Irmgard IrmingerIrmgard Irminger--FingerFinger

Biology of Aging Laboratory and Monitoring LaboratoryUniversity and University Hospitals of Geneva, Switzerland

Functions of BARD1 and associated proteins and its

application in cancer prognostics and treatment

Tumor suppressor BARD1Tumor suppressor BARD1patented:patented:

1.Use as vaccine1.Use as vaccine2. Use for cell killing2. Use for cell killing

3.Use as prognostic marker3.Use as prognostic marker

Irmgard IrmingerIrmgard Irminger--FingerFingerUniversity and University Hospitals GenevaUniversity and University Hospitals Geneva

Clinical application of BARD1: background and principlesThe “normal” functions of BARD1 BARD1 expression in tumors:

in breast and ovarian cancersin lung cancer

BARD1 expression the human cytotrophoblast:a non-malignant cells with an invasive phenotype

AND in amnion fluid-secreted by the cytotrophoblast?

Detection of BARD1 in serum for cancer screening?

Immunogenic BARD1 applied for tumor cell killing

Genes implicated in epithelial cancers: predisposition genes (tumor suppressor genes)

• p53 (lung, colon, breast, prostate)• BRCA1 (breast, ovary, prostate) • BRCA2 (breast, ovary)• BARD1 (breast, ovary, uterus,

– and other proliferative tissues)

Function: repair, apoptosis (cell death)

Old Age Cancers are derived from epithelial cells

BCC Basal cell carcinomaSCC Squamous cell carcinoma

DePinho, 2000

Accumulation of damage during aging

agingaging

environmentalinsult

apoptosis

long-term loss offunctional tissue(Alzheimer, heart

failure etc.)

decisionmaking

cancercancer

Accumulation of endogenous

damage

damaged cellsabnormal function

abnormal activationabnormal growth

damage

repairrepair

BARD1BARD1

BARD1BARD1

BARD1 and its interacting proteins:a converging vehicle

Irminger-Finger et al., Bioch. 1999; Irminger-Finger et al., Mol Cell 2001; Feki et al. & Irminger-Finger, BOR 2005

P53DNA-PKapoptosis

BARD1- BRCA1interaction

p53

Rad51

RNA-Pol II

RHA

PCNA?

BRCTdomain

Ankyrin repeats

Ubiquitin-ligaseactivity

BRCA2

BAP1BACH1

PolyadenylationNFk-B binding

Phospho-binding

Transcription activation

RING

BARD1 and BRCA1 mutations predisposing to breast cancer

HumanBARD1 RING ANK BRCT

Q513HC557S

N295SK312N

358D364G1765C

G1743CA1009T

A957G 1144del21

A1481G

G1592A

HumanBRCA1

RING BRCT

C>GC>G Y>AY>AMissense mutations

No one like BARD1

BARD1

BRCA1

Arabidopsis2 hypoth.p.

ArabidopsisCAC05430C. elegans3 hypoth. p.C. elegans6 hypoth. p.

C. elegansT15566

C. elegansT15564

RINGRING ANKANK BRCTBRCT

Joukov, Livingston, PNAS, 2001

Xenopus

BRCA1BARD1

Irminger-Finger and Leung, IJBCB 2002

transfection of BARD1

BARD1 induces apoptosis in a p53-mediatedpathway

B pcBARD1 pcBQ>HS P S P

p53

IP: α-BARD1

BARD1

non-treat. doxoS P S P

AIP: α -p53

BARD1

PCNA

IP: α -BARD1 non-treat. doxo

S P S P

p53

PCNA

p53+/+ p53-/-

BARD1 binds to p53BARD1 binds to p53

BARD1 stabilizes p53BARD1 stabilizes p53

Irminger-Finger et al., Mol Cell 2001

Mapping of the apoptotic regionpc

DNA3

BARD1

∆BR

CT∆

ANK

RING

∆RI

NG

ANK2

APORING ANKYRIN

BARD1BRCT

NLS131 NLS 408 NLS 693BARD1∆ RING∆ BRCT

ANK2RING

∆ ANK

Q564HQ564HC557SC557S

yes

yesyes

yes

yesno

noBARD1 Q564H

X?

Deletion mutants lacking the BRCA1 interaction domain are efficient in apoptosis induction and have increased protein stability

BARD1 is upregulated in response to cellular stress

DoxorubicinTAC-2 cells

On On the protein levelthe protein level

Western

On On the mRNA levelthe mRNA level

RT-PCR

UV - 1 4 - mindoxo - - - 10 µg

BARD1

p53

ES cells

Mechanism of apoptosis induction by BARD1

• Exogenous expression of BARD1 leads to apoptosis

• BARD1-induced apoptosis depends on functional p53

• Upregulation of BARD1 results in stabilization of p53 protein

• Repression/deletion of BARD1 results in apoptosis resistence

BARD1 deficient tumor cells: NuTu-19 resistent to apoptosis

BARD1

ANKRING BRCT

H-300PVC JH-3

0.8KB BARD1In1

In2 In3 In4 765 8 In9

In1In2 In3 In4

In7In6In5 In8 In9

InInIn In

2KB BARD1

C29

3T

H-300 JH3PVC

293T

NuTu-

1929

3TNuT

u-19

NuTu-

19BARD1

A

0.8 kb

2.2 kb2 kb

M p53 BARD1B

Feki et al., & Irminger-Finger, Oncogene 2005

BARD1 required for p53 phosphorylation

C

doxoNuTu-19TAC2

- ++-

p53

BARD1

p-p53

actin

Apoptotic treatment

HEK NuTu-19 BARD1

BARD1

p53

p-p53

- ++-

actin

BARD1 transfection

Exogenous expression of BARD1 can induce apoptosis in NuTu-19 cells

0

5

10

15

20

25

30

35

40

45

50

NuTu-19 NuTu-19 + Doxo

NuTu-19+ BARD1

% o

f apo

ptos

is

TAC-2 TAC-2+ Doxo

NuTu-19

TAC2

BARD1 colocalizes with phospho-p53

DAPI FITC Phospho-p53-Alexa 555 MERGE

A DAPI FITC P53-Alexa 555 MERGE

Mapping p53 binding regionIP p53

BARD1 ∆-RING ∆-HIND ∆-BRCT ANK RING EGFP

S P S P S P S P S P S P S P

G215CG176CG1743CA1009T

A957G

1144del21 A1481G

G1592A G2354A

apoapo

yes

yesyes

yes

noyes

100

80

60

100

0

p53p53bindingbinding

60

mBARD1

RING (aa 1-266) EGFP

ANK (aa 394-604) EGFP

∆ BRCT (aa 1-604) EGFP

∆ HIND (aa 394-765) EGFP

∆ RING (aa 137-765) EGFP

BARD1 (aa 1-765) EGFP

RING BRCT

NLS 131 NLS 408 NLS 693

ANK

Feki et al., and Irminger-Finger, Oncogene 2005

BARD1 induces apoptosis by catalyzing phosphorylation of p53

• BARD1 binds to p53 and to phosphorylated p53 • BARD1 binds to Ku-70, the catalytic subunit of

DNA-PK but not ATM• Exogenous expression of BARD1 in BARD1-

deficient cells induces p53 phosphorylation• Exogenous expression of BARD1 in BARD1

deficient cells restores apoptosis sensitivity

Co-localization of BRCA1 and BARD1 with repair proteins in “nuclear dots”

+BARD1

+BARD1

Jin, Baer, JBC 1997; Scully, Livingston, Cell 1997

pEGFP BARD1-EGFP ∆RING-EGFPA

....but not only “nuclear dots”

∆BRCT-EGFP ANK-EGFP RING-EGFP

RING-EGFP

0102030405060

EGFP-N1

BARD1-EGFP

RING-EGFP

∆-RING-EGFP

∆-HIND -EGFP

∆-BRCTEGFP

ANK-EGFP

%ap

opto

tic G

FP+

cells

B

BARD1 translocates to the cytoplasm during apoptosis

T=6 T=12

T=24 T=48

Jefford et al., & Irminger-Finger, Oncogene 2004

Intracellular localization of BARD1 upon apoptosis induction

non treated UV

BA

RD

1B

RC

A1

BARD1 is translocated to the cytoplasm during apoptosis

Ab: PVC

Total cell extract0 3 4 5 6 7 14 22 24 h

*

**

Nucl. extract

Ab: C20

Cytopl. extract

Ab: C20

0 3 4 5 6 7 14 22 24 h 0 3 4 5 6 7 14 22 24 h

p67

The C-terminal p67 is translocated to the cytoplasm and is immunogenic and anti-tumorigenec in an animal model of colon cancer (Gautier, Irminger-Finger et al., Cancer Res 2000).

Apoptotic function of BARD1 in vivo

• In morphogenesisspermatogenesis

• In homeostasisSpleen, bone

• In tumor suppression

Cytoplasmic localization of BARD1 in vivo

BARD1 N-19A BARD1 PVC

B BARD1 N-19

BARD1 upregulation in vivo by ischemia

Core region Penumbra

Left hemisphere

Left hemisphere

BRCA1 BARD1BRCA1

Apoptosis and (meiotic) repair during spermatogenesis

Bcl-xPMB4SCFIL-4LIFGas6bFGF

SCFBcl-xLtestosterone

LIFCNTF

PGCSpermatogonia Spermatocytes Spermatids Spermatozoa

Apoptosis Apoptosis Apoptosis Apoptosis

- +

TGFß Bax

+

Bax

- +-Bcl-xLtestosterone

Gonocytes

- -

BARD1?BARD1? Modified from:

Print, Bioassays, 2000

Schematic diagram of seminiferous tube

BARD1 expression is correlated with apoptosis markers

A

b

aa

b

anti-BARD1

TUNEL

Feki et al., & Irminger-Finger, BOR 2004

B activated caspase 3 anti-BARD1

N-19b

b

a

a

c d

d

N-19

JH3

c

e

ef

f

BARD1 and BRCA1 expression overlap partially

Ab

a

baBAR

D1

5x 20x 20x

B

c

c dd BR

CA1

5x 40x 40x

C

5x

Different BARD1 isoforms are expressed at different stages

A PPjrG PL SR

97 kD

C-2074 kD Western blot

97 kDJH-3

74 kD

97 kD

74 kDJH-2

B G P PjrL P SR

2300 bp2000 bp

RT-PC

R

BARD1

GAPDH

BARD1β lacks the RING finger

BARD1βIn1

In2 In3 In4In7In6In5 In8 In9

BARD1ANKRING BRCT

Q>HQ>H

N-19 C-20JH-2 JH-3PVC

BARD1β is an efficient apoptosis inducer

0102030405060708090

100

% c

elld

eath

CGR8 BARD1b BARD1bBRCA1

BARD1 BARD1BRCA1

Doxo

p53BARD1b BARD1

BRCA1BARD1bBRCA1

BARD1CGR8 Doxo

BARD1 expression associated with abortive spermatogenesis

Obstructive Azoospermia Sertoli cell SyndromeA

B d

12

3

ccryptorchidy

Presumed tumor suppressor pathways ofBARD1

DNA repair pathways(e.g.ubiquitination)

p53

Cell cyclearrest

Other stabilizingsignals?

BRCA1

11BARD1

BARD1BARD1

BRCA1

BARD1

22

BARD1

Cleavageor isoformof BARD1

33

Irminger-Finger et al., Mol Cell 2001

p53

BARD1 p53

p53p53

apoptosisapoptosis55

66 immunogenic

44

BARD1

Expected loss of BARD1 in tumorigenesis

Carcinogenic stresstranscription activation

apoptosisBARD1BARD1 p53p53

p53 turnover

PPser15ser15

- inhibition of degradation - direct stabilization

Carcinogenesis

-loss of BARD1 -loss of p53

BRCA1mutatedor deleted

However, BARD1 is upregulated in tumors

N19 40x

b

N19 40x

a

N19 5x b

a

CC

CEn

CSe

CM

uCN19 C20

5x5x

5x5x

5x5x

5x5x

BARD1 expression is specific for tumor type

A

Expression of N- and C-terminal epitopes

40x

5x 5x

40x

N19 C20

5x

40x 40x

5x

N19 C20

0

10

20

30

40

50

60

70

80

90

100OB18 OB41OB42 OB15OB16 OB44OB43 OB11OB35 OB12OB38 OB39OB40 OB14OB13 OB36OB37 OB31OB33 OB29OB34 OB30OB32 OB2OB3 OB4OB9 OB17OB23 OB28OB24 OB25OB5 OB27OB22 OB26OB21 OB10OB19 OB20OB1 OB6OB7 OB8

CCC EnC SeC MuC

% o

fN19

pos

itive

cel

ls

Correlation of BARD1 expression with tumor grade

OB16 A1 Clair cell carcinoma C20 x40

BARD1 but not p53 is correlated with tumor type

40x 40x

40x40x

N19 p53A

010

20

3040

5060

7080

90

100

CCC EnC SeC MuC

N19C20p53

% o

fpos

itive

cel

ls

BCorrelation of BARD1 and p53 with histological type of

ovarian carcinoma

Figure 3

Correlation of BARD1 expression with tumor grade and stage

0

5

10

15

20

25

30

35

G1 G2 G3

N19C20p53

% o

fpos

itive

cel

ls

Correlation of BARD1 and p53 with differentiation in sporadic breast cancer

0

10

20

30

40

50

60

70

T1 T2 T3 T4

N19C20p53

% o

fpos

itive

cel

ls

Correlation of BARD1 and p53 with tumor size in sporadic breast cancer

D

No correlation of BARD1 and p53 expression in NSCLC

0102030405060708090

100

adenosquam

adenosqu large

6159 103566609 80408315 83411708 1274012748 11761659 85328664 1227811638 127566634 80759977 1276711601 11031531 83668930 103627987 901810319 66617723 865012111 98446699 81008981 114007993 1120311605 12500

Distribution of N19 expression in different pathological types

5x

N19 40x

5x

p53 40x

A B

% o

fN

19 p

o si ti

v e c

ells

Figure 5

No correlation of BARD1 expression with tumor grade and

stage in NSCLC

BARD1(N19/C20) and p53in different pathological type

0

10

20

30

40

50

60

70

80

90

100

G1 G2 G3 G1-3

N19C20p53

0

10

20

30

40

50

60

70

80

90

100

stage I stage II stage III stage IV

N19C20P53

BARD1(N19/C20) and p53in different pathological grade

BARD1(N19/C20) and p53in different stages

C

% o

fp o

sitiv

e c e

lls

% o

fp o

sitiv

e c e

lls

% o

fp o

sitiv

e c e

lls

0102030405060708090

100

adenosquam

adensqularge

N19C20P53

A

C-terminus

1441-2252

N-terminus

74-1481

hGAPDH

OB C+ 8 8* 9 21 23 24 24* 25 33 33* 34 35 37 C-

Tumors express an aberrant form of BARD1

2252

74

1441

1481783

G1765C

G1743CA1009T

A957G 1144del21

A1481G

G1592A

Figure 6

BARD1 is transcriptionally altered in rat ovarian cancer cells

and in human cancers?

BARD12.2 kb

ANKRING BRCT

H-300 PVC JH-3

BARD1δ0.8 kb

8 In9

In1

In2

In3

In4

In7

In6

In5

In8 In

9

InIn

1In

2

804-826

1618-1641

BARD1γ2.0 kb

2252

74

1441

1481783

Figure 5A-C

BARD1 expression in human cancers

Elevated expression of BARD1 in cancerCytoplasmic localization of BARD1 in tumorsLoss of 5’region Expression is correlated with tumor type, grade and stage in breast and ovarian cancerNo correlation in NSCLCNo correlation of BARD1 and p53 expression

BARD1 expression in human cytotrophoblast

5x40x 40x

BARD1 N-19 BARD1 WFS

BARD1ANKRING BRCT

Q>HQ>H

N-19 C-20JH-2 JH-3WFS

BARD1 expression in placenta at 12 weeks of pregnancy

20x 40x

BARD1 N-19

Immunohistochemistry of human placenta at 12th weeks of pregnancy. BARD1 N-19 antibody was used on sections of human placentas of the first trimeter of pragnancy. Representative images at 12 weeks of pregnancy demonstrate BARD1 redistribution to the brush border membrane. This subcellular localization suggests that BARD1 might be secreted.

BARD1 expression is correlated with apoptoticmarkers

40x5x

Activated Caspase 3

Identification of potential apoptotic cells. Since BARD1 expression can induce apoptosis in vitro and in vivo, activating a p53-caspase pathway, we stained placenta sections with antibodies against acitvated caspase 3. A complete correlation of BARD1 expression and activated caspase 3 expression can be observed. However, whether the BARD1 expressing cells actually undergo apoptosis can not be determined by these experiments.

BARD1 expression in invasive cells

Only BARD1γ expressed in invasive cells

Ab: PVCAb: WFS

5 weeks 12 weeks

BARD1-C20 40xBARD1-C20 40x

BARD1-PVC 40xBARD1-PVC 40x

BRCA1 40x BRCA1 40x

Differentially spliced isoform of BARD1 in cytotrophoblast and placenta lack RING

finger

T P M CRT-PCR

BARD1

actin

2,3kb2kb

A

ANKRING BRCT

Q>HQ>H

JH-2 JH-3

BARD1γIn1

In2 In3 In4In7In6In5 In8 In9

BARD1

N-19 C-20PVC WFS

B

BARD1 is secreted from CTBs in cultureB

AR

D1

(UA

/mio

cells

/ml)

0

20

40

60

80

100

120

CTB (2) MCF-7 MDA-MB231CTB (1)

In cell lysatesA

CTB (2) MCF-7 MDA-MB231BA

RD

1 (U

A/m

ioce

lls/m

l)

0.01.02.03.04.05.06.07.08.09

In medium of cell culturesB

PMAdoxocontrol

0.01.02.03.04.05.06.07.08.09

0-24 h 24-28 h

In medium of CTBsC

Quantification of BARD1 by ELISA

Coating C 20 (1/200) Coating N19 (1/200)Tampon

Pool Liq. Amn. 1/4

Pool Liq. Amn. 1/20Pool Liq. Amn. 1/100MC CTB (conc 8x)

Serum PB 2.10.03 ndlSerum pool Femme enceinte ndlSerum AM 2.10.03 ndl

C20 biot1/1000

C20 biot1/1000

WFS 1/100

WFS 1/100

PVC 1/100

PVC 1/100

Conclusions• BARD1 has a novel cellular function in

CTBs and placenta– Regulated at different times of pregnancy– Regulated expression of different epitopes

• Cytotrophoblast is expressing full length and splice variant γ of BARD1

• BARD1 is secreted from CTBs• A function of BARD1

– in invasion?– in angiogenesis?

Animal cancer model for tumor cell killing

NuTu-19 ovarian cancer cells

After 5 weeks 100%

Metastases detection by photo medicine

A

GFPGFPretroviral vector

liver

diaphragma

digestive tube

B

visual light

omentum

FITC

peritoneum

Transduction of NuTu-19 cells with lentiviralvectors expressing BARD1 or GFP

Packaging plasmids: pCMV DR8.92, pCMV DR8.93 (core and enzymatic components of HIV-1)Envelop plasmid: pMDG (VSV-G protein)Transducing vectors: pLOX/TW-BARD1 or pLOX/TW-rTTAh

Ovarian cancer Model

GFPGFP--cancer cancer cellscells

Gene therapyfor cell killing byviral delivery of

Inducible BARD1

pCMV DR8.92, pCMV DR8.93pMDGpLox TW BARD1 or pLox/TW-rTTAh

293T cells

Transduced NuTu cellsexpressingGFP or

BARD1 and rTTAhproteins

Apoptosis induction by inducible BARD1

A BNuTu-19 cells

NuTu-19 cells transduced with

pLOX/TW-gfp lentiviral vector

NuTu-19 cells transduced with 50X concentrated pLOX/TW-gfp lentiviralvector

GFP expression

GFP expression

NuTu-19 cells

NuTu-19 cells transduced with

pLOX/TW-gfp lentiviral vector

NuTu-19 cells transduced with

pLOX/TW-gfp lentiviral vector

and induced with Doxycyclin (2µg/ml)

0

10

20

30

40

50

60

NuTu NuTu-PLOX/BARD1

+ 2.5 ugDoxycycline

+5ugDoxycycline

+10 ugDoxycycline

+15 ugDoxycycline

+20ug ofDoxycycline

C 50

0 1 ug/ml 2ug/ml 4ug/ml 8ug/ml 16ug/mlDoxycyclin

Death rate after GFP induction with Doxycyclin

Death rate after BARD1 induction with Doxycyclin 40

30

20

10

0

Diaphragm

Thymus

Omentum

Nutu-19-EGFP tumors in Fisher rat

Diaphragm Omentum Thymus

Presumed tumor suppressorpathways of BARD1

DNA repair pathways(e.g.ubiquitination)

BRCA1

apoptosisapoptosis

11

22BARD1

p53

Cell cyclearrest

Other stabilizingsignals?

BARD1

BARD1

BRCA1

BARD1

BARD1BARD1

Cleavageor isoformof BARD1

p53

immunogenic

BARD1

33

Irminger-Finger et al., Mol Cell 2001

p53

p53p53

4455

66

Viral BARD1 injection causes tumor shrinking

NuTu-19: epithelial ovarian cancer cells

2 weeks later

Fisher rat 344: immuno-competent

4 weeks later

Left RightBA

RD

1EG

FP

0.00

0.50

1.00

1.50

2.00

2.50

BIG in

jectio

n

Ctl righ

t

GFP injec

tion

Ctl righ

t

NuTu lef

tNuT

u right

L R L R L R

cm

BARD1 expression in tumors causes immune response

BARD1

40x

Nutu

GFP

NuTu and GFP

40x

NuTu and SNGFP

CD3

40x

40x

BARD1 for tumor cell killing

• Ovarian cancer model• BARD1 deficient tumor cells• Viral delivery of functional BARD1• Replace viral BARD1 by small

molecules• Use BARD1 in anti-tumor vaccination

Irminger-Finger labChantal GenetAurélie Caillon

Francoise PiottonLaura Ortolan

Stephan Stephan RyserRyser

Charles Edward Jefford

Igor Igor BondarevBondarev

FabriceCallabria

Philipe Philipe BerardiBerardi

MamadouHady Sow

Anis Feki

Visiting scientists

JianJian YuYu WuWu

Collaborations

Paul Bischof (Geneva)Geoff Laurent (London)Andrea Bianco (Naples)Jean Harb (Nantes)Maria-Adelaide Caligo (Pisa)Marie-Francoise Pelte (Geneva)Anne-ThereseVlastos (Geneva)

FundingFunding: : JnJJnJ, SNSF, , SNSF, EuroPrEuroPr, JT, GR , JT, GR