ORIGINAL ARTICLE An integrated genomic approach identifies ARID1A as a candidate tumor-suppressor gene in breast cancer A Mamo 1,9 , L Cavallone 2,9 , S Tuzmen 3 , C Chabot 1 , C Ferrario 1 , S Hassan 1 , H Edgren 4,5 , O Kallioniemi 4,5 , O Aleynikova 6 , E Przybytkowski 1 , K Malcolm 1 , S Mousses 3 , PN Tonin 2,7,8 and M Basik 1 1 Department of Oncology, Lady Davis Institute, Sir Mortimer B Davis Jewish General Hospital, McGill University, Montreal, Que´bec, Canada; 2 Department of Human Genetics, McGill University, Montreal, Que´bec, Canada; 3 Pharmaceutical Genomics Division, The Translational Genomics Research Institute, Scottsdale, AZ, USA; 4 University of Helsinki, Institute for Molecular Medicine (FIMM), Helsinki, Finland; 5 VTT Technical Research Centre of Finland and University of Turku, Medical Biotechnology, Turku, Finland; 6 Department of Pathology, Jewish General Hospital, Montreal, Que´bec, Canada; 7 Department of Medicine, McGill University, Montreal, Que´bec, Canada and 8 The Research Institute of the McGill University Health Centre, Montreal, Que´bec, Canada Tumor-suppressor genes (TSGs) have been classically defined as genes whose loss of function in tumor cells contributes to the formation and/or maintenance of the tumor phenotype. TSGs containing nonsense mutations may not be expressed because of nonsense-mediated RNA decay (NMD). We combined inhibition of the NMD process, which clears transcripts that contain nonsense mutations, with the applica- tion of high-density single-nucleotide polymorphism arrays analysis to discriminate allelic content in order to identify candidate TSGs in five breast cancer cell lines. We identified ARID1A as a target of NMD in the T47D breast cancer cell line, likely as a consequence of a mutation in exon-9, which introduces a premature stop codon at position Q944. ARID1A encodes a human homolog of yeast SWI1, which is an integral member of the hSWI/SNF complex, an ATP- dependent, chromatin-remodeling, multiple-subunit enzyme. Although we did not find any somatic mutations in 11 breast tumors, which show DNA copy-number loss at the 1p36 locus adjacent to ARID1A, we show that low ARID1A RNA or nuclear protein expression is associated with more aggressive breast cancer phenotypes, such as high tumor grade, in two independent cohorts of over 200 human breast cancer cases each. We also found that low ARID1A nuclear expression becomes more prevalent during the later stages of breast tumor progression. Finally, we found that ARID1A re- expression in the T47D cell line results in significant inhibition of colony formation in soft agar. These results suggest that ARID1A may be a candidate TSG in breast cancer. Oncogene (2012) 31, 2090–2100; doi:10.1038/onc.2011.386; published online 5 September 2011 Keywords: ARID1A; nonsense-mediated mRNA decay; tumor-suppressor gene; breast cancer Introduction Cancers occur as a result of dysregulation in the function of oncogenes and tumor-suppressor genes (TSGs). TSGs have been classically defined as genes whose loss of function in tumor cells contributes to the formation and/or maintenance of the tumor phenotype (Presneau et al., 2003). Various mechanisms of inactiva- tion of TSGs have been reported and can include deletion of one allele through chromosomal non- dysjunction and/or intra-chromosomal rearrangement, and mutational inactivation of the remaining allele (Presneau et al., 2003). TSGs have been identified in cancer both as a result of a search for germline mutations in hereditary cancers and the molecular characterization of somatic deletions and mutations in tumor tissue RNA and DNA (Vogelstein and Kinzler, 2004). However, TSGs containing nonsense mutations may not be detectable using RNA-based next-genera- tion re-sequencing because RNA containing such aberrant transcripts is eliminated by the nonsense- mediated RNA decay process (NMD) (Noensie and Dietz, 2001). Indeed, an interesting strategy to discover potential TSG candidates was elaborated by Noensie and Dietz (2001) and adapted by Huusko et al. (2004). Noensie and Dietz (2001) proposed that it would be possible to discover nonsense mutation containing mRNA by inhibiting the NMD process. In this way, transcripts containing nonsense mutations are stabilized and selectively increase in quantity relative to non- mutated transcripts. Huusko et al. (2004) then combined NMD inhibition with array comparative genomic hybridization (aCGH) in order to enrich for the selection of candidates mapping to deleted regions in prostate cancer cell lines, thereby identifying EPHB2 as a novel candidate TSG in prostate cancer. We have adapted the approach of Huusko et al. by integrating the allelic content inferred from high-density genotyping single-nucleotide polymorphism (SNP) arrays to identify candidates TSGs in five breast cancer cell lines. Here we report the discovery of ARID1A as a candidate TSG in the T47D breast cancer cell line by Received 16 February 2011; revised 6 July 2011; accepted 26 July 2011; published online 5 September 2011 Correspondence: Dr M Basik, Department of Oncology, Lady Davis Institute, Sir Mortimer B Davis Jewish General Hospital, McGill University, 3755 Cote Ste-Catherine, Montre´ al, Que´ bec, Canada H3T 1E2. E-mail: markbasik@gmail.com 9 These authors contributed equally to this work as first co-authors. Oncogene (2012) 31, 2090–2100 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc