TYPES OF GENETIC INSTABILITY IN CANCER

Aneuploidy

Chromosome breakageDeletions and translocationsGene Amplification

HSRs - homogeneously staining regionsDMs - double minutes

Elevation of mutation rates

Epigenetic changes"CIMP" (CpG island methylator) phenotype

Chromosomal Instability in Cancer

MSI Negative MSI Positive

APC(D5S346)

BAT25

BAT26

D2S123

Mfd15CA(D17S250)

N T N T

Microsatellite Instability in Cancer

GENETIC BASIS OF INSTABILITY

Mismatch-repair defects

Base-excision repair defect

Cell-cycle checkpoint defects

p53 mutations (Li-Fraumeni syndrome)

Spindle-checkpoint defects (BUB1, MAD2)

Other defects in repair or recombination

Chromosome breakage syndromes

Pathways of DNA repair

O6-alkylguanine DNA alkyltransferase

suicide protein transfers methyl group

Base excision repair

glycosylase, AP endonuclease, DNA polymerase, ligase

Nucleotide excision repair

transcription-coupled and global

Cross-link repair

Fanconi’s anemia genes and BRCA2

Double-strand break repair

Homologous recombination and non-homologous end- joining

Heteroduplex and mismatch repair

Post-replication repair; translesion synthesis by pol eta, zeta, iota or kappa

Familial cancer syndromes with defects in DNA damage response

ataxia telangiectasia (AT), Nijmegen breakage syndrome, AT-like disorder

- cell cycle checkpoints and DNA repair (dsb repair). ATM, NBS1, MRE-11

Fanconi’s anemia (FA) - DNA repair (cross-link repair). BRCA2 and FANC-A, B, C, D1, and E

Hereditary non-polyposis colorectal cancer (HNPCC) – DNA repair (mismatch repair) and cell cycle checkpoints. hMSH2, hMLH1, PMS2, hMSH6

xeroderma pigmentosum (XP) – DNA repair (nucleotide excision repair, translesion synthesis) and transcriptional regulation. XPA, B, C, D, E, F, G and V

Familial breast cancer I – cell cycle checkpoints. BRCA1

Li-Fraumeni syndrome (LFS) - cell cycle checkpoints, DNA repair and transcriptional regulation. p53, Chk2

Bloom’s syndrome, Werner syndrome, Rothmund-Thompson syndrome – DNA repair and cell cycle checkpoints. Blm, Wrn, RecQL

HUMAN MICROSATELLITES

Repeat units of 1 - 5 base pairs

Tracts of ~ 6 - 30 repeat units

Highly interspersed

>100,000 tracts/genome

Many are polymorphic

Lynch & de la Chapelle, New Eng. J. Med. 348:919 (2003)

MISMATCH-REPAIR PROTEINS

Bacteria: MutH, MutS, MutL

Human: hMSH2 hMSH3 = MutS homologues hMSH6

hMLH1 hPMS2

= MutL homologues

mplexMSH2-MSH3 complex MSH2-MSH6 complex

MLH1-PMS2 complex

Familial cancer syndromes

Ataxia telangiectasia - cell cycle checkpoint function, DNA repair. ATM, NBS1, MRE-11

Fanconi’s anemia - DNA repair. BRCA2 and 5 other genes

HNPCC - mismatch repair, hMSH2, hMLH1, PMS2, hMSH6

Xeroderma pigmentosum - nucleotide excision repair and post-replication repair. 8 XP genes

Familial breast cancer I - S and G2 checkpoint responses. BRCA1

Li-Fraumeni syndrome – cell cycle checkpoint function and DNA repair. P53, Chk2

Bloom’s syndrome, Werner syndrome, Rothmund-Thompson syndrome – chromosomal instability. Blm, Wrn, RecQ

Checkpoint genes and cancer

ATM – mutated in ataxia telangiectasia, a familial cancer syndrome; related to DNA-PK and ATR; phosphorylates NBS1, Chk2, p53, Abl, BRCA1

P53 – mutated in Li-Fraumeni syndrome with early onset breast cancer and fibrosarcoma; transactivates p21Waf1, p48/XPE, 14-3-3

BRCA1 – familial breast cancer; interacts with BASC, ATM, ATR, BRCA2, Rad51 and Rad52

Checkpoints and carcinogenesis

Cancer is characterized by enhanced growth and genetic instability

Cell cycle checkpoints slow growth and preserve genetic stability

Defects in cell cycle checkpoint function enhance growth and genetic instability, thereby fueling malignant progression

Inactivation of p53-dependent G1 checkpoint function in diploid human

fibroblasts

HPV16E6 ablates p53 function by ubiquitin-mediated proteolysis

Use replication-defective retrovirus to transduce HPV16E6 or dominant-negative p53 alleles

p53 dominant-negative alleles (V143A, H179Q) compete for substrates

Flow cytometric analysis of cell cycle checkpoint response to DNA damage:

normal human fibroblasts

G1 checkpoint G2 checkpoint

DNA/PI

Pho

spho

-his

tone

H3

FIT

C-a

nti-B

rdU

P53-dependent G1 checkpoint function

20

40

60

80

100

120

140

10/2

7/200

0

11/3

/2000

11/1

0/200

0

11/1

7/200

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4/200

0

12/1

/2000

12/8

/2000

12/1

5/200

0

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2/200

0

12/2

9/200

0

1/5/2

001

1/12/

2001

1/19/

2001

1/26/

2001

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001

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001

2/16/

2001

2/23/

2001

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001

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001

3/16/

2001

3/23/

2001

3/30/

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001

4/13/

2001

4/20/

2001

4/27/

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001

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2001

5/18/

2001

5/25/

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001

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001

6/15/

2001

6/22/

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001

7/13/

2001

7/20/

2001

7/27/

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8/3/2

001

8/10/

2001

8/17/

2001

8/24/

2001

8/31/

2001

9/7/2

001

Weeks in Culture

Cu

mu

lati

ve P

DL

NHF1 HIT/LXIN NHF1 HIT/p53-A143VNHF1 HIT/p53-H179Q NHF1 HIT/E6

Inactivation of p53 extends cellular lifespan but leads to crisis; immortal

lines may emerge from crisis

Chromosomal aberrations in aging E6-expressing fibroblasts

Genetic instability in human fibroblasts

Finite lifespan Extended lifespan Immortal

G1 + G1 -G1 -

G2 + G2 +/- G2 +/-

diploid aneuploid,polyploid

aneuploid

What is the source of polyploidy?

E6-expressing cells start with diploid stable genomes

Attenuation of DNA damage G2 checkpoint function in ataxia telangiectasia is not associated with polyploidy

P53-defective cells are prone to polyploidy when they experience stress on the mitotic spindle

Polyploidization in aging E6-expressing cells

DNA inputs to the G2 checkpoint

Decatenation checkpoint monitors chromatid separation by topoII

Model of ATR/ATM-dependent G2 checkpoints

Catenated Chromatids

ATR

DNA dsb

BRCA1

G2 M

Chk1

Cdc25C

Cyclin B1/Cdk1Activity

Plk1

ATM/ATR

Cyclin B1/Cdk1Localization

Attenuation of decatenation checkpoint in aging E6-expressing cells

Expression of telomerase suppresses chromosomal

destabilization in aging E6-expressing cells

Aberrations per 50 cells• Cell Strain PDL Dicentrics/ Breaks/ %Polyploidy %Evading G2

Rings Exchanges Delay

• F7-neo 10 0 2 8 7 5

30 0 2 4 0, 2

• F7-E6 10 0 1 2 17 5

30 19 6 4 22, 48

58 25 38 33 133 66

• F7-E6-TRT(-) 58 40 18 36 88 11

• F7-E6-TRT(+) 58 0 2 3 9 7

0

20

40

60

80

100

120

140

160

normal

lym

phoblast

s

normal

fibro

blast

s

HeLa

DLD1

HCT116

DU145

HEC59

LoVo

"norm

al" b

reas

t epith

elia

Sum10

2

Sum14

9

Sum44

Sum18

5

normal

bla

dder

epith

eliaRT4

UM-U

C-3

TCC-SUP

J82

T24

per

cen

t o

f ce

lls

evad

ing

G2

del

ay

DNA damage Decatenation

Defective G2 checkpoint function in cancer lines

0

10

20

30

40

50

60

mutation status

per

cen

t ev

adin

g G

2 d

elay

wt N B

Figure 5. DNA damage G2 checkpoint function in normal human melanocytes and melanoma cell lines. Upper left panels: flow cytometric quantification of mitotic cells labeled with Anti-phospho-histone H3 antibody. Upper right panel: percent of cells in mitosis 2 h after 1.5 Gy IR, equivalent to the percent of cells evading IR-induced G2 delay. Bottom left panel: G2 checkpoint function in melanoma lines with wildtypeN-Ras and B-Raf alleles (wt, n=4), with mutant N-Ras (N, n=8) or mutant B-Raf (B, n=6).

Summary

• Cancers display two different types of genetic instability affecting microsatellites and chromosomes

• Microsatellite instability is due to defects in mismatch/heteroduplex repair and increases mutation rates

• Chromosomal instability is due to defects in cell cycle checkpoint function and may be driven by telomere erosion to the point of crisis

• Genetic instability increases the risk that clones activate oncogenes and inactivate tumor suppressor genes