘lock and key’ processes described be activated either by cleavage or conformational above. These are more appropriately thought of as a change before they can cleave their substrates (Earn- ‘stimulus–response model’ in which a cell needs recep- shaw et al., 1999; Nicholson, 1999). Caspase 3, a so- tors to sense an alteration, signal transduction pathways called ‘downstream’ or ‘effector’ caspase that is respon- to pass on the information, and effector mechanisms to sible for most of the cleavages that occur during mediate a response. For example, exposure of a cell to apoptosis (Slee et al., 2001; Kottke et al., 2002), is ionizing radiation leads to detection of DNA lesions, activated when it is cleaved by ‘upstream’ or ‘initiator’ activation of a series of kinases, and recruitment and/or caspases such as caspases 8, 9, or 10. In addition to

Oncogene Apoptosis and anticancer drug resistance SH Kaufmann and DL Vaux 7416 cleaving vital protein substrates within the cell, effector caspases can cleave and activate initiator caspases in an amplification loop (Slee et al., 1999). Mechanisms of caspase activation are remarkably conserved from C. elegans to Drosophila to man (see Figure 2 and Table 1). In each case, the initiator caspases are activated by adaptor proteins. For example, in mammalian cells, the adaptor Fas-associated polypeptide with death domain (FADD) can bind to and activate caspases 8and 10, and the adaptor Apoptotic protease activating factor-1 (Apaf-1) can bind to and activate caspase 9. These procaspase– adaptor protein interactions involve specialized protein interaction domains called death effector domains and caspase recruitment domains in the prodomains of the initiator caspases (Earnshaw et al., 1999; Nicholson, 1999; Hengartner, 2000). In the case of caspase 9, it is now clear that the activating event is an Apaf-1-induced dimerization that drives a conformational change at the Figure 2 Comparison of initiator caspase activation across species. Conformational changes are indicated by changes in active site of the zymogen (Renatus et al., 2001). Signal- shape, and increased activity is indicated by a cross-hatched fill induced activation of other initiator caspases presum- pattern. Adaptor/active caspase complexes have been shown to be ably involves similar changes (Boatright, 2003). multimers referred to as ‘apoptosomes’ in Drosophila and Many different signal transduction pathways can lead mammals. Question marks indicate current uncertainty as to whether DARK exists in an inactive conformation that requires to activation of the initiator caspases. For convenience, activation by binding to cytochrome c or another mitochondrial these are often grouped together (Budihardjo et al., component (Martin, 2002) 1999; Hengartner, 2000). Ligation of certain tumor necrosis factor-a (TNF) receptor family members, for example, TNFR1, Fas, or death receptors 4 and 5, often is translocated in indolent lymphomas (Adams and leads to apoptosis involving FADD and caspases 8and Cory, 1998; Gross et al., 1999; Cory and Adams, 2002). 10 as a consequence of signaling through what is termed These Bcl-2 family members can be divided into three the ‘extrinsic pathway’ or the ‘death receptor pathway’ groups. The first group, which includes Bcl-2, Bcl-xL (Ashkenazi, 2002). Apoptosis induced in this way is and others, inhibits cell death. They contain four rarely inhibitable by overexpression of antiapoptotic conserved regions termed BH (‘Bcl-2 homology’) Bcl-2 family members (see below) (Strasser et al., 1995; domains that appear to be involved in various homo- Newton and Strasser, 2000). On the other hand, typic and heterotypic protein–protein interactions. The apoptosis induced by radiation, p53, glucocorticoids, second group includes proapoptotic proteins such as cytokine deprivation, and most chemotherapeutic drugs Bax and Bak, which also contain multiple BH domains. usually leads to apoptosis that involves Apaf-1- Family members in the third group, which includes Bim, mediated caspase 9 activation (Kaufmann and Earn- Bik, Bid, Bad, Hrk, Noxa, Puma, Bmf, and others, are shaw, 2000). In mammalian cells, signals upstream of also proapoptotic, but they only contain a BH3 domain caspase 9 activation involve mitochondria and can be and are often referred to as ‘BH3-only polypeptides’ inhibited by Bcl-2, leading to designation of these (Adams and Cory, 1998; Huang and Strasser, 2000). signals as the ‘Bcl-2-inhibitable pathway,’ the ‘intrinsic Developmentally regulated cell death in C. elegans pathway,’ or the ‘mitochondrial pathway’ (Zimmer- (Figure 2 and Table 1) provides elegant examples of the mann et al., 2001). roles of Bcl-2 family members in the control of cell Both caspase activation and caspase activity are death. Activation of CED-3, a caspase required for tightly controlled. As described below, caspase 9 demise of all 131 somatic cells that ordinarily die during activation is inhibited by antiapoptotic Bcl-2 family development, requires binding to the Apaf-1 homolog members and facilitated by proapoptotic Bcl-2 family CED-4. This CED-3/CED-4 interaction is ordinarily members (Cory and Adams, 2002). In addition, active prevented by the binding of the antiapoptotic Bcl-2 caspases 3, 7, and 9 can be inhibited by XIAP, a member homolog CED-9 to CED-4. In cells fated to die, of the IAP family of proteins (Salvesen and Duckett, expression of the BH3-only family member EGL-1 leads 2002). to formation of EGL-1/CED-9 complexes, displacing CED-4 and allowing it to facilitate activation of CED-3 (Conradt and Horvitz, 1998; Chen et al., 2000). In mammalian cells, most of the short-term regulation Bcl-2 family and control of the mitochondrial pathway of ‘Bcl-2-inhibitable’ apoptotic pathways is likewise achieved through activation of BH3-only proteins, Mammalian cells express B20 different polypeptides although the activating events can be post-translational that share some degree of similarity with Bcl-2, an as well as transcriptional (Huang and Strasser, 2000; antiapoptotic protein initially identified because its gene Puthalakath and Strasser, 2002). Thus Bad is activated

Oncogene Apoptosis and anticancer drug resistance SH Kaufmann and DL Vaux 7417 Table 1 Comparison of apoptotic pathway components in model organisms Component Presumed function Examples in

C. elegans Drosophila Human

Initiator caspase Convert other signal to protease activity Ced-3 DRONC Caspase 8 Caspase 9 Adaptor Induce activating conformational change Ced-4 Dark/Dapa-1 FADD in initiator caspase zymogen DREDD Apaf-1

Antiapoptotic Bcl-2 Inhibit initiator caspase activation Ced-9 ?Buffy Bcl-2, Bcl-xL family regulator Proapoptotic Bcl-2 Initiate the apoptotic process in a tissue- Egl-1 Bid, Bim, Bad, family regulator and temporally regulated manner Noxa, Puma Caspase inhibitor Inhibit caspases — DIAP1 XIAP ML-IAP IAP antagonist Regulate IAP activity — Grim, Hid, Reaper, Sickle Smac/Diablo, HtrA2/Omi

by dephosphorylation, Bid by proteolytic cleavage, Bim it requires the existence of an adaptor and a caspase for by release from sequestration, and Noxa and Puma by which there is currently little evidence. transcription. According to current models, these polypeptides promote apoptosis by binding to antiapoptotic Bcl-2 family members (CED-9 homologs) or, IAP proteins and a second level of regulation in the case of cleaved Bid, to Bax and its homologs (Huang and Strasser, 2000). While Bcl-2 family members regulate the mitochondrial Even though human Bcl-2 can at least partially pathway prior to caspase activation, IAP proteins are complement loss of its worm homolog Ced-9, demon- thought to regulate apoptosis after caspase activation strating the high degree of conservation of these (Salvesen and Duckett, 2002). Originally identified components (Hengartner et al., 1992; Vaux et al., because of their ability to inhibit apoptosis in virus- 1992), there are important differences between apoptotic infected insect cells (Crook et al., 1993), this polypeptide pathways in worms and mammals. First, CED-4 family contains several species, including XIAP (X appears to be capable of activating CED-3 without chromosome-linked mammalian inhibitor of apoptosis)/ any accessory proteins (Irmler et al., 1997). In contrast, MIHA (Mammalian IAP homolog A), cIAP1 (cellular full-length mammalian Apaf-1 requires interaction with IAP1)/MIHB, cIAP2/MIHC, ML-IAP (melanoma cytochrome c, which is released from mitochondria IAP)/Livin and neuronal apoptosis inhibitory protein during apoptosis, to induce a conformational change (NAIP), some of which are capable of inhibiting active that facilitates the binding and activation of procaspase caspases 3, 7, and 9 in vitro (Deveraux and Reed, 1999; 9 (Boatright, 2003; Li et al., 1997). Second, CED-9 binds Salvesen and Duckett, 2002). While low nanomolar directly to and inhibits CED-4 (Spector et al., 1997). In concentrations of XIAP can inhibit caspases, cIAP1 and mammalian cells, in contrast, neither Bcl-2 nor Bcl-x 2 are estimated to be 1000-fold less potent and might not binds to Apaf-1 (Moriishi et al., 1999; Hausmann et al., exist in cells at high enough concentrations to directly 2000). Instead, they inhibit Apaf-1 activation indirectly inhibit caspases in vivo. In addition, the RING finger by preventing mitochondrial release of cytochrome c. domains at the C termini of many of these antiapoptotic There are two current models for Bcl-2 function IAPs can transfer ubiquitin to associated polypeptides (Figure 3). One suggests that Bcl-2, together with other such as caspases, IAP antagonists and the IAP proteins family members, acts by directly preventing release of themselves, thereby marking them for proteasome- polypeptides from mitochondria (Cheng et al., 2001). mediated degradation (Huang et al., 2000; Yang et al., The second suggests that Bcl-2 acts on a yet to be 2000; Suzuki et al., 2001). identified CED-4 homolog to prevent activation of a Some of the strongest evidence for regulation of caspase that can cleave substrates, including those on apoptosis by IAP proteins has come from genetic studies the mitochondria, causing release of cytochrome c in Drosophila (Hay, 2000; Martin, 2002). As summar- (Adams and Cory, 2001; Marsden et al., 2002). The ized in Figure 2, the fly caspase-9 homolog DRONC is first model requires mammalian Bcl-2 to have evolved activated by the Apaf-1 homolog DARK/HAC-1/Dapa- activities different from those of its nematode homolog 1. The ability of DRONC to kill cells during develop- and implies that the role of caspases in apoptosis is ment, however, is inhibited by the Drosophila IAP vestigial, with loss of mitochondrial function and release homolog DIAP1. On the other hand, this antiapoptotic of mitochondrial polypeptides such as apoptosis-indu- activity of DIAP1 is antagonized by four genes, reaper cing factor (AIF) (Daugas et al., 2000) being the (rpr), head involution defective (hid), grim and sickle, important steps. While the second model is consistent which control virtually all developmental cell death in with what we know about apoptosis in C. elegans and Drosophila. Importantly, the polypeptides encoded by suggests that caspases are the key effectors of apoptosis, rpr, hid, grim, and sickle contain a short region of

Oncogene Apoptosis and anticancer drug resistance SH Kaufmann and DL Vaux 7418 Cellular Damage and antagonizing IAP proteins in the same way as Grim, HID, Reaper, and Sickle bind DIAP1 (Verhagen and Damage Receptors, e.g., RADs, p53 Vaux, 2002), there are again important differences.

Signal First, unlike the insect polypeptides, the mammalian Death Transduction Death Receptors Receptors IAP antagonists are sequestered in mitochondria and BH3-only proteins come in contact with XIAP only after Bcl-2-regulated FADD release (Du et al., 2000; Verhagen et al., 2000; Adrian BID et al rpr hid grim, Caspase-8 ., 2001). Second, unlike deletion of , ,or BAX BAX tBID BCL-2 FADD BAK BAK deletion of Smac/Diablo did not result in a develop- BCL-2 Adaptor ? mental phenotype or alter sensitivity to a range of Caspase-2 Other caspases ? Caspase-8 apoptotic stimuli (Okada et al., 2002), raising the ? ? ? possibility that the mammalian IAP antagonists might be much less important in the regulation of cell death, just like the mammalian IAP proteins.

Smac/ Smac/ Cyt c Diablo Cyt c AIF ENDO-G Diablo

Apaf-1 Apaf-1 Inhibition of cell death during oncogenesis Caspase-9 Caspase-9 IAP IAP Downstream Why has such a complicated cell death process evolved Caspases Downstream Caspases ? in metazoans? It might have arisen initially as a defence Cell Death against intracellular parasites, such as viruses; however, Cell Death in larger animals it also acts to precisely counterbalance Figure 3 Two models for apoptosis. Available and emerging data cell replication and maintain constant cell number in support two distinct models for control of apoptotic pathways. The normal adult tissues. Any imbalance between cell model on the left invokes an essential role for mitochondria in most production and cell destruction will, over time, drama- cases of apoptosis and suggests that, even if caspases are inhibited, cells will die from loss of mitochondrial functions and release of tically alter the number of cells in a tissue, with mitochondrial enzymes such as AIF, an oxidoreductase (Miramar potentially deleterious consequences. et al., 2001), and endonuclease G (Li et al., 2001b). In the model on Although several changes are required for develop- the right, caspases are required for all apoptotic cell death, and ment of the full-blown neoplastic phenotype (Hanahan mitochondria play a nonessential role that serves to accelerate cell death. Although the model on the left has been widely accepted, and Weinberg, 2000), one of the earliest changes recent evidence places activation of caspases, particularly caspase- required for a cancer to develop is an excess of cell 2, upstream of mitochondrial dysfunction in some models of production relative to cell death. In theory, this could be chemotherapy-induced apoptosis (Guo et al., 2002b; Lassus et al., achieved by increasing cell division, decreasing apopto- 2002; Marsden et al., 2002; Paroni et al., 2002; Robertson et al., sis, or both. Histological examination of tumors is 2002), raising the possibility that another adaptor, possibly regulated by Bcl-2 family members in a manner analogous to consistent with this theory. An increase in mitotic figures CED-4, regulates caspase activation upstream of mitochondria is a classical feature of most cancers. Apoptotic cells are also common in solid tumors (Wyllie, 1985), possibly reflecting the deleterious effects of some of the proproliferative changes (see below). Nonetheless, as long as the death rate does not exceed the rate of cell homology at their amino termini. Recent studies division, the tumor will not regress. indicate that these amino-terminal polypeptides bind The discovery of activated oncogenes in human DIAP1, blocking its interaction with DRONC and cancers, and the formation of cancers in transgenic facilitating DIAP1 degradation (Wu et al., 2001; Martin, mice overexpressing oncogenes, has confirmed the 2002). This interruption of DIAP1 function is required importance of increased cell proliferation in oncogenesis for normal developmental death in Drosophila. (Hanahan and Weinberg, 2000; Evan and Vousden, Data supporting similar roles for IAP proteins and 2001). The finding that one of the genes translocated in their antagonists in the control of mammalian apoptosis follicular lymphomas, Bcl-2, encodes a cell death are not as strong. While overexpression of XIAP can inhibitor suggested that inhibition of apoptosis could inhibit apoptosis (Duckett et al., 1996; Liston et al., also lead to malignancy (Vaux et al., 1988). Consistent 1996; Uren et al., 1996), targeted XIAP disruption fails with this possibility, Bcl-2 transgenic mice develop to yield a developmental phenotype in mice (Harlin neoplasms, although it should be noted that tumors et al., 2001), either because of compensation by other arise in Bcl-2 transgenic mice only with long latency IAP proteins or because the regulation of apoptosis by (Strasser et al., 1993). IAP proteins has become vestigial in mammals. Like- As p53, which is the gene most commonly mutated in wise, the importance of IAP antagonists in mammalian human cancers, promotes apoptosis via the ‘Bcl-2- cells is less clear-cut. Even though mammalian cells inhibitable’ pathway, most likely by increasing tran- contain Smac (second mitochondrial activator of scription of the BH3-only proteins Puma and Noxa caspases)/Diablo (direct IAP binding protein with low (Huang and Strasser, 2000; Puthalakath and Strasser, pI) and HtrA2/Omi, which are capable of binding to 2002), there has been considerable interest in ascertain-

Oncogene Apoptosis and anticancer drug resistance SH Kaufmann and DL Vaux 7419 ing whether the antiapoptotic effects of p53 mutation First, it is important to keep in mind that studies of contribute to carcinogenesis. Careful examination has clinical samples establish a correlation, not causality. It demonstrated that p53À/À mice commonly develop has been observed, for example, that Bax is often neoplasms, including T-cell lymphomas, at an early upregulated in breast cancer (Reed, 1997). Given the age, whereas Bcl-2 transgenic mice hardly ever do even postulated role of Bax as a proapoptotic protein, it is though Bcl-2 transgenic thymocytes are at least as difﬁcult to argue that Bax upregulation plays a role in resistant to apoptosis as their p53À/À counterparts the pathogenesis of this disorder. Instead, Bax appears (Strasser et al., 1994). These observations suggest that to be upregulated through a poorly understood feed- the proapoptotic activity of p53 plays at best a minor back loop that sometimes increases Bax levels when Bcl- role in tumor suppression in mice in vivo. 2 rises (Miyashita et al., 1995; Kaufmann et al., 1998). In Is there evidence for inhibition of apoptotic pathways short, not every change in apoptotic components in clinical cancer? Recent studies have demonstrated necessarily contributes to the pathogenesis of cancer. changes that could affect either the death receptor Second, there is little evidence to date that alterations pathway or the Bcl-2-inhibitable pathway in a wide in apoptotic pathways similar to those observed in variety of neoplasms (Table 2). Along the mitochondrial human cancers lead to spontaneous cancers when pathway, for example, Bcl-2 upregulation is observed in introduced into mice. To the contrary, only a few of indolent lymphomas and a variety of additional these alterations have been shown to hasten the onset of neoplasms (Reed, 1999), Bax mutations have been malignancy even in cancer-prone mice. No increase in observed in mismatch repair-deﬁcient colon and gastric spontaneous tumors has been reported in mice lacking cancers (Rampino et al., 1997; Ionov et al., 2000), and genes for Fas, FasL (Cohen and Eisenberg, 1991), Bax, loss of Apaf-1 expression (Soengas et al., 2001) as well as Bak (Lindsten et al., 2000), Bim (Bouillet et al., 1999), overexpression of the IAP ML-IAP/Livin (Vucic et al., Apaf-1 (Honarpour et al., 2000), caspase 2 (Bergeron 2000; Kasof and Gomes, 2001) has been observed in et al., 1998) or caspase 3 (Zheng and Flavell, 2000), for melanomas. Observations such as these have led to example, or mice transgenic for XIAP (Conte et al., speculation that alterations in apoptotic pathways might 2001), the caspase 8inhibitor CrmA (Smith et al., 1996), be involved in the origin of human cancers and might or the broad-spectrum caspase inhibitor p35 (Izquierdo contribute to resistance to chemotherapy. These con- et al., 1999). Thus, inhibition of cell death alone appears clusions, however, need to be treated cautiously for to be very weakly oncogenic. Although mice harboring several reasons. Bcl-2 or Mcl-1 transgenes do develop lymphomas

Table 2 Examples of apoptotic pathway alterations in clinical cancer Alteration Presumed effect Cancer type References

Mitochondrial pathway Bcl-2 overexpression Inhibition of mitochondrial polypeptide release/ Follicular lymphoma Reed (1995) caspase activation Bcl-xL overexpression Inhibition of mitochondrial polypeptide release/ Multiple myeloma Catlett-Falcone et al. (1999) caspase activation Bax mutation Inability to express at least one Bax allele Mismatch-repair-deﬁcient Rampino et al. (1997), colon and gastric cancer Ionov et al. (2000) APAF-1 methylation Apaf-1 downregulation, leading to deﬁcient Melanoma Soengas et al. (2001) caspase 9 activation XIAP overexpression Inhibition of caspases 3, 7, and 9 AML Tamm et al. (2000) ML-IAP overexpression Inhibition of caspases 3, 7, and 9 Melanoma Kasof and Gomes (2001), Vucic et al. (2000)

Death receptor pathway Fas downregulation Insensitivity to Fas ligand-expressing cytotoxic Hepatoma Strand et al. (1996) lymphocytes Fas or FADD mutation Insensitivity to Fas ligand-expressing cytotoxic Non-small cell lung cancer Shin et al. (2002b) lymphocytes TRAIL receptor mutation Possible decreased sensitivity to TRAIL-expres- Breast cancer Shin et al. (2001) sing cells of the immune system, for example, natural killer cells and macrophages Decoy receptor 3 Decreased sensitivity to Fas ligand-expressing Colon and lung cancer Pitti et al. (1998), overexpression or ampliﬁ- cytotoxic lymphocytes Bai et al. (2000) cation Caspase 8 deletion or Decreased sensitivity to death ligand-induced Medulloblastoma, Grotzer et al. (2000), methylation apoptosis neuroblastoma, small cell Shivapurkar et al. (2002), lung cancer Eggert et al. (2001), Teitz et al. (2000) Caspase 10 mutation Decreased sensitivity to death ligand-induced Gastric cancer, lymphoma Park et al. (2002), apoptosis Shin et al. (2002a) c-FLIP overexpression Decreased sensitivity to death ligand-induced Melanoma, pancreatic Grifﬁth et al. (1998), apoptosis cancer Elnemri et al. (2001)

Oncogene Apoptosis and anticancer drug resistance SH Kaufmann and DL Vaux 7420 (Strasser et al., 1993; Zhou et al., 2001), they do so with What about other neoplasms and other treatments? very long latency, with few arising before the mice are After institution of therapy for acute leukemia, apop- 12–18months old. This long latency highlights the tosis can also be detected in circulating blasts (Li et al., importance of secondary, cooperating mutations that 1994; Seiter et al., 1997; Stahnke et al., 2001), providing must accompany the inhibition of apoptosis in order for evidence that apoptotic pathways are also activated in tumors to develop. In contrast, stimulation of cell these disorders in vivo. In the solid tumor setting it is division by c-myc in transgenic mice generates lympho- more difficult to demonstrate an increase in apoptosis mas much more quickly, with half the mice succumbing because (i) the poor response of many solid tumors to at 10 weeks (Adams et al., 1985). The fact that mice existing agents limits the amount of apoptosis that bearing both a myc and a bcl-2 transgene develop might be expected and (ii) efficient phagocytosis of tumors when only days old indicates that the combina- apoptotic cells makes it possible for extensive tumor tion of increased proliferation and inhibition of cell regressions to occur with only modest increases in the death is very potent indeed (Strasser et al., 1993). number of apoptotic cells present at any point in time This last observation also implies that tumors arise (Kyprianou et al., 1990). Nonetheless, increased apop- more slowly in the myc-only mice than in the myc þ bcl- tosis has been demonstrated in murine solid tumors after 2 mice because potential transformed cells in the myc- effective antineoplastic treatment (Martin et al., 1994). only mice are being removed by apoptosis. Although Despite the current fascination with apoptotic path- some have interpreted these results as suggesting that c- ways, it is important to stress that other processes might myc has intrinsic, direct, proapoptotic activity (the ‘lock also contribute to cancer cell (and normal cell) demise and key’ model) in addition to its cell cycle effects (Juin (Houghton, 1999). With the possible exception of et al., 1999), another interpretation is that cells can glucocorticoids and the abl kinase inhibitor imatinib detect cell cycle dysregulation caused by myc over- mesylate (STI571), practically all of the cancer therapies expression and respond by undergoing p53-mediated that have proven clinical utility have direct toxic effects (Zindy et al., 1998), Bcl-2-inhibitable (Eischen et al., on cellular metabolism. In addition, virtually all 2001) apoptosis (the ‘stimulus–response’ model). established and investigational antineoplastic agents In summary, currently available information suggests can induce apoptosis in susceptible cell types in vitro that the process of carcinogenesis usually requires at (Kaufmann and Earnshaw, 2000). These observations least two cellular changes, one inhibiting apoptosis and raise the question of how much of the killing by another that facilitates cell proliferation (Green and chemotherapy in vivo is due to direct toxicity vs Evan, 2002). The fact that apoptotic cells can be found induction of apoptosis. This question is extremely in cancers, even those with p53 mutations (Wachenfeld difficult to answer. Studies performed in vitro often et al., 2001), suggests that tumors in vivo may be close to look at short-term effects using drug concentrations that equilibrium, with both high proapoptotic signals (e.g. are different from those achievable in vivo. In addition, due to disrupted cell cycle regulation from inappropriate while studies have been performed in vitro using cells myc expression) and high antiapoptotic activity (e.g. lacking one or two apoptotic pathway components, increased Bcl-2 expression). To the extent that this is the none of the studies has completely eliminated all case, cancer cells might be just as easy to tip into apoptotic pathways (e.g. by knocking out FADD, apoptosis as normal cells despite the presence of Bak, and Bax in a single cell line). These studies are alterations in their apoptotic pathways. even less feasible in vivo because it is impossible to generate a mouse in which all of the apoptotic pathways are eliminated. Chemotherapy and apoptosis

Consistent with the speculation that cancer cells remain susceptible to the induction of apoptosis, indolent How does chemotherapy induce apoptosis? lymphomas and chronic lymphocytic leukemia (CLL), which almost universally express high Bcl-2 levels Inhibition of any vital metabolic process can lead to (Hanada et al., 1993; Reed, 1995), respond with stress responses, including apoptosis. For some thera- dramatic and sometimes durable remissions after treat- pies, the detection and signal transduction pathways ment with a variety of agents, including glucocorticoids, have been elucidated. Damage to DNA caused by purine nucleoside analogues, and the anti-CD20 anti- ionizing radiation or drugs, for example, can lead to body rituximab (Rai et al., 2000; Keating et al., 2002; stabilization of p53 (Giaccia and Kastan, 1998; Abra- Kipps, 2002). Although one might argue whether the ham, 2001) and consequent transcriptional activation of effects of these agents reﬂect a direct toxic effect (‘lock genes encoding a number of polypeptides, including Bax and key’ model) or induction of apoptosis, recent studies (Miyashita and Reed, 1995), Apaf-1 (Fortin et al., 2001; have demonstrated caspase activation and other apop- Kannan et al., 2001; Robles et al., 2001), the BH3-only totic changes in circulating CLL cells after rituximab polypeptides Noxa and PUMA (Oda et al., 2000a; Yu treatment (Byrd et al., 2002). Accordingly, these et al., 2001), and p53AIP1 (Oda et al., 2000b). Each of indolent B-cell neoplasms provide examples where these polypeptides can potentially activate the Bcl-2- apoptosis can be readily triggered by effective therapies inhibitable pathway. Because of the involvement despite high Bcl-2 expression. of other transcriptional activators and repressors in

Oncogene Apoptosis and anticancer drug resistance SH Kaufmann and DL Vaux 7421 expression of these polypeptides, not all of these First, there is frequent misunderstanding of this polypeptides are regulated to the same extent by p53 hypothesis. What is being postulated is resistance of in all model systems. The role of Bax transcription in cells relative to what their sensitivity would be if apoptotic p53-induced death, for example, has been questioned pathways were not altered, not absolute resistance to because it is difficult to demonstrate DNA damage- therapy. Other changes in cancer cells, for example, their induced Bax upregulation in many cells, thymocytes aberrant entry into the cell cycle and loss of checkpoints from BaxÀ/À mice undergo p53-dependent apoptosis (Hartwell and Kastan, 1994; Sherr, 2000), enhance normally, and the putative p53 binding elements in the sensitivity to anticancer drugs and undoubtedly con- Bax promoter are not conserved between man and tribute to the (admittedly limited) therapeutic index of mouse (Knudson et al., 1995; Brady et al., 1996; Igata these agents. What is being hypothesized is that the et al., 1999). Likewise, the extent and importance of therapeutic index would be greater if neoplastic cells did Noxa upregulation has been questioned (Yu et al., not have antiapoptotic changes. 2001). Second, there is considerable uncertainty regarding Because transfecting p53À/À cells with bcl-2 causes a the best way to test the hypothesis that alterations in further reduction in apoptosis of irradiated cells apoptotic pathways affect drug sensitivity. To take the (Strasser et al., 1994), p53 cannot be the only mediator example of the role of Bcl-2 in drug resistance, if an of DNA damage-induced apoptosis. For example, in experiment merely measures apoptosis at a particular mitogen-stimulated T cells, IRF-1 rather than p53 has point in time, it will fail to address the concern that Bcl- been implicated as the mediator of radiation-induced 2 might delay apoptosis without altering the ultimate apoptosis by the Bcl-2-inhibitable pathway (Tamura fate of the cells (Yin and Schimke, 1995). Colony- et al., 1995). forming assays can measure longer term effects on cell In addition to DNA damage, apoptosis can be survival (Martins et al., 1997) but are subject to other induced by inhibition of transcription, translation, limitations. Since clonogenic assays measure the ability DNA replication, or microtubule function. Apoptosis of cells to proliferate, they do not distinguish between in response to these types of stresses is reduced in cells or prolonged cell cycle arrest and cell death (Waldman transgenic mice overexpressing Bcl-2 (Sentman et al., et al., 1997). Moreover, if a clonogenic assay is 1991; Strasser et al., 1991) or cells containing targeted performed under conditions where only a small percen- deletion of both Bax and Bak (Lindsten et al., 2000). In tage of cells form colonies, it is possible that the results contrast, apoptosis in response to these agents still will reflect unique properties of the colony-forming cells occurs in cells or mice lacking Fas, FasL, or caspase 8 rather than the fate of the bulk population (Schmitt and and in cells expressing the caspase 8inhibitor CrmA, an Lowe, 2001). An alternative approach is to measure the inhibitor of FADD, or activated protein kinase C ability of a population to recover and proliferate at late isoforms that inhibit the death receptor pathway time points after drug treatment in vitro or in vivo. This (Eischen et al., 1997; Villunger et al., 1997; Varfolomeev approach is considerably more labor intensive but has et al., 1998; Yeh et al., 1998; Newton and Strasser, 2000, provided some of the most convincing evidence that Bcl- 2001; Meng et al., 2002). Experiments such as these 2 overexpression affects drug sensitivity (Schmitt and suggest that, with a few possible exceptions such as 5- Lowe, 2001). fluorouracil (Tillman et al., 1999), chemotherapy-in- Finally, the study of drug resistance in clinical duced apoptosis proceeds via the Bcl-2-inhibitable material raises its own set of methodological issues. In pathway. Nevertheless, increased expression of death any study of clinical samples, it is important that the receptors and their ligands has often been observed in percentage of neoplastic cells in the population be cells treated with chemotherapeutic agents (Kaufmann estimated and stated. Inclusion of samples that contain and Earnshaw, 2000; Herr and Debatin, 2001). This small percentages of neoplastic cells, for example, can upregulation of death receptor pathway components potentially lead to erroneous conclusions if assays may reflect part of a generalized stress response, but that measure a parameter in the entire population there is little evidence that the death receptor pathway is (e.g. RT–PCR or immunoblotting) are applied (Estrov required for the cells to undergo apoptosis in response et al., 1998). For this reason, assays that measure to most chemotherapy. parameters on a single-cell basis (e.g. flow cytometry or immunohistochemistry) might be preferable in some settings; however, it is important to realize the inherent difficulty of quantifying immunohistochemical assays How to study the role of apoptosis in drug resistance and the potential for variation in results in different areas of a large tumor due to emergence of clones with If anticancer drugs kill cells by inducing apoptosis, and varying properties. Once samples have been assayed, cancer cells have alterations that inhibit apoptotic it is also important that results be analysed in pathways, then it stands to reason that these alterations statistically valid ways, including application of suitable might contribute to drug resistance. Although this corrections for the effects of multiple comparisons hypothesis has been articulated by a number of authors (Matthews and Farewell, 1988). Finally, as mentioned (e.g. Green et al., 1994; Lowe, 1995; Hannun, 1997; above, it is important to keep in mind that analyses of Reed, 1999), several concerns have stood in the way of clinical material can only establish correlations, not its widespread acceptance. causality.

Oncogene Apoptosis and anticancer drug resistance SH Kaufmann and DL Vaux 7422 A role for altered apoptotic pathways in multidrug over, it is important to emphasize that BaxÀ/À cells resistance? represent an extreme situation that might not occur in human cancers in vivo. Instead, mismatch repair- When the preceding considerations are taken into deficient tumors appear to have inactivating mutations account, is there any evidence that alterations in in a single BAX allele (Rampino et al., 1997), and it apoptotic pathways contribute to the drug resistance? remains to be established whether the resulting cells or We believe that a large and growing body of evidence cell lines are deficient in Bax protein. If Bax is simply supports the claim that alterations in certain Bcl-2 downregulated, then models in which Bax is down- family members might contribute in some settings. This regulated might be more informative than those evidence is strongest for Bcl-2 itself. containing BAX deletions. Similar comments apply to Studies in clinical samples have demonstrated a Bak, which is currently thought to cooperate with Bax in correlation between high expression of Bcl-2 and poor facilitating the release of cytotoxic polypeptides from prognosis in a number of neoplasms, including acute mitochondria after many cellular insults (Wei et al., myelogenous leukemia, acute lymphocytic leukemia, 2001). non-Hodgkin’s lymphomas, and postate cancer (Reed, There is even less information available regarding 1995, 1999). In further support of the hypothesis that alterations in BH3-only proteins. In perhaps the best- Bcl-2 contributes to drug resistance, forced overexpres- studied example, BIM deletion renders thymocytes sion of Bcl-2 in tissue culture cells has been observed to selectively resistant to paclitaxel-induced apoptosis in prevent apoptosis due to several hundred different vitro (Puthalakath et al., 1999). It remains to be treatments, including the majority of agents used as determined whether BimÀ/À cells are resistant to cancer chemotherapy (Miyashita and Reed, 1992; Yang paclitaxel in vivo and whether quantitative Bim altera- and Korsmeyer, 1996; Johnstone et al., 2002). Deter- tions that fall short of complete deletion also affect drug mining whether the decreased number of apoptotic cells sensitivity. at any point in time translates into altered long-term Since p53 induces cell death by engaging the Bcl-2- cellular survival has been more problematic. Colony- inhibitable apoptotic pathway, a discussion of this forming assays have yielded conflicting results, with pathway would be incomplete without at least mention- some studies showing that Bcl-2 enhances clonogenic ing p53. p53 alterations appear to affect sensitivity to survival (Miyashita and Reed, 1993) and others ques- both ionizing radiation and drugs. Studies in tissue tioning this conclusion (Yin and Schimke, 1995; Lock culture have indicated that p53 deletion can render both and Stribinskiene, 1996; Kyprianou et al., 1997). One E1A/ras-transformed fibroblasts (Lowe et al., 1993) and potential factor complicating the use of colony-forming HCT116 colon cancer cells (Bunz et al., 1999) resistant assays in this context has been the low efficiency of to the induction of apoptosis by various anticancer colony formation in some of the model systems (Schmitt drugs in vitro. When the same cell lines are grown as and Lowe, 2001). Assays that compare the ability of xenografts, responses are diminished in the absence of isogenic cells to regrow after drug treatment in vivo, p53. Consistent with these results, studies have also however, have recently established that forced Bcl-2 established that ionizing radiation or high-dose 5- overexpression can enhance the ability of lymphoma fluorouracil induces less apoptosis in epithelial cells of cells to recover after a variety of drug treatments the small and large intestines of p53À/À mice compared (Schmitt and Lowe, 2001). to p53 þ / þ littermates (Merritt et al., 1997; Pritchard Similar sorts of observations support the view that et al., 1998). Collectively, these results suggest that p53 Bcl-xL might contribute to drug resistance. High Bcl-xL status can affect drug sensitivity. As indicated above, levels correlate with a poorer prognosis in patients with however, the relative importance of various p53 intermediate-grade lymphomas (Bairey et al., 1999), regulated proapopotic proteins remains to be deter- squamous cell carcinoma of the oropharyx (Aebersold mined. The entire issue of p53 and drug resistance is et al., 2001), soft-tissue sarcomas (Kohler et al., 2002), reviewed more comprehensively by El-Deiry (2003) in and myelodysplastic syndrome (Boudard et al., 2002). this issue. Assays examining the ability of cells to regrow after drug treatment in vitro and in vivo have not only established the ability of Bcl-xL to enhance tumor cell repopulation after cytotoxic insults (Simonian et al., Other alterations in apoptotic pathways might or might 1997; Liu et al., 1999), but also suggested that Bcl-xL is a not affect drug sensitivity more potent inducer of cell survival under these conditions (Simonian et al., 1997). The effects of other Current data supporting the role of alterations in other antiapoptotic Bcl-2 family members remain to be apoptotic regulators are not as strong. NAIP, XIAP, established in a similar fashion. cIAP1, and cIAP2, were reported to diminish apoptosis The effects of alterations in proapoptotic Bcl-2 family triggered by a number of agents, including camptothecin members also require further study. Although it has and menadione in vitro (Liston et al., 1996). Whether been reported that BAX deletion inhibits drug-induced these effects result in enhanced long-term survival after apoptosis in HCT116 cells in vitro (Zhang et al., 2000; drug treatment still remains to be established. A recent Theodorakis et al., 2002), the effects of this alteration on attempt to correlate expression of these polypeptides drug sensitivity in vivo remain to be established. More- with drug sensitivity in the 60 cell lines used by the

Oncogene Apoptosis and anticancer drug resistance SH Kaufmann and DL Vaux 7423 National Cancer Institute to screen potential anticancer mocytes (Yoshida et al., 1998), the Apaf-1-deficient drugs failed to demonstrate the postulated negative melanoma cells exhibited diminished apoptosis in correlation (Tamm et al., 2000). Unfortunately, ‘drug response to doxorubicin (Soengas et al., 2001). Whether sensitivity’ in the latter study was measured only in 48h Apaf-1 deficiency also affects long-term viability after assays, which might be too short to allow apoptotic drug treatment remains to be determined. In contrast to changes to occur in many cell lines, and was quantified alterations in antiapoptotic Bcl-2 family members or by calculating the drug concentration that inhibits p53, which clearly alter long-term survival after drug proliferation by 50%, which reflects antiproliferative treatment, it is possible that downregulation of Apaf-1, effects rather than cell killing. Accordingly, further caspase-9, or the effector caspases will inhibit the rapid studies, preferably using isogenic model systems, are development of apoptotic changes without affecting required to determine whether enhanced expression of long-term survival. Observations of Apaf-1, caspase 9, IAP proteins enhances the ability of tumor cell or caspase 3 knockout mice have shown that, with the populations to survive drug treatment. exception of certain neurons, developmental cell death It is important to emphasize that not all BIR- occurs normally in the absence of these components containing polypeptides (BIRPs) are necessarily caspase (Zheng et al., 1999; Honarpour et al., 2000). It could be inhibitors (Silke and Vaux, 2001). Survivin, a human that release of proteins from mitochondria kills cells by protein containing a single BIR, illustrates this point. a caspase-independent process, or that other caspases Survivin was originally reported to be overexpressed in a kill cells in the absence of Apaf-1 and caspase 9 (see variety of neoplasms (Ambrosini et al., 1997). A Figure 3). growing number of studies have also indicated a correlation between elevated survivin levels and a poor prognosis, for example, in acute myelogenous leukemia, neuroblastoma and malignant glioma as well as cancers Do not throw out the baby of the colon, prostate, ovary, breast, pancreas, and esophagus (reviewed in Mow et al., 2001; Kamihira Despite the strength of recent evidence showing that et al., 2001; Chakravarti et al., 2002). Several additional alterations in Bcl-2, Bxl-xL, and p53 can affect drug observations, however, have suggested that survivin sensitivity, it is important to emphasize two points. might not be an inhibitor of apoptosis. First, the group First, these conclusions are based on the examination in that originally suggested an IAP function for survivin vivo of a total of four pairs of neoplastic cell lines, only has been unable to demonstrate that it inhibits caspase 3 two of which are carcinoma models. It is, therefore, (Banks et al., 2000). Second, survivin localizes to the unclear how widely applicable these conclusions are to mitotic apparatus, where it behaves like a typical other cellular contexts (Brown and Wouters, 1999). chromosomal passenger protein (Adams et al., 2001). Second, it is important to stress that these changes result Third, targeted deletion of the mouse survivin gene is in relative resistance to induction of apoptosis, not embryonic lethal but, unlike deletion of Bcl-xL, results in absolute resistance. As indicated above, cancer cells altered ability of cells to complete mitosis rather than an have a variety of defects in DNA repair and cell cycle excess of apoptosis (Uren et al., 2000). Given this checkpoint pathways that might counterbalance the information suggesting that survivin is a component of effect of upregulated Bcl-2 or Bcl-xL by enhancing the the mitotic machinery, how is one to intrepret the sensitivity of cells to drug-induced apoptosis. Moreover, reports of poorer prognoses in patients with survivin- while Bcl-2 upregulation might prevent the rapid expressing neoplasms? Additional studies have demon- apoptotic death of cells, it is important to keep in mind strated transcriptional activation of the survivin pro- that it would not prevent killing of cells by agents that moter under conditions where cells are actively cycling have intrinsic toxicity, as long as the cells were treated (Li and Altieri, 1999). One possibility, therefore, is that with a sufficient dose for a sufficient time. A crucial issue survivin expression in a tumor reflects the number of is whether the apoptotic response of neoplastic cells to cells that are actively cycling. If patients are treated with toxins occurs at a significantly lower dose of drug than is ineffective therapeutics (as is the case with many solid required for direct lethal effects. tumors), it is perhaps not surprising that patients with As a result of these outstanding issues, it is essential more proliferative neoplasms will have a poorer prog- that improved but incomplete understanding of apop- nosis. According to this interpretation, the clinical totic pathways not be utilized prematurely to withhold association between poor prognosis and high survivin or alter treatment. CLL provides an informative expression might not reflect any effect of survivin on example. Based on the fact that high levels of Bcl-2 apoptosis at all. Further studies are required to are observed in the vast majority of CLL cases (Hanada determine whether this interpretation is correct or et al., 1993), one might predict that CLL would be whether survivin’s role in mitosis affects tumor cell highly resistant to chemotherapy. Taken to its extreme, biology in some other way. such a prediction would suggest that it is fruitless to The effects of alterations in levels of Apaf-1 and treat CLL with currently available agents. Nothing caspases also require further study. Silencing of APAF-1 could be further from the truth. CLL cells also appear to has been reported in melanoma cell lines and clinical contain additional alterations, for example, mutations in samples (Soengas et al., 2001). Consistent with prior the DNA damage-responsive kinase ATM (Bullrich observations in Apaf-1À/À murine fibroblasts and thy- et al., 1999; Pettitt et al., 2001), that increase their

Oncogene Apoptosis and anticancer drug resistance SH Kaufmann and DL Vaux 7424 sensitivity to various agents. As a consequence, CLL phosphatidylinositol-3 (PI-3) kinase/Akt pathway remains sensitive to the induction of apoptosis by a (Skorski et al., 1995; Neshat et al., 2000), which can variety of agents in vitro (Thomas et al., 1996; Chandra potentially inhibit the mitochondrial pathway in a et al., 1998) and in vivo (Byrd et al., 2002). number of ways (Kaufmann and Gores, 2000; Vivanco Put another way, a diverse and useful group of and Sawyers, 2002). Treatment with the somewhat chemotherapeutic drugs has been identified over the past selective Bcr/abl kinase inhibitor imatinib mesylate 50 years by empirical means. In retrospect it is clear that (Druker et al., 1996) results in downregulation of Bcl- most if not all can induce apoptosis in susceptible cells xL and inhibition of the PI-3 kinase/Akt pathway in Bcr/ (Kaufmann and Earnshaw, 2000). Some, such as abl-positive cells (Horita et al., 2000; Neshat et al., dexamethasone, probably work only by inducing 2000). These changes sensitize Bcr/abl-positive cells to apoptosis. This new understanding has sometimes the cytotoxic effects of other agents in vitro (reviewed in obscured the fact that most of these drugs can kill cells La Rosee et al., 2002). More surprising, however, has independent of the apoptotic response (Figure 1). been the demonstration that imatinib by itself induces Whether they work by inducing apoptosis or not, there complete hematological and cytogenetic responses in a is abundant evidence that these drugs can have clinically substantial fraction of patients with chronic-phase useful effects even in cells overexpressing Bcl-2 or with chronic myelogenous leukemia (Druker et al., 2001; mutant p53. Kantarjian et al., 2002). Studies in tissue culture have In the late 1990s there was similar debate about demonstrated that imatinib can induce apoptosis whether p53 mutations were responsible for resistance to through activation of the mitochondrial pathway in radiotherapy (Finkel, 1999). The demonstration that Bcr/abl-positive cell lines (Dan et al., 1998; Fang et al., p53 was required for rapid interphase death of 2000; Mow et al., 2002). Whether the effects of imatinib particular cells was extrapolated by some to indicate on apoptotic pathways play a role in its clinical effects, that treating patients with p53-mutant tumors was however, remains somewhat controversial. A recent hopeless. Luckily, radiation oncologists who had study of the effects of imatinib on CML marrow extensive experience successfully treating patients with progenitors suggests that the predominant effect of p53 mutant tumors ingored this advice. clinically achievable drug doses is inhibition of Bcr/abl- induced proliferation (Holtz et al., 2002). Importantly, however, cells from patients with the best clinical response to imatinib also exhibited apoptotic changes Enhancing the apoptotic response in response to low drug concentrations in vitro, a point that was ignored in the discussion of Holtz et al. Further As illustrated in the preceding sections, the complexity studies are required to define not only the extent to of human cancers can make it difficult to extract which imatinib-induced apoptosis plays a role in the clinically pertinent conclusions from controlled experi- successful treatment of CML, but also to determine ments in the lab. Nonetheless, it is important not to whether imatinib-induced downregulation of Bcl-xL and completely ignore the biochemical findings. If altera- the PI-3/Akt pathway will translate into improved tions in apoptotic pathways render cancer cells more therapy for Bcr/abl-driven acute leukemias. resistant than they would be in the absence of those alterations, then enhanced understanding of apoptotic pathways might suggest ways of sensitizing cancers to Altering effective levels of Bcl-2 existing therapies or even entirely new approaches for In view of the widespread expression of Bcl-2 in human the treatment of cancer. Although space limitations cancers (Reed, 1999), there has been considerable preclude a comprehensive discussion of novel therapeu- interest in downregulating or inhibiting this poly- tic approaches that are being tested, a number of peptide. In theory, this approach could be used to treat examples are described in the following sections. follicular lymphoma as well as increase the propensity of other cells to undergo apoptosis in response to Imatinib mesylate (STI571) as a paradigm chemotherapy. A series of studies (reviewed in Cotter, 1999) has Imatinib mesylate provides an excellent example of an demonstrated that a phosphorothioate oligonucleotide agent that might act, at least in part, by altering complementary to the first 18nucleotides of the Bcl-2 apoptotic pathways. Studies dating to the early 1990s coding sequence (now called G3139, augmerosen, or demonstrated that transfection with p190Bcr/abl or p210Bcr/ Genasense) is particularly effective at downregulating abl diminished the apoptotic response of cell lines to Bcl-2 in lymphoma cells containing the t(14;18) chro- agents that activate the Bcl-2-inhibitable pathway mosomal translocation and inhibiting engraftment of (Laneuville et al., 1994; McGahon et al., 1994; Cortez these cells in nude mice. Based on these results, a phase I et al., 1995). Subsequent analysis has demonstrated that study of G3139 administered as a 14-day continuous this reflects Bcr/abl-mediated phosphorylation of infusion was completed in 21 patients with relapsed non- STAT5 (signal transducer and activator of transcrip- Hodgkin’s lymphoma (Cotter, 1999; Waters et al., tion-5), which upregulates BCL-X transcription (Shuai 2000). G3139 caused downregulation of Bcl-2 protein et al., 1996; Amarante-Mendes et al., 1998; Horita et al., in 58% of cases and responses in 14% of patients, with 2000), as well as Bcr/abl-mediated activation of the one complete response lasting X36 months. Ongoing

Oncogene Apoptosis and anticancer drug resistance SH Kaufmann and DL Vaux 7425 trials are examining G3139 as a single agent in mela- enhance apoptosis in tissue culture cells treated with a noma, prostate, breast, and colon cancer (Banerjee, 2001). wide variety of agents (Arnt et al., 2002; Fulda et al., On the other hand, current understanding of Bcl-2 as 2002; Guo et al., 2002; Yang et al., 2003). Importantly, a regulator of the mitochondrial pathway (see above) the same approach enhances the antineoplastic effects of suggests that Bcl-2 downregulation might be more TRAIL in a brain tumor model in vivo (Fulda et al., effective when combined with other proapoptotic 2002). Future studies will undoubtedly determine stimuli. Several studies have shown, for example, that whether the effects of cytotoxic chemotherapeutic agents cytarabine-induced apoptosis can be enhanced in vitro are also enhanced in vivo and whether the N-terminal by treating leukemia cells with Bcl-2 antisense oligonu- Smac sequence can be turned into an efficacious small- cleotides (Reed et al., 1990; Campos et al., 1994). Based molecule peptidomimetic. on this rationale, recently reported pilot studies have evaluated G3139 in combination with dacarbazine, Death receptor ligands paclitaxel, docetaxel, or mitoxantrone (Chi et al., 2001; Tolcher, 2001; Jansen et al., 2000; Rudin et al., 2002). The possibility of killing cancer cells by ligating death One concern with these types of combinations would be receptors has been investigated for a long time. that Bcl-2 downregulation might sensitize normal tissues Although TNF-a, the founding member of the DR to the chemotherapy, thereby enhancing toxicity. ligand family, kills a number of cancer and leukemia cell Enhanced toxicity has not been a major problem in lines, the septic shock-like syndrome caused by this the combination trials even though G3139 doses were agent limits its ability to be administered systemically sufficient to downregulate Bcl-2 levels in accessible (Lejeune et al., 1998). Accordingly, TNF-a is reserved surrogate tissues (Jansen et al., 2000; Chi et al., 2001; for local infusions into isolated, perfused limbs. Inter- Rudin et al., 2002). Phase III trials are required to estingly, recent analysis has demonstrated that TNF-a- determine whether this approach enhances the efficacy induced antitumor effects require TNF receptors on of the conventional agents. tumor endothelial cells, not on the cancer cells themselves (Stoelcker et al., 2000). These results, which BH3 mimetics suggest that TNF-a kills cancer cells indirectly by Antiapoptotic Bcl-2 family members are antagonized by causing constriction of their blood supply, provide a BH3-only proteins in vitro and in vivo. This antagonism nice explanation for the observation that TNF causes depends on the interaction between the BH3 domain, a necrosis in vivo rather than apoptosis. region containing 9 amino acids, on the BH3-only Several observations raise the possibility that the protein and a cleft in antiapoptotic Bcl-2 family more recently identified TNF family member TNF-a- members (Huang and Strasser, 2000). This raises the related apoptosis inducing ligand (TRAIL) might be possibility that small-molecule BH3-only protein mi- more suitable for systemic treatment of cancer and metics could be identified and used to antagonize Bcl-2 leukemia (Ashkenazi, 2002). First, TRAIL is utilized by interferon-g-stimulated lymphocytes and natural killer or Bcl-xL function. A number of such compounds have been reported (Wang et al., 2000; Degterev et al., 2001; cells to kill their targets, which include transformed cells Tzung et al., 2001). Whether these can be turned into (Walczak and Krammer, 2000). Second, the limited useful drugs for indolent B-cell lymphomas or agents expression of DR4 and DR5 in normal adult cells that modulate the action of other anticancer treatments (Schulze-Osthoff et al., 1998) raises the possibility that remains to be seen. the TRAIL might exhibit some selectivity for solid tumors and leukemia. Consistent with this view, Modulation of antiapoptotic IAP protein function recombinant TRAIL kills neoplastic cell lines in vitro and shrinks tumors in animals (Ashkenazi et al., 1999; As indicated above, data linking elevated XIAP, cIAP1, Walczak et al., 1999). Alternatively, it has also been or cIAP2 to drug resistance in the clinical setting are reported that certain antibodies to TRAIL receptors can currently relatively limited. Based on studies showing activate receptor-mediated signaling and kill target cells that elevated XIAP expression can inhibit apoptosis in (Ichikawa et al., 2001). Clinical trials of these agents are cell lines in vitro, several groups have investigated the awaited with interest. effects of XIAP antisense approaches in model systems. In short, recent advances in understanding apoptotic Antisense molecules that reduce XIAP levels sensitize pathways and their regulation have led to the testing of non-small cell lung cancer cells to radiation (Holcik a number of approaches designed to alter the apoptotic et al., 2000) and ovarian cancer cells to cisplatin-induced sensitivity of cells or directly induce apoptosis. Ongoing apoptosis (Li et al., 2001a). Whether this approach can preclinical and clinical studies should provide important selectively sensitize cancer cells to proapoptotic stimuli information about the efficacy of these approaches over in vivo remains to be demonstrated. the next several years. Alternatively, small molecules that mimic the N- termini of the IAP antagonists Smac/Diablo or HtrA2/ Omi might be useful to counter IAP activity in vivo. Inhibiting apoptosis to treat cancer Recent studies have shown that the N-terminal 6–8 amino acids of Smac, when coupled to a sequence that An alternative approach would be to inhibit therapy- carries them through the plasma membrane, can induced apoptosis in normal tissues. The radiation or

Oncogene Apoptosis and anticancer drug resistance SH Kaufmann and DL Vaux 7426 chemotherapy dose that can be administered is usually and quantitative changes in apoptotic pathways actually limited by toxic side effects, such as damage to the do contribute to drug resistance in a clinically mean- gastrointestinal system and bone marrow. Curative ingful sense, that is, in the ability of cells to withstand doses often cannot be given because of lethal side drug treatment and subsequently repopulate their effects, many of which appear to result from the environment. Insightful, statistically rigorous analyses apoptosis of normal cells. In view of the role of wild- of clinical material are required to identify qualitative type p53 in these toxic effects, it has been proposed that and quantitative alterations in apoptotic pathways in p53 inhibitors might protect normal cells, whereas various tumor types. Moreover, carefully controlled cancer cells lacking functional p53 would not be preclinical and clinical studies are needed to determine protected (Komarov et al., 1999). Preliminary analysis whether interventions that alter the apoptotic threshold has demonstrated that one p53 inhibitor, pifithrin-alpha, truly enhance the efficacy of anticancer therapies. Until inhibits the toxic effects of lethal g-irradiation (Komar- these types of studies are performed, the idea that ov et al., 1999). Studies demonstrating that this altered apoptotic pathways contribute to clinical drug approach will improve the therapeutic index in p53- resistance must be considered an attractive hypothesis deficient tumors are awaited with interest. The possibi- rather than an established fact. lity that other antiapoptotic drugs, such as caspase inhibitors, might also reduce the side effects of chemotherapy, or allow higher doses to be tolerated, Acknowledgements might also merit further investigation. We acknowledge helpful suggestions of Greg Gores, Wei Meng, and Christina Arnt as well as the secretarial assistance of Deb Strauss. Work in our laboratories is supported by Summary grants from the National Cancer Institute (SHK) and Leukemia and Lymphoma Society and Australian National Recent studies have provided increased understanding Health and Medical Research Couuncil (DLV). DLV is a of the manner in which apoptotic pathways are Leukemia and Lymphoma Society Scholar. regulated. At the same time, various alterations in these apoptotic pathways have been identified in cancer cell Note added in proof: We apologize to the many authors whose most recent work is not cited. Since the submission of this lines and clinical tumor specimens. The idea that these review in August, 2002, there has been considerable progress in alterations simultaneously contribute to the processes of understanding the regulation of apoptotic pathways. Inter- carcinogenesis and drug resistance is an appealing one. ested readers are referred to more timely reviews, including Additional rigorous experiments in suitable model Coultas and Strasser (2003), Newmeyer and Ferguson-Miller systems are required to determine whether qualitative (2003) and Nicholson and Thornberry (2003).

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