Programmed Cell Death
Vassiliki Karantza-Wadsworth/ Devita Cancer Book
Eileen P. White
Type I programmed cell death, also known as apoptosis, is a genetic pathway for rapid and efficient killing of unnecessary or damaged cells; it was initially described by Kerr et al.,1 Vogt,2 and Wyllie et al.3 They detailed a novel morphologic process for cell death that included swiftly executed cell shrinkage, blebbing of the plasma membrane, chromatin condensation, and intranucleosomal DNA fragmentation, after which cell corpses were engulfed by neighboring cells and degraded. Apoptosis (commonly pronounced ap-a-tow'-sis), is a term coined from the Greek apo, or from, and ptosis, or falling, to make the analogy of leaves falling off a tree. Although underappreciated at the time, once the genes that controlled apoptosis were identified in model organisms and humans, and it was shown that perturbation of this program disturbed development and provoked disease, the importance of apoptosis was generally realized.
Cell death by apoptosis is required for sculpting tissues in normal development and is part of the host defense system against disease.4,5,6 These developmental cell deaths span the removal of the interdigital webs and tadpole tales, to selection for and against specific B- and T-cell populations essential for controlling the immune response. Proper regulation of apoptosis is critical in that excessive apoptosis is associated with degenerative conditions, and deficient apoptosis promotes autoimmunity and cancer. Furthermore, apoptosis is required for eliminating damaged or pathogen-infected cells as a mechanism for limiting disease, especially cancer. In turn, tumors and pathogens have also evolved elegant mechanisms for disabling apoptosis to facilitate their persistence and disease progression. In human cancers, mechanisms to disable apoptosis include loss of function of the apoptosis-promoting p53 tumor suppressor and gain of function of the apoptosis-inhibitory and oncogenic Bcl-2. It became apparent that cancer progression was aided not only by increasing the rate of cell multiplication through activation of the c-myc oncogene, for example, but also by decreasing the rate of cell elimination through apoptosis, exemplified by gain of Bcl-2 expression (Fig. 7.1). Indeed, activation of oncogenes such as c-myc can promote apoptosis, providing an explanation for the necessity for inactivation of the apoptotic pathway in many tumors. Furthermore, the effectiveness of many existing anticancer drugs involves or is facilitated by triggering the apoptotic response. Thus, a detailed understanding of the components, molecular signaling events, and control points in the apoptotic pathway has enabled rational approaches to chemotherapy aimed at restoring the capacity for apoptosis to tumor cells. Identification of the molecular means by which tumors inactivate apoptosis has led to cancer therapies directly targeting the apoptotic pathway.7 These drugs are now entering the clinic to specifically reactivate apoptosis in tumor cells in which it is disabled to achieve tumor regression. We review here the key aspects of apoptosis and how they relate to cancer development, progression and treatment response.
Model Organisms Provide Mechanistic Insight into Apoptosis Regulation
Key to elevating the field of programmed cell death from a descriptive to a mechanism-based process was the discovery of genes in the nematode Caenorhabditis elegans (C. elegans) that control cell death, the cell death defective or ced genes.8 Genetic analysis revealed that ced-4 and ced-3 promote cell death as worms with defective mutations in these genes possessed extra cells. In contrast, the ced-9 gene product inhibits the death-promoting function of the ced-4 and ced-3 gene products, thereby maintaining cell viability.9 The proapoptotic egl-1 gene product inhibits the ced-9 gene product, creating a linear pathway controlled upstream by cell-specific death specification regulators, and downstream by cell corpse engulfment and degradation mechanisms (Fig. 7.2).10 These findings help propel work in mammalian systems when it became apparent that Ced-9 was homologous to Bcl-2,11 Ced-3 was homologous to interleukin 1-ฮฒ converting enzyme, a cysteine protease that would later be classified as a member of the caspase family of aspartic acid proteases,12 Egl-1 was a BH3-only protein homologue,10 and that the proapoptotic apoptotic protease-activating factor-1 (APAF-1) identified in mammals was homologous to Ced-4.13 A similar cell death pathway in the fruit fly Drosophila melanogaster identified Reaper, Hid, and Grim as inhibitors of the inhibitors of apoptosis proteins (IAPs) that negatively regulate caspase activation.14 This eventually led to the identification of their mammalian counterpart second mitochondrial-derived activator of caspase (SMAC), also known as direct IAP-binding protein with low pI (DIABLO).15 These and other studies established the paradigm whereby proapoptotic BH3-only proteins inhibit antiapoptotic Bcl-2 proteins that prevent both APAF-1-mediated caspase activation by cytochrome c, and inhibition of caspase inhibitors (IAPs) (Fig. 7.2). The resulting caspase activation and proteolytic cellular destruction leads rapidly to cell death.

Figure 7.1. Role of apoptosis in tumor progression. Tumor progression occurs through cooperation of proliferative and antiapoptotic functions. In normal cells in epithelial tissues (green cells) initiating mutational events, such as deregulation of c-myc expression, deregulate cell growth control and promote abnormal cell proliferation (yellow cells) while triggering a proapoptotic tumor suppression mechanism (red apoptotic cells) that can restrict tumor expansion. Subsequent acquisition of mutations that disable the apoptotic response, exemplified by bcl-2 overexpression, prevents this effective means of killing emerging tumor cells, thereby favoring tumor expansion. Similar oncogenic events occur in lymphoid tissues.
Discovery of Bcl-2 and its Role as an Apoptosis Inhibitor in B-Cell Lymphoma
To identify mechanisms of oncogenesis, the bcl-2 gene was cloned from the site of frequent chromosome translocation t(14;18): (q32;q21) in human follicular lymphoma.16,17,18 This chromosome rearrangement places bcl-2 under the transcriptional control of the immunoglobulin heavy chain locus causing abnormally high levels of bcl-2 expression. Distinct from other oncogenes at the time, instead of promoting cell proliferation, bcl-2 promoted B-cell tumorigenesis by the novel concept of providing a survival advantage to cells stimulated to proliferate by c-myc.19 Indeed, engineering high Bcl-2 expression in the lymphoid compartment in mutant mice promotes follicular hyperplasia that progresses to lymphoma on c-myc translocation, and bcl-2 synergizes with c-myc to produce lymphoid tumors, paralleling events in human follicular lymphoma.20,21 Bcl-2 localizes to mitochondria22 where it has broad activity in promoting cell survival through suppression of apoptosis provoked by numerous events including oncogene activation (c-myc, E1A), tumor suppressor activation (p53), growth factor and cytokine limitation, and cellular damage.5,6,23 It also became clear that inactivation of the retinoblastoma tumor suppressor pathway promotes a p53-mediated apoptotic response, suggesting that apoptosis was part of a tumor suppression mechanism that responded to deregulation of cell growth


(Figs. 7.1 and 7.3).24,25 Indeed, apoptotic defects acquired by a variety of means are a common event in human tumorigenesis.

Figure 7.2. Analogous pathways regulate programmed cell death/apoptosis in metazoans. Regulation of programmed cell death in the nematode Caenorhabditis elegans (top) and in mammals (bottom). Shaded regions highlight corresponding homologous genes and protein families. In C. elegans, numerous cell death specification genes can up-regulate the transcription of the BH3-only protein Egl-1, which interacts with the antiapoptotic Bcl-2 homologue Ced-9, inhibiting its interaction with Ced-4. Ced-4, the Apaf-1 homologue, in turn, activates the caspase Ced-3, leading to cell death. A variety of engulfment gene products are then responsible for apoptotic corpse elimination and nucleases degrade the genome. In mammals, many survival, damage, and stress events impinge on the numerous members of the BH3-only class of proapoptotic proteins to either activate them to promote apoptosis, or suppress their activation to enable cell survival. BH3-only proteins interact with and antagonize the numerous Bcl-2-related multidomain antiapoptotic proteins that serve to sequester proapoptotic Bax and Bak, and may also contribute directly to Bax/Bak activation. Bax or Bak is essential for signaling apoptosis by permeabilizing the outer mitochondrial membrane to allow the release of cytochrome c and SMAC. Cytochrome c acts as a cofactor for Apaf-1-mediated caspase activation in the apoptosome, and the second mitochondrial-derived activator of caspase (SMAC) amino-terminal four amino acids bind and antagonize the inhibitors of apoptosis proteins (IAPs). IAPs interact with and suppress caspases, and inhibition of IAPs by interaction with SMAC facilitates caspase activation, widespread substrate cleavage, and cell death. Many engulfment gene products are responsible for corpse elimination and caspase-activated nucleases in the apoptotic cell itself, and additional nucleases within the engulfing cell are responsible for degradation of the genome.

Figure 7.3. Regulation of apoptosis by the Bcl-2 family of proteins in mammals. A: Schematic of apoptosis regulation by the Bcl-2 family. Cytotoxic events activate while survival signaling events suppress the activity of the BH3-only class of Bcl-2 family members (orange). BH3-only proteins are controlled at the transcription level and also by numerous posttranscriptional events that modulate phosphorylation, proteolysis, localization, sequestration, and protein stability. Once activated, BH3-only proteins disrupt functional sequestration of Bak and Bax by the multidomain antiapoptotic Bcl-2-like proteins (blue) and may also directly facilitate Bax/Bak activation. Although Bak is commonly membrane-associated in a complex with Mcl-1 and Bcl-xL in healthy cells, Bax resides in the cytoplasm as an inactive monomer with its carboxy-terminus occluding the BH3-binding hydrophobic cleft.75 Bax activation thereby additionally requires a change in protein conformation and membrane translocation by an unknown mechanism that may be facilitated by tBid binding. Binding specificity among BH3-only proteins for antiapoptotic Bcl-2-like proteins determines which complexes are disrupted, with some BH3-only proteins having broad specificity and others not. Survival and death-signaling events can also modulate apoptosis by targeting the multidomain antiapoptotic proteins either by antagonizing their antiapoptotic function or by stimulating their function to promote survival. ABT-737 is a rationally designed Bad BH3-mimetic that can bind Bcl-2, Bcl-xL, and Bcl-w but not Mcl-1, and can promote apoptosis where survival does not depend on Mcl-1. Once activated, Bax or Bak oligomerization promotes apoptosis. B: Tumor necrosis factor (TNF)-ฮฑ plus cyloheximide (TNF/CHX) apoptotic signaling induces mitochondrial membrane translocation and a conformational change exposing the amino-terminus of Bax (visualized here by the Bax-NT antibody) and apoptosis, which is blocked by sequestration of Bax by the antiapoptotic viral Bcl-2 homologue E1B 19K. The human cancer cell line (HeLa cells), with or without E1B 19K expression, were then left untreated or treated with TNF/CHX. The localization of conformationally altered Bax (Bax-NT) and cytochrome c (left and middle panels), or E1B 19K and cytochrome c (right panel), are shown. The proapoptotic stimulus (TNF/CHX) induces Bax activation, mitochondrial translocation, and cytochrome c release from mitochondria that leads to caspase activation and apoptotic cell death, whereas expression of E1B 19K sequesters Bax, thereby blocking cytochrome c release from mitochondria, caspase activation, and apoptotic cell death. The yellow and red arrows, respectively, mark cells with partial or complete cytochrome c release from mitochondria on TNF/CHX treatment.
Control of Apoptosis by Bcl-2 Family Members
Bcl-2 is the first member of what is now a large family of related proteins that regulate apoptosis and are conserved among metazoans including worms, flies, and mammals, and also viruses.5,6,23,26 Multidomain Bcl-2 family members containing Bcl-2 homology regions 1-4 (BH1-4) are either antiapoptotic (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, Bfl-1/A1, and virally encoded Bcl-2 homologues such as E1B 19K), or proapoptotic (Bax and Bak), and antiapoptotic proteins block apoptosis primarily by binding and sequestering Bax and Bak (Fig. 7.3A).27,28,29 Bax and Bak are functionally redundant and required for signaling apoptosis.30 In healthy cells, Bak is bound and sequestered by Mcl-1 and Bcl-xL at cellular membranes, whereas Bax resides in the cytosol in a latent form and requires conformational activation and translocation to membranes where it is either sequestered by antiapoptotic Bcl-2-like proteins or otherwise induces apoptosis (Fig. 7.3A,B).
Control of Multidomain Bcl-2 Family Proteins by the BH3-Only Proteins
BH3-only Bcl-2 family members (Bim, Bid, Nbk/Bik, Puma, Bmf, Bad, and Noxa) are proapoptotic and primarily required to antagonize the survival activity of antiapoptotic Bcl-2-like proteins by displacing Bax and Bak to allow apoptosis.29 The different BH3-only proteins respond to specific stimuli to activate apoptosis (Fig. 7.3A). For example, Bim induces apoptosis in response to taxanes,31 Puma and Noxa are transcriptional targets of and mediate apoptosis in response to p53 activation,32 Bad signals apoptosis on growth factor withdrawal, Bid is required for apoptosis signaled by death receptors, Bmf is regulated by the cytoskeleton,33 and Nbk/Bik promotes apoptosis in response to inhibition of protein synthesis.34 The BH3 of BH3-only proteins bind to a hydrophobic cleft in the multidomain Bcl-2 family members that also supports Bax and Bak binding,7,35 causing their displacement.29 Differential binding specificities among the BH3s of different BH3-only proteins determine whether they bind one or more Bcl-2-related proteins and displace Bax or Bak or both.36 Noxa binds and antagonizes Mcl-1, whereas Bad binds and antagonizes Bcl-2 and Bcl-xL, necessitating cooperation between Noxa and Bad function for efficient apoptosis. In contrast, Bim, Bid, and Puma have broader binding specificity and antagonize Mcl-1, Bcl-2, and Bcl-xL to release both Bax and Bak to induce apoptosis.4 Although Bid, Bim, and Puma may in some cases directly promote Bax and Bak activation, this is not yet been found to be essential for apoptosis.29 Importantly, it is this BH3 interaction with Bcl-2 that is the molecular basis for the BH3-mimetic class of proapoptotic, Bcl-2 antagonizing anticancer drugs (Fig. 7.4).7,37,38,39 This detailed understanding of the Bcl-2 family member protein interactions and function is allowing rational, apoptosis-targeted therapy.

Figure 7.4. Three-dimensional structure of Bcl-xL with bound Bad BH3 ligand and ABT-737. Space-filling model of Bcl-xL illustrating the hydrophobic cleft binding the 25-mer peptide (green helix) of the Bad BH3 (left) or the rationally designed BH3-mimetic ABT-737 in green (right). (From Fesik S. Nature Publishing group. Promoting apoptosis as a strategy for cancer drug discovery. Nat Rev Cancer 2005;5:880, with permission.)
Role of Mitochondrial Membrane Permeabilization in Apoptosis
Once activated, Bax and Bak oligomerize in the mitochondrial outer membrane rendering it permeable to proapoptotic mitochondrial proteins cytochrome c (Fig. 7.3B) and SMAC.40,41,42,43,44 How Bcl-2 family members permeabilize membranes is not entirely clear but it is likely related to a change in topology of the proteins in the membrane and formation of a channel or pore.45 Once released into the cytoplasm, cytochrome c interacts with the WD40 domains of APAF-1 in the apoptosome, a wheellike particle with sevenfold symmetry that serves as a scaffold for caspase-9 activation.46 SMAC functions to antagonize the caspase inhibitors, the IAP proteins, to facilitate caspase activation. The amino-terminus of SMAC binds to IAPs, neutralizing their caspase-inhibitory function. Subsequent effector

caspase activation (e.g., caspase-3) leads to the rapid, orderly dismantling of the cell and cell death without activating the innate immune response.47 Execution of apoptotic cell death is extremely rapid and efficient, resulting in cell death in under 1 hour in mammalian cells.48
Control of Apoptosis by Death Receptors
One of the apoptotic pathways being modulated in cancer therapies is that belonging to the death receptors. Ligands related to tumor necrosis factor-ฮฑ (TNF-ฮฑ) including Fas ligand and tumor necrosis factor-related ligand (TRAIL) and their cognate receptors were identified as potent activators of apoptosis, and this pathway is critical for regulating the immune response.49 Engagement of the receptors with soluble or membrane-bound ligand activates the death-inducing signaling complex composed of adaptor proteins such as FADD, which promotes activation of caspase-8 (Fig. 7.5). Caspase-8, in turn, cleaves the BH3-only protein BID to its truncated or activated form tBid, which then antagonizes the antiapoptotic function of Bcl-2-like proteins promoting Bax and Bak activation.4 This process signals cytochrome c and SMAC release from mitochondria and caspase-9 and -3 activation and cell death. In some cell types that do not require this Bcl-2 family protein-regulated mitochondrial amplification step, active caspase-8 can directly cleave and activate effector caspases to cause cell death by apoptosis (Fig. 7.5).
Modulation of the Death Receptor Pathway in Cancer Therapy
The ability of soluble ligands to activate the apoptotic response has stimulated interest in using this pathway to therapeutically induce apoptosis preferentially in tumor cells. Although TNF-ฮฑ and Fas ligand proved highly toxic to both normal and tumor cells, the latter display preferential sensitivity to TRAIL, which has now entered clinical trials (Fig. 7.5).49 Moreover, in cases in which apoptosis is blocked at the mitochondrial level in tumors, SMAC mimetics have proved useful in stimulating the activity of TRAIL by antagonizing the caspase-inhibitory function of IAPs to facilitate direct caspase-3 activation by caspase-8 (Fig. 7.5).50,51 Thus, defining this pathway of apoptosis regulation has revealed novel opportunities to rational therapy designed to activate apoptosis preferentially in tumor cells.

Figure 7.5. Therapeutic modulation of the apoptotic pathway downstream of death receptors. Tumor necrosis factor-related ligand (TRAIL) and related death-promoting ligands engage their cognate death receptors and activate caspase-8, which then cleaves Bid to active tBid. tBid can bind Bcl-2 and related antiapoptotic proteins to release Bax and Bak and may also directly promote their activation to permeabilize the mitochondrial outer membrane to release the APAF-1 cofactor cytochrome c, and the inhibitors of apoptosis protein (IAP) antagonist second mitochondrial-derived activator of caspase (SMAC) that promote caspase-9 and -3 activation and cell death. BH3-mimetics such as ABT-737 can promote apoptosis induction by TRAIL by relieving the protective capacity of the antiapoptotic Bcl-2-like proteins. In cells that do not depend on the mitochondrial apoptotic signal, TRAIL-mediated caspase-8 activation can directly promote downstream caspase activation and can synergize with SMAC mimetics in this case.
Drugs Targeting the Bcl-2 Family for Chemotherapy
In addition to the Bcl-2 up-regulation in B-cell lymphoma previously described, there are other mechanisms for directly or indirectly inactivating apoptosis in tumors that facilitate tumor progression and treatment resistance. Inactivation of the p53 tumor suppressor, or the p53 pathway through the gain of function of the p53 inhibitor MDM-2, is a common occurrence in tumors that results in the loss of the proapoptotic and growth-arrest functions of p53.24,25 The BH3-only proteins Puma and Noxa are transcriptional targets of p53, the loss of which prevents induction of the p53-mediated response to genotoxic stress in tumors as part of the mechanism of tumor suppression.32 Various means for restoration of p53 function in tumors are, therefore, an attractive therapeutic approach.
Activation of the MAP kinase pathway is common in tumors and results in stimulation of tumor cell proliferation, but also the phosphorylation and proteasome-mediated degradation of the BH3-only protein Bim. This Bim inactivation promotes tumor growth by preventing apoptosis while also producing resistance to the taxane class of chemotherapeutic drugs. Evidence suggests that this loss of Bim function resulting from its phosphorylation and proteasome-mediated degradation is rectified by blocking Bim degradation using a proteasome inhibitor (bortezomib)

(Fig. 7.6).31 Similarly, direct inhibition of MAP kinase pathway signaling with inhibitors (sorafenib, UO126) can also restore apoptotic function in addition to suppressing the proliferative response (Fig. 7.6). Receptor tyrosine kinase pathway activation in tumors also promotes tumor cell proliferation in part through MAP kinase pathway activation in addition to inhibiting apoptosis through Bim inactivation. In chronic myelogenous leukemia, in which chromosomal translocation and activation of the Bcr/Abl tyrosine kinase leads to Bim inhibition, blocking kinase signaling with imatinib mesylate restores Bim and also Bad apoptotic function as a therapeutic strategy (Fig. 7.6).52 Similarly, activation of the PI-3 kinase pathway commonly through loss of PTEN tumor suppressor function and AKT activation results in phosphorylation and inactivation of the BH3-only protein Bad and reduction of Bim transcription through inhibition of forkhead factors, resulting in down-regulation of apoptosis.53 Thus, inhibitors of the PI-3 kinase pathway can restore apoptosis and facilitate tumor regression. NF-ฮบB, a cytokine-responsive transcription factor, also promotes tumor growth while turning on the expression of antiapoptotic regulators Bcl-xL, Bfl-1, and IAPs (Fig. 7.3A). Strategies to inhibit NF-ฮบB are likely to promote tumor regression in part through restoration of apoptotic function.54

Figure 7.6. Therapeutic regulation of Bim and the MAP kinase pathway in cancer chemotherapy. Bim protein stability is regulated by Erk phosphorylation and proteasome-mediated degradation. Therapeutic modulation of the MAP kinase pathway (imatinib mesylate, sorafenib, and UO126) or proteasome function (bortezomib) can restore Bim protein levels and apoptosis function. Taxanes also stimulate Bim expression and promote Bim-mediated apoptosis, synergizing with the aforementioned inhibitors.
Direct Modulation of Bcl-2 with BH3-Mimetics
The observation that antiapoptotic Bcl-2 family members bound and sequestered BH3-regions in a hydrophobic cleft as a means to suppress apoptosis activation (Fig. 7.4), revealed the opportunity for the rational design of small molecules that occlude the cleft, thereby promoting apoptosis.7,39 One such approach resulted in ABT-737, which binds the BH3 binding pocket of Bcl-2, Bcl-xL, and Bcl-w but not Mcl-1, similarly to the BH3 of the BH3-only protein Bad (Fig. 7.4). ABT-737 exhibits single-agent activity against some human lymphoma and small cell lung cancer cell lines in vitro and in mouse xenographs in vivo, and in primary patient-derived cells, and has recently entered clinical trials. Thus, deciphering the mechanisms of apoptosis regulation in tumor cells is yielding novel opportunities for rational drug design and therapeutic intervention. These analyses can help predict which tumors have the potential to respond to apoptosis modulation and which types of drug combinations are likely to produce that response.
Killing the Unkillable Cells: Alternate Approaches to Achieving Tumor Cell Death
An apoptotic response to therapy in tumors may not always be possible to achieve; therefore, it is important to determine alternate cell death processes and how to access them, specifically in tumor cells. One intrinsic difference between normal and tumor cells is their metabolic reliance on aerobic glycolysis, which is an inefficient means for generation of adenosine triphosphate (ATP) required for sustaining homeostasis.55 This tumor cell-specific reduced metabolic capacity is frequently coupled with high energy demand because of a rapid rate of cell growth, with the potential to render tumor cells susceptible to cell death resulting from metabolic catastrophe in which cellular energy consumption exceeds production.48,56 One means to specifically drive tumor cells toward metabolic catastrophe is through therapeutic nutrient deprivation that may be an additional consequence of the use of angiogenesis inhibitors. As the catabolic process of autophagy is central to mitigation of metabolic stress, this has suggested that metabolic catastrophe can be promoted in tumor cells through internal nutrient deprivation by inhibiting autophagy. Alternatively, tumor cells are also expected to be preferentially sensitive to therapeutic stimulation of energy consumption. Importantly, induction of cell death by metabolic catastrophe can occur independently of an intact apoptotic response, suggesting that modulation of tumor cell metabolism may be therapeutically advantageous.

Role of Autophagy in Promoting Cell Survival to Metabolic Stress
Autophagy (commonly pronounced aw-tof'—je), a term coined from the Greek auto, or oneself, and phagy, or eating, is an evolutionarily conserved catabolic lysosomal pathway that results in degradation of long-lived proteins and organelles. This process involves formation of the โ€œautophagosome,โ€ a double-membrane vesicle in the cytosol that engulfs organelles and cytoplasm, and then fuses with the lysosome to form the โ€œautolysosome,โ€ where the sequestered contents are degraded and recycled for protein and ATP synthesis.57,58 Autophagy can promote survival to nutrient deprivation through recycling of intracellular nutrients in the short term, and can potentially induce cell death through progressive cellular consumption in the long term; hence its designation as type II programmed cell death.
Autophagy is regulated by mTOR in the PI3-kinase/AKT pathway that functions to link nutrient availability to cellular metabolism. Under conditions of nutrient limitation, normal

cells use this pathway to turn down protein synthesis while activating the catabolic process of autophagy to maintain homeostasis. In solid tumors, autophagy localizes to regions of metabolic stress and is a survival mechanism during nutrient starvation, as self-digestion provides an alternative energy source (Fig. 7.7).59 This process of autophagy during nutrient deprivation allows recovery of growth and proliferative capacity with remarkably high fidelity when nutrients are restored.48,59 Similarly, in hematopoietic cells, growth factor deprivation activates autophagy, which is essential for maintenance of ATP production and cellular survival.60 In normal mouse development, amino acid production by autophagic degradation of โ€œselfโ€ proteins allows maintenance of energy homeostasis and survival during neonatal starvation.61 Thus, both normal and tumor cells use autophagy to buffer metabolic stress, thereby mitigating potentially damaging effects of fluctuations or interruptions in external nutrient or growth factor availability. This also allows maintenance of cell metabolic function and continuation of normal cellular activities in the short term, which is likely an integral part of homeostasis.

Figure 7.7. Role of autophagy in enabling survival of tumor cells to metabolic stress. As epithelial tumor cells proliferate and multiple cell layers accumulate, the initial absence of a blood supply produces metabolic stress in regions most distal to nutrients and oxygen in the center of the tumor mass. In tumor cells with apoptosis defects, this allows tumor cells in these metabolically stressed tumor regions to survive through autophagy. Subsequent angiogenesis relieves metabolic stress, obviating the need for autophagy, fueling tumor growth.
Autophagy is not only involved in recycling of normal cellular constituents, but is also essential for damaged protein and organelle removal, as defects in this process result in accumulation of ubiquitin-positive aggregates and deformed cellular structures, which may promote cellular degeneration.62,63,64 Autophagy contributes to innate immunity by protecting cells against infection with intracellular pathogens,65 and to acquired immunity by promoting T-lymphocyte survival and proliferation.66 Moreover, autophagy is involved in cellular development and differentiation, and may have a protective role against aging.57
Autophagy is also a form of cell death, when allowed to proceed to completion and when cells unable to undergo apoptosis are triggered to die. It is often unclear whether autophagy is directly involved in initiation and/or execution of cell death or if it merely represents a failed or exhausted attempt to preserve cell viability. Recent studies indicate that autophagy may play an active role in programmed cell death, but the conditions under which autophagy promotes cell death versus cell survival remain to be resolved.67 The apparently conflicting prosurvival and prodeath functions of autophagy can, however, be reconciled if one considers autophagy a prolonged but interruptible pathway to cell death on starvation, in which nutrient restoration prior to its culmination can provide cellular salvation. This contrasts the death processes of apoptosis and necrosis, which are executed rapidly and are irreversible.48
Role of Autophagy in Tumor Suppression
Defective autophagy has been implicated in tumorigenesis because the essential autophagy regulator beclin1 is monoallelically deleted in 40% to 75% of human breast, ovarian, and prostate tumors, resulting in decreased Beclin1 levels.68 beclin1 is the mammalian ortholog of the yeast atg6/vps30 gene, which is required for autophagosome formation.69 beclin1 complements the autophagy defect present in atg6/vps30-disrupted yeast and in human MCF7 breast cancer cells, the latter in association with inhibition of MCF7-induced tumorigenesis in nude mice.68 beclin1-/- mice die early in embryogenesis, whereas aging beclin1+/- mice display preneoplastic changes in mammary tissue and increased incidence of lymphoma and carcinomas of the lung and liver.70,71 Tumors forming in beclin1+/- mice express wild type beclin1 mRNA and protein, indicating that beclin1 is a haploinsufficient tumor suppressor.71
Recent studies revealed that autophagy enables tumor cell survival in vitro and in vivo when apoptosis is inactivated,59 as commonly occurs in human cancers. How inactivation of a survival pathway promotes tumorigenesis is intriguing, and represents an area of great scientific interest with potentially significant clinical implications. In addition to providing an alternate means to energy generation during periods of starvation, autophagy may also

have a role in maintaining homeostasis through protein and organelle quality control, especially under conditions of metabolic stress in which ATP is limiting and cellular damage can accumulate. This function of autophagy may be particularly critical in tumors, which are regularly subjected to metabolic stress because of their dependence on the inefficient process of aerobic glycolysis and because of their intermittently limited blood supply during rapid tumor growth or metastasis (Fig. 7.7). Thus, autophagy defects in tumors reduce cellular fitness and render cancer cells prone to damage, which may in turn contribute to tumor progression, if persistent.56
As apoptosis is commonly disabled in oncogenesis, which can confound successful treatment, means to activate alternate pathways for cell death may be therapeutically advantageous. Because it has now become clear that tumor cells, particularly those with defects in apoptosis, rely on autophagy to survive metabolic stress, autophagy inhibitors may render cancer cells more susceptible to cell death. Several antineoplastic agents have been observed to induce autophagy in human cancer cell lines. However, whether autophagy that is induced by anticancer drugs actively contributes to cancer cell death or is a manifestation of the cancer cell's effort to maintain metabolism during treatment, or even represents a mechanism of cell death resistance, is unclear in most cases. Specific inhibitors targeting the autophagy pathway are under development, and their potential utility in cancer therapy remains to be tested.
Therapeutic Induction of Necrotic Cell Death
Recent evidence suggests that tumor cells in which apoptosis has been disabled can be diverted to necrosis, which has traditionally been considered an unregulated (and thus, not programmed) form of cell death implicated in pathologic states, such as ischemia, trauma, and infection, although this notion is now being challenged.72,73 Necrosis is derived from the Greek word nekros, for corpse, and it involves rapid swelling of the cell, loss of plasma membrane integrity, and release of the cellular contents into the extracellular environment, resulting in an acute inflammatory response.48,72 Treatment with alkylating agents results in DNA damage, which activates the DNA repair protein poly(ADP-ribose) polymerase (PARP). Cell fate is ultimately determined by PARP-mediated stimulation of ฮฒ-nicotinamide adenine dinucleotide and ATP consumption to which glycolytic tumor cells are preferentially sensitive. The glycolytic state (Warburg effect) and inefficient mode of energy production in most cancer cells results in rapid ATP depletion and necrotic cell death of apoptosis-defective tumor cells in response to PARP activation.74 Tumor cells with defects in both apoptosis and autophagy are particularly susceptible to death by necrosis under metabolic stress, as loss of autophagy potential deprives these cells of an alternate energy source for maintenance of metabolism and viability in conditions of oxygen and nutrient limitation.59
Manipulation of tumor cell metabolism is an appealing therapeutic approach as it can be used to induce cancer cell death by metabolic catastrophe.48 This is particularly relevant for tumors with increased proliferative capacity and high bioenergetic requirements, such as tumors with constitutive activation of the PI3-kinase/Akt pathway, which are unable to down-regulate metabolism and to activate autophagy in response to starvation. Thus, the very properties that confer cancer cells with the capacity for rapid growth may also render them susceptible to metabolic stress pharmacologically induced by a wide variety of means, including nutrient deprivation, angiogenesis inhibition, glycolysis inhibition, accelerated ATP consumption, or autophagy inhibition.