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:

a4

to a7

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 an, 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 patient’s 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-INK4Cp16-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 inhibitorsDecitabine

(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 lesionCervix

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 2nd

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 1014

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 106

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 ‘‘multipotent’’or

‘‘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 valuableresource 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

NeuralStem Cells(nestin)

Microvessels

Lung Metastasis

CD44 CD24CSC

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 106

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 andin 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 populationsand 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 generatetransit-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 onceagain 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 ssDNABRCA1:

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 3ʹ-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 pathwayStrand 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 3ʹ

ssDNA

forms double-helical complexes with the DNA strands of the sister chromatid.DNA synthesisLigation

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

Triradialchromosomes

QaudriradialChromosomes

• 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 EMSThis 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-linkMonoadduct

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).

TranslocationsLocal 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 pointThe 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 spindleSpindle

Attached

Analysis of Breast cancer genomes

Gain

Loss