Apoptosis, Necrosis, and Autophagy

In the simple arithmetic of life, a tissue grows if cells divide more frequently than they die, whereas one in which cell death is more frequent than cell division shrinks. This arithmetic, trivial as it seems, is at the heart of the understanding of cancer and the efforts to effectively treat it. However, the mechanisms that control this relationship are far from trivial.

One theory of cancer, in fact, suggests that an evolutionary “hard-wiring” of the machineries of tissue expansion and cell death is fundamental to tumor suppression. This concept, called antagonistic pleiotropy ( Figure 14-1 ), states that any molecular event in a multicellular organism that directs a cell to divide also instructs it to die, and that the latter will occur unless additional signals (either exogenous or endogenous) promote cell survival.

It is critical to note that this idea is fundamentally different from the prevalent (and incorrect) notion that “cancer cells are resistant to cell death,” which as we will see is not only inaccurate, but patently untrue. Antagonistic pleiotropy only demands that the specific cell death pathways engaged by signals promoting clonal expansion be sufficiently muted that the rate of division exceeds the rate of cell death. Other pathways of cell death not only remain available, but may be sensitized in the process, providing us with the opportunity to shift the balance by therapies.

The Ways Cells Die

There are different ways in which cells die, and these are grouped on the basis of the morphologies of the dying cells ( Figure 14-2 ) and more recently by the mechanisms of cell death as we understand them. By general agreement, there are three modalities of cell death: apoptosis (type I), autophagic cell death (type II), and necrosis (type III). Within each of these modes there are additional varieties of morphologies and/or pathways that engage death.

Apoptosis is characterized by condensed chromatin, membrane blebbing, and cell shrinkage, and the cell often breaks into smaller, membrane-bound bodies (see Figure 14-2 ). The DNA within chromatin is cleaved by the action of a nuclease that cuts the DNA between nucleosomes, producing the degraded DNA hallmark of apoptosis. Before the loss of plasma membrane integrity, the lipids of the membrane “scramble,” resulting in the appearance of phospholipids on the surface, which are normally preferentially associated with the inner leaflet. All of these events are due to the action of caspase proteases that are activated during apoptosis. Usually, apoptotic cells are effectively cleared by phagocytosis (“cell eating”) by macrophages and other cell types, before the contents of the dying cell can be released. This involves the recognition of a phospholipid, phosphatidylserine, which appears on the outer leaflet as a consequence of the scrambling event just noted. As a result, apoptotic cell death does not usually engage an inflammatory response and is sometimes described as being immunologically “silent.”

Necrosis, in contrast, involves swelling of the cell and organelles. As plasma membrane integrity is disrupted and the contents of the cell spill out, some components act as signals to other cells that damage has occurred (see Figure 14-2 ). These signals, called damage-associated molecular patterns (DAMPs), can trigger inflammatory responses.

The third mode of cell death, autophagic cell death, is problematic. It is characterized by the formation of large vacuoles (often enlarged lysosomes) and the activation of autophagy (see Figure 14-2 ). As with necrosis, caspases are not activated. However, there is considerable disagreement regarding the role of autophagy in the cell death itself. In most cases, disruption of the autophagy machinery accelerates the cell death, and therefore this is viewed as “cell death accompanied by autophagy.” There are examples in Drosophila , however, in which the autophagic machinery is fundamentally involved in the cell death process itself, although it is unclear to what extent such bona fide autophagic cell death occurs in mammals.

In thinking about the different modes of cell death, a useful distinction can be made between cell death that is “passive,” meaning that the cell is killed, and that which is “active,” that is, the cell participates in its own demise. In the first case, cell death can only be prevented by removing or blocking the toxic insult, or by repairing damage more quickly than it occurs. In cases of active cell death, this quietus may also be blocked if the cellular pathways involved are inhibited or disrupted.

It is important to note, however, that just because a cell death process is active, this does not mean that it represents a specialized cell death mechanism that was evolutionarily selected as a physiological pathway of cell death. Much as a running but not a motionless train can be destroyed by derailing, a cellular activity can be “sabotaged” to inflict lethal damage on the cell. In contrast, there are specialized cell death pathways that are indeed mechanisms of physiological cell death, and thus can be regarded as cellular “suicide.” Figure 14-3 illustrates these distinctions, although in many cases we can only speculate as to whether a process is sabotage versus suicide.

Figure 14-3, Active and passive cell death

From the perspective of cancer biology, however, the distinction is particularly useful. When cell death is engaged as a mechanism of tumor suppression, it may be regarded as suicide, whereas the effects of therapeutic intervention may manifest either by recruiting such a suicide pathway or by effectively sabotaging a process in the cancer cell to result in its death.

The best example of active cell suicide is apoptosis, although there may be others (as we note in Figure 14-3 and in the following discussion). In contrast, the lethal overproduction of reactive oxygen species (ROS) by disruption of the mitochondrial electron transport chain, the engagement of membrane NADH oxidases, or the loss of ROS scavenging mechanisms may be regarded as cellular sabotage (see Figure 14-3 , and other examples discussed later).

This distinction blurs when we consider the evolution of cell death. Any mechanism of cellular sabotage could well be selected to become a process of cellular suicide, and indeed, many scenarios for the evolution of apoptosis begin with this premise. At this point, it may be best to remind the reader that these distinctions are, like many classifications, merely a starting point for understanding the intricacies of the processes. Nevertheless, it might be interesting to think about why, of all the possible cell suicide mechanisms that might have evolved, apoptosis has emerged as the predominant form of active cell death in the animal kingdom.

Apoptosis

Caspase Activation

Apoptosis is orchestrated by the actions of caspases, which are cysteine proteases (that is, they have a cysteine at the active sites). There are at least 17 caspases that have been described in mammals, and we understand the functions of only a few of them. The process of apoptosis occurs through the activation of a subset of these, called the executioner caspases (caspase-3, caspase-6, and caspase-7 in mammals), and once activated, these cut up to a thousand different cellular substrates in the cell to precipitate the changes associated with this form of cell death. The “cuts” are at specific sites in the substrates and occur at aspartate residues. This is the origin of “caspase,” a c ysteine protease with Asp-ase activity.

The executioner caspases are present in cells in an inactive, dimeric form, which becomes activated when cut at specific aspartate residues, resulting in a conformational change that forms the active enzyme. The active enzyme now cuts its substrates, some of which are responsible for the changes associated with apoptosis.

In general, this activating cleavage is mediated by other caspases, called initiator caspases . Unlike the executioner caspases, the inactive forms of the initiator caspases present in cells are monomeric and contain large prodomains that include sites for protein-protein interaction. Adapter molecules, which form “caspase activation platforms,” bind to the prodomains of initiator caspases, forcing them into contact, and the latter then fold into active enzymes. These can then process the executioner caspases, thereby activating them to cleave their substrates and precipitate apoptosis.

The formation of caspase activation platforms and the caspases they activate define the apoptotic pathways. Here we consider in detail only two major pathways of apoptosis, the mitochondrial pathway and the death receptor pathway. It should be noted, however, that there are others, reviewed elsewhere. One of these involves the formation of caspase activation platforms for the engagement and activation of caspase-1, which is critically involved in inflammatory responses by processing specific cytokines and promoting their release. The activation of caspase-1 can also kill cells by a process resembling apoptosis (often called pyroptosis ). The activation of caspase-1 occurs in response to many infectious agents and has been implicated in promoting forms of cancer that are associated with inflammatory conditions. Caspases can directly signal apoptosis or can use mitochondria as an intermediate, and additional point of regulation, in signaling of apoptosis.

The Mitochondrial Pathway of Apoptosis

Starting in the 1980s, the intracellular molecules that controlled the morphology referred to as apoptosis began to be identified. Cell death had previously been thought of as a passive event (as noted earlier): a universally negative occurrence that cells were constructed to avoid at all costs. Careful observations in human pathological samples as well as in model systems revealed that instead, cell death in multicellular organisms was often a carefully controlled event with stereotypical morphologic and temporal patterns. Moreover, when it was observed that altering the function of certain genes could alter the commitment, phenotype, and progression to cell death, it became clear that cells contained within them genetic programs to perform a function of choosing death as a cell fate, and then committing cell suicide. Although several different forms of programmed cell death have been identified, the first to be characterized was apoptosis.

The mitochondrial (sometimes called intrinsic ) pathway of apoptosis responds to a diverse group of initiating events, including treatment with a wide variety of cytotoxic drugs, growth factor withdrawal, and oncogene activation. The mitochondrial pathway of apoptosis get its name because of the centrality of this organelle in coordinating both cell fate decision making as well as execution. Key features of apoptosis via the mitochondrial pathway include mitochondrial outer membrane permeabilization (MOMP), followed by release to the cytosol of molecules that facilitate execution of the death program, including cytochrome c, SMAC, and Omi. Cytochrome c is essential to the formation of one of the caspase activation platforms, the apoptosome, which is formed when cytochrome c activates APAF-1 to oligomerize, and the latter then binds and activates caspase-9. Although caspases may perform normal physiological functions, they are best known for cleavage of proteins during apoptosis important for cell integrity, including cytoskeletal proteins and PARP-1. Among the caspase-dependent phenomena commonly observed in association with apoptosis are extracellular exposure of the phosphatidylserine that is usually found only on the inner leaflet of the plasma membrane (which results in recognition by Annexin V) and internucleosomal cleavage of chromosomal DNA (which results in laddering of DNA on electrophoresis and nuclear condensation). In vivo in a multicellular organism, an important consequence of these changes is recognition and clearance of the apoptotic cell by phagocytic cells. It may well be that commitment to cell death is already made at the point of MOMP, whereas many of the downstream morphologies we associate with apoptosis might be most important mainly in facilitating the clearance of apoptotic cells, minimizing inflammation, and optimizing cannibalization of their macromolecules.

The BCL-2 Protein Family

The B cell leukemia/lymphoma 2 (BCL-2) family of proteins controls commitment to apoptotic cell death via the mitochondrial pathway. The discovery and characterization of this important family is intimately linked to cancer, as BCL-2 was initially cloned from the t(14;18) chromosomal translocation that is present in nearly all cases of follicular lymphoma. The translocation places the bcl-2 gene on chromosome 18 under the control of regulatory elements of immunoglobulin genes, yielding overexpression of the BCL-2 protein in cells of the B-lineage.

The key step in apoptosis commitment that is controlled by the BCL-2 family is MOMP. In simplest terms, there are pro- and anti-apoptotic members of the BCL-2 family. When the pro-apoptotic proteins overwhelm the anti-apoptotic ones, MOMP occurs, and the cell is irreversibly committed to apoptosis. The details of this commitment involve complicated interactions among the BCL-2 family proteins that number greater than a dozen. BAX and BAK are obligate pro-apoptotic “effectors,” proteins that homo-oligomerize to form the pores that cause MOMP. In order to form the oligomers required to cause MOMP, BAX and BAK must be activated. Proteins of the pro-apoptotic “activator” BH3-only protein subfamily, which include BIM, BID, and perhaps PUMA, can directly interact with BAX and BAK to cause an allosteric change that functionally corresponds to activation. There may also be other ways that BAX and BAK are activated, perhaps even independent of interaction with other proteins.

Anti-apoptotic BCL-2 family proteins include BCL-2, BCL-XL, BCL-w, MCL-1, and BFL-1 (A1). These proteins inhibit apoptosis by binding and sequestering activator proteins or monomeric “activator” BAX or BAK proteins before they can oligomerize. Another subfamily, the pro-apoptotic “sensitizer” BH3-only proteins, lack the ability to directly activate BAX and BAK with high efficiency, but promote apoptosis by competitively inhibiting the ability of anti-apoptotic proteins to bind activators, BAX, or BAK ( Figure 14-4 ).

Figure 14-4, Control of MOMP by the BCL-2 family of proteins

BH3-only proteins get their name from their possession of the BCL-2 homology 3 (BH3) region, but of no other BCL-2 homology region. The BH3 domain is essential for the pro-death activity of all of the pro-apoptotic BCL-2 family proteins. It is a roughly 20 amino acid amphipathic α-helix that binds into a hydrophobic BH3 binding cleft present in all of the anti-apoptotic proteins. There is a selective pattern of interaction of BH3-only proteins with anti-apoptotic proteins. For instance, the BAD BH3 domain interacts selectively with BCL-2, BCL-XL, and BCL-w, whereas the NOXA BH3 peptide interacts selectively with MCL-1. Small-molecule BH3 domain mimetics that compete for these interactions, effectively inhibiting anti-apoptotic protein function, are being tested in cancer clinical trials (see later discussion).

Mitochondrial Priming and Chemotherapy Response

The concept has emerged from many studies, especially in murine models of cancer, that apoptosis serves as a natural barrier to carcinogenesis. In several murine models, defects in apoptosis facilitate carcinogenesis, supporting this concept. Many of the changes that commonly occur in oncogenesis, including genomic instability and oncogene activation, contribute to pro-apoptotic signaling, which may well result in deletion of nascent malignant clones. Malignant clones that do survive must therefore have selected for mechanisms of evading apoptotic death provoked by this pro-apoptotic signaling by some combination of fostering anti-apoptotic signaling and attenuating pro-apoptotic signaling.

However, as it is commonly used, the oft-made assertion that resistance to apoptosis is a fundamental property of all cancer begs the question, “Compared to what?” It is difficult to find experimental or clinical evidence that cancers generally are more resistant to apoptosis than the normal nonmalignant cells in the human body. The fact that cancer cells have successfully buffered prior pro-apoptotic signaling does not necessarily mean that they are well prepared to evade subsequent pro-apoptotic signaling.

In fact, many conventional chemotherapies used in the clinic kill cells via apoptosis. The specific mechanisms may vary widely. Taxanes may increase levels of BIM, alter subcellular localization of BH3-only proteins, and decrease levels of MCL-1. Cytotoxic agents can induce a decrease in MCL-1 levels, though whether this is a necessary and sufficient step for commitment to apoptosis in these cases is less clear. DNA-damaging agents, by activating p53, can activate transcription of pro-apoptotic PUMA, NOXA, and BAX (see also Chapter 3, Chapter 15 ). In addition, p53 may promote apoptosis via direct interaction with BCL-2 family proteins. Space does not permit detailing all of the relationships that have been discovered between conventional chemotherapy agents and the BCL-2 family. Although there are sometimes attempts to identify the key BCL-2 family dictating response to a particular agent, it is likely that following most chemotherapies, more than one BCL-2 family protein takes part in determining the cell fate decision.

The ability of chemotherapy to selectively kill cancer cells makes it likely that in many cancer cells the apoptotic pathway is not only intact, but even more sensitive to pro-apoptotic signaling than in normal cells. This hypothesis has been directly tested using a mitochondrial assay called BH3 profiling, an assay that systematically compares mitochondrial response to a standardized panel of synthetic BH3 domain peptides. These studies have shown that chemosensitive cancer cells are indeed more sensitive to pro-apoptotic signals than normal cells, suggesting mitochondrial apoptotic priming as an important determinant of the therapeutic index in vivo and in vitro. Normal hematopoietic cells are the most primed of normal tissues, consistent with their perennial role as the site of dose-limiting toxicity for most cytotoxic regimens. Measuring pretreatment mitochondrial priming may even be useful as a predictive biomarker, because it correlates well with clinical response.

From a perspective of protein biochemistry, cells that are highly primed have little anti-apoptotic reserve to buffer subsequent pro-apoptotic signaling. Very often, a highly primed cell will express abundant anti-apoptotic BCL-2 family proteins, but these will already be occupied by pro-apoptotic activators such as BIM. In these cases, the cells are dependent on the continuous function of the anti-apoptotic protein(s) for survival. Inhibition of one or more of these can result in MOMP and apoptosis in highly primed cells ( Figure 14-5 ).

Figure 14-5, Primed versus unprimed mitochondria

It is worth considering the distinction between expression of an anti-apoptotic protein, and dependence on that anti-apoptotic protein. If one enforces BCL-2 overexpression via transfection of a plasmid vector in a healthy cell line already well established in culture, one will not necessarily obtain a BCL-2–dependent cell line, because there is likely no dependence on the additional BCL-2. The cell line was doing fine without it before transfection, and inhibition of BCL-2 would just restore the status quo ante. In oncogenesis, however, increased expression of BCL-2 is selected for, not extrinsically induced. This implies that the increased BCL-2 that was selected for is required for the continuous buffering of pro-apoptotic signals. In biochemical terms, that means that BCL-2 is largely already in complex with pro-apoptotic BCL-2 family members such as BIM or BAX. In the instance of the transfected cell line, however, BCL-2 is mainly empty and ready to buffer subsequent pro-apoptotic signaling. In the case where BCL-2 expression has been positively selected in the face of extant pro-apoptotic signaling, the BCL-2 is mainly full. Rather than providing anti-apoptotic reserve, the BCL-2 in the latter case is actually storing pro-apoptotic proteins at the mitochondrion, priming it for sensitivity to apoptosis. The cancer cell just discussed is thus dependent on continuous BCL-2 function for survival. In other cancer cells, similar to the transfected cell line, BCL-2 expression may be incidental, rather than selected for, and the BCL-2 not required tonically. Protein and transcript levels of multiple BCL-2 family members, as well as functional assays such as BH3 profiling, have been used to identify cells that are dependent on BCL-2 and hence likely to be sensitive to antagonists of BCL-2 (see later discussion).

Targeted Therapies and Apoptosis

In the past decade, modern “targeted” therapies have occupied the lion’s share of attention in preclinical development, with several achieving approval for clinical use in cancer. Most of these agents also kill by inducing signaling via the mitochondrial apoptotic pathway. However, in this case, selective killing of cancer cells depends less on differences in pretreatment mitochondrial priming and more on selective dependencies of cancer cells. For instance, imatinib, the first small-molecule kinase inhibitor to be approved for use in cancer, inhibits activity of the Abl kinase, an activity that is enhanced by the BCR/ABL fusion protein created by the t(9;22) chromosomal translocation present in chronic myelogenous leukemia (CML). The selective killing of malignant CML cells over normal cells depends on the selective dependence of CML cells on activity of the BCR/ABL kinase. Another family of tyrosine kinase inhibitors, those directed against epidermal growth factor receptor (EGFR), are selectively effective in lung cancers with activating mutations in EGFR.

A general property of kinase inhibitors in cancer, whether tyrosine kinase or serine/threonine kinase inhibitors, is their utilization of the mitochondrial apoptotic pathway to kill cancer cells. Interestingly, although doubtless other BCL-2 family members also contribute, BIM, a pro-apoptotic activator BH3-only protein, is regulated by many different kinase inhibitors. In turn, lethality of kinase inhibition depends on the upregulation of BIM. BIM upregulation is required for killing by imatinib whether in CML or in gastrointestinal stromal tumors (GIST) where the constitutively active c-KIT kinase is the relevant target. Similar observations have been made for inhibitors of EGFR, MEK, ALK, and b-RAF. A fascinating human genetic study found that a germline polymorphism of BIM that removes the BH3 domain that is required for pro-apoptotic function is common in an Asian population. Intriguingly, this polymorphism conferred inferior clinical response to inhibitors of both BCR-ABL in CML and of EGFR in non–small-cell lung cancer, apparently confirming its importance in cancer cell–fate decision making in clinical use of kinase inhibitors.

Proteasome inhibitors such as bortezomib have become a mainstay of multiple myeloma therapy and have found application in other diseases such as mantle-cell lymphoma. These also apparently kill via the mitochondrial apoptotic pathway. In this case, the most often cited mechanism of response is the pro-apoptotic BH3-only family protein NOXA, which can selectively bind and inactivate MCL-1, an anti-apoptotic protein also in the BCL-2 family. Of course, proteasome inhibitors alter the levels of thousands of proteins, so that there may be several proteins that are affected that are important for cell-fate decision after proteasome inhibitors.

Directly Targeting the BCL-2 Family in Cancer Treatment

Given the centrality of the BCL-2 family of proteins in determining cell fate in cancer, considerable effort has been expended on inhibition of anti-apoptotic BCL-2 family proteins to induce cancer cell death.

These efforts are in their infancy, and important questions need to be resolved. For example, should the agent have a narrow or broad spectrum of inhibition of anti-apoptotic BCL-2 family proteins? Broad-spectrum agents will likely be more toxic to more kinds of cancers; however, there is the possibility of a narrowed therapeutic index that could limit clinical utility. Will these agents be best used as single agents or in combination with others? Although some diseases may demonstrate clinical sensitivity to single agents, it may well be that combination with the powerful, albeit less selective, pro-death signaling induced by conventional cytotoxic agents will be necessary to provide the significant benefit in long-term clinical outcomes that is so badly needed in many cancers. In addition, combinations with targeted agents such as kinase inhibitors have proved promising in vitro.

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