Apoptosis In Cancer.pdf

Carcinogenesis vol.21 no.3 pp.485–495, 2000

Apoptosis in cancer

Scott W.Lowe1 and Athena W.Lin Cold Spring Harbor Laboratory, 1 Bungtown Road, PO Box 100, Cold Spring Harbor, New York, NY 11724, USA 1To

whom correspondence should be addressed E-mail: [email protected]

In the last decade, basic cancer research has produced remarkable advances in our understanding of cancer biology and cancer genetics. Among the most important of these advances is the realization that apoptosis and the genes that control it have a profound effect on the malignant phenotype. For example, it is now clear that some oncogenic mutations disrupt apoptosis, leading to tumor initiation, progression or metastasis. Conversely, compelling evidence indicates that other oncogenic changes promote apoptosis, thereby producing selective pressure to override apoptosis during multistage carcinogenesis. Finally, it is now well documented that most cytotoxic anticancer agents induce apoptosis, raising the intriguing possibility that defects in apoptotic programs contribute to treatment failure. Because the same mutations that suppress apoptosis during tumor development also reduce treatment sensitivity, apoptosis provides a conceptual framework to link cancer genetics with cancer therapy. An intense research effort is uncovering the underlying mechanisms of apoptosis such that, in the next decade, one envisions that this information will produce new strategies to exploit apoptosis for therapeutic benefit. Introduction Apoptosis was initially described by its morphological characteristics, including cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation (1–3). The realization that apoptosis is a gene-directed program has had profound implications for our understanding of developmental biology and tissue homeostasis, for it implies that cell numbers can be regulated by factors that influence cell survival as well as those that control proliferation and differentiation. Moreover, the genetic basis for apoptosis implies that cell death, like any other metabolic or developmental program, can be disrupted by mutation. In fact, defects in apoptotic pathways are now thought to contribute to a number of human diseases, ranging from neurodegenerative disorders to malignancy (4). The notion that apoptosis might influence the malignant phenotype goes back to the early 1970s. Kinetic studies of tumor growth implied that cell loss from tumors could be massive; indeed, observed tumor growth rates could be ⬍5% of that predicted from proliferation measurements alone (1,2). In principle, changes in this ‘cell loss factor’ could have a major impact on tumor growth or regression. Although most Abbreviations: IGF, insulin-like growth factor; NF-κB, nuclear factor κB; PTP, permeability transition pore; TNF-α, tumor necrosis factor α; TRAF-2, TNF receptor-associated factor. © Oxford University Press

of this death was assumed to arise from necrosis—a catastrophic (and easily discernible) type of cell death—Kerr et al. raised the possibility that a large percentage of cell loss from tumors was due to apoptosis (1). Subsequent studies revealed a high frequency of apoptosis in spontaneously regressing tumors and in tumors treated with cytotoxic anticancer agents (3). Together, these observations suggested that apoptosis contributed to the high rate of cell loss in malignant tumors and, moreover, could promote tumor progression. Nevertheless, the importance of apoptosis in cancer remained under-appreciated for ⬎15 years. Apoptosis and tumorigenesis The cloning and characterization of the bcl-2 oncogene established the importance of apoptosis in tumor development. bcl2 was first identified at the chromosomal breakpoint of t(14; 18) in a human leukemia line and was later shown to be a common event in follicular lymphoma (5,6). At this time, oncogenes were classified as either ‘transforming’ or ‘immortalizing’ oncogenes based on their properties in rodent cell transformation assays. However, bcl-2 did not behave like a typical oncogene: instead of disrupting normal proliferation controls, Bcl-2 promoted cell survival by blocking programmed cell death (7–9). Moreover, in transgenic mice, Bcl-2 overexpression promoted lymphoproliferation and accelerated c-Mycinduced lymphomagenesis (8,10). To date, at least 15 Bcl-2 family member proteins have been identified in mammalian cells, including proteins that promote apoptosis and those that prevent apoptosis (11). In addition to Bcl-2, Bcl-xL is a potent death suppressor that is upregulated in some tumor types (12). Conversely, Bax is a death promoter that is inactivated in certain types of colon cancer and in hematopoietic malignancies (13,14). p53 was the first tumor suppressor gene linked to apoptosis. p53 mutations occur in the majority of human tumors and are often associated with advanced tumor stage and poor patient prognosis (15). By 1992, p53 was clearly established as a checkpoint protein involved in cell-cycle arrest and maintaining genomic integrity following DNA damage. However, p53 could induce apoptosis when overexpressed in a myeloid leukemia cell line, suggesting that p53 might also regulate cell survival (16). Studies using p53 knockout mice demonstrated that endogenous p53 could participate in apoptosis: p53 was required for radiation-induced cell death in the thymus, but not cell death induced by glucocorticoids or other apoptotic stimuli (17,18). Hence, the role of p53 in apoptosis was indirectly linked to DNA damage and could be stimulus(radiation) and tissue-specific (thymocytes). It is now known that other stimuli can activate p53 to promote apoptosis, including hypoxia and mitogenic oncogenes (see below). Moreover, several upstream and downstream components of the p53 pathway (e.g. Mdm-2, ARF and Bax) are mutated in human tumors (15). Although the initial studies on Bcl-2 and p53 established 485

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the importance of apoptosis in carcinogenesis, it is now clear that mutations in many cancer-related genes can disrupt apoptosis. For example, the Fas/CD95 receptor normally controls cell numbers in the immune system by eliminating cells through apoptosis, and disruption of this pathway can lead to lymphoproliferative disorders and even cancers (19). In addition, several signal transduction pathways promote cell survival in response to growth and/or survival factors, and these pathways may be crucial in controlling cell numbers in vivo. One critical pathway involves signaling through PI-3 kinase (20), which can be activated by Ras and is downregulated by the PTEN tumor suppressor (21). Ras activation and PTEN loss are common in human tumors. Studies using transgenic and knockout mice provide direct evidence that disruption of apoptosis can promote tumor development. In addition to the lymphomas (10), bcl-2 transgenes accelerate SV40 large T antigen-induced mammary carcinogenesis (22). Similarly, p53 loss produced by gene ‘knockout’ accelerates tumor progression in many settings, including the murine retina, lens, choroid plexus and in the lymphoid compartment (23). In many cases, p53 loss is associated with reduced apoptosis in situ. Additionally, mouse studies reveal the crucial role of extracellular survival factors in supporting tumor progression. For example, large T antigeninduced pancreatic tumors require the upregulation of insulin-like growth factor 2 (IGF-2) for progression to carcinomas—in fact, tumors arising in IGF-2 animals remain hyperplastic and display excessive apoptosis (24). Moreover, inactivation of PTEN promotes tumorigenesis and cell survival when disrupted in mice (21), and disruption of the pro-apoptotic bax gene accelerates brain and mammary tumorigenesis in large T antigen transgenic mice (25,26). Because molecules that regulate apoptosis can have other activities, it is often difficult to demonstrate that mutations in tumors actually confer a survival advantage. For example, p53 can promote apoptosis, cell-cycle arrest and senescence such that loss of p53 function increases viability, chromosomal instability and cellular lifespan. However, compelling evidence indicates its apoptotic activity is important in tumor suppression. First, marked decreases in apoptosis correlate with the occurrence of p53 mutations in some transgenic mice (23) and in clonal progression of Wilms’ tumor (27). Second, disruption of several p53 effectors in apoptosis (e.g. bax, apaf-1 and casp-9) can promote oncogenic transformation and tumor development in mouse model systems (25,28,29). In colon cancer, bax and p53 mutations appear mutually exclusive, consistent with a pathway relationship (30). In contrast, p21, which is essential for p53-mediated arrest, is rarely mutated in human tumors (31). Finally, some tumor-derived p53 mutants remain capable of promoting cell-cycle arrest while losing their apoptotic potential (32,33). Nevertheless, these data do not imply that the other p53 activities are dispensible for tumor suppression; rather, they simply argue that its apoptotic activity is important. What triggers apoptosis during tumor development? A variety of signals appear important. Extracellular triggers include growth/survival factor depletion, hypoxia, radiation and loss of cell-matrix interactions. Internal imbalances can also trigger apoptosis, including DNA damage (produced by cell-cycle checkpoint defects or exogenous toxins), telomere malfunction and inappropriate proliferative signals produced by oncogenic mutations. In some instances the apoptotic ‘trigger’ actually alleviates an anti-apoptotic signal. For 486

example, IGF-1 promotes cell survival through the PI-3 kinase pathway (34), and depletion of IGF-1 or other survival factors can trigger ‘death by default’ (35). In contrast, other stimuli involve true pro-apoptotic factors; as an example, many forms of cellular stress can activate p53, which promotes apoptosis through pro-apoptotic molecules like Bax (25,28,36). The identification of apoptotic ‘triggers’ provides insight into the forces of tumor evolution (see also next section). For example, in the skin, excessive exposure to UV radiation induces apoptosis, which presumably serves to eliminate heavily damaged cells. UV radiation induces apoptosis, and loss of p53 function leads to the survival of these damaged cells thereby initiating tumor development (37). Other apoptotic triggers are important in tumor progression. As developing tumors outgrow their blood supply, they encounter hypoxia (low oxygen), which can activate p53 to promote apoptosis (38). Cells acquiring apoptosis defects (e.g. p53 mutations) can survive hypoxic stress, leading to their clonal expansion within the tumor (38). Similarly, as developing tumor cells undergo repeated divisions, telomeres are shortened until some malfunction triggers either senescence or apoptosis. Like hypoxia, p53 is required for apoptosis induced by telomere malfunction; thus p53 mutant cells survive this response and are genomically unstable (39). Indeed, suppression of the apoptotic response to telomere malfunction may explain why combined loss of telomerase and p53 stimulates tumor development (40). These studies may explain why p53 mutations are usually late events in tumor development—a cell acquiring a p53 mutation might not have a selective advantage until the developing tumor encounters hypoxic conditions or achieves sufficient telomere erosion. Disruption of apoptosis may also contribute to tumor metastasis. To metastasize, a tumor cell must acquire the ability to survive in the bloodstream and invade a foreign tissue. Normally, this process is prevented by the propensity of epithelial cells to die in suspension, or in the absence of the appropriate tissue survival (41). Clearly, the fact that these processes trigger apoptosis creates selective pressure to mutate apoptotic programs during tumor development. Apoptosis in suspension is controlled by a number of other molecules, including the focal adhesion kinase and signal transduction pathways (42). Some studies argue that p53 and Bcl-2 can also influence cell death in suspension (43), and others observe enrichment for p53 mutations or Bcl-2 overexpression in metastases (44,45). Hence, loss of apoptosis can impact tumor initiation, progression and metastasis. Mechanisms of apoptosis Although the primary focus of this review is on the biology of apoptosis in cancer, much is known about the biochemical action of many apoptotic players, and key components are being assembled into pathways. At the molecular level, the best understood cell death pathways involve those initiated by ‘death receptors‘, including Fas/CD95, TNFR1, DR3, DR4 and DR5 (46). Upon binding, tumor necrosis factor α (TNFα) trimerizes its ligand TNFR1 and results in the subsequent recruitment of the signal transducing molecules TRADD through conserved protein interaction regions known as ‘death domains’. TRADD recruits RIP and TNF receptor-associated factor (TRAF-2), leading to activation of nuclear factor κB (NF-κB), which suppresses TNF-α-induced apoptosis (47). While the recruitment of FADD by TRADD results in apoptosis

Apoptosis in cancer

through activation of a cell death protease, caspase-8. Activated caspase-8 initiates a protease cascade that cleaves cellular targets and results in apoptotic cell death (46). Hence, disruption of FADD can prevent activation of caspase-8, thereby producing defects in receptor-mediated cell death (48). This pathway is rarely the target of oncogenic mutations, but, if anything, it is enhanced during tumor development (see below). Growth factors, cytokines and DNA damage appear to signal cell death through the mitochondria, and this pathway is the target of many oncogenic mutations. These diverse signals affect the function of Bcl-2 family members which, in turn, can modulate the mitochondrial function though the permeability transition pore (PTP), a proposed channel evolved in mitochondria following necrotic or apoptotic signals. The PTP is thought to be composed of clustered components of the mitochondrial membranes, including the voltage-dependent anion channel and adenine nucleotide translocator; and the opening of PTP results in the release of cytochrome c from mitochondria (49). Consistent with this idea, the crystal structure of Bcl-xL is reminiscent of pore-forming proteins of some bacterial toxins (50). Enforced expression of the pro-apoptotic molecules Bax or Bak can result in increased mitochondria membrane potential and release of cytochrome c, which can be blocked by overexpression of Bcl-2 (51). Cytosolic cytochrome c can interact with Apaf-1 and pro-caspase-9 to initiate a protease cascade similar to that described above (52–54). As eluded to above, a series of enzymes known as caspases are considered the engine of apoptotic cell death. Caspases are cysteine proteases that are expressed as inactive proenzymes, and can be broadly classified into ‘signaling’ or ‘effector’ caspases (55). Signaling pro-caspases associate with specific adapter molecules that facilitate caspase activation by induced proximity (56–58). For example, caspase-9 associates with Apaf-1, and oligomerization of this complex in the presence of cytochrome c can activate the downstream caspase cascade. Other adapter/caspase complexes include FADD/ caspase-8 and RAIDD/caspase-2 (46). In mitochondrial pathways, most caspases act downstream of cytochrome c release, and some evidence suggests that disruption of caspases only delays cell death (59,60). However, in other circumstances, loss of these proteases produces pathological increases in cell numbers (29,61–65). To date, little is known about the involvement of caspase mutations in cancers. Nevertheless, disruption of Apaf-1 is associated with Noonan’s Syndrome, caspase-10 mutations contribute to autoimmune lymphoproliferative syndrome type II (65,66), and frameshift mutations in caspase-5 can occur in hereditary nonpolyposis colorectal cancers, gastrointestinal and endometrial tumors (67). Certain signal transduction pathways alter the probability with which pro-apoptotic signals induce apoptosis. For example, cytokines such as IL-6 can suppress p53-induced apoptosis in certain cell types (16). Also, TNF-α-induced apoptosis is modulated by TRADD’s ability to bind TRAF2, which facilitates NF-κB-mediated cell survival (47,68). Finally, PI-3 kinase pathway mediates cell survival signaling from extracellular cytokines receptors. These receptors activate Ras and a kinase cascade involving PI-3 kinase and Akt leading to the ultimate phosphorylation and inactivation of pro-apoptotic molecules such as BAD and caspase-9 (69,70). PTEN acts as a lipid phosphatase to inactivate 3-phosphorylated phosphoinositides, thereby downregulating this pathway (71,72). Together, these studies imply that the ultimate decision to initiate

apoptosis results from a complex integration of internal and external pro- and anti-apoptotic signals. Apoptotic programs cannot simply be described as two parallel programs converging on a common caspase machinery. First, genetic studies using caspase-deficient mice demonstrate that the requirement for different death effector molecules during apoptosis is highly variable, being cell-type and stimulus specific (61–65). Second, a large degree of ‘cross-talk’ can exist between pathways. For example, p53 can transactivate genes encoding death receptors (73). Also, receptor-mediated activation of caspase-8 can cleave and activate BID, a proapoptotic Bcl-2 family member that can facilitate cytochrome c release from the mitochondria (74,75). Animal studies indicate that the caspase-8/BID pathway is highly cell-type dependent (76). It is likely that more complexities will be identified; although confounding to the experimentalist, cell type and stimuli specificities provide avenues for designing selective therapeutics. Oncogenic mutations can promote apoptosis Some oncogenic changes promote, rather than suppress apoptosis. Although this finding has ramifications for multistep carcinogenesis and cancer therapy (see below), the initial insights came from studies on adenovirus. Adenovirus encodes several oncoproteins that, when expressed together, transform rodent cells (77). The E1A oncoprotein induces quiescent cells to enter S phase (presumably to make the host cell permissive for virus replication) and, as a result, acts as a potent oncogene. The E1B 19 and 55K oncoproteins are required for efficient virus replication and cooperate with E1A in transformation. Adenovirus mutants lacking the E1B 19K protein induce an E1A-dependent ‘cytocidal’ phenotype that is associated with ‘degradation’ of both viral and host-cell DNA (78,79). As a result, E1B mutant adenoviruses produce poor virus yield. Subsequent studies explained these puzzling observation: E1A induces apoptosis, and the E1B 19K oncoprotein acts like Bcl2 to suppresses apoptosis (80). In virus-infected cells, E1B prevents E1A-induced apoptosis, allowing viral replication to proceed. This implies that apoptosis can act to counter virus replication and provides a biologic basis for cooperation between E1A and E1B in adenovirus transformation. Studies on the c-myc oncogene highlight the importance of oncogene-induced apoptosis in human cancer (81). In normal cells, ectopic c-Myc expression drives proliferation and prevents cell-cycle arrest upon serum withdrawal. However, while c-Myc-expressing cells continue to proliferate in low serum, cells do not accumulate because they die by apoptosis (82). Importantly, survival factors such as IGF-1 can suppress cMyc-induced cell death without producing substantial effects on c-Myc-induced proliferation (83). The observation that cMyc actively promotes apoptosis explains the potent cooperative effects observed between c-myc and bcl-2 in murine lymphomagenesis (10). In fact, much like E1A and E1B, c-Myc cooperates with Bcl-2 to transform rodent fibroblasts (84,85). The ability of c-Myc to cooperate with Bcl-2 in transformation could be viewed very much like E1A and E1B; Bcl-2 allows c-Myc-induced proliferation to proceed without apoptosis. Why do some oncogenes promote apoptosis? Studies on cMyc have been unable to separate its proliferative functions from those which promote apoptosis, implying that the processes are coupled (86). Similarly, in normal fibroblasts, the 487

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ability of E1A to promote apoptosis depends on its ability to inactivate the retinoblastoma tumor suppressor, and cannot be separated from its transforming functions (87). However, Rb loss leads to increased apoptosis in animal models (88,89), and reintroduction of Rb into cells lacking functional protein can suppress apoptosis (90). Both E1A and loss of Rb deregulate the E2F transcription factors, and E2F-1 overexpression itself can induce apoptosis (91–93). Hence, the pro-apoptotic activities of E1A and c-Myc appear directly or indirectly coupled to their hyperproliferative activity, implying that apoptosis acts as part of a cellular fail-safe mechanism to limit the consequences of aberrant mitogenic signaling. The critical involvement of p53 in oncogene-induced cell death underscores the importance of this response as a protective measure against oncogenic transformation. Although p53 is not required for normal apoptotic deaths during development, E1A can stabilize p53 protein (94), and p53-deficient cells are resistant to E1A-induced apoptosis (95,96). p53 loss substitutes for the E1B proteins in promoting the growth, survival and transformation of E1A-expressing cells (95). This implies that the primary function of the E1B proteins in adenovirus transformation is to circumvent this program. c-Myc also induces apoptosis in a p53-dependent manner (97,98). In vivo, loss of p53 function can dramatically accelerate tumorigenesis induced by mitogenic oncogenes such as large T antigen, E7 and c-Myc, which is associated with decreased apoptosis in situ (99–102). Importantly, in MMTV/c-myc transgenic mice, loss of p53 function accelerates thymic lymphoma but not mammary cancers, suggesting the involvement of p53-indepenedent mechanism. Hence, oncogenes can induce apoptosis in both p53-dependent and -independent manners, depending on cellular context. Recent studies indicate that p19ARF acts as an essential intermediate in oncogene signaling to p53 (103). For example, oncogenes such as E1A or c-myc induce ARF message and protein in normal mouse embryo fibroblasts, which correlates with their ability to activate p53 and promote apoptosis. Recently, ARF was shown to be a transcriptional target of c-Myc (104). In contrast, these oncogenes fail to activate p53 in ARF-null cells, and promote proliferation without substantial apoptosis (105,106). Together, these studies indicate that p19ARF acts as part of a p53-dependent fail-safe mechanism to counter hyperproliferative signals, and predict that disruption of ARF, or the INK4a/ARF locus, should cooperate with mitogenic oncogenes during tumor development. Studies using c-myc transgenic mice support this view (102). One question is whether oncogenes directly induce apoptosis or ‘sensitize’ the cell to various apoptotic stimuli. Clearly, oncogenes can induce apoptosis directly if expressed at sufficient levels, and E1A-expressing cells possess an oncogenegenerated activity capable of activating apoptosis in cell free systems (107). However, cells stably expressing E1A or cMyc are ‘sensitized’ to diverse apoptotic stimuli, including serum depletion, DNA damaging agents, hypoxia, Fas, TNFα and other stimuli (81). One target of oncogene-induced sensitization is the mitochondria. Both E1A and c-Myc facilitate cytochrome c release from mitochondria, and cytochrome c alone is sufficient to sensitize cells to diverse agents (107,108). Oncogenes can facilitate cytochrome c release independently of p53 (108), although it is worth noting that p53 can induce Bax and proteins that affect mitochondrial function (109,110). Hence, p53 may be involved in sensitization 488

Fig. 1. Oncogene-induced apoptosis. Oncogenes such as E1A and c-myc induce apoptosis through p53-dependent and independent pathways, and both pathways may facilitate cytochrome c release from mitochondria. In any case, the Apaf-1/caspase-9 death effector complex appears important for oncogene-induced death. Current evidence has not ruled out the possibility that oncogenes and/or p53 influence Apaf-1 and/or caspase-9 independent of cytochrome c, but this remains a possibility. Components of the oncogeneinduced cell-death program that are mutated in human tumors are shown in black, candidate tumor suppressors are shown in gray.

as well as active induction of cell death in oncogene-expressing cells (Figure 1). Caspase-9 and its adapter Apaf-1 are essential downstream components of p53 in oncogene-induced death (29). Hence, disruption of either Apaf-1 or caspase-9 in fibroblasts prevents c-Myc-induced death without affecting p53 accumulation or activation. In fact, inactivation of either caspase-9 or apaf-1 completely substitutes for p53 loss in promoting the oncogenic transformation of cells by c-Myc and Ras (29). Concordantly, Apaf-1 has been identified in cell free systems as an ‘oncogenegenerated activity’ important for apoptosis in E1A-expressing cells (107,111). Because cytochrome c is an essential co-factor that is required for activation of the Apaf-1/caspase-9 protease complex, these studies suggest how oncogenes are linked to the effector phase of apoptosis (Figure 1). Of note, several molecules that modulate oncogene-induced apoptosis are mutated in human tumors, and the data described above identify Apaf-1 and caspase-9 as candidate tumor suppressors (Figure 1). However, to date, no mutations in either apaf-1 or caspase9 have been described. Perhaps these components act too late in the cell death program to provide a long-term survival advantage; alternatively, the mutational status of these genes may not have been thoroughly examined. Apoptosis and cancer therapy Most anticancer agents now in use were developed using empirical screens designed to identify agents that selectively kill tumor cells. Until recently, most research into drug action focused on their intracellular targets, the nature of the cellular damage produced by the drug–target interaction, or resistance mechanisms that prevent the drug target interaction. However,

Apoptosis in cancer

Fig. 2. Anticancer agents induce apoptosis. Hematoxylin and eosin staining of lymph nodes from lymphoma-bearing mice (B cell lymphoma) left untreated (A) or isolated 5 h after treatment with cyclophosphamide (B). Apoptotic cells are identified by their reduced size and by the presence of highly condensed chromatin. Massive apoptosis occurs in response to cyclophosphamide.

in the 1970s pathologists noticed that radiation and chemotherapy can induce cell death with morphological features of apoptosis (112) (Figure 2), although the significance of these observations was not widely appreciated. In particular, the premise that anticancer agents induce apoptotic cell death implies that cellular responses occurring after the drug–target interaction can have impact on drug-induced cell death (113– 115). It is now well-established that anticancer agents induce apoptosis, and that disruption of apoptotic programs can reduce treatment sensitivity (116). Since agents with distinct primary targets can induce apoptosis through similar mechanisms, mutations in apoptotic programs produce multi-drug resistance (113,117). For example, many agents activate p53, and that p53 loss can attenuate drug-induced cell death (15). Moreover, p53 mutations reduce therapy-induced apoptosis and tumor regression in experimentally generated and spontaneous murine tumors (102,118), whereas re-introduction of normal p53 to p53 mutant tumor lines and xenographs cooperates with chemotherapy to induce apoptosis and tumor regression (15,119). p53 is not strictly required for drug-induced cell death; indeed, at sufficient doses virtually all anticancer agents induce apoptosis (and other types of death) independently of p53 (15). In fact, the contribution of p53 to drug-induced apoptosis is determined by a variety of factors, including agent, dose, tissue and mutational background of the tumor (15,120,121). In short-term assays, Bcl-2 can promote resistance to a wide range of anticancer agents (122,123) and can even prevent p53-independent deaths (124). Because Bcl-2 is considered as a general apoptosis inhibitor, these results argue for the broad importance of apoptosis in treatment sensitivity. Additionally, death receptor pathways may also contribute to therapy-induced apoptosis (125), although the relative contribution of these effects is controversial (126). In human cancer, the most compelling links between apoptosis and treatment sensitivity occur in patients with leukemia or lymphoma. In these malignancies, p53 mutations correlate with short remissions and drug resistance following therapy (15). Also, INK4a/ARF mutations, which reduce cyclophosphamide-induced death in murine lymphomas (102), are associated with poor treatment outcome in acute lymphoblastic leukemia (127). The extent to which apoptosis contributes to

treatment sensitivity in carcinomas is less clear: whereas some studies identify striking correlations between p53 mutations and poor treatment response, others see no effect (116,128). Few studies have associated Bcl-2 with drug resistance in patients and, in fact, high Bcl-2 levels may be a good prognostic indicator for breast cancer. Finally, in some settings loss of Bcl-2 and p53 delays therapy-induced apoptosis but does not enhance long-term survival in clonogenic assays (128). What could explain these discrepancies? Though it is possible that apoptosis does not contribute to treatment sensitivity in solid tumors, some caveats are worth mentioning. First, clinical studies typically examine single alterations (e.g. p53 mutation) relying on detection methods that are not perfect and cannot exclude extragenic mutations in the same pathway (15,116). This makes it virtually impossible to determine negative results. As an example, murine lymphomas harboring INK4a/ARF mutations are chemoresistant, display defective p53 function, but retain wild-type p53 genes (102). These tumors would be mistakenly classified as ‘p53 normal’ by current technologies. Secondly, while clonogenic survival is often considered the ‘gold-standard’ of cytotoxicity assays, this readout does not always reflect the in vivo response (129). It is possible that extracellular survival factors—influenced by cell density or microenvironment—can affect drug-induced death (130). Anticancer agents induce apoptosis in normal tissues as well as in tumors. In fact, many of the pathologists who identified apoptosis in tumors realized that apoptotic cell death was induced in a subset of normal tissues (e.g. bone marrow and intestine), and it was suggested that the process might contribute to the ‘toxicity’ associated with chemotherapy (112). Studies using mouse models provide strong support for this idea. For example, moderate doses of radiation and chemotherapy induce apoptosis in the murine thymus, spleen, bone marrow and intestine, the same tissues that account for the deleterious side-effects of chemotherapy. However, these tissues in p53 ‘knockout’ mice display much reduced apoptosis and cell loss following radiation or chemotherapy (17,18,131– 134), and these animals are resistant to otherwise lethal doses of ionizing radiation (135). Similarly, ectopic expression of Bcl-2 in bone marrow cells achieves a similar effect (136). Together, these studies strongly suggest that drug-induced apoptosis causes loss of normal cells and contributes to the side effects of cancer therapy. Apoptosis: a biological link between cancer genetics and cancer therapy The studies described above highlight the fact that disruption of apoptosis can promote tumor initiation, progression and treatment resistance. Indeed, it is remarkable that the same genetic alterations that influence apoptosis during tumorigenesis also modulate treatment sensitivity. For example, c-Myc enhances apoptosis in low concentrations of survival factors or oxygen and following treatment with diverse cytotoxic agents (38,82,137), conversely, loss of p53 and overexpression of Bcl-2 suppress apoptosis induced by oncogenes, depletion of survival factors, hypoxia and cytotoxic drugs (17,118,138). As a result, anti-apoptotic mutations arising during the course of tumor development can simultaneously select for chemoresistant cells. This pattern of co-selection may explain the phenomenon of de novo drug resistance, i.e. 489

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tumors that initially respond poorly to therapy despite having never been selected in the presence of drug. Studies on c-Myc-induced lymphomagenesis support directly link disruption of apoptosis during tumor development to de novo resistance (102). In a mouse lymphoma model, INK4a/ARF or p53 mutations dramatically accelerate c-Mycinduced lymphomagenesis, owing to a defect in oncogeneinduced apoptosis. Moreover, these tumors respond poorly to therapy; whereas mice harboring tumors with an intact p53 pathway are cured, the vast majority harboring tumors with INK4a/ARF or p53 mutations relapse shortly after treatment. This pattern of ‘resistance’ is particularly common in advanced solid tumors. These tumor-types probably encounter many ‘triggers’ of apoptosis during tumor evolution (e.g. survival factor depletion, hypoxia, loss of cell-matrix interactions), and it seems likely that one or more apoptotic programs must be lost to reach the malignant state. Perhaps this explains why many advanced solid tumors are inherently difficult to treat. Non-apoptotic forms of ‘programmed cell death’ Although apoptosis is a cell death program, not all programmed ‘deaths’ are apoptotic. In addition to cell death, other programmed responses contribute to the deletion of potentially cancerous cells. Senescence is an irreversible program of cellcycle arrest that is disrupted in many tumors or tumor-derived lines (139). Replicative senescence was originally defined by the observation that primary cells have a genetically determined limit to their proliferative potential in cell culture, after which they permanently arrest with characteristic features. Owing to the ‘end-replication problem’, telomeres shorten during each cell division unless telomerase is expressed, and it is thought that some aspect of excessive telomere shortening activates cell-cycle arrest and other characteristics of senescence (139). However, other stimuli can induce phenotypes suggestive of senescence, including mitogenic oncogenes and ionizing radiation (140–143). These observations imply that cellular senescence can be induced by diverse stimuli leading to the engagement of a common cell-cycle arrest program. In this view, senescence is conceptually similar to apoptosis, which is induced by diverse stimuli leading to the engagement of a common cell death program. Consequently, the biological roles of cellular senescence may go beyond the control of cellular or organismal aging, and reflect a global anti-proliferative response to a variety of cellular stresses. In this view, senescence may represent an important form of programmed cellular ‘death’ that limits tumor development. Recent evidence suggests that anticancer agents induce cellular senescence in human tumorderived lines treated in culture or as xenographs (144,145). Together, the non-apoptotic forms of ‘programmed cell death’, such as senescence, might also be important for therapy. Apoptosis and new therapeutic strategies Since apoptotic programs can be manipulated to produce massive changes in cell death, the genes and proteins controlling apoptosis are potential drug targets. As indicated above, many empirically derived cytotoxic drugs already may target apoptosis, albeit indirectly and non-exclusively. They are also mutagenic and toxic to normal tissues. In contrast, agents that directly induce apoptosis may provide less opportunity for acquired drug resistance, decrease mutagenesis and reduce toxicity. Two observations suggest that such strategies are 490

feasible. First, most anti-apoptotic mutations act relatively upstream in the program (e.g. PTEN and p53 loss; Ras and NF-κB activation), implying that tumor cells retain the ‘machinery’ and latent potential for apoptosis. Secondly, tumorspecific alterations in apoptotic programs provide opportunities to target cell death in a selective manner. Several current strategies are discussed below. Targeting anti-apoptotic activities In instances where apoptosis is disabled by dominant oncogenes, agents that disrupt their anti-apoptotic function can produce remarkable increases in cell death. Overexpression of anti-apoptotic Bcl-2 family members can promote tumorigenesis and chemoresistance, suggesting that functional inhibition of these proteins might be lethal to cancer cells. Indeed, adenoviral gene transfer of Bax results in cytotoxicity in human ovarian cancer cell lines, and administration of AdDF3-Bax following tumor inoculation eradicated ⬎99% of tumor implants in nude mice (146). Adenovirus-mediated gene transfer of Bcl-xS, a dominant-negative repressor of Bcl-2 and Bcl-xL, can synergize with chemotherapy and promote tumor regression in xenographs (147,148), but produces minimal apoptosis when introduced into normal epithelial cells (149). Some current anticancer agents may unwittingly target the Bcl-2 family; for example, taxanes may induce phosphorylation and inactivation of Bcl-2 (150). As more is learned about Bcl-2 structure and function, small molecule inhibitors of Bcl2 action might become feasible. Hyperactivation of cell survival signaling may accompany tumor development, and these pathways are particularly exciting targets for small molecule inhibition. NF-κB activity can be induced by oncogenes or anticancer agents to promote cell survival, such that inactivating NF-κB enhances cell death in response to tumor necrosis factor and certain chemotherapeutic agents (151). In fact, inhibiting NF-κB activity using I-κB or proteosome inhibitors synergizes with radiation and chemotherapy to induce cell death in tumor lines and xenographs (152,153). The PI-3 kinase/Akt pathway involves a series of enzymes that transit the survival signals. In principle, small molecule inhibition of any of these molecules might restore apoptosis to tumor cells, or synergize with more classic agents to induce cell death (154). Oncogenic ras mutations deregulate normal growth control but can also signal cell survival (34). Therefore, agents that interfere with Ras function might be cytostatic or cytotoxic. To be biologically active, Ras must be modified by a farnysltransferase, and many groups have developed farnysltransferase inhibitors as anti-tumor agents. Although these agents were predicted to be cytostatic, they can induce massive apoptosis and tumor regression of mammary carcinomas arising in ras transgenic mice (120). In addition, inhibition of Ras-GAP induces apoptosis specifically in tumor, but not in normal cells, suggesting Ras-GAP as a novel target for cancer therapy (155). Why would these agents induce apoptosis and, moreover, why would they be selective? The answer is unknown, but it is possible that tumor cells become dependent on survival signaling through the PI-3 kinase pathway, which could be suppressed by Ras inhibition. Restoring pro-apoptotic activities In circumstances where apoptosis is lost by a recessive mutation, restoring the dysfunctional gene or activity can promote massive cell death. For example, reintroduction of p53 into p53 mutant tumor cells can directly induce apoptosis

Apoptosis in cancer

or enhance treatment sensitivity in tumor cell lines or in xenographs (156,157). Indeed, strategies using this approach are currently in clinical trials (158). As a general rule, tumor cells are inherently more sensitive to p53 inhibition than normal cells, perhaps because mitogenic oncogenes can activate p53 to promote apoptosis (117). Hence, there is some rationale for selectivity of this approach. Of note, strategies to counter recessive anti-apoptotic mutations need not rely on gene or protein therapy. In some instances, inactivation of a proapoptotic gene might actually promote cell survival by relieving inhibition of a downstream death suppressor (e.g. PTEN loss de-represses Akt). In such instances, the downstream effector may be a more suitable drug target (154). Death ligands Although mutations in death receptors are rare events in human tumors, changes accompanying tumorigenesis can alter the regulation of these pathways. Mitogenic oncogenes like c-myc and E1A can increase sensitivity to Fas and TNF-α in cultured cells (159,160), and many human tumors have alterations in TRAIL-mediated death pathways (19). TRAIL is a TNFrelated protein that initiates p53-independent apoptosis by binding to its receptors DR4 or DR5. Normal cells are resistant to TRAIL-induced apoptosis, apparently because these cells express ‘decoy’ receptors that compete with DR4 and DR5 for TRAIL but do not transmit a death-inducing signal (161,162). Through an unknown mechanism, the expression of the decoy receptor 1 seems to be widely lost on tumor cells, making them exquisitely susceptible to TRAIL-mediated cell death. Because DR5 is a p53-responsive gene, combination therapy with TRAIL and classic cytotoxic agents may be particularly effective in treating tumors with functional p53 (73). A conceptually related strategy for selective tumor-cell killing involves a viral protein known as ‘apoptin’, the VP3 product of chicken anemia virus (CAV). This protein is responsible for the cytopathic effects of CAV (163) and induces apoptosis in tumor cells but not normal cells (164). Although its mechanism of action remains unknown, apoptin-induced apoptosis is independent of p53 and is enhanced by Bcl-2 (165,166). At the very least, the remarkable effects of apoptin underscore the point that selective induction of apoptosis in tumor cells is achievable. Enhancing the effects of pro-apoptotic mutations Basic studies suggest that it is possible to directly harness the pro-apoptotic forces produced by certain oncogenic mutations to selectively kill tumor cells. As mentioned earlier, oncogenes like c-myc and inactivation of tumor suppressors such as Rb force proliferation but at the same time promote apoptosis, presumably as a cellular safe-guard against tumorigenesis. In normal cells, E1A promotes apoptosis and enhances chemosensitivity through a dual mechanism that involves inactivation of the retinoblastoma protein and binding the p300/CBP transcription co-activators. As a result, E1A mutants unable to bind Rb (but retaining the p300/CBP interaction) promote apoptosis in Rb-deficient cells but not normal cells (87). In principle, these E1A mutants, or small molecules which mimic their action, may provide tumor-specific anticancer agents by exploiting the fact that the Rb pathway is disrupted in the majority of human cancers. Rb mutations lead to increased E2F activity, and this promotes both proliferation and apoptosis (91–93). Whereas Rb represses E2F activity during G0/G1, cdk2 kinase can repress E2F activity during S phase (167). It has been suggested

that simultaneous inactivation of Rb and cdk2 would be particularly pro-apoptotic, and produce a synthetic lethal effect in cells with a mutant Rb pathway (168). Consistent with this hypothesis, cell-permeable peptides that interfere with cdk2 activity readily induce apoptosis in tumor cells while having little effect on normal cell lines (168). Interestingly, although E2F-induced apoptosis is potentiated by p53 (91), these peptides can induce apoptosis in p53-deficient tumor cell lines. Therefore, this strategy has widespread potential. Chemoprotection As indicated above, the propensity of certain tissues (e.g. bone marrow and intestine) to drug-induced apoptosis may limit the effectiveness of many current therapies. Studies using mouse models have clearly documented the importance of p53 for apoptosis in thymocytes, bone marrow and intestinal stem cells (see above); consequently, agents which suppress p53 function may be effective radio- or chemoprotective agents and/or allow dose intensification of current regimens. Since most advanced solid tumors have lost p53 function, these inhibitors should not interfere with cell death of most tumor cells. Recently, a small molecule (designated pifithrin-α) has been identified that inhibits p53-mediated transcriptional responses and p53-induced apoptosis in cultured cells (169). In mice, pifithrin-α is a potent radioprotective agent; pifithrinα treated mice survive otherwise lethal doses of ionizing radiation. Several caveats exist with respect to p53 inhibition in patients. First, the premise is based on animal studies, and it is not yet known the extent to which p53 contributes to anticancer agent toxicity in humans. Secondly, although the protective effects in mice are striking, the magnitude of the protective effect over repeated doses and time is unknown. Thirdly, p53 inhibition may promote mutations by allowing the survival of mutated cells. This is clearly a concern, since p53 ‘knockout’ mice are extremely sensitive to radiationinduced carcinogenesis. However, transient inhibition of p53 may be less mutagenic (169). In any case, most current anticancer agents are directly mutagenic, and it is not clear whether p53 inhibitors (which would only indirectly promote mutations) would be worse. At the very least, this strategy warrants further consideration. Conclusion The last decade has seen an extraordinary increase in our understanding of apoptosis, and its contribution to cancer and cancer therapy. Furthermore, the molecular mechanisms that control and execute apoptotic cell death are coming into focus. Although there is much more to learn, our current understanding of apoptosis provides new avenues for cancer diagnostics, prognosis and therapy. In the coming years, it seems likely that rational strategies to manipulate cell suicide programs will produce new therapies that are less toxic and mutagenic than current treatment regimens. Acknowledgements The authors apologize to those investigators whose work was not cited due to an oversight or space constraints. We thank M.McCurrach for editorial comments and support. S.W.L. is a Rita Allen Scholar; S.W.L. and A.W.L. receive support from grants CA13106 and AG16379 from the National Institutes of Health.

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