apoptosis and cancer

1. Therapeutic strategies:
Targeting apoptosis in cancer
Last updated: September 2020

2. Introduction
• Apoptosis, a form of programmed cell death, is a key aspect of cellular homeostasis1
• In cancer, the processes and signals that promote apoptosis are inhibited, allowing
cancer cells to survive and proliferate in a dysregulated manner2,3
• A deeper understanding of cell death pathways in cancer is essential for the development
of precisely targeted treatments or synergistic treatment combinations4
• A number of different strategies are under investigation to optimise outcomes with
pro-apoptotic agents3
1. Fuchs Y, Stellar H. Cell 2011;147(4):74258; 2. Sharma A, et al. Cancers 2019;11:1144; 3. Jan R, Chaudry G. Adv Pharm Bull 2019;9(2):20518; 4. Ricci MS, Zong WX. Oncologist
2006;11(4):34257.

3. Cell death and apoptosis

4. Cell death is a necessary biological function involved in
growth, homeostasis and maintenance of healthy cells
• Programmed cell death plays a fundamental role in animal development
and tissue homeostasis
1. Fuchs Y, Steller H. Cell 2011;147(4):74258; 2. Jan R, Chaudhry G. Adv Pharm Bull 2019;9:205–18.
Elimination of abnormal cells
during pathogenesis
Arrangement of cells
during morphogenesis
Regulation of cell number
during homeostasis

5. Cell death typically occurs as a result of either necrosis or a
programmed cell death such as apoptosis*
Necrosis occurs when cells die in
response to injury or cellular stress,
by swelling and rupturing1,2
1. Jacobson MD, et al. Cell 1997;88(3):34754; 2. Jan R, Chaudhry G. Adv Pharm Bull 2019;9:205-18; 3. Van Cruchton S, Van den Broeck W. Anat Histol Embryol 2002;31:214–23.
*Apoptosis is one example of programmed cell death; other examples include autophagy and necroptosis.2
Figures adapted from Van Cruchton S, Van den Broeck W. 2002.3
Apoptosis occurs when cells die during
(e.g.) homeostasis, by condensation –
without losing membrane integrity - and
removal by phagocytosis1,2
Injured cell Cell swells Cell becomes
‘leaky’ (blebbing)
Cell breaks
down
Redundant
cell
Cell and
chromatin
condense
and shrink
Cell ‘buds’ into
apoptotic bodies
Apoptotic bodies
removed by
phagocytosis

6. 1. Fuchs Y, Steller H. Cell 2011;147(4):74258; 2. Elmore S. Toxicol Pathol 2007;35(4):495–516.
OH
HO
H
H
H
Apoptosis
Apoptosis can be triggered by a range of internal
and external circumstances1,2

7. • there are two alternative pathways
that initiate apoptosis one is mediated by death
receptors on the cell surface sometimes
referred to as the 'extrinsic pathway' the other
is mediated by mitochondria referred to as the
'instrinsic pathway'.
• In both pathways, cysteine
aspartyl-specific proteases (caspases) are
activated that cleave cellular substrates, and
this leads to the biochemical and morphological
changes that are characteristic of apoptosis.

8. • Cells may be induced to undergo apoptosis by extracellular signals, socalled
“death factors,” or by internal physical/chemical insults such as DNA
damage or oxidative stress. Subsequently, two non-exclusive molecular
pathways, the extrinsic and the intrinsic, respectively, may be activated.
• Caspases are specific proteases that act like molecular scissors to cleave
intracellular proteins at
• “caspases” derives from three of the characteristics of the enzymes: they
are cysteine-rich aspartate proteases. More than 13 mammalian caspases
have been identified. They are synthesized as inactive enzymes called
procaspases that need to be cleaved at aspartate residues in order to be
activated. Although for the most part procaspases are considered inactive,
procaspases possess some activity—about 2% of the proteolytic activity of
fully activated caspases. aspartate residues (one of the 20 amino acids).

9. • Moreover, as caspases cleave at aspartate residues and procaspases are
themselves activated by cleavage at aspartate residues, caspases
participate in a cascade of activation whereby one caspase can activate
another caspase in a chain reaction.
• This mechanism, whereby caspases activate procaspases, leads to amplifi
cation of an apoptotic signal: only a few initially activated caspase molecules
can produce the rapid and complete conversion of a pool of procaspases.

11. The extrinsic pathway: mediated by
membrane death receptors
• A death factor such as Fas ligand or tumor necrosis factor (TNF) is
received by a transmembrane death receptor such as Fas receptor or TNF
receptor, respectively. TNF is a soluble factor while Fas ligand is bound to
the plasma membrane of neighboring cells. When ligands bind to the death
receptors, the receptors undergo a conformational change and oligomerize
(several come together) in order to transduce the signal into the cell.
• The conformational change exposes so-called death domains that are
located on the receptors’ cytoplasmic tail and enable intracellular adaptor
proteins such as FADD (Fas-associated death domain protein) and TRADD
(TNF receptor-associated death domain protein) to bind via their death
domains

12. • Caspase-8 is known as an initiator caspase as it is the first link between the
receptor and the apoptotic proteases and it is key to the extrinsic pathway.
Together the death ligands, receptors, adaptors, and initiator caspase are
called the death inducing signaling complex (DISC).
• Caspase-8 initiates a cascade of caspase activation: one activated caspase
cleaves and activates other caspases, called executioner caspases
(caspase-3, -6, and -7). The cascade ultimately causes the cleavage of
specifi c protein targets and results in apoptosis.
• This process can be inhibited by c-Flip an inhibitor of apoptosis. c-Flip can
bind to adaptor FADD via a DED and inhibit caspase-8 recruitment and
activation.

13. • The breakdown of the cell results from the proteolysis of the target proteins.
Target proteins include nuclear lamins allowing for nuclear shrinkage,
cytoskeletal proteins such as actin and intermediate filaments for
rearranging cell structure, specific kinases for cell signaling, and other
enzymes such as caspase-activated DNase for the cleavage of chromatin.
• Caspases also cleave the tumor suppressor protein, RB and this cleavage
results in the degradation of RB protein. This event is required for apoptosis
induced by TNF and points to a role for RB in the inhibition of apoptosis

15. The intrinsic pathway: mediated by the
mitochondria
• Stimuli from inside the cell, such as DNA damage and oxidative stress,
induce the intrinsic pathway of apoptosis through the Bcl-2 family of proteins
that act at the outer mitochondrial membrane.
• There are two groups within the Bcl-2 family that have opposing functions:
one group of Bcl-2 proteins inhibits apoptosis and another group promotes
apoptosis

17. p53 and apoptosis
• means. As a transcription factor, p53 induces the expression of genes that
code for death receptors and pro-apoptotic members of the Bcl-2 family.
• Examples include Fas receptor, Bax, and Bak. These genes contain a
consensus p53-binding site in their promoter regions. p53 can also repress
the expression of anti-apoptotic factors, such as Bcl-2 and Bcl-x and IAPs.
Recent evidence demonstrates that a member of the Bcl-2 family called
PUMA (p53 upregulated modulator of apoptosis), a target of p53, is
essential for apoptosis induced by p53.

18. Evasion of apoptotic processes
in cancer

19. Cancer cells survive and proliferate via
evasion of apoptogenic triggers
• Abnormal regulation of programmed cell
death is associated with many diseases,
including cancer1
• Defects in DNA repair and chromosome
segregation normally trigger cell death to
remove these genetically unstable cells2
• Defects in apoptosis:
– Allow genetically unstable cells to survive and allow
selection of progressively aggressive clones2,3
– Allow neoplastic cells to survive beyond their typical
lifespan, providing protection from hypoxia and
oxidative stress as the tumor mass expands3
– Allow epithelial cells to survive in a suspended state,
detached from the extracellular matrix, which
facilitates metastasis3
1. Fuchs Y, Stellar H. Cell 2011;147(4):74258; 2. Hassan M, et al. Biomed Res Int 2014;2014:150845; 3. Reed JC. Cancer Cell. 2003;3(1):17–22.
OH
HO
H
H
H
Apoptosis

20. Cancer cells inhibit the intrinsic pathway by
upregulating anti-apoptotic Bcl-2 proteins
Bak, Bcl-2 antagonist/killer; Bax, Bcl-2 associated X protein; Bcl, B-cell lymphoma; BH, Bcl-2 homology; BIM, Bcl-2 interacting mediator of cell death; MOMP, mitochondrial outer membrane
permeabilization; PUMA, p53-upregulated modulator of apoptosis; tBID, truncated BH3 interacting domain death agonist.
Figure adapted from Baig et al. 2016.4
1. van Delft MF, Huang DCS. Cell Res 2006;16:203–13; Happo L, et al. J Cell Sci 2012;125(5):1081–7; 3. Sharma A, et al. Cancers 2019;11:1144; 4. Baig S, et al. Cell Death Dis 2016;7:e2058.
BH3
protein
Bax/Bak
SMAC
XIAP
IAP
Mitochondrion Cytochrome C
Apoptosis
Procaspase-9
Apoptosome
Apaf-1
Caspase 3,6,7
Stimulus, e.g. ROS
Release of ‘activators’
(e.g.) PUMA
Activation of
apoptotic
pathway via
MOMP
• Bcl-2 proteins – all of whom contain BH1-4
domains – are key regulators of the intrinsic
pathway1,2
• The intrinsic pathway is initiated by release
of pro-apoptotic BH3 ‘activators’ (i.e. BIM,
PUMA, tBID) that activate multi-domain
‘effectors’ (e.g. Bax; Bak) to promote MOMP2
• Cancer cells often express elevated levels of
pro-apoptotic BH3-only proteins3
– Thus, cancer cells will typically upregulate anti-
apoptotic Bcl-2 proteins in order to sequester
unbound pro-apoptotic proteins, preventing
MOMP and activation of the apoptotic cascade3

21. Cancer cells inhibit the intrinsic pathway
by suppressing p53 expression
• The p53 tumor suppressor protein upregulates
pro-apoptotic BH3-only proteins such as PUMA
and NOXA1
• The p53 gene is mutated or deleted in many
human cancers, inactivating its suppressor activity2
• In some cancers with wild-type p53 status, its
function is effectively inhibited by amplified
expression of MDM2, which is the primary cellular
inhibitor of p531,2
• Thus, by inhibiting or deleting p53 activity,
malignant cells remove a key driver for apoptosis
and maintain their proliferative state
BH, Bcl-2 homology; MDM2, murine double minute 2; PUMA, p53 upregulated modulator of apoptosis.
1. Aubrey BJ, et al. Cell Death Differ 2018;25:104–13; 2, Shangary S, Wang S. Clin Cancer Res 2008;14(17):5318–24. 3. Nag S, et al. J Biomed Res 2013;27(4);254–71; 4. Zhao Y, et al. Acta Biochim
Biophys Sin 2014;46:180–9.
Tumor cell3,4

22. Tumor cell
Cancer cells inhibit the intrinsic pathway
by modifying cell metabolism
• Specific Bcl-2 proteins can be regulated by
metabolite stresses1
– Glucose deprivation may induce PUMA via
p53 induction1
– p53 inhibits glycolysis through down-regulation
of glucose transporters, glycolytic enzymes, and
inhibition of hypoxic-inducible factors1
• Activation of oncogenes (e.g. RAS, AKT,
MYC) as well as loss of genes such as p53
drive aerobic glycolysis in cancer cells and
promote a glycolytic phenotype,1,2 thereby
subverting micronutrient-driven apoptotic
activation
1. Sharma A, et al. Cancers 2019;11:1144; 2. Zheng J. Oncol Lett 2012;4:1151–7.
Bcl, B-cell lymphoma; PUMA, p53-upregulated modulator of apoptosis; tBID, truncated BH3 interacting domain death agonist.
p53
Oncogene
Glycolytic
environment

23. Cancer cells inhibit the intrinsic pathway
by offsetting oxidative stress mechanisms
• Higher ROS can upregulate cell death
pathways1
– Low ROS may upregulate proliferative
pathways and, thus, promote tumorigenesis2
• Due to their high metabolic activity, cancer
cells often contain increased ROS2,3
• Consequently a cancer cell must synthesize
antioxidants in order to retain the cell’s redox
homeostasis2,3
• Cancer cells may also increase the uptake
of antioxidant nutrients and metabolic
enzymes2 to moderate intracellular ROS
ROS, reactive oxygen species.
1. Redza-Dutordoir M, et al. Biochim Biophys Acta 2016;1863:2977–92; 2. Sharma A, et al. Cancers 2019;11:1144; 3. Liou GY, Storz P. Free Radic Res 2010;44(5):479–96.
Increased ROS =
increased cell death
activation
Decreased ROS =
increased tumorigenesis

24. Cancer cells have evolved strategies to
evade extrinsic pathway-induced apoptosis
• The extrinsic pathway is mediated by the
death receptors TNFR1, CD95, FasR, APO-1,
DR4 and DR51
• Death receptors are activated by regulatory
ligands such as TRAIL, which induce DISC
formation and initiate the apoptotic pathway1
• In cancer cells, TRAIL resistance may be
caused by various genetic mutations
(resulting in altered apoptotic signaling
proteins), or overexpression of
anti-apoptotic Bcl-2 proteins1
APO-1, apoptosis antigen 1; Bcl, B-cell lymphoma; CD95, cluster of differentiation 95; DISC, death-inducing signaling complex; DR, death receptor; FasR, Fas receptor; TNFR1, tumor necrosis
factor receptor 1; TRAIL, TNF-related apoptosis-inducing ligand.
Figure adapted from Baig et al. 2016.2
1. Ukrainskaya VM, et al. Acta Naturae 2017;9(3):55–63; 2. Baig S, et al. Cell Death Dis 2016;7:e2058.
Activated death
receptor
DISC
Caspase 8
Procaspase-3,6,7
Caspase 3,6,7
Apoptosis

25. Targeting apoptosis in cancer therapy

26. Apaf, apoptotic protease activating factor; Bak, Bcl-2 antagonist/killer; Bax, Bcl-2-associated X protein; Bcl, B-cell lymphoma; DISC, death-inducing signaling complex; DR, death receptor; IAP,
inhibitor of apoptosis proteins; MDM2, murine double minute 2; SMAC, second mitochondrial-derived activator of caspases (also known as DIABLO, direct IAP-binding protein with low pI); X-linked
inhibitor of apoptosis protein.
*These are investigational compounds and have not been approved. Their efficacy and safety have not been established.
Figures adapted from Baig et al. 2016.3
1. Ricci MS, Zong WX. Oncologist 2006;11(4):342–57; 2. Jan R, Chaudry G. Adv Pharm Bull 2019;9(2):20518; 3. Baig S, et al. Cell Death Dis 2016;7:e2058.
DISC
Caspase 8
Procaspase-3,6,7
Caspase 3,6,7
Apoptosis
DR ligands2
BH3
protein
Bax/bak
SMAC
XIAP
IAP
Mitochondrion Cytochrome C
Apoptosis
Procaspase-9
Apoptosome
Apaf-1
Caspase 3,6,7
p53/MDM2
expression2
Attenuation of
Bcl-2 proteins2
SMAC expression2
Targeted induction of apoptosis in cancer cells
A deeper understanding of cell death pathways in cancer is essential for the development of
precisely targeted treatments or synergistic treatment combinations1*

27. Bcl, B-cell lymphoma; BH, Bcl-2 homology; Mcl-1. myeloid cell leukemia-1; XIAP, X-linked inhibitor of apoptosis protein.
*These are investigational compounds and have not been approved. Their efficacy and safety have not been established.
1. NCT02427451. Available at: https://clinicaltrials.gov/ct2/show/NCT02427451. Accessed July 2020; 2. NCT04277637. Available at: https://clinicaltrials.gov/ct2/show/NCT04277637. Accessed July
2020; 3. Baig S, et al. Cell Death Dis 2016;7:e2058; 4. Lu AQ, et al. Int J Clin Exp Med 2018;11(7):6767–75; 5. Boffo S, et al. J Exp Clin Cancer Res 2018;37:36; 6. Jan R, Chaudry G. Adv Pharm Bull
2019;9(2):205–18; 7. Mukherjee N, et al. Cell Death Dis 2018;9:907.
• Bcl-2 inhibitors1,2*
• Antisense oligonucleotides*
to enhance sensitivity to
cytotoxic drugs3,4
• BH3-mimicking agents6,7*
• Cyclin-dependent kinase inhibitors*
to downregulate expression of factors
such as Bcl-2, Mcl-1 and XIAP5
Strategies
used to inhibit
the anti-apoptotic
Bcl-2 family include:
Approaches to target the intrinsic
pathway: Bcl-2 attenuation

28. Spotlight on p53/MDM2
• The tumor suppressor gene p53 impacts both
cell cycle arrest and apoptosis1
• p53 activity is inhibited by MDM2 upregulation
– Antisense oligonucleotides and MDM2 inhibitors*
have been developed to reduce MDM2
overexpression and trigger wild-type p53 activity1,2
• Another approach uses small molecules
that reactivate wild-type function* of
mutant p531,3
MDM2, mouse double-minute 2; wt, wild-type.
*These are investigational compounds and have not been approved. Their efficacy and safety have not been established.
1. Jan R, Chaudry G. Adv Pharm Bull 2019;9(2):205–18; 2. Rayburn ER, et al. Anticancer Agents Med Chem 2009;9(8):882–903; 3. Di Agostino S, et al. J Exp Clin Cancer Res 2019;38(1):290;
4. Rudolph D, et al. Presented at the Annual Meeting of the American Association for Cancer Research 2018. Abstract 4868 and poster; 5. Nag S, et al. J Biomed Res 2013;27(4);254–71; 6. Zhao Y,
et al. Acta Biochim Biophys Sin 2014;46:180–9.
Interaction inhibitor*
Tumor cell4–6

29. Spotlight on SMAC
• SMAC is released in response to Bcl-2 activity
on the mitochondrion, where it binds to and
induces IAP degradation to promote apoptosis1
– In cancer cells, elevated IAP expression increases cell
survival, tumor growth and metastasis1
• SMAC mimetics* bind to cellular IAPs to
restore effector caspase function and are
under investigation alone or as
combination therapy2,3
• However, SMAC is overexpressed in some
cancers, suggesting a role in non-apoptotic
cancer cell survival and a possible role for
reducing SMAC expression in some cancers4,5
1. Bai L, et al. Pharmacol Ther 2014;144(1):82–95; 2. Jan R, Chaudry G. Adv Pharm Bull 2019;9(2):205–18; 3. Derakshan A, et al. Clin Cancer Res 2017;23(6):1379–87; 4. Zhao XY, et al. Cells
2020;9:1012; 5. Paul A, et al. Mol Ther 2018;26(3):680–94; 6. Baig S, et al. Cell Death Dis 2016;7:e2058.
Bcl, B-cell lymphoma; IAP, inhibitor of apoptosis protein; SMAC, second mitochondrial-derived activator of caspases (also known as DIABLO, direct IAP-binding protein with low pI).
*These are investigational compounds and have not been approved. Their efficacy and safety have not been established.
Figure adapted from Baig et al. 2016.6
BH3
protein
Bax/bak
SMAC
XIAP
IAP
Mitochondrion Cytochrome C
Apoptosis
Procaspase-9
Apoptosome
Apaf-1
Caspase 3,6,7

30. Spotlight on TRAIL ligands
DR, death receptor; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand.
*These are investigational compounds and have not been approved. Their efficacy and safety have not been established.
Figure adapted from Garcia-Martinez et al. 2019.5
DR4 and DR5 receptors are candidates for
targeted tumor therapy, due to their high
expression levels in cancer cells1
• DR4 and DR5 are activated by TRAIL ligands,
which are widely expressed on cells.2
Approaches to targeting TRAIL in cancer
therapy include:
– TRAIL-R novel forms3*
– TRAIL-R1 and -R2 antibodies1,4*
– Peptide agonists1*
• A key feature of targeting TRAIL is its reduced
cytotoxicity to normal cells versus, for
example, Fas ligands or TNF1
1. Ukrainskaya VM, et al. Acta Naturae 2017;9(3):55–63; 2. Ashkenazi A, et al. J Clin Invest 1999;104(2):155–62; 3. Leng Y, et al. Cancer Chemother Pharmacol 2017;79(6):1141–49;
4. NCT04137289. Available at https://clinicaltrials.gov/ct2/show/NCT04137289. Accessed July 2020; 5. Garcia-Martinez JM, et al. American Association for Cancer Research Annual Meeting 2019;
Abstract 2051.
TRAILR2/CDH17
antibody*
Tumor-specific induction of apoptosis

31. Summary
• Abnormal regulation of apoptosis is associated with many diseases,
including cancer1
• Apoptosis is a complex process with numerous points of regulation that
may be targeted to provide therapeutic benefit in cancer2
• Future cancer treatments may include modulation of strategic points of the
intrinsic and extrinsic apoptotic pathways, such as:
– Inhibition of anti-apoptotic Bcl-2 family3*
– Upregulation of tumor suppressor gene p534*
– Regulation of SMAC activity3*
– Application of TRAIL antibodies5*
Bcl, B-cell lymphoma; SMAC, second mitochondrial-derived activator of caspases (also known as DIABLO, direct IAP-binding protein with low pI); TRAIL, TNF-related apoptosis-inducing ligand.
*These are investigational compounds and have not been approved. Their efficacy and safety have not been established.
1. Fuchs Y, Stellar H. Cell 2011;147(4):74258; 2. Fox JL, Macfarlane M. Br J Cancer 2016;115:5–11; 3. Jan R, Chaudry G. Adv Pharm Bull 2019;9(2):205–18; 4. Rayburn ER, et al. Anticancer Agents
Med Chem 2009;9(8):882–903; 5. Ukrainskaya VM, et al. Acta Naturae 2017;9(3):55–63.