Acquired Capability: Evading Apoptosis

The ability of tumor cell populations to expand in number is determined not only by the rate of cell
proliferation but also by the rate of cell attrition. Programmed cell death—apoptosis—represents a major
source of this attrition. The evidence is mounting, principally from studies in mouse models and cultured cells,
as well as from descriptive analyses of biopsied stages in human carcinogenesis, that acquired resistance
toward apoptosis is a hallmark of most and perhaps all types of cancer.

Observations accumulated over the past decade indicate that the apoptotic program is present in latent form
in virtually all cell types throughout the body. Once triggered by a variety of physiologic signals, this program
unfolds in a precisely choreographed series of steps. Cellular membranes are disrupted, the cytoplasmic and
nuclear skeletons are broken down, the cytosol is extruded, the chromosomes are degraded, and the nucleus
is fragmented, all in a span of 30–120 min. In the end, the shriveled cell corpse is engulfed by nearby cells in a
tissue and disappears, typically within 24 hr (

Wyllie et al. 1980

).

The apoptotic machinery can be broadly divided into two classes of components—sensors and effectors. The
sensors are responsible for monitoring the extracellular and intracellular environment for conditions of
normality or abnormality that influence whether a cell should live or die. These signals regulate the second
class of components, which function as effectors of apoptotic death. The sentinels include cell surface
receptors that bind survival or death factors. Examples of these ligand/receptor pairs include survival signals
conveyed by IGF-1/IGF-2 through their receptor, IGF-1R, and by IL-3 and its cognate receptor, IL-3R (

63

, 

12

). Death signals are conveyed by the FAS ligand binding the FAS receptor and by TNFα binding TNF-R1 (

Ashkenazi and Dixit 1999

). Intracellular sensors monitor the cell's well-being and activate the death pathway in response to detecting
abnormalities, including DNA damage, signaling imbalance provoked by oncogene action, survival factor
insufficiency, or hypoxia (

Evan and Littlewood 1998

). Further, the life of most cells is in part maintained by cell–matrix and cell–cell adherence-based survival
signals whose abrogation elicits apoptosis (

50

, 

36

). Both soluble and immobilized apoptotic regulatory signals likely reflect the needs of tissues to maintain their
constituent cells in appropriate architectural configurations.

Many of the signals that elicit apoptosis converge on the mitochondria, which respond to proapoptotic signals
by releasing cytochrome C, a potent catalyst of apoptosis (
Green and Reed 1998

). Members of the Bcl-2 family of proteins, whose members have either proapoptotic (Bax, Bak, Bid, Bim) or
antiapoptotic (Bcl-2, Bcl-XL, Bcl-W) function, act in part by governing mitochondrial death signaling through
cytochrome C release. The p53 tumor suppressor protein can elicit apoptosis by upregulating expression of
proapoptotic Bax in response to sensing DNA damage; Bax in turn stimulates mitochondria to release
cytochrome C.

The ultimate effectors of apoptosis include an array of intracellular proteases termed caspases (

Thornberry and Lazebnik 1998

). Two “gatekeeper” caspases, −8 and −9, are activated by death receptors such as FAS or by the cytochrome C
released from mitochondria, respectively. These proximal caspases trigger the activation of a dozen or more
effector caspases that execute the death program, through selective destruction of subcellular structures and
organelles, and of the genome.

The possibility that apoptosis serves as a barrier to cancer was first raised in 1972, when Kerr, Wyllie, and
Currie described massive apoptosis in the cells populating rapidly growing, hormone-dependent tumors
following hormone withdrawal (

Kerr et al. 1972

). The discovery of the bcl-2 oncogene by its upregulation via chromosomal translocation in follicular

lymphoma (reviewed in 

Korsmeyer 1992

) and its recognition as having antiapoptotic activity (

Vaux et al. 1988

) opened up the investigation of apoptosis in cancer at the molecular level. When coexpressed with
a myc oncogene in transgenic mice, the bcl-2 gene was able to promote formation of B cell lymphomas by
enhancing lymphocyte survival, not by further stimulating their myc-induced proliferation (

Strasser et al. 1990

); further, 50% of the infrequent lymphomas arising in bcl-2 single transgenic transgenic mice had somatic
translocations activating c-myc, confirming a selective pressure during lymphomagenesis to upregulate both
Bcl-2 and c-Myc (

McDonnell and Korsmeyer 1991

).

Further insight into the myc-bcl-2 interaction emerged later from studying the effects of a myc oncogene on
fibroblasts cultured in low serum. Widespread apoptosis was induced in myc-expressing cells lacking serum;
the consequent apoptosis could be abrogated by exogenous survival factors (e.g., IGF-1), by forced
overexpression of Bcl-2 or the related Bcl-XL protein, or by disruption of the FAS death signaling circuit (

Hueber et al. 1997

). Collectively, the data indicate that a cell's apoptotic program can be triggered by an overexpressed
oncogene. Indeed, elimination of cells bearing activated oncogenes by apoptosis may represent the primary
means by which such mutant cells are continually culled from the body's tissues.

Other examples strengthen the consensus that apoptosis is a major barrier to cancer that must be
circumvented. Thus, in transgenic mice where the pRb tumor suppressor was functionally inactivated in the
choroid plexus, slowly growing microscopic tumors arose, exhibiting high apoptotic rates; the additional
inactivation of the p53 tumor suppressor protein, a component of the apoptotic signaling circuitry, led to
rapidly growing tumors containing low numbers of apoptotic cells (

Symonds et al. 1994

). The role of extracellular survival factors is illustrated by disease progression in transgenic mice prone to
pancreatic islet tumors. If IGF-2 gene expression, which is activated in this tumorigenesis pathway, was
abrogated using gene knockout mice, tumor growth and progression were impaired, as evidenced by the
appearance of comparatively small, benign tumors showing high rates of apoptosis (

Christofori et al. 1994

). In these cells, the absence of IGF-2 did not affect cell proliferation rates, clearly identifying it as an
antiapoptotic survival factor. Collectively, these observations argue that altering components of the apoptotic
machinery can dramatically affect the dynamics of tumor progression, providing a rationale for the inactivation
of this machinery during tumor development.

Resistance to apoptosis can be acquired by cancer cells through a variety of strategies. Surely, the most
commonly occurring loss of a proapoptotic regulator through mutation involves the p53 tumor suppressor
gene. The resulting functional inactivation of its product, the p53 protein, is seen in greater than 50% of human
cancers and results in the removal of a key component of the DNA damage sensor that can induce the
apoptotic effector cascade (

Harris 1996

). Signals evoked by other abnormalities, including hypoxia and oncogene hyperexpression, are also funneled
in part via p53 to the apoptotic machinery; these too are impaired at eliciting apoptosis when p53 function is
lost (

Levine 1997

). Additionally, the PI3 kinase–AKT/PKB pathway, which transmits antiapoptotic survival signals, is likely
involved in mitigating apoptosis in a substantial fraction of human tumors. This survival signaling circuit can be
activated by extracellular factors such as IGF-1/2 or IL-3 (

Evan and Littlewood 1998

), by intracellular signals emanating from Ras (

Downward 1998

), or by loss of the pTEN tumor suppressor, a phospholipid phosphatase that normally attenuates the AKT
survival signal (

Cantley and Neel 1999

). Recently, a mechanism for abrogating the FAS death signal has been revealed in a high fraction of lung and
colon carcinoma cell lines: a nonsignaling decoy receptor for FAS ligand is upregulated, titrating the death-
inducing signal away from the FAS death receptor (
Pitti et al. 1998

). We expect that virtually all cancer cells harbor alterations that enable evasion of apoptosis.

It is now possible to lay out a provisional apoptotic signaling circuitry (Figure 2); while incomplete, it is evident
that most regulatory and effector components are present in redundant form. This redundancy holds
important implications for the development of novel types of antitumor therapy, since tumor cells that have
lost proapoptotic components are likely to retain other similar ones. We anticipate that new technologies will
be able to display the apoptotic pathways still operative in specific types of cancer cells and that new drugs will
enable cross-talk between the still intact components of parallel apoptotic signaling pathways in tumor cells,
resulting in restoration of the apoptotic defense mechanism, with substantial therapeutic benefit.

 Resisting Cell Death

The apoptotic machinery is composed of both upstream regulators and downstream
effector components (Adams and Cory, 2007). The regulators, in turn, are divided
into two major circuits, one receiving and processing extracellular death-inducing
signals (the extrinsic apoptotic program, involving for example the Fas ligand/Fas
receptor), and the other sensing and integrating a variety of signals of intracellular
origin (the intrinsic program). Each culminates in activation of a normally latent
protease (caspases 8 and 9, respectively), which proceeds to initiate a cascade of
proteolysis involving effector caspases responsible for the execution phase of
apoptosis, in which the cell is progressively disassembled and then consumed, both
by its neighbors and by professional phagocytic cells. Currently, the intrinsic
apoptotic program is more widely implicated as a barrier to cancer pathogenesis.
The “apoptotic trigger” that conveys signals between the regulators and effectors is
controlled by counterbalancing pro- and antiapoptotic members of the Bcl-2 family
of regulatory proteins (Adams and Cory, 2007). The archetype, Bcl-2, along with
its closest relatives (Bcl-xL, Bcl-w, Mcl-1, A1) are inhibitors of apoptosis, acting
in large part by binding to and thereby suppressing two proapoptotic triggering
proteins (Bax and Bak); the latter are embedded in the mitochondrial outer
membrane. When relieved of inhibition by their antiapoptotic relatives, Bax and
Bak disrupt the integrity of the outer mitochondrial membrane, causing the release
of proapoptotic signaling proteins, the most important of which is cytochrome c.
The released cytochrome c activates, in turn, a cascade of caspases that act via their
proteolytic activities to induce the multiple cellular changes associated with the
apoptotic program. Bax and Bak share protein-protein interaction domains, termed
BH3 motifs, with the antiapoptotic Bcl-2-like proteins that mediate their various
physical interactions. The activities of a subfamily of related proteins, each of
which contains a single such BH3 motif, are coupled to a variety of sensors of
cellular abnormality; these “BH3-only” proteins act either by interfering with
antiapoptotic Bcl-2 proteins or by directly stimulating the proapoptotic members of
this family (Adams and Cory, 2007, Willis and Adams, 2005).
Although the cellular conditions that trigger apoptosis remain to be fully
enumerated, several abnormality sensors that play key roles in tumor development
have been identified (Adams and Cory, 2007, Lowe et al., 2004). Most notable is a
DNA-damage sensor that functions via the TP53 tumor suppressor (Junttila and
Evan, 2009); TP53 induces apoptosis by upregulating expression of the Noxa and
Puma BH3-only proteins, doing so in response to substantial levels of DNA breaks
and other chromosomal abnormalities. Alternatively, insufficient survival factor
signaling (for instance inadequate levels of interleukin-3 in lymphocytes or of
insulin-like growth factor 1/2 [Igf1/2] in epithelial cells) can elicit apoptosis
through a BH3-only protein called Bim. Yet another condition leading to cell death
involves hyperactive signaling by certain oncoproteins, such as Myc, which
triggers apoptosis (in part via Bim and other BH3-only proteins) unless
counterbalanced by antiapoptotic factors (Junttila and Evan, 2009, Lowe
et al., 2004).
Tumor cells evolve a variety of strategies to limit or circumvent apoptosis. Most
common is the loss of TP53 tumor suppressor function, which eliminates this
critical damage sensor from the apoptosis-inducing circuitry. Alternatively, tumors
may achieve similar ends by increasing expression of antiapoptotic regulators (Bcl-
2, Bcl-xL) or of survival signals (Igf1/2), by downregulating proapoptotic factors
(Bax, Bim, Puma), or by short-circuiting the extrinsic ligand-induced death
pathway. The multiplicity of apoptosis-avoiding mechanisms presumably reflects
the diversity of apoptosis-inducing signals that cancer cell populations encounter
during their evolution to the malignant state.
The structure of the apoptotic machinery and program, and the strategies used by
cancer cells to evade its actions, were widely appreciated by the beginning of the
last decade. The most notable conceptual advances since then have involved other
forms of cell death that broaden the scope of “programmed cell death” as a barrier
to cancer.