APOPTOSIS & CANCER How is the initiator caspase frist activated in response to an apoptotic signal? The role of pRB in controlling transcription of genes The Role of p53: Guardian of the Genome Mutations in the PI3K/Akt/mTOR Pathway Drive Cancer Cells to Grow

1. APOPTOSIS & CANCER
1010–1011 cells in the human body die every day by apoptosis. Self reacting T lymphocytes are
eliminated by apoptosis. Apoptosis appears to be involved in neurodegenerative diseases
CED-3 gene product play a crucial role in the process of apoptosis in C.elegans.
In mammals Apoptosis associated intracellular proteases are called CASPASES.
These proteases have a cysteine at their active site and cleave their target proteins at specific aspartic
acids; they are therefore called caspases (c for cysteine and asp for aspartic acid).
In programmed cell death, the cell surface often bulges outward and, it breaks up into membrane-enclosed
fragments called apoptotic bodies. The surface of the cell or apoptotic bodies becomes chemically altered,
so that macrophage rapidly engulfs them, before they can spill their contents. In contrast to apoptosis,
Necrotic cells swell and burst, spilling their contents over their neighbors and eliciting an inflammatory
response.
Apoptosis vs Necrosis
CASPASES
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References:
1. Albert, Molecular –cell biology 6th ed.
2. Karp, cell & molecular biology 7th ed.
3. Cooper, the cell 8th ed.
4. Lodish, molecular cell biology 6th ed.

2. Target Proteins of Caspases:
FAK, PKB, PKC, and Raf1. Inactivation of FAK, for example, is presumed to disrupt cell adhesion.
Lamins - Cleavage leads to the disassembly of the nuclear lamina and shrinkage of the nucleus.
Proteins of the cytoskeleton- Cleavage of these proteins lead to changes in cell shape.
Caspase activated DNase (CAD), an endonuclease, attacks DNA, severing it into fragments.
During apoptosis, a phospholipid “scramblase” moves
phosphatidylserine molecules to the outer leaflet of the
plasma membrane where they are recognized as an “eat me”
signal by specialized macrophages.
This entire apoptotic program can be
executed in less than an hour. The
apoptotic bodies are recognized by the
presence of phosphatidylserine on their
surface. Phosphatidylserine is a
phospholipid that is normally present
only on the inner leaflet of the plasma
membrane. 2

3. Caspase activation during Apoptosis
Caspases are synthesized in the cell as inactive precursors and are activated only during apoptosis. There
are two major classes of apoptotic caspases: initiator caspases and executioner caspases.
The major function of the initiator caspases is to activate the executioner caspases. Once activated,
executioner caspases catalyze the widespread protein cleavage events that kill the cell.
Executioner caspases are initially formed as inactive dimers. Presence of
initiator caspase, the executioner caspase dimer undergoes an activating
conformational change. The executioner caspases then cleave a variety of
key proteins, leading to the controlled death of the cell.
An initiator caspase contains a protease domain in
its C-terminal region and a small protein interaction
domain near its N- terminus. It is initially made in
an inactive, monomeric form, sometimes called
procaspase. Apoptotic signals trigger the binding to
the adaptor proteins, initiator caspases dimerize and
activated, leading to cleavage of a specifc site in
their protease domains.
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4. The Extrinsic Pathway of Apoptosis
How is the initiator caspase frist activated in response to an
apoptotic signal?
The two best-understood activation mechanisms in
mammalian cells are called the extrinsic pathway and the
intrinsic, or mitochondrial, pathway .
Extracellular signal proteins binding to cell-surface death
receptors trigger the extrinsic pathway of apoptosis. Death
receptors contain an extracellular ligand-binding domain, a
single transmembrane domain, and an intracellular death
domain.The receptors are homotrimers and belong to the
tumor necrosis factor (TNF) receptor family, which includes a
receptor for TNF itself and the Fas death receptor.
When TNF binds to a TNF receptor (TNFR1), the activated
receptor binds two different cytoplasmic adaptor proteins
(TRADD and FADD-Fas associated death domain) and
procaspase-8 to form a multiprotein complex at the inner surface
of the plasma membrane .
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5. The Intrinsic Pathway of Apoptosis
Activation of the intrinsic pathway is regulated by members of the Bcl-2 family of
proteins, which are characterized by the presence of one or more BH domains.
Bcl-2 family members subdivided into 3 groups:
(1) Proapoptotic members ... promote apoptosis (e.g., Bax and Bak),
(2) Anti-apoptotic members (e.g., Bcl-xL, Bcl-w, and Bcl-2)
(3) BH3-only proteins.... promote apoptosis (e.g., Bid, Bad, Puma and Bim)
(So-named because they share only one small domain—the BH3 domain—with other Bcl-2 family members)
Internal stimuli, such as irreparable genetic damage, lack of oxygen (hypoxia),
extremely high concentrations of cytosolic Ca2+, viral infection, or severe
oxidative stress trigger apoptosis by the intrinsic pathway .
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6. The intrinsic (mitochondria-mediated)
pathway of apoptosis.
In mammalian cells, internal cell death signals
induce apoptosis as a result of damage to
mitochondria. When active, the proapoptotic
effector proteins Bax and Bak form oligomers
in the outer membrane of mitochondria,
resulting in the release of cytochrome c from
the intermembrane space. Release of
cytochrome c leads to the formation of
apoptosomes containing Apaf-1 and caspase-9
in which caspase-9 is activated initiator.
Caspase-9 then activates downstream caspases,
such as caspase-3, by proteolytic cleavage.
Under normal conditions of cell survival,
cytochrome c is localized to the mitochondrial
intermembrane space while Apaf-1 and caspase-9
are found in the cytosol, so caspase-9 remains
inactive.
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7. CANCER
THE CAUSES OF CANCER
DNA tumor viruses and RNA tumor viruses:
DNA viruses are... polyoma virus, simian virus 40 (SV40), adenovirus, and
herpes-like viruses.
Human papilloma virus (HPV) causes cervical cancer.An effective vaccine against
this virus is now available.
Epstein Barr virus– casuses Burkitt’s
lymphoma in areas where malaria is
common. Herpes virus (HHV-8),
Kaposi’s sarcoma.
Helicobacter pylori is responsible for
ulcers and gastric lymphomas
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8. THE GENETICS OF CANCER
The most common solid tumors—such as those of the breast, colon, prostate, and
lung—arise in epithelial tissue.
Leukaemia develops in rapidly dividing blood-forming tissues
Expression of telomerase have a tremendous growth advantage of cancer cells.
The Pap smear is a test for detecting precancerous cells in the epithelial lining of the
cervix.
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9. Tumor-Suppressor Genes and Oncogenes
The oncogene is not a viral gene, but a cellular gene that had become incorporated into
the viral genome during a previous infection (e.g. Src).
Normal cells possess a variety of proto-oncogenes, functions in a cell’s normal
activities
Proto-oncogene to Oncogene Conversion
1. By gene mutation
2. By gene Duplication one or more times
3. By Chromosome rearrangement
Genes class, in which a gain-of-function mutation can drive a cell toward cancer,
are called protooncogenes; their mutant, overactive or overexpressed forms are
called oncogenes.
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10. 10

11. Cancer-critical mutations –Dominant & Recessive
(A)Oncogenes act in a dominant
manner: a gain-of-function
mutation in a single copy of the
cancer-critical gene can drive a
cell toward cancer.
(B) Mutations in tumor suppressor
genes, on the other hand,
generally act in a recessive
manner: the function of both
alleles of the cancer critical
gene must be lost to drive a
cell toward cancer.
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12. Tumor-Suppressor Genes
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13. The transformation of a normal cell to a cancer cell is accompanied by the loss of
function of one or more tumor-suppressor genes.
Protein encoded by the RB gene, pRB, helps regulate the passage of cells from the G1
stage of the cell cycle into S phase.
E2F family of transcription factors are required for G1 to S phage transition.
E2F proteins are normally bound to pRB and prevents them from activating a number
of genes encoding proteins required for S-phase activities (e.g., cyclin E and DNA
polymerase α).
The first tumor-suppressor gene to be studied and eventually cloned—and one of
the most important—is associated with a rare childhood cancer of the retina of the
eye, called retinoblastoma.
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14. The role of pRB in controlling transcription of genes
During most of G1, the unphosphorylated pRB is bound to the
E2F protein. The E2F–pRB complex binds to the promoter
regions of numerous genes involved in cell cycle progression,
acting as a transcriptional repressor.
Activation of the cyclin-dependent kinase (Cdk) leads to the
phosphorylation of pRB, which can no longer bind the E2F
protein (step 2). Loss of the bound pRB converts the DNA-
bound E2F into a transcriptional activator, leading to expression
of the genes being regulated (step 3).
The mRNA is translated into proteins (step 4) that are required
for the progression of cells from G1 into S phase of the cell
cycle (step 5).
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15. The Role of p53: Guardian of the Genome
p53 is a transcription factor that activates the expression of a large number of genes
involved in cell cycle regulation and apoptosis.
p53 increases the expression of the protein p21, that inhibits the cyclin-dependent
kinase that normally drives a cell through the G1 checkpoint.
p53 rises in the damaged G1 cell and progression through the cell cycle is arrested.
p53 can direct a genetically damaged cell to death by apoptosis.
p53 can bind to Bax proteins at the outer mitochondrial membrane and triggers
apoptosis.
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16. p53 degradation is facilitated by a protein called MDM2 ( a ubiquitin ligase).
DNA damage trigger the phosphorylation of p53 (by ATM related pathway), that is no
longer able to interact with MDM2, which stabilizes existing p53 molecules in the
nucleus.
DNA damage lead to stabilization of p53?
A model for the function of p53. Action of p53 p53 is required for both cell cycle arrest and apoptosis
induced by DNA damage. Cell cycle arrest is mediated by induction of
the Cdk inhibitor p21 and apoptosis by induction of the proapoptotic
Bcl-2 family members PUMA and Noxa. The MDM2 oncogene
protein is a ubiquitin ligase that targets p53. 16

17. Another tumor-suppressor gene, APC associated with colon cancer.
APC suppresses the Wnt pathway, which activates the transcription of genes (e.g., MYC and
CCND1) that promote cell proliferation.
Two genes, BRCA1 and BRCA2 are responsible for the
majority of the inherited cases of breast cancer.
The BRCA proteins respond to DNA damage and activate
DNA repair by means of homologous recombination.
DNA damage initiates activity of both tumor-
suppressor genes and proto-oncogenes
DNA damage is seen to cause double-strand breaks in
the DNA (step 1) that are repaired by a proposed
multiprotein complex that includes BRCA1 and
BRCA2 (step 2a). Mutations in either of the genes that
encode these proteins can block the repair process
(step 2b). If DNA damage is not repaired, a checkpoint
is activated that leads to a rise in the level of p53
activity (step 3a).
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18. Mutations in the PI3K/Akt/mTOR Pathway Drive Cancer Cells to Grow
The phosphoinositide 3-kinase (PI 3 kinase)/
Akt/mTOR intracellular signaling pathway is
critical for cell growth control.
Extracellular signal proteins, including insulin and
insulin-like growth factors, activate this pathway.
The abnormal activation of the protein kinases Akt
and mTOR not only stimulates protein synthesis,
but also greatly increases both glucose uptake and
glycolysis (Warburg effect) in tumor cells.
Tumor-suppressor gene, PTEN (phosphatase)
suppresses the PI 3-kinase/Akt/mTOR pathway
by dephosphorylating the PI (3,4,5) P3 molecules
that the PI 3-kinase in normal cells.
In obese or type 2 diabetes, insulin levels are abnormally high, driving cancer cell growth
without need of mutation in the PI 3-kinase/Akt/mTOR pathway.
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19. The oncogene mutated most frequently in human tumors is RAS, a GTPase.
Oncogenes That Encode Growth Factors or Their Receptors
simian sarcoma virus oncogene (sis) derived from the cellular gene for platelet-derived growth factor
(PDGF).
Avian erythroblastosis virus oncogene (erbB) encodes an EGF receptor.
Oncogenes That Encode Cytoplasmic Protein Kinases
Raf is a serine-threonine protein kinase.
Oncogenes That Encode Nuclear Transcription Factors
Transcription factor MYC is a oncogen
Myc regulates the expression of a huge number of proteins and miRNAs involved in cell growth and
proliferation.
Oncogenes
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20. These include ..
growth factors (1),
receptors for growth factors (2),
protein kinases and the proteins that activate them (3),
proteins that regulate the cell cycle (4),
transcription factors (5),
proteins that modify chromatin (6),
Metabolic enzymes (7), and
proteins that inhibit apoptosis (8).
Proteins involved in mitosis, tissue invasion, and
metastasis are not included.
Proteins encoded by proto-oncogenes
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21. Tumor suppressors and tumor suppression are shown in
red, whereas oncogenes and tumor stimulation are shown
in blue. Arrows indicate activation, perpendicular lines
indicate inhibition. Among the proteins depicted in
this figure are transcription factors (p53, MYC, and E2F), a
transcriptional coactivator or corepressor (pRB), a lipid
kinase (PI3K) and lipid phosphatase (PTEN), a cytoplasmic
tyrosine kinase (RAF) and its activator (RAS), a GTPase
activating protein for RAS (NF1), a protein kinase that
promotes cell survival (PKB/AKT), a protein that senses
DNA breaks (BRCA), subunits of a cyclin-dependent kinase
(CYCLIN D1 and CDK4), a Cdk inhibitor (p21), an
antiapoptotic protein (BCL-2), a ubiquitin ligase (MDM2), an
enzyme that elongates DNA (telomerase), and a protein
that binds growth factors (e.g., EGFR). The arrows and lines
do not necessarily represent direct activation or inhibition.
For example, PTEN inhibits PKB through removal of a
phosphate from PIP3 and EGFR activates RAS via GRB2 and
SOS. The dashed line indicates indirect action by activation
of expression of the MYC gene.
An overview of several of the signaling pathways involved in tumorigenesis
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22. Pathways affected by human oncogenes and tumor suppressor gene
The oncogenes and tumor
suppressor genes mutated in
human cancers can be organized
into 12 pathways that confer a
selective growth advantage by
regulating cell survival, genome
maintenance, and cell fate
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23. Molecular Approaches to Cancer Treatment
Oncogene-targeted drugs
Fifty percent of cancers
could be prevented by
changes in lifestyle.
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24. Many cancers may be treatable by enhancing the immune response against the specifc tumor.
Immunotherapy
(A) Tumor cells will produce many
mutant proteins which are displayed
on MHC complexes on the tumor-cell
surface and would normally activate a
T cell response that destroys the tumor
but the cancer cells have evolved
immunosuppressive mechanisms
that protect them from such killing.
(B) Several monoclonal antibodies against PD1 (programmed cell death protein 1) and a hypothetical
protein X have been developed that prevent the downregulation of T cells, and these antibodies (such as
pembrolizumab or Keytruda) have been found to be effective against some types of cancer,
including melanoma and some kinds of lung cancer.
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