Apoptosis

For a long time, instances of cell death have been catalogued as apoptotic on the basis of purely morphologic manifestations, including cytoplasmic and nuclear shrinkage (pyknosis), nuclear breakdown (karyorrhexis), and plasma membrane blebbing.⁵ This morphologic definition, reflecting the original observations by Kerr, Wyllie, and Currie,³ is progressively being abandoned in favor of a biochemical one.⁴ Thus apoptosis can occur as a result of the activation of either of two distinct but not mutually exclusive signaling pathways. On one hand, extrinsic apoptosis depends on a peculiar class of cysteine proteases called caspases and is always initiated by an extracellular stimulus acting on plasma membrane receptors. On the other hand, intrinsic apoptosis invariably follows the permeabilization of mitochondrial membranes but does not always require caspases.⁴ The enzymatic activity of executioner caspases, encompassing caspase-3, -6 and -7, is responsible for multiple (but not all) of the classic morphologic and biochemical manifestations of apoptosis, including karyorrhexis and the internucleosomal degradation of DNA.¹⁵

Extrinsic Apoptosis

Extrinsic apoptosis is frequently elicited by the ligand-induced activation of plasma membrane proteins of the death receptor family, such as CD95/FAS and the tumor necrosis factor α receptor 1.¹⁶ Alternatively, apoptotic signals can be transduced by the so-called dependence receptors (such as patched 1) when the extracellular concentration of trophic factors, such as netrin-1, falls below a critical threshold level. This means that—at odds with death receptors—dependence receptors deliver proapoptotic signals in the absence, rather than in the presence, of their ligands.¹⁷ Death receptors undergo spontaneous trimerization because of the so-called preligand assembly domain.¹⁸ Ligand binding stabilizes this configuration and hence allows for the recruitment of several proteins at the cytoplasmic tails of death receptors, including (but in some instances not limited to) pro-caspase-8, receptor-interacting protein kinase 1 (RIPK1, also known as RIP1), FAS-associated protein with a death domain, and cellular inhibitor of apoptosis proteins (cIAP1 and cIAP2, which are E3 ubiquitin ligases that also inhibit apoptosis as a result of their ability to interfere with caspase activation).¹⁹ This multiprotein platform is known as “death-inducing signaling complex” and stimulates the conversion of pro-caspase-8 into caspase-8, which proteolytically activates caspase-3 and hence initiates the degradative cascade that mediates the execution of extrinsic apoptosis.²⁰ Of note, the components of the signaling pathways that emanate from specific death receptors exhibit some degree of variation, yet all converge (in apoptosis-permissive conditions, as described later) to the activation of caspase-8. The molecular mechanisms that link dependence receptors to caspase-3 activation are not precisely understood, yet they appear to involve caspase-9 and the adaptor proteins DRAL and TUCAN (Fig. 5-1).²¹

Necrosis

Classically, necrosis was defined as an instance of cell death lacking the peculiar morphologic manifestations of apoptosis and the accumulation of cytoplasmic vacuoles that characterize autophagic cells. Somehow, this definition was in line with the belief that necrosis would always proceed in an unregulated fashion and would only terminate accidental instances of cell death.⁵ In the late 1990s, the group of investigators led by Peter Vandenabeele demonstrated that the engagement of FAS does not always lead to cell death via extrinsic apoptosis.^40,41 This observation instilled in some researchers the suspicion that, similar to apoptosis, necrosis also might be orchestrated by a refined molecular machinery and ignited an intense wave of research that has not yet come to an end.⁶

The best-characterized pathway of regulated necrosis, which is also known as necroptosis, is elicited by the ligation of death receptors in conditions in which caspase-8 is inhibited (either by pharmacologic or by genetic interventions). In this context, death-inducing signaling complex–bound RIPK1 does not get degraded by caspase-8 as it occurs during extrinsic apoptosis; rather, it recruits and functionally interacts with its homolog RIPK3, generating the so-called necrosome.44–44 Recently it has been shown that the mixed-lineage kinase like protein and the mitochondrial phosphatase PGAM5 may contribute to the execution of regulated necrosis downstream of RIPK1 and RIPK3.^45,46 These results confirmed previous suspicions regarding the involvement of mitochondria in regulated necrosis, driven by the prominent role exerted by reactive oxygen species and by metabolic circuitries that (at least in part) localize to mitochondria, such as glutaminolysis.^44,47 Stimuli other than death receptor ligands (e.g., alkylating DNA damage) reportedly trigger regulated necrosis.⁴⁸ However, it remains unclear whether these instances of regulated necrosis also require RIPK1 and RIPK3 and proceed via PGAM5-dependent mitochondrial fragmentation (Fig. 5-2).⁴⁶ Regulated necrosis occurs during mammalian development, in particular at the bone growth plate (i.e., the zone of the bone that controls its length), as well as during adult tissue homeostasis, for instance, in the lower regions of intestinal crypts.^49,50 Moreover, RIPK1/RIPK3-dependent necroptosis has been involved in the pathophysiology of several diseases, including viral infection, neurodegeneration, ischemia, and others.⁵¹

Autophagy

Autophagy entails the engulfment of intracellular structures (including organelles, protein aggregates, and portions of cytoplasm) by double-membraned vacuoles called autophagosomes. Autophagosomes normally fuse with lysosomes, leading to the degradation of their content by lysosomal hydrolases. Baseline levels of autophagy contribute to the maintenance of intracellular homeostasis by ensuring the removal of old and damaged (and hence potentially dangerous) organelles, notably mitochondria.52,53 In addition, autophagy is upregulated in response to a wide array of stressful conditions, including nutrient deprivation, hypoxia, and the presence of xenobiotics, such as anticancer agents.⁵⁴

For a while it was thought that the continuative activation of autophagy eventually would lead to the exhaustion of cellular resources and cell death. Such an “autophagic cell death” was defined morphologically by an extensive vacuolization of the cytoplasm, representative of an elevated number of autophagosomes and autolysosomes (the organelles that are generated by the autophagosomal-lysosomal fusion).5,14 However, the association between the accumulation of autophagosomes and cell death has rarely if ever been proved to be causal, in particular in settings of stress-induced cancer cell death. Indeed, the inhibition of autophagy by pharmacologic or genetic means often accelerates (rather than inhibits) cell death, suggesting that autophagy constitutes a stress response mechanism that attempts (but fails) to avoid the cellular demise.⁵⁵ These results cast doubts on the appropriateness of the term “autophagic cell death,” which inadvertently suggests a cause-effect relationship between these two phenomena.¹⁴ Thus far autophagy has been demonstrated to mediate cell death in several developmental scenarios, notably during the metamorphosis of insects.^56,57 Moreover, at least in some instances, autophagy appears to contribute to the execution of human cancer cells succumbing to specific experimental cell death inducers in vitro.⁵⁸ These observations de facto justify the use of the expression “autophagic cell death” under selected circumstances.⁴ Defects of the autophagic machinery have been associated with a plethora of human pathophysiologic conditions, including accelerated aging, neurodegeneration, and cancer.^59,60 However, this association appears to be more strictly related to the role that autophagy exerts in the regulation of intracellular homeostasis rather than as a bona fide cell death mechanism⁵² and hence will not be treated here in further detail.

Oncogenes and Cell Death Regulation

Oncogenes (i.e., genes that stimulate malignant transformation) were originally identified in tumorigenic viruses and then were shown to exist as inactive variants (or proto-oncogenes) in the human genome.⁶³ Proto-oncogenes including MYC and NRAS are involved in the regulation of mitogenic signals and hence play a critical role in the control of tissue homeostasis. Proto-oncogenes are not intrinsically tumorigenic and must acquire gain-of-function alterations to become so. This process can originate from alterations as gross as chromosomal translocations that bring proto-oncogene coding sequences in the proximity of strong transcriptional regulators (as in the case of MYC, which is often rearranged in persons with lymphoma) or as specific as point mutations that render proto-oncogene products constitutively active (as in the case of NRAS, which is affected by hyperactivating mutations in 20% to 25% of all cancers).^64,65 By transducing strong and persistent mitogenic signals, constitutively active MYC and NRAS—as well as other oncoproteins such as the epidermal-growth factor receptor (which is often overactivated in lung and colon cancer) and the Abelson tyrosine kinase (ABL, which is fused to BCR upon the t(9;22) translocation in chronic myelogenous leukemia)—facilitate the escape of malignant precursors from the growth control that usually guarantees tissue homeostasis. As previously mentioned, however, one single alteration of this kind is insufficient to convert normal cells into their malignant counterparts. The overactivation of mitogenic pathways indeed causes consistent functional alterations and drives cells into a state of constant stress, featuring significant metabolic rewiring and increased generation of reactive oxygen species. Because oncogenic stress normally would lead to cell death, only premalignant cells in which the cell death signaling pathways are blocked can progress toward tumorigenesis.⁶¹

Premalignant cells resist oncogenic stress by activating one (or more) among several pathways that (1) directly counteract the execution of cell death (because of the deregulation of additional proto-oncogenes) or (2) facilitate the management of intracellular stress (as a result of the elicitation of per se nononcogenic stress response pathways). This response often leads to two distinct phenomena of dependence, which are called “oncogene addiction” and “nononcogene addiction.”⁶² In the former scenario, not only the tumorigenic phenotype but also the survival of cancer cells becomes dependent on the overactivation of one or more proto-oncogenes. This dependency has provided a rationale for the use of multiple oncogene-targeted agents in anticancer therapy, some of which (such as the BCR-ABL–targeting compound imatinib) have been associated with impressive rates of therapeutic responses.⁶¹ In the latter scenario, cancer cells become dependent on the activation of intrinsically nononcogenic mechanisms of intracellular stress management, such as those centered around molecular chaperones of the heat-shock protein family.⁶² Major examples of prosurvival systems that often get overactivated throughout (and contribute to) oncogenesis are provided by antiapoptotic BCL-2 family members, the nuclear factor–κB (NF-κB) pathway, and the serine/threonine protein kinase AKT.

Antiapoptotic BCL-2 proteins have been shown to regulate cell death by multiple mechanisms, including the sequestration of their proapoptotic counterparts into inactive complexes, as well as Ca²⁺-related and metabolic effects (Fig. 5-3).^29,66,67 Following the discovery that the overexpression of BCL-2 as a result of the t(14;18) translocation underlies multiple instances of lymphomagenesis,¹⁰ preclinical and clinical evidence has accumulated to demonstrate that not only BCL-2 but also its antiapoptotic homologues BCL-X_L and MCL-1 exert bona fide oncogenic functions.⁶⁸ The overexpression of BCL-2 shortens the latency period required for transgenic mice that overexpress MYC in B cells to develop lymphomas.¹¹ Along similar lines, both BCL-X_L and MCL-1 have been shown to cooperate with MYC in murine models of hematopoietic malignant transformation.^69,70 However, consistent with the notion of multistep oncogenesis previously described, mice engineered for the overexpression of BCL-2, BCL-X_L, or MCL-1 in hematopoietic cells exhibit perturbed myelopoiesis or lymphopoiesis but are only slightly more prone to the development of age-related lymphomas than are their normal counterparts.^69,71,72 Of note, the protein expression levels of BCL-2, BCL-X_L, and MCL-1 are elevated in a wide range of human tumors.⁷³

NF-κB is a transcription factor that participates in the cellular response to a wide array of conditions, including (but not limited to) cytokine stimulation, infection, and oxidative stress. Under normal circumstances, NF-κB subunits such as p65^RELA and p50^NFKB1 are held in check in the cytoplasm by inhibitory interactions with IκBα. In the presence of NF-κB–inducing conditions, however, IκBα is phosphorylated by the so-called IκB kinase (IKK, a multiprotein complex including two catalytic subunits, i.e., IKKα and IKKβ, and one regulatory component, i.e., IKKγ/NEMO), resulting in its proteasomal degradation and in the nuclear translocation of NF-κB.⁷⁴ Nuclear NF-κB homodimers and heterodimers control the expression of more than 150 target genes, including genes that code for mitogens, other transcription factors (including MYC), inhibitors of extrinsic apoptosis (e.g., the so-called FLIPs), IAPs, BCL-2 and BCL-X_L (Fig. 5-4).⁷⁵ Thus, the activation of NF-κB orchestrates the response of cells to stress while delivering a strong prosurvival (and in some cases mitogenic) signal. Multiple lines of evidence demonstrate that the NF-κB system exerts genuine oncogenic functions. First, REL genes are homologues of the avian reticuloendotheliosis virus v-rel oncogene, which is strictly required for virus-induced malignant transformation.^76,77 Second, the human leukemia/lymphoma virus type I can transform target cells via a mechanism that depends, at least in part, on the activation of cellular IKK by the viral protein Tax.⁷⁸ Finally, several components of the NF-κB pathway get overexpressed or overactivated during malignant transformation. For instance, REL genes are amplified or affected by gain-of-function mutations in a constituent proportion of B-cell lymphomas,⁷⁹ and the IkB-related protein BCL-3 (which stimulates the nuclear translocation and activity of NF-κB dimers)⁸⁰ is involved in a chromosomal rearrangement, t(14;19), that is found in some hematologic malignancies.⁸¹ Of note, the NF-κB system appears to play a critical role in the handling of oncogenic stress, as demonstrated by the fact that multiple oncoproteins, including RAS and BCR-ABL, stimulate NF-κB nuclear translocation and transcriptional activity.^82,83

One of the hallmarks of cancer cells as originally formulated by Hanahan and Weinberg in 2000 consists of their ability to circumvent the absence of extracellular growth factors and de facto emancipate from homeostatic tissue regulation.⁸⁴ Several growth factors, including interleukin-2, the platelet-derived growth factor, and the insulin-like growth factor, promote proliferation and survival via transmembrane receptors that activate the phosphoinositide-3-kinase (PI3K, also known as phosphatidylinositol-3-kinase). Active PI3K generates plasma membrane–bound phosphatidylinositol-3,4,5-trisphophate, in turn stimulating the activation of the serine/threonine kinase AKT (also known as protein kinase B). By phosphorylating multiple substrates, AKT transduces prosurvival signals via distinct mechanisms. For example, AKT inhibits the BH3-only protein BAD by facilitating its sequestration by 14-3-3 proteins, stimulates the degradation of the oncosuppressor protein p53 (discussed later), promotes glucose uptake and hence favors the maintenance of bioenergetic homeostasis, and blocks the nuclear translocation of proapoptotic Forkhead transcription factors such as FKHRL1.⁸⁵ Moreover, AKT indirectly activates the mammalian target of rapamycin (mTOR), thus stimulating cell growth and proliferation while inhibiting autophagy (Fig. 5-5).⁸⁶ Several preclinical and clinical studies have demonstrated the importance of AKT in tumorigenesis. In transgenic mice expressing constitutively active AKT under the control of a T cell–restricted promoter, lymphoma develops early in life.⁸⁷ Amplification of AKT genes or gain-of-function AKT mutations have been detected in several neoplasms, including (but not limited to) breast and gastric cancers. In addition, loss of function of the oncosuppressor PTEN (coding for the phosphatase that directly antagonizes PI3K and hence inhibits AKT, discussed later) is common across a wide array of tumors.⁸⁸ Of note, the inhibition of mTOR limits tumor formation in Pten+/- mice,⁸⁹ demonstrating that AKT-driven oncogenesis relies, at least in part, on mTOR overactivation. This observation is particularly interesting in view of the fact the mTOR inhibition stimulates autophagy, which is considered a legitimate oncosuppressor mechanism during early tumorigenesis.⁹⁰

Oncosuppressors and Cell Death Regulation

The existence of tumor suppressor genes (also known as oncosuppressor genes), that is, genes whose products actively counteract tumorigenesis, was first suspected in the 1960s, when Harris and colleagues observed that highly malignant Ehrlich ascites cells lose the ability to form tumors upon fusion with virtually nontumorigenic cells.⁹¹ However, the molecular characterization of the first tumor suppressor gene lagged until the 1980s, when the complementary DNA encoding the protein product of the RB1 gene (a negative regulator of cell cycle progression involved in the development of pediatric retinoblastoma) was isolated and sequenced.⁹² Since then, several other oncosuppressor proteins have been identified that encompass negative regulators of mitogenic signals, such as PTEN, pro-apoptotic BCL-2 family members like BAX, and proteins that couple the management of cellular stress to cell death induction, such as ATM and p53. Oncosuppressor genes are inactivated during oncogenesis not only upon loss-of-function mutations and deletions but also after epigenetic gene silencing. Thus promoter hypermethylation at CpG islands or histone deacetylation can turn off oncosuppressor gene expression as a result of local chromatin remodeling and impaired access for essential transcription factors.⁹³ These instances are exemplified by the loss of caspase-8 expression in pediatric neuroblastomas or by the loss of p14^ARF (an upstream activator of p53; discussed later) expression in multiple tumor types.^94,95 Irrespective of the underlying mechanisms, in most cases, both alleles of one particular tumor suppressor gene must be inactivated for the establishment of tumor-permissive conditions. In this context, germline mutations in oncosuppressor genes are associated with familial tumor syndromes (as one copy of the gene is lost in the whole organism), whereas somatic mutations are detected frequently in sporadic tumors (in this case, both alleles have been inactivated during tumorigenesis).⁹⁶

As previously mentioned, the protein product of PTEN (phosphatase and tensin homolog) functions as a phosphatase, catalyzing the conversion of phosphatidylinositol-3,4,5-trisphosphate into phosphatidylinositol-4,5-diphosphate, hence essentially antagonizing the enzymatic activity of PI3K and negatively regulating AKT (Fig. 5-5).⁹⁷ PTEN was discovered in the search for an oncosuppressor gene on chromosome 10 that often is lost in glioblastoma, breast, endometrial, and prostate cancer.⁹⁸ Subsequent preclinical and clinical observations supported the notion that PTEN loss of function sustains tumorigenesis in many different organs.^99,100 Recently the oncosuppressive functions of PTEN have been shown to be particularly sensitive to gene dosage, even beyond the classic concept of haploinsufficiency.¹⁰¹ Of note, the frequency of PTEN inactivation in human tumors is exceeded only by that of p53 (discussed later).⁸⁸

Pro-apoptotic BCL-2 family members, in particular BAX and BAK, exert crucial oncosuppressive functions as they mediate the execution of cell death in response to adverse conditions, including oncogenic stress (Fig. 5-3). Results from multiple murine models support the notion that BAX and BAK operate in vivo to counteract oncogenesis independent of p53. For instance, murine cells expressing adenoviral E1A (a proliferative factor) and dominant negative p53 are unable to form tumors in mice unless Bax and Bak are lost.¹⁰² In addition, loss-of-function mutations in BAX and BAK have been detected in many hematopoietic and solid malignancies.^73,103 By acting as stress sensors that activate intrinsic apoptosis, BH3-only proteins also could exert, at least theoretically, prominent oncosuppressive functions. That said, evidence implicating them as legitimate tumor suppressors is scant, and only a few articles report their loss in patients with cancer.¹⁰⁴ The reasons underlying this apparent discrepancy have not yet been clarified but perhaps are linked to the fact that whereas BAX and BAK operate at the convergence of multiple lethal signaling pathways, single BH3-only proteins are involved in highly specific signaling cascades and perhaps are relatively redundant among each other.

p53 originally was identified in 1979 by two methodologically distinct approaches, that is, as a cellular protein coimmunoprecipitating with the large-T antigen in SV40-transformed cells105,106 and as the target of antibodies isolated from the serum of immunodeficient mice bearing human tumors.^107,108 Since then, p53 has been subjected to an intense wave of investigation, leading to the discovery that p53 regulates an ever-growing list of cellular processes.¹⁰⁹ The first (and perhaps best) characterized function of p53 is that of a stress-responsive transcription factor. In physiologic conditions, the intracellular levels of p53 are regulated by HDM2, an E3 ubiquitin ligase that polyubiquitinates p53 and hence targets it to proteasomal degradation.¹¹⁰ In response to a variety of adverse conditions, including DNA damage and oncogenic stress, p53 escapes HDM2-mediated degradation and accumulates in the cytoplasm. This response can be due to posttranslational modifications of p53 that reduce its affinity for HDM2 or to the expression of HDM2 inhibitors such as p14^ARF.¹¹¹ Irrespective of the underlying molecular mechanisms, stabilized p53 assembles into transcriptionally proficient tetramers that can regulate the expression of distinct sets of genes, leading to highly diverse functional outcomes.¹¹² Among the most common p53-regulated responses are reversible cell cycle arrest, senescence, and cell death. The latter is accomplished via the upregulation of multiple genes involved in the execution of apoptosis, including APAF1, BAX, FAS, and PUMA,^112–115 as well as via transcription-independent mechanisms whereby cytoplasmic p53 stimulates MOMP by interacting with both proapoptotic and antiapoptotic members of the BCL-2 family (Fig. 5-6).^116–119 The inactivation of p53, be it mutational, due to the overexpression/overactivation of p53 negative regulators such as HDM2, or resulting from the inactivation of upstream p53-activating factors such as p14^ARF, is the most common molecular alteration in human cancer, affecting more than 50% of all neoplasms. In addition, the p53 status has been shown to influence prognosis and/or therapeutic outcome in multiple oncologic settings, including (but not limited to) breast, lung, and colorectal cancer.¹²⁰ These clinical data reflect a huge number of preclinical observations, demonstrating that p53 exerts legitimate oncosuppression in vivo.¹²¹ Of note, recent research has focused on physiological aspects of the p53 biology, unveiling a critical role for baseline p53 levels in the maintenance of bioenergetic, redox, and genomic homeostasis.^122–125 Thus p53 appears to exert oncosuppressive functions both as it regulates intracellular homeostasis, thereby preventing malignant transformation, and as it orchestrates the elimination of potentially tumorigenic cells.