Cancer Biology: Apoptosis

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IGP Feb. 16, 2004 Elizabeth Yang
Cancer Biology Apoptosis
Apoptosis - what is it? Bcl-2 - prototype
anti-apoptotic oncogene C. elegans
apoptosis Mammalian apoptosis Mitochondrial and
death receptor pathways Apoptosis in
cancer Therapeutic apoptosis targets
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What is apoptosis?
apoptosis
1. normal cell
2. compaction, segregation of chromatin,
convolution of nuclear outline, organelles intact
necrosis
3. nucleus fragments, cytoplasm condenses, cell
surface protrusion, membrane-bound apoptotic
bodies
7. irregular chromatin clumping, gross
swelling of mitochondria
4. phagocytosis of apoptotic bodies
8. cellular components disintegrate,
membrane rupture
5. degradation by lysosomal enzymes
6. digestion within lysosomes
Kerr JFR, Wyllie AH, Currie AR. Br J. Cancer
26239-257, 1972
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apoptosis
necrosis
Apoptosis was originally described as
characteristic EM appearance.
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BCL-2 is an anti-apoptosis gene deregulated in
t(1418) lymphomas
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Increased T-cell lymphomas in lck-Bcl2 transgenic
mice
control n56
lckpr-bcl2 n68
Linette et al, Blood 1995
Bcl2 is deregulated in t(1418) follicular B
cell lymphomas. Overexpression of Bcl2 increases
the propensity to develop tumors. Not all Bcl2
overexpressing mice develop tumors. Inappropriate
survival (anti-apoptosis) is one primary event
in oncogenesis, other hits are necessary for
cancer to develop.
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C. elegans as a genetic model for determining
apoptotic pathways
All dying cells
Specific cells
engulfing cell
healthy cell committed to die
healthy cell
dead cell
Killing
Initiate death
Engulfment
Degradation
Decision to die
nuc-1
ced-1 ced-6 ced-7 ced-2 ced-5 ced-10
NSM sister cells ces2
ces-1
ced-9
ced-4
ced-3
HSN neurons in hermaphorodites egl-1
Apaf
caspases
CAD
Bid/Bad BH3
Bcl2
ced-9 loss of function too many cells die CED-9
gain of function too many cells live CED-3
gain of function all cells dead ced-3 or ced-4
loss of function cells cant die, too many
cells live
Bcl2-/- cells die more Bcl2 overexpression
cells live better caspase activation cells
die caspases-/- too many cells live
Hentgartner and Horvitz. Cell 76665, 1994
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/BMF
Noxa
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BCL2/BCL-xL
-transmembrane protein, BH1-4 domains
-localizes to outer mitochondrial membrane, ER,
perinuclear membrane, but not plasma
membrane. -heterodimerizes with pro-apoptotic
family members such as Bax. Ratio of Bcl2Bax
important in determining susceptibility to death
(rheostat model). -crystal structure homologous
to bacterial toxins-pore-forming (permeability
transition). -functions to repress many, but not
all, apoptotic pathways. -homologous to
C.elegans Ced-9.
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CASPASES/Ced-3
precursor
active enzyme
QACRG
prodomain
p20
p10
Asp
Asp
High homology
Variable Regulation of activation
catalytic sites
Synthesized as precursors. Cysteine at active
site. Regulate their own activation
auto-catalytic. Cleaves after Asp
residues. Tetrapeptide substrate recognition
sites. Activity can be inhibited by peptides such
as zVAD. Can activate other caspases caspase
cascade.
NA Thornberry. Cell Death and Diff 61023, 1999
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Caspase substrates
P4
P3
P2
P1
hydrophobic
I
Caspases-1, 4, 5, 13
D
E
X
D
II
Caspases-2, 3, 7, CED-3
aliphatic
III
Caspases-6, 8, 9, 10
Caspase 2
Caspase 8
Caspase 9
Caspase 10
Caspase 6
Caspase 3
Caspase 7
apoptosis
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apoptosome
Release of mitochondrial intermembrane proteins
BCL2
cytochrome c
caspase cascade
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STRUCTURE OF APOPTOSOME
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BH3 proteins act as sensors
(Noxa, PUMA)
(Bid)
(Bim, Bmf)
(Bad, Bim)
Yang E et. al. Cell 80285, 1995 Puthalakath H
et.al. Mol Cell 3287, 1999 Oda E et.al. Science
2881053, 2000 Nakano K and Vousdan KH. Mol Cell
7683, 2001 Yu J et.al. Mol Cell 7673,
2001 Puthalakath H et. al. Science 2931829, 2001
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Core apoptotic pathway
mammalian
Bcl-2 Bcl-xL Bax Bak
Bid Bim Bad Noxa
Apaf-1
Caspase 9, 3
Mitochondrial dysfunction
C. elegans
egl-1
ced-9
ced-3
ced-4
effector
BH3-only
Multi-domain Bcl2
adaptor
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IAP family - bind and inhibit caspases (Inhibitor
of apoptosis)
Baculoviral
Op-IAP
BIR (baculoviral IAP repeat) essential for
anti-apoptotic activity Block caspases or
activation of caspases Not all BIRPs are
IAPs Survivin is expressed in G2/M, associates
with microtubules Some BIRPs are required for
cytokinesis
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Mitochondrial pathway
AIF Endo G
nuclear degradation
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Fas(CD95)/TNFR mediated apoptosis
I. Fas engagement activates caspases
directly
II. Fas activates caspases via
mitochondrial amplification loop through
cleavage of Bid
Bid
Fas (receptor) is a member of the TNFR receptor
family. Fas and TNFR share a region of
intracellular homology--the death
domain--required to signal apoptosis. FasL
(ligand) is a member of the TNF family. Fas/FasL
(TNF/TNFR) interaction results in activation of
apoptosis, through recruitment of caspase 8 into
the cell death complex (DISC) at the membrane.
Xu L et.al. Cell 94481, 1998 Li H et. al. Cell
94491, 1996
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(No Transcript)
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Mitochondrial pathway - Permeability transition
pore PTP
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Mitochondrial pathway - respiratory complexes
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Drosophila apoptosis
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Oncogene cooperation bcl2 cooperates with myc to
increase lymphomagenesis
control
bcl2-Ig
(50 myc rearranged)
bcl2-Ig E?-myc
E?-myc
Marin et al, Exp Cell Res 1995
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Cell proliferation and apoptosis --- Myc
Dual signal model
Proliferation
Apoptosis
serum factors IGF-1
BCL-2
Myc is expressed in proliferating cells, absent
in quiescent cells. Myc is deregulated in
t(814) Burkitts lymphoma, and many other
cancers. Ectopic Myc expression induces cell
cycle entry. Ectopic Myc expression also induces
apoptosis in the absence of serum survival
factors. Mycs mitogenic and pro-apoptotic
activities are genetically inseparable. They
both require intact amino transcription
activation domain DNA binding domain dimerizatio
n with Max
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Cell cycle arrest and apoptosis --- p53
p53 required for some apoptotic pathways
Repair
Cell cycle arrest
DNA damage
Damage response
p53
Apoptosis
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p53 mutations abrogate apoptosis and contributes
to oncogenesis
Transcription activation
DNA binding
Oligomer
x x x
Transcriptional activation domain required for
both arrest and efficient apoptosis. Arrest
requires p21, apoptosis does not require p21. p53
can induce Bax expression, but p53-mediated
apoptosis still occurs in bax-/- mice. p53
mutations in human tumors cluster in DNA binding
domain---block arrest and apoptosis. Li-Fraumeni
families constitutional p53 mutation in one
allele--cancer prone. p53 mutations seen in 50
of human cancers.
Cell cycle arrest
X
p53 mutations in tumors
DNA damage
Damage response
X
Apoptosis
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Cell proliferation and apoptosis --- Rb
Loss of Rb function (Rb mutation or E2F mutation)
G1
M
S
Excessive cell proliferation Apoptosis (p53
dependent)
G2
Rb phosphorylation releases E2F---G1
S. Loss of Rb function (retinoblastomas)
defective cell cycle arrest. E2Fs mitogenic and
apoptotic functions are separable. E2F
transactivation mutants are defective for cell
proliferation, but still induce apoptosis that
is suppressible by Rb.
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Cell proliferation and apoptosis --- Rb and p53
Rb deleted in retinoblastomas. Rb and p53 both
deleted (mutated) in secondary osteosarcomas
arising in retinoblastoma survivors.
Cell proliferation
Rb mutation
Cell cycle arrest
Apoptosis
X
X
p53 mutation
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Differentiation and apoptosis
chrm 17
chrm 15
PML
nuclear hormone receptor binds retinoic
acid transcription repression leads to
differentiation
RING finger suppresses growth required for
apoptosis
TNF
irradiation
Fas
IFN
PML
Apoptosis
t(1517) in acute promyelocytic leukemia
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t(1517) in APL (acute promyelocytic leukemia)
Differentiation
RXR
RA
RA
RA
RA
PML/RARa inhibits RARa transcription repression
function

differentiation blocked
apoptosis blocked

PML/RARa inhibits PMLs apoptotic function
leukemogenic
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Akt is a survival kinase --- often activated in
tumors
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PI3K/AKT signaling and apoptosis --
PTEN/MMAC (phosphatase and tensin homolog deleted
on chromosome ten/ mutated in multiple advanced
cancers)
(PI4P, PI4,5P2)
(PI3,4P2 PI3,4,5P3)
(PI4P, PI4,5P2)
Receptor Tyr kinase
PI3K
SH2
PTEN
PH
PH
AKT kinase
PDK1
BAD FKHRL-1 Caspase 9 Other targets
PKC p70S6kinase
GSK3
Cyclin D1
Cell survival
Cell Cycle Entry
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Fas/FasL in cancer immune surveillance
Tumor cell counterattack
Immune attack against tumor cells
Cytotoxic T cell/NK cell
Cytotoxic T cell/NK cell
FasL gene
Apoptosis
activation
T cell receptor/ NK receptor
FasL
Fas
Fas
FasL
MHC/antigen
Apoptosis
FasL gene
Tumor cell
Tumor cell
Nagata, Nature Medicine 1996
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Immune evasion by tumor cells
T cell
FasL
Fas
soluble decoy receptors
Fas
FasL
FADD
FLIP
Tumor cell
evade apoptosis
caspase 8
1. c-FLIP interact with adaptor protein FADD,
block Fas-mediated caspase 8 activation and
cell death. 2. Proteolytic shedding of FasL from
cell surface. 3. Soluble decoy receptor for Fas
L (DcR3). 4. Downregulation of Fas by tumor cell.
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What are some of the therapeutic targets of
apoptosis?
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BH3-mimetics as therapeutics
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BH3 peptides Letai et al. Cancer Cell 2002
BAD BH3 peptide is potent against Bcl-2, causes
cytochrome c release and cell death
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HA14-1 A non-peptidic organic compound that binds
Bcl-2 protein
Effect of HA14-1 on the viability of HL-60 cells.
The cells were incubated with HA14-1 at different
concentrations for 4 h. The cell viability was
determined by using MTS method in 96-well plate.
Structure-based discovery of an organic compound
that binds Bcl-2 protein and induces apoptosis of
tumor cells Jia-Lun Wang, Dongxiang Liu,
Zhi-Jia Zhang, Simei Shan, Xiaobing Han,
Srinivasa M. Srinivasula, Carlo M. Croce, Emad S.
Alnemri, and Ziwei Huang Kimmel Cancer Center,
Jefferson Medical College, Thomas Jefferson
University, Philadelphia, PA 19107
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Small molecule inhibitors of Bcl-xL Fluorescence
polarization high-throughput screen of 16,320
chemicals from ChemBridge Corporation
Degterev et al. Nature Cell Biology 2001
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Antimycin A mimics a cell-death-inducing Bcl-2
homology domain 3 Shie-Pon Tzung et al. Nature
Cell Biology 2001
Only Bcl-xL overexpressing cells are sensitive
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Inhibition of human Colo 205 tumor growth in nude
mice by anti-DR4 (TRAIL-R1) mAbs.
Chuntharapai et al. J Immunol 2001
Effect is moderate and many tumors are
TRAIL-resistant (because they have upregulated
FLIP).
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ONYX-015 Adenovirus lacking E1B55K, cannot bind
and inactivate p53, replicates only in
p53-deficient cells and kills them.
Frank McCormick
2001
Issues ONYX-015 can replicate in some cells with
low p53 E1B55K serves other viral
functions.
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Key Points

• Apoptosis (programmed cell death)--cellular
  response to physiological changes. Death results
  from a genetically encoded suicide mechanism.
• Anti-apoptosis is not the same as cell
  proliferation.
• Conservation of biological pathways. C. elegans
  apoptosis pathway egl1-------ced-9------ced-4-
  ------ced3
• BH3------Bcl2-------Apaf1-------caspases
• Intrinsic (Mitochondrial) and Extrinsic
  (Receptor-mediatied) apoptosis pathways.
• Anti-apoptosis is oncogenic many oncogenes
  affect apoptosis pathways.