BIOLOGY OF CANCER

SC/BIOL 4010 3.0

Lecture 8
i.

Cancer progression
ii.

Cancer Stem Cells
iii.

Genomic instability
November 21, 2013
MR Stratton, PJ Campbell and PA
Futreal

(2009) The Cancer Genome.
Nature 458:719
All cancers arise as the result of changes that have occurred in

the DNA sequence of the genomes of cancer cells.
passenger mutations
driver mutations
Most cancers take time to develop
Epidemiology shows that the chance to develop most epithelial cancers increases with age.

Due to the late onset, even if we were to cure all cancer this would only extend the average
life span by 3.83 years for men and 3.38 years for women, due to

other age related disease.
Conversely, if we prevented all other age-related diseases we would all eventually contract
cancer because the development of some sort of malignancy will be inevitable given enough
time.
The Age of death from a cancer can model the number of rate limiting
steps it requires to develop.
Mortality from cancer can
be modelled by rate that
is:
a
4

to a
7
Where a=patient age at
initial diagnosis.

The chance of death from epithelial cancers usually increases to the 5 or 6 power of elapsed
time.

If the probability of outcome is a
n
, this means that n+1 independent random events of the
same probability are required before the final outcome is possible (Death from cancer).
Thus an n=5 (slope of 5 on the graph) requires 6 independent events.

Each of these events likely takes years to decades, and thus the final outcome is not
achieved until the latter decades of life.
Multistep tumorigenesis
The Succession of various epithelial carcinomas follow similar histological progression.
Each stage of histological presentation of the disease is thought to give rise to the next.

However each stage can take a variable amount of time depending

on the cancer type,
nature of mutations involved, etc.

In reality the information to prove this progression is scarce,

and some cancers may skip
particular steps on the road of tumorigenesis.
Adenoma to Carcinoma

Besides the histopathological

identification of the stage of the cancer, the progression
of some cancers can be easily monitored over time. i.e. Colon cancer and polyps (FAP).
Here precancerous adenomas have been identified in a patients lungs.
By 19 months one of these lesions has progressed to a carcinoma.
While several others have disappeared

Interestingly all 3 of the initial spots had a rare p53 mutation, indicating a common
origin.
A common set of mutations are responsible for colon cancer progression
The LOH was examined by looking at markers on particular chromosomal regions.

Many chromosome arms lose LOH during tumour progression, the ones shown generally
occur in at least 50% of the cancers examined, and likely indicate a tumour suppressor.
equally important is the activation of a oncogene

such as K-ras
These regions were later to be shown to be LOH of APC, 18q TSG,

and p53.
Generally loss of tumour suppressor genes far exceed activation

of oncogenes.
Another feature is the global loss of methyaltion

of DNA.
Findings from The Cancer Genome Atlas study
NF1 is a member of the
RAS-GAP family of
proteins that stimulate the
GTPase

activity of RAS
Findings from The Cancer Genome Atlas study
Findings from The Cancer Genome Atlas study
p15-INK4B p18-INK4C p16-INK4A
Findings from The Cancer Genome Atlas study

74% of tumours

contained mutations in all 3
pathways (p53, RB and RTK/PI3K pathways)
suggesting that deregulation of the 3 pathways is
a core requirement for glioblastoma

development

mutual exclusivity of mutations within each of
the 3 pathways (p53, RB and RTK pathways)
Methylation

and CpG

islands
In genomic DNA, a methyl group
may be added to cytosine to form
5-methylcytosine. This process is
known as DNA methylation

and only
occurs in cytosines

that are
followed by a guanine (5' CG 3').

Many genes in the human genome
have upstream CG-rich regions
called CpG

islands. DNA
methylation

of a gene's CpG

island
represses gene expression.

Different cell types have
different methylation

patterns,
which contributes to the
differences in gene expression in
different cell types.
During DNA replication, the
parent's methylation

pattern is
copied to the newly synthesized
strand. This ensures that a parent
strand passes on its methylation

pattern (and therefore, its gene
expression) to its daughter strand.
Gene Suppression

In normal mammalian cells, CpG

islands in proximal gene promoter regions are largely
protected from DNA methylation

(cytosines

are shown as open lollipops) and reside in
regions of open chromatin favorable for gene transcription.

In contrast cytosines

in CpG

dinucleotides

which are methylated

(black lollipops) are
packaged as closed chromatin unfavorable for transcription.
Methylation

of tumour suppressor genes
in cancer

In contrast, cytosines

in CpG

dinuleotides

in other regions of the genome display
hypomethylation

and are associated with aberrantly loosened chromatin.
The overall result is abnormal chromatin packaging with the potential for underpinning an
abnormal cellular memory for gene expression and for conveying abnormal structural
function for chromosomes.

In cancer cells, there
tends to be a reversal
of the methyaltion

pattern of normal cells.

Proximal promoter CpG

islands for many
abnormally silenced
genes (the tumor
suppressor genes listed)
become DNA
hypermethylated

and
reside in a closed
chromatin, which is not
favorable for
transcription (red X).
DNA methyl transferase

enzymes
When DNA replicates, the bulk of methylation

copying is done by DNMT1
DNMT3A and DNMT3B are de novo methyltransferases

involved in embryonic development
and in the establishment of genomic imprints
Also soon after DNA replication, DNMT3A and DNMT3B complete the methylation

process
and correct errors that are left by the DNMT1 enzyme. These enzymes are closely
associated with CpG

islands.
Normal colon
Adenocarcinoma Aggressive Colon Cancer
Methylation

of the RASSF1A promoter

From this analysis of normal vs. tumour tissue it is apparent that methylation

is part of
the suppression of this tumour suppressor. Interestingly this disregulation

started with
some modification in adjacent normal tissue.
Many cancer types have upregulated

DNMT3 enzymes
Methylation

could occur on both alleles or on one allele followed by LOH.
DMNT3a/b inhibitors
Decitabine

(5-

azacytidine , or AZA)
dose-response plots of nanaomycin

A against DNMT1 and
DNMT3B. The IC50 concentrations were determined by
biochemical DNMT assays under identical conditions
Shows specificity for DMNT3B rather than DNMT1
Reactivation of a tumour
suppressor promoter

Illustrated are the relative
demethylation

levels in percentage of
treated cells to untreated cells for 31
CpG

sites within the RASSF1 promoter.
Nanaomycin

A treatment resulted in
demethylation

that was restricted to a
few CpG

sites
This was sufficient to de-repress the
RASSF1A tumour

suppressor.
AZA is in clinical trials for AML.
DNA methylation

in situ

Methylation

of critical growth
controlling genes often occur
early during the development of
a tumour.
This can occur before there is
any histological evidence of
tumour.

These areas are than fertile
grounds from which cancer can
emerge.

p16 promoter methylation

can
be assed in situ with a specific
probe.

p16 suppression is obvious in
high grade cervical cancer but
can also be detected is
apparently normal breast tissue.
Low grade lesion
High Grade lesion
Cervix
Normal Breast lobules
Methylation

of multiple genes
within tumour cell genomes
Most of these genes are normally un-methylated.
They become methylated

and therefore repressed during cancer progression.
Alternate Paths during cancer progression
The sequence of mutations that lead to colon cancer are not invariant in colon cancer

For example the loss of APC function is a common feature in the

initiation of 80% of colon
cancers, and can be duplicated by mutations with in the same pathway that regulates -

catenin

and

which lead to polyps.

However the 2
nd

mutation in sequence is variable and could be any of 1 of 5 genetic
changes. These may also influence future genetic change creating even more alternate
pathways (not shown).
The hierarchy of pathways is supported by only fragmentary evidence since, 1-the exact
number of alterations to cause colon cancer is unknown 2-All 4 of these categories may not
be required and 3-whehter a change in 1 functional group is mutually excluded from

another.
Colon
Pancreas
Cancer Cell Phenotype vs. Genotype

Each hallmark of cancer represents an
acquired phenotype of malignancy.

Although tempting to assign a single
genetic alteration to each hallmark, this
is not the case.
Many of the alterations we have covered
in the course can often effect multiple
Hallmarks.

Additionally some of the acquired
capabilities can only be acquired
collaboratively by multiple genetic
alterations.
With what we know can we transform a
normal cell?

Normal cells start off with wild type genomes which than acquire sporadic mutations
or alterations.

Each stage along the pathway of tumourigensis

must therefore counteract or disable
each defence that guards against such alterations.

Colon epithelial cells are highly successful at protecting against such alteration, since
during our lifetimes we generate 10
14

such cells and have a surprising low rate of
cancer.

Organs affected by sporadic tumours can occasionally sprout multiple apparent
independent tumours, a phenomenon called field cancerization.
These tumours erupt in close proximity but have a different set of genetic alterations.

Imagine that a single cell undergoes the initial step of tumourigensis

but is
phenotypically

normal. This may lead to a clonal

patch of cells with the same mutation,
which may also acquire another mutation in a single cell leading

to large portion of
genotypically

altered cells.

This patch may than acquire 2 independent alterations in different cells leading to
emergence of genetically distinct tumors.
Clues from field cancerization

provide insight to multi-step tumorigenesis
Field Cancerization

Genetic analysis of normal epithelial tissue reveals that patches of the tissue may
contain gene alterations, such as chromosomal LOH.
17p LOH: detected by mutant p53 specific antibody.

This p53 mutant patch may expand over time to become a Field. Cells with in this
field often have LOH at different loci.

When cells independently in this field acquire mutations including LOH at 11q
independent tumours can arise at different parts of the field.
There may be wide distribution of
altered cells throughout a tissue.

Although p53 is mutated in many of
the cells of the lung tissue, they
have not spawned a cancer.

These single alterations are
inalienable with time, but only rarely
acquire the secondary and tertiary
mutations required for full
malignancy.

Over time it became clear
that a series of genetic
alterations was behind the
phenotypic changes seen at
the morphological level.
In a type of micro-evolution
cells can acquire a survival
advantage over the normal
cells around it.

Each mutation occurs in a
single progenitor of a
population and confers the
ability of mutated cells expand
by proliferation.
Clonal

Expansion

Each clonal

expansion of mutated cells will contain all the genetic alterations from the
previous expansion and thus will have the cumulative selective advantage of each.

Each succeeding mutation has the ability to dominate the local tissue environment
overshadowing the original clonal

expansions.
One advantageous mutation can occur once every 10
6

cells.

A sequence of 4 to 6 such populations can be used to explain cancer at the genetic and
cellular level.
These events are likely spaced far apart in time.
Clonal

diversification due to high mutation rates

As tumour progression
proceeds the genome often
becomes more unstable.
Thus at some point the
clonal

expansion of
multiple independent
mutations can occur.
These can lead to multiple
parallel clonal

expansions.

These subclones

do not
form distinct regions with
in the tumour, rather each
subclone

intermingles with
the other subclones

in the
tumour mass.
As a consequence of genetic instability many tumours have multiple lineages of mutations.

In addition with the increased rate of mutation the chances of more advantageous clonal

expansions to occur are increased.
These sub populations are of course just a prone to generate even more subclones.
Adult Stem Cells
Cells residing in adult tissues,
such as adult stem cells, can only
give rise to cell types within their
lineage and are called either
multipotentor

unipotent,

depending on the number of
developmental options they have.
Upon terminal differentiation,
cells entirely lose their
developmental potential.
Adult SCs

are present in the
bone marrow, blood, cornea &
retina, skin, skeletal muscles,
dental pulp, liver, and brain.

As adult SCs

can be propagated
in large quantities without losing
their ability to differentiate into
different tissue types, they
represent a highly valuable
resource for the development of
cellular therapies.
Science 327, 542 (2010)
Stem cells and gastrointestinal crypts
A)

Stem cells in red, slowly divide and
each replenish about 150 transit
amplifying cells (yellow and green).
Rapidly proliferating transit
amplifying cells give rise to 3500
enterocytes

(blue). The cells migrate
up the vilus

and eventually apoptose.
The Stem cells are protected from
the environment by mucus and physical
location at the bottom of the crypt.
Cell position 4 also has a stem cell
population which is normally quiescent,
but responds to injury. Other stem
cells are found in between Paneth

cells.
B)

Lgr5 marker used to identify stem
cells (green).
C)

Tritium labelled Nuclei migrating up
the vilus

from the bottom of the
crypts.
Cancer stem cells are the core of a tumour

Brain tumour stem cells can be
identified by expression of CD133.

Such cells can be identified within a
section of the tumour (red).

They can also be separated roughly into
2 sub populations using FACS, based on
the amount of CD133 expressed.

The Majority of the cells do not express
high levels of the stem cell marker and
do not rapidly grow in soft agar.
However the high expressing CD133
population grows rapidly in soft agar
forming tumour spheres (3D colonies).
The high CD133 population gives rise to
both low and high CD133 cells in the same
distribution as the original tumour.
Thus like the epithelial cells it is derived
from the cancer stem cell is acting to
restore the cell lineages of the original
tissue, albeit in aberrant fashion.

Most of the cells originating from the
cancer stem cell are more differentiated
and incapable of unlimited division.
Identification of stem cells in Tumours
Human Glioma
Neural
Stem Cells
(nestin)
Microvessels
Lung Metastasis
CD44
CD24
CSC
Colon Carcinoma
Chromosomal
amplification
ALDH1

Cancer stem cells (CSCs) often share the same markers as stem cells, and thus are
useful in identifying these cells with in the tumour.
The is little commonality between the organization of these cells within a tumour.
Self Renewal of stem cells

Pools of stem cells divide asymmetrically, 1 daughter becomes a

stem cell and the
other becomes a post-mitotic cell or a transit amplifying cell.

In this way stem cells are capable of self renewal for many generations and seed
future lineages of more differentiated cells which have a limited capacity to divide.
Inedibility through some misshape a stem cell will eventually be

lost and thus needs
to be replaced for normal tissue maintenance.
These replacement mechanisms may be what becomes deregulated in cancerous
tissue.
Origin of Cancer stem cells

A stem cell gives rise to a flock of cells (Transit amplifying progenitor cells) which
symmetrically divide and ultimately differentiate into the mature tissue.

Mutations that effect the regulation of stem cell renewal or revert a progenitor cell
back to stem like state could potentially induce CSCs.
The cancer stem cell than resembles the original adult stem cell

and acts to maintain
the mature tissue, in this case the tumour.

This of course implies that the stem cell population in a tissue is what is the primary
target of mutagens.
Clonal

expansion of CSCs

While this scheme is logical, it holds
several inherent difficulties that
cannot be reconciled with the actual
biology.

It implies that the targets of
mutation at each stage of tumour
progression are stem cells.

stem cells represent only a small
proportion of the cells within a tissue,
normal or neoplastic.
Since mutations occur at a low rate
per cell generation (1 per 10
6

cell
divisions), this means that tumor-

promoting mutations are
mathematically unlikely.
i.e.in

the colonic crypt

Additionally mutations generally
occur in actively dividing cells and in
part are associated with mistakes in
DNA replication,

If stem cells divide relatively
infrequently this also decreases the
likelihood that they represent the
targets of mutation.
Transit amplifying cells as CSC origin

non-stem cells can, with a certain
frequency, dedifferentiate into stem cells.

This dedifferentiation can occur in both
immortalized epithelial cell populations and
in their transformed derivatives, both in
vitro and in vivo.

multi-step tumour progression could
actually proceed when particular, mutations
strike a cell in the transit-amplifying/
progenitor cell populations, which are far
larger than the stem cell populations
and are actively dividing.
Thus, the dedifferentiation of this cell
into a stem cell ensures that the new
mutation can now be is in the stem cell pool.
The mutant cell with this new mutation
gradually displaces the other stem cells in
this population, and these now generate
transit-amplifying and more-differentiated
cells carrying this new mutation.

A second mutation that may trigger a
subsequent round of clonal

expansion will, as
before, strike a cell in the newly formed
pool of transit-amplifying cells and once
again will be introduced into the stem cell
pool via dedifferentiation.
DNA Repair Mechanisms

Errors introduced in the replication process are the simplest
source of damage to the double helix.

With the addition of each base, there is the possibility that
an incorrect base might be incorporated, forming a non-

Watson

Crick base pair.

These locally distort the DNA double helix and can be
mutagenic; resulting in permanent changes in the DNA.

A mutation in DNA pol-

that prevents proof reading (D400A)
leads to increased cancer incidence in mice.
These include lymphomas, skin carcinomas, and lung cancer.

Some hereditary cancers have also been identified to be due
to mutation of pol

or

in humans.
Mismatch Repair

Sequences containing tandem arrays of
repeated triplet sequences are prone to
expansion by the stuttering of DNA pol

causing the

looping out of some of the repeats.
Or the DNA it self loops out before
replication.
The double helix formed from the red
template strand will contain additional
sequences encompassing the looped-

out region.

Mismatch-repair systems consist of at least two
proteins, one for detecting the mismatch and the
other for recruiting an endonuclease

that cleaves
the newly synthesized DNA strand close to the
lesion and a polymerase fills the gap.
The mismatch-

repair proteins are called MutS

and MutL.

some adenine bases of the parent strand of
DNA are methylated, whereas the newly
synthesized daughter strand is not yet
methylated.
Thus, the repair machinery recognizes that the
base attached to the methylated

DNA is correct.
Microsatellite instability
Defects in mismatch repair are responsible for the accumulation of mutations in the genome.
Microsatellites are highly repetitive tracts of DNA which are prone to contract or expand
during the process of replication, unless mismatch repair is active.
The failure of mismatch repair leads to many expansions of microsatellites through the
genome.
A)

A microsatellite repeat BAT25 has expanded in a case of colon cancer, but remains
unchanged from normal in a breast cancer. This indicates one of the genetic alterations
in the colon is failure of mismatch repair.
B)

Mutations recorded for the 518 kinases

in 220 tumours. Those with defective mismatch
repair showed a high degree of mutation, comparable to gliomas

treated with a known
mutagenic chemotherapy agent.
The majority of theses changes
are sporadic rather than
familial).

Generally as a result of
promoter methylation

or somatic
mutation of mismatch repair
genes.
In fact 15% of sporadic gastric
colorectal and endometrial
cancer can be traced back to the
methyaltion

of the MLH1 (Mut

L
Homolog 1) promoter.
The silencing of this gene
silences mismatch repair leading
microsatellite instability.
TGF-

and microsatellite instability Suppression of MLH1 in endometrial tissue
A hereditary mismatch repair disease Hereditary
nonpolyposis

colorectal cancer (HNPCC) is defective in
MLH1 (MutL

in E Coli)
The inability to detect mismatches leads to high
mutation rates of genes with microsatellite DNA.
One of these genes is TGF-

Receptor II undergoes a
deletion of 2 AAs

in a A stretch.
The resulting protein is truncated by frame shift.
The other allele is also subject to LOH.
The loss the TGFR II circumvents the normal anti-

apoptotic signals associated with the pathway.
Further studies have found that TGFR II is often
mutated in colon carcinomas that have microsatellite
instability.
The endometrium

normally has strong
nuclear staining for MLH1 (brown staining: N
upper arrow).

However this is lost in the tumour (T) and
in surrounding normal tissue (N lower arrow).

Further analysis showed the MLH1
promoter to be methylated

in both normal
and tumour tissue.
This is indicative that loss of MLH1 is an
early event in tumourigenesis.
Base-excision repair.

Sometimes damage to DNA can be repaired
directly, without having to remove any
fragments of the DNA, a process called direct
repair.

Modified bases, such as 8-

oxyguanine

or 3-

methyladenine, are excised by the E. coli enzyme
AlkA.

The binding of this enzyme to damaged DNA
flips the affected base out of the DNA double
helix and into the active site of the enzyme.

The enzyme then acts as a glycosylase, cleaving
the glycosidic

bond to release the damaged base.
At this stage, the DNA backbone is intact, but a
base is missing.

This hole is called an AP site because it is
apurinic

( devoid of A or G) or apyrimidinic

(
devoid of C or T).

An AP endonuclease

recognizes this defect and
nicks the backbone adjacent to the missing base.

DNA polymerase inserts an undamaged
nucleotide, as dictated by the base on the
undamaged complementary strand.

Finally, the repaired strand is sealed by DNA
ligase
The nucleotide-

excision repair system
recognizes distortions in the DNA double
helix caused by the presence of a damaged
base.

excision repair is utilized for the excision
of a pyrimidine

dimer.

First, an enzyme complex consisting of the
proteins encoded by the uvrABC

genes
detects the distortion produced by the DNA
damage.
The UvrABC

enzyme, an excinuclease

then
cuts the damaged DNA strand at two sites
on both the 5

side and 3

side.

DNA polymerase enters the gap to carry
out repair synthesis.
The 3

end of the nicked strand is the
primer, and the intact complementary strand
is the template.

Finally, the 3

end of the newly synthesized
stretch of DNA and the original part of the
DNA chain are joined by DNA ligase.
Nucleotide-

excision repair
p53 can control DNA repair
The p53 protein when
activated can lead to the
transcriptional up regulation
of repair genes.

It also halts the cell cycle
in order to give the cell time
to allow these repairs.

The loss of p53 therefore
can lead to mutation in DNA
by both mismatch repair and
nucleotide excision repair.
Large Scale genome sequencing reveals diverse patterns of mutagenesis
Frequency of mutation by cancer for 1035 cancer genomes.
Distributions of point mutations shown below for each cancer.
Figure 12.10 The Biology of Cancer (

Garland Science 2007)
Damage at DNA replication forks
Collapsed replication fork
The repair of ss

breaks at replication forks
occurs primarily by HR
Unlike some of the previous sources
of DNA damage seen in the course
damage due to replication is an
endogenous source of mutation.

However this mechanism could also
be how particular mutagens end up
causing DNA damage, because the
polymerase is unable to properly
replicate damaged bases.

During DNA replication the DNA
molecules are vulnerable to breakage
in single stranded portions near the
replication fork.
If DNA pol

stalls at a damaged or
incorrect base it leaves a vulnerable
single stranded region to possible
breakage.

This break is the equivalent of a
double stranded break, since it has
occurred in an already formed helix.
Figure 12.32 The Biology of Cancer (

Garland Science 2007)
Repair of DSBs

by
Homologous Recombination

HR requires a homologous DNA
sequence (provided by a sister
chromatid) as a template

HR leads to error free (high
fidelity) repair

HR occurs in a cell cycle
restricted manner: occurs in the S
and G2 phases of the cell cycle
These phases of the cell cycle can
provide a sister chromatid

and are
more likely to experience double
stranded breaks.
DNA replication results in 2
chromatids

that are separated at
the next mitosis.
Nonhomologous

end joining

NHEJ is used to restore a DNA
double helix following a double-strand
break when a sister chromatid

is not
available.
Occurs in G1

The template for the repair is
through base-pairing of the processed
end of the DNA (by a limited degree of
base pairing).
The resulting DNA is missing
information and is thus an error prone
process.
When HR fails NHEJ is the default
pathway.
NHEJ has a normal role in the
rearrangement of genes which encode
antibodies.
About 5

10% of breast cancers and 10 -

15% of ovarian cancers are inherited.
Inherited mutations in the BRCA1 and
BRCA2 genes account for over 80% of
inherited breast and ovarian cancers.
Mutations in either BRCA1 or BRCA2
significantly increase risk of breast
cancer and ovarian cancer (50 to 85
percent chance of developing breast
cancer by age 70; i.e. about 5-fold
increased risk).
BRCA1 and BRCA2 are tumour suppressor
genes. WHY?
Breast cancer susceptibility genes: BRCA1 or BRCA2
Recall Lecture 6
Angelia Jolie
BRCA1 and BRCA2 proteins are involved in the repair of DNA double-

strand breaks through the homologous recombination pathway and play
critical roles in the maintenance of genomic stability.
BRCA1 and BRCA2
Act molecularly as a scaffold
for various proteins in the
DNA damage response.
BRCA2:

8 BRC domains which bind Rad51.
These form filaments and coat ssDNA
BRCA1:

Coordinates the proteins involved in
recognition of the DNA damage response
RAD50/Mre11/Nbs leading to ATM activation.

Loss of different partners of
BRCA1 affects different check
points and HR.

The S check point ensures that
cells with damaged DNA halt and
repair their DNA before proceeding
HR molecular pathway

Recognition.

After a DSB occurs the
lesion is recognized and bound by the
heterotrimeric

MRN complex:
Mre11,/Rad50/Nbs1
Nuclease-mediated resection

This MRN
complex recruits CtIP

(CtPBinteracting

protein) and, in complex with BRCA1 and
BARD1, undertakes exonuclease

activity to
generate two 3-overhanging single-strand
(ss) DNA ends.
These ssDNA

ends are rapidly coated by
RPA (replication protein A; green).
BRCA1, acting through PALB2, then
recruits BRCA2, and the latter loads
Rad51 (orange) onto the ssDNA, thereby
displacing the previously bound RPA
protein.
HR pathway
Strand Invasion

This Rad51 protein, which
acts as a recombinase

then facilitates
strand invasion into the undamaged
homologous region of the sister chromatid.
This depends on unwinding the double-

helical DNA of the sister chromatid;
Rad51 also participates in the search for
binding to the proper complementary
sequence in the sister chromatid

(termed a
homology search).
A displacement (D-) loop is produced when
the 3

ssDNA

forms double-helical
complexes with the DNA strands of the
sister chromatid.
DNA synthesis
Ligation
BRCA1 and the DNA damage response

Hydroxyurea

causes replication fork collapse. BRCA1 localizes to these areas as
can be seen by its co-localization with PCNA .

Micro-irradiation (using a UV laser on living cells) causes localized DSB in the
lasers path. The DNA damage response includes phosphorylation

of H2AX (known as
H2AX) and localization of BRCA1 to area of DNA damage.
Karotypic

alterations due to loss of BRCA2
Chromatid

breaks
Triradial
chromosomes
Qaudriradial
Chromosomes
Karotype

of Mouse embryonic fibroblasts (MEFs) that are BRCA2 -/-

Deficient HR can lead to fusions between chromosome arms leading to translocations
and aberrant chromosomal pairings at metaphase.
The fusions are caused by incorrectly repaired DSB likely as a consequence of NHEJ.
Breast Cancer studies indicate that common cancers are
caused by defects in caretaker genes

Although hereditary BRCA mutations strongly
influence the development of breast cancer
many questions remain.
Why are these mutations not 100% penetrant

?

Why are breast and ovarian the main tissues
that develop cancer?

A study using identical twins suggests that
women that develop breast cancer have
multigenic

factors that predispose them to
cancer.

Normal lymphocytes from breast cancer
patients show a high degree of aberrations when
exposed to ionizing radiation, much more so than
lymphocytes from a volunteer control group.
Interestingly, relatives of the breast cancer
patients also showed a high degree of hyper-

senstivity

to the radiation in their lymphoblasts.

The assumption here is that a segment of the
population are more predisposed to cancer
because there cells less capable of repairing
DNA damage.
The caretaker genes that protect genomic
integrity are less efficient in these women.
Most Chemotherapies Target dividing DNA and the damage response
Conventional chemotherapies target rapidly growing cancer cells by:
1) Damage to the DNA of the affected cancer cells.
2) Inhibit the synthesis of new DNA strands to stop the cell from replicating.
3) Stop mitosis.
Unfortunately, the majority of drugs are not specific, which leads to many of the common
side effects associated with cancer chemotherapy. The side effects are seen in tissues
with a rapid turnover of cells including skin, hair, gastrointestinal, and bone marrow.
These normal cells, also end up damaged by the chemotherapy program.
Fluorouracil (5FU) inhibits DNA and RNA metabolism in
dividing cells
5FU is metabolized to FUMP, FUDP, FUTP, FdUMP

FdUMP

inhibits thymidylate

synthase

(required for production of dTMP

from dUMP

leading to depletion dTTP)
FUTP inhibits RNA synthesis when it is incorporated into RNA
Epirubicin
Doxorubicin

Such drugs take advantage of
the fact that many cancer cells
disable G2/M controls such as HR.

The drugs cause DNA damage
that remains unrepaired as cells
progress into mitosis.

The cells enter into mitotic
catastrope

that results in
aneuploidy, polyploidy, and
micronuclei instability that
directly leads to apoptosis.

These Drugs intercalate between DNA base
pairs resulting in inhibition of DNA synthesis
inhibitors of topoisomerase

II preventing re-

ligation of cleaved DNA molecules during DNA
synthesis
generates DNA double-strand breaks
Cyclophosphamide

is a bifunctional

alkylating

agent

has two reactive groups and each molecule can react with two sites on DNA
leading to intrastrand

cross-links or interstrand

cross-links
MMS EMS
This induces inhibition of DNA replication, leading
to cell death. CYC exerts its cytotoxic

effect on
both resting and dividing lymphocytes.
Cisplatin

is a DNA cross-linking agent

the chloride ligands

are displaced by purine

nitrogen atoms (N7), most commonly in guanine, on
two adjacent bases on the same strand.

The formation of these bonds causes a severe
kink in the structure of the DNA
Interstrand

cross-links prevent DNA strand
separation and block DNA replication and
transcription
Moreover, research results suggest that certain
nuclear proteins bind to the cisplatin-

damaged
DNA and prevent access to DNA-

repair enzymes.
The net effect of the cisplatin

treatment is that
the cell undergoes apoptosis, killing the cancer
cell.
Interstrand

cross-link
Intrastrand

cross-link
Monoadduct
Poly (ADP-ribose) polymerase 1
PARP is activated by DNA ss

breaks
required for ss

break repair and BER

When the enzyme binds to these breaks it proceeds to ADP
ribosylate

it self and other surrounding proteins.
The poly ADP tails serve as docking sites for recruiting the repair
enzymes needed to fix the ssDNA

break.
When PARP is inhibited these breaks persist
and on the next round of replication generate
double stranded breaks

The inhibition of PARPs

leads to
the accumulation of DNA single-

strand breaks, which can lead to
DNA double-strand breaks at
replication forks.

In most cells, PARP inhibition is
compensated by increased
homologous recombination

The inhibition of PARP-1 is a
potential synthetic lethal

therapeutic strategy for the
treatment of cancers arising in
carriers of a BRCA1 or BRCA2
mutation.

In the absence of HR (due to the
absence of BRCA function), BER
allows the cancer cell to recover
from DNA damage; the addition of
a PARP inhibitor results in what is
termed "synthetic lethality".
Poly (ADP-ribose) polymerase 1
Synthetic lethality:
A gene interaction in which single-gene defects are compatible with cell
viability, but the combination of gene defects results in cell death
Comen

and Robson (2010)
.
Ashworth A JCO 2008;26:3785-3790
2008 by American Society of Clinical Oncology
Fig 4.BRCA2 mutant cells are
exquisitely sensitive to a
potent PARP inhibitor.
Clonogenic

survival curves of
BRCA2 wild-type, heterozygous,
and deficient cells after
treatment with the poly(ADP)
ribose polymerase inhibitor
KU0058948.

BRCA2-deficient cells are more
than 1,000-fold more sensitive
than wild-type or heterozygous
cells to KU0058948.
Chromosomal Translocations
Aberrant chromosomal structure in cancer cells has been known for almost a century.
Causes we have covered in the course so far:
-Unrepaired DSB during replication can lead NHEJ i.e

BRCA1/2
-Misfiring of mechanisms for re-arranging immunoglobulin i.e. BCR-ABl

& Myc
-Breakage-fusion cycles at crisis when telomeres are critically short. i.e. p53
However many chromosomal translocations cannot be accounted for by the above
mechanisms: since the number of translations can vary immensely between cancers and
certain cancers have common translocation hot spots (not random breakage as predicted
in Breakage fusion cycles or NHEJ).
Translocations
Local
Re-arrangements
Circos

Plots
Localized firestorms of Chromosomal translocations
Large scale genome sequencing has
allowed the identification of hot spots
of chromosomal translocations in
particular cancers.

Here a thyroid carcinoma has a cluster of 77 distinct re-arrangements centered around
the short arm of chromosome 9.
Each of the lines represent a specific fusion event.
A catastrophic chromosome breakage (or shattering ) of the chromosome occurs in a
localized area and NHEJ attempts to repair the damage.
The reasons for Chromothripsis

(chromosome shattering remains obscure)
Chromosomal instability in cultured cancer cells

In addition to mutation and translocations cancer cells often shown changes in
the number of chromosomes (aneuploidy).
These can have just as dramatic effects on cellular behaviour
Chromosomal instability refers to condition in which 85% of carcinomas acquire
the inability to regulate chromosome segregation at mitosis.
Cultured cancer cells maintain this property when propagated in tissue culture.

In fact the chromosomal aneuploidy

will continue to affect future expansions of
cancer cell line to he point where they may no longer resemble the initial tumour.
Normal Cells Breast Cancer
Chromosomal vs. Microsatellite
Instability
Microsatellite instability is mutually exclusive from chromosomal instability.

Colon and rectal cancers that show high chromosomal instability

appear to have low
microsatellite instability and vice versa.

Thus it appears that a particular cancer must acquire some sort

of mechanism to
increase genome mutability in order to favour neoplastic

development.
Spindle assembly check point
The complex process of chromosome segregation is monitored by a series of check point
controls to ensure that non-disjunction does not occur.
Spindle assembly check point.
The cell does not commit itself
to anaphase

before it is fully
prepared.
In most cell types, a spindle-

attachment checkpoint

mechanism operates to ensure
that all chromosomes

are properly
attached before sister-chromatid

separation.
The checkpoint

depends on a
sensor mechanism that monitors
the kinetochore, the specialized
region of the chromosome

that
attaches to microtubules

of the
spindle.
Any kinetochore

that is not
properly attached to the spindle
sends out a signal to block Cdc20-

APC

dependent sister-chromatid

separation.
Several proteins, including Mad2,

are recruited to unattached
kinetochores

and are required for
the spindle-attachment
checkpoint.
CENP-E the motor protein recognizes that a microtubule is
unattached, by recruiting a group of proteins.
Mad 1 is phosphorylated

by recruited kinases

which disrupts its
interaction with Mad2. Mad2 is capable of disrupting APC-cdc20.
Temporarily halting the mitotic cycle.
Properly attached microtubules prevent Mad2 activity, since Mad1

is no longer recruited and phosphorylated. APC is free to continue
the cycle.
A single unattached spindle is sufficient to halt the mitotic cycle.
Unattached spindle
Spindle
Attached
Analysis of Breast cancer genomes
Gain
Loss