Prosurvival mechanisms
I. Endogenous prosurvival mechanisms
II. Autophagy
III. Induced prosurivival mechanisms
MECHANISMS OF CARCINOGENESIS
SPRING SEMESTER
(Dean Tang, April 27, 2011)
Two
Apoptotic
Pathways
Prosurvival mechanisms: BH3-only proteins are tightly regulated
Bad
Bax Cyto c
Bak
Apaf-1
Caspase-9
Noxa
Caspase-3
BH3-only
BCL-2
1.
2.
anti-apoptotic
Bcl-2 proteins
BH1-3
pro-apoptotic
proteins
adaptor
caspase
Like Egl-1, some BH3-only proteins (e.g., Puma, Noxa, Hrk, and Bim) are normally not expressed in
some cell types and need transcriptional activation.
Some BH3-only proteins are expressed but are not lethal. For example, Bim in many cells is expressed
but remains bound to tubulin or tubulin-based dynein motor complexes and therefore is not
proapoptotic. Bid is expressed in many cells but generally needs a post-translational cleavage to
generate proapoptotic tBid. Bad is abundant in many cells but inactive due to PHOSPHORYLATION
and sequestration by 14-3-3 proteins.
BAD: Phosphorylation sequesters it in the cytosol
A Major Prosurvival Pathway: PI3K-Akt
*Phosphotidylinositol 3-kinase, or PI3K, is activated by multiple mitogens and survival
factors. Prototypical examples include: NGF in neuronal cells, IL-3 and other cytokines in
lymphocytes, PDGF and EGF in mesenchymal cells, and IGF-1 in epithelial cells.
*These tyrosine kinase receptors, as well as seven transmembrane G protein-coupled
receptors and activated Ras, recruit PI3K to the membrane, which then catalyzes the transfer
of phosphate from ATP to the D3 position of the inositol ring of membrane-localized
phosphoinositides.
*The main products, PI3,4P and PI3,4,5P, function as signaling molecules that can activate
TK, small G proteins, and ser/thr kinases such as atypical PKCs, pp70S6K, and c-Akt.
Activation of c-Akt is the major survival function.
*Akt (PKB) has a central kinase domain with ser/thr substrate specificities, an amino terminal PH
(which mediates protein-lipid and/or protein-protein interactions), and a C-terminal hydrophobic
and proline-rich domain.
*Direct binding of PI3,4,5P or related lipids to the PH domain in Akt leads to its translocation to
plasma membrane. At this location and with PIP bound, Akt is phosphorylated (at Thr308 and
Ser473) and activated by PKDs (3-phosphoinositide-dependent protein kinases). Integrin-linked
kinase (ILK) is also a PIP-dependent Akt kinase (Ser473).
*Activated Akt has three main apoptosis-related targets: Bad, caspase-9, and forkhead (FKH)
transcription factors. Phosphorylation of Bad (at Ser136) and of caspase-9 inactivate these proapoptotic proteins. Phosphorylation of FKH and Bad sequesters them in the cytosol by binding to
14-3-3.
*Transfection with constitutively active Akt blocks apoptosis induced by deprivation, UV irradiation,
matrix detachment, cell-cycle disruption, DNA damage, TGFb, anti-Fas, etc.
*Conversely, trasnfection with dominant-negative alleles of Akt (catalytically inactive Akt, a mutant
Akt consisting only of the PH domain) blocks the pro-survival functions of survival factors.
*PTEN is a lipid phosphatase that dephosphorylates PI3K-generated 3’-phosphorylated
phosphotidylinositides.
*Mitogens and survival factors activate a parallel survival pathway: Raf-MEK-MAPK pathway.
Ras-dependent survival is mediated by both PI3K and Raf-MEK-MAPK pathway.
Prosurvival mechanisms: BH3-only proteins are tightly regulated
Bad
Bax Cyto c
Bak
Apaf-1
Caspase-9
Noxa
Caspase-3
BH3-only
BCL-2
1.
2.
3.
anti-apoptotic
Bcl-2 proteins
BH1-3
pro-apoptotic
proteins
adaptor
caspase
Like Egl-1, some BH3-only proteins (e.g., Puma, Noxa, Hrk, and Bim) are normally not expressed in
some cell types and need transcriptional activation.
Some BH3-only proteins are expressed but are not lethal. For example, Bim in many cells is expressed
but remains bound to tubulin or tubulin-based dynein motor complexes and therefore is not
proapoptotic. Bid is expressed in many cells but generally needs a post-translational cleavage to
generate proapoptotic tBid. Bad is abundant in many cells but inactive due to PHOSPHORYLATION
and sequestration by 14-3-3 proteins.
Bim undergoes biallelic deletion in mantle cell lymphoma and promoter methylation in other B-cell
lymphomas. A few human lymphomas also have Noxa gene mutations. Bik/Nbk is mutated in renal cell
carcinomas.
BH3-only proteins may have physiological/prosurvival functions
*BID is required for myeloid homeostasis: Bid-deficient mice, as they age, spontaneously develop a
myeloproliferative disorder resembling CMML (chronic myelomonocytic leukemia).
*Bid plays a role in DNA-damage response and in promoting cell survival (Zinkel SS, et al., Cell 122,
579-591, 2005; Kamer I et al., Cell 122, 593-603, 2005).
*Bad is involved in normal glycolysis and in promoting cell survival (Danial NN et al., Nature 424,
952-956, 2003; Seo et al., JBC 279, 42240-42249, 2004).
*Most bim-deficient mice die embryonically, suggesting Bim may also have a normal physiological
function. Bim may have a role in regulating cytoskeletal integrity.
*bid-/- mice (Yin et al., Nature 400:886-891, 1999.)
--- Animals develop normally
--- Resistant to anti-Fas induced liver failure
*bim-/- mice (Bouillet et al., Science 286, 1735-1738, 1999.)
--- A significant number of bim-null, and even some of the bim+/mice die in utero prior to E9.5 for unknown reasons.
--- Bim deficiency causes an SLE (systematic lupus erythematosus)like autoimmune disease.
Anti-apoptotic Bcl-2 proteins function as
endogenous prosurvival molecules
Bad
Bax Cyto c
Bak
Apaf-1
Caspase-9
Noxa
Caspase-3
BH3-only
BCL-2
1.
2.
3.
anti-apoptotic
Bcl-2 proteins
BH1-3
pro-apoptotic
proteins
adaptor
caspase
Bcl-2 and Bcl-xL are indispensable for stem cell development and are critical for the
survival of stem cells and many mature cell types. Bcl-2 and/or Bcl-xL are overexpressed
perhaps in all tumor cells.
Mcl-1 is critical for the survival of HSCs, neutrophils (not macrophages), fibroblasts,
keratinocytes, and many other cell types. Mcl-1 is also overexpressed in several cancers.
Bcl-w is expressed in a majority of invasive gastric adenocarcinomas.
Knockouts of anti-apoptotic Bcl-2 family proteins
*bcl-2-/- mice (Nakayama et al., Science 261: 1584, 1993; Veis et al., Cell 75:229-240, 1993)
--- Defects in renal development (essential for kid. stem cells)
--- Premature loss of B/T lymphoid cells: Immunodeficiency
--- Defects in melanocyte development (essential for the survival of melanocyte
stem cells): Loss of hair pigmentation
*bcl-xL-/- mice (Motoyama et al., Science 267:1506-1510, 1995.)
--- Animals die during embryogenesis
--- Massive apoptosis in developing post-mitotic cells of the embryonic spinal cord,
brain stem, and DRG
--- Extensive apoptosis is also observed within the liver and hematopoietic systems
--- Haploinsufficiency for Bcl-xL causes thrombocytopenia (Mason KD et al., Cell 128,
1173-1186, 2007).
*mcl-1-/- mice
--- defects in survival and implantation of zygote (Rinkenberger et al., G&D 14:23, 2000)
--- in conditional KO, critical in early hematopoiesis and B/T lymphopoiesis
(Opferman JT et al., Nature 426, 671-676, 2003; Science 307, 1101-4, 2005)
*bcl-w-/- mice
--- defects in spermatogenesis (Print CG et al., PNAS 95, 12424-31, 1998; Ross AJ
et al., Nat Genet. 18, 251-6, 1998)
Bcl-2 proteins control apoptosis
Bad
Bax Cyto c
Bak
Apaf-1
Caspase-9
Noxa
Caspase-3
BH3-only
BCL-2
anti-apoptotic
Bcl-2 proteins
BH1-3
pro-apoptotic
proteins
adaptor
caspase
Bax and/or Bak knockouts
*bax-/- mice (Knudson et al., Science 270:96-99, 1995)
--- Animals develop rather normally
--- Increased numbers of sympathetic and motor neurons
--- In vitro, bax-/- sympathetic neurons are resistant to cell death
upon trophic factor withdrawal
--- Male homozygous mice are sterile due to a paradoxical increase
in the death of spermatogonia
*bak-/- mice (Lindsten et al., Mol. Cell 6:1389-1399, 2000)
--- Animals develop normally
--- bak-/- cells show normal apoptotic responses to both extrinsic
and intrinsic apoptotic stimuli
*bax-/-bak-/- mice:
--- bak-/- bax-/- mice die perinatally with <10% animals reaching adulthood
--- Adult bak-/- bax-/- mice display multiple phenotypic abnormalities (retain
interdigital webs, imperforate vaginas, neurological abnormalities
including deafness, and circling behavior, increased numbers of neuronal
and hematopoietic progenitors; leading to elevated numbers of
granulocytes and mature T/B lymphocytes and enlarged spleen and LN
and infiltration of parenchymal organs; bak-/- bax-/- cells are resistant to
death
induced by neglect or irradiation but sensitive to Fas.)
Bax and Bak may have non-apoptotic and physiological functions
*Unstimulated, Bax is monomeric and inactive in the cytosol, probably due to its concealed amino
(or carboxy) termini. Bax is also normally “sequestered” in the cytosol as a result of binding to
multiple proteins including 14-3-3 proteins, Ku70, the peptide humanin, HSP70, crystallins, and
ARC or Apoptosis Repressor with Caspase recruitment domain).
*Recently, Bax has been shown to be retro-translocated by Bcl-xL to block its proapoptotic effect
(Edilich et al., Cell 145: 104-116, 2011).
*Different from Bax, most Bak is constitutively expressed in the mitochondria by associating with
VDAC2.
*Bax and Bak modulate the UPR by a direct interaction with IRE1 (inositol-requiring enzyme 1)
(Hetz et al., Science 312, 572, 2006)
•Bax and Bak play an important role in mitochondrial morphogenesis (Karbowski M, Norris KL,
Cleland MM, Jeong SY, Youle RJ. Nature 443:658-62, 2006)
*Bak has neuroprotective effect (Fannjiang Y et al., Dev. Cell. 4, 575-585, 2003).
*In some cancer cells, Bax and Bak are mutated.
Bcl-2 proteins control apoptosis
Bad
Bax Cyto c
Bak
Apaf-1
Caspase-9
Noxa
Caspase-3
BH3-only
BCL-2
anti-apoptotic
Bcl-2 proteins
BH1-3
pro-apoptotic
proteins
adaptor
caspase
Chandra D, Liu J-W, Tang DG. Early mitochondrial activation and cytochrome c
up-regulation during apoptosis. J. Biol. Chem. 277: 50842-50854, 2002.
Holo-cyto. c
1
Apaf-1
99-109
1248
415
NH2-
-COOH
CARD
NOD
WD-40 repeats (13)
Shi, Y., Mol. Cell, 9, 459-470, 2002
Apoptosome Formation can be Reconstituted using Recombinant Proteins
--- A Process that Requires (d)ATP
Holo-cyto. c
+
1
Apaf-1
99-109
NH2-
-COOH
CARD
+(d)ATP
(0.1-0.5 mM)
1248
415
NOD
WD-40 repeats (13)
Physiological
(mM) levels
of (deoxy)N
Holo-cyto. c
1
Apaf-1
99-109
1248
415
NH2-
-COOH
CARD
NOD
WD-40 repeats (13)
Caspase-9 activation
Chandra D, Bratton SB, Person MD, Tian Y, Martin AG, Ayers M, Fearnhead HO,
Gandhi V, and Tang DG. Intracellular nucleotides act as critical prosurvival
factors by binding to cytochrome c and inhibiting apoptosome. Cell 125: 1333-1346, 2006.
Holo-cyto. c
1
Apaf-1
99-109
1248
415
NH2-
-COOH
CARD
NOD
WD-40 repeats (13)
Cytochrome c
Apaf-1
Apoptosome
Caspase-9
Caspase-3
Apoptosis
Physiological
(mM) levels
of (deoxy)N
*Physiological levels of K+ also inhibits cytochrome c-dependent
apoptosome formation (JBC, 276, 41985-990, 2001).
*Bao Q, Lu W, Rabinowitz JD, Shi Y. Calcium blocks
formation of apoptosome by preventing nucleotide
exchange in Apaf-1. Mol Cell. 2007 Jan 26;25(2):18192.
Caspases are executioners of apoptosis
Bad
Bax Cyto c
Bak
Apaf-1
Caspase-9
Noxa
Caspase-3
BH3-only
BCL-2
anti-apoptotic
Bcl-2 proteins
BH1-3
pro-apoptotic
proteins
adaptor
caspase
Types of caspases:
-Initiator caspases: caspase-2, -8, -9 and -10
-Executioner Caspases: caspase-3, -6 and -7
Caspase Structure:
p3-26
Prodomain
p20
p10
Large subunit
Small subunit
Linker
Caspase prodomains:
Prodomains:In executioner caspases: ~3kd
In initiator caspases: 10-26 kd
D. Chandra
Mol. Cell
9, 459-470,
2002
Caspase substrates
Proteosome components (Mol Cell 14, 81-93, 2004)
Mitochondrial complex I p75 subunit (Cell 117, 773-786, 2004)
Bcl-2 proteins control apoptosis
Bad
Bax Cyto c
Bak
Apaf-1
Caspase-9
Noxa
Caspase-3
Death???
IAPs
Inhibitor-of-Apoptosis Proteins
497
1
XIAP
BIR1
BIR2
BIR3
RING
236
BIR3
ILP-2
Livin
RING
298
BIR
RING
604
cIAP-1
BIR1
BIR2
BIR3
CARD
RING
cIAP-2
BIR1
BIR2
BIR3
CARD
RING
618
1403
NAIP
BRUCE
Survivin
BIR2
BIR1
BIR
BIR3
4845
UBC
142
BIR
S. Bratton
IAPs
IAPs function as critical prosurvival molecules
*IAPs (especially XIAP) are generally overexpreessed
in multiple types of cancer cells
apoptotic
stimulation
Smac (second mitochondrial
activator of caspases)
Shi, Y. Mol. Cell, 9, 459-470, 2002
Caspases may have apoptosis-independent/physiological
functions
Drosophila caspases (Cell 126, 583-596, 2006)
--- Non-apoptotic caspase activity is required for proper sensory organ precursor development.
Caspase-1
--- Promotes cell survival upon toxin challenge possibly through membrane repair (Cell 126,
1135-1145, 2006).
--- Connects immunity (host defense) and apoptotic machinery (Bruey J-M., Cell 129, 45-56, 2007).
Caspase-3
--- Caspase-3 activation is involved in cancer cell invasion (Cancer Res, 65, 9121-9125, 2005).
Caspase-8 (Nature 419, 395, 2002; Genes & Dev 17, 883, 2003; Science 307, 1465, 2005)
--- Targeted disruption of casp-8 in T-cell lineage leads to marked decrease in peripheral T-cells
and impaired T-cell response ex vivo to activation stimuli.
--- Casp-8 ablation protected thymocytes and activated T-cells from CD95 ligation but not
anti-CD3-induced apoptosis, or apoptosis activated by the mitochondrial stimuli
--- Casp-8 mutant mice were unable to mount an immune response to viral infection
--- Casp-8 required for NF-kB activation by antigen receptor
--- Casp-8 promotes cell motility and calpain activity (Cancer Res., 66, 4273-4278, 2006)
--- However, loss of caspase-8 potentiates neuroblastoma metastasis (Nature 439, 95-99, 2006)
--- Caspase-8 interacts with androgen receptor (AR) and regulates AR-driven gene expression
(Qi W et al., EMBO J. 26, 65-75, 2007)
Prosurvival mechanisms
I. Endogenous prosurvival mechanisms
II. Autophagy
III. Induced prosurivival mechanisms
Autophagy
*First recognized under EM early 1960s.
*Also called macroautophagy: Self-eating to survive.
*A unique form of membrane trafficking in which membrane compartments (autophagosomes) engulf
both organelles and cytosolic macromolecules and deliver them to the lysosome for degradation.
Mizushima N. CDD, 12, 1535-1541, 2005
Autophagy
*Molecular insights first came from studies in yeast, which expresses >20 autophagy-related genes
(ATGs).
*In yeast, two ubiquitin-like conjugation systems, Atg8-PE (phosphatidylethanolamine; Atg8 = LC3 in
mammals) and Atg12-Atg5, are involved in forming the initial autophagic vesicles.
Yorimitsu and Klionsky. CDD, 12, 1542-1552, 2005
Autophagosome nucleation requires a complex containing
Atg6 (Beclin 1 in mammals) that recruits the class III PI3K
VPS34 to generate PI3P. Expansion of autophagosome membranes
involves Atg12 and Atg8 (or LC3).
Autophagy: A Major Prosurvival Mechanism
- In yeast, the primary function of autophagy is to maintain viability during starvation.
- In mice, autophagy supports newborn survival at birth - in the absence of Atg5, mice do not survive
neonatal starvation and die within a day after birth (Kuma et al., Nature 432, 1032, 2004).
-In fact, autophagy-defective embryos fail to reach the blastocyct stage (Science 321:117, 2008).
- In IL-3-dependent lymphocytes, cells without Bax and Bak survive for weeks due to autophagy
- knockdown of Atg7 significantly reduces this survival (Lum et al., Cell 120, 237-48, 2005).
- Autophagy-supported cell survival probably results from the products of self-degradation in
lysosomes (e.g., amino acids), which support the TCA cycle to generate ATP.
- Animal cells exhibit low levels of constitutive autophagy - a defense mechanism to eliminate
aggregation-prone proteins (inclusion bodies) in, e.g., neurons and liver cells (absence of Atg5
and Atg7 in mouse brain induces neurodegenerative diseases (Hara T et al., Nature 441, 885,
2006; Komatsu M et al., Nature 441, 880, 2006).
-Autophagy sequesters and kills the invading pathogens (Nakagawa I et al., Science 306, 1037, 2004).
- Autophagy helps eliminate superfluous organelles (peroxisomes, mito., ER, etc) and maintain
cellular homeostasis).
- Defects in autophagy have been linked to liver diseases, neurodegeneration, Crohn’s disease, aging,
metabolic syndrome and cancer
*Indeed, autophagy functions as prosurvival mechanisms in cancer cells and mediate their resistance
to therapeutics (Albedin MJ, et al., CDD, 14, 500-510, 2007; Katayama M et al., CDD, 14,
548-558, 2007; Amaravadi RK et al., JCI, 117:326-336, 2007).
Autophagy & Metabolism
Rabinowitz JD & White E. Autophagy & Metabolism. Science 330: 1344-1348, 2010.
Autophagy & Metabolism
Rabinowitz JD & White E. Autophagy & Metabolism. Science 330: 1344-1348, 2010.
Autophagy (Type II PCD) & Apoptosis (Type I PCD)
*Autophagic cell death: nonapoptotic PCD (type II)
*A large number of autophagosomes are present in dying cells: Cause-and-effect relationship unclear
*The first example: mouse L929 fibroblasts treated with zVAD  cells undergo nonapoptotic cell
death  knockdown of Atg7 or Beclin-1 reduced cell death + zVAD treatment somehow
caused degradation of catalase resulting in increased ROS production followed by cell
death (Yu L et al., Science 304, 1500-1502, 2004).
*In Bax/Bak-DKO MEFs treated with chemicals such as etoposide cells die through ‘autophagy’ cells do not die when Atg5 and Beclin-1 were silenced (Shimizu et al., NCB 6, 1221-1228,
2004).
*’Autophagy’ might act upstream of apoptosis: apoptosis of CD4 T cells by HIV envelope glycoproteins inhibited when Beclin-1 and Atg7 were silenced (Espert L et al., JCI, 116,
2161-72, 2006).
*Problems: 1) Autophagy is a morphological and descriptive term;
2) The Atg proteins may have autophagy-unrelated functions;
3) Truncated Atg5 can bind Bcl-xL in mitochondria and causes apoptosis (Yousefi S
et al., NCB 8, 1124-32, 2006).
4) Bcl-2 negatively regulates autophagy by binding to Beclin-1 (Pattingre S et al., Cell
122, 927-39, 2005).
5) ‘Autophagic cell death’ might be caused by excessive nonselective autophagy itself.
Levine B and Yuan J. JCI, 115, 2679-2688, 2005
Yoshimori T et al., Cell 128, 833-836, 2007
Yoshimori T et al., Cell 128, 833-836, 2007
Qu X et al., Cell 128, 931-46, 2007
Prosurvival mechanisms
I. Endogenous prosurvival mechanisms
II. Autophagy
III. Induced prosurivival mechanisms
Apoptotic stimulation
Cell
Survival
Death
Two
Apoptotic
Pathways
Induction of prosurvival
molecules during apoptosis induction
Liu et al., ONCOGENE 2005
FasL (EMBO J. 23, 3175, 2004)
--- FasL promotes tumor cell migration and invasion.
Micheau O. & Tschopp J. Cell 114, 181-190, 2003
Barnhart & Peter, Cell 114, 148-150, 2003
Algeciras-Schimnich et al., PNAS 100, 11445, 2003
Barnhart et al., EMBO J. 23, 3175-3185, 2004
Prosurvival mechanisms
I. Endogenous prosurvival mechanisms
II. Autophagy
III. Induced prosurivival mechanisms
Apoptotic stimulation
Cell
Survival
Death