BARD1 functions in malignant transformation and in therapeutic application IrmgardIrmgard IrmingerIrminger--FingerFinger Biology of Aging Laboratory and Monitoring Laboratory University and University Hospitals of Geneva, Switzerland BARD1 and BRCA1 (and 2) mutations associated with breast and ovarian cancer A1009T A1481G G1743C A957G 1144del21 G1592A G1765C Q513H 358D364 C557S K312N N295S Human BARD1 RING ANK BRCT RING BRCT Human BRCA1 Missense and mutations C>G Y>A Human BRCA2 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/BRCA1 heterodimer: 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 activation NFk-B binding Irminger-Finger et al., Bioch. 1999 Presumed tumor suppressor pathways of BARD1 BARD1 apoptosis death pathway BARD1 BRCA1 DNA repair pathways (e.g.ubiquitination) Cell cycle BRCA1 BA arrest RD1 Survival pathway In normal cell life? Role of BARD1, BRCA1, and BRCA2 in normal life • BARD1, BRCA1 and BRCA2 ko are embroynic lethal • BARD1 and BRCA1 ko cells are cellular lethal • Depletion of BARD1, BRCA1 or BRCA2 cause genetic instability • Irminger-Finger et al., 1998 • Jukov et al., 2002 • McCarthy et al., 2003 • Genetic instability is the hallmark of tumors with mutations in BARD1, BRCA1 and BRCA2 BARD1 is upregulated in mitosis HeLa TAC-2 PC-3 BARD1 (H-300) red Alexa555 DNA blue DAPI BARD1 localizes to spindle microtubles DNA β-Tubulin BARD1 Merge HeLa PC-3 TAC-2 A S-phase Metaphase Anaphase Telophase Cytokinesis DNA -tubulin β 1 D BAR merge BARD1 a relay player in mitosis B Anaphase Telophase Cytokinesis BARD1/BRCA1 BARD1/BRCA2 Ubiquitin ligase control of targeting cytokinesis γtubulin Who are the partners? IP anti- A Cell lysate TACC1 Beads only NS G1/S G2/M NS G1/S G2/M NS G1/S G2/M BARD1* 83 IP anti- B Cell lysate BARD1 Beads only NS G1/S G2/M NS G1/S G2/M NS G1/S G2/M 175 TACC*1 C Aurora A Aurora B 32,5 IP anti- D Cell lysate BARD1 Beads only NS G1/S G2/M NS G1/S G2/M NS G1/S G2/M BRCA2 siRNA depletion of BARD1 AB 3.5 ion 3 s s 3 i c 2.s 5 ab al 2 rm 1.5 abno s 1 Microtubule BARD1 Nucleus 0.38 0. cell 5 % 0 Mock siRNA BARD1 C Aurora B remains at the midbody D wildtype siRNA BARD1 Numerical chromosomal instability in BARD1 depleted cells chromosome X chromosome X 70 90 HeLa 80 60 MCF-7 HeLa siBARD1 70 50 MCF-7 siBARD1 60 40 50 30 40 20 30 20 10 10 0 0 12345678 123456 chromosome 18 chromosome 18 60 90 80 50 MCF-7 70 MCF-7 siBARD1 40 60 50 30 40 20 30 20 10 10 0 0 12345678 1234567 Role of BARD1 in normal life • BARD1 increased during mitosis • Localizes to mitotic spindle via TACC1 • Controls centromer duplications via BARD1/BRCA1 ubiquitin ligase activity • Controls cytokinesis via interaction with Aurora B and BRCA2 • Failure of doing all these jobs leads to genetic instability Presumed tumor suppressor pathways of BARD1 BARD1 apoptosis death pathway BARD1 BRCA1 DNA repair pathways (e.g.ubiquitination) Cell cycle BRCA1 BA arrest RD1 Survival pathway In normal cell life? Progression through mitosis BARD1 as tumor suppressor: induction of p53-mediated apoptosis 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 A 1 T G D C K N IN 2 R R N G K D N R c A B A I N C557S Q564H 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. BRCA1 competes apoptosis by BARD1 in co-transfection assays. 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 Exogenous expression of BARD1 is sufficiant for apoptosis induction in apoptosis-resistant NuTu-19 cells 50 45 NuTu-19 40 TAC2 35 30 apoptosis 25 20 % of 15 10 5 0 NuTu-19 NuTu-19 NuTu-19 TAC-2 TAC-2 + Doxo + BARD1 + Doxo Apoptosis resistant NuTu-19 cells lack functional BARD1 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 BARD1 colocalizes with phospho-p53 A DAPI FITC P53-Alexa 555 MERGE DAPI FITC Phospho-p53-Alexa 555 MERGE Mapping the 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., Oncogene 2005 BARD1 brings the kinase to its target A 293T 293T + BARD1 IP Ku-70 SPSP p53 BARD1 IP p53 p53 Ku-70 IP BARD1 p53 ATM B NuTu NuTu + BARD1 NuTu + beads IP BARD1 S P S P S P Ku-70 Presumed tumor suppressor pathways of BARD1 DNA-PK p53 BARD1 1 BARD p53 apoptosis p53 p53 BARD1 BARD1 BARD1 BRCA1 DNA repair pathways 1 (e.g.ubiquitination) Cell cycle BA arrest RD1 Survival pathway BRCA1 In normal cell life? Progression through mitosis BARD1 upregulation in vivo by ischemia Left hemisphere Penumbra Core region Stroke area Stroke area BARD1 BARD1 tunel Left hemisphere BRCA1 BRCA1 BARD1 BARD1 expression is correlated with apoptosis in spermatogenesis anti-BARD1 a a TUNEL b b Feki et al., & Irminger-Finger, BOR 2004 Mechanism of apoptosis induction by BARD1 • Excess BARD1 over BRCA1 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 Co-localization of BRCA1 and BARD1 with repair proteins in “nuclear dots” +BARD1 +BARD1 Jin, Baer, JBC 1997; Scully, Livingston, Cell 1997 BARD1 translocates to the cytoplasm upon apoptosis induction non treated UV BARD1 BRCA1 BARD1 is translocated to the cytoplasm during apoptosis Total cell extract Nucl. extract Cytopl. extract Doxo 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 appears in the cytoplasm. p67 is immunogenic and anti-tumorigenec in an animal model of colon cancer (Gautier, Irminger-Finger et al., Cancer Res 2000). Presumed tumor suppressor pathways of BARD1 Cleavage by caspase caspase p53 BARD 1 1 BARD p53 apoptosis p53 p67 p53 stable BARD1 BARD1 BARD1 BRCA1 DNA repair pathways 1 (e.g.ubiquitination) Cell cycle BA arrest RD1 Survival pathway BRCA1 In normal cell life? Progression through mitosis Colon cancer model: immunotherapy A Metastases detection by photo medicine Colon After 5 weeks 100% cancer cells B Apoptotic treatment Colon Immunized cancer cells vaccination survivors After 5 weeks Colon cancer cells Gautier, Irminger-Finger et al., Cancer Research 2000 Apoptotic BARD1 isoform p67 is immunogenic Western blot p67 Identification of Immunized Epitopes: survivors Screening of Expression library BARD1 partial sequence RING ANK BRCT F1 rBARD1 NLS 131 NLS 408 NLS 693 Immunized F1 survivors Colon cancer cells Gautier, Irminger-Finger et al., Cancer Research 2000 Animal cancer model for tumor cell killing A Metastases detection NuTu-19 ovarian After 5 weeks 100% cancer cells GFP-tagged 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 NuTu-GFP NuTu-19 cells or NuTu-19 cells transduced with pLOX/TW-gfp lentiviral vector NuTu-BARD1 NuTu-19 cells transduced with 50X concentrated pLOX/TW-gfp lentiviral vector GFP expression Nutu-19-EGFP tumors in Fisher rat Diaphragm Omentum Thymus Diaphragm Omentum Thymus Viral delivered BARD1 causes tumor shrinking A Tumor detection NuTu-19 ovarian subcutaneously cancer cells L and R BARD1-GFP retroviral vector left B Left Right BARD1 2.50 2.00 1.50 m c 1.00 0.50 LR LR LR EGFP BIG/ - GFP -/ NuTu/NuTu 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 • Anti-tumor vaccination Several tumor suppressor functions of BARD1 immunogenic Cleavage by caspase caspase p53 1 BARD1 BARD 1 p67 BARD p53 apoptosis p53 stable p53 BARD1 BARD1 BARD1 1 BRCA1 DNA repair pathways (e.g.ubiquitination) Cell cycle BA arrest RD1 Survival pathway BRCA1 In normal cell life? Progression through mitosis 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 Sarantis Gagos (Athens) Daniel Birnbaum (Marseille) Gianni del Sal (Trieste) Geoff Laurent (London) Jean Harb (Nantes) Attila Major (Geneva) Funding:Funding: JnJ,JnJ, SNSF,SNSF, EuroPr,EuroPr, JT,JT, GRGR .