cancer genetics part 1

tumor suppressor examples

p53, Rb, APC, BRCA1

oncogene

accelerator
mutated genes that cause cancer

genome maintenance mutations lead to cancer because _____

mutations in DNA cause oncogenes
chromosome loss
more mutations = more likely to hit consequential genes contributing to cancer
BRCA1 repairs damaged DNA

cell fate mutations lead to cancer because _____

defects in differentiation cause uncontrolled growth
stem cells trapped in transit amplifying state

cell growth and survival genes lead to cancer because _____

signals that say divide are unleashed when they shouldnt be
no apoptosis
p53 signaling hub - normally inhibits cell cycle
cdk1 - normally promotes cell cycle
braf - pro mitogenic growth

passenger mutation

not directly contributing to cancer
silent mutation (no change in AA)
unrelated, benign
not going to be listed on cards

driver mutation

consequential in inducing cancer
common/important pathways
listed on cards

different combinations of mutations can ________

contribute to the same type of cancer

mutations in the same gene can be associated with ________

different types of cancers

how do normal cells become cancer cells

cancer relevant genes → altered function

hallmarks of cancer

Sustaining proliferative signaling
Evading growth suppressors
Resisting cell death
Enabling replicative immortality
Inducing angiogenesis
Activating invasion and metastasis

how is cancer a genetic disease

normal cell → (mutations, epigenetics) → cancer cell

mutations - direct changes to nucleotide
epigenetics - changing what genes are on/off

genetic changes - sequencing + genetic studies

2 things that cancer cells do are ____

1. grow/divide
2. spread (metastasis, goes through basement membrane)

cancer is caused by ___

dysregulation of genes

Two major ways in which genes become dysregulated

1. genetic - mutations
2. epigenetic - histone mods, DNA methylation

genetic causes of cancer

inherited/somatic
induced by mutagens (UV radiation, chemical, viruses, smoking, cells dividing)

epigenetic causes of cancer

histone modification, DNA methylation

mutagens

cause mutations
always carcinogenic

carcinogens

cause cancer
not always mutagens (could be epigenetic)

cancer cells are _____clonal in origin

monoclonal
1 cell was the source

2 pieces of evidence for monoclonal origin

1. myeloma: tumor of B cell precursors (antibody producing cells)
2. chromosomal aberrations

evidence for monoclonal origin:
myeloma

each B cell produces a different antibody
Antibody diversity is produced by VDJ recombination

a monoclonal myeloma cancer cell will express that antibody in high amounts

evidence for monoclonal origin:
chromosomal aberrations

translocations
all cells in tumor tissue will have the same translocation

stain using FISH

multistep process of cancer:
histopathological

normal
hyperplasia - excess # of normal cells
dysplasia - abnormal size/shape
^^^BENIGN^^^

neoplastic - breaks basement membrane

multistep process of cancer:
genetic

increasingly neoplastic
increasing # altered genetic loci

normal → loss APC → hyperplastic → hypomethalyation/activation → carcinoma

tumor progression takes ____

time
could be years after initiated to develop into cancer

tumor evolution

evolving units are cells
leads to clonal expansions

high mutation rates =

clonal diversification

monoclonal origin, but subsequent ____ can lead to ______

mutations can lead to heterogeneity

Where do cancer causing mutations come from?

inherited pre-disposition
knudson's two hit hypothesis

mutation terminology based on phenotypic effects

- Null, loss-of-function, gain-of-function, reduction-of-function
- hypermorphic (increase gene function), hypomorphic (decreased), neomorphic (new)

- Recessive
- Dominant
- dominant negative
- Haploinsufficient
- Autosomal -vs- X-linked
- Lethal, sterile

- Conditional (WT phenotype under one condition, mutant phenotype under other envi condition)

mutation terminology based on phenotypic effects:
hypermorphic
hypomorphic
neomorphic

increase gene function
decrease gene function
new gene function

mutation terminology based on molecular lesion

- Point mutation: change of 1 base
- missense: change aa
- nonsense: change to stop
- silent: no change in aa
- neutral: occurs in intergenic region
Indel (insertion/deletion)
Sometimes called DIPs (deletion/insertion) polymorphism
Point mutations
transitions: purine->purine, pyrimidine->pyrimidine
transversions: purine

missense mutation

based on molecular lesion
point mutation

change aa

nonsense mutation

based on molecular lesion
point mutation

change to stop

silent mutation

based on molecular lesion
point mutation

no change in aa

neutral mutation

based on molecular lesion
point mutation

occurs in intergenic (non-coding) region

indel mutation

based on molecular lesion
point mutation

insertion/deletion

Sometimes called DIPs (deletion/insertion) polymorphism

transition mutation

purine → purine
pyrimidine → pyrimidine

transversion mutation

purine → pyrimidine
vice versa

Where do cancer causing mutations come from?

Inherited Pre-disposition

ovarian has highest %

in the two hit hypothesis, initially all cells are ___

heterozygous
cell cycle control is normal
1 hit

in the two hit hypothesis, a mutation becomes ___

homozygous
cell cycle control is abnormal

inherited predisposition

For most inherited pre-dispositions to cancer the mutation is recessive at the cellular level, but leads to a dominant inheritance pattern.

Why?

cancer is familial

two hit hypothesis

Spectrum of genetic instability in cancer

Nucleotide-level alterations
- DNA damage repair pathways
- MMR or NER deficiency
- herediatry or somatic

Gross Chromosomal Rearrangements
- telomere erosion
- ionizing radiation
- non-allelic HR
- replication stress
- chromothripsis

Whole Chromosome Instability
- loss/gain of whole chrom
- spindle checkpoint dysfunction
- centrosome overduplication
- chromatid cohesion defects
- merotelic attachments

Discuss the evidence that most variability in cancer incidence between tissues is due to numbers of cell division undertaken

PAPER #0

Random mutations occurring during normal DNA replication of stem cell division explains the variation in cancer risk among tissues.

This is shown by the strong correlation between the number of stem cell divisions and the lifetime risk of cancer.

This explains why certain parts of the body are more likely to develop cancer, regardless of hereditary factors or exposure to environmental mutagens.

why is cell division mutagenic

Most dangerous thing a cell can do: DNA replication
not perfect

- mis-incorporation of bases (mistakes, presence of tautomers in DNA)
- incorporation of base analogs (more tautomeric shifts)
- breakage of unwound DNA (one backbone broken progresses into full DSB)

importance of proofreading

B: Pol delt synthesizes lagging strand
Q: Does proofreading activity contribute to genome maintenance?
A: mutate proofreading domain in mice
O: RECESSIVE (need 2 mutant alleles, low survival rate)
C: proofreading in important for survival

Normally, mutations missed by proof-reading can be ____

repaired

Where do cancer causing mutations come from?

- DNA replication
- Inherited

- Induced: mutagen exposure
- spontaneous

categories of mutagen + examples

physical: UV, x-rays, gamma rays, cosmic rays
chemical: ethidium bromide, smoking
infectious: HPV

How do viruses cause cancer?

1. carry oncogenes (RSV and src)
2. insertional mutagenesis
3. illegitimate recombination

How do viruses cause cancer?
1. carry oncogenes

do not have promoter/enhancer/silencer

RSV (Rous sarcoma virus)
has src oncogene - drives human cell to replicate

How do viruses cause cancer?
2. insertional mutagenesis

insertion next to, and activation of, cellular proto-oncogenes

random - may be next to genes with no proliferative activity
sometimes convert proto-onco to oncogene

Myc oncoprotein dysregulation

How do viruses cause cancer?
3. illegitimate recombination

full/partial integration of viral genome
eg. HPV = misregulation of viral genes

episome - circular extrachromosomal DNA
linearization & recomb with host cell

The nature of viral propagation affects the way in which a virus causes cancer

2 types of cancer causing viruses:

- Retrovirus: RNA genome. [Copied to DNA and
inserts into host chr]
TRANSCRIPTION

- DNA tumor viruses: DNA genome carried in host
cell, doesn't integrate into genome
TRANSLATION

Physical and Chemical mutagens:
Types of changes

Chemical:
endogenous - produced in body
1. metabolic products, reactive oxygen species
2. breakdown of toxins, EtOH -> formaldehyde

exogenous - external

Procarcinogens undergo _________ and become ______

cellular processing = ultimate carcinogen

Mutational signatures

- Pattern of mutations produced by a particular
mutational process
- Mutational process = DNA damage PLUS DNA repair/replication

- Different damaging agents -> different pattern
- History of past exposure to specific chemicals can sometimes be inferred by the mutational signature observed

EX: smokers have more G:C to T:A

Exposure to a mutagen may leave a
specific _______ in the DNA

signature

1) How has the profile of mutations been altered by smoking?
- more G:C to T:A

2) Come up with an explanation.
chemicals in smoke interact with those specific bases and produce a mutational signature

Where are mutations found
Within the body?

epithelial ells
exposed to mutagens
# of divisions

Where are mutations found
Within the genome?

CpG islands
- methylates C's get deaminated and looks like T

late-replicating genes - less time to be replicated = accumulate mut

regions associated with repressive chromatin marks - transcription coupled repair (better NER, nucleotide excision repair)
- NER mutations lead to other muts spread across genome

transcription factor binding sites - prevent access to repair

Where are mutations found
Within the genome?
CpG islands

methylates C's get deaminated and looks like T

Where are mutations found
Within the genome?
late-replicating genes

less time to be replicated = accumulate mutation

Where are mutations found
Within the genome?
repressive chromatin marks

regions associated with repressive chromatin marks

- transcription coupled repair (better NER, nucleotide excision repair)
- NER mutations lead to other muts spread across genome

Where are mutations found
Within the genome?
transcription factor binding sites

prevent access to repair

Does this graph show that some
nucleotides are more prone to damage
than others?

NO

sequencing of cancer

if mutation is neutral, there is no advtg for cancer
if mutation decreases function of p53, there is an advtg
= accumulation of inactive p53

= selection, more likely to have this
evolutionary process

tumor suppressor

brakes
inhibit cell growth

oncogene examples

Ras-Raf-MEK-ERK

protooncogenes

Normal cellular genes that regulate cell proliferation and differentiation that can become oncogenes.

Mutator Hypothesis

Loss of DNA repair genes leads to genome instability
increase mutation rate
cellular transformation

Modern sequencing techniques suggest that defects in DNA repair contribute to cancer when

repair pathway
- inactivated
- temporarily overwhelmed (high doses)

DNA editing
- enzymes deregulated

2 ways DNA double strand breaks can be repaired by ____

DSB rejoining (NHEJ, non-homologous end joining)
- used when no sister chromatid available (G1, S phase)

homologous recomb (HDR, homology directed repair)
- late S, G2, mitosis
BRCA1

NHEJ

non-homologous end joining
- error prone
- alignment between segments getting joined isnt informed in WT
- used when no sister chromatid/template is available (G1, S phase)


• Ends are recognized and processed
• Ends are bridged and then ligated

HDR

Homology Directed Repair
- Use undamaged homologous DNA in
sister chromatid
- used during late S, G2, mitosis

- Break recognized
- Resection (3'overhang)
- Rad51 binds ssDNA = invasion

BRCA1

BRCA1 functions in DSB repair
Evidence

1. localization
2. expose cells to DNA damaging agents (cisplatin) = death

3. LOF mutation in mice = illegitimate recombination
4. interaction (immunoprecipitation)

BRCA1 helps cells choose _____

NHEJ
- default if ∆BRCA1
- 1st proteins jump on, then pause, and BRCA kicks them off

or

HDR
- more common

diagram DNA repair pathways

Removal of NORMAL bases
- MisMatch Repair (MMR)

Repair of ABNORMAL bases
- Direct Repair eg. MGMT
- BER
- NER

diagram DNA repair pathways:
Removal of NORMAL bases

Mismatch repair
- Recognize normal structure, wrong location
- Especially important for repairing mistakes made by strand slippage

diagram DNA repair pathways
MisMatch Repair (MMR)

- Recognize mismatch - MutSα
- Identify recently synthesized strand - MutLα
- Excise nucleotide
- Resynthesize

diagram DNA repair pathways
Repair of ABNORMAL bases

Direct repair

Direct Repair
- MGMT - DNA alkyltransferase
- suicide enzyme - only used once
- expression in mice = resistance to MNU

diagram DNA repair pathways
Repair of ABNORMAL bases

BER

Base excision repair (BER)
- Recognizes altered bases with minimal helix distorting effects
- DNA glycosylases specialized to recognize a specific abnormal base
- Cleavage: APE 5', AP lyase 3'
- DNA pol, ligase

diagram DNA repair pathways
Repair of ABNORMAL bases

NER

- Entire nucleotide cut out
- Repairs lesions caused by exogenous agents eg. UV
- Large multiprotein complex.
- DNA pol, ligase

THYMINE DIMERS - bulky

Determining karyotype using the metaphase
spread/FISH technique

find sequence of interest in nucleus

1. block cells in metaphase

2. fix using chemical
- metaphase spread - break apart on glass slide

3. (myc. gene) probe from sequence
- normal or amplified?
DNA seq → probe → DNA comp. seq → label → fluorscent dye

*denature so probe can bind
should have: 4 dots, 2 copies of each gene
X X
(1) (1)

polyploid

genomes in which chromosomes are present in more than 2 copies

triploid

addition of haploid # of chromosomes

tetraploid

addition of diploid # of chromosomes

aneuploid

loss or gain of chromosomes

many cancer cells are aneuploid
- 90% of solid tumors, 75% hematopoietic cancers

- some are STABLY aneuploid - gained/lost but happy
- others UNSTABLE
(chromosome instability - CIN)
constantly restructuring
unstable karyotype

effects of aneuploidy

gene expression
Gene dosage imbalance
Protein deregulation
Metabolic alterations
Replication stress
Senescence

effects of aneuploidy
gene expression

Chromosome gains and losses typically result in a proportional change in the expression of genes on an affected chromosome

- i.e. an increase in gene expression from trisomic chromosomes
- a decrease in expression from monosomies

effects of aneuploidy
protein deregulation

- proteins function in complexes
- lead to proteotoxic stress inducing protein aggregation

effects of aneuploidy
replication stress

- more DNA that needs to be replicated (no time)
- hyper-recombination
- chromosome mis-segregation

effects of aneuploidy
senescence

- removed from cell cycle but not dead
- secretes molecules, modulates microenvironment

Does aneuploidy provide a selective
advantage?
trisomy 21

trisomy 21 - ALL CELLS ARE ANEUPLOID

tumor suppressive
- dec angiogenesis
- dec risk solid tumor

oncogenic
- inc myeloproliferation
- inc risk haematopoetic tumor

Does aneuploidy provide a selective
advantage?

its complicated

tumor suppressive
- Aneuploid cells are slowgrowing/unhealthy

oncogenic
- many CIN mouse models are cancer prone

How can Aneuploidy be both pro- and anti-
tumorigenic?

Aneuploid cells likely accumulate mutations to restore their fitness.

How might Aneuploidy aid tumorigenesis?

- Evolutionary flexibility: Increased genomic instability
-> increased genomic diversity
-> rare combos
-> selective advantage

• Senescent cells modify microenvironment
• Amplification of oncogenes/normal genes.
• Loss of tumor suppressors
• neoantigen-independent mechanism to promote tumorigenesis
(change protein structures, immune system doesn't recognize itself)

evidence that aneuploidy causes cancer

- aneuploidy appears early in tumor transformation
- CIN mice - cancer prone, dont have to treat with other mutagens
- mut that cause chrom missegreation in tumor cells are inc in inherited cancers

How does aneuploidy arise?

mis-segregation of chromosomes during mitosis.

Mechanisms that aid appropriate chr
segregation

• The SAC remains active until all chr are aligned.
• Sister chromatids stay attached until anaphase.

• Bipolar attachments of chr.
• Bipolar spindle allows equal chr segregation to 2 daughter cells.

• Cell cycle regulation