Molecular and Cellular Pathobiology Deubiquitination of g-Tubulin by BAP1 Prevents Chromosome Instability in Breast Cancer Cells

Microtubule nucleation requires the g-tubulin ring complex, and during the M-phase (mitosis) this complex accumulates at the centrosome to support mitotic spindle formation. The posttranslational modification of g-tubulin through ubiquitination is vital for regulating microtubule nucleation and centrosome duplication. Blocking the BRCA1/BARD1-dependent ubiquitination of g-tubulin causes centrosome amplification. In the current study, we identifiedBRCA1-associated protein-1 (BAP1) as a deubiquitination enzyme for g-tubulin. BAP1 was downregulated in metastatic adenocarcinoma breast cell lines compared with noncancerous human breast epithelial cells. Furthermore, low expression of BAP1 was associated with reduced overall survival of patients with breast cancer. Reduced expression of BAP1 in breast cancer cell lines was associated with mitotic abnormalities. Importantly, rescue experiments including expression of full length but not the catalytic mutant of BAP1 reduced ubiquitination of g-tubulin and preventedmitotic defects. Our study uncovers a newmechanism for BAP1 involved in deubiquitination of g-tubulin, which is required to prevent abnormal mitotic spindle formation and genome instability. Cancer Res; 74(22); 6499–508. 2014 AACR. Introduction Posttranslational modification of proteins by covalent attachment of ubiquitin controls many essential cellular processes by targeting the proteins for assembly into complexes, transport, and degradation (1, 2). The type of polyubiquitin chains, including lysine 48 (K48) versus lysine 63 (K63), or monoubiquitination can induce different signaling pathways that regulate different aspects of cellular functions. The most prominent function of polyubiquitination chains through K48 linkage degrades the substrate through the mechanism of the proteasome, whereas K63-linked ubiquitin chains add new functional properties to the modified protein, except for proteosomal degradation (1, 2). Monoubiquitination or multiple monoubiquitination generally leads to transcription regulation as well as DNA repair and cell-cycle progression (1, 2). The action of ubiquitin ligation enzymes responsible for the attachment of the ubiquitin moieties to the substrate is reversed by the action of deubiquitination enzymes (DUB; ref. 3). The human genome encodes a large number of putative DUBs, and accumulating evidence indicates that most of these enzymes regulate a limited number of proteins (4). The dysregulation of DUB function is highly related to human diseases, especially cancer, with examples of hyperactivation or inactivation due to genetic or epigenetic changes. In this aspect, it is vital to identify substrate-specific DUBs to understand the signaling pathways that are dysregulated in cancer cells. Although it is becoming increasingly clear that DUBs play critical roles in various cellular pathways, physiologic roles for most of the human DUBs remain unknown. BRCA1-associated protein-1 (BAP1) is a nuclear-localized DUB that was originally identified as a BRCA1-binding protein in a yeast two-hybrid screen (5). BAP1 is ubiquitously expressed in different human tissues, especially in the testis, placenta, and ovaries, with varying levels detected in other tissues, including human breast. In mice, BAP1 expression is upregulated in the breast during puberty and pregnancy (6). BAP1 belongs to the ubiquitin C-terminal hydrolase (UCH) family of DUBs, and the activity of BAP1 is restricted to the Nterminal UCH domain, which executes binding and cleavage of the ubiquitin isopeptide bond (5). The BAP1 gene is frequently mutated in lung, breast, melanoma, and renal cell carcinoma (5, 7) and overexpression of BAP1 in lung cancer cells reduces both tumor formation inmice and cell growth in culture. It has thus been suggested that BAP1 acts as a tumor suppressor gene (6). The tumor suppressor property of BAP1 is dependent on its nuclear localization and deubiquitin activity (8). In MCF7 breast cancer cell lines, it was shown that the binding of BAP1 to breast cancer type 1 susceptibility protein (BRCA1) enhances BRCA1-mediated growth suppression (5). However, in contrast to this result, it has been shown that growth suppression is independent of BRCA1 and that BAP1 disrupts the BRCA1/BRCA1-associated ring domain 1 (BARD1) heterodimer, but cannot reverse its autoubiquitination (6, 9, 10). Department of LaboratoryMedicine, Translational Cancer Research, Lund University, Medicon Village, Lund, Sweden. Department of Clinical Sciences,SectionofOncology-Pathology, LundUniversity, Lund,Sweden. Department of Oncology, Ska ne University Hospital, Lund, Sweden. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Ramin Massoumi, Department of Laboratory Medicine, Molecular Tumor Pathology, Lund University, Medicon village, Scheelev€ agen 8, SE-223 63 Lund 33381, Sweden. Phone: 0046-462226430; Fax: 0046-4033-7063; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-14-0221 2014 American Association for Cancer Research. Cancer Research www.aacrjournals.org 6499 on July 22, 2017. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst September 16, 2014; DOI: 10.1158/0008-5472.CAN-14-0221