Abeloff's Clinical Oncology, 4th Edition

Part I – Science of Clinical Oncology

Section A – Biology and Cancer

Chapter 5 – Cell Life and Death

Rebecca L. Elstrom,Craig B. Thompson




Apoptosis control mechanisms seem to be impaired in virtually all tumors, suggesting that a required step in carcinogenesis is to disengage the apoptotic machinery.



Two basic pathways of apoptosis have been described: the extrinsic or death receptor-mediated pathway and the intrinsic or mitochondrial pathway.



The fate of a cell is determined by the balance of proapoptotic and antiapoptotic factors within the cell.



Oncogenic transformation promotes proapoptotic pathways. Cancer cells must disable tumor suppressor molecules and/or activate survival signals to evade programmed cell death.



Therapeutic strategies aimed at restoring tumor suppressors and interfering with survival factors are playing increasingly important roles in antineoplastic treatments.



There is a growing understanding of the role of alternative forms of cell death, particularly necrosis, in the response of cancer cells to treatment.


The evolution of a normal cell into cancer involves disruption and deregulation of several basic cellular processes. Multicellular organisms, in their evolution from simple, single cells, have developed redundant controls through which the homeostasis between different cell types is maintained. One of the safeguards that prevents excess cell accumulation is the presence of cell-intrinsic programs that can induce programmed cell death, the best studied being apoptosis. The growing understanding that transforming mutations can activate this intrinsic death response has emphasized the importance of this process in preventing cancer cell development. Apoptosis control mechanisms seem to be impaired in virtually all tumors, suggesting that a required step in carcinogenesis is to disengage the apoptotic machinery.

The concept that genes regulating cell death could play a role in tumorigenesis arose in the mid-1980s, when investigators first discovered that a translocation commonly found in follicular lymphoma, between chromosomes 14 and 18, brings a region on chromosome 18 called breakpoint cluster region 2 (Bcl-2) into close proximity with the immunoglobulin heavy chain enhancer on chromosome 14, resulting in overexpression of the Bcl-2 gene.[1] Later work showed that the Bcl-2 gene product promotes oncogenesis by a novel mechanism. [2] [3] Instead of inducing cell proliferation or invasion, the Bcl-2 protein inhibits the normal programmed death of B cells, resulting in the failure to eliminate the clonal B cells as they accumulate in excess. These findings demonstrated for the first time that disarming death pathways within a cell could predispose to development of malignancy.

Since the description of Bcl-2, extensive progress has been made in understanding both the mechanisms of apoptosis and the ways in which this process contributes to tumorigenesis. The identification of a family of genes related to Bcl-2 that contribute to the balance between life and death, together with the discovery of the critical role of mitochondria in cellular homeostasis and apoptosis, have broadened our understanding of the dynamic interplay of forces determining the fate of cells.

In addition to progress in elucidating apoptotic mechanisms and deregulation, there has been an increasing understanding of the role of nonapoptotic mechanisms of cell death in both organismal homeostasis and pathogenic states such as cancer. Necrosis, a death process traditionally considered as unregulated and catastrophic, may instead represent a regulated event that contributes to organismal development and homeostasis. Autophagy, in which a cell “eats itself,” is also under investigation as a form of nonapoptotic cell death, although the function of this process, whether to induce death or, conversely, to maintain survival, is controversial.

Harnessing these forces will improve our capability not only to understand the mechanisms by which normal cells become malignant but also to prevent and treat cancer in humans.


Cell death occurs by two general classes of mechanism: apoptosis, or programmed cell death, and necrosis. Autophagy has been proposed as a third potential mechanism of cell death, although the physiologic functions of this process remain under study.

The process of apoptosis involves a cell-intrinsic suicide program that not only kills the cell but also stimulates the clearance and complete degradation of the corpse without inducing an inflammatory reaction. Apoptosis differs from other forms of cell death, such as necrosis, in that clearance of the cell is controlled through the activity of caspases—cysteine proteases with aspartate specificity that normally exist in an inactive, zymogen form. The initiator caspases 8 and 9 are activated by cellular signals and subsequently cleave downstream effector caspases, such as caspases 3, 6, and 7. These effector caspases set into motion the degradation of cellular components such as structural proteins, cell cycle machinery, and DNA. These processes result in the characteristic morphology of apoptotic cells, membrane blebbing, cell shrinkage, and DNA fragmentation. The end result is the disposal of the cell in a controlled manner, allowing turnover and phagocytosis without the inflammatory reaction to intracellular substances that accompanies death by necrosis.

As noted in the preceding discussion, the caspase cascade is initiated through specific cellular signals. These signals come through one of two major pathways: the extrinsic, receptor-mediated pathway, or the intrinsic, mitochondrial pathway. Although these pathways often are considered separately, extensive cross-talk exists between them.

Cell Death by Murder

In some cases, apoptosis is initiated through ligation of specific cell-surface receptors, the death receptors ( Fig. 5-1 ). These molecules are members of the tumor necrosis factor receptor (TNFR) family. The best studied of these include Fas and TNFR1. [4] [5] These receptors exist as trimers at the cell surface that are activated on binding of ligand, Fas ligand, and TNF, respectively. The intracellular domains of these receptors contain death domains which, on activation of the receptor, can recruit a death-inducing signaling complex, which leads to activation of a caspase cascade. The Fas death domain binds the Fas-associated death domain (FADD) adapter protein, which directly recruits caspase 8 and allows its cleavage and activation.


Figure 5-1  Death receptors can initiate apoptosis or promote cell survival. On ligation of the death receptor, adaptors are recruited. For TNFR1, the outcome of ligand binding depends on the state of the cell. TRADD might bind the FADD molecule, which recruits caspase 8, resulting in its oligomerization and activation. Active caspase 8 in turn cleaves and activates effector caspases and the BH3-only molecule Bid, initiating an apoptotic cascade. This process is inhibited by IAPs. Alternatively, binding of TNFR1 might induce recruitment of RIP and IκB kinase (IKK), resulting in release of the NF-κB transcription factor from its inhibitor, IκB. NF-κB then enters the nucleus, activating transcription of survival genes such as IAPs, therefore antagonizing the proapoptotic factors.



Activation of TNFR1, on the other hand, has multiple potential downstream effects.[6] TNFR1 binds the TNFR-associated death domain (TRADD) adapter protein, which in turn might recruit FADD, resulting in caspase 8 activation and apoptosis, as with Fas. TRADD also, however, can bind TNFR-associated factor-2, which may recruit inhibitor of apoptosis proteins (IAPs), which bind the death-inducing signaling complex and inhibit activation of caspase 8. Alternatively, TNFR-associated factor-2 may recruit components of the mitogen-activated protein kinase (MAPK) cascade, leading to activation of Jun-N-terminal kinase (JNK) and c-jun. Although in some systems JNK seems to promote TNFR1-induced apoptosis, these findings are inconsistent, and the role of JNK in receptor-mediated apoptosis is controversial. Finally, TRADD can bind receptor-interacting protein (RIP), resulting in activation of NF-κB, which antagonizes the apoptotic program. NF-κB is a transcription factor that activates expression of survival molecules such as IAPs and c-FLIP, a direct inhibitor of caspase 8 activation.[7] IAPs were first described in baculoviruses, where they were shown to inhibit apoptosis of host cells following viral infection.[8] Homologs such as XIAP and c-IAP-1 and -2 have since been identified in mammals; they seem to function by inhibiting caspase activation in both the death receptor and mitochondrial pathways. [9] [10] [11]

Another ligand-receptor pair that functions in both immune regulation and control of cancer is the TNF-related apoptosis-inducing ligand, TRAIL, and its receptors, TRAIL-R1/DR4 and TRAILR2/DR5.[12] [13] TRAIL induces apoptosis in a variety of transformed cells but is much less toxic to normal cells than to abnormal ones. The intracellular signaling pathways induced by TRAIL are similar to those of the TNFR1. TRAIL is expressed as a cell-surface molecule on natural killer cells, and it can inhibit tumor growth.[14] TRAIL also binds to decoy receptors TRAILR3/DcR1 and TRAIL-R4/DcR2, which sequester TRAIL from the signaling receptors, blocking TRAIL-induced apoptosis. [15] [16] Expression of these decoy receptors might provide the mechanism by which normal cells escape TRAIL-induced death, in that many tumor cells show lower expression of the decoy receptors.

Cell Death by Suicide

The importance of mitochondria in apoptosis was demonstrated in 1996, when Liu and colleagues[17] demonstrated that cytochrome c, a component of the electron transport chain normally contained in the mitochondrial intermembrane space, could initiate programmed cell death when present in the cytosol. This finding led to the demonstration that the intrinsic, or mitochondria-dependent, cell death program resulted from loss of mitochondrial integrity with release of intermembrane space contents ( Fig. 5-2 ). Cytochrome c, on release into the cytosol, forms a complex with Apaf-1 and adenosine triphosphatase (ATP). This complex, known as the apoptosome, binds and activates procaspase 9.[18] Other mitochondrial contents also participate in apoptosis. For example, Smac/DIABLO, [19] [20] Htra2,[21] apoptosis-inducing factor (AIF), and endonuclease (endo) G also contribute to the cell death program. [22] [23] [24] Inhibitory proteins such as XIAP and cIAPs-1 and -2 bind to the apoptosome and inhibit activation of caspases. Smac/DIABLO and Htra2 function by inhibiting the inhibitors, which allows apoptosis to proceed. The progression of the cell death program seems to depend on the relative ratios of apoptosis promoters and inhibitors. Evidence suggests that AIF, on the other hand, might function independently of caspase activity. Upon release from mitochondria, AIF translocates to the nucleus, where, in cooperation with endo G, it can initiate large-scale fragmentation of DNA.


Figure 5-2  Mitochondria-dependent apoptosis. Under conditions of cellular stress, the proapoptotic molecules Bax and/or Bak oligomerize at the mitochondria and induce release of mitochondrial contents, including cytochrome c. Cytochrome c then binds Apaf-1, and in combination with ATP forms the apoptosome. The apoptosome induces cleavage and activation of caspase 9, which activates a caspase cascade culminating in cell death. This process might be stimulated by DNA damage, which results in activation of p53, which, among several functions, promotes transcription of the BH3-only molecules Noxa and PUMA. The apoptotic pathway is antagonized by Bcl-2, which can block cytochrome c release and mitochondrial dysfunction. Growth factor stimulation might also antagonize apoptosis, in part through activation of such survival factors as Akt, which can phosphorylate and inactivate proapoptotic molecules such as the BH3 protein, Bad.



Controversy exists over the mechanism by which mitochondria lose integrity, resulting in release of their contents and initiation of the caspase cascade. General mechanisms proposed include mitochondrial dysfunction leading to matrix swelling and outer membrane rupture. Alternatively, loss of mitochondrial integrity could involve formation of specific pores large enough to release the intermembrane components. Another controversy exists over the importance of caspases in the actual death of cells following mitochondrial compromise as opposed to their role of simply orchestrating cellular disposal. Some investigators have shown that once mitochondria lose integrity, cells will die even in the absence of caspase activation.[25] This finding is consistent with the idea that release of mitochondrial contents proceeds from large-scale catastrophe to the mitochondria, with cellular viability impossible in the absence of mitochondrial function. Others have suggested that, under some circumstances, cells can recover even after cytochrome c release if caspases are held in check.[26] The function of IAPs in preventing apoptosome activity suggests that in some cases death can be prevented, but the role of caspases probably varies with different apoptotic stimuli.

Bcl-2 Family

Bcl-2 family proteins play a key role in regulation of mitochondrial integrity and programmed cell death. The Bcl-2 family includes proteins with both antiapoptotic and proapoptotic function. Bcl-2, the first identified member of this family, acts to prevent apoptosis, as do Bcl-XL and Mcl-1. Bax and Bak antagonize the function of the antiapoptotic family members and are critical in promoting apoptosis through the mitochondrial pathway. All these family members have multiple domains, termed Bcl-2 homology (BH) domains. A third type of Bcl-2 family member, exemplified by Bad, Bim, and others, consist of a single BH domain and are termed BH3-only molecules. The BH3-only family members also play a key role in promoting apoptosis.

Extensive progress has been made in understanding the mechanisms by which these Bcl-2 family members regulate mitochondrial integrity and apoptosis, although many questions remain. The antiapoptotic molecules Bcl-2 and Bcl-XL localize to the mitochondria and maintain mitochondrial integrity. This could occur through promotion of exchange of metabolic substrates across the mitochondrial membrane, allowing maintenance of respiration.[27] Alternatively, antiapoptotic Bcl-2 family members might bind and inhibit the function of proapoptotic family members.[28] The multidomain proapoptotic molecules, Bax and Bak, are required for mitochondrial apoptosis initiated through most stimuli.[29] Mice with targeted knockouts of either of these molecules show largely normal apoptotic function, but loss of both Bax and Bak results in a severe defect in apoptosis.[30] This finding suggests that although these two molecules might have largely redundant functions, presence of one or the other is critical to allowing apoptosis to proceed. The BH3-only molecules, in contrast, seem to have a signaling function. Various apoptotic stimuli induce expression and/or activation of specific BH3-only family members, which translocate to the mitochondria and initiate Bax/Bak-dependent apoptosis ( Fig. 5-3 ). They could operate either by activating Bax and Bak or by inhibiting the antiapoptotic function of Bcl-2 and Bcl-Xl. It has been suggested that different members of this group could have distinct functions—some directly binding and activating Bax/Bak, and others indirectly activating these proapoptotic molecules by binding and antagonizing Bcl-2/Bcl-XL.[31]


Figure 5-3  BH3-only proteins initiate apoptosis. Several BH3-only proteins exist and could function in various ways to promote apoptosis. Bid is cleaved by caspase 8 on death receptor stimulation and subsequently translocates to mitochondria, where it can activate oligomerization of Bax and Bak. Other proteins, such as Bad and Noxa, function by inhibiting antiapoptotic Bcl-2 molecules. Noxa is transcriptionally regulated by p53, and Bad is regulated by inhibitory phosphorylation, which is growth factor dependent.



The BH3-only protein Bid provides an example of cross-talk between the receptor-mediated and mitochondrial pathways of apoptosis. Bid is a target of active caspase 8. [32] [33] Once cleaved, this truncated form of the protein, tBid, can translocate to mitochondria, inducing cytochrome c release and amplification of the apoptotic signal. Although in some cell types death receptor engagement can kill cells independently of mitochondrial participation, in other cell types, this amplification step is critical to effect cell death.


In contrast to apoptosis, necrosis has largely been considered an uncontrolled, default form of cell death. Morphologically, necrosis is characterized by swelling of organelles and loss of plasma membrane integrity, and it can be induced by exposure of cells to overtly pathologic conditions such as extreme temperature or pH, or mechanical force. Increasingly, however, it is being recognized that at least in many cases, necrosis can occur through a highly regulated process, dependent, as in apoptosis, on specific signaling pathways. The critical components of this process include effectors that induce irreversible bioenergetic compromise in the cell, and those that result in release of inflammatory mediators from the cell, inducing a host response.

Necrosis can be activated both by death receptor signaling and by cell-intrinsic events. The absence of specific caspase activities, such as caspase 8, or the presence of apoptosis inhibitors, such as may be present in viral infection, may block apoptotic death in response to TNFR or Fas ligand stimulation, but induce a necrotic program. The regulated nature of this necrotic stimulus is illustrated by the fact that blocking the activity of the RIP kinase blocks necrotic cell death.[34]

Programmed necrosis may also be an important way in which organisms dispose of cells that have accumulated DNA damage. Although cells sustaining DNA damage in many cases die an apoptotic death, a functioning apoptotic pathway is not required for DNA damage–induced death. Zong and colleagues have shown that the activation of poly(ADP-ribose) polymerase (PARP) in response to DNA damage plays a critical role in the death of actively growing cells sustaining such damage.[35] PARP binds to DNA strand breaks, catalyzing the synthesis of poly(ADP-ribose) polymers on histones, promoting recognition of strand breaks by DNA repair enzymes. β-nicotinamide adenine dinucleotide (NAD) is a critical substrate of this reaction, and therefore PARP activation leads to depletion of NAD from the cytosol. Because cytosolic NAD is required for glycolytic metabolism, this consumption of NAD in response to DNA damage compromises the production of ATP in cells that are dependent on glycolysis, rather than mitochondrial metabolism, for energy production, such as rapidly proliferating cells and most cancer cells. In contrast, vegetative cells, deriving the bulk of their ATP from oxidative phosphorylation, remain bioenergetically intact. This necrotic death via PARP activation does not depend on apoptotic mediators such as Bak and Bax.

The activation of programmed necrosis might act as a warning system to the organism, resulting in release of proinflammatory mediators and activation of the immune system. This could be of particular benefit in the cases discussed previously, viral infection and cancer, in allowing the organism to mount an effective, global response to the insult.


Autophagy is a cellular process in which cytoplasmic components, including proteins and organelles, are sequestered into acidic vacuoles for degradation. The degradation products then become available as a source to support biosynthesis and energy production. Autophagy is promoted by metabolic stress and forms a response to metabolic emergencies within the cell.

The role of autophagy in cell death is not clear and is under active investigation. Some have hypothesized that autophagy is in and of itself a mechanism of cell death. Other investigators, however, have suggested that autophagy is instead a protective mechanism, allowing the cell to maintain bioenergetic integrity in the face of an inability to access nutrients. In this scenario, death would occur as a result of the depletion of intracellular resources, despite, not because of, autophagy.

Support for both these hypotheses can be found in the experimental literature. Lum and colleagues demonstrated that cells deprived of growth factors, leading to a defect in nutrient uptake, could maintain survival through activation of autophagy.[36] Inhibition of autophagy induction in this system led to rapid cell death. On the other hand, the loss of beclin-1, a gene product necessary for the induction of autophagy in mammals, predisposes to cancer in animal models, [37] [38] and loss of beclin-1 is seen in some tumor types. The mechanism by which beclin-1 functions as a tumor suppressor is unclear, however. Beclin-1 associates with Bcl-2 and therefore might have a more direct role in apoptosis. Further investigation will be needed to clarify the role of autophagy in cancer.


The events that can lead to mitochondrial apoptosis are varied. These include loss of normal survival-promoting extrinsic signals, DNA damage, metabolic stress such as hypoxia and nutrient limitation, oncogenic stresses, and toxins. Under normal circumstances, cells require extrinsic signals to promote cellular homeostasis and survival. These signals include growth factors and cell-cell or cell-matrix contact. Survival signals demonstrate to the cell that it is in an appropriate location and that cells like it are present in an appropriate number. Loss of these signals can occur if the cell finds itself in an ectopic position (loss of cell-cell or cell-matrix contact) or when specific cell types are in excess numbers (causing competition for growth factors), resulting in apoptosis.

Oncogenes as Triggers of Apoptosis

Oncogenic stresses, such as activation of Myc or loss of Rb with subsequent uncontrolled activation of the cell cycle machinery, can induce apoptosis. The mechanisms by which this occurs are not clear, but it is well demonstrated that oncogenesis through these pathways requires the additional step of inhibition of programmed cell death. In that Myc promotes activity of biosynthetic pathways, one possibility is that it promotes metabolic stress. [39] [40] Other oncogenic stresses, such as loss of the Rb tumor suppressor or DNA damage, promote activity of the tumor suppressor p53.[41]

Research performed in recent years has demonstrated clearly the importance of apoptotic pathways in control of tumorigenesis. Cancer cells, through inappropriate growth and proliferation, outstripping of resources, and translocation to environments to which they are not adapted, subject themselves to death triggers and therefore must disable the apoptotic response to survive. A critical point in understanding the role of apoptosis in cancer is that lack of death alone does not suffice to make a cancer cell. Rather, tumors must activate proliferative, growth, and invasion programs—the targets of traditional oncogenes. It is these programs and their tendency to overwhelm the cell's survival signals that place the cell under apoptotic stress. Disabling of apoptotic pathways makes the cancer cells intrinsically defective in initiation of programmed cell death; such disabling promotes resistance to antineoplastic therapy but also suggests that many cancer cells live constantly “on the edge” of death. It is possible that restoration of apoptotic function could suffice for, or at least contribute to, the elimination of a tumor.

Tumor Suppressors Promote Apoptosis


p53 is one of the best studied tumor suppressors, and its function is lost in at least half of human solid tumors. p53 activity is induced through stabilization of the protein in response to various oncogenic signals (including DNA damage), resulting in inhibition of cell growth through either cell cycle arrest or induction of apoptosis.[42] The specific mechanisms by which one or the other response occurs are not completely clear but could include duration of activity or the prevailing state of the cell. The activity of p53 seems to be mediated largely through its ability to act as a transcription factor, playing the roles of both transcriptional activator and repressor for different targets.

p53 activity and levels are controlled in large part by its upstream regulator, MDM2. MDM2 protein binds p53 and exports it from the nucleus, blocking its ability to act as a transcriptional regulator. MDM2 also targets p53 for proteasome-dependent degradation through its activity as a ubiquitin ligase. MDM2, in turn, is inhibited in its inhibition by p14ARF. In the absence of loss of p53 itself, overexpression of MDM2 can act as an oncogene, functionally suppressing p53 activity. MDM2 is overexpressed in multiple human tumor types, including lung cancer, brain cancers, and breast cancers.[43] [44] p14ARF, conversely, has been shown to be lost in various tumors, including colon cancers.[45] Abnormalities of these upstream regulators tend to occur in tumors that retain wild-type p53, demonstrating that dismantling of the p53/MDM2/p14ARF pathway plays a key role in tumorigenesis.

In addition to regulation by MDM2, post-translational modifications of p53 also influence its biologic activity. Phosphorylation and acetylation affect p53 stabilization and DNA-binding activity, and it seems that both these modifications promote p53 function. Mutant p53 proteins found in cancer cells are in some cases highly acetylated and phosphorylated, leading to stabilization and accumulation of the mutant protein.[46] This stabilized mutant protein can then act as a dominant negative, forming inactive complexes with residual wild-type protein.

The antitumor activity of p53 is mediated largely through its transcriptional effects. p53-dependent genes play multiple roles in apoptosis. For example, several proapoptotic Bcl-2 family members are transcriptionally activated by p53, including Bax and the BH3-only proteins Noxa and PUMA. [47] [48] Expression of Apaf-1, another important element in the mitochondrial pathway, is induced through p53 activity. p53 promotes death receptor pathways through activation of Fas transcription, and it inhibits survival signaling through induction of PTEN (see later discussion).[42] Although this list of antitumor effects of p53 is far from exhaustive, the foregoing examples provide insight into the importance of this pathway in blocking tumorigenesis. Nontranscriptional roles for p53 in apoptotic regulation have also been proposed, including direct binding and inhibition of the activity of Bcl-XL and Bcl-2. [49] [50] [51]

Proapoptotic Bcl-2 Family Members

In contrast to the oncogenic effects of the antiapoptotic Bcl-2 family members, proapoptotic family members, particularly Bax, have been implicated as tumor suppressors. Bax and its functional homolog, Bak, are critical in mediating apoptosis through the mitochondrial pathway induced by many cellular stresses. [30] [46] Experimental models have suggested that Bax and Bak have p53-independent function in suppression of tumorigenesis and act as tumor suppressors.[52] In one study, murine cells expressing adenoviral E1A, a proliferative factor, and dominant negative p53 were unable to form tumors in mice. Additional loss of Bax and Bak, however, resulted in the formation of highly invasive tumors, emphasizing the capability of these molecules to inhibit carcinogenesis. Furthermore, mutations in Bax and Bak have been identified in many colon and gastric cancers. [53] [54]

BH3-only proteins could also be important in preventing tumorigenesis. Although evidence implicating them as bona fide tumor suppressors is scant, BH3-only proteins play a role in the response to apoptotic stimuli of various death pathways, including p53 and death receptors.


The activity of the survival pathway mediated through PI3K and Akt, discussed subsequently, is antagonized by phosphatase and tensin homolog on chromosome 10 (PTEN), a dual-specificity (protein and lipid) phosphatase that degrades phosphatidylinositol-3,4,5-triphosphate [Ptd(3,4,5)P3] back to the bisphosphate form, terminating the signal of PI3K. PTEN was discovered in the search for a tumor suppressor on chromosome 10 that is frequently lost in glioblastoma and prostate cancer. Since its discovery, researchers have shown that PTEN acts as a negative regulator of Akt.[55] Tumorigenesis in response to loss of PTEN depends in part on deregulation of Akt activity.[56]

The frequency of PTEN loss in human tumors is exceeded only by that of p53. PTEN function is abnormal in the majority of glioblastomas and prostate cancers and has been described in many other human cancers, including breast cancer and endometrial cancer. Furthermore, mice bearing an inactive allele of PTEN develop tumors in multiple organ systems. [57] [58] The loss of a single allele of PTEN seems to be sufficient to promote tumorigenesis, as haploinsufficient mice frequently do not lose the second allele upon development of tumors, and loss of the second allele is a late event in many tumors.

Survival Factors Prevent Apoptosis in Cancer Cells

Antiapoptotic Bcl-2 Family Members

The central role of Bcl-2 family members in control of apoptosis suggests that these proteins may be appropriate targets of dysregulation in tumorigenesis, and, as predicted, many tumors show alterations in these proteins. The earliest description of antiapoptotic activity in cancer was that of overexpression of Bcl-2 in follicular lymphoma. This overexpression is brought about by the t(14;18) translocation, which brings the Bcl-2 gene locus into juxtaposition with the immunoglobulin heavy-chain enhancer, an abnormality found in at least 85% of follicular lymphomas. Since this discovery, Bcl-2 overexpression has been found in a multitude of different cancers, including other types of lymphoma and solid tumors such as breast cancer.[59]

The importance of Bcl-2 in the pathogenesis of cancer has also been demonstrated in experimental models. For example, mice expressing transgenic c-Myc in B cells develop lymphoma with a long latency period, suggesting the need for other transforming mutations for tumorigenesis. Coexpression of Bcl-2 markedly shortens the latency period, demonstrating synergy of these two molecules in lymphomagenesis.[2] Myc activation in cell lines induces apoptosis, in part through activation of p53. These experiments imply that a critical step in Myc-induced transformation is inhibition of apoptosis, and that Bcl-2 can provide this function. Bcl-Xl, another antiapoptotic family member with function similar to Bcl-2, also seems to play a role in both experimental and naturally occurring human tumors.[60] [61] [62]

Mice expressing transgenic Bcl-2 and Bcl-XL illustrate the important concept that inhibition of apoptosis alone does not induce tumorigenesis. Enforced expression of these molecules in B lymphocytes of mice leads to accumulation of lymphocytes, but lymphoma develops only rarely.[63] Instead, Bcl-2 and Bcl-XL facilitate lymphomagenesis by inhibiting the death that normally accompanies oncogenic activation. Likewise, lymphocytes bearing the t(14;18) can be detected in some healthy people with no evidence of lymphoma.[58] Taken together, these points of evidence emphasize that although suppression of apoptosis is an important step in transformation, it is not sufficient to drive carcinogenesis.


Another pathway through which tumor cells might suppress apoptosis is the nuclear factor-κB (NF-κB) pathway. As discussed previously, activation of NF-κB during death receptor stimulation sets into motion a transcriptional program that inhibits apoptosis and promotes survival. NF-κB, under normal circumstances, is held in check by binding of the inhibitor of NF-κB, IκB. NF-κB is released on phosphorylation of IκB through activity of two IκB kinases, IKK-α and IKK-β.[64] Phosphorylation targets IκB for ubiquitination and degradation by the 26S proteasome, releasing NF-κB to translocate to the nucleus and activate its target genes ( Fig. 5-4 ). Oncogenic stimuli, as well as survival signals, promote NF-κB activation. For example, Ras-mediated transformation stimulates transcriptional activity of NF-κB.[65] Furthermore, the Bcr-Abl fusion protein, a causative mutation in chronic myeloid leukemia, activates NF-κB by promoting nuclear translocation.[66] Virally induced transformation by human leukemia/lymphoma virus type 1 is dependent in part on Tax-mediated activation of IKKs.[67]


Figure 5-4  NF-κB is a transcriptional activator of multiple antiapoptotic genes. Under resting conditions, NF-κB is bound by its inhibitor, IκB, which prevents its transcriptional activity by maintaining it in the cytoplasm. Activation signals stimulate activity of IκB kinases (IKK), which phosphorylate IκB. Phosphorylated IκB can then be ubiquitinated and targeted to the proteasome for degradation. This process releases NF-κB to enter the nucleus, where it activates transcription from its target promoters.



The proto-oncogene Bcl-3 was identified on one arm of a translocation found in some lymphoid malignancies, t(14;19). This translocation brings the Bcl-3 gene in proximity to the immunoglobulin heavy-chain enhancer, resulting in its overexpression. The Bcl-3 gene encodes a member of the IκB family, but this gene product seems to function differently from other IκBs.[68] Specifically, Bcl-3 localizes to the nucleus and modifies the function of NF-κB subunits.[69] When overexpressed in normal T cells, Bcl-3 promotes survival following cellular activation, and expression seems to be induced on treatmentwith immunologic adjuvants, suggesting a physiologic survival role for it in the immune system.[70] Mice expressing a Bcl-3 transgene in B lymphocytes do not develop lymphoma, but they do show accumulation of B cells and hyper-responsiveness of the immune system, similar to the findings in Bcl-2 transgenic mice.


Many tumors lose extracellularly derived survival signals during pathogenesis, either by outstripping limited growth factors or by translocating to inappropriate environments. One signaling pathway that has been implicated in provision of survival signals is the phosphatidylinositol-3-kinase (PI3K) pathway. [71] [72] Many growth factors, including interleukin-2, interleukin-3, platelet-derived growth factor, and insulin-like growth factor, signal in part through PI3K. PI3K phosphorylates phosphatidylinositide-4,5-bisphosphate (Ptd(4,5)P2) to Ptd(3,4,5)P3, which acts as a second messenger, activating downstream effectors such as the serine/threonine kinase Akt, also known as protein kinase B (PKB; Fig. 5-5 ). Akt seems to be a critical survival factor in many cell types, and its activity might promote survival through multiple functions, such as phosphorylation and inactivation of the BH3-only protein Bad, and through the Forkhead family transcription factor FKHRL1.[73] In addition, Akt acts in the insulin signaling pathway to promote glucose uptake, and it seems to play a similar role in non-insulin-responsive cells, promoting glucose uptake and glycolysis upon growth factor stimulation. It has been proposed that this metabolism-promoting effect of Akt might protect mitochondrial integrity through maintenance of substrate availability, thereby preventing apoptosis ( Box 5-1 ). [74] [75]


Figure 5-5  The PI3K-Akt pathway is activated by multiple growth factor receptors and oncogenes and plays a critical role in promoting cell survival. PI3K is activated by growth factor stimulation or intracellular signals such as activated Ras or the oncogene BCR-Abl.Active PI3K phosphorylates phosphatidylinositols to phosphatidylinositol-3,4,5-triphosphate (PIP3) at the plasma membrane. PIP3 recruits Akt and its activating kinases, PDK1 and an uncharacterized PDK2, to the membrane, where Akt is phosphorylated and activated. Akt then promotes survival functions such as Bad and Forkhead inactivation, activation of NF-κB and MDM2, and glucose metabolism. PI3K activity is antagonized by the phosphatase PTEN, which degrades PIP3.



Box 5-1 


Cells that lose survival signals fail to maintain themselves and undergo progressive atrophy. Loss of survival-inducing signal transduction leads to downregulation of cell-surface nutrient transporters (e.g., glucose transporters) and to a decreased rate of glucose metabolism, as indicated by decreased levels of hexokinase and phosphofructokinase, two key regulatory enzymes in the glycolytic pathway. [69] [100] [101] [102] The loss of glycolytic products reduces delivery of substrate to the mitochondria, resulting in mitochondrial damage.

Nutrient limitation might have similar metabolic effects when cells accumulate in excess of the existing vascular supply.

In the 1920s, Warburg observed that cancer cells metabolize glucose at a higher rate than their normal counterparts. Furthermore, he found that the malignant cells relied on glycolysis for a disproportionate amount of their ATP production, with comparatively little energy produced by oxidative phosphorylation. Warburg hypothesized that this shift to aerobic glycolysis resulted from defects in mitochondrial function in the cancer cells.

Other researchers have subsequently confirmed Warburg's findings of increased aerobic glycolysis in cancer cells. The high rate of glucose uptake in tumors has formed the basis of a novel imaging modality, positron emission tomography (PET), using a fluorine-18-labeled glucose analog. A study evaluating PET scanning in lymphoma showed that more than 90% of lymphomas—including very indolent tumors—metabolize glucose at an abnormally high rate.[103] Although several research groups have found mutations in genes that encode mitochondrial enzymes in cancer cells, the fact that normal lymphocytes are unable to maintain glucose uptake and glycolysis in the face of dropping ATP levels suggests an alternative hypothesis. In this scenario, normal cells lack the ability to take up sufficient glucose to maintain themselves and instead are dependent on extrinsic signal transduction to maintain the expression and function of nutrient transporters. As a corollary, mutations that activate such signaling pathways could permit the cell to take up glucose in excess of that needed for bioenergetic or synthetic activities. Under such conditions, cells would secrete the excess glucose as lactate and have sufficient bioenergetic reserves to support entry into and progression through the cell cycle.

These findings raise the possibility that cancer cells, in the process of transformation, turn on signaling pathways that allow autonomous access to nutrients and metabolic pathways, rendering the cells independent of the extracellular signals normally required to maintain nutrient uptake. This facilitated access to nutrients would provide substrate to mitochondria, allowing maintenance of mitochondrial function and suppression of apoptosis even in the absence of growth factor signaling. If this hypothesis is true, autonomous access to nutrients is probably accomplished in multiple ways by different tumors.

One potential contributor to this goal is Akt. Akt is critical in the insulin signaling pathway to activate glucose uptake in insulin-responsive tissues, and it seems to play a similar role in non-insulin-responsive cells on growth factor stimulation. Although the role of Akt as an oncogene might include several functions, it clearly has the potential to promote glucose transporter expression and activity of glycolytic enzymes.

The role of metabolic control in tumorigenesis is poorly understood. Elucidation of this fundamental process in cancer might offer greater appreciation for the mechanisms of carcinogenesis.

Furthermore, the recognition of the importance of metabolic control in cancer could offer new therapeutic targets, improving our chances of defeating cancer in the future.

Another important mediator in the PI3K/Akt pathway is the mammalian target of rapamycin (mTOR). Akt indirectly activates mTOR through inhibitory phosphorylation of the TSC1/TSC2 complex,[76]leading to a complex interplay of proteins culminating in mTOR activation. Once active, mTOR promotes protein translation, leading to cell growth and proliferation. The complexity of this pathway, however, including multiple interacting protein partners and feedback mechanisms, makes it clear that the exact effects of mTOR activity on cell fate and cancer development require further elucidation.

Multiple studies have demonstrated the importance of Akt activity in tumorigenesis, either through amplification of one of the three AKT genes, or through loss of PTEN function. Amplification of Aktresults in a similar phenotype to PTEN loss and has been found in gastric cancers, breast cancers, and other tumor types. [77] [78] [79] Furthermore, animal models also have demonstrated the role of Akt in tumorigenesis. Mice expressing constitutively active Akt in T cells develop thymic lymphoma at a high rate.[80] Furthermore, inhibition of mTOR activity diminishes tumor formation in mice heterozygous for PTEN, demonstrating the importance of mTOR in tumorigenesis mediated by this pathway.[81]

Epigenetic Gene Silencing

Inhibition of expression of tumor suppressor genes through epigenetic mechanisms is emerging as an important mechanism by which tumor cells might disable proapoptotic pathways. Promoter methylation at CpG islands could repress transcription of genes in the absence of mutation ( Fig. 5-6 ). In addition, histone deacetylation might also turn off gene expression, possibly by inhibiting access of transcription factors (TF). For example, Soengas and associates[82] have shown that many melanoma cells (both primary tumors and cell lines) suppress Apaf-1 expression. This results in inhibition of p53-dependent apoptosis in these cells and renders them resistant to chemotherapy. Apaf-1 suppression is mediated not by mutation of Apaf-1 but instead through methylation, because treatment with the methylation inhibitor 5-azacytidine restores both Apaf-1 expression and chemosensitivity. Methylation seems to play an important role in suppression of tumor suppressors in other tumors as well. For example, childhood neuroblastomas show loss of expression of caspase 8 through methylation of its promoter at high frequency.[83] Finally, promoter methylation of the p14ARF locus might suppress its expression in multiple tumor types.[84]


Figure 5-6  Epigenetic gene silencing is a mechanism by which cancer cells turn off tumor suppressor gene expression. In the process of development or oncogenesis, genes might be silenced through promoter methylation. In tumors, promoters of tumor suppressor genes such as Apaf-1 and caspase 8 are frequent targets of methylation. Methylation inhibitors such as 5-azacytidine reactivate expression of these tumor suppressor genes, potentially contributing to anticancer therapy.



Acetylation of histones can also repress transcription of genes important in regulation of cell death. Global changes in histone acetylation have been found in cancer cells and are hypothesized to contribute to aberrant gene expression promoting survival and malignancy.[85]


As discussed previously, cancer cells must dismantle or inhibit apoptotic pathways to maintain their transformed phenotype. Many transformation-inducing mutations also have the effect of promoting programmed cell death, and cells are unable to pass through these initial changes to become cancer unless apoptosis is inhibited. This fact has two correlates. First, most traditional cancer chemotherapies act through induction of apoptosis, and the intrinsic apoptotic defects of these cells could make them inherently resistant to chemotherapy. On the other hand, cancer cells are constantly living “on the edge,” pushed beyond the normal limits of cell viability. This fact might make tumor cells profoundly susceptible to apoptosis if either the defect can be corrected or another death pathway can be activated. This reasoning is the basis of the many attempts currently in progress to design therapies that will attain one of these objectives.

Restoration of Apoptotic Capability

Restoration of lost apoptotic pathways can be accomplished by several methods. First, if a proapoptotic gene, such as p53, is mutated, gene therapy provides a direct way in which to restore expression of the missing protein. One approach has been to attempt to deliver the gene in question via an adenoviral vector, for example with p53 in tumors of the head and neck, and in lung tumors [86] [87]; some success has been seen with intratumoral injections. Systemic therapy poses an additional challenge, however, in terms of both feasibility and safety. The effect of gene therapy with p53 would be expected to be seen only in cells in which the transgene is expressed, requiring that every cell be infected by the vector. Furthermore, the safety of adenovirus vectors remains at issue.

Another, similar approach has been to target cancer cells lacking p53 by taking advantage of the fact that adenovirus must inactivate p53 to replicate in cells. Usually this is accomplished through the activity of the virus's E1Bp55 protein, which binds and inactivates p53. A virus that lacks E1Bp55 is unable to replicate in normal cells. Cancer cells that lack p53 present a viable target, however, allowing the virus to accomplish its lytic life cycle and killing the cell. This approach is being used clinically with the drug ONYX-015, which, similar to p53 gene therapy, has shown success with intratumoral injection in combination with chemotherapy in head and neck cancers.[88] Once again, systemic delivery seems more problematic.

In a strategy that is growing in importance in cancer therapy, small molecules targeting the p53 pathway have been designed to restore p53 function. One approach takes advantage of the fact that, in most cancers, p53 is inactivated not by deletion but rather through a point mutation that results in accumulation of inactive protein. CP-31398 is a drug found in a screen for therapeutic agents that restore wild-type conformation to mutant p53 in tumor cells.[89] Since its identification, researchers have found conflicting data regarding its ability to restore p53 function in tumor cells, with some studies finding evidence of restoration of p53 function but others reporting nonspecific toxicity. [90] [91]

Another approach has been to target the interaction of p53 with the inhibitory molecule MDM-2. Small-molecule inhibitors have been identified and have shown promise in preclinical studies.[92] Early-phase clinical studies using this approach are expected to be underway shortly.

The realization that many cancer cells turn off expression of proapoptotic genes by epigenetic mechanisms has provided another approach to the restoration of tumor suppressor function. Histone deacetylase inhibitors have entered clinical trials and have shown evidence of activity. [93] [94] One of these, suberoylanilide hydroxamic acid, has shown activity in several tumor types and has been approved for use in cutaneous T-cell lymphomas. DNA methylation inhibitors are also in development.[95] The lack of specificity of these treatments raises theoretical concerns that genes which have been silenced in differentiation (e.g., hTERT, the human telomerase gene that might play a role in tumor promotion) or other tumor-promoting genes could be turned on through demethylation or deacetylation. Silencing of TRAIL decoy receptors through methylation has been demonstrated in cancer cells.[96] The end result of these therapies could depend on the balance of genes silenced through epigenetic mechanisms in each cancer, but early clinical trials are promising.

Inhibition of Survival Factors

Small-molecule inhibitors might show promise in the inhibition of survival factors expressed in cancer cells. The 26S proteasome is important in myriad cellular pathways, but its importance in activation of NF-κB, through degradation of IκB, has raised the possibility that inhibition of proteasomal degradation could have proapoptotic effects in cancer cells. Akt has also been reported to phosphorylate the tumor suppressor genes TSC1 and TSC2 and target them for proteasomal degradation.[97] Bortezomib, a proteasomal inhibitor, has shown activity in several hematologic malignancies, notably multiple myeloma,[98] in which it has been approved by the FDA for use, and mantle cell lymphoma. The activity of bortezomib as a sensitizing agent, lowering the threshold for apoptosis in response to other cytotoxic agents, is also under active investigation.

Small-molecule inhibitors targeting several other components of survival pathways are under development. Inhibitors of mTOR and Akt are currently in clinical trials, and a small-molecule inhibitor of Bcl-2 is undergoing preclinical testing at the time of this writing.[99] Inhibitors of IAP molecules are also drawing interest and under preclinical development.

Antisense strategies have been explored to target antiapoptotic molecules. Antisense oligonucleotides act by binding to specific messenger RNAs, forming double-stranded RNA complexes. These complexes might inhibit expression of the target messenger RNAs, either by blocking translation or through targeting them for destruction by the cell through recognition of abnormal double-stranded RNA. The most developed of these antisense oligonucleotides is one targeted against Bcl-2, [100] [101] [102] oblimersen, also known as Genasense. Trials in hematologic malignancies and in several solid tumors have shown some activity and it might be particularly useful as a sensitizing agent when used in combination with other cytotoxic therapies.

Death Receptor Activation

As discussed in previous sections, death receptor signaling seems to play an important role in some tumors. Initial studies examined the use of Fas and TNF as death-inducing ligands. Yet, although these molecules demonstrated antitumor activity, their utility as therapeutic agents has been compromised by toxicity, with both normal cells and malignant cells targeted for death.

Investigations of TRAIL have raised the possibility that this ligand might be more selective for tumor cells. Most normal cells express decoy receptors that sequester TRAIL, preventing it from sending an intracellular death signal. Tumor cells seem to be uniquely sensitive to the apoptotic stimulus of TRAIL; one reason might be downregulation of decoy receptor expression, possibly by promoter methylation.[87] Studies in mice and nonhuman primates have demonstrated minimal toxicity to normal cells with administration of TRAIL. [103] [104] When researchers examined the effects of TRAIL on human hepatocytes in vitro, however, it was found to cause significant cell death, raising the question whether TRAIL, like its counterparts Fas and TNF, might be too toxic for use in humans at therapeutic doses.[105] Early-phase clinical trials of TRAIL are underway.

Induction of Necrosis

The recognition of necrosis as a regulated, physiologic process raises the possibility that induction of necrotic cell death might present advantages in targeting cancer cells. Some currently used therapeutics might act in large part through inducing necrosis, and a strategy priming cells for necrotic cell death could contribute to the effectiveness of these treatments. As discussed previously, the metabolic state of a cancer cell seems to predispose it to necrosis in response to DNA damage through loss of the ability to produce ATP through glycolysis. Cancer cells might be sensitized to the effects of DNA-damagingagents if also treated with agents that interfere with glycolysis. Whereas all cells depend to some extent on glycolytic metabolism, a modest inhibition of glycolysis in cells that are highly dependent on this pathway could potentially enhance the effects of DNA damage-based treatments. Other strategies to induce necrosis, such as induction of reactive oxygen species and promotion of RIP activity, might also be optimized as our understanding of these pathways increases. A potential advantage of necrosis as a treatment strategy lies in the potential to promote an immune reaction, and therefore possibly recruit the immune system to assist in fighting the tumor.


Understanding of mechanisms of cell death and their importance in tumorigenesis is evolving rapidly. Our interpretation of these findings will certainly undergo revision as more information constantly becomes available. The current appreciation for these processes, however, already has provided opportunities for advancement of patient care.

In the future, research further clarifying the basic mechanisms of programmed cell death—including events mediating mitochondrial demise along with a better understanding of the nature of death signals—will continue to improve our arsenal of potential weapons against cancer. As has been found with traditional antineoplastic therapies, success will most likely be found with a combination of therapeutic approaches. These might include the addition of apoptosis-based therapies to traditional agents, combining proapoptotic with antisurvival approaches, along with strategies to manipulate both forms of cell death that await development as knowledge of these complex processes evolves.


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