ORIGINAL ARTICLE Clinicopathologic significance of BubR1 and Mad2 overexpression in oral cancer JH Teixeira 1 , PMA Silva 1,2 , J Faria 1 , I Ferreira 1 , P Duarte 1 , ML Delgado 1 , O Queir os 1,3 , R Moreira 1,3 , J Barbosa 1 , CA Lopes 4 , JB do Amaral 5 , LS Monteiro 1, *, H Bousbaa 1,6,7, * 1 Instituto de Investigac ß~ ao e Formac ß~ ao Avanc ßada em Ci ^ encias e Tecnologias da Sa ude, CESPU, Gandra PRD; 2 Centre for Molecular and Structural Biomedicine, CBME/IBB, University of Algarve, Faro; 3 CBMA - Center of Molecular and Environmental Biology, Department of Biology, University of Minho, Braga; 4 Molecular Pathology and Immunology Department, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto; 5 Stomatology Department, Hospital de Santo Ant onio, Centro Hospitalar do Porto, Porto; 6 Centro de Qu  ımica Medicinal da Universidade do Porto (CEQUIMED-UP), Porto; 7 Centro Interdisciplinar de Investigac ß~ ao Marinha e Ambiental (CIIMAR/CIMAR), Universidade do Porto, Porto, Portugal OBJECTIVES: BubR1 and Mad2 are central components of the mitotic checkpoint complex that inhibits ana- phase onset until all chromosomes are correctly aligned at the metaphase plate. We propose to analyse the combined expression of BubR1 and Mad2 and assess its significance to oral squamous cell carcinoma (OSCC) diagnosis and prognosis. MATERIALS AND METHODS: BubR1 and Mad2 expression was assessed by real-time PCR in OSCC cell lines and in normal human oral keratino- cytes, and by immunohistochemistry in 65 patients with OSCC. The results were compared regarding clinicopathological parameters, proliferative activity and survival. RESULTS: BubR1 and Mad2 transcripts were overex- pressed in OSCC cell lines which also exhibited attenu- ated spindle assembly checkpoint activity. BubR1 and Mad2 were also overexpressed in patients with OSCC. BubR1 expression was associated with advanced stages and larger tumour size in univariate analysis, and with shorter overall survival both in univariate and multivari- ate analysis. Mad2 overexpression was associated with that of BubR1 and, importantly, high expression of Mad2 and BubR1 was associated with increased cellular proliferation. CONCLUSION: Our data propose a role for BubR1 and Mad2 in OSCC cellular proliferation, progression and prognosis. Oral Diseases (2015) doi: 10.1111/odi.12335 Keywords: oral cancer; BubR1; Mad2; spindle assembly checkpoint; proliferation; prognosis Introduction Despite advances in detection and therapy modalities, oral squamous cell carcinoma (OSCC) remains the most com- mon head and neck cancer worldwide, with a survival rate still dismally low (Neville and Day, 2002; Jemal et al, 2011). There is a strong interest in understanding the molecular mechanism underlying oral cancer development and progression and identifying specific biomarkers for early detection, effective prognosis, better management and improved treatment outcomes of OSCC. Chromosomal instability (CIN) plays a key role in tumorigenesis and is often found in OSCC, with complex structural and numerical variations (Lengauer et al, 1998; Bockmuhl and Petersen, 2002). In OSCC cell lines, it was reported that abnormal karyotype arises from defects in chromosome segregation, frequently in the form of lag- ging chromosomes (Saunders et al, 2000). In normal cycling cells, errors in chromosome attachments to the mitotic spindle activate the spindle assembly checkpoint (SAC) which prevents anaphase onset until the attachment errors are corrected (Logarinho and Bousbaa, 2008; Silva et al, 2011). This SAC activity is mediated by the mitotic checkpoint complex (MCC) that forms between the SAC proteins BubR1, Mad2 and Bub3 in association with the Cdc20 protein, an activator of the anaphase promoting complex or cyclosome (APC/C Cdc20 ) (Sudakin et al, 2001). The APC/C Cdc20 is thus inactivated and unable to target key mitotic proteins such as securin and cyclin B for degradation by the proteasome, and the cells are retained in mitosis. Several studies reported that defects in SAC activity are linked to an increased rate of aneuploidization during Correspondence: Hassan Bousbaa, Instituto de Investigac ß~ ao e Formac ß~ ao Avanc ßada em Ci^ encias e Tecnologias da Sa ude, CESPU, Rua Central de Gandra, 1317, 4585-116 Gandra PRD, Portugal. Tel: +351 224157186, Fax: +351 224157102, E-mail: hassan.bousbaa@iscsn.cespu.pt *These authors contributed equally to this work. Received 16 January 2015; revised 1 March 2015; accepted 1 March 2015 Oral Diseases (2015) doi:10.1111/odi.12335 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd All rights reserved www.wiley.com