F.

05 MOLECULAR BASIS OF ONCOGENESIS  Pro-apoptotic family

(PART 2) o don‟t have any BH 4
Dr. Adefuin| April 26, 2018 o Bax, Bak, Bok; have 3 BH domains (BH 1, 2 3)

 “BH3 only”proapototic proteins:

OUTLINE o Bik, Blk, Hrk, Bnip3, Bid, Bad, Bim, Puma, Noxa and
I. APOPTOSIS Bmf
1. Morphological Events
2. Apoptosis vs. Necrosis  Remember Bid, Box, Bak, & bcl2
3. Caspases (Know which are anti-apoptotic and pro-apoptotic)
o Activation
o Targets
4. Scheme of Apoptosis
o Pathways
5. Regulation
II. ANGIOGENESIS
A. Types
B. Angiogenic switch
C. Tumor blood vessels
D. Steps
III. METASTASIS C. CASPASES

I. APOPTOSIS

If the problem cannot be regulated in the cell cycle, the cell will
instead go to apoptosis or programmed cell death.
1. derived from a Greek word meaning “dropping off” or
“falling off” of petals from the trees
2. programmed cell death

A. MORPHOLOGICAL EVENTS
a) Chromatin condensation
b) DNA fragmentation
c) Nuclear breakdown
d) Membrane "blebbing"
e) Cell fragmentation (forming apoptotic bodies)
f) Phagocytosis
B. APOPTOSIS vs. NECROSIS
Apoptosis
- Internal / external signals
- Energy dependent
- Single cells
- The cell shrinks and is
engulfed by macrophages or
neighboring cells
- Normally, no inflammatory
reaction
 ICE subfamily - usually involved in inflammation
-Caspase 13, 5
 the CED-3 subfamily were first studied in nematodes
Necrosis o involved in apoptosis
- Ischemia/ toxins/ rad„n o Caspases 7, 3, 6
- Non-energy dependent o Caspases 8, 10 2, 9
- Groups of cells o With caspase recruitment domain and death
- The cells swell and release effector domain (like policemen)
their content into the ‒ Activated via proteolytic cleavage
surroundings and the ‒ Target several proteins
circulation
- Marked inflammatory
reaction

Apoptosis deals with our bcl2 family

I. Anti-apoptotic
a. bcl-2, bcl-X, bcl-w
b. they have BH (BCL homologue domains) 1, 2 ,3, 4 and
membrane anchors (TM domain)
c. all have 4 BH domains involved in regulation and pore formation

‒ Caspases are very powerful in effective cell death

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a) ACTIVATION OF CASPASES
Death Initiator Signaling Complex (DISC):
Procaspase (inactive form) -> cleavage -> activation to  Ligand receptor DD
tetramer Caspase  FAS associated DD
 initiator caspases
b) TARGETS OF CASPASES o Activated caspase 8 and 10
 Action: It will activate 3, 6 ,7 (executioner caspases)
1. Protein kinases Results: act on their target –> apoptosis
o inactivation of FAK (focal adhesion kinase) ->
disruption of cell adhesion ->detachment of apoptotic
cell from neighboring cells
2. Lamins
o in inner lining of nuclear envelope; results in cleavage
o disassembly of nuclear lamina & shrinkage of nucleus
3. Proteins required for cell structure
1. cleavage of intermediate filaments, actin, gelsolin ->
changes in cell shape (remember in apoptosis they
usually shrink)
4. Endonuclease (CAD)
2. caspase-activated DNase -> attacks DNA, severing it
into fragments (DNA fragmentation)
5. Enzymes involved in DNA repair

D. SCHEME OF APOPTOSIS
II. The overall process of apoptosis is complex and it is
tightly regulated. It includes proteins that act as
receptors and, adapters, procaspases and caspases, and
pro- and antiapoptotic factors.
III. There are extrinsic and intrinsic pathways, with the
mitochondria being important participants in the
intrinsic pathway.
IV. There are two major events in apoptosis: the death
receptor (extrinsic) pathway and the mitochondrial
(intrinsic) pathway.

TWO PATHWAYS IN APOPTOSIS

1. EXTRINSIC PATHWAY  When death signals bind to receptors what happens is your
 dependent on death signals (aka “death ligands”): cell recruit death domains (DD), it will recruit FADD(FAS
 Fas ligand (“CD95”), TNF, DR4, DR5 associated death domains)
 External signals initiating apoptosis  FADD is an adaptor protein which will activate your
 outside the cell or on the membrane are receptors initiator caspases (8 and 10).
 The ligand receptor death domain, adaptor protein FADD,
Death signals / Ligands Receptor and activated caspases is your DISC (death induced
CD95/ FasL FAS signaling complex).
TNF (tumor necrosis factor) TNF-R o it will activate your executioner caspases (6, 3 and 7).
DR4, DR5 and TRAIL (TNF TRAIL-R o Caspase-3 (an effector) is activated. It digests important
related apoptosis inducing structural proteins such as, various cytoskeletal
ligand) proteins, and enzymes involved in DNA repair, causing
cell death.
 Extrinsic pathway have initiator caspasesand executioner
STEPS IN EXTRINSIC PATHWAY (Refer to Figure)
caspases which directly causes target apoptosis.
1. Ligand binds to receptor (ex. DR4, DR5 binds to TRAIL-R)
2. Recruits death domains (DD)
2.INTRINSIC PATHWAY
3. Recruit FAS associated DD then changes in conformation
 Apoptosis via intrinsic pathway can be triggered by
4. Activate Initiator caspases: Caspase 8, 10
presence of internal stimuli:
5. Activate Executioner caspases: Caspase 3, 6, 7 which
- DNA damage
would now act on the target to cause,
- Oncogene induced proliferation
6. APOPTOSIS
- Loss of attachment to extracellular membrane
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- Chemotherapy
- Radiation therapy
 Can be initiated by exposure to reactive oxygen species,
DNA damage and other stimuli.
 Not involved with TNF and DISC related families
 Involved with the mitochondria
 bind to outer membrane of mitochondria - > APAF-1
 Pro-apoptotic proteins,Bak and Baxare situated near at
the membrane of the mitochondria, and are dimerized to
either Bax-Bax or Bak-Bak or Bak-Bax to become active
 Pro apoptotic proteins Bax, Bak, Bid, etc. oligomerize and
bind to the outer mitochondrial membrane, creating
pores
 Mitochondria becomes permeable via the action of these
pro-apoptotic dimerized proteins; inducing
mitochondrial outer membrane permeabilization
or MOMP
 Cytochrome C, coupled with ApAF (Apoptosis activating
Factor) will be released from the mitochondria due to
MOMP and will activate caspase 9 (initiator caspase)
 In the cytoplasm, cytochrome c interacts with APAF-1,
procaspase-9, and ATP to form a multi-protein
complex known as an apoptosome.
 Apoptosome
 APAF-1 + Caspase 9
 will activate the executioner caspases (3, 6, &
7)
 Executioner caspases will target nuclear and
cytoplasmic elements -> APOPTOSIS
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BID
 the bridge between intrinsic and extrinsic pathway
 both pathways can be happening at the same time
because of the caspase 8 action on Bid (pro apoptotic
protein)
 When Bid is truncated via caspase 8 it will result to
functions of Bid:
1. It will cause the Bax translocation to the
mitochondria
2. Bax, Bak oligomerization- cause Bax, Bak and
other pro-apoptotic proteins to dimerize,
resulting to pore formation (MOMP) (go to
intrinsic pathway)
3. Cytochrome c release – It will go to the
membrane of the mitochondria that will cause
the release of cytochrome c ->couples with
APAF 1 -> apoptosome –>executioner
caspases ->apoptosis
4. It will inactivate the anti-apoptotic bcl family
PAREDES|ROMERO|GARCIA, K.
E. REGULATION OF APOPTOSIS -DNA damage due to UV rays, gamma rays,
 Pro-apoptotic (prodeath) chemotherapy
 Anti-apoptotic (prolife) -ribonucleotide depletion
 p53-downstream activates caspase 2 –>caspase 2 will -telomere shortening
activate executioner caspases (3, 6, 7) to cause
3. center of everything
apoptosis
4. Inactivated: by mdm2
5. When Activated -> cell cycle arrest ->cell can repair
 Inhibitors of apoptosis proteins (IAP)
before proceeding to the cell cycle.
 released from mitochondria
6. If damage is irrepairable, induces apoptosis
 Inhibit caspase 9, 3 and 7
7. In the cell cycle, it causes the release of P16
 Inihibit 1 initiator caspase and 2 executioner 8. P16 -inhibitor of CDKs.
caspases
 Prolife / survival
o In oncogenes checkpoint
o P14 will inhibit mdm2,
 SMAC (Second Mitochondrial Activator of Caspases) o Inhibition of mdm2 will activate p53,
coupled with DIABLO o Resulting to transcriptional activation of p53
o it inhibits IAP
responsive genes
o prodeath / apoptosis  NOXA, PUMA, etc and all the pro
apoptotic proteins
 Inhibition of initiators and executioners of apoptosis o p21, cannot be induced unless p53 is activated
o no apoptosis occurs
o IAP (inhibitor of apoptosis protein)

 pro-apoptotic proteins (Bax, Bak, etc.) by themselves

can inhibit anti-apoptotic proteins (Bcl family)
 anti-apoptotic proteins by themselves can inhibit
oligomerization of pro-apoptotic proteins and the
release of cytochrome C
 TRAF 2 - part of death domain
 NFkB
o Released from Death Domains (DD)
o involved in inflammation and apoptosis
o activates anti-apoptic protein, IAP and FLIP(Fliase
like protein)
 FLIP inhibits caspase 8(initiator caspase)
 FLIP inhibits the conversion of procaspase-8
to its active form.
 Prodeath
 PI3k (PI3 kinase) II. ANGIOGENESIS
o downstream of Ras (transcription factor)
o Stimulates anti-apoptotic protein -normal (for wound healing) but also has a role in
 AKT malignancies.
o Downstream of PI3k Tumor- cannot grow beyond 2 mm3 . it needs nutrients and
o inhibits pro apoptotic proteins oxygen. Without these, it cannot grow. So it produces more
o activates NFkB and MDM2 blood vessels to access more nutrients and oxygen.
 MDM2 o Vasculogenesis
o Holds p53 in captivity, preventing it from o formation of vascular structures from
translocation circulating or tissue-resident endothelial stem
 Ras-Raf –Mek pathway cells (from angioblast)
o In cancer, this pathway is notorious so we have o Which proliferate into de novo endothelial
Ras inhibitors and PI3k inihibitors cells
 P10 (phosphatase-tensin in chromosome 10) o Arteriogenesis
o Inihibits AKT
o formation of medium sized blood vessels
possessing tunica media plus adventitia
p53 INDUCTION o Angiogenesis
- Involve in cell cycle and apoptosis o formation of new blood vessels (formation of
1. guardian of the genome thin-walled endothelium-lined structure with or
2. Induced in the presence of: without muscular smooth muscle wall and
- hypoxia

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 pericytes (fibrocytes)) from pre-existing D. STEPS IN ANGIOGENESIS
vessels.
o They form sprouts
o Relates to repair mechanism and in spread of
cancer

A. TWO TYPES OF ANGIOGENESIS

1. Sprouting angiogenesis – more common in cancer

spread
2. Intussusceptive or splitting angiogenesis – blood
vessels then splits into 2 branches or more
B. ANGIOGENIC SWITCH

o Phase in tumor development when proangiogenic

factors > anti-angiogenic factors to have balance
 Pro-angiogenic: VEGF(vascular endothelial
growth factor), bFGF(basal fibroblast growth
factor), TGF (transforming growth factor), EGF
(epidermal growth factor), TNF (Tumor
necrosis factor), Angiogenin, IL-8, Angiopoietin
 Anti-angiogenic: Angiostatin, Endostatin,
Vasostatin, IFN (Interferon), Prolactin, IL-12,
Tumstatin
*IF Pro-angiogenic > Anti-angiogenic = Angiogenesis

C. TUMOR BLOOD VESSELS

o Abnormal architecture
o Tortuous, dilated, uneven diameter, excessive
branching, shunting, are “monsters”
o Vessel walls have: numerous openings, widened
interendothelial junctions, absent, or discontinuous
basement membrane
o Lack perivascular cells (pericytes, smooth muscle cells)
which regulate flow in response to tissue metabolic
needs
(endothelial cells + tumor cells)
o Vascular lining= Endothelial Cells (highly proliferative) +
tumor cells
o Sometimes, tumor cells themselves mimic the
endothelial cells and form part of the new blood vessels.
o There is variable blood flow: have areas which are
hypoxemic and acidotic
o resistant to hypoxemia-induced apoptosis
o that‟s why they can evade apoptosis
o If they are resistant to that, then they could
continue sprouting and growing new blood
vessels
o End Result: Endothelial Cell Proliferation and survival.
Cancer cell grows.
1. Stimulation of endothelial cells by growth factors (VEGFs)
2. Degradation of extracellular matrix by proteases->degradation
of basement membrane -> escape of endothelial cells
3. Proliferation of endothelial cells and migration into the tumor
4. Formation of new capillary tubes – cancer cells can also
migrate and form part of new vessels
*Tumor grows- Hypoxia Induced Factor (HIF) usually silent, is
activated  results in release of growth factors
VEGFA- main growth factor involved in angiogenesis
VEGF-binds to its receptors VEGFR2 and tumors cause the
Elaboration of growth factors and participate in angiogenesis.
When VEGF binds to VEGFR2:
Ang2 + Tie 2= pro-angiogenesis -> Tie2 + Integrins -> they act
on the extracellular matrix (degradation) and cause migration of
endothelial cells, forming a new sprout
Ang1 + Ang2
Ang1 + Tie 2= anti-angiogenesis
5. Highlight: vascular mimicry of the tumor cells
o Tumor cells form part of the vessel wall
o Tumor have now access to oxygen, and to all its
nutients, it can now grow
o In region of hypoxia, you have your HIF factor (Hypoxia
Induced Factor) -> this will stimulate your growth
factors (VEGF, VEGF) -> Growth Factors will bind to
their receptors, so what happens?

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 o Ang2 will interact with Tie 2 1. Tumor cell
 Area in that part is destabilized o It elaborates snail and twists
o ANG 1 – is anti angiogenesis o what happens is you have new integrins expressed, this
o Your avβ3, avβ5 and a5β1 will act on your ECM
o Eventually what we end up with are leaky vessels,
dilated tumor vessels and even your tumor may form
part of the vessel walls, that way they grow….ok? 
o This has to do with these factors, VEGFR,
VEGF, Tie 2, Ang2,Ang 1 and avBeta family

III. METASTASIS

Major Features of Tissue Invasion

o Cell adhesion to basement membrane then destroy it
o Local proteolysis of the membrane
o Movement of cell thru the tear/ rent in the membrane
time you expressed N-cadherin instead of of E-cadherin
and the ECM (ability to survive in new
o There will be EMT-> Epithelial to messenchymal
microenvironment)
transition
o Only known receptor: Met-HGF
o Normal epithelial phenotype now, goes into your
o Rate limiting step: ability for tumor cells to survive
messenchymal phenotype
and expand in the novel microenvironment of the
metastatic state . Some cells die when they travel
2. Tumor associated fibroblast and Tumor associated
because they are unable to survive in the new
macrophages elaborate:
environment. If they are able to survive ->
METASTASIS a. MMP(matrix metanoprotease), cytokines, and
growth factors
 The problem is, the cancer cells go to the
b. which eventually lead to expression of your
metastatic site, and would they be able to survive
TGF-B(transforming growth factor B) and your
there for life
HGF (hepatic growth factors)
1. Elaborating of growth hormone (P-cadherin and N-
cadherin)
P-Cadherin- involved in adherence junctions. Cells are TGF-B -> TGF-B receptor intumor cell
tight and non-mobile. HGF -> C-MET receptor in tumor cell
During metastasis: P-cadherin is downregulated by factors that *HGF- only known ligand that binds to C-MET
induce metastasis. These interactions cause the increase in Snail and
N-cadherin upregulated-> loosening of cells-> increase in snail Twist
and twist

Epithelial to mescenchymal transition

 Central to metastasis
 Results in loose and mobile cells leading to metastasis

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For further understanding refer to Harrison‟s Internal Med book:
Part 7 Oncology and Hematology (Dra. Adefuin‟s reference on
Molecular Biology of Oncogenes topics)

REMEMBER(bonus points): SAN PAOLO BOTTLE OF

WATER/SAN PAOLO PURIFIED SPRING WATER!!!
(official water of Notredame de Chartres Hospital – sabi
ni doc )

REFERENCES for the quiz: Lippincott and Harrison

CHECKPOINT

A. Normal blood vessel

B. Tumor blood vessel

1. Acidosis
2. Hierarchical blood flow
3. Absent or few pericytes
4. Normoxic
5. Increase permeability

A. Prodeath
B. Prolife

1. SMAC DIABLO
2. NFkB
3. PI3 kinase
4. AKT
5. IAP
TRUE OR FALSE:

1. Bax, Bak, Bok are pro-apoptotic proteins that do not have BH 4.

2. Extrinsic pathway of apoptosis involves the mitochondria.
3. Caspase 8 and 10 are initiator caspases that is part of your DISC.
4. MOMP is when you anti-apoptotic proteins dimerizenear the membrane of your mitochondria,
creating pores for cytochrome c to exit
5. BID is the bridge between intrinsic and extrinsic pathway
6. The guardian genome is activated by mdm2
7. Angiogenesis is formation of new blood vessels from existing vessels.
8. Intussuceptive angiogenesis is more common in cancer spread
9. Tumor blood vessels are tortous, and has an abnormal architecture
10. BES NATAPOS MO! Congrats!!

Key answers: BABAB; AABAB; TFTFTFTFT

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