University of Dundee

Chronic p53-independent p21 expression causes genomic instability by deregulatingreplication licensingGalanos, Panagiotis; Vougas, Konstantinos; Walter, David; Polyzos, Alexander; Maya-Mendoza, Apolinar; Haagensen, Emma J.Published in:Nature Cell Biology

DOI:10.1038/ncb3378

Publication date:2016

Document VersionPeer reviewed version

Link to publication in Discovery Research Portal

Citation for published version (APA):Galanos, P., Vougas, K., Walter, D., Polyzos, A., Maya-Mendoza, A., Haagensen, E. J., Kokkalis, A., Roumelioti,F-M., Gagos, S., Tzetis, M., Canovas, B., Igea, A., Ahuja, A. K., Zellweger, R., Havaki, S., Kanavakis, E.,Kletsas, D., Roninson, I. B., Garbis, S. D., ... Gorgoulis, V. G. (2016). Chronic p53-independent p21 expressioncauses genomic instability by deregulating replication licensing. Nature Cell Biology, 18(7), 777-789.https://doi.org/10.1038/ncb3378

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Download date: 18. Feb. 2022

b.

p21WAF1 Ki67 DAPI p21WAF1 Ki67

Figure 1 H

ead

&N

eck

Ca

L

un

g C

a

Uro

thel

ial

Ca

Cumulative data

p21WAF1 Ki67 Ki67 p21WAF1 DAPI

p21WAF1 Ki67 p21WAF1 Ki67 DAPI

a.

c.

40% 3.24

45% 1.58

10% 1.22 5% 1.58

39% 3.08

39% 4.30 15% 1.58

7% 2.55

38% 5.05

34% 6.20 19% 4.24

7% 1.58

p21-/ki67+

p21-/ki67-

p21+/ki67-

p21+/ki67+

p21WAF1-/Ki67+

p21WAF1-/Ki67-

p21WAF1+/Ki67-

p21WAF1+/Ki67+ Serial section IHC analysis IF analysis

a.

e.

Figure 2

OFF ON 4d-siCdt1siCdc6 ON 4d

G1

57.97 S

5.99

G2

19.02

>4N

8.89

G1

71.71 S

5.35

G2

17.68

>4N

0.78

b.

G1

29.94

S

26.31

G2

36.59

>4N

0.59

PI

EdU

Li-Fraumeni fibroblasts

(p53mut/null)

-

Cdc6

Cdt1

actin

p21WAF1

+ pBabe p21WAF1

+ - pBabe

actin

Cdc6

HT1080 p21WAF1 –IPTG-ON

(wt p53)

p21WAF1

Cdt1

H1299 p21WAF1 –Ponesterone-ON

(p53null)

actin

Cdc6

p21WAF1

Cdt1

:

:

0d 2d 4d 0d 2d 4d Timeline:

Transcriptomics ProteomicsCDT1

CDC6

ORC1

DNA Replication Licensing

CENPF

INCE

SPDLY

PLK1

KIF23

BUB1B

BOREA

ERC6L

Mitotic Machinery

Strong Overexpression

No Differentiation

Strong Suppression

KIF2C

Saos2 p21WAF1 TetON

OFF ON 96h

+shp53 -shp53

Ap G1 S G2/M

2.53 45.19 17.51 28.25

Ap G1 S G2/M

26.07 55.03 6.06 10.14

Ap G1 S G2/M

4.14 70.54 5.65 15.95

p21WAF1

p53

Cdt1

Cdc6

actin

OFF

-sh

p5

3

+sh

p5

3

ON 96h

HT1080 p21WAF1 –IPTG-ON

Saos2 p21WAF1 TetON

d.

PI PI PI

c.

Cdt1

SET8

actin

p21WAF1

Saos2

0 2 4 6 8 10

% cells with DNA content 4N

OFF

ON 4d

ON 4d-siCdt1/Cdc6

0 10 20 30 40

05

101520253035

<0.2 <0.4 <0.6 <0.8 <1 <1.2 <1.4 <1.6 <2 >2

OFF

ON 4d

CIdU/IdU ratio fork asymmetry

% R

epli

cati

on

fo

rks

OFF ON48h ON 96h

ctl

si

siC

dc6

si

Cd

t1

siC

dc6

+si

Cdt1

Saos2 p21WAF1 TetON

0 hours

24 hours

96 hours

Tail moment (arbitrary units)

Vinculin

Cdc6

Cdt1

p21WAF1

γH2AX

DNA damage

Replication fork asymmetry Replication fork speed

CldU/IdU *p (T-test) n

OFF 1.03 0.24 498

OFF vs ON 4d 1.09 0.36 0.001885 377

Total fork speed:

((CldU+IdU)/40min)

CldU 20min + IdU 20min

OFF ON 4d

0

5

10

15

20

25

30

35

<0.2 <0.4 <0.6 <0.8 <1 <1.2 <1.4 <1.6 <2 <3

OFF

ON 4d

Kb/min

% R

epli

cati

on

fo

rks

CldU kb/min Idu Kb/min Total speed p (T-test) n

OFF 1.16 0.26 1.17 0.33 1.16 0.27 498

ON 4d 0.9 0.43 0.88 0.47 0.89 0.43 7.50E-24 377

OFF+ctl si ON 96h+ctl si

ON 96h

+siCdc6/siCdt1

DA

PI

p2

1W

AF

1

53

BP

1

Mer

ged

Cdc6

Cdt1

p21WAF1

0h 12h 48h 96h

ctl si

48h 96h

siRNAs

0h

actin

γH2AX

siCd

c6

γH2AX

siCd

t1

actin

H2AX

H2AX

Cdc6

γH2AX

H2AX

Cdt1

p21WAF1

siCd

c6+

siCd

t1

No of 53BP1 foci per cell

γH2AX

Ed

U

OΝ 48h

16.42

33.07

38.65

6.42

ON 96h

73.69 0.73

19.70 5.89

OFF

29.56 9.13

53.30 9.01

0

20

40

60

80

100

% o

f E

dU-p

osit

ive

cell

s w

ith

incr

ease

dγH

2AX

OFF

ON48h

ON96h

*

*

OFF+ctl si ON 96h+ctl si OΝ 96h+siCdc6/siCdt1

PI

γH

2A

X

2n 4n 2n 4n

2n 4n

S-phase

0

10

20

30

40

50

% o

f γH

2A

X c

ell

s gate

d in f

ram

e

OFF+ctl si

ON96h+ctl si

ON96h +siCdc6/siCdt1

* *

Figure 3

a. b.

d.

c.

e.

* p < .002

0 10 20 30

OFF+ctl si

ON96h+ctl si

ON96h +siCdc6/siCdt1

0 10 20 30 40

0 10 20 30 40

0 10 20 30 40

0 10 20 30 40

0 10 20 30 40 50

% EdU positive cells with increased γH2AX

Saos2 p21WAF1 TetON

MUS81

actin

p21WAF1

ctl si siMUS81

γH2AX

Ed

U

OFF+ctl si

49,66

45,63

3,19

1,52

ON96h+ctl si

44,18

18,87

33,97

2,99

OΝ96 h+siMUS81

8,45

74,63

4,72

12,20

OFF+ctl si

ON96h+ctl si

ON96h +siMUS81

OFF 96h

ON 96h

ON 96h

ctl

si

ctl

si

siM

US

81

OFF ON96h

Brd

U

p2

1W

AF

1

DA

PI

OFF ON96h

a. b. Non-denaturating conditions

DA

PI

c.

d.

e.

Figure 4

p2

1W

AF

1

0

1

2

3

4

5

6

ON 96h OFF

*

Ra

tio

γH

2A

X/H

2A

X

(arb

itra

ry u

nit

s)

ctl si

siRad51

0

2

4

6

8 *

Ra

tio

γH

2A

X/H

2A

X

(arb

itra

ry u

nit

s)

ctl si

siRad52

g.

E2F4 ChIP

Fo

ld c

ha

ng

e

(arb

itra

ry u

nit

s)

OFF

ON 96h

0

20

40

60

80

100

120

h.

p21WAF1

γH2AX

0 20 40 60

% cells with increased γH2AX

ctl si

siRad52

OFF

ON 96h

γH

2A

X

PI

3,80

OFF

27,84

ON 96h

si ctl

22,05

OFF

45,61

ON 96h

siRad52

γH

2A

X

PI

OFF+ctl si

ON96h+ctl si

ON96h +siMUS81

f.

Rad52

actin

H2AX

ctl

si

ctl

si

siR

ad5

2

siR

ad5

2

OFF ON 96h

ON 96h OFF

OFF ON 96h

85

85

E2F4

input

OFF ON 96h

130

130

NC

input

E2F4 (85bp):

1000Rad51

1000Rad51NC (130bp):

RPA

Rad51

p21WAF1

γH2AX

actin

H2AX

ctl

si

ctl

si

siR

ad5

1

siR

ad5

1

OFF ON 96h

0 10 20 30 40

0 10 20 30 40

OFF 96h ON 96h

siC

dc6

/siC

dt1

ct

r si

si

Cdc6

si

Cdt1

Sao

s2 p

21

WA

F1 T

et-O

N

sip73

% senescent cells

Figure 5

96h

0h

% senescent cells

96h

0h

% senescent cells

OFF

Saos2- p21WAF1

p14ARF

p21WAF1

actin

ON 96h

p16INK4A

a. b.

c.

p21WAF1

actin

p73

p21WAF1

actin

p73

0 10 20 30 40

96h

0h

% senescent cells

96h

0h

% senescent cells

0 10 20 30 40

96h

0h

0 10 20 30 40

0

20

40

60

80

OFF ON 2d ON 4d ON 20d

% B

rdU

inco

rpora

tion

4 d

ays

20 d

ays

ON

20 d

ays

OF

F

DAPI

Escape

p21WAF1 Cyclin A

2 d

ays

Saos2 p21WAF1 Tet-ON

ON 96h

53.33 22.86

1.16 22.65

ON 20 days

28.49 55.63

2.98 12.90

OFF ON (Escape)

20 days

0 50 100

+/-

-/+

+/+

% of CyclinA/p21WAF1 cells

a.

d.

g.

c.

0

0,25

0,5

0,75

1

OFF ON

Nu

clea

r are

a (μ

m2)

0

10

20

30

40

50

60

70

OFF ON 4d ON 20d

% o

f E

dU

/p21

WA

F1

posi

tive

cell

s

p21WAF1

EdU

OFF

55.28 0.24

0.10 44.04

OFF ON 4 d

ays

20

day

s 0 d

ays

2 d

ays

Escape

*

b.

e. f.

Cdk2-pT160

p21WAF1

Cdk2-total

OFF ON 4d ON 20d

actin

p73

j.

Figure 6

0 1 2 3 4 5 6

Normalized TM

OFF

ON 4 d

ON 10 d

ON 20 d

ON 25 d

OFF ON 4 d ON 10 d ON 20 d ON 25 d

DA

PI

p2

1W

AF

1 B

rdU

ON 20 days

% total p21WAF1 cells

% total BrdU/p21WAF1 cells

0

20

40

60

80

100

h.

OF

F

ON

45

d

0 20 40

Number of colonies per 2500 cells

OFF ON 45d

0 1 2

Clone diameter (mm)

0 0,5 1 1,5 2 2,5 3

IC50 (μg/ml)

Taxol Cis-platinum Doxorubicin

OFF

ON 20d

ON 20d +

OFF 10d

i.

0 100 200 300 400

OFF ON 45d

Invasion (% of control)

Novel translocations (N: 103) in “escaped” vs OFF cells

FRA3B

Lo

g2

Rat

io

Large scale loss

Log2R

atio

Figure 7

Chr. 1 Chr. 2 Chr. 3 Chr. 4 Chr. 5 Chr. 6 Chr. 7 Chr. 8 Chr. 9 Chr. 10 Chr. 11 Chr. 12 Chr. 22 Chr. 21 Chr. 20 Chr. 19 Chr. 18 Chr. 17 Chr. 16 Chr. 15 Chr. 14 Chr. 13

Probe gain Probe loss Potential chromoanasynthesis Potential chromothripsis

Chro

moso

mes

5.92 Kbp

20.1 Kbp

8.38 Kbp

14 Kbp

105.4 Kbp 90.64 Kbp 147.42 Kbp

50.05 Kbp

13.67 Kbp

15.06 Kbp

42.98 Kbp

71.89 Kbp 350.66 Kbp 128.18 Kbp

Chr. X

a.

b.

0

10

20

30

40

50

60

70

80N=100 nuclei

OFF

Tota

l N

o o

f m

icro

nucl

ei

ON 30d

c. d.

p53-independent overproduction of p21WAF1/Cip1CRL4Cdt1

SCFSkp2

Cdt1

E2F1/2

+

Cdc6+

Replication Stress: Re-replication DNA damage Rad52 error-prone repair

oversatu

ration

Genomic

Instability

and

Cancer

Progression

e.

Micronuclei in “escaped” cells

aCGH analysis of “escaped” vs OFF cells

Concordance between aCGH and deep sequencing (NGS)

Chromosome 1

NGS aCGH

Chromosome 5

aCGH NGS

Chromosome X

aCGH NGS

Chromosome 8

aCGH NGS