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 gene 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 p53 BAP1 Nuclear localization BACH1 RHA signals Granin BRCA2 motif BARD1 interaction Ankyrin 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 Ubiquitin 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 BARD1 repression results in loss of contact inhibition of growth TAC-2 TAC-2/ABI 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 protein 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 isindependentof A Marker pcDNA BARD1 BRCA1 B Marker control wt doxo BARD1 control Brca 1-/ doxo - BARD1 % TUNEL pos. cells 10 15 20 25 30 0 5 C c o ntrol TUNEL-assay Brca wt 1-/ - dox o B A RD1 BRCA1 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 STRES S BARD1 BARD1 BARD1 B A BARD1 R D BARD1 1 BARD1 B A p53 R D 1 apoptosis BRCA1 BARD1 BRCA1 DNA repair 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 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 CGR8 CGR8-tBARD1 CGR8-tBARD1- CGR8-pcBARD1 CGR8-pcBARD1- CGR8-Doxorubicin BRCA1 BRCA1 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 proteins – 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 ovarian cancer 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.