Role of BARD1 in cell life and death

Irmgard Irminger-Finger Biology of Aging Laboratory

Department of Geriatrics University of Geneva Switzerland Age is the biggest risk factor for cancer

DePinho, 2000 Cancer Risks for Men

prostate

From1950 1990 Cancer Risks for Women

breast

From1950 1990 Cancers of old age are different from young age cancers Cancer in old age has a different face than cancer in young age

BCC Basal cell carcinoma SCC Squamous cell carcinoma

DePinho, 2000 What could link epithelial cell derived cancers to aging? BARD1, not just another cancer predisposition Common pathways for cancer and aging?

Accumulation of damage

DNA damage DNA damage proliferation- DNA damage arrest repair senescence

proliferation cancer

elimination apoptosis Cellular aging BARD1 Summary

• BARD1 structure • BARD1 repression studies • BARD1 overexpression studies • BARD1 dynamic localization • BARD1 induced upon stress • Non-correlated expression of BARD1 and BRCA1 • Role in spermatogenesis • BARD1 upregulation upon stress • Role in tumorigenesis • BARD1 a tumor antigen • BARD1 in cancer vaccine and genetherapy Conserved structures in BARD1 and BRCA1

Q>H

RING ANK BRCT BARD1 66 95 53 97 77 91 conservation RING BRCT BRCA1

C>G 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 BARD1- BRCA1 heterodimer

Rad51 PCNA ? RNA-Pol II BAP1 Nuclear localization BACH1 RHA signals Granin BRCA2 motif

BARD1 interaction repeats BRCT BRCA1 domain interaction

Ubiquitin-ligase Polyadenylation activity A B C D BARD1 BRCA1 BARD1

BRCA1 BARD1 Bcl-3

Pol II/Holo CstF-50 NF-kB Replication? S-phase dots Transcription: mRNA Regulation of Pol II/Holo Interaction Processing? transcription? [Scully et al. 1997}

HU/UV DNA ? HU/UV ? damage?

BARD1

BRCA1 INHIBITION OF MODULATION mRNA of NF-κB processing activity? DNA REPAIR: TRANSCRIPTION [Kleiman &Manley, [Dechend et al. 1999] PCNA/Rad51 BLOCK? 2000, 2001] co-localization ligase [Jin et al., 1997; activity Scully et al., 1997] [Parvin 2001] Does tumor suppressor function of BARD1 depend on BRCA1-BARD1 heterodimer? BARD1- BRCA1 Heterodimer

Nuclear localization signals Granin motif

BARD1 interaction Ankyrin repeats BRCT BRCA1 domain interaction Transcription Ubiquitin-ligase Polyadenylation activity Activation? BARD1 repression in vitro

• mouse mammary epithelial cells

• repression of BARD1 with antisense and ribozyme constructs

• analysis of phenotype: MORPHOLOGY, CELL CYCLE, MORPHOGENETIC PROPERTIES Altered morphology of BARD1 antisense and ribozyme expressing cells

TAC-2 AB-I AB-K

enlarged nuclei Flat, ruffled mulitnuclear Genomic instability in BARD1-repressed cells

8n

G2/M G2/M 4n G2/M 2n G1 G1 G1 TAC-2/ABI inhibition of growth repression results in loss of contact TAC-2

BARD1 BARD1 overexpression studies

• Constitutive expression of mouse BARD1 cDNA

• Based on the model of functional BARD1-BRCA1 heterodimer, overexpression should have no phenotype unless BARD1 has a BRCA1 independent function

•Result: – cells over-expressing exogenous BARD1 are not viable BARD1 overexpression leads to apoptosis TUNEL staining pcDNA3 pcBARD1 pcp53

d 50%

40

30

20

10

pcDNA pcBard1 pcp53 BARD1 is upregulated in response to cellular stress

On the level On the mRNA level

Doxorubicin UV - 1 4 - min RT-PCR doxo -- -10 µg TAC-2 cells BARD1

tubulin

UV 0 1 4 min ES cells BARD1 BARD1-induced apoptosis is independent of BRCA1

A B C o o RD1 RD1 A A 1 ontrol ontrol A B c dox c dox B A TUNEL-assay RD1 DN

A 30 pc BRC Marker

B wt Brca1-/- Marker wt 25 Brca1-/- lls

e 20 . c 15

10 TUNEL pos

% 5

0 control doxo BARD1 What is the mechanism of BARD1- induced apoptosis ? BARD1-induced apoptosis mediated by p53

Induction of apoptosis after transfection of BARD1 p53+/+ p53-/- BARD1 stabilizes p53 by direct interaction

A non-treat. doxo B IP: α -p53 S P S P pcBARD1 pcBQ>H BARD1 IP: α-BARD1 S P S P

PCNA p53 non-treat. doxo IP: α -BARD1 S P S P BARD1 p53

PCNA Localization of BARD1 during apotosis

A. nuclei (DAPI) B. BARD-GFP C. Anti-BARD1

A+B overlay B+C overlay A+C overlay Localization of BARD1-GFP deletion mutant during apoptosis

A. nuclei (DAPI) B. ∆RING-BARD-EGF C. Anti-BARD1

A+B overlay B+C overlay A+C overlay Mapping of the apoptotic region

C557S Q564H RING ANKYRIN BRCT 3 G A 1 T K N N D C N I 2 BARD1 APO D R R A G R K B c A ∆ N N p I ∆ B ∆ R A BARD1 NLS131 NLS 408 NLS 693 yes ∆ RING yes ∆ BRCT yes ANK2 yes no RING yes ∆ ANK D X no BARD1 Q564H ? BARD1 acts in p53-mediated apoptotic pathway

Stress transcription activation

BARD1 p53 apoptosis

P53 turnover - inhibition of degradation - direct stabilization Non-correlated expression of BARD1 and BRCA1 - independent functions? BARD1 isexpressed inmostrapidlydividing

mBARD1 ytRNA Tac-2 Placenta Thymus tissues Spleen Liver Stomach Lung Muscle CNS BARD1 Correlated expression ofBARD1andBRCA1

TAC-2 mg. virgin mg. 4 d postl.

mg. 5 d in mosttissues mg 10 d ovary o. 7d gest o. 20 d gest. prostate young prostate adult thymus spleen BRCA1 BARD1 Non-correlated expression of BARD1 and BRCA1 in some tissues

BARD1 expression in absence of BRCA1 is consistent with BARD1 but not BRCA1 mutations found in uterine tumors [Thai, Baer, Hum Mol Gen 1998]. Bimodal function of BARD1

STRESS RD1 D1 BA BAR 3 BA p5 apoptosis RD BA RD1 1 RD D1 BA 1 BAR

RD1 D1 BA BAR

D1 BAR DNA repair BRCA1 BRCA1 Does bimodal function of BARD1 exist in vivo? Apoptosis and (meiotic) repair during spermatogenesis

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

BARD1 N-19 antibody TUNEL BARD1 immuno-localization in testis

Premeiotic (N-19) Postmeiotic (JH-3) A B BARD1 absent in human Cryptorchidy

C D

E

Nucleus phagocytosed by Sertoli cell Sertoli cells Apoptotic bodies BARD1 staining smooth muscle cells layer BARD1 isoforms in purified cells from testis

GPLPjr P SRS

97 kD JH3 73 kD

97 kD C-20 73 kD

Western blot Western 97 kD 73 kD JH2 Differentially spliced BARD1 in testis

PCSR Pjr PL G S

BARD1

Actin

P: Pachytene spermatocyte (3 months old) SR: Round spermatid (3 months old) Pjr: Pachytene spermatocyte (23 days old) PL: Preleptotene spermatocyte (23 days old) G: Spermatogonia (9 days) S: Sertoli (3months old) C: Control tBARD1 is lacking the RING finger

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

Q>H BARD1 RING ANK BRCT

In5 In6 In7 In8 In In2 In3 In4 9 In1 tBARD1 tBARD1 more efficient in apotosis induction

A 90

80 eath

d 70

60

cell 50 % 40

30

20

10

0 CG R8 CG R8 - t B A R D 1 C G R 8 - t B A R D 1 - CG R8 - p c B A R D 1 CG R 8 - p c B A R D1 - CG R8 - D o x o r u b i c i n BR C A 1 B RCA 1 B p53 BARD1 expression is hormonally controlled

Preleptotene spermatogonia Round spermatid Sertoli conditioned media Sertoli conditioned media

F-/T- F-/T+ F+/T- F+T+ F-/T- F-/T+ F+/T- F+T+

BARD1

BRCA1

P53

β Tubulin tBARD1 the superkiller

Hormones? t BA RD 3 tBARD p5 apoptosis tBARD D AR tB STRESS tBARD tBARD D1 BARD1 BAR 3 p5 apoptosis D1 apoptosis BAR BA BA D1 RD RD BAR 1 1

RD1 D1 BA BAR

1 ARD B DNA repair

BRCA1 BRCA1 Two in vivo functions of BARD1

• in complex with BRCA1 and other repair – Cell cycle control and genomic stability – Repair functions

• BRCA1-independent: apoptotic functions – in response to stress – in maintenance of tissue homeostasis – in development (to be shown) Does stress-dependent upregulation of BARD1 exist in vivo? BARD1 expression in the brain after ischemia

Penumbra Core region BARD1 but not BRCA1 is expressed in penumbra BARD1 induction in response to cellular stress Upregulation of BARD1 associated with pre-malignant abnormal proliferation?

Control Bleomycin

BARD1

TUNEL BARD1 upregulation in tumorigenesis?

benign ovarian tumor malignant ovarian tumor Lack of BARD1 permissive of tumorigenesis

Carcinogenic stress transcription activation apoptosis BARD1 p53 -loss of BARD1 -loss of p53 P53 turnover - inhibition of degradation Carcinogenesis - direct stabilization Loss of BARD1 function in malignant cancer cells NuTu-19

Expression of BARD1 in NuTu cells Apoptotic response to cellular stress

TAC2 NuTu-19 45 IP (N-19) T SPT S P 40 35

30

25

20 97 15 BARD1 10

66 5

0 (PVC) NuTu +doxo +BARD1 Defect of BARD1 pathway in NuTu-19 cells

BARD1-p53 interaction in NuTu-19 cells

TAC2 NuTu-19 IP: anti BARD1 SP S P Western: anti-phospho p53 66 p53 47

NuTu-19 nt doxo 66 p53 47

Conclusion: NuTu-19 cells express truncated form of BARD1 phosph p53 (ser37) is absent in NuTu-19 cells p53 is present in NuTu-19 and co-IP with BARD1 BARD1 a tumor antigen Animal model to test BARD1 function in tumorigenesis

Animal model of

Metastases detection by photo medicine

NuTu-19 ovarian cancer After 5 weeks 100% cells

Rose et al. 1996 Search for tumor antigens

Animal model of colon cancer

Metastases and death after 5 weeks PROb colon cancer cells: wt BARD1

Apoptotic treatment

Apoptotic bodies Immunized survivors Identification of BARD1 as tumor antigen

Identification of p67 on Western blots RING ANK BRCT Immunized survivors Serum F1 Screening of rat Identification of cDNA encoding cDNA expression libraries C-terminal portion of BARD1

Vaccination with F1 renders rats partially immune to tumorgenesis Gautier, Irminger-Finger, Cancer Res. 2000 BARD1 upregulation and cleavage associated with apoptosis

Gautier, Irminger-Finger, Cancer Res. 2000 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

** *

Ab: PVC Ab: C20 Ab: C20 Cleavage of BARD1 3 p53 p53 BARD1 p67 p67 p53 p53 2 p53 apoptosis BARD1 immunogenic BARD1 Other p53 stabilizing signals? BARD1 1 DNA repair BRCA1 functions (e.g.ubiquitination) Cell cycle arrest BRCA1 BARD1 Applications

Cancer vaccine based on p67BARD1 Gene therapy: killing of unwanted cells Antisense: promote cell survival Application: Immuntherapy

F1F1

Metastases detection by i) photo medicine, ii) tagged cells

Reduced death NuTu-19 ovarian rate? PROb colon cancer Application: Gene therapy

Metastases detection by NuTu-19 ovarian or i) photo medicine, PROb colon cancer ii) tagged cells

Reduced death Viral delivery of BARD1 rate? Adeno or retroviral vector Tools: lentiviral BARD1

Tet ON BARD1 Tet ON BARD1-GFP CMV BARD1 iresGFP CMV BARD1-GFP Tet ON BARD1 iresGFP Tools: Nutu-GFP cells Monitoring of metastatic spreading

Liver and peritoneum Immune therapy and gene therapy

ImmunizationImmunization p67BARD1p67BARD1

TumorTumor inductioninduction Metastases detection by i) photo medicine, ii) tagged cells NuTu-19 ovarian or PROb colon cancer Reduced or delayed TumorTumor cellcell killingkilling tumor formation? Viral delivery of BARD1 Reduced death rate? Adeno or retroviral vector Tumor specific promoter

SensitivitySensitivity toto chemotherapychemotherapy Collaborators

• Antibodies • Biology of Aging LAB - J Harb, Nantes • RNA expression and • Cancer and Aging Group repression – Anis Feki - P Sappino, G Vaudan, R – Charles Edward Jefford Montesano, J Sorinano, – Nicola Molarfi Geneva – Mamadu Hadi • Pathologies – Auréllie Caillet - G Laurent, London, - W-C Leung, New Orleans Stem cells group - H. Lucas, A. Major, Jian Li Brain ischemia - F.M. Pelte, Geneva Michel Dubois-Dauphin

Karl-Heinz Krause