Published OnlineFirst January 27, 2009; DOI: 10.1158/0008-5472.CAN-08-2134 Research Article Distinct Roles of BARD1 Isoforms in Mitosis: Full-Length BARD1 Mediates Aurora B Degradation, Cancer-Associated BARD1B Scaffolds Aurora B and BRCA2 Stephan Ryser,1,2 Eva Dizin,1,2 Charles Edward Jefford,1,2 Be´ne´dicte Delaval,3 Sarantis Gagos,4 Agni Christodoulidou,4 Karl-Heinz Krause,2 Daniel Birnbaum,3 and Irmgard Irminger-Finger1,2 1Molecular Gynecology and Obstetrics Laboratory, Department of Gynecology and Obstetrics and 2Department of Medical Genetics and Laboratory Medicine, University Hospitals Geneva, Geneva, Switzerland; 3Laboratoire d’Oncologie Mole´culaire, Centre de Recherche en Cance´rologiede Marseille, UMR891 Inserm-Institut Paoli-Calmettes, Marseille, France; and 4Laboratory of Genetics, Foundation of Biomedical Research, Academy of Athens, Athens, Greece Abstract NH2 terminal RING finger domains (4), and either Bard1 or Brca1 The BRCA1-associated ring domain protein 1 (BARD1) deletion induces breast cancer in tissue-specific conditional interacts with BRCA1 via its RING finger domain. The knockout mice (5). BARD1 is the major protein-binding partner BARD1-BRCA1 complex participates in DNA repair, cell cycle of BRCA1, with whom it forms a stable heterodimer and acts in control, genomic stability, and mitotic spindle formation tumor suppressor functions but has also BRCA1-independent through its E3 ubiquitin ligase activity. Cancer cells express functions in apoptosis (6, 7). The BARD1-BRCA1 heterodimer is an severalBARD1 protein isoforms, includingthe RING finger– E3 ubiquitin ligase implicated in DNA repair and homologous deficient variant BARD1B. Here, we show that BARD1 has recombination (8, 9), centrosome duplication (10), and mitotic BRCA1-dependent and BRCA1-independent functions in spindle assembly (11), which are essential functions for maintain- mitosis. BARD1, but not BRCA1, localizes to the midbody at ing genomic stability (10, 12). telophase and cytokinesis, where it colocalizes with Aurora B. During S-phase, BRCA1, BRCA2, and BARD1 are partially The 97-kDa full-length (FL) BARD1 coimmunoprecipates with colocalized to distinct nuclear dots (13, 14). During mitosis, BRCA1 BRCA1, but the 82-kDa BARD1B coimmunoprecipitates with localizes to spindle poles (15), and BRCA2 localizes to the midbody Aurora B and BRCA2. We used selective small interfering RNAs during telophase and cytokinesis, wherein it is involved in to distinguish the functions of FL BARD1 and BARD1B. contractile ring and midbody formation and completion of Depletion of FL BARD1 had only minor effects on cell growth cytokinesis (16). Depletion of BRCA2 impedes abscission of the and did not abolish midbody localization of BARD1 staining, midbody and cell separation (16), providing an explanation for the but resulted in massive up-regulation of Aurora B. In contrast, genetic instability observed in cancers associated with mutations suppression of FL BARD1 and BARD1B led to growth arrest in BRCA2. Midbody formation and abscission are also controlled by and correlated with various mitotic defects and disappearance the microtubule-binding protein transforming acidic coiled coil– of midbody localization of BARD1 staining. Our data suggest a containing protein 1 (TACC1) and the mitotic kinase Aurora B (17). novelfunction of FL BARD1 in Aurora B ubiquitination and TACC1 was reported to interact with BARD1 in Caenorhabditis degradation, opposing a proproliferative function of BARD1B elegans (18). BARD1 expression is up-regulated during mitosis (19), in scaffolding Aurora B and BRCA2. Thus, loss of FL BARD1 presumably due to phosphorylation by cyclin-dependent kinase/ and up-regulation of Aurora B, as observed in cancer cells, can cyclin complexes (20), but its subcellular localization during be explained by an imbalance of FL BARD1 and BARD1B. mitosis has not been investigated. [Cancer Res 2009;69(3):1125–34] The mitotic functions of BRCA1 and BARD1 (11) could explain why normal proliferating cells are not viable without BRCA1 or BARD1. However, mouse trophoblast giant cells, which Introduction undergo endomitosis, a nuclear division not followed by cell Breast and ovarian cancers with mutations in BRCA1 and BRCA2 division, are not affected in the Bard1 knockout mouse (3), show a severe genomic instability phenotype (1). Genomic suggesting that the lethal phenotype of BARD1 depletion is linked instability and a premalignant phenotype are also features of to a function at exit of mitosis, similar to functions attributed BRCA1-associated ring domain protein 1 (BARD1)–deficient cells to BRCA2 (16). (2), causing early embryonic death of BARD1 knockout mice (3). A genetic link between BRCA2 and BARD1 was found with the BARD1 and BRCA1 form a stable heterodimer via their respective BRCA2 mutation 999del5, which, when combined with the BARD1 variant Cys557Ser, results in 100% probability for developing breast and/or ovarian cancer for carriers of the BRCA2 999del5/BARD1 Cys557Ser double mutation (21). Thus, BRCA2 and BARD1 might Note: Supplementary data for this article are available at Cancer Research Online act in a common pathway. (http://cancerres.aacrjournals.org/). Whereas mutations in BARD1 are rare in cancers (7), aberrant S. Ryser and E. Dizin contributed equally. Requests for reprints: Irmgard Irminger-Finger, University Hospitals Geneva (HUG), up-regulation of isoforms is associated with poor prognosis in Blvd de la Cluse, Geneva, CH-1211, Geneva, Switzerland. Phone: 41-22-382-4327; Fax: breast and ovarian cancer (22, 23). We found aberrantly expressed, 41-22-382145; E-mail: [email protected]. I2009 American Association for Cancer Research. differentially spliced BARD1 isoforms, which lack the BRCA1- doi:10.1158/0008-5472.CAN-08-2134 interacting RING finger, in breast, ovarian, and endometrial cancer www.aacrjournals.org 1125 Cancer Res 2009; 69: (3). February 1, 2009 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst January 27, 2009; DOI: 10.1158/0008-5472.CAN-08-2134 Cancer Research cells (23, 24) and human cytotrophoblasts (25). Repression of these on a logarithmic scale after determining the cell number along the isoforms in cancer cells that lack full-length (FL) BARD1 led to exponential phase and calculating the mean population-doubling time. growth arrest (23), which suggests that BRCA1-independent Immunofluorescence microscopy. Cells were fixed with 2% parafor- proproliferative, presumably mitotic, functions are retained in maldehyde for 15 min at room temperature (RT) or with methanol for 6 min at À20jC and rinsed in acetone for 30 s. Paraformaldehyde-fixed cells were cancer-associated BARD1 isoforms. permeabilized in 1% Triton/PBS for 15 min at RT and then blocked in 1% We therefore designed this study to understand the different serum/PBS for 30 min. Coverslides were incubated with appropriate roles of FL BARD1 and cancer-associated isoforms in mitosis. We antibodies for 1 h at RT in 1% FCS/PBS, washed, and stained with 4¶,6- show that FL BARD1 and the RING finger–deficient p82 isoform diamidino-2-phenylindole for 3 min. Coverslips were mounted using BARD1h have different functions and protein-protein interaction fluorogard solution and analyzed under a Nikon epifluorescence micro- properties. FL BARD1 interacts with BRCA1 and is involved in scope, and images were captured with a 3.3-megapixel CCD camera. Images Aurora B ubiquitination and degradation during mitosis. In were processed with Metamorph software (Visitron). Primary antibodies contrast, BARD1h stabilizes Aurora B and forms a complex with used were BRCA1 (Ab-1; Calbiochem), BARD1 (H-300; Santa Cruz), BARD1 BRCA2 and Aurora B, proteins known to be involved in midbody (BL518; Bethyl Laboratories), Aurora B (AIM-1; BD Biosciences). PVC was formation (16, 17). These findings establish an unexpected link reported previously (2). Antibody p25 was produced in rabbits against between BARD1 and Aurora B and BRCA2, which are aberrantly peptide sequence MVAVPGTVAPRC encoded in alternative open reading frame (ORF) of exon 1. Antibody specificity was probed in inversed ELISAs overexpressed or mutated in cancer. We suggest a molecular with different peptides and on human cancer cell lines (data not shown). pathway that explains the function of FL BARD1 as tumor Immunoprecipitation and Western blot. Protein lysates used for suppressor together with BRCA1 and loss of FL BARD1, but up- immunoprecipitation were prepared from HeLa cells, unsynchronized and regulated expression of RING finger–deficient isoforms with synchronized in G1-S or G2-M. At 24 h after plating, cells were arrested in S proproliferative functions, in cancer. phase using 2 mmol/L thymidine for 18 h, released from the arrest for 9 h, arrested a second time using thymidine (2 mmol/L) for 18 h (G1-S), and released from the arrest for 5 h (G2-M). Materials and Methods Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer [50 mmol/L Tris (pH 8), 150 mmol/L NaCl, 2 mmol/L EDTA, 0.5% NP40, Plasmid constructs. BRCA1 and BARD1 small interfering RNA (siRNA) 10% glycerol] supplemented with protease inhibitors (complete EDTA-free, constructs were generated by annealing complementary oligonucleotides Roche Applied Science). Protein concentrations were determined by the and inserting them into the pSuperScript vector as BglII/HindIII fragment: Bradford procedure (Bio-Rad). Lysates containing 400 Ag of protein were human BRCA1-siRNA