Oncology exam 2
Lectures and book Molecular Biology of Cancer
Chapter 7 till 14 WITHOUT SEMINARS
Gezondheid en Leven & Biomedical Sciences
Inhoud
Lecture 1 & Chapter 7 – Apoptosis ................................................................................................................................... 2
Lecture 2 & Chapter 7 – Apoptosis and Cancer ................................................................................................................ 6
Lecture 3 & Chapter 8 –Stem cells, Differentiation and Cancer ....................................................................................... 8
Lecture 4 & Chapter 9 – Metastasis ................................................................................................................................ 13
Lecture 5 & Chapter 14 – Technology, Targeted Drug and Diagnostic Development .................................................... 17
Lecture 6 & Chapter 10 – Angiogenesis .......................................................................................................................... 19
Lecture 7 & Chapter 2,11 – Nutrients, Hormones and Gene Interactions ..................................................................... 23
Lecture 8 & Chapter 12,13 – Inflammation, Immunotherapy and cancer...................................................................... 27
Lecture 9 & Chapter 13 – Infection agents and Cancer .................................................................................................. 36
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,Lecture 1 & Chapter 7 – Apoptosis
Apoptosis is the regulated and orderly destruction of a cell through a genetically encoded process known as
programmed cell death (PCD). Apoptosis is a type of cell suicide that is intrinsic to the cell, a trigger can activate it.
25 million cells die of apoptosis per second, so this is a very common process. It is an organized, neat and tidy
process, leaving behind little evidence of the preexisting cell. The cell undergoing apoptosis is swept clean during
phagocytosis by macrophages, neighboring cells can recognize molecular flags (due to the recognition of
phosphatidyl serine which flips to the outside of the membrane of the dying cells).
Function of apoptosis
Apoptosis is an ATP-dependent process and is physiological in many organs. This process is active during
developmental morphogenesis in embryogenesis, of which the development of toes and fingers is an example; it
controls cell numbers; it removes damaged cells; it has a negative and positive selection effect on the selection of
lymphocytes and it creates the cytotoxic effect of radio- and chemotherapy. Apoptosis is part of the mechanisms
which control the net number of cells in the body, therefore it is a crucial tumor suppressor mechanism.
Apoptosis and necrosis
The most important difference is that apoptosis does not give an inflammation while necrosis does. Necrosis attacks
cytokines due to the cell lysis, by which the cell contents are released in the surrounding tissue, and thereby induces
inflammation. Necrosis be induced by external factors such as infections and trauma, while apoptosis is mostly
induced intrinsically or due to extracellular signals.
Apoptosis Necrosis
Cell shrinkage Cell swells
Membrane blebbing Membranes become leaky
Organelles stay intact Organelles are damaged
Apoptotic bodies are formed Cell lyses
Chromatin condensation and fragmentation Chromatin damaged
No inflammation is induced Inflammation is induced
Process of apoptosis changes in morphology changes is shown: after the cell
shrinkages, the outside of the membrane forms blebs this eventually results in
apoptotic bodies which can be recognized and cleaned up by macrophages.
Induction of apoptosis, used in cancer therapy
This is used in, for example, T-cell leukemia. Chemo can give rise to apoptotic
bodies, which can be recognized by the condensation of the spheres. This
chance in morphology can be used to test whether the tumor cells are sensitive
to chemotherapy.
When does apoptosis normally occur?:
- Programmed apoptosis, for example during embryogenesis
- Due to the loss of growth factors or cell adhesions, this is seen a lot in the gut
- Activation of death receptors or the TNFR family
- Activation of T- and B cell antigen receptors
- Due to programming of CTLs
- Due to DNA damage caused by irradiation or chemo
- Due to stress conditions on the cell
Molecular mechanisms of apoptosis
Apoptosis leads to mitochondrial changes, which activate the caspase family, proteolytic cleavage of structural and
functional proteins. This gives a loss of function and this leads to an apoptosis morphology. Caspases are cysteine-
rich aspartate proteases, which are synthesized as zymogens (= are considered not active, but show 2% activity) this
form is called pro-caspase. There is a requirement for an aspartic acid (an amino acid) at the P1 position, at this
position the zymogens can be cleaved. 14 family members are known, of which caspase 2,3,6,7,8,9 and 10 are
involved in apoptosis. This can be divided into 3 groups of caspases:
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,Initiator caspases, which can activate effector caspases by cleavage: 2,8,9 and 10
Effector caspases, which can activate apoptosis: 3,6 and 7
Inflammatory caspases, involved in neurodegenerative diseases: 1,4 and 5
Between the initiator caspases and effector caspases a caspase cascade is formed. This happens via proteolytic
cleavage of the pro-caspases, leading to following activation until one of the effector caspases is activated.
Caspase activation
Caspase proteins exist of a pro-domain, a large subunit domain, a spacer and a
small subunit domain. The spacer domain combines the large and small
subunit domain. After signaling of a trigger the spacer is being cut out, this
causes a direct binding between the large and small subunit domain. Secondly,
the pre-domain is being cut out, leaving the small and large subunit domain
behind, this is the fully active caspase.
4 ways to induce apoptosis
1. Intrinsic/stress-induced or mitochondrial apoptosis pathway
2. Extrinsic/death receptor mediated apoptosis pathway
➔ These two are investigated most for cancer and are discussed in the lecture
3. Granzyme B mediated apoptosis, which is mediated in inflammatory diseases
4. ER mediated apoptosis, which is induced in neurodegenerative diseases.
The 2 important apoptotic pathways
The overview shows the combined pathways of the extrinsic and
intrinsic apoptosis, which will be discussed in detail. The result of both
pathways is the breakdown of the cell. This is caused by the
proteolysis of target proteins, which includes nuclear Lamins,
cytoskeletal proteins like actine, intermediate filaments which make
up the cell structure, specific kinases for cell signaling and other
enzymes like caspase-activated DNases (= which cuts DNA between
nucleosomes and generates a DNA ladder that can be detected). The
blue molecules are important regulators of apoptosis and will be
discussed later on.
Regulators of the apoptosis pathways are:
BCL-2 family:
- Inhibitors: BCL-2, BCL-xl
- Activators: Bax, Bak,
- Activators containing the BH3-only members of the BCL-2 family: Bid, BAD, NOXA, PUMA
C-flip:
- C-flip short: inhibitor of activation of caspase 8
- C-flip long: controversial, inhibition or inducer of apoptosis
IAPs: inhibitors of apoptosis proteins
- cIAP1,2, XIAP
Extrinsic apoptosis
This pathway is activated by the binding of extracellular ligands like, TRAIL, FAS, TNF-a, which can bind to the death
receptor. TNF is a soluble factor, while FAS ligand is bound to the plasma membrane of neighboring cells. When the
ligands bind to the receptor it undergoes a conformational change, due which the receptors form a homotrimer in
order to transduce the signal into the cell. The homotrimer is formed in the cytoplasm and has intracellular adaptor
proteins, like FADD and TRADD, these proteins transduce the signal from the receptor to the caspases. The adaptors
recruit pro-caspase-8 via the death effector domains (DED). All pro-caspase 8 molecules are now in close proximity,
and because they still have the 2% activity, they cleave and activate each other. Caspase 8 then initiates a cascade of
caspase activation, which eventually leads to the cleavage of specific protein targets and results in apoptosis.
➔ DISC complex is involved: death ligands, receptors, adaptors and initiator caspase
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,This process can be inhibited by C-Flip, a catalytically inactive caspase-8 homolog. C-Flip can bind to FADD or DED
and this inhibits caspase-8 recruitment and activation.
Caspase 8 needs to dimerize in order to be activated. There are two ways of
inhibiting this activation using C-flips.
- Short C-flip does not have a caspase domain, due which caspase 8 is
not forming a dimer.
- long C-flip is more difficult, discussion whether this form is anti-
apoptotic or pro-apoptotic is going on. In the lecture we concluded
that the effect is dependent of the concentration of the long C-flip.
This molecule does have a caspase domain; therefore, it can form a
dimer. Nevertheless, the capacity of the dimer is reduced, due to
another domain on the long C-flip. But, short C-flip has a higher
reducing capacity.
Intrinsic pathway
The intrinsic pathway does not depend on external stimuli, stimuli like DNA damage,
oxidative stress, chemotherapy and oncogene activation can activate this pathway. The
internal stress is noticed by P53, which is stabilized due to the signals. P53, activates pro-
apoptotic Bcl-2 proteins, Bax and Bak. This happens in combination with activation of
BH3-only proteins, these proteins form a transient interaction with Bax and thereby
induce a conformational change. This change helps to translocate Bax from the cytoplasm
to the nucleus. Bax dimerizes and later oligomerizes, by which it inserts into the outer
mitochondrial membrane. The intermembrane space is a supply cabinet of the apoptotic
mediators. The pro-apoptotic proteins cause a release of Cytochrome C from the
mitochondria. The release of BCL-2 pro-apoptotic members is therefore called
mitochondrial outer membrane permeabilization (MOMP). Cytochrome C, Apaf-1, ATP
and procaspase 9 form an apoptosome. The binding of Cytochrome C to Apaf-1 triggers
the formation of a wheel-like heptameric complex, that recruits pro-caspase-9. The
complex activates caspase 9 which again induces a caspase cascade.
Bcl-2 family
This is a large family of proteins with sequence homology.
These form heterodimers or homodimers through shared
domains like Bcl-2 homology domain (BH3), of which every
member has at least 1 and most even have 3 or 4. These
molecules regulate the mitochondrial permeabilization. They
can be divided in anti and pro apoptotic groups. Between the
pro-apoptotic group 2 groups are possible: the normal
morphology group and the BH3 only group.
➔ BH3 only: functions via induction of the pro-apoptotic members (=
activators) or inhibits the anti-apoptotic BCL2 proteins (sensitizers).
Upstream signals inducing apoptosis (= via the mitochondria)
When bax and bak are translocated to the mitochondria, the Bcl-2 family decides
which effect is given: anti or pro apoptotic. There is a balance between the
different signals, both the pro-apoptotic signals as the anti-apoptotic signals are
considered. This is caused by the protein-protein interactions, and the ratio of
activity determines the outcome:
➔ BIM, BID and PUMA are non-selective regulators of the Bcl-2 family and of
MCL1 and AL (the last two are cell proliferating proteins)
➔ BAD is a selective regulator for the Bcl-2 family
➔ NOXA is selective for MCL1 & AL.
In normal healthy cells, bak and bax are present and therefore they can form a
heterodimer when damage is sensed. In lymphoma’s Bcl-2 is overexpressed due to a
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, translocation. Therefore, dimers are formed in combination with Bcl-2 due which the heterodimer does not have any
activity.
Bim is also present; this protein can bind Bcl-2 and thereby blocks its activity. Therefore, Bax and Bak can still form a
heterodimer. Bim is a pro-apoptotic protein.
Downstream effects of apoptosis: IAP family members
The IAP family members, fully named: inhibitors of apoptosis proteins, can be
divided into three groups, based on the different side groups like BIR groups
(Baculovirus IAP repeat).
- cIAP1/2: mostly inhibits downstream of the extrinsic pathway.
- Surviving: mostly inhibits downstream of the intrinsic pathway.
- XIAP: binds directly to, and inhibits, the activity of caspase 3 and 7. It
also inhibits caspase-9 but does so by binding to monomeric caspase-
9 and locking the active site for conformation.
Other downstream regulators of the apoptosis pathway are: SMAC, second mitochondria-derived activator, and
DIABLO. These molecules are also released from the mitochondria and these can
eliminate the inhibition which is caused by IAPs.
Crosstalk between the extrinsic and intrinsic pathway
This forms an important connection between the two apoptosis inducting pathways, this
is an important effect to consider in selecting cancer therapy. This connection is formed
by the conversion at the level of the activation of downstream caspases.
Example: Caspase 8, from the extrinsic pathway, can cut and activate Bid, by which Bax
and Bak are activated, which are part of the intrinsic pathway. This causes the
connection between the pathways, by which the extrinsic pathway can activate the
intrinsic pathway.
P53 and apoptosis
P53 induces apoptosis in response to DNA damage and cellular stress. This can lead to transcription-dependent and
independent activation.
- Transcription dependent takes place in the nucleus: p53 induces expression of genes that code for death
receptors and pro-apoptotic members of the Bcl-2 family, for example FAS, BAX and BAC. These genes
contain a consensus p53-binding site in their promotor regions. Thereby it represses the expression of anti-
apoptotic factors, such as BCL-2 and IAPs. And p53 takes care of the upregulation of Puma expression (=
necessary for the induction of apoptosis. Puma is a protein which interacts with Bcl-2, which is thereby
inhibited (get rid of the blockage of apoptosis)).
- Transcription independent takes place in the cytoplasm: p53 activation of Bax in the cytoplasm and
subsequent release of cytochrome C and caspase activation leads to apoptosis. This is caused by PUMA,
because this protein acts as an enabler to release P53 from BCL-xl in the cytoplasm, so that p53 can activate
BAX.
➔ PUMA links the functions of the dependent and independent means.
Detection of apoptosis:
- Using the morphology of the cells, like shown above. This is the golden standard
- Checking for DNA damage
- Viability studies: which compares the number of cells alive with the number of died cells.
- RT-PCR, next generation sequencing: checking the whole exome or genome
- Caspase activation
➔ Western blot, immunoprecipitation (used a lot)
Proteins, like BCL-xl, p53 or PUMA can be identified by using antibodies against those proteins.
➔ Immunohistochemistry
➔ FACS
➔ Fluorescence
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