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Apoptosis Mechanisms: Implications for Cancer Drug Discovery

Apoptosis Mechanisms: Implications for Cancer Drug Discovery

ABSTRACT: Defects in the regulation of apoptosis (programmed cell death) make important contributions to the pathogenesis and progression of most cancers and leukemias. Apoptosis defects also figure prominently in resistance to chemotherapy, radiotherapy, hormonal therapy, and immune-based treatments. Apoptosis is caused by activation of intracellular proteases, known as caspases, that are responsible directly or indirectly for the morphologic and biochemical events that characterize the apoptotic cell. Numerous proteins that regulate these cell death proteases have been discovered, including proteins belonging to the Bcl-2, inhibitor of apoptosis, caspase-associated recruitment domain, death domain, and death effector domain families. These caspase-regulating proteins provide mechanisms for linking environmental stimuli to cell death responses or to maintenance of cell survival. Alterations in the expression and function of several apoptosisregulating genes have been demonstrated in cancer, suggesting targets for drug discovery. Knowledge of the molecular details of apoptosis regulation and the three-dimensional structures of apoptosis proteins has revealed new strategies for identifying small-molecule drugs that may yield more effective treatments for malignancies. Apoptosisregulating genes are also beginning to find utility as targets for antisense oligonucleotides.

Defects in the mechanisms of
programmed cell death
(apoptosis) play important
roles in many aspects of tumor pathogenesis
and progression. For example,
apoptosis defects allow neoplastic
cells to survive beyond their normally
intended life spans.[1] Thus, the need
for exogenous survival factors is subverted.
Protection is provided against
hypoxia and oxidative stress as tumor
mass expands; and time is allowed for
accumulative genetic alterations that
deregulate cell proliferation, interfere
with differentiation, promote angiogenesis,
and increase cell motility and
invasiveness during tumor progression.

In fact, apoptosis defects are recognized
as an important complement
to proto-oncogene activation, because
many deregulated oncoproteins that
drive cell division also trigger apoptosis
(eg, Myc, E1a, Cyclin-D1).[2] Similarly,
defects in DNA repair and chromosome
segregation normally trigger
cell suicide as a defense mechanism
for eradicating genetically unstable
cells; thus, apoptosis defects permit
survival of the genetically unstable
cells, providing opportunities for selection
of progressively more aggressive
clones.[3]

Apoptosis defects also facilitate
metastasis by allowing epithelial cells
to survive in a suspended state, without
attachment to an extracellular matrix.[
4] These defects also promote
resistance to the immune system because
many of the weapons that cytolytic
T cells and natural killer cells
use for attacking tumors depend on the
integrity of the apoptosis machinery.[
5] Finally, cancer-associated defects
in apoptosis play a role in
chemoresistance and radioresistance,
increasing the threshold for cell death
and thereby requiring higher doses for
tumor killing.[6] Thus, defective regulation
of apoptosis is a fundamental
aspect of the biology of cancer.

Because apoptosis defects permit a
wide variety of aberrant cellular behaviors,
as exhibited in cancer cells,
therapeutic strategies that negate the
apoptosis advantage for tumors are
predicted to selectively kill cancer
cells as opposed to normal cells. Fundamentally,
cancer cells should be
more dependent on apoptosis defense
mechanisms than normal cells and
thus should be proportionally more
sensitive to interventions that target
apoptosis proteins and genes. To date,
efforts to bring apoptosis-based strategies
into animal models or human
clinical trials have provided support
for this concept of selective vulnerability
of neoplastic cells as opposed to
normal cells.

A solid knowledge base now exists
about the mechanisms of apoptosis
regulation, the proteins involved, their
3D structures, and biochemical
mechanisms. Over the past 2 decades,
a clearer understanding has emerged
of the defects in expression or function
of apoptosis-regulating genes and
proteins relating to cancer. This information
can now be exploited for
devising strategies for small-molecule
drug discovery toward the goal of
revolutionary treatments for cancer
and leukemia.

Apoptosis Pathways

Apoptosis is caused by proteases
known as caspases, which stands for
cysteine aspartyl-specific proteases.[
7,8] Caspases constitute a family
of intracellular cysteine proteases
that collaborate in proteolytic cascades,
where caspases activate themselves
and each other.[9,10] Within
these proteolytic cascades, caspases
can be positioned as either upstream
"initiators" or downstream "effectors"
of apoptosis.[11] Eleven caspases have
been identified in the human genome.

Several pathways for activating
caspases probably exist, though details
remain sketchy for some of them (Figure
1). The simplest pathway is exploited
by cytolytic T cells and natural
killer cells, which inject apoptosisinducing
proteases, particularly
granzyme B, into target cells via
perforin channels.[12,13] Unlike the
caspases, granzyme B is a serine protease,
but similar to the caspases,
granzyme B specifically cleaves substrates
at Asp residues. Granzyme B
is capable of cleaving and activating
multiple caspases and some caspase
substrates. Endogenous and viral inhibitors
of granzyme B have been
identified, accounting for resistance to
this apoptotic inducer.[14-16]

Another caspase-activation pathway
is represented by the tumor necrosis
factor (TNF)-family receptors.
Of the approximately 30 known members
of the TNF family in humans, 8
contain a so-called death domain in
their cytosolic tails.[17] Several of
these death domain-containing TNFfamily
receptors use caspase activation
as a signaling mechanism, including
TNFR1/CD120a, Fas/APO1/CD95,
DR3/Apo2/Weasle, DR4/TrailR1,
DR5/TrailR2, and DR6. Ligation of
these receptors at the cell surface results
in the recruitment of several intracellular
proteins, including certain
procaspases, to the cytosolic domains
of these receptors, forming a "deathinducing
signaling complex" (DISC)
that triggers the activation of caspases
and leads to apoptosis.[18,19] The
specific caspases summoned to the
DISC are caspase-8 and, in some
cases, caspase-10. These caspases contain
so-called death effector domains
in their N-terminal prodomains that
bind to a corresponding death effector
domain in the adapter protein, Fasassociated
death domain (FADD), thus
linking them to the TNF-family death
receptor complexes.

Mitochondria also play important
roles in apoptosis, releasing cytochrome
c into the cytosol, which then
causes assembly of a multiprotein
caspase-activating complex, referred
to as the "apoptosome."[20,21] The
central component of the apoptosome
is Apaf-1, a caspase-activating protein
that oligomerizes on binding cytochrome
c and that specifically binds
procaspase-9. Apaf-1 and procaspase-
9 interact via their caspase-associated
recruitment domains (CARDs). Such
a CARD-CARD interaction plays important
roles in many steps in the pathways
of apoptosis.

The mitochondrial pathway for
apoptosis is activated by myriad
stimuli, including growth factor deprivation,
oxidants, Ca2+ overload,
DNA-damaging agents, and others.
Mitochondria can also participate in
cell death pathways induced via TNFfamily
death receptors, through crosstalk
mechanisms involving proteins
such as Bid, BAR, and Bap31.[22-25]
However, mitochondrial (intrinsic)
and death receptor (extrinsic) pathways
for the activation of caspases are
fully capable of independent operation
in most types of cells.[26] In addition
to cytochrome c, mitochondria also
release several other proteins of relevance
to apoptosis, including endonuclease
G, AIF (an activator of
nuclear endonucleases), and inhibitor
of apoptosis protein (IAP) antagonists
Smac (Diablo) and Omi (HtrA2).

Pathways of apoptosis linked to
damage in the endoplasmic reticulum
and Golgi, as well as a pathway linked
to nuclear structures called PODs
(PML oncogenic domains) or nuclear
bodies, have also been described but
are poorly characterized to date.

Suppressors of Apoptosis

Several antagonists of the caspaseactivation
pathways have been discovered,
and multiple examples of
dysregulation of their expression or
function in cancers have been obtained.
Because our current knowledge
is greatest where the mitochondrial
("intrinsic") and TNF-family death
receptor ("extrinsic") pathways for
apoptosis are concerned, most available
information about antagonists
centers on these two apoptotic pathways.
In this article, three types of
apoptosis-suppressing proteins known
to be overexpressed in tumors, including
prostate cancers, are considered:
IAPs, FLIP, and Bcl-2.

Inhibitor of Apoptosis Proteins

Inhibitor of apoptosis proteins represent
an evolutionarily conserved
family of suppressors of apoptosis.
Members of the IAP family, originally
identified in baculoviruses, contain
one or more copies of a domain called
the baculoviral IAP repeat (BIR).
These BIR domains are sometimes
accompanied by other domains, including
RING domains, ubiquitinconjugating
enzyme folds (E2s), and
NACHT-family nucleotide-binding
domains. The human genome encodes
eight IAP-family members: XIAP,
cIAP1, cIAP2, Naip, Apollon (Bruce),
ILP2 (Ts-IAP), ML-IAP (K-IAP;
Livin), and Survivin.

The BIR domains of several IAPfamily
proteins were originally shown
by our laboratory to be responsible for
directly binding and specifically inhibiting
caspases, thus identifying
IAPs as endogenous inhibitors of cell
death proteases.[27-31] Multiple
other laboratories have confirmed and
extended these findings, providing
conclusive evidence that many IAPfamily
proteins operate as caspase
suppressors.[32-41] However, IAPs
vary in the specific caspases they inhibit.
For example, XIAP suppresses
both downstream effector caspases
that operate at points of convergence
of apoptosis pathways and caspase-
9, the apical protease in the mitochondrial
pathway for apoptosis.[27,29,30]

In contrast, ML-IAP is a potent
suppressor of only caspase-9. No examples
of IAP-mediated suppression
of proteases that operate in the upstream
portions of the apoptosis pathway
activated by TNF-family receptors
have been found (Figure 2).

Evidence of overexpression of
IAPs in cancer has been obtained, suggesting
a role for these suppressors of
apoptosis in malignancy.[31,42] For
example, the IAP-family member
Survivin is overexpressed in most cancers[
43] and has become a topic of
considerable attention for its dual role
as a regulator of cell division (chromosome
segregation and cytokinesis)
and apoptosis.[44-46] Similarly, the
IAP-family member ML-IAP is rarely
expressed in normal tissues but is
found at elevated levels in melanomas
and some renal cancers.[33,40,47]
Moreover, XIAP has been reported by
our group to be overexpressed in a
substantial proportion of acute
myelogenous leukemias, with higher
levels correlating with shorter remission
durations and shorter overall
patient survival.[48] Evidence of
overexpression of XIAP has also been
reported for renal and lung cancers[
49,50]; overexpression of cIAP1
has been associated with ovarian cancer.
Chromosomal translocations
activating cIAP2 are found in some
lymphomas.[51] Thus, various IAPfamily
proteins are overexpressed in
specific types of cancer.

However, more than one member
of the IAP family can be overexpressed
simultaneously by some
tumors. For example, in prostate
cancers, we found evidence that
protein levels of XIAP, cIAP1, cIAP2,
and Survivin can sometimes become
simultaneously increased in tumors,[
52] suggesting redundancy in
expression of these antiapoptotic proteins.
We have also found evidence of
apparent simultaneous overexpression
of cIAP1, cIAP2, and Survivin in colon
cancer (manuscript in preparation).
The observation of overexpression of
multiple IAP-family members implies
that perhaps some aspects of their
regulation are shared.

Indeed, during a screen of the National
Cancer Institute panel of 60
human tumor cell lines, assessing IAP
expression at the messenger RNA
(mRNA) and protein levels, we obtained
evidence that mRNA levels of
XIAP, cIAP1, and cIAP2 do not correlate
with their protein levels,[48]
suggesting that posttranscriptional
regulation of these IAP-family proteins
is important. Interestingly, all
three of these IAP-family proteins contain
a RING domain that binds E2s
(ubiquitin-conjugating enzymes), implying
that alternations in the turnover
rate of IAP-family proteins may occur
in cancers that overexpress multiple
family members simultaneously.

The functional importance of
overexpressed IAPs for apoptosis suppression
in cancers has been supported
by antisense experiments.[53-57] In
these experiments, knocking down
expression of Survivin, XIAP, or other
IAPs has been shown to induce
apoptosis of tumor cell lines in culture
or to sensitize tumor cell lines to
apoptosis induced by anticancer
drugs.[53-57] In contrast, gene knockout
studies in mice imply that normal
cells are possibly less dependent on
IAPs than tumor cells because targeted
disruption of the genes for xiap, ciap1,
and ciap2, both individually and in
combination, produces little phenotype.[
58; personal communication, T.
Mak, 2004]

Implications for Treatment
Taken together, these observations
imply that drugs that interfere with the
action of IAPs could be useful for the
treatment of cancer. Recently, a strategy
for devising small-molecule inhibitors
of IAPs has been suggested
by the discovery of natural antagonists
of IAPs.[35,38] Proteins such as Smac
and Omi (HtrA2) have been shown to
bind IAPs and suppress them, releasing
caspases to kill cells.[35,38,59] A
7'mer peptide corresponding to the Nterminus
of Smac is reported to be
sufficient to bind IAPs and block their
association with caspases.[60]

Moreover, we have confirmed that
peptides as short as tetramers can potently
reverse caspase inhibition by
IAPs, functioning in a stoichiometric
manner at micromolar concentrations.[
61; unpublished data] By fusing
membrane-penetrating peptides
onto Smac or Omi peptides, it is possible
to induce apoptosis of cancer cell
lines in culture as well as to suppress
tumor formation in xenograft models
in mice.[62-65] Thus, these data provide
proof-of-concept evidence that
small molecules that mimic the effects
of these IAP-binding peptides could
potentially be exploited as drugs for
cancer treatment.

Drug Discovery Strategies
Structural analysis of the interactions
of IAPs with caspases and of
IAPs with Smac has helped to lay a
foundation for such drug-discovery
efforts. First, our structure-function
studies of IAP-family member XIAP
showed that, although this protein contains
three tandem BIR domains, a
single BIR is sufficient to bind and
suppress caspases. These studies demonstrated
that the BIR2 domain specifically
inhibits caspase-3 and
caspase-7, whereas the BIR3 domain
of XIAP blocks the activity of caspase-
9.[29,30] Thus, discrete domains in
IAPs are responsible for binding and
inhibiting caspases.

Second, the 3D structure of the
BIR3 domain complexed with Smac
revealed that the N-terminal 4 amino
acids of the mature Smac protein binds
in the same crevice normally occupied
by the N-terminus of the small subunit
of caspase-9, thus suggesting
completion for binding.[37,60,66,67]
Consequently, small-molecule compounds
that mimic the Smac 4?mer
peptide should dislodge active
caspase-9 from BIR3, thus inducing
apoptosis (Figure 3).

The structural details regarding the
interaction of BIR2 of XIAP with
caspases and its relation to Smac are
less clear due to poor atomic resolution
of the N-terminus of the smallsubunit
of caspases-3 or -7 complexed
with BIR2, as determined by x-ray
crystallography by scientists at our
institution and elsewhere.[32,39] In
the crystal structure of the XIAP
BIR2-caspase-3 complex, the NH2-
terminus of the caspase-3 p10 subunit
interacts with the surface of BIR2,[32]
which may be an artifact of crystallization.
Though, to date, the mechanism
of inhibition of XIAP by Smac
remains unclear, modeling studies
suggest the presence of a similar
Smac-binding pocket on BIR2.

In addition to chemical inhibitors
of IAPs based on mimicking Smac,
other strategies can also be envisioned
and have begun to be exploited. For
example, using an enzyme derepression
assay where screens were performed
to identify compounds capable
of dislodging XIAP from
caspase-3 and restoring protease activity,
we and other investigators have
identified small-molecule antagonists
of XIAP.[68,69] These compounds
target a non-Smac site on XIAP,
which remains to be defined at the
structural level.

Interestingly, in addition to Smac
and Omi (HtrA2), other endogenous
antagonists of IAPs have been
reported, including XAF1, NRAGE,
and ARTS, which operate through an
alternative mechanism.[70-72] Thus,
it is conceivable that the aforementioned
small-molecule antagonists
of IAPs mimic one or more of these
endogenous antagonists of IAPs,
a concept awaiting experimental
testing.

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