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 +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 3 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 a N19 5x b a b N19 40x N19 40x BARD1 expression is specific for tumor type N19 C20 A C C C 5x 5x C n E 5x 5x C e S 5x 5x uC M 5x 5x Expression of N- and C-terminal epitopes N19 C20 N19 C20 5x 5x 5x 5x 40x 40x 40x 40x 100 OB18 OB41 OB42 OB15 90 OB16 OB44 OB43 OB11 80 OB35 OB12 OB38 OB39 70 OB40 OB14 OB13 OB36 OB37 OB31 60 OB33 OB29 OB34 OB30 50 OB32 OB2 OB3 OB4 19 positive cells 40 OB9 OB17 N OB23 OB28 OB24 OB25 30 OB5 OB27 OB22 OB26 % of 20 OB21 OB10 OB19 OB20 10 OB1 OB6 0 OB7 OB8 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 CCCEnC SeCMuC 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 N19 N19 60 30 C20 C20 p53 s p53 25 l 50 s l l l e e c c 20 40 e e v i v t i 15 30 i t i s s o o 10 20 p p f f o o 5 10 % % 0 0 G1 G2 G3 T1 T2 T3 T4 No correlation of BARD1 and p53 expression