Molecular control of apoptosis

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Molecular control of apoptosis

• David J. McConkey, Ph.D.
• Dept. Cancer Biology
• U.T. M.D. Anderson Cancer Center

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Modes of physiological cell death

• Apoptosis cellular shrinkage, suppresses
  inflammation
• Necrosis cellular swelling, loss of
  intracellular contents, inflammation
• Autophagy auto-digestion, can allow for survival
  in face of limited nutrient availability, may
  also control tumor expansion

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Autophagy

• Macroautophagy - disposal of whole organelles
  (mitochondria, peroxisomes) and protein
  aggregates
• Chaparone-mediated autophagy disposal of
  specific proteins and possibly protein aggregates
• Both complement the activity of the proteasome in
  bulk protein turnover

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Knockout mice

• Mice lacking crucial autophagy genes (Atg5, Atg7)
  develop neurodegenerative disease that is similar
  to certain human pathologies (Alzheimers,
  Huntingtins, etc)
• Neurons develop large protein aggregates
  (inclusion bodies)
• Similar effects are observed in cells with
  disrupted proteasome function

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Autophagy in yeast

• Activated when nutrients are in limited supply
• Allows cells to recycle the energy that is
  stored in cellular structures until a food supply
  is provided

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Autophagy in cancer

• Beth Levine showed that tumors develop defects
  in autophagy (beclin-1) as they progress
• Craig Thompson showed that tumors with defective
  apoptosis use autophagy to produce ATP when their
  growth factors are withdrawn

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Autophagy and signal transduction

• Key regulator mTOR (downstream target of the
  PI-3 kinase/AKT pathway)
• Active AKT activates mTOR, which then inhibits
  autophagy
• Another input phosphorylation of eIF2??activates
  autophagy via mechanisms that are still being
  identified

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Binding of growth factor ligands activates kinase
receptors leading to recruitment of PI3K to
receptor complex
Sansal, I. et al. J Clin Oncol 222954-2963 2004
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The unfolded protein response
Szegezdi et al, EMBO Rep. 7(9) 880-85, 2006
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Caspase structures
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tBID
SMAC
IAPs
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Release of Apoptotic Factors from the Mitochondria
Smac
Mitochondria
IAP
Caspase 9
Smac
IAP
AIF
Apaf-1
dATP
Cytochrome C
To Nucleus
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Inhibitor of apoptosis proteins

• Originally identified in viruses that infect
  insect cells (Lois Miller)
• Subsequently found in C. elegans, mammalian cells
• Inhibit apoptosis by direct binding to caspases
  (procaspases and active caspases)
• Also regulate death receptor signaling

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IAP structural features
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(IAPs)
(Caspase)
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The BCL-2 family

• BCL-2 Cloned at the t(1418) breakpoint in
  follicular lymphoma
• Cory, Korsmeyer Inhibits apoptosis
• Family consists of cell death inhibitors and cell
  death inducers
• Recent studies Regulate release of
  mitochondrial factors

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Bcl-2 Family Members Domains
Anti-Apoptotic
Bcl-2 Bcl-xL
BH4
BH2
BH3
BH1
TM
Pro-Apoptotic
Bax Bak
BH2
BH3
BH1
TM
Bid Bad
BH3
Adapted from Chao and Korsmeyer, 1998
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How do BCL-2 family proteins regulate cytochrome
c release?

• Korsmeyer Tetramerization of Bak, Bax in
  mitochondrial membrane forms a pore large enough
  to accommodate release
• Kroemer, Tsujimoto Proteins interact with key
  components of a structure called the PT pore
• Can also regulate ion fluxes in ER, mitochondria

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Bcl-2 Family Member Regulation of the Mitochondria
PTP
VDAC
Bax
Bak
Bak
Bak
Bid
Bcl-2
Bak
BH3
Bax
Cyt c
Mitochondria
Cyt c
Bax
Bax
Bax
Bax
Bax
Anions
Bcl-2
Bcl-2
Bcl-2
Bax
Bax
Cations
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Cell death signals in cancer progression/metastasi
s

• Hypoxia/hypoglycemia (Thompson)
• Growth factor withdrawal
• Detachment from basement membrane, adjacent cells
  in tissues (aniokis)
• Death-promoting signals from the environment
  (death receptors)

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Early work Evan and Wyllie

• Expressed an estrogen receptor-Myc fusion protein
  in Rat-1 fibroblasts
• Under normal circumstances Serum withdrawal
  stimulates growth arrest (G1)
• However, estrogen-mediated activation of Myc
  abrogated growth arrest
• However, under these conditions rates of cell
  death were also increased

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Evan Myc couples proliferation to cell death

• In normal cells Expression of Myc
  simultaneously promotes S phase progression and
  cell death
• Suppression of the latter requires the presence
  of exogenous survival factors
• These factors often take the form of cytokines
  (IGF-1, PDGF, etc)

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Effects of enforced Myc expression in mice

• Transgenic mice expressing Myc under the control
  of the immunoglobulin promoter/enhancer
• Stimulates hyperplasia in mature B cells
  associated with increased apoptosis
• Progression to lymphoma requires a second genetic
  event that suppresses apoptosis (mutation of p53,
  BCL-2 expression)

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DNA viruses also engage the apoptotic pathway

• Adenovirus E1A protein functions to promote an
  S-phase-like state in infected cells
• In mutant viruses lacking E1B, infection drives
  S-phase and apoptosis
• E1B Encodes a BCL-2 homologue (E1B-19K) and a
  p53-binding protein (E1B-52K)

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Coupling of cell cycle dysregulation and cell
death p53

• Oncogene-mediated cell death (Myc, Ras, E1A)
  appears to be driven by p53 (p63, p73)
• Cell death in Rb-/- mice is also p53-dependent
• Mechanism may involve p14/p19ARF-mediated
  activation of p53

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The first p53 death target Bax

• Miyashita and Reed, Cell 80 293-299, 1995
• Identified p53 binding site in Bax promoter
• Observation has been confirmed by numerous groups
• However, Bax is also regulated by BH3-only
  proteins and sometimes changes in subcellular
  localization

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Viral disruption of p53 pathway regulation
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DQMD
baculovirus p35
(effector caspases)
p10
p25
LVAD
cowpox virus crmA
p5
p33
(caspases 1 and 8)
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Survival signals and apoptosis

• Cells beyond the blastomere stage of development
  require continuous exposure to environmental
  survival signals to suppress apoptosis
• Can be provided by soluble factors (growth
  factors, cytokines) and adhesion molecules
  (integrins, coreceptors)

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Binding of growth factor ligands activates kinase
receptors leading to recruitment of PI3K to
receptor complex
Sansal, I. et al. J Clin Oncol 222954-2963 2004
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The AKT pathway is negatively regulated by PTEN

• PTEN phosphatase on chromosome 10
• Also known as MMAC mutated in multiple
  adenocarcinomas
• Lipid phosphatase

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PTEN (blue), a lipid phosphatase
Sansal, I. et al. J Clin Oncol 222954-2963 2004