DNA repair and the mutational basis of cancer

Cancer Biology 553

Thomas E. Wilson
Departments of Pathology and Human Genetics

A classical view of clonal
expansion in cancer
Cancers are:

Clonal

Arise by mutation

Multi-step in origin

Genetically heterogeneous


Define mutation:
A mutation alters the information encoded in genomic
DNA, either by changing the base sequence OR the
number of times it is represented.



Define epigenetic:
Epigenetic changes alter the way information in the
genome is expressed without altering the sequence
information itself (not our subject here).



Cancer: a disease of DNA
A tumor-specific point mutation
normal tumor
JAK2 V617F mutation in polycythemia vera
Activates a tyrosine kinase
Note: heterozygous mutation in this instance since it is dominant
Systematic sequencing of cancer genes
The Cancer Genome Atlas (NIH, US)
Cancer Genome Project (Sanger, UK)
COSMIC database
Etc.
Systematic sequencing of cancer genes
CML and the Philadelphia chromosome
Carcinomas show multiple gross chromosomal rearrangements
Systematic sequencing of cancer genes


Do cancer cells have deranged (i.e. mutated) genomes?
" Unequivocally YES! 100%.
" Most mutations acquired somatically (some inherited).


Is genomic instability a phenotype of cancer cells?
" Thats a tougher one!
" Requires an understanding of the genesis of mutations.

?
?
Types of tumor suppressors

Caretakers
" Genes whose normal function is to maintain the integrity of
the genome, i.e. to repair damage and prevent mutations.
" DNA damage repair and response genes

Gatekeepers
" Genes whose normal function is to prevent the outgrowth of
cells with deranged genomes.
" Apoptosis genes, cell cycle, etc.

Kinzler and Vogelstein, Nature. 1997;386:761-763.

Telomere-dependent transient genomic instability
Cancers have non-clonal mutations too
Observed
experimentally
(Larry Loeb)
Suggest ongoing
high rate of
mutagenesis
Cancer predisposition syndromes:
highly penetrant DNA repair defects lead to cancer
You are not normal (its OK, no one is!)
cancer mutation genome lesion
DNA damage
DNA
repair
Cancer: a disease of DNA
The relationship of DNA damage, repair and mutation
Mutations are NOT created directly, but represent the inaccurate
chemical repair of DNA damage/lesions.
Mutagenesis/cancer is a balance between forces that damage the
genome, and the efficiency and accuracy of DNA repair mechanisms.
Increased carcinogenesis is expected with either increased DNA
damage or inherited or acquired DNA repair dysfunction.
Nature
Nurture
Chemicals DNA damaging subset of carcinogens
Exposure is systemic or topical any organ system potentially at risk.
Typical DNA lesion is an adduct of some portion of the chemical agent
with the bases of the DNA adducts can be small (CH3) to quite big
(polycyclic aromatics).


-CH
3
Benzo[a]pyrene-
diol epoxide
Radiation and the electromagnetic spectrum
-----------------------------increasing energy---------------------------->
(E = h#)
Ultraviolet light
Includes UVA (320-400 nm), UVB (280-320 nm) and UVC (200-280 nm).
UVB causes most human mutations (UVA lower energy, UVC filtered by
atmosphere).

UV is non-penetrating, and so skin is the relevant tissue. Melanin in skin
protects against UV damage of skin cell DNA.

The DNA lesion caused by UV light is dimers of dipyrimidine sequences
in the DNA.


UV-induced DNA damage
Ionizing Radiation
Includes x-rays, $ rays, % particles, etc. Radiant energy surrounds us at
low levels!

As a tissue-penetrating agent, ionizing radiation leads to tumors of deep
tissues, most notably hematologic.

Effects caused by direct ionization of DNA with subsequent decay, or
secondary oxidation of DNA via induced oxygen radicals.

DNA lesions are strand breaks, usually with partially degraded sugars at
the termini, and base lesions. Base lesions and single-strand breaks
predominate, but double-strand breaks are most cytotoxic.


Endogenous/spontaneous DNA damage
Spontaneous Decay: in particular G residues are subject to a low rate
of spontaneous depurination. C residues are subject to spontaneous
deamination to U.

Reactive Oxygen Species (ROS)
H
2
0
2
, superoxide (O
2
-) and hydroxyl radical (.OH) are normally produced
in the body.

Lead to base damage (e.g. 8-oxo-G), base loss, and strand breaks.

~10
4
oxidative lesions occur/human genome each day!
Examples of common oxidative base lesions
Endogenous/spontaneous DNA damage
Replication Errors: Base substitutions, slippage, and strand breaks
can all occur during normal replication.

Microsatellites: Short mono- or di-nucleotide repeats subject to
slippage. Unrepaired slippage results in increased expansion or
contraction known as Microsatellite Instability (MSI).



Endogenous/spontaneous DNA damage
Telomere failure: because somatic cells lack telomerase, chromosome
ends eventually lose their protective telomere repeat, unmasking them
as double-strand breaks subject to breakage-fusion-bridge cycles.


Normal mitosis
Anaphase bridge
i.e. fused telomeres
Common events in DNA repair
Recognition that a DNA lesion exists.

Cessation of cell cycle.

Activation/recruitment of the repair machinery.

Removal of any damaged bases, adducts, etc.

Resynthesis of any required bases (polymerization).

Rejoining of broken DNA strands (ligation).
Checkpoint
Repair
Base Excision Repair
Lesions = small,
non-distorting base damage
Repair = very narrow excision
and resynthesis (1-5 nt)
Highly accurate
Base Excision Repair
Failure of BER leads to double-strand breaks during replication (also
forms the basis of recent therapeutic interventions):



No hereditary disorders of bases-excision repair are known frank BER
deficiency likely not compatible with life.

Dominant BER mutations in sporadic cancers may be linked to the
mutator phenotype of cancers (e.g. gastric cancer, prostate cancer).

Importance of inter-individual variability? XRCC1 polymorphisms, a
BER gene, are often claimed to be associated with cancer occurrence,
but the data are variable.
Nucleotide Excision Repair
Lesions = bulky, helix-distorting
base damage
Repair = somewhat wider excision
and resynthesis (12-30 nt)
Nucleotide Excision Repair
Failure of NER leads to mutations largely through the action of
replicative bypass polymerases that allow nucleotide addition in the
absence of base-pairing (e.g. PolN, Rev3)

Nucleotide Excision Repair
Global genomic NER: Repair of lesions in intergenic regions or non-
transcribed strands.
Xeroderma pigmentosum (XP) Seven complementation groups (A-G) of
recessive human NER deficiency characterized by photosensitivity and
dramatically increased rate of skin cancers. No general cancer
predisposition.

Transcription-coupled NER: Actively transcribed strands of DNA are
repaired preferentially, i.e. faster than non-transcribed strands.
Cockaynes Syndrome (CS) a recessive human disorder of growth failure
and premature aging, which, strikingly, does NOT include a predisposition
to cancer specific despite loss of transcription coupled NER.


Mismatch Repair
Lesions = base mismatch/loop

Recognition of damaged
strand as newly synthesized
strand (nick or methyl (M))
Repair = very wide excision
and resynthesis
(100s of nts)
Mismatch Repair
Failure of repair simply allows fixation of replication errors in the next
replication cycle.

Hereditary Non-polyposis Colon Cancer (HNPCC) Autosomally-
dominantly inherited hMSH2 and hMLH1 > hPMS1 and hPMS2
mutations act as classical tumor suppressors, leading to familial
colon cancer >> other cancer.

Microsatellite instability phenotype is noted in sporadic cancers also
(colon (15%) > others), revealing acquired MMR defects.
Double-strand break repair
Unlike all other DNA lesions, DSBs require the interaction of molecules
(i.e. the DSB ends) that are potentially quite distant.






Failure of repair leads to chromosome fragmentation and loss, and/or
rearrangement due to secondary repair pathways. (CIN = chromosomal
instability)

Thus, DSBs are the most difficult to repair and most severe DNA lesions.
The two main pathways of DSB repair
Homologous recombination (HR), in which a DSB is repaired using an
intact homologous donor chromosome (i.e. a second copy of the same
sequence).




Nonhomologous end joining (NHEJ), in which a DSB is repaired without a
donor chromosome by simply putting the end back together.



Of the two, NHEJ is comparatively less accurate because less
information is used to guide repair.
Homologous recombination
Familial Breast Cancer: BRCA1 and BRCA2 tumor suppressor genes
act in nuclear complexes with RAD51 and function in HR pathways.
Tumors from these patients show extensive chromosomal abnormalities.

Fanconis Anemia: FANCD2 gene, when ubiquitylated, is important for
activating HR repair of interstrand crosslinks, although the precise
mechanism is not well understood.
Dysfunction of the Fanconis pathway (numerous factors, all leading to
FANCD2 ubiquitylation) is observed in cancers such as ovarian cancer
known to be highly susceptible to cross-linking agents.


Nonhomologous end joining
Severe Combined Immune Deficiency (SCID) mutations
of some NHEJ proteins (DNA-PK, XLF, Artemis) are one
cause of failure of V(D)J recombination during B and T cell
development. Patients also have increased risk for
lymphoid malignancy.



Other very rare patients have been described with more
severe NHEJ or HR defects and cancer predisposition.
However, complete loss of either DSBR pathway is not
compatible with life.

How important are inter-individual variations/
polymorphisms?
Summary
cancer mutation genome lesion
DNA damage
DNA
repair
Nature
Nurture??
chemotherapy
radiation therapy
cell death