BARD1 and Associated Proteins and Its Application in Cancer Prognostics and Treatment

Functions of BARD1 and associated proteins and its application in cancer prognostics and treatment

IrmgardIrmgard IrmingerIrminger--FingerFinger

Biology of Aging Laboratory and Monitoring Laboratory University and University Hospitals of Geneva, Switzerland TumorTumor suppressorsuppressor BARD1BARD1 patented:patented:

1.Use as vaccine 2. Use for cell killing 3.Use as prognostic marker

IrmgardIrmgard IrmingerIrminger--FingerFinger University and University Hospitals Geneva Clinical application of BARD1: background and principles

The “normal” functions of BARD1 BARD1 expression in tumors: in breast and ovarian cancers in 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 carcinoma SCC Squamous cell carcinoma

DePinho, 2000 Accumulation of damage during aging

agingaging

damaged cells long-term loss of abnormal function functional tissue abnormal activation (Alzheimer, heart abnormal growth failure etc.)

apoptosis cancer environmental insult damage BARD1 Accumulation decision of endogenous BARD1 making damage repairrepair BARD1 and its interacting proteins:a converging vehicle

Rad51 PCNA ? RNA-Pol II p53 BAP1 BACH1 RHA BRCA2

Ankyrin BARD1- BRCA1 RING repeats Phospho-binding BRCT interaction domain

Transcription Ubiquitin-ligase Polyadenylation activity P53 activation NFk-B binding DNA-PK apoptosis Irminger-Finger et al., Bioch. 1999; Irminger-Finger et al., Mol Cell 2001; Feki et al. & Irminger-Finger, BOR 2005 BARD1 and BRCA1 mutations predisposing to breast cancer

A1009T A1481G G1743C

A957G 1144del21 G1592A G1765C Q513H 358D364 C557S K312N N295S

Human BARD1 RING ANK BRCT

RING BRCT Human BRCA1

C>G Missense mutations Y>A

No one like BARD1

RING ANK BRCT BARD1

BRCA1

Arabidopsis 2 hypoth.p. Arabidopsis CAC05430 C. elegans 3 hypoth. p. C. elegans 6 hypoth. p. C. elegans T15564 C. elegans T15566

Xenopus BARD1 Joukov, Livingston, PNAS, 2001 BRCA1

Irminger-Finger and Leung, IJBCB 2002 BARD1 induces apoptosis in a p53-mediated pathway BARD1 binds to p53 A non-treat. doxo IP: α -p53 S P S P

transfection of BARD1 BARD1

p53+/+ p53-/- PCNA

non-treat. doxo IP: α -BARD1 S P S P

p53

PCNA

BARD1 stabilizes p53 B pcBARD1 pcBQ>H IP: α-BARD1 S P S P

p53

BARD1

Irminger-Finger et al., Mol Cell 2001 Mapping of the apoptotic region

3 1 T G A C K N D N 2 R R N G I K C557S Q564H D N R c A B A I N p B ∆ ∆ R ∆ A RING ANKYRIN BRCT BARD1 APO NLS131 NLS 408 NLS 693 BARD1 yes ∆ RING yes ∆ BRCT yes ANK2 yes RING no ∆ ANK yes X no BARD1 Q564H ?

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

On the protein level On the mRNA level TAC-2 cells ES cells Doxorubicin UV - 1 4 - min doxo -- -10 µg

BARD1

p53

Western RT-PCR 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

A H-300 PVC JH3

9 9 9 -1 -1 -1 u u u C T T T T T T 93 u 93 u 93 u 2 N 2 N 2 N H-300 PVC JH-3 BARD1

BARD1 RING ANK BRCT In In In In B In In In 5 6 7 8 In In 2 3 4 9 1 M p53 BARD1 2KB BARD1 In In In In In In In 5 6 7 8 In In 2 3 4 9 1 2.2 kb 0.8KB BARD1 2 kb

0.8 kb

Feki et al., & Irminger-Finger, Oncogene 2005 BARD1 required for p53 phosphorylation

Apoptotic treatment BARD1 transfection

TAC2 NuTu-19 HEK NuTu-19 doxo - + -+ BARD1 - + -+

BARD BARD1 1 C p53 p53

p-p53 p-p53

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

50 45 NuTu-19 40 TAC2 35 is 30 25 20

of apoptos 15 % 10 5 0 NuTu-19 NuTu-19 NuTu-19 TAC-2 TAC-2 + Doxo + BARD1 + Doxo BARD1 colocalizes with phospho-p53

A DAPI FITC P53-Alexa 555 MERGE

DAPI FITC Phospho-p53-Alexa 555 MERGE Mapping p53 binding region

IP p53

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

1144del2 G1592A G2354A A1481G A1009T1 G1743C G215C A957G G176C RING ANK BRCT mBARD1 p53 NLS 131 NLS 408 NLS 693 binding apo

BARD1 (aa 1-765) EGFP 100 yes ∆ RING (aa 137-765) EGFP 60 yes ∆ HIND (aa 394-765) EGFP 80 yes ∆ BRCT (aa 1-604) EGFP 100 yes ANK (aa 394-604) EGFP 60 yes RING (aa 1-266) EGFP 0 no

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

Jin, Baer, JBC 1997; Scully, Livingston, Cell 1997 ....but not only “nuclear dots” pEGFP BARD1-EGFP ∆RING- A EGFP

∆BRCT-EGFP ANK-EGFP RING-EGFP

s l 60

B cel 50 40 GFP+ c RING-EGFP30 20 optoti p

a 10

% 0 EGFP- BARD1- RING- ∆-RING- ∆-HIND - ∆-BRCT ANK- N1 EGFP EGFP EGFP EGFP EGFP EGFP 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 BARD1 BRCA1 BARD1 is translocated to the cytoplasm during apoptosis

Total cell extract Nucl. extract Cytopl. extract 0 3 4 5 6 7 14 22 24 h 0 3 4 5 6 7 14 22 24 h 0 3 4 5 6 7 14 22 24 h

** * p67

Ab: PVC Ab: C20 Ab: C20

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 morphogenesis spermatogenesis •In homeostasis Spleen, bone • In tumor suppression Cytoplasmic localization of BARD1 in vivo

A BARD1 PVC BARD1 N-19

B BARD1 N-19 BARD1 upregulation in vivo by ischemia

Left hemisphere

Penumbra Core region

Left hemisphere

BRCA1 BRCA1 BARD1 Apoptosis and (meiotic) repair during spermatogenesis

Bcl-x PMB4 SCF IL-4 LIF SCF Gas6 LIF Bcl-xL Bcl-xL bFGF TGFß CNTF testosterone Bax testosterone Bax - + - --+ - +

Spermatogonia Spermatocytes Spermatids Spermatozoa PGC Gonocytes Apoptosis Apoptosis Apoptosis Apoptosis

BARD1? Modified from: Print, Bioassays, 2000 Schematic diagram of seminiferous tube BARD1 expression is correlated with apoptosis markers A anti-BARD1 a a

TUNEL b b

Feki et al., & Irminger-Finger, BOR 2004 B activated caspase 3 anti-BARD1 N-19 a b a b

c d N-19

c d

f JH3 e f e BARD1 and BRCA1 expression overlap partially a b

A BARD1 a b

5x 20x 20x

B c d d BRCA1

c 5x 40x 40x C

5x Different BARD1 isoforms are expressed at different stages A GPLPjr P SR 97 kD 74 kD C-20 Western blot

97 kD JH-3 74 kD

97 kD JH-2 74 kD

B GPLPjr P SR

2300 bp RT-PCR 2000 bp BARD1

GAPDH BARD1β lacks the RING finger

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

Q>H BARD1 RING ANK BRCT In In5 6 In7 In8 In In2 In3 In4 9 In1 BARD1β BARD1β is an efficient apoptosis inducer

100 90 80 70

eath 60 d 50

cell 40 % 30 20 10 0 CGR8 BARD1b BARD1b BARD1 BARD1 Doxo BRCA1 BRCA1

p53 CGR8 BARD1b BARD1b BARD1 BARD1 Doxo BRCA1 BRCA1 BARD1 expression associated with abortive spermatogenesis

A Obstructive Azoospermia Sertoli cell Syndrome

cryptorchidy B c d

2 1 Presumed tumor suppressor pathways of BARD1

6 Cleavage immunogenic or isoform of BARD1 4 3 p53 5 BARD1 1 ARD1 p53 BARD B apoptosis p53 2 p53

BARD1 BARD1 Other p53 stabilizing signals? BARD1

1 DNA repair pathways BRCA1 (e.g.ubiquitination)

Cell cycle arrest BRCA1 BARD1

Irminger-Finger et al., Mol Cell 2001 Expected loss of BARD1 in tumorigenesis

Carcinogenic stress transcription activation Pser15 apoptosis BARD1 p53 -loss of BARD1 -loss of p53

BRCA1 p53 turnover mutated or deleted - inhibition of degradation Carcinogenesis - direct stabilization However, BARD1 is upregulated in tumors

N19 5x b a b

N19 40x N19 40x ype t 20 C for tumor 5x 5x 5x 5x specific N19

5x 5x 5x 5x

C C C M uC C E n S e C A BARD1 expression is Expression of N- and C-terminal epitopes

N19 C20 N19 C20

5x 5x 5x 5x

40x 40x 40x 40x

100 OB 1 8 OB 4 1 OB 4 2 OB 1 5 90 OB 1 6 OB 4 4 OB 4 3 OB 1 1 80 OB 3 5 OB 1 2 OB 3 8 OB 3 9 70 OB 4 0 OB 1 4 OB 1 3 OB 3 6 OB 3 7 OB 3 1 60 OB 3 3 OB 2 9 OB 3 4 OB 3 0 50 OB 3 2 OB 2 OB 3 OB 4 19 positive cells 40 OB 9 OB 1 7 N OB 2 3 OB 2 8 OB 2 4 OB 2 5 30 OB 5 OB 2 7

% of OB 2 2 OB 2 6 20 OB 2 1 OB 1 0 OB 1 9 OB 2 0 10 OB 1 OB 6 0 OB 7 OB 8 CCC EnC SeC MuC Correlation of BARD1 expression with tumor grade OB16 A1 Clair cell carcinoma C20 x40 BARD1 but not p53 is correlated with tumor type

A N19 p53 B Correlation of BARD1 and p53 with histological type of ovarian carcinoma

100 90 N19 C20 80 p53 40x 40x 70 60 50 40 positive cells 30 20 % of 10 40x 40x 0 CCCEnCSeCMuC

Figure 3 Correlation of BARD1 expression with tumor grade and stage

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

D 35 70 N1 9 N1 9 60 30 C20 C20

p53 s p53

25 l 50

c 20 40

t i 15 30 i

o 10

p 20

o 5 10

% % 0 0 G1 G2 G3 T1 T2 T3 T4 No correlation of BARD1 and p53 expression in NSCLC

AB Distribution of N19 expression in different pathological types

5x 5x 100

l l 6159 10356

e 90 6609 8040

c 8315 8341

1708 12740 80 12748 1176 e 1659 8532

i 8664 12278

t 70 11638 12756 i 6634 8075

s 9977 12767 60 11601 1103 o 1531 8366

p 8930 10362 50 7987 9018

9 10319 6661 7723 8650 40 12111 9844 6699 8100 N1 8981 11400 7993 11203 f 30 11605 12500

o 20 % 10 N19 40x p53 40x 0 o m qu ge en ua os ar ad sq en l ad

Figure 5 No correlation of BARD1 expression with tumor grade and stage in NSCLC

C BARD1(N19/C20) and p53 BARD1(N19/C20) and p53 BARD1(N19/C20) and p53 in different pathological type in different pathological grade in different stages

100 100 100 N19 N19 N19 90 90 90 C20 C20 C20 P53 80 P53 80 p53 80

s 70

s l 70

s 70

e 60

60 60 c

e 50 v 50

v i

v 50

40 s 40

o 40

p p 30 30

30 f

o 20

% 20

% % 10 10 0 10 0 0 no m u e stage I stage II stage III stage IV e ua sq arg G1 G2 G3 G1-3 ad sq en l ad Tumors express an aberrant form of BARD1 A OB C+ 8 8* 9 21 23 24 24* 25 33 33* 34 35 37 C- N-terminus 74-1481

C-terminus 1441-2252

hGAPDH

A1009T A1481G G1743C A957G 1144del21 G1592A G1765C

74 783 1481

1441 2252

Figure 6 BARD1 is transcriptionally altered in rat ovarian cancer cells and in human cancers?

H-300 PVC JH-3

BARD1 2.2 kb RING ANK BRCT

In In In In In In In In In 5 6 7 8 1 2 3 4 9

BARD1γ 2.0 kb 804-826 In In In In 8 9 1 2

BARD1δ 0.8 kb

1618-1641

74 783 1481

1441 2252

Figure 5A-C BARD1 expression in human cancers

Elevated expression of BARD1 in cancer Cytoplasmic localization of BARD1 in tumors Loss of 5’region Expression is correlated with tumor type, grade and stage in breast and ovarian cancer No correlation in NSCLC No correlation of BARD1 and p53 expression BARD1 expression in human cytotrophoblast

40x 5x 40x

BARD1 N-19 BARD1 WFS

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

Q>H BARD1 RING ANK BRCT 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 apoptotic markers

5x 40x 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: WFS Ab: PVC 5 weeks 12 weeks

BARD1-C20 40x BARD1-C20 40x

BARD1-PVC 40x BARD1-PVC 40x

BRCA1 40x BRCA1 40x Differentially spliced isoform of BARD1 in cytotrophoblast and placenta lack RING finger

A RT-PCR T P M C

BARD1 2,3kb 2kb actin

B N-19 JH-2 JH-3 C-20 PVC WFS Q>H BARD1 RING ANK BRCT

In5 In6 In7 In8 In9 In2 In3 In4 In1 BARD1γ BARD1 is secreted from CTBs in culture

A In cell lysates 120

100 ells/ml) c 80

20 BARD1 (UA/mio 0 CTB (1) CTB (2) MCF-7 MDA-MB231

B In medium of cell cultures C In medium of CTBs .09 .09 .08 .08

ells/ml) .07 .07 c .06 .06 .05 .05 .04 .04 .03 .03 control .02 .02 doxo .01 .01 PMA 0 0 BARD1 (UA/mio CTB (2) MCF-7 MDA-MB231 0-24 h 24-28 h Quantification of BARD1 by ELISA

Coating C 20 (1/200) Coating N19 (1/200) Tampon Pool Liq. Amn. 1/4 Pool Liq. Amn. 1/20 Pool Liq. Amn. 1/100 MC CTB (conc 8x) Serum PB 2.10.03 ndl Serum pool Femme enceinte ndl Serum AM 2.10.03 ndl C20 biot WFS PVC C20 biot WFS PVC 1/1000 1/100 1/100 1/1000 1/100 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 A Metastases detection by photo medicine

NuTu-19 ovarian After 5 weeks 100% cancer cells GFP retroviral vector

B liver

peritoneum

omentum digestive tube

diaphragma

visual light FITC Transduction of NuTu-19 cells with lentiviral vectors 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

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

ells 293T c Transduced NuTu cells expressing GFP or BARD1 and rTTAh Gene therapy proteins for cell killing by Ovarian cancer Model viral delivery of GFP-cancer cells Inducible BARD1 Apoptosis induction by inducible BARD1

NuTu-19 cells 60 A NuTu-19 cells transduced with B pLOX/TW-gfp lentiviral vector 50 NuTu-19 cells transduced with 50X concentrated pLOX/TW-gfp lentiviral 40 vector 30

0 GFP expression NuTu NuTu- + 2.5 ug +5ug +10 ug +15 ug +20ug of PLOX/BARD1 Doxycycline Doxycycline Doxycycline Doxycycline Doxycycline

NuTu-19 cells NuTu-19 cells transduced with Death rate after GFP induction with Doxycyclin 50 pLOX/TW-gfp lentiviral vector C NuTu-19 cells transduced with Death rate after BARD1 induction with Doxycyclin 40 pLOX/TW-gfp lentiviral vector and induced with Doxycyclin (2µg/ml) 30

0 GFP expression 0 1 ug/ml 2ug/ml 4ug/ml 8ug/ml 16ug/ml Doxycyclin Diaphragm Thymus Omentum Nutu-19-EGFP tumors in Fisher rat

Diaphragm Omentum Thymus Presumed tumor suppressor pathways of BARD1

6 Cleavage immunogenic or isoform of BARD13 4 5 p53

BARD1 1 ARD1 p53 BARD B p53 2 p53 apoptosis BARD1 BARD1 Other p53 stabilizing signals? BARD1

1 DNA repair pathways BRCA1 (e.g.ubiquitination)

Cell cycle arrest BRCA1 BARD1

Irminger-Finger et al., Mol Cell 2001 Viral BARD1 injection causes tumor shrinking

2 weeks later

NuTu-19: epithelial ovarian cancer cells Fisher rat 344: immuno-competent

4 weeks later

Left Right BARD1

2.50

2.00

1.50

m 1.00

0.50 EGFP 0.00 LR LR LR t n t t t on h h f h ti g io g le g ri ct ri ri jec je u n u i Ctl Ctl G P in NuT F NuT BI G BARD1 expression in tumors causes immune response

GFP BARD1 CD3 Nutu

NuTu and GFP

40x

NuTu and SNGFP

40x 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 Anis Feki Charles Edward Fabrice Jefford Mamadou Callabria Hady Sow Irminger-Finger lab Chantal Genet Aurélie Caillon Francoise Piotton Laura Ortolan JianJian YuYu WuWu StephanStephan RyserRyser IgorIgor BondarevBondarev PhilipePhilipe BerardiBerardi Visiting scientists Collaborations

FundingFunding:: JnJJnJ,, SNSF,SNSF, EuroPrEuroPr,, JT,JT, GRGR

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